Giao Trinh Cong Nghe Te Bao

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Li ni u
Cng ngh t bo l mt b phn quan trng ca cng ngh sinh hc, ch yu nghin cu cc
qu trnh nui cy t bo ng-thc vt v vi sinh vt sn xut sinh khi, sn xut cc hp cht c
hot tnh sinh hc (enzyme, vaccine, cc cht th cp), lm m hnh thc nghim kho st cc tc
ng ca ho cht, lm nguyn liu ghp t bo v c quan
Mc d, cc k thut nui cy t bo ch c pht trin vo na u th k 20, nhng n nay
cc ng dng ca chng c nhng bc tin vt bc nh s ng gp ca cng ngh DNA ti t
hp.
Bn cnh cc gio trnh nh: sinh hc phn t, nhp mn cng ngh sinh hc, cng ngh DNA
ti t hp, cng ngh chuyn gen gio trnh cng ngh t bo s gip sinh vin tip cn thm mt
lnh vc khc ca cng ngh sinh hc thng qua vic cung cp nhng kin thc c bn v cc vn
sau:
- Sinh trng v ng hc sinh trng ca t bo.
- Thit k cc h ln men.
- Nui cy t bo v cc ng dng ca chng.
Gio trnh cng ngh t bo c bin son theo hng kho st mt qu trnh sinh hc mang
tnh cng ngh nhiu hn c l qu trnh ln men ng dng cho c t bo vi sinh vt, ln t bo
ng-thc vt trong cc thit b nui cy (bioreactor/fermenter). Do , mt s ng dng khc ca cc
k thut nui cy m v t bo ni chung chng ti khng a vo gio trnh ny.
Lnh vc cng ngh t bo rt rng v a dng, hn na gio trnh ny mi c xut bn ln
u tin nn kh trnh khi thiu st hoc cha p ng c yu cu bn c. V th, chng ti rt
mong nhn c nhiu kin ng gp ln xut bn sau c hon thin hn.
Tc gi
Phan Thanh Quyn - http://violet.vn/phanthanhquyen
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Chng 1
M u
I. Cng ngh sinh hc
n nay c rt nhiu nh ngha v cch din t khc nhau v cng ngh sinh hc ty theo
tng tc gi v t chc. Tuy nhin, cng ngh sinh hc (biotechnology) c th c nh ngha
mt cch tng qut nh sau:
Cng ngh sinh hc l cc qu trnh sn xut quy m cng nghip m nhn t tham gia
trc tip v quyt nh l cc t bo sng (vi sinh vt, thc vt v ng vt). Mi t bo sng ca
c th sinh vt hot ng trong lnh vc sn xut ny c xem nh mt l phn ng nh.
Nu cng ngh sinh hc c nh ngha theo hng trn th n khng th c tha nhn l
mt lnh vc khoa hc mi. Bi v, t xa xa loi ngi bit s dng cc vi sinh vt ln men
bnh m v thc phm, cho d h khng bit c ch ca nhng bin i sinh hc ny l nh th no.
Loi ngi cng bit t rt lu vic lai to ng vt v thc vt ci thin nng sut vt nui v
cy trng c tt hn. V th, cng ngh sinh hc c nh ngha nh trn c xem nh cng ngh
sinh hc truyn thng.
Tuy nhin, trong nhng nm gn y thut ng cng ngh sinh hc thng c s dng nhm
cp n nhng k thut mi nh DNA ti t hp v dung hp t bo, v c xem l lnh vc cng
ngh sinh hc hin i.
1. Cng ngh DNA ti t hp (DNA recombinant technology)
L nhng k thut cho php thao tc trc tip nguyn liu di truyn ca cc t bo ring bit, c
th c s dng pht trin cc vi sinh vt sn xut cc sn phm mi cng nh cc c th hu ch
khc. Nhng k thut ny cn c gi l k thut di truyn (genetic engineering), cng ngh di
truyn (genetic technology), thao tc gen (gene manipulation), k thut gen (gene engineering) hay
cng ngh gen (gene technology)... Mc tiu chnh ca cng ngh DNA ti t hp l gn mt gen
ngoi lai (foreign gene) m ha cho mt sn phm mong mun vo trong cc dng DNA mch vng
(plasmid vector) v sau a chng vo trong mt c th vt ch, sao cho gen ngoi lai c th biu
hin sn xut sn phm ca n t c th ny.
2. Dung hp t bo (cell fusion)
L qu trnh hnh thnh mt t bo lai n (single hybrid cell) vi nhn v t bo cht t hai loi
t bo ring bit t hp cc c im mong mun ca c hai loi t bo ny. Chng hn, cc t bo
c bit ca h thng min dch c th sn xut ra cc khng th hu ch. Tuy nhin, cc t bo ny
thng kh nui cy v tc sinh trng ca chng rt chm. Mt khc, cc t bo khi u nht nh
no c cc c im bt t v phn chia nhanh. Bng cch dung hp hai t bo ny, mt t bo lai
hybridoma c th c to ra mang c hai tnh trng trn. Cc khng th n dng (monoclonal
antibodies-Mabs) c sn xut t cc t bo lai, c dng chn on, iu tr bnh v tinh sch
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protein.
3. ng dng ca cng ngh sinh hc hin i
Cc ng dng ca cng ngh sinh hc hin i l rt nhiu (Bng 1.1). Cc dc phm him v
t trin trc y nh insulin cha bnh i tho ng, hormone sinh trng ngi iu tr
bnh ci ca tr em, interferon chng vim nhim, vaccine phng bnh v cc khng th n dng
dng chn on... c th c sn xut bng cc t bo c bin i di truyn hoc cc t bo lai
r tin vi s lng ln. Cc con ging sch bnh hoc kho mnh hn, cc vt nui dng lm thc
phm c sn lng cao c th c pht trin, cc loi cy trng quan trng c th c bin i di
truyn c cc tnh trng chng chu stress, chng chu cht dit c v khng cn trng. Hn na,
cng ngh DNA ti t hp c th c ng dng pht trin cc vi sinh vt c bin i di truyn
(genetically modification) sao cho chng c th sn xut cc hp cht ha hc khc nhau vi sn lng
cao hn cc vi sinh vt bnh thng.
Bng 1.1. Cc ng dng ca cng ngh sinh hc hin i.
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II. Cng ngh t bo
Cc cng ngh DNA ti t hp hoc dung hp t bo c khi u bi nhng nghin cu thun
ty v cc kt qu cui cng c th pht trin thnh mt loi t bo mi c th sn xut sn phm vi
s lng t i qui m phng th nghim. Tuy nhin, cc kt qu ni trn li rt c ngha thng mi
v v th n i hi phi pht trin thnh quy trnh cng nghip vi mt cng ngh kh thi v c hiu
qu kinh t. pht trin mt qu trnh sn xut quy m phng th nghim thnh mt quy trnh cng
nghip ln, chng ta khng th ch n thun tng kch thc ca bnh nui cy (vessel) ln.
V d: quy m phng th nghim l 100 mL, mt bnh tam gic nh nui trn mt my lc l
phng thc l tng nui cy t bo. Nhng i vi hot ng quy m ln 2.000 L, chng ta
khng th s dng mt bnh nui khc c th tch ln hn v lc n, m cn phi thit k mt h ln
men (fermenter) hay cn gi l ni phn ng sinh hc (bioreactor) hiu qu nui cy t bo trong
nhng iu kin ti u nht. V th, cng ngh t bo (mt trong nhng lnh vc chnh ca cng ngh
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sinh hc) c vai tr rt quan trng trong thng mi ha cc sn phm ca n.
minh ha vai tr ca cng ngh t bo, c th xem mt qu trnh sinh hc c trng bao gm
cc t bo vi khun nh trnh by hnh 1.1. Cc nguyn liu th (thng l sinh khi) c x l v
trn vi cc thnh phn cn thit khc t bo c th sinh trng tt trong mt hn hp dch lng,
mi trng nui cy c kh trng loi b tt c cc c th sng v a vo bnh nui cy hnh tr
ln, thit b c trng vi cnh khuy, vch ngn, h thng thng kh v cc b phn cm bin iu
chnh cc iu kin ln men. Mt chng vi sinh vt thun khit c a vo trong mt bnh nui cy.
Cc t bo khi u sinh sn theo hm m sau mt thi gian nht nh ca pha lag v t ti nng
t bo cc i khi mi trng b s dng ht. S ln men s dng li v cc thnh phn s c ht
ra thu hi sn phm v tinh sch chng. Qu trnh ny c hot ng theo kiu ln men m (batch
culture) hoc lin tc (continuous culture).
Khi tin hnh mt qu trnh sinh hc (bioprocessing) trn quy m ln cn lu :
- Phi thu c cc cht xc tc sinh hc tt nht (vi sinh vt, t bo ng vt, t bo thc vt,
hoc enzyme) cho mt qu trnh mong mun.
- To ra mi trng tt nht c th cho s xc tc bng cch thit k cc bioreactor/fermenter
thch hp v cho n hot ng trong mt phng thc ti u.
- Phn tch cc sn phm mong mun t hn hp phn ng trong mt phng thc kinh t nht.
Cc nhim v t ra bao gm thit k v pht trin mt qu trnh sinh hc. Cc vn c bn
c i hi cho cng vic ny nh sau:
Hnh 1.1. Mt qu trnh sinh hc c trng.
1. Chng ta mong i thay i ci g
tr li cu hi ny, cn phi c nhng hiu bit v cc khoa hc c bn ca qu trnh cng
ngh. l vi sinh vt hc, ha sinh hc, di truyn hc, sinh hc phn t... Chng ta cn phi tm hiu
cc vn ny trong mt phm vi nht nh. iu quan trng y l cc cht xc tc sinh hc c
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chn lc hoc sa i di truyn phi thch hp cho cc hot ng sn xut quy m ln.
2. Qu trnh sinh hc xy ra vi mt tc nh th no
Nu mt qu trnh nht nh c th sn xut mt sn phm, th iu quan trng cn bit l qu
trnh s xy ra vi tc nh th no. ng hc ca qu trnh s chi phi cc tc phn ng di
nh hng ca cc iu kin vt l v ha hc nht nh. Chng ta cn nm vng ha ng hc
(chemical kinetics) thit k ni phn ng (reactor) thch hp. Cc k thut tng t c ng dng
gii quyt ng hc enzyme (enzyme kinetics) hoc ng hc t bo (cell kinetics). thit k mt
h ln men hiu qu cho cc cht xc tc sinh hc hot ng, iu quan trng cn bit l tc phn
ng b nh hng nh th no bi cc iu kin hot ng khng ging nhau. iu ny bao gm c
nghin cu v nhit ng hc (thermodynamics), cc hin tng vn chuyn, cc tng tc sinh hc,
kh nng n nh ca cc dng t bo vi sinh vt (hoc t bo ng vt v thc vt) dng lm nguyn
liu sn xut...
3. H thng c hot ng v iu chnh nh th no t c hiu sut ti a
s hot ng v iu chnh h thng c ti u, chng ta cn phi pht trin cc b cm
bin trc tuyn (on-line sensor) chnh xc. Thut ton ti u trc tuyn cn c xy dng v ti u
ha tng cng kh nng hot ng ca cc qu trnh sinh hc v m bo rng nhng qu trnh
ny c hot ng mt cch kinh t nht.
4. Cc sn phm c phn tch nh th no c c s tinh sch cc i v gi thnh ti thiu
i vi bc ny, qu trnh bio-downstream (phn tch sinh hc), chng ta c th s dng cc
k thut phn tch khc nhau c pht trin trong cc qu trnh ha hc nh chng ct, hp th, tch
chit, hp ph, sy kh, lc, kt ta v ngm chit. Hn na, song song vi cc k thut phn tch tiu
chun ny, chng ta cn thit pht trin cc k thut mi thch hp phn tch cc nguyn liu sinh
hc. Nhiu k thut c pht trin phn tch hoc phn tch cc nguyn liu sinh hc quy m
phng th nghim, nh l sc k (chromatography), in di (electrophoresis) v thm tch (dialysis).
Cc k thut ny cn c nghin cu thm sao cho chng c th hot ng hiu qu trn quy m
cng nghip.
III. Qu trnh sinh hc
Cc ng dng cng nghip ca cc qu trnh sinh hc l s dng cc t bo sng hoc thnh
phn ca chng thc hin nhng thay i vt l v ha hc. So vi cc qu trnh ha hc truyn
thng, cc qu trnh sinh hc c nhng u im v nhc im nh sau:
1. Cc u im
- iu kin phn ng nh nhng. iu kin phn ng cho cc qu trnh sinh hc l nh nhng-
n ha. c trng l nhit phng, p sut kh quyn v pH mi trng kh trung tnh. Kt qu, s
hot ng t nguy him v iu kin sn xut t phc tp hn so vi cc qu trnh ha hc c bit.
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- Tnh c hiu. Mt cht xc tc enzyme c tnh c hiu cao v xc tc ch mt hoc mt s t
cc phn ng ha hc. S a dng ca cc enzyme hin c c th xc tc cho mt phm vi rt rng cc
phn ng khc nhau.
- Tnh hiu lc. Tc ca mt phn ng c xc tc bng enzyme thng nhanh hn nhiu
so vi khi phn ng ny thc hin nh cc cht xc tc khng phi sinh hc. Ch mt lng nh
enzyme c yu cu cng sn xut mt hiu qu mong mun.
- Cc ti nguyn c th i mi. Nguyn liu th ch yu ca cc qu trnh sinh hc l sinh
khi (biomass) cung cp c b khung carbon ln nng lng cn cho s tng hp cc ha cht hu c.
- Cng ngh DNA ti t hp. L nhng k thut sa i h thng di truyn nhm nng cao
nng sut sinh hc. S pht trin ca nhng k thut ny ha hn cc kh nng khng l ci thin
cc qu trnh sinh hc.
2. Cc nhc im
- Cc hn hp sn phm phc tp. Trong cc trng hp nui cy t bo (vi sinh vt, thc vt
hoc ng vt). Cc phn ng a enzyme xy ra trong mt chui tun t hoc song song, hn hp sn
phm cui cng cha khi lng t bo, nhiu sn phm trao i cht ph, v mt phn cn li ca cc
cht dinh dng ban u. Khi lng t bo cng cha cc thnh phn khc nhau ca t bo.
- Cc mi trng nc long. Cc thnh phn c gi tr thng mi ch c sn xut vi mt
lng nh trong mi trng nc nn s phn tch chng l rt t tin. Bi v cc sn phm ca cc
qu trnh sinh hc thng mn cm vi nhit, do cc k thut phn tch truyn thng khng th s
dng m phi pht trin cc k thut phn tch mi cho cc mc ch sn xut trn quy m ln.
- S nhim bn. H thng ln men c th d dng b nhim bn, do nhiu vi khun v nm mc
c th sinh trng rt mnh trong hu ht cc mi trng nui cy. Vn tr nn kh khn hn khi
nui cy t bo ng vt v thc vt bi v chng cn mt thi gian sinh trng di ngy v tc
sinh trng ca chng chm hn rt nhiu so vi tc sinh trng ca vi khun v nm mc trong
mi trng nhim bn.
- Khuynh hng hay bin i. Cc t bo c khuynh hng t bin do s thay i mi trng
v c th mt i mt vi c im gy thit hi cho s thnh cng ca qu trnh sn xut. Cc enzyme
tng i mn cm hoc l cc phn t khng n nh v i hi s cn thn trong khi s dng chng.
IV. nh ngha s ln men
Thng thng, s ln men (fermentation) c nh ngha l qu trnh sn xut ethanol hoc
lactic acid t glucose (C
6
H
12
O
6
).
- Qu trnh sn xut ethanol. L qu trnh m mt s nm men phn gii cc loi ng trong
mi trng ym kh sn xut ru ethanol.
- Qu trnh sn xut lactic acid. L qu trnh m mt s enzyme nh lactodehydrogenase phn
gii cc cht trung gian nh NADH (trong ng phn ym kh) thnh lactic acid ch khng thnh
ethanol. Ln men lactic c dng trong cng ngh ch bin sa lm phomt v sa chua.
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Tuy nhin, ngy nay ngi ta m rng nh ngha cho khi nim ny nh sau: Ln men l
qu trnh s dng cc enzyme bin i nhng hp cht hu c theo Websters New College
Dictionary (A Merriam-Webster 1977) v y l nh ngha m chng ti s dng trong gio trnh ny
dng m t cc qu trnh nui cy cc t bo vi sinh vt, ng vt v thc vt trong cc h ln men
hay cc ni phn ng sinh hc.
Ti liu tham kho/c thm
1. Atkinson B and Mavituna F. 1991. Biochemical Engineering and Biotechnology Handbook. 2
nd
ed.
Stockton Press, New York, USA.
2. Flickinger MC and Drew SW. 1999. Encyclopedia of Bioprocess Technology: Fermentation,
Biocatalysis and Bioseparation. John Wiley & Sons, New York, USA.
3. Lee JM. 2001. Biochemical Engineering. Prentice Hall, Inc. USA.
4. Ratledge C and Kristiansen B. 2002. Basic Biotechnology. Cambridge University Press, UK.
5. Shuler ML and Kargi F. 2002. Bioprocess Engineering-Basic Concepts. 2
nd
ed. Prentice Hall, Inc.
New Jersey, USA.
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Chng 2
Sinh trng v bt ng ca t bo
I. Xc nh sinh trng ca t bo
Trong cc h thng sinh hc, mi s sinh trng u c th c nh ngha l s tng tun t
ca cc thnh phn ha hc. Tng n thun khi lng khng th phn nh y s sinh trng, do
t bo c th ch tng hm lng cc sn phm d tr ca chng nh l glycogen, poly--
hydroxybutyrite. S sinh trng cn bng (balanced growth) c nh ngha l s sinh trng m
trong sut qu trnh s nhn i sinh khi xy ra cng vi s nhn i ca tt c cc c tnh xc
nh khc ca qun th nh l protein, DNA, RNA v nc ni bo. Mt khc, qu trnh nui cy tri
qua s sinh trng cn bng duy tr mt thnh phn ha hc khng i. Trong mt mi trng dinh
dng thch hp m sau t bo s tr nn thch nghi th n s trong trng thi sinh trng cn
bng.
Tip theo qu trnh sinh trng, cn phi xc nh s lng t bo. S sinh trng ca t bo c
th c xc nh bng s lng t bo, sinh khi t bo hoc hot tnh t bo.
1. Xc nh s lng t bo
1.1. m bng knh hin vi
S lng t bo trong qun lc c th c m di knh hin vi bng cch m cc t bo
c a vo trong mt bung m c bit. C hai loi bung m c dng m s lng t
bo trong mt mu dch lng:
- Haemocytomete. Bung m t bo mu dng cho nhng t bo c ng knh 3 m (Hnh
2.1).
- Petroff-Hausser counting chamber. Bung m Petroff-Hausser c dng ch yu cho vi
khun.
C hai loi bung m c cc ng k vung c trng trn b mt ca tm knh (lam knh).
Khung trn mi mt ca tm m (grid) gi mt tm knh ph (cover glass) cch tm m mt
khong cch bit (v d: 0,1 mm) sao cho th tch ca vung c bit chnh xc. Mt mu dch
huyn ph t bo cn m c cho chy qua di tm ph v lm y bung m. Sau , m s
lng t bo trn mt n v din tch ca ng k di knh hin vi. Cc dch huyn ph m c
cng c th m c nu chng c pha long thch hp.
Mt s u im ca phng php m trc tip:
- Ch cn cc thit b ti thiu.
- Cc kt qu thu c nhanh.
- C th quan st cc c im hnh thi ca c th.
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Hnh 2.1. Bung m haemocytometer.
Mt s nhc im ca phng php m trc tip:
- Thng rt kh phn bit cc t bo cht v t bo sng.
- Khng thch hp cho cc dch huyn ph c mt thp.
- Cc t bo c kch thc nh thng kh quan st di knh hin vi v c th khng thy khi
m.
- Phng php m thc t gy mi mt v nhm ln trong qu trnh m.
- Khng thch hp i vi cc t bo xp thnh cm nh l mycelium (th si nm).
1.2. m cc t bo pht trin trn a nui cy (petri dish)
Cc t bo pht trin c nh ngha l t bo c th phn chia v to ra khun lc. C hai cch
thc hin phng php m trn a petri: phng php a tri (spread plate) v phng php a
rt (pour plate).
Vi phng php a tri, mt th tch nh hn 100 L c tri khp b mt agar. Vi phng
php a rt, mu vt c trn vi agar nng chy ( ngui n khong 60
o
C) v c rt trn a
v trng. Cc a sau c nui cho n khi xut hin khun lc, v s lng khun lc c m
trc tip. iu quan trng l s lng cc khun lc pht trin trn a phi khng qu ln hoc khng
qu nh. thu c mt s lng t bo thch hp trn mt n v din tch th mu vt phi c
pha long. Trng hp cn pha long nhiu, ngi ta thng s dng k thut pha long tun t (serial
dilution). V d: thc hin pha long 1/10
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, th c th thc hin ba ln pha long lin tip 1/100
hoc su ln pha long lin tip 1/10.
1.3. m bng my m
trnh s n iu khi m trc tip bng knh hin vi, c th s dng phng php m bng
my m. K thut ny cho php khng ch m c s lng t bo, m cn o c kch thc t
bo. Nhc im ca phng php ny l n khng th phn bit gia t bo v cc phn t bn khc.
K thut ny cng kh s dng vi cc c th dng chui v khng em li kt qu tt vi cc c th
dng h si (v d nh nm).
2. Xc nh sinh khi t bo
2.1. Trng lng kh ca t bo
Trng lng kh t bo c th c xc nh trc tip bng cch ly mt lng ti thiu ca dch
huyn ph t bo ly tm. Sau khi th ni, t bo c ra bng nc ct loi b tt c cc cht
ha tan. Dch huyn ph c ly tm mt ln na v cc t bo sau khi kt c li c sy kh trong
t sy v cn xc nh trng lng. y l phng thc trc tip nht xc nh s lng sinh
khi t bo. Tuy nhin, cch xc nh nh th tn nhiu thi gian v d b qua nhng thay i nh ca
sinh khi t bo. K thut ny ch c th s dng i vi nhng dch huyn ph dy c v t bo phi
c ra sch hon ton khi nhng cht ngoi sinh bm vo.
2.2. c ca dch huyn ph t bo
Sinh khi t bo cng c th c xc nh bng phng php quang hc thng qua xc nh
lng nh sng b tn x bi dch huyn ph t bo. K thut ny da trn c s lp lun rng cc phn
t nh tn x nh sng mt cch tng xng, trong cc gii hn nht nh, ti nng ca chng. Khi
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tia sng xuyn qua dch huyn ph ca t bo, th lng nh sng truyn qua b gim i do kt qu ca
s tn x, nh vy chnh l phng php xc nh mt t bo.
Vic o c ca dch huyn ph t bo thng c thc hin trn my quang ph c cc
n v hp th (A). Kh nng hp th (absorbency) c nh ngha l s logarithm ca t l gia
cng nh sng va chm vo dch huyn ph t bo (I
o
) v cng nh sng c truyn qua bi
dch huyn ph (I):
ng cong chun (standard curve) c th thu c bng cch o hp th (A) ca mu vi
nng t bo bit trc. Vic o thng c thc hin bc sng t 600-700 nm.
3. Cc phng php gin tip
Phng php gin tip xc nh sinh khi t bo da trn php tnh h s t lng ton phn
(overall stoichiometry) cho s sinh trng v to thnh sn phm, c th c trnh by trong mt
dng chung nh sau:
ngun carbon + ngun nitrogen + phosphate + O
2
sinh khi t bo + CO
2
+ H
2
O + sn phm + nhit
S thay i sinh khi t bo c th c kim sot gin tip bng cch xc nh s tiu th cht
dinh dng, to thnh sn phm, cc thnh phn t bo, gii phng nhit hoc cc tnh cht vt l khc
ca dch nui cy t bo.
3.1. S tiu th cht dinh dng
Cn chn mt cht dinh dng m cht ny khng bao gi c s dng tng hp sn phm
trao i cht. Phosphate, sulfate hoc magnesium c th l nhng chn la tt. Khi sinh khi t bo l
sn phm chnh, th nng ca ngun carbon cn li trong mi trng c th c o nh gi
sinh khi t bo.
3.2. To thnh cc sn phm
iu quan trng l cn kim tra sn phm to thnh c c kt hp vi s sinh trng hay
khng. Mt s sn phm c to thnh sau khi sinh khi t bo t ti pha tnh (stationary phase) ca
chu k sinh trng v v th, khng c kt hp vi s sinh trng. S gii phng CO
2
c th c
xc nh v n c quan h t lng i vi s sinh trng ca t bo. Mt sn phm hon ton chung
cho mi phn ng ln men l ion H
+
. Lng kim b sung vo dch ln men s duy tr pH thch hp
cho sinh trng.
3.3. Cc thnh phn t bo
i vi cc nui cy tri qua s sinh trng cn bng, th cc thnh phn t bo thuc nhm i
phn t nh l protein, RNA v DNA c th c xc nh thay cho sinh khi t bo. Tuy nhin, cn
phi thn trng do t l ca nhng nguyn liu ny trong t bo c th thay i theo thi gian nu nui
cy khng tri qua s sinh trng cn bng.
13
3.4. Gii phng nhit
S sinh trng ca t bo pht ra nhit. Lng nhit pht ra ty thuc vo hiu sut s dng
nng lng carbon. V th, vic xc nh nhit ca h ln men c th kt hp gin tip vi s sinh
trng ca t bo. Tuy nhin, lng nhit ton phn tch ly trong h ln men ph thuc vo hiu qu
phi hp ca cc ngun sinh nhit v mt nhit khc nhau nh: nhit t qu trnh khuy v bay hi,
nhit tiu hao xung quanh thnh ca h ln men v nhit nhy (sensible heat) trong lung khng kh.
V th, xc nh sinh trng bng s gii phng nhit, cn phi thit lp cn bng nng lng ca
h ln men mc d y mt cng vic khng d dng.
3.5. nht
Sinh trng ca c th h si (nm) hoc s to thnh polysaccharide lm tng nht ca
dch ln men (fermentation broth). V th, vic xc nh nht ca dch ln men rt hu ch trong cc
qu trnh ln men quy m cng nghip. nht biu kin o c tc dch chuyn c nh c
th c dng nh gi nng t bo hoc nng sn phm.
II. Bt ng t bo
Phng php bt ng (immobilization) cc t bo hon chnh c nhiu u im hn cc k
thut nui cy truyn thng. Bng cch bt ng t bo, vic thit k quy trnh n gin hn khi cc t
bo c gn vi cc phn t ln hoc trn cc b mt c phn tch d dng khi dng sn phm.
iu ny m bo hot ng lin tc ca h ln men khng b nguy c ra tri t bo. S bt ng
cng c th cung cp cc iu kin c li cho s phn ha t bo v s truyn t thng tin gia cc t
bo, bng cch y thc y sn phm c sn lng cc cht trao i th cp cao. S bt ng c th
bo v t bo bng cch lm gim cc s c lin quan ti lc trt (shear forces) gy tn thng t
bo.
Cc phng php bt ng t bo c th c phn chia thnh bn nhm chnh c tm tt
bng 2.1.
1. Gn ln b mt
Cc t bo c th gn ln b mt ca mnh g nh, collagen, microcarrier, hoc cc nha tng
hp trao i ion (resin). Mt v d ca kiu bt ng ny l s dng cc microcarrier cho cc t bo
ng vt. u im chnh ca microcarrier l n cung cp mt din tch b mt ln gn t bo. Vt
liu cho microcarrier bao gm cc nha tng hp trao i ion, cc ht nh da trn c s dextran c
bc bng gelatin, cc ht polyacrylamide, cc ht polystyrene, cc ht thy tinh rng, cc ht cellulose
hnh tr, v cc git floruacarbon nh c n nh bng polylysine. Hin nay, cc microcarrier da
trn dextran c s dng rng ri nht bt ng t bo ng vt.
2. To th xp
14
Phng php ny cho php cc t bo khuch tn trong cc th xp c sn nh cordierite v
pore glass (ht thy tinh c nhiu l rng nh), trong chng s sinh trng v c gi li. u
im chnh ca phng php ny l cc vt liu to th xp c sn chng chu c s phn hy
trong cc bnh nui c khuy t hn cc loi vt liu to th xp khc (alginate, acrylamide), v th
xp thng khng c hi i vi t bo. Tuy nhin, phng php ny gp kh khn trong vic hng
ti nng cao ca t bo do th tch l thy tinh (pore) b gii hn bi chng c lm sn bng cc
loi vt liu to th xp c trng.
Bng 2.1. Cc phng php bt ng t bo.
Mt phng thc khc l bc cc t bo bng th xp c to thnh trong iu kin in situ. Cc
vt liu thuc gelatin khc nhau nh acrylamide, alginate, collagen, v -carrageenan, c th c trn
vi dch huyn ph t bo v to gel trong cc dng v kch thc khc nhau.
Mt phng thc n gin khc l to cc ht hnh cu dng gel alginate-calcium l nh sau:
Cc t bo dch huyn ph sau khi c li s c trn vi alginate to ra mt nng alginate cui
cng t 1-3% (w/v) v hn hp alginate-t bo c bm bng kim tim thuc vo dung dch calcium
chloride. Cc ht c to thnh ngay tc thi c ng knh t 1-5 mm ty thuc vo nng t bo
v alginate ca dung dch v kch thc ca mi kim tim. Cn lu thm l phi duy tr s v trng
trong sut qu trnh bt ng t bo.
Nhc im chnh ca vic s dng alginate bt ng t bo l lt cc t bo t s phn
chia t bo xut hin bn trong cc ht ring r. Vic lt t bo c th c gim thiu hoc bng cch
tng nng ca alginate hoc calcium chlorite trong cc ht hoc bng cch to ra cc ht nh. Tuy
nhin, vic tng nng ca alginate hoc calcium chloride trong ht c th lm gim tc khuch
tn c cht thng qua gel v c th nh hng n kh nng sng st ca cc t bo c bao bc.
3. S dng bao vi th
Cc t bo c th c bt ng bng cc bao vi th (microcapsule) c mng hoc mng bn
thm khng c nh hoc c nh. u im ca k thut ng v bao l to mt din tch b mt ln
cho s tip xc ca c cht v t bo. Mng bn thm ch cho i qua mt cch chn lc nhng thnh
phn c trng lng phn t thp.
Cc mng dng si rng to thnh mt cu trc hnh ng thng c sp hng nh l cc b si
song song bn trong mt bung hnh tr. Cc t bo c gi li trn thnh ca cc si rng trong khi
15
mi trng dinh dng c thng kh lun chuyn quanh cc si. Loi mng ny c th cung cp
thm s bo v chng nhim bn mi trng. Tuy nhin, nhc im chnh ca h thng mng ny l
gi thnh cao, s tc nghn ca mng lm tr ngi cho vic chuyn khi v gy kh khn trong vic
thng kh.
4. T kt khi
Cc t bo t kt khi hoc kt thnh cm nh len cng c th c xem nh l cc t bo c
bt ng do kch thc ln ca chng c u im tng t nh s bt ng bng cc phng php
khc. Trong khi nm mc s to ra cc tiu th t nhin, th cc t bo vi khun hoc nm men li cn
n s kt cm. Cc nhn t kt thnh cm nhn to hoc cc nhn t lin kt ngang (cross-linkers) c
th c b sung tng cng qu trnh.
III. Mt s th nghim in hnh
1. ng cong sinh trng ca nm men
Trong th nghim ny, chng nm men s c nui cy trong bnh tam gic thu tinh, v s
thay i nng t bo s c kim sot bng cch dng ba k thut khc nhau: m di knh hin
vi, xc nh khi lng kh, c ca dch huyn ph t bo.
1.1. Nguyn liu
- Mt chng nm men bt k sinh trng trong nui cy dch huyn ph. Chng ta c th thu
chng nm men t mt phng th nghim vi sinh vt hoc mua mt chng c bit t cng ty.
- Glucose, dch chit nm men, NH
4
Cl, MgSO
4
, CaCl
2
v cht chng to bt pha mi trng.
- Hai bnh tam gic 125 mL
- Pipette v trng
- n Bunsen
- Ni kh trng
- T m
- Hemocytometer
- Ly tm
- Cn ha cht
- My quang ph
1.2. Phng thc tin hnh
- Chun b mi trng nui cy theo bng 2.2. Rt 50 mL/bnh vo 2 bnh tam gic loi 125 mL.
16
Bng 2.2. Mi trng sinh trng c trng ca nm men.
- Nt bnh tam gic bng mt trong s vt liu sau: giy nhm, nt plastic chu nhit, np inox,
hoc bng khng thm nc.
- Kh trng bnh tam gic ng mi trng 121
o
C trong 20 pht.
- Tip mu (inoculate) 1 mL dch nui cy nm men trc vo bnh tam gic cha mi trng
v trng. Tin hnh cn thn khi b nhim bn pipette, np y v bnh tam gic trong sut qu
trnh tip mu. gim thiu c hi nhim bn, nn t np y v c ca bnh tam gic sau khi ly
np ra cy nm men vo.
- t bnh tam gic vo trong t m 37
o
C.
- Ly 2 mL mu cc khong thi gian khc nhau trong qu trnh nui cy xc nh nng
t bo bng cch m trn knh hin vi, xc nh trng lng kh v o c (mt quang) bng
my quang ph. Thi gian ly mu phi c sp xp sao c th thu c cho ng cong sinh trng
tt th hin c ba phase sinh trng (xem chng 3). Trc khi ly mu phi trn tt c cc thnh phn
trong bnh tam gic bng cch lc.
2. ng cong sinh trng ca thc vt
2.1. Nguyn liu
- Mt dng t bo dch huyn ph ca thc vt sinh trng tt. Phng thc sau y da trn c
s nui cy t bo thuc l. Nu chn dng t bo khc th cn thay i mi trng.
- Hn hp mui khong ca Murashige-Skoog (1962) (Bng 7.2), 2,4-dichlorophenoxyacetic
acid (2,4-D), KH
2
PO
4
, inositol, thiamine.HCl, v sucrose pha ch mi trng.
- Hai bnh tam gic 125 mL
- Pipette loi ming rng v trng (10 mL)
- T nui t bo thc vt km my lc
- Cn ha cht
- Ly tm
- Eppendorf tube
- Chun b mi trng mui khong ca Murashige-Skoog, 0,2 mg/L 2,4-D, 0,18 g/L KH
2
PO
4
,
0,1 g/L inositol, 1 mg/L thiamine.HCl v 30 g/L sucrose
5
. iu chnh pH ti 5,8 bng KOH 1N v
17
phn phi mi trng vo hai bnh tam gic loi 125 mL, sao cho mi bnh tam gic cha 30 mL.
- y np bnh tam gic v kh trng 121
o
C trong 20 pht.
- Cy vo bnh tam gic cha 30 mL mi trng vi 1,5 mL ca dch huyn ph t bo 7 ngy
tui v t trong t nui t bo thc vt c my lc v lc 150 vng/pht, nhit nui 27
o
C, chiu
sng 8 gi/ngy cng 2.000 lux.
- Ly 1,5 mL dch nui cy mi ngy c trn k v cho vo Eppendorf tube cn, ly tm
tube 9.000 vng/pht trong 4-5 pht v loi th ni. Cn li tube v tnh ton phn trm trng lng
t bo m. t tube trong t sy 70
o
C trong hai ngy v cn li tnh ton trng lng kh.
- V th s thay i nng t bo kh v ti (m) theo thi gian.
3. Bt ng t bo thc vt
3.1. Nguyn liu
- 30 mL t bo dch huyn ph thuc l sinh trng bng phng php m t trong th nghim
trc.
- Alginate
- CaCl
2
- Bm nhu ng
- Ni p sut
- Cn ha cht
- Trn 0,875 g alginate, 5 mL mi trng nui cy thc vt v 25 mL nc v kh trng.
- i cho 30 mL dch nui cy huyn ph ca t bo thc vt lng xung, loi b th ni. Bc
ny thng mt khong 10 pht.
- B sung hn hp alginate v trn vi cc t bo c c li.
- Bm hn hp alginate-t bo qua mt ng silicon v trng (1,6 mm ID) v cung cp tng git
vo trong bnh tam gic cha 200 mL dung dch v trng ca CaCl
2
0,12 M. Cc git nh s phn ng
ngay lp tc vi CaCl
2
to ra cc ht hnh cu c ng knh khong 3,75-4,5 mm.
- Gi cc ht trong dung dch CaCl
2
khong 1 gi m bo phn ng kt ta xy ra hon
ton.
- Cy vo bnh tam gic cha 30 mL mi trng thc vt vi khong 30 ht t bo thc vt
c bt ng v t n trong t nui t bo thc vt c my lc v lc 150 vng/pht, nhit nui
27
o
C, chiu sng 8gi/ngy cng 2.000 lux.
- Chng ta c th xc nh nng t bo kh v m ca cc t bo t do trong dch huyn ph
v cc t bo c bt ng trong sut qu trnh nui cy m. xc nh nng t bo ca cc t
bo c bt ng, cn phi ha tan cc ht trong potassium phosphate 1 M trong 24 gi.
1. Atkinson B and Mavituna F. 1991. Biochemical Engineering and Biotechnology Handbook. 2
nd
ed.
Stockton Press, New York, USA.
18
4. Ratledge C and Kristiansen B. 2002. Basic Biotechnology. Cambridge University Press, UK.
nd
ed. Prentice Hall, Inc.
New Jersey, USA.
6. Vogel HC and Todaro CL. 1997. Fermentation and Biochemical Engineering Handbook (Principles,
Process Design, and Equipment). 2
nd
ed. Noyes Publications. New Jersey, USA.
1
Collagen: cht to keo.
2
Microcarrier: mt tiu th c kch thc hin vi (thng l mt ht polymer ng knh khong 200 m)
cc t bo trong nui cy dch huyn ph gn vo v sinh trng.
3
Emulsion: dng nh tng.
4
Polyelectrolytes: cc cht a in phn.
5
Mi trng ny ch c tnh cht tham kho. Thng thng cc loi thc vt khc nhau vi cc mc ch nui
cy khc nhau, s c cc nhu cu dinh dng khc nhau. Khi , chng ta phi thit k cc mi trng nui cy
c tnh c hiu cao hn.
19
Chng 3
ng hc sinh trng ca t bo
I. M u
Hiu bit y ng hc sinh trng ca cc t bo thc vt, ng vt v vi sinh vt l rt
cn thit thit k v hot ng cc h ln men. ng hc t bo c quan h vi tc sinh
trng t bo v chu nh hng ca cc iu kin vt l v ha hc.
ng hc t bo l kt qu ca h thng cc phn ng ha sinh v cc qu trnh vn
chuyn phc tp, bao gm nhiu pha v cc h thng nhiu thnh phn. Trong sut thi gian
sinh trng, hn hp khng ng nht ca cc t bo gi v non thay i lin tc v t thch nghi
vi mi trng dinh dng l yu t cng thay i lin tc trong cc iu kin vt l v ha hc.
Ni chung, m hnh ton hc chnh xc ca ng hc sinh trng l khng c th c c. Thm
ch mt m hnh thc t cng kh tip cn bi v n c th cha nhiu thng s khng th xc
nh.
V th, chng ta cn gi nh c th t c nhng m hnh n gin nh vy s hu ch
hn cho vic thit k h thng ln men (xem chng 4) v d bo hiu sut. Cc m hnh khc
nhau c th c pht trin trn c s cc gi nh v cc thnh phn v qun th t bo nh
trnh by trong bng 3.1.
Ngoi cc gi nh i vi t bo, mi trng c thit k sao cho ch mt thnh phn c
th gii hn tc phn ng, cn tt c cc thnh phn khc hin din cc nng cao m
nhng thay i nh ca chng khng nh hng r rt n tc phn ng. Cc h thng ln men
cng c kim sot sao cho cc thng s mi trng nh pH, nhit v nng oxygen ha tan
c duy tr mt mc khng i.
Trong chng ny, cc phng trnh ng hc t bo bt ngun t m hnh c phn
phi, khng cu trc. Cc phng trnh ny c ng dng thit k v phn tch cc h ln
men l tng.
Bng 3.1. Cc m hnh khc nhau ca ng hc t bo.
20
II. nh ngha
Trc tin, chng ta hy nh ngha mt s thut ng dng cho sinh trng ca t bo. Nu
cp n nng t bo m khng km theo bt k mt ghi ch c im no, th n c th c
hiu theo nhiu ngha khc nhau. c th l s lng t bo, trng lng ti t bo, hoc trng
lng kh t bo trn mt n v th tch. Trong chng ny, chng ta thng nht cc thut ng sau:
C
X
: nng t bo, trng lng kh t bo trn mt n v th tch.
C
N
: mt s lng t bo, s lng t bo trn mt n v th tch.
: mt t bo, trng lng ti t bo trn mt n v th tch ca khi lng t bo.
T , c th nh ngha tc sinh trng ca t bo theo mt s cch khc nhau nh sau:
dC
X
/dt: s thay i nng kh ca t bo theo thi gian.
r
X
: tc sinh trng ca t bo trn c s trng lng kh.
dC
N
/dt: s thay i mt s lng t bo theo thi gian.
r
N
: tc sinh trng ca t bo trn c s s lng.
o: tc phn chia ca t bo trn c s s lng dlog
2
C
N
/dt
Dng nh dC
X
/dt v r
X
lun lun ging nhau, nhng thc ra khng phi nh vy. Gi tr
dC
X
/dt l s thay i nng t bo trong h ln men, l yu t c th bao gm hiu qu ca tc
dng chy i vo v i ra, s ti sinh t bo, v cc iu kin hot ng khc ca h ln men. Trong
khi r
X
l tc sinh trng thc t ca t bo. Hai gi tr ny ch ging nhau trong trng hp
hot ng ln men m.
Tc sinh trng da trn s lng t bo v tc sinh trng da trn trng lng t bo
khng nht thit phi ging nhau, bi v kch thc trung bnh ca cc t bo c th rt khc nhau khi
chuyn t pha sinh trng ny n pha sinh trng khc. Khi sinh khi ca mt t bo ring bit
tng ln m khng c s phn chia, th tc sinh trng da trn trng lng t bo cng tng ln,
trong khi tc sinh trng da trn s lng t bo li gi nguyn. Tuy nhin, trong sut thi gian
sinh trng theo hm m, pha sinh trng m chng ta quan tm nht di quan im ca cng
ngh, th tc sinh trng da trn s lng t bo v tc sinh trng da trn trng lng t
bo c th c gi nh l tng ng nhau.
Trong mt s trng hp tc sinh trng b nhm ln vi tc phn chia, l khi nim
c nh ngha nh l tc phn chia t bo trn mt n v thi gian. Nu tt c t bo trong
21
bnh nui cy thi im t = 0 (C
N
= C
No
) phn chia ch sau mt thi gian nht nh, th qun th t
bo s tng ln C
No
2 ln. Nu cc t bo c phn chia n ln sau thi gian t, th s lng tng s
ca t bo s l:
V tc phn chia trung bnh l:
V n = log
2
C
N
- log
2
C
No
theo phng trnh 3.1, nn tc phn chia trung bnh s l:
V tc phn chia thi gian t l:
V th, tc sinh trng (c nh ngha l s thay i s lng t bo theo thi gian) chnh
l dc ca ng cong C
N
theo t. Trong khi , tc phn chia l dc ca ng cong log
2
C
N
theo t. Nh gii thch, tc phn chia l hng s trong sut thi gian sinh trng theo hm m,
trong khi tc sinh trng li khng nh vy. V th, hai khi nim ny khng c nhm ln vi
nhau.
III. Chu k sinh trng ca nui cy m
Nu nui cy cc vi sinh vt n bo trong mi trng v trng sch v o mt s lng t
bo theo thi gian th trn th ca n ta c th thy c su pha sinh trng v cht ca t bo
(Hnh 3.1), l:
- Pha lag. L thi gian khi s thay i s lng t bo bng khng.
- Pha sinh trng nhanh. S lng t bo bt u tng v tc phn chia t n cc i.
- Pha sinh trng theo hm m. S lng t bo tng theo hm m khi t bo bt u phn
chia, tc sinh trng tng ln trong sut pha ny, nhng tc phn chia t l vi dlnC
N
/dt, l
hng s gi tr cc i ca n, nh c minh ha hnh 3.1.
- Pha sinh trng chm. Khi tc sinh trng t n cc i, th giai on tip theo l pha
sinh trng chm trong c hai tc sinh trng v tc phn chia u gim.
- Pha tnh. Qun th t bo t n gi tr cc i v s khng tng thm na.
- Pha cht. Sau khi cc cht dinh dng ca t bo cn kit, t bo s bt u cht v s
lng t bo sng st s gim.
1. Pha lag
Pha lag (hoc pha tnh khi u hoc tim tng) l thi k khi u ca qu trnh nui cy,
trong sut thi k ny s thay i s lng t bo l bng khng hoc khng ng k. Mc d s
22
lng t bo khng tng ln, nhng t bo c th sinh trng bng cch tng kch thc trong sut
thi k ny.
Hnh 3.1. ng cong sinh trng c trng ca cc c th n bo. (A) pha lag, (B) pha sinh
trng nhanh, (C) pha sinh trng theo hm m, (D) pha sinh trng chm, (E) pha tnh, (F) pha
cht.
di ca pha lag ty thuc vo nhiu nhn t, chng hn nh loi v tui ca c th vi sinh
vt (hoc t bo ng-thc vt), v cc iu kin nui cy. Pha lag thng xut hin do t bo phi
iu chnh vi mi trng mi trc khi s sinh trng c th bt u. Nu vi sinh vt c cy t
mi trng c nng cht dinh dng thp vo mi trng c nng cht dinh dng cao, th
pha lag thng ko di. Nu n c chuyn t ni c nng cao n ni c nng thp th
thng khng xut hin pha lag.
Mt nhn t quan trng khc nh hng n di ca pha lag l lng mu c a vo nui
cy (inoculum size). Nu mt lng nh t bo c a vo mt th tch ln th chng s c mt pha
lag di. trng hp nui cy t bo trn quy m ln, th thi gian ca pha lag cng ngn cng tt. V
th, a mu vo (cn gi l tip mu) quy trnh ln men cng nghip, chng ta cn phi c mt
dy cc ni ln men c lng mu ln dn gim thiu nh hng ca pha lag.
Vo giai on kt thc pha lag, khi s sinh trng ca t bo bt u, th tc phn chia t bo
tng ln t t v t n gi tr cc i thi k sinh trng theo hm m, nh trnh by bng s tng
23
ln gc un cong B trong hnh 3.1. Thi k chuyn tip ny c gi chung l pha sinh trng
nhanh v thng c xem nh l mt phn ca pha lag.
2. Pha sinh trng theo hm m (pha logarithm)
cc c th n bo, s nhn i tng dn ca s lng t bo cho kt qu tc sinh
trng tng ln lin tc trong qun th. Nui cy vi khun tri qua s sinh trng cn bng kiu nh
phn ng ha hc bc mt t xc tc. V th, tc tng trng ca qun th t bo mi thi im
t l vi mt s lng (C
N
) ca t bo hin din ti thi im .
Trong : hng s c bit nh l tc sinh trng c trng (gi
-1
). Khng nn nhm ln
tc sinh trng c trng vi tc sinh trng (c cc n v v ngha khc hn). Tc sinh
trng l s thay i ca mt s lng t bo theo thi gian, trong khi tc sinh trng c
trng l:
l s thay i theo logarithm t nhin ca mt s lng t bo theo thi gian. So snh
phng trnh (3.4) v (3.6) cho thy:
V vy, tc sinh trng c trng bng ln2 ln tc phn chia o.
Nu l hng s theo thi gian trong sut thi k sinh trng theo pha hm m, th phng
trnh (3.5) c th c ly tch phn t t
0
ti t khi :
Trong : C
No
l mt s lng t bo t
0
khi s sinh trng hm m bt u. Phng trnh
(3.9) cho thy s tng ln ca s lng t bo theo hm m i vi thi gian.
Thi gian cn thit gp i qun th, c gi l thi gian nhn i (t
d
), c th c lng t
phng trnh (3.9), bng cch t C
N
= 2C
No
v t
0
= 0, gii theo t ta c:
Thi gian nhn i t l nghch vi tc sinh trng c trng v bng s nghch o ca tc
phn chia.
24
3. Cc nhn t nh hng n tc sinh trng c trng
3.1. Nng c cht
Mt trong nhng phng trnh c s dng rng ri nht th hin nh hng ca nng c
cht (cht dinh dng) ln l phng trnh Monod:
Trong : C
S
l nng ca c cht gii hn (limiting substrate) trong mi trng v K
S
l h
s h thng. Mi quan h ny c trnh by bng th trong hnh 3.2. Gi tr ca K
S
tng ng
vi nng ca cht dinh dng khi tc sinh trng c trng bng mt na gi tr cc i ca
n (
max
).
Theo phng trnh Monod, s tng ln v sau ca nng cht dinh dng khi t n
max
khng nh hng n tc sinh trng c trng, nh trnh by hnh 3.2. Tuy nhin, tc
sinh trng c trng s gim xung khi nng cht dinh dng tng ln vt qu mt mc
nht nh.
3.2. Nng sn phm
Khi cc t bo sinh trng, chng s sn xut ra cc sn phm trao i cht v c th tch ly
trong mi trng. Sinh trng ca cc vi sinh vt thng b c ch bi cc sn phm ny, nh
hng ca cc sn phm ny c th c b sung vo phng trnh Monod nh sau:
Trong : K
P
l h s nng sn phm v C
P
l nng sn phm.
C hai phng trnh trn m t s c ch sn phm kh tt. C
Pm
c k hiu l nng
cc i ca sn phm, l yu t lm cho cc t bo khng th sinh trng do s c ch sn phm.
3.3. Cc iu kin khc
Tc sinh trng c trng ca cc t bo cng b nh hng bi pH mi trng, nhit v
s cung cp oxygen. Nhit v pH ti u ca cc loi t bo khc nhau (ng-thc vt v vi sinh
vt) l cng khc nhau.
25
Hnh 3.2. S ph thuc ca tc sinh trng c trng vo nng ca cht dinh dng
gii hn sinh trng.
max
= 0,935/gi; K
S
= 0,2210
-4
mol/L.
4. Pha tnh v pha cht
Sinh trng ca qun th t bo thng b hn ch hoc do s dng ht ton b cc cht dinh
dng c sn hoc do s tch ly cc sn phm c ca s trao i cht. Kt qu l tc sinh
trng gim v s sinh trng cui cng dng li. thi im ny nui cy c gi l pha tnh.
Giai on chuyn tip gia pha hm m v pha tnh bao gm mt thi k sinh trng khng cn
bng v trong sut thi k ny cc thnh phn khc nhau ca t bo c tng hp cc tc
khng bng nhau. Kt qu l cc t bo trong pha tnh c mt thnh phn ha hc khc vi cc t
bo trong pha hm m.
Pha tnh thng c tip theo bi pha cht m trong cc c th trong qun th b cht. S
cht xut hin hoc do s suy yu ca vic bo qun nng lng ca t bo, hoc do s tch ly cc
sn phm c t. Ging nh s sinh trng, s cht l mt hm m. Trong mt s trng hp, c
th khng ch cht m cn phn hy, mt qu trnh cn c gi l s phn gii.
IV. Cc k hiu
C nng , khi lng trn mt n v th tch nui cy, kg/m
3
C
N
mt s lng t bo, s lng t bo/m
3
C
No
mt s lng t bo ti thi im t
0
, s lng t bo/m
3
C
P
nng sn phm
C
Pm
nng cc i ca sn phm
C
S
nng c cht
C
X
nng t bo, trng lng kh t bo trn th tch kg/m
3
dC
X
/dt s thay i nng kh ca t bo theo thi gian
dC
N
/dt s thay i mt s lng t bo theo thi gian
K
P
h s nng sn phm
K
S
h s h thng cho ng hc Monod, kg/m
3
n s lng t bo
r tc
r
X
tc sinh trng ca t bo trn c s trng lng kh
26
r
N
tc sinh trng ca t bo trn c s s lng
t thi gian, s
t
d
thi gian nhn i, s
o tc phn chia ca t bo, s
-1
tc phn chia t bo trung bnh, s
-1
tc sinh trng c trng, s
-1
hoc kg/m
3
/s
max
tc sinh trng cc i, s
-1
hoc kg/m
3
/s
mt t bo, kg/m
3
1. Asenjo JA and Merchuk JC. 1995. Bioreactor System Design. Marcel Dekker, Inc. New
York, USA.
2. Atkinson B and Mavituna F. 1991. Biochemical Engineering and Biotechnology Handbook.
2
nd
ed. Stockton Press, New York, USA.
3. Chia TF. 2003. Engineering Applications in Biology. Updated 1
st
ed. McGraw-Hill Education,
Singapore.
6. Ratledge C and Kristiansen B. 2002. Basic Biotechnology. Cambridge University Press,
UK.
nd
ed. Prentice
Hall, Inc. New Jersey, USA.
8. Vogel HC and Todaro CL. 1997. Fermentation and Biochemical Engineering Handbook
(Principles, Process Design, and Equipment). 2
nd
27
Chng 4
Thit k h ln men
I. H ln men thng khuy
Ni phn ng sinh hc (bioreactor) hay cn gi l h ln men (fermenter) l loi thit b m
trong n s bin i ha sinh c tin hnh bi cc t bo sng hoc cc thnh phn t bo in vivo
(enzyme). Trong chng ny, ni phn ng sinh hc nui cy cc t bo sng c gi l h ln
men phn bit cc ni phn ng sinh hc dng cho cc enzyme. Trong phng th nghim, cc t
bo thng c nui cy trong cc bnh tam gic trn my lc. Lc nh bnh tam gic rt hiu qu
to ra dch huyn ph t bo, tng cng s oxy ha thng qua b mt cht lng v tr gip s
chuyn khi (mass transfer) ca cc cht dinh dng m khng gy nguy him cho cu trc t bo.
Hnh 4. 1. S h ln men dng cho sn xut penicillin.
i vi hot ng sn xut quy m ln, th h thng ln men thng khuy (stirred-tank
fermenter, STF) c s dng rng ri nht thit k cho qu trnh ln men cng nghip. N c th
c dng cho c hai trng hp ln men hiu kh (aerobic) v ym kh (anaerobic) trong mt phm
vi rng cc loi t bo khc nhau bao gm vi sinh vt, ng vt v thc vt.
Hnh 4.1 gii thiu s h ln men dng trong sn xut penicillin. Cng pha trn (mixing
intensity) c th rt khc nhau bng cch chn loi cnh khuy (impeller) thch hp v cc tc
khuy khc nhau. Vic sc kh v khuy c hc trong h ln men rt tt cho nui cy dch huyn ph
t bo, s oxy ha, s pha trn mi trng v truyn nhit. STF cng c th c dng cho cc mi
trng c nht cao. N l mt trong nhng h ln men quy m ln u tin c pht trin trong
cng nghip dc. c im v tim nng ca STF c nghin cu rng ri. Do h ln men thng
khuy thng c lm bng thp khng r v hot ng trong iu kin n ha nn tui th ca
thit b rt lu.
Nhc im ca h ln men thng khuy bt ngun t u im ca n. B phn (cnh) khuy
rt hiu qu trong vic pha trn cc thnh phn ca h ln men, nhng li tiu th mt lng ln
cng sut v c th gy nguy him cho nhng h thng t bo nui cy mn cm vi lc trt (shear
28
force) nh t bo ng vt c v hoc t bo thc vt. Lc trt ca cht lng trong hn hp c
to ra bi gradient tc ca cc thnh phn tc (hng tm v tip tuyn) ca cht lng khi ri
khi vng cnh khuy. Khi cht lng ri khi vng trung tm, th tc ca n v tr trn v di
cnh khuy (c khong cch bng chiu rng cnh khuy) s gim khong 85% v to ra mt vng
trt cao. Khi t l chiu rng cnh khuy trn ng knh ca n tng th profile tc t c dng
c trng ca parabol m tr nn t hn v n to ra lc trt t hn do gradient tc ln dn ln.
V th, bng cch tng chiu rng cnh khuy, c th ng dng thnh cng STF trong nui cy t
bo ng vt hoc t bo thc vt.
Nhiu h ln men quy m phng th nghim c lm bng thy tinh c np bng thp khng
r. Cc thng ln men ln hn c lm bng thp khng r. T l chiu cao trn ng knh ca
thng ln men (vessel) hoc l 2/1 hoc l 3/1 v thng c khuy bng hai hoc ba turbine
khuy (cnh khuy). Trc cnh khuy c gn trn np hoc t y ca thng bng gi . T l
ng knh cnh khuy (D
I
) trn ng knh ca thng (D
T
) thng l t 0,3-0,4. Trong trng hp
h ln men c hai cnh khuy, th khong cch gia cnh khuy th nht vi y ca vessel v
khong cch gia hai cnh khuy bng 1,5 ng knh cnh khuy. Khong cch ny gim xung
cn 1,0 so vi ng knh cnh khuy trong trng hp h ln men c ba cnh khuy. Bn vch
ngn (baffles) cch u nhau thng c thit k ngn cn s hnh thnh dng xoy lm gim
hiu sut pha trn. Chiu rng ca vch ngn thng bng 1/10 ng knh ca thng (tank).
trng hp h ln men hiu kh (aerobic fermenter), th mt b phun l n (single orifice sparger)
hoc mt b phun vng c s dng sc kh cho h ln men. B phn phun c t v tr
gia cnh khuy cui cng v y ca vessel. pH trong h ln men c th c duy tr bng cch
dng dung dch m hoc b iu chnh pH (pH controller). Nhit c iu chnh bng h thng
gia nhit v lm lnh t ng.
1. H ln men dng nt (plug-flow fermenter, PFF) hoc m (batch fermenter)
Mt h ln men khuy l tng phi c kh nng pha trn tt sao cho cc thnh phn ng
nht trong mt kt cu mi thi im. Mt h ln men l tng khc l h ln men dng nt, mt
dng tng ng ca h ln men m.
Trong h ln men dng ng (tubular-flow fermenter), cht dinh dng (c cht) v t bo i vo
mt u ca ng hnh tr v t bo s sinh trng trong khi chng i qua ng ny. Do ng di v
thiu b phn khuy nn ngn cn s pha trn hon ton ca cht lng, v th tnh cht ca dng
chy thay i trong hai chiu tip tuyn v hng tm. Tuy nhin, s bin thin trong chiu hng
tm nh hn chiu tip tuyn. Mt h ln men dng ng m khng c nhng bin thin hng tm
th c gi l h ln men dng nt (PFF).
Thc t, h ln men PFF rt kh xy dng. Cho d h ln men PFF trng thi n nh (steady
state) c hot ng trong mt kiu lin tc, th nng t bo ca h ln men m l tng sau
thi gian t s ging nh nng t bo ca h ln men PFF trng thi n nh v tr chiu dc ni
m thi gian lu (residence time) t bng t (Hnh 4.2). V th, s phn tch sau y ng dng cho c
hai, h ln men m l tng v PFF trng thi n nh.
Nu mi trng lng c tip mu bng nui cy kt ht (seed culture), th t bo s bt u
sinh trng theo hm m sau pha lag. Trong h ln men m, s thay i nng t bo bng tc
sinh trng t bo:
29
Hnh 4.2. S (a) h ln men thng khuy m v (b) h ln men dng nt.
thu c phng trnh hiu sut ca ln men m, chng ta cn ly tch phn phng trnh
(4.1) s c:
Cn lu rng, phng trnh (4.2) ch c ng dng khi r
X
> 0. V th, t
0
(trong phng trnh
4.2) khng phi l thi gian ca nui cy ban u sau khi tip mu, m l thi gian t bo khi ng
sinh trng, l giai on pha sinh trng bt u tng nhanh.
Theo phng trnh (4.2), thi gian sinh trng tng m t-t
0
chnh l din tch pha di ng
cong 1/r
X
theo C
X
gia C
Xo
v C
X
(Hnh 4.3). ng cong lin tc hnh 4.3 c tnh ton bng
phng trnh Monod v vng c mu ti bng t-t
0
. Thi gian sinh trng tng m t khi c c
lng bng th ny v xc nh n th da vo ng cong t theo C
X
l n gin hn. Tuy
nhin, biu din bng th s thun tin trong vic so snh tim nng ca cc cu hnh h ln men
khc nhau (s c tho lun sau). Lc ny ch lu rng, ng cong c mu ti dng ch U l
c trng ca cc phn ng xc tc t ng:
S + X X + X
30
Hnh 4.3. th ca thi gian sinh trng tng m t-t
0
(vng ti). ng cong lin tc biu
din m hnh Monod vi
max
= 0,935/gi; K
S
= 0,71 g/L; Y
X/S
= 0,6; C
Xo
= 1,6 g/L v C
So
= 10 g/L.
Tc khi u ca phn ng xc tc t ng chm do nng ca X thp. Tc phn
ng tng ln khi cc t bo sinh sn v sau s t n tc ti a. Khi lng c cht gim v
cc sn phm c c tch ly, th tc phn ng gim xung gi tr thp hn.
Nu ng hc Monod (Monod kinetics) biu din thch hp tc sinh trng trong sut pha
hm m, th chng ta c th thay th phng trnh (3.11) chng 3 vo phng trnh (4.2) c
c:
Phng trnh (4.3) c th tnh c tch phn nu chng ta bit mi quan h gia C
S
v C
X
.
Ngi ta quan st thy rng s lng sinh khi t bo c sn xut t l vi lng c cht gii
hn c tiu th. Hiu sut sinh trng (Y
X/S
) c nh ngha nh sau:
Thay phng trnh (4.4) vo phng trnh (4.3), tch phn ca phng trnh tng hp ny s
a ra mi quan h gia nng t bo v thi gian:
2. H ln men thng khuy lin tc (continuous stirred-tank fermenter-CSTF) l tng
Qun th t bo c th tip tc giai on sinh trng hm m trong mt thi gian di bng
cch duy tr h thng nui cy lin tc. Hnh 4.4 trnh by s h ln men thng khuy lin tc
(CSTF). Bung sinh trng (thng ln men hay bnh nui) c kt ni vi bnh cha mi trng v
trng. Khi qu trnh sinh trng bt u th mi trng sch c cung cp lin tc t bnh cha mi
trng.
H thng nui cy lin tc c th hot ng nh l mt chemostat (th n nh ha tnh) hoc
31
turbidostat (th n nh c). Trong chemostat tc dng chy c ci t mt gi tr c bit
v tc sinh trng ca nui cy s iu chnh tc dng chy ny. Ni chung, hot ng
chemostat d dng hn turbidostat, do n c th c thc hin bng cch t my bm mt tc
dng chy khng i, trong khi turbidostat i hi mt thit b cm quang (optical sensing device)
v mt b iu chnh (controller). Tuy nhin, turbidostat c gii thiu khi h ln men lin tc cn
c tin hnh cc tc pha long cao gn vi im ra tri (washout point), khi ta c th ngn
cn s ra tri bng cch iu ha tc dng chy trong trng hp tht thot t bo thng qua
dng chy ra ngoi vt qu s sinh trng t bo trong h ln men.
Hnh 4.4. S h ln men thng khuy lin tc (CSTF).
Cn bng nguyn liu cho t bo trong CSTF (Hnh 4.4) c th c vit nh sau:
Trong : r
X
l tc sinh trng t bo trong h ln men v dC
X
/dt biu din s thay i nng
t bo trong h ln men theo thi gian.
i vi CSTF hot ng trng thi n nh, th s thay i nng t bo theo thi gian l
bng khng (dC
X
/dt) do cc t bo trong bnh nui ch sinh trng nhanh thay th nhng t
bo b hao ht theo dng chy ra ngoi, v phng trnh (4.6) tr thnh:
Phng trnh (4.7) cho thy thi gian lu cn thit (t
m
) bng din tch hnh ch nht c chiu
rng C
X -
C
Xi
v chiu cao 1/r
X
trn ng cong 1/r
X
theo C
X
.
Hnh 4.5 biu din ng cong 1/r
X
theo C
X
. Din tch hnh ch nht c t m trong hnh
bng thi gian lu trong CSTF khi dng chy vo l v trng. Minh ha thi gian lu bng th c
th gip chng ta so snh hiu qu ca cc h ln men. H ln men c thi gian lu ngn hn (
t ti mt nng t bo nht nh) l hiu qu hn. Hot ng ti u ca h ln men da trn s
minh ha th ny s c tho lun trong phn tip theo.
32
Hnh 4.5. Minh ha bng th c lng thi gian lu cho CSTF. ng biu din m
hnh Monod vi
max
= 0,935/gi; K
S
= 0,71 g/L; Y
X/S
= 0,6; C
Si
= 10 g/L v C
Xi
= 0.
Nu dng chy vo l v trng (C
Xi
= 0) v t bo trong CSTF ang sinh trng theo hm m
(rX = X
) th phng trnh (4.7) s tr thnh:

Trong : D c bit nh l tc pha long v c gi tr bng nghch o ca thi gian lu
(t
m
). V th, i vi CSTF trng thi n nh c cht dinh dng v trng, th tc sinh trng c
trng bng tc pha long. Mt khc, tc sinh trng c trng ca t bo c th c iu
chnh bng cch thay i tc dng chy mi trng. Nu tc sinh trng c th c biu din
bng phng trnh Monod, th sau :
T phng trnh (4.9), C
S
c th c tnh ton bng thi gian lu bit v cc thng s
ng hc Monod nh sau:
Tuy nhin, cn ch rng phng trnh (4.10) ch c gi tr t
m
max
>1. Nu t
m
max
<1, tc
sinh trng ca t bo s thp hn tc t bo tht thot theo dng chy ra ngoi. Do , tt c t
bo trong h ln men s b ra tri, v phng trnh (4.10) s khng c gi tr.
Nu hiu sut sinh trng (Y
X/S
) l hng s, th sau :
Thay phng trnh (4.10) vo phng trnh (4.11) s cho hiu sut tng quan i vi C
X
nh
sau:
33
Tng t:
Trong : C
P
l nng sn phm, C
Pi
l nng sn phm a vo.
Mt ln na, phng trnh (4.12) v (4.13) ch c gi tr khi t
m
max
>1.
Trong phn ny, chng ta t cn bng nguyn liu cho nng t bo v thu c cc
phng trnh khc nhau cho CSTF. Cc phng trnh tng t cng c th thu c bng cch t
cc cn bng nguyn liu cho nng c cht v nng sn phm.
3. c lng cc thng s ng hc Monod
ng thc tc sinh trng c trng v tc pha long ca CSTF trng thi n nh
(phng trnh 4.9) tin li trong nghin cu nh hng ca cc thnh phn khc nhau ca mi
trng ln tc sinh trng c trng. Bng cch o nng c cht trng thi n nh vi cc
tc dng chy khc nhau, cc m hnh ng hc khc nhau c th c th nghim v gi tr ca
cc thng s ng hc c th c c lng. Sp xp li phng trnh (4.9) c th thu c mi
quan h tuyn tnh nh sau:
Trong : bng tc pha long (D) cho chemostat. Nu mt t bo nht nh tun theo ng
hc Monod, th th 1/ theo 1/C
S
s em li gi tr
max
v K
S
(bng cch c phn b chn v
dc ca ng thng). th ny c u im cho thy mi quan h gia bin c lp (C
S
) v bin
ph thuc . Tuy nhin, 1/ s tin ti nu nng c cht gim dn n trng lng vt qu
mc o khi nng c cht thp v trng lng khng o khi cc nng c cht cao.
Phng trnh (4.9) c th sp xp li a ra cc mi quan h tuyn tnh ng dng thay cho
phng trnh (4.14) nhm c lng tt hn cc thng s trong nhng trng hp nht nh:
Tuy nhin, gii hn ca php tnh gn ng ny ( xc nh cc thng s ng hc) gp kh
khn khi s dng CSTF. i vi trng hp vn hnh theo tng m, chng ta thm ch c th dng
bnh tam gic lc trn my lc vn hnh nhiu m vi cc iu kin khc nhau trong cng mt thi
gian. Vn hnh theo tng m trong ni ln men c khuy cng khng kh khn lm, do khng c cc
34
kt ni i vo v i ra (ngoi tr b phn cung cp khng kh) v thi gian vn hnh ngn, t c nguy
c ca s nhim bn h ln men.
vn hnh CSTF, chng ta cn c cc ngun cung cp dinh dng v tch tr sn phm
c kt ni v trng vi h ln men. Tc ca cc dng chy vo v ra khi h ln men cn c
kim sot mt cch chnh xc. Thnh thong, vic kim sot tc dng chy ra c th gp kh khn
do s to bt v kt khi ca cc t bo. Do thi gian vn hnh t nht mt vi ngy hoc thm ch c
tun t ti trng thi n nh (cng gy ra s bin i tc pha long), cho nn lun c ri ro
cao i vi h ln men do b nhim bn. Thng xuyn gp kh khn trong vic t ti trng thi n
nh bi t bin ca t bo v kh nng thch nghi vi mi trng mi ca chng.
Hn na, do hu ht cc h ln men quy m ln c tin hnh trong kiu tng m, cho nn
cc thng s ng hc c xc nh bi nghin cu chemostat phi d bo c s sinh trng
trong kiu ln men ny. Tuy nhin, bng chng (kim tra v xc minh) m hnh ng hc v c
lng cc thng s ng hc bng cch vn hnh chemostat l phng php ng tin cy nht do
iu kin mi trng khng thay i ca n.
Cc s liu ca vn hnh theo tng m c th c dng xc nh cc thng s ng hc,
cho d n khng phi l phng thc c gii thiu cao. Tc sinh trng c trng trong sut
qu trnh vn hnh theo tng m c th c c lng bng cch o dc ca ng cong nng
t bo theo thi gian cc im khc nhau. Nng c cht cn thit c o cng cc im
ni m dc c c. Sau cc th theo cc phng trnh (4.14), (4.15) v (4.16) c th
c xy dng xc nh cc thng s ng hc. Tuy nhin, gi tr ca cc thng s thu c
trong phng php ny cn thit c kho st cn thn xem chng c trong phm vi hp l cho
cc t bo c kim tra hay khng.
4. Hiu sut ca CSTF
Thng thng, hiu sut ca h ln men c hiu nh l s lng sn phm c sn xut
trn mt n v thi gian v th tch. Nu dng chy vo l v trng (C
Xi
= 0) th hiu sut sinh khi t
bo bng C
X/
t
m
, chnh l dc ca ng thng ca ng cong C
X
theo t
m
(Hnh 4.6).
Hnh 4.6. S thay i nng t bo v c cht nh l mt hm ca thi gian lu. Hiu sut
bng dc ca ng thng . ng cong c v bng m hnh Monod vi
max
=
35
0,935/gi; K
S
= 0,71 g/L; Y
X/S
= 0,6; v C
Si
= 10 g/L.
Hiu sut im A bng hiu sut im B. im A nng t bo ca dng chy ra thp
nhng thi gian lu li ngn, v th mi trng c th chy qua d dng hn. Ngc li, im B
nng t bo ca dng chy ra cao nhng thi gian lu li di v th ch c mt lng nh ca mi
trng chy qua. im A l vng khng n nh v rt gn vi im ra tri D, v v ch cn mt s
dao ng nh trong thi gian lu cng c th em li mt s thay i ln trong nng t bo. Khi
dc ca ng thng tng ln th hiu sut s tng v di ca gim. dc ca ng
thng s t gi tr cc i khi n l ng tip tuyn ca ng cong C
X
. V th, gi tr hiu sut
cc i bng dc ca ng . Hiu sut cc i s t c im D.
iu kin hot ng t hiu sut cc i CSTF c th c lng theo th bng cch
dng ng cong 1/r
X
theo C
X
. Hiu sut cc i c th thu c khi thi gian lu l ti thiu. V thi
gian lu bng din tch ca hnh ch nht vi chiu rng C
X
v chiu cao 1/r
X
trn ng cong 1/r
X
theo C
X
, cho nn n s t ti thiu khi 1/r
X
l ti thiu (Hnh 4.7).
Hnh 4.7. Minh ha bng th CSTF vi hiu sut cc i. ng lin tc biu din cho m hnh
Monod vi
max
= 0,935/gi; K
S
= 0,71 g/L; Y
X/S
= 0,6; C
Si
= 10 g/L; v C
Xi
= 0.
iu cn lu l iu chnh cc phng trnh cho nng t bo v thi gian lu sao cho
hiu sut t bo t cc i. Hiu sut t bo cho CSTF trng thi n nh vi cht dinh dng v
trng l:
Hiu sut t cc i khi drX/dCx = 0, sau khi thay th C
S
= C
Si
- C
X
/Y
X/S
vo phng trnh
(4.17), ly tch phn theo C
X
v t phng trnh tng hp bng 0, chng ta thu c nng t bo
ti u (C
X,opt
) cho hiu sut cc i nh sau:
36
Thay phng trnh (4.20) vo phng trnh (4.17) thu c mt thi gian lu ti u (t
m,opt
)
nh sau:
5. So snh nui cy ca h ln men m v h ln men thng khuy lin tc
Nh cp, thi gian lu cn thit nui cy m hoc PFF trng thi n nh t ti mt
nng t bo nht nh l:
Trong : t
0
l thi gian cn thit t ti pha sinh trng theo hm m. Din tch bn di
ca ng cong 1/r
X
theo C
X
, gia C
Xi
v C
X
l bng t
b
- t
1
nh c trnh by hnh 4.3.
Mt khc, thi gian lu CSTF c biu din bi phng trnh (4.17) bng din tch hnh
ch nht vi chiu rng C
X
- C
Xi
, v chiu cao 1/r
X
.
V ng cong 1/r
X
theo C
X
c dng hnh ch U nn chng ta c th c mt vi nhn xt cho h
ln men n nh sau:
- Hu ht cc h ln men sn xut l mt CSTF hot ng vi nng t bo m gi tr
ca 1/r
X
l ti thiu (Hnh 4.8 a) do n i hi thi gian lu ngn nht.
- Nu nng cui cng ca t bo c hng ti trong pha tnh, th h ln men m l
chn la tt hn CSTF, v thi gian lu cn thit cho nui cy m (Hnh 4.8 b) l ngn hn ca
CSTF.
37
Hnh 4.8. Minh ha bng th thi gian lu c yu cu (vng ti) cho: (a) CSTF v (b) h
ln men m.
II. Thu hi t bo
i vi hot ng lin tc ca PFF v CSTF, cc t bo tht thot cng vi dng chy ra
(outlet) hn ch hiu sut ca h ln men. V th, hiu sut c th c ci thin bng cch thu
hi (recycling) t bo t dng chy ra a tr li h ln men.
1. Thu hi t bo PFF
PFF i hi s hin din ban u ca t bo trong dng chy vo (inlet) nh l mt h ln men
m i hi a mu vo ban u. Phng thc kinh t nht cung cp t bo trong dng chy vo
l thu hi mt phn ca dng chy ra a tr li dng chy vo vi (hoc khng c) thit b tch ri
t bo.
Hnh 4.9 m t s thu hi t bo PFF. Khng ging nh CSTF, PFF khng i hi thit b
tch ri t bo thu hi, v s hin din ca n khng lm tng ng k hiu sut ca h ln men.
Phng trnh hiu sut ca PFF vi ng hc Monod c th c vit nh sau:
Trong : t
p
l thi gian lu da trn tc dng chy ca ton b h thng. Thi gian lu
thc t trong h ln men ln hn t
p
do tc dng chy tng ln nh thu hi t bo.
38
Hnh 4.9. S thu hi t bo PFF.
Nu hiu sut sinh trng l khng i th:
Thay phng trnh (4.24) vo trong phng trnh (4.23) cho C
S
v ly tch phn ta s c kt
qu sau:
Trong : C
X
v C
S
c th c c lng t s cn bng t bo v c cht im phi trn
ca dng chy vo v dng chy thu hi nh sau:
Nng t bo ca dng chy ra, c th c c lng t ton b s cn bng t bo nh
sau:
Nng t bo ca dng chy thu hi c th c c lng t s cn bng t bo trn b
lc nh sau:
Trong : | l t l x (bleeding) c nh ngha nh sau:
Hnh 4.10 trnh by hiu qu ca tc thu hi (R) trn thi gian lu ca h thng PFF c thu
hi. Lu rng thi gian lu c tnh ton da trn tc dng chy vo, l thi gian lu thc
s ca h ln men. Thi gian lu thc t trn h thng PFF l khng quan trng bi v n s gim
xung khi tng tc thu hi.
Khi | = 1, tc x s bng tc dng chy, v tc dng chy ca phn c lc L l
bng 0, v th dng chy thu hi khng c lc. Thi gian lu s l v hn nu R bng 0 v gim r
rt khi R tng ln. Trong trng hp ny t l thu hi ti u c th trong khong 0,2.
39
Mt ng cong khc trong hnh 4.10 l cho = 1,8. Thi gian lu cn thit c th gim bng
cch tp trung dng chy thu hi t 25-40% khi R trong khong 0,2-1,0. Khi R s 1,2, th mt on
ca ng cong c biu din bng du chm, bi v kh c th gim t l thu hi xung di 0,2
khi | = 0,8.
Hnh 4.10. nh hng ca tc thu hi (R) v t l x ( ln thi gian lu (
p
=V/F gi)
ca h thng PFF c thu hi. ng cong c v bng m hnh Monod vi
max
= 0,935/gi; K
S
=
0,71 g/L; Y
X/S
= 0,6; C
Si
= 1,3 g/L; v C
Xi
= 0.
Vic phn tch trong phn ny v cc phn sau cng c th c ng dng trong cc b lng
t bo nh l mt b phn phn tch t bo. Dng chy ra ca b lng t bo s bng F = B+L v
nng ca n s l (B/F)C
Xi
=|C
Xi
.
2. Thu hi t bo CSTF
Hiu sut t bo trong CSTF tng ln cng vi vic tng tc pha long v t n gi tr cc
i. Nu tc pha long tng ln qu im cc i, th hiu sut ca h ln men s gim t ngt
v t bo s bt u b pha long do tc sinh sn t bo km hn s hao ht t bo dng chy
ra. Mt phng thc ci thin hiu sut h ln men l thu hi t bo bng cch tch ri t bo khi
dng chy sn phm bng h lc dng chy ngang (cross-flow filter unit) (Hnh 4.11).
Nng cao ca t bo (c duy tr bng cch thu hi t bo) s lm tng hiu sut t bo
khi tc sinh trng t l tng ng vi nng t bo. Tuy nhin, phi c gii hn trong vic tng
hiu sut t bo vi vic tng nng t bo bi v trong mi trng c nng t bo cao, th tc
chuyn khi cht dinh dng s b gim do vic dn vo mt ni qu ng v gy kt khi ca t
bo. Vic duy tr nng qu cao ca t bo cng khng c li bi v b phn lc s thng xuyn
b hng hn trng hp nng t bo cao.
40
Hnh 4.11. S thu hi t bo CSTF.
Nu tt c t bo c thu hi tr li trong h ln men, th nng t bo s tng lin tc theo
thi gian v trng thi n nh s khng bao gi t c. V th, hot ng ca CSTF c s thu
hi trng thi n nh, chng ta cn c mt dng x (Hnh 4.11). Phng trnh cn bng nguyn
liu cho t bo trong h ln men c b phn thu hi t bo c dng nh sau:
Cn lu rng, tc dng chy thc t i vo v i ra khi b phn lc khng quyt nh
hon ton n s cn bng tt c nguyn liu. i vi CSTF trng thi n nh c s thu hi t bo
v cht dinh dng v trng, th:
Lc ny D thay cho D v bng tc sinh trng c trng. Khi D = 1 t bo khng c thu
hi, v th = .
Nu tc sinh trng c th biu din bng ng hc Monod, th thay th phng trnh (3.11)
chng 3 vo phng trnh (4.32) ta c:
C
S
ch c ngha khi t
m
max
> . Nng t bo trong h ln men c th c tnh ton t gi tr
ca C
S
nh sau:
Hnh 4.12 cho thy nh hng ca t l x ln hiu sut t bo i vi m hnh Monod. Khi b
41
gim xung t 1 (khng thu hi) ti 0,5 th hiu sut t bo c tng ln gp i.
Hnh 4.12. nh hng ca t l x ln hiu sut t bo (DC
X
).
III. Cc h ln men khc
Nhiu h ln men khc c xut v th nghim. Cc h ln men ny c thit k
ci thin hoc nhc im ca h ln men thng khuy (tiu th cng sut ln) hoc cc yu cu
c bit ca mt qu trnh ln men nht nh nh: sc kh tt hn, chuyn nhit hiu qu, tch hoc
gi li t bo, bt ng t bo, gim bt thit b v gi thnh ca sn phm, v thng khng c
thit k cho quy m ln.
Cc h ln men thng c phn loi da trn c s cc kiu bnh nui ca chng nh l
thng, ct, hoc cc h ln men vng (loop). C hai h ln men thng v ct c xy dng trn c
s bnh nui hnh tr. C th phn loi da theo t l chiu cao (H) trn ng knh (D) nh sau:
H/D < 3 cho h ln men thng.
H/D > 3 cho h ln men ct.
H ln men vng l h ln men thng hoc ct c vng lu thng cht lng, (c th l ng
thng gi (draft) gia hoc l mt ci vng gn bn ngoi h ln men).
C th phn loi cc h ln men theo mt cch khc da trn c s cc thnh phn ca h ln
men c phi hp nh th no: bi kh nn, bi b phn chuyn ng c hc bn trong, hoc bi
bm cht lng bn ngoi. Cc h ln men tiu biu trong mi loi c trnh by bng 4.1, v cc
u im v nhc im ca ba loi h ln men c bn c trnh by bng 4.2.
Bng 4.1. Phn loi cc h ln men.
42
1. H ln men ct (column fermenter)
H ln men n gin nht l h ln men ct bong bng (cn gi l h ln men thp-tower
fermenter), thng bao gm mt bnh tr di, c b phn phun kh di y (Hnh 4.13 a-c). Cc
thnh phn ca h ln men c trn bng cch tng s lng bong bng ln, cng l yu t c th
cung cp oxygen cn thit cho t bo. Khi cc t bo lng xung, nng cao ca t bo c th
c duy tr phn thp hn ca ct m khng c bt k mt thit b no tch ri chng.
Tuy nhin, h ln men ct bong bng thng b hn ch trng hp ln men hiu kh v vic
tng cc bong bng khng th cung cp mt s pha trn y cho s sinh trng ti u. Ch c
phn thp hn ca ct c th duy tr nng t bo cao dn n s ln men ban u nhanh c
tip theo bi s ln men chm hn do cc c cht mong mun b gim i. Khi nng t bo tng
ln trong h ln men, cn c lu tc khng kh cao duy tr dch huyn ph t bo v s pha trn.
Tuy nhin, lu tc khng kh tng ln c th gy ra s to bt nhiu v vic duy tr cc bong bng kh
trong ct dn n lm gim hiu sut ca h ln men. Khi bong bng tng ln nhiu trong ct chng
c th kt thnh mt khi nhanh chng lm gim tc chuyn oxygen. V th, cc h ln men ct c
th khng thay i c v hn ch mt phm vi kh hp cc iu kin hot ng.
Bng 4.2. u im v nhc im ca cu hnh ba h ln men c bn.
43
khc phc nhc im ca h ln men ct, mt vi kiu thit k khc c xut. H
ln men ct hnh chp ngc (Hnh 4.13 b) c th duy tr lu tc khng kh cao trn mt n v din
tch phn thp hn ca h ln men m c nng t bo cao. Mt vi khay sng lc c th
c ci t trong ct (Hnh 4.13 c) tng hiu qu tip xc kh-cht lng v ph v s kt khi ca
bong bng kh. tng cng s pha trn m khng c cc phn chuyn ng bn trong, dch ln
men (mi trng) c th c bm ra ngoi v quay vng (tun hon) bng cch dng mt bm
cht lng bn ngoi (Hnh 4.13 d v e).
Hnh 4.13. Cc h ln men ct: (a) ct bong bng (bubble column), (b) ct hnh nn (tapered
column), (c) ct bong bng c khay sng lc (sieve-tray bubble column), (d) ct bong bng c khay
sng lc vi bm bn ngoi, (e) ct nhi (packed-bed) vi bm bn ngoi.
2. H ln men vng (loop fermenter)
H ln men vng l h ln men thng (tank fermenter) hoc ct (column fermenter) c vng lu
thng cht lng, n c th l mt ng thng gi gia hoc l mt ci vng bn ngoi. Ty thuc
44
vo s lu thng cht lng c to ra nh th no, m h ln men c phn loi thnh ba kiu:
lc y khng kh (air-lift), vng khuy (stirred loop) v vi phun (jet loop) (Hnh 4.14).
S lu thng cht lng ca h ln men dng lc y nh vo vic phun khng kh to ra s
khc nhau v mt gia phn giu bong bng ca cht lng trong tm ng (riser) v phn c
rt ht bong bng nng hn ca cht lng trong y thng (downcomer) (Hnh 4.14 a). S pha trn
v lu thng cht lng trong thng ln men c th c tng cng bng cch gn thm mt b
phn bm bn ngoi (Hnh 4.14 b). Tuy nhin, vic b sung bm lm gim u im ca h ln
men nh lc y khng kh l hiu sut nng lng thp v n gin.
H ln men p lc chu k ICI (Imperial Chemical Industries Ltd., England) l mt h ln men
dng lc y khng kh vi mt vng bn ngoi (outer loop) c pht trin cho ln men hiu kh i
hi c s chuyn nhit. Mi trng v khng kh c a vo trong cc phn cao hn v thp hn
(Hnh 4.14 c). Khng kh phc v cho hai mc ch: cung cp oxygen cn thit cho s sinh trng
ca t bo v to ra s lu thng t nhin ca cht lng trong h ln men thng qua mt ci vng.
B phn trao i nhit lm lnh mi trng lng c ci t vo trong ci vng . H ln men
ny c chng minh l to ra mt tc hp th oxygen cao trn mt n v th tch.
Hnh 4.14. Cc h ln men vng: (a) lc y khng kh, (b) lc y khng kh c bm bn ngoi, (c)
p lc chu k ICI.
IV. Cc k hiu
B tc chy ca dng x, m
3
/s
t l x, c nh ngha nh B/F
C
P
nng sn phm
C
Pi
nng sn phm a vo
C
S
nng c cht
C
S
nng c cht im phi trn ca dng chy vo v dng chy thu hi
C
So
nng c cht ti thi im t
0
nng c cht sau khi ra khi h ln men
C
Si
nng c cht a vo
C
S,opt
nng c cht ti u
C
X
nng t bo
45
C
X
nng t bo im phi trn ca dng chy vo v dng chy thu hi
C
Xo
nng t bo ti thi im t
0
nng t bo sau khi ra khi h ln men
C
Xi
nng t bo a vo
nng t bo thu hi qua lc
nng t bo ca dng chy thu hi
C
X,opt
nng t bo ti u
D tc pha long, s
-1
F tc dng chy, m
3
/s
t
m
thi gian lu, s
t
m,opt
thi gian lu ti u, s
t
p
thi gian lu da trn tc dng chy ca ton b h thng
t
b
thi gian lu cn thit nui cy m hoc PFF trng thi n nh t ti mt nng t bo nht
nh
K
S
h s h thng
L tc dng chy qua lc, m
3
/s
tc sinh trng c trng, s
-1
hoc kg/m
3
/s
max
tc sinh trng cc i
R tc thu hi
r
X
tc sinh trng t bo
V th tch lm vic ca h ln men, m
3
Y
P/S
hiu sut sn phm/c cht
Y
X/S
hiu sut sinh trng/c cht
X t bo trn c s trng lng kh
York, USA.
2
nd
nd
ed. Prentice
Hall, Inc. NJ, USA.
nd
46
Chng 5
Nui cy t bo vi sinh vt
Mc d cho n th k 19 vai tr ca vi sinh vt trong nhng bin i sinh hc vn khng c
tha nhn, nhng con ngi s dng vi sinh vt t rt lu trong vic ch bin thc phm, thc
ung c cn, sn xut sa, dt vi... Ngy nay, vic s dng vi sinh vt rng ri hn trc y rt
nhiu. Chng khng ch c dng trong cc qu trnh vi sinh vt truyn thng m cn cho cc qu
trnh mi nh sn xut dc phm, ha cht cng nghip, enzyme, ha cht nng nghip, x l
nc thi, lc khong v cc cng ngh DNA ti t hp. Chng ny ch tp trung gii thiu cc ng
dng ca nui cy t bo vi sinh vt trong sn xut dc phm (c bit l dc phm DNA ti t
hp) v sn xut enzyme.
I. T bo vi sinh vt
Tt c cc c th sng c Haekel (1866) phn loi thnh gii ng vt (animal kingdom),
gii thc vt (plant kingdom) v sinh vt n bo (protist) nh trnh by trong bng 5.1.
Cc protist c xem l cc c th sng tng i n gin so vi thc vt v ng vt.
Chng bao gm to, ng vt nguyn sinh, nm v vi khun. S pht trin ca knh hin vi in t
cho php cc nh khoa hc tha nhn rng cu trc n v ca tt c cc c th sng c phn
chia trong hai loi: sinh vt tin nhn (prokaryotes) v sinh vt nhn tht (eukaryotes).
Cc t bo tin nhn l n v cu trc trong hai nhm vi sinh vt: vi khun v to lam (cn gi
l vi khun lam-cyanobacteria). Cc t bo tin nhn c kch thc nh v n gin nh trnh by
hnh 5.1, n khng c chia thnh ngn bi cc h thng mng n v (unit membrane systems).
Cc t bo ch c hai vng bn trong c phn bit cu trc l: t bo cht v vng nhn (hoc dch
nhn). T bo cht c cc chm dng ht mu ti chnh l thnh phn ribosome, bao gm protein v
ribonucleic acid (RNA). Ribosome l ni xy ra cc phn ng ha sinh quan trng cho s tng hp
protein. Vng nhn c dng khng u, rt bit lp mc d n khng c gii hn bi mng. Vng
nhn cha deoxyribonucleotic acid (DNA) mang thng tin di truyn xc nh s sn xut protein v
cc cht khc ca t bo cng nh xc nh cc cu trc ca n.
Bng 5.1. Phn loi cc c th sng.
47
Hnh 5.1. Minh ha mt t bo c trng ca sinh vt tin nhn.
Cc t bo prokaryote c bao quanh bi thnh t bo (cell wall) v mng t bo (cell
membrane), thnh t bo thng dy hn mng t bo, bo v t bo khi cc nh hng t bn
ngoi. Mng t bo (hoc mng t bo cht) l mt hng ro chn lc gia phn bn trong t bo v
mi trng bn ngoi. Cc phn t ln nht c bit i qua mng ny l cc on DNA v cc
protein c trng lng phn t thp. Mng t bo c th c gp li v m rng trong t bo cht.
Mng t bo nh l b mt m trn cc cht khc ca t bo c gn vo v gi nhiu chc
nng quan trng ca t bo.
48
Hnh 5.2. Minh ha mt t bo c trng ca sinh vt nhn tht.
Cc t bo eukaryote phc tp hn, chng l cu trc n v ng vt, thc vt, protozoa,
nm v to. T bo eukaryote c cc h thng mng n v bn trong tch bit nhiu thnh phn
chc nng ca t bo nh trnh by hnh 5.2. Cc t bo eukaryote ln hn v phc tp hn cc
t bo prokaryote t 1.000-10.000 ln. Nhn c bao bc chung quanh bi mt mng i c cc l
nh rng t 40-70 m, bn trong nhn cha cc nhim sc th. Nhn iu ha cc tnh cht di truyn
v tt c cc hot ng sng ca t bo. Nhim sc th di v l cc th si mnh, cha cc gen
sp xp trn mt chui mch thng trong cc nucleoprotein (protein cng nucleic acid). T bo cht
cha mt s ln cc ht gi l ribosome cn thit trong cc phn ng lin tc tng hp cc
nguyn liu t bo. Ribosome c tp trung mt cch c bit dc theo b mt x x ca li ni
sinh cht, mt mng li khng u ca cc knh ni lin nhau c phn nh vi mng. Ty th
cha cc enzyme vn chuyn in t s dng oxygen trong qu trnh sn sinh ra nng lng. Khng
bo v lysosome l cc c quan t gip cch ly cc phn ng ha hc khc nhau trong t bo.
II. Vi khun
1. Hnh dng
Vi khun l nhng c th sng n bo c kch thc hin vi. Hin nay, ngi ta bit c
khong 1.500 loi trong tt c mi trng t nhin. ng knh c trng ca t bo vi khun trong
khong 0,5-1 m. Chiu di ca vi khun rt khc nhau. Vi khun xut hin trong cc dng nh sau:
cocci (c dng hnh cu hoc hnh trng), bacilli (c dng hnh tr hoc hnh que), spirilla (c dng
cun xon c).
2. Kiu sinh trng
Vi khun sinh sn ch yu theo phng thc phn i (binary fission) nh c minh ha
trong hnh 5.3. Qu trnh ny bao gm mt s bc sau: ko di t bo, lm vo ca thnh t bo,
phn phi nguyn liu ca nhn, bt u hnh thnh mt vch ngn ngang, phn phi nguyn liu t
bo vo trong hai t bo, v phn chia thnh hai t bo mi. y l qu trnh sinh sn v tnh.
3. Cc iu kin vt l nh hng n sinh trng
Ba nhn t vt l ch yu nh hng n sinh trng ca vi khun l nhit , oxygen v
pH.
Do s sinh trng v hot tnh ca vi khun biu th s hot ng ca enzyme, v do tc
ca cc phn ng enzyme tng ln cng vi vic tng nhit , cho nn tc sinh trng ca vi
khun s ph thuc vo nhit . Ty thuc vo phm vi nhit m chng sinh trng, vi khun s
c gi l psychrophiles
4
, mesophiles
5
hoc thermophiles
6
. Phm vi nhit m mi nhm c kh
nng sinh trng v nhit ti u c trnh by trong bng 5.2.
Cc kh quan trng ch yu trong nui cy vi khun l oxygen v CO
2
. C th chia ra bn loi vi
khun ty theo s phn ng ca chng i vi oxygen nh sau:
- Vi khun hiu kh sinh trng trong s c mt ca oxygen t do.
49
- Vi khun k kh sinh trng trong iu kin khng c oxygen t do.
- Vi khun k kh ty sinh trng trong iu kin c oxygen t do hoc khng.
- Vi khun a t oxygen sinh trng trong s c mt mt lng rt t oxy t do.
Hnh 5.3. Vi khun sinh sn bng cch phn i.
Bng 5.2. Phm vi nhit thch hp cho sinh trng ca cc loi vi khun khc nhau.
i vi hu ht vi khun, pH ti u cho sinh trng nm trong khong 6,5-7,5. Mc d, mt
vi vi khun c th sinh trng phm vi pH cc oan, nhng hu ht cc loi gii hn cc i v
cc tiu nm trong khong gia pH 4 v pH 9.
III. Vi nm
Nm ni chung l cc thc vt khng c chlorophyll v v th khng th t tng hp cc cht
dinh dng cho chng. Chng rt khc nhau v kch thc v hnh dng, t nm men n bo
(single-cell yeast) n nm n a bo (multicellular mushroom). Trong s chng, nm men v nm
mc l nhng loi nm cng nghip quan trng.
1. Nm men
Nm men c phn b rng ri trong t nhin. Chng c tm thy trong trong qu, ht v
cc loi thc phm c cha ng khc. Chng cng c trong t, trong khng kh, trn da v
trong rut ng vt. Do nm men khng c chlorophyll (dip lc t), cho nn chng ph thuc vo
thc vt bc cao v ng vt c c nng lng cho cc hot ng sng. Nm men ni chung
l cc c th n bo, c hnh dng t hnh cu n hnh trng. Kch thc ca chng rng t 1-5
m v di t 5-30 m. Thnh t bo t bo non thng mng v tr nn dy t bo gi.
Kiu sinh trng ph bin nht nm men l ny chi (budding), y l mt qu trnh sinh sn
50
v tnh nh minh ho hnh 5.4. Mt chi nh (hoc t bo con-daughter cell) c to thnh trn
b mt ca t bo trng thnh. Chi sinh trng v c lm y cc nguyn liu nhn v t bo
cht t t bo b m. Khi chi ln bng b m, th b my nhn c hai t bo c thay i v t
bo c phn ct. T bo con c th bm vo t bo b m, thng l ngay sau khi t bo phn
chia.
Nm men quan trng nht l cc chng ca Saccharomyces cerevisiae c dng trong sn
xut ru vang, bia v bt n bnh m.
Hnh 5.4. Kiu sinh trng c trng ca nm men bng cch ny chi.
2. Nm mc
Nm mc l loi nm dng si (Hnh 5.5). Mt t bo sinh sn n hoc bo t (bo t ht
nh-conidia) c ny mm to thnh mt si di gi l si nm (hyphae) phn cnh lp li khi
n ko di mt cu trc sinh dng gi l h si nm (mycelium). H ny bao gm mt khi t bo
cht a nhn trong mt h thng cc ng phn cnh nhiu v cng. Do h si nm c kh nng
sinh trng v hn, cho nn n c th t ti cc kch thc v m.
Hnh 5.5. Kiu sinh trng ca nm si bng cch phn cnh.
Cc loi nm mc quan trng nht trong cng nghip l Aspergillus v Penicillium. Mc c
s dng trong sn xut khng sinh, ha cht cng nghip, enzyme, v cc thc phm b sung.
51
IV. Mi trng nui cy
S sinh trng ca qun th vi khun trong mi trng dinh dng nhn to c gi l nui
cy (cultivation). Mt kiu nui cy ch cha mt loi vi sinh vt c gi l nui cy thun khit (pure
culture). Nui cy hn hp (mixed culture) l mt loi nui cy cha nhiu hn mt loi vi sinh vt.
Cc bc cn thit cho nui cy vi sinh vt l nh sau:
- Chun b mi trng nui cy cho php vi sinh vt c th sinh trng tt nht.
- Kh trng mi trng loi b tt c cc c th sng c trong bnh nui cy.
- Cy vi sinh vt vo trong mi trng chun b.
nui cy vi sinh vt, mi trng nui cy phi c chun b trong nhng loi bnh nui
c s dng ph bin nht, chng hn ng nghim, bnh tam gic, a petri hoc ni ln men. C
hai loi mi trng nui cy chnh: mi trng t nhin (da trn kinh nghim) v mi trng tng
hp (thnh phn ha hc xc nh). Ni chung, cc mi trng dinh dng rt khc nhau v hnh
thi (dng rn hoc lng) v thnh phn, ty thuc vo loi vi sinh vt c nui cy v mc ch
nui cy.
1. Mi trng t nhin
L nhng loi mi trng da trn c s ca kinh nghim, m khng da trn s hiu bit
chnh xc v thnh phn cc cht dinh dng v tc ng ca chng. Mi trng t nhin thng
cha peptone, dch chit tht b, hoc dch chit nm men. Khi s dng mi trng rn, c th b
sung cc tc nhn lm rn vo mi trng nh gelatin hoc agar. V d v mi trng rn v mi
trng lng dng cho sinh trng ca nhiu loi vi sinh vt d dng l tng i n gin, ph bin
l canh dinh dng (nutrient broth) v agar dinh dng (nutrient agar). Thnh phn ca chng nh
sau:
- Canh dinh dng: 3 g nc chit tht b, 5 g peptone, 5 g dch chit nm men v nc b
sung ti 1 L.
- Agar dinh dng: cng thnh phn nh canh dinh dng nhng c thm 15 g agar v nc
b sung ti 1 L.
2. Mi trng tng hp
Mi trng cha cc dung dch pha long ca ha cht tinh khit, c bit l cc hp cht
hu c v/hoc v c. Chng thng c s dng cho cc mc ch nghin cu hn l sn
xut. Mi trng c th n gin nh l mui ammonium v c cng vi cc mui khong v ng,
hoc phc tp nh l casein tinh sch c b sung thm cc vitamin, mui khong v ng.
3. Kh trng
Mi trng dinh dng thch hp c chn nui cy mt loi vi sinh vt c bit s c
rt vo cc bnh nui cy. Nu s dng cc ng nghim hoc bnh tam gic, th chng phi c p
bng mt np y thch hp cho php c s trao i kh vi kh quyn nhng vn ngn cn cc c
th ngoi lai ri vo mi trng. Cc loi np khc nhau c dng trong phng th nghim bao
gm: nt bng, bt plastic, np vn, np kim loi v giy nhm.
Mi trng sau phi c kh trng loi b tt c cc c th sng trong bnh cha mi
52
trng. Phng php kh trng ph bin nht l kh trng bng hi nc di p sut cao trong
ni kh trng (autoclave). Ni chung, autoclave hot ng p sut 15 psi 121
o
C. Thi gian kh
trng ty thuc vo bn cht ca nguyn liu, loi bnh cha v th tch ca mi trng. V d, i
vi ng nghim cha mi trng lng c th kh trng t 15-20 pht 121
o
C.
4. Nui cy
Nui cy l s tip mu (vi sinh vt) vo bnh cha mi trng dinh dng v trng. S tip
mu khi nui cy trn mi trng rn c agar c thc hin bng que cy c vng kim loi u
(metal wire hoc loop) c kh trng nhanh trc khi s dng bng cch t nng trn n cn.
Cy chuyn trong nui cy lng thng c thc hin bng Pasteur pipette. S tip mu
thng c tin hnh trong t cy v trng (laminar flow cabinet) gim thiu nguy c nhim bn.
iu quan trng l phi nm vng cc k thut ht pipette thch hp cho vic tip mu v ly mu
trong qu trnh nui cy.
V. Sn xut khng sinh
1. Sn xut penicillin
Sn xut penicillin l mt trong nhng ng dng thng mi quan trng thnh cng trong
vic pht trin cc qu trnh nui cy vi sinh vt quy m phng th nghim thnh quy m sn xut
cng nghip.
Gia nm 1941 v 1945, sn xut penicillin ca M tng t ch hu nh khng c g n 650 t
n v
7
trn mt thng, trong khi gi ca n gim t 20 USD trn 100.000 n v xung cn 60
cent. Hiu sut ca penicillin cng c tng ln hng ngn ln nh vo nhng yu t sau:
- Ci thin thnh phn mi trng-nc chit ngm ng c dng lm cht khi ng sinh
trng, dng lactose thay cho glucose, v b sung phenylacetic acid.
- Pht trin cc k thut nui cy chm.
- To ra cc chng t bin ca Penicillium chrysognum bng phng php chiu x tia cc
tm v tia X.
- Cc phng php hin i ca cc k thut phn tch v tinh sch u ra (downstream
processing)
8
.
Ngy nay, hiu sut ca penicillin thm ch cn cao hn na bi k thut chn lc cc th t
bin tt hn, cng nh ci thin mi trng v cc k thut ln men hp l hn.
1.1. Cc bc chnh ca k thut sn xut penicillin
- Chun b v kh trng mi trng. Mi trng c trng cha nc chit ngm ng (4-5%
trng lng kh), b sung ngun nitrogen nh l bt u nnh, dch chit nm men, cht lng ging
nc sau khi sa chua ng li; ngun carbon l lactose, v mt s loi m khc.
- Tip mu (cy gy). Cc bo t ng kh sinh trng trn mi trng thch nghing
c cy gy vo mi trng lng trong bnh tam gic nui cy lc, tip theo l nui cy kt ht
th cp v s cp, v nui cy trong h ln men quy m ln c th tch tng ln nhiu ln. Vic tng
dn th tch ca nui cy kt ht c thc hin to ra mt lng mu ln a vo nui cy
sao cho mi bc c rt ngn hp l v thit b quy m ln c s dng hiu qu.
53
- Nui cy. H ln men c cnh khuy c s dng trong kiu ln men m c cung cp dinh
dng (fed-batch culture) l glucose v nitrogen trong sut qu trnh nui cy. Kch thc c trng
ca bnh nui khong t 40.000-200.000 L. Oxygen c cung cp bng phng php phun khng
kh tc 0,5-1,0 th tch khng kh/th tch cht lng/pht. Cng sut a vo quay turbine v
phun kh l khong 1-4 W/L. pH c duy tr 6,5. Trong ln men penicillin c trng hu ht
sinh khi t bo cn thit c thu trong sut 40 gi u tin. Penicillin bt u c sn xut pha
sinh trng hm m v tip tc c sn xut cho ti khi n t ti pha tnh. S sinh trng phi
c tip tc mt tc ti thiu nht nh duy tr sn lng penicillin cao. y l l do ti sao
glucose v nitrogen c cung cp lin tc trong sut qu trnh ln men mc d chng c b
sung vo mi trng ngay t u. Penicillin sau khi c t bo sn xut tit vo mi trng nui
cy.
- Qu trnh downstream. Sau khi loi b h si nm mc, penicillin c phn tch t canh
trng (mi trng nui cy) bng phng thc chit dng nc ngc hai giai on lin tc vi
amyl hoc butyl acetate.
2. Sn xut streptomycin
Streptomycin l mt khng sinh dng ph bin trong y hc, th y v bo v thc vt.
Streptomycin c pht hin vo nm 1944 t dch nui cy mt chng x khun Streptomyces
griseus (cn gi l Actinomyces streptomycin). Ging x khun sinh streptomycin khi nui cy chm
pht trin thnh hai pha:
- Pha th nht (pha sinh trng mnh). Cc bo t ny chi v mc thnh si sau 6-8 gi, mi
bo t mc mt chi, khun ty thng thng v phn nhnh rt yu, t bo cht a kim.
- Pha th hai (khun ty khng pht trin). Cui ngy th ba si x khun b chia nh v bt u
t phn.
Nhng ging sinh streptomycin thng khng n nh. Do , trong tng lai cn c s can
thip ca cng ngh DNA ti t hp (xem chng 8) nhm to ra nhng ging c hot lc cao v n
nh a vo sn xut. Gi bo t dng ng kh trong khong nm nm c th cn 96-99%
hot lc, trong ct thch anh ti ba nm, trn mi trng thch nc u 5
o
C ti mt nm. Cc
ngun carbon m ging Streptomyces c th ng ha c v sinh khng sinh l glucose, tinh bt,
dextrin, maltose, fructose, galactose, manose. Trong thc t, glucose v tinh bt c dng lm
ngun nguyn liu trong sn xut streptomycin.
2.1. Cc phng php sn xut streptomycin
Ln men streptomycin c thc hin theo phng php nui cy chm. Qu trnh ln men ny
cng ging nh ln men cc loi khng sinh khc, bao gm cc giai on: nhn ging v ln men
chnh.
- Nhn ging. Ging x khun c bo qun dng bo t. Cy bo t vo mi trng nhn
ging trong bnh tam gic, lc 180-220 vng/pht 26-28
o
C/30-70 gi, sau cho tip vo cc ni
nhn ging (c sc kh v khuy), nui tip cho pht trin sinh khi 20-40 gi. Nhim v chnh trong
giai on nhn ging l to ra mt khi lng ln khun ty x khun a kim c kh nng pht trin
mnh trong giai on ln men chnh v to thnh mt lng ln khng sinh.
- Ln men. Ln men streptomycin l qu trnh ln men hai pha in hnh. Nhit ln men
khong 26-28
o
C, thi gian ln men 96 gi. Trong thi gian ln men cn phi thng kh v khuy trn
mi trng. Lng khng kh thi qua mi trng trung bnh l 1 th tch/1 th tch mi trng.
Khuy mi trng lin tc trong sut c qu trnh ln men (k c khi nhn ging) nu ngng khuy
ch trong mt thi gian ngn s lm gim hiu sut streptomycin. pH trong nhng gi u c gim
54
cht t sau tng dn.
VI. Sn xut thuc bng cng ngh DNA ti t hp
1. Insulin
Ngy nay, vic sn xut insulin quy m cng nghip c l l mt trong nhng thnh cng ni bt
nht, sm nht ca cng ngh sinh hc hin i.
Insulin l mt protein c tuyn ty tit ra nhm iu ha lng ng trong mu. C th
thiu ht insulin trong mu s lm ri lon hu ht cc qu trnh trao i cht c th, dn n tch
t nhiu ng trong nc tiu. Trong c th, insulin c tng hp di dng proinsulin gm ba
chui polypeptide: A, B v C. Khi proinsulin chuyn thnh insulin, chui C c loi b, hai chui A
v B ni vi nhau bi hai cu disulfide (-S-S-). iu tr bnh ny ngi ta thng tim insulin cho
ngi bnh. Ch phm insulin ny c tch chit t tuyn ty ca gia sc. Tuy nhin, c c
100 gram insulin ngi ta phi s dng ty ca 4.000-5.000 con b. V vy, gi thnh ca insulin
trc y l rt cao. Mt khc, v cu to ca insulin b hi khc vi insulin ngi nn trong mu ca
bnh nhn c iu tr bng insulin b bao gi cng xut hin khng th i vi insulin b. iu ny
gy ra mt s hu qu khng mong mun lm gim mt phn hot tnh ca insulin cng nh gim
thi gian tc ng ca thuc.
Boger (1978) ln u tin s dng cng ngh DNA ti t hp thng qua vi khun E. coli thu
nhn c mt lng ln insulin. C th, ngi ta chuyn gen m ha tnh trng sn xut insulin
ca ngi sang cho E. coli. Vi khun E. coli ti t hp gen c nui cy trong ni ln men c dung
tch 1.000 L, sau mt thi gian ngn c th thu c 200 g insulin, tng ng vi lng insulin
chit rt t 8.000-10.000 con b. Thnh cng ny cho thy gen ca ngi c th lm vic mt
cch hiu qu trong genome ca vi sinh vt.
Hai sn phm thng mi ca insulin (c sn xut t nm men S. cerevisiae ti t hp) hin ang
c s dng l: Actrapid v Novolog (NovoNordisk).
2. Interferon
Interferon c bn cht protein, l yu t min dch khng c hiu, gip c th chng li nhiu
loi bnh do interferon c ph tc dng khng virus rng.
Thng thng thu nhn interferon, ngi ta phi tch chit chng t huyt thanh ca mu
nn rt tn km.
Bng phng php cng ngh DNA ti t hp tng t nh insulin, hin nay ngi ta c th
thu nhn mt lng ln interferon thng qua cc c th vi sinh vt c ti t hp gen phc v
cho vic iu tr cc bnh nhim trng nh vim gan B, vim gan C, mt s bnh ung th do virus...
Gilbert (1980) ot gii thng Nobel nh thnh cng trong vic thu nhn interferon t E.
coli c ti t hp gen m ha interferon. n nm 1981, i hc Washington (M) thnh
cng trong vic thu nhn interferon t nm men S. cerevisiae c hiu sut cao gp 10.000 ln so vi
t bo E. coli ti t hp.
3. Hormone
3.1. Hormone sinh trng ngi (human growth hormone-HGH)
Thng thng, HGH ca ng vt c v c sn xut t tuyn yn ca cc ng vt non v
trong sut thi gian trc khi chng trng thnh. HGH c tc dng tng tc sinh trng v kch
thch c th tng khi lng c. ngi sau 30 tui, sn xut hormone sinh trng s dng li, nu
tim HGH sau tui ny s gy ra s pht trin c bp v lng m gim xung.
55
Hormone sinh trng ngi l protein cha khong 191 amino acid, thiu n c th ngi s
b ln. Trc y hormone pht trin c tch t tuyn yn ca ngi cht. Mi t thi cho khong
4-6 mg HGH v mun cha khi cho mt ngi ln phi cn lng HGH thu c t 100-150 t thi.
iu ny cho thy mt tr ngi rt ln khi cha tr chng ln cho tr em. Hin nay, HGH c sn
xut bng cng ngh DNA ti t hp thng qua E. coli vi mt hiu sut rt ln (1 L dch ln men ca
E. coli thu c HGH tng ng vi lng cht ny thu c t 60 t thi). y l mt trong
nhng protein c sn xut bng cng ngh sinh hc hin i sm nht. HGH ti t hp khc vi
HGH bnh thng bi mt amino acid, do E. coli khng c kh nng loi b gc methionine khi u
m n thng b loi sau khi dch m trong t bo ngi.
Insulin v hormone sinh trng ngi l cc protein tng i n gin v khng b glycosyl
ha v bin i nhiu hu dch m, nn c th dng E. coli (prokaryote) lm t bo vt ch sn
xut chng. i vi nhng protein i hi qu trnh glycosyl v bin i hu dch m th khng th s
dng t bo prokaryote lm t bo vt ch trong cng ngh DNA ti t hp m phi s dng cc t
bo eukaryote nh: nm men, nm mc, thc vt hoc ng vt.
3.2. Somatostatin
y l loi hormone c bit, thng c tng hp trong no ng vt v ngi vi mt hm
lng v cng thp. Somatostatin c vai tr iu ha hormone sinh trng v insulin i vo mu,
kim tra s tng hp hai loi hormone ny. Quy trnh sn xut loi sn phm sinh hc c bit qu
ny cng bao gm cc bc ch yu sau y:
+ Phn lp gen m ha somatostatin hoc tng hp trong ng nghim.
+ To dng gen somatostain bng cch gn gen ny vo vector plasmid chuyn gen vo E.
coli. Mi m nui cy 7,5 L vi khun E. coli ny s cho ra 5 mg somatostatin nguyn cht. Trc y,
mun c khi lng hormone ny phi tin hnh c nm trn nguyn liu ly t na triu no cu.
4. Vaccine
Trong sn xut vaccine, cho n thi gian gn y, ngi ta vn s dng vaccine bt hot
hoc vaccine sng nhc c lm khng nguyn kch thch to khng th cn thit trong c th
ngi v vt nui. Nhng vaccine c sn xut theo cch ny c mt vi hn ch, chng hn
vaccine sng nhc c c kh nng quay tr li dng c hoc hot lc ca n gim kh nhanh
trong c th ngi v vt nui.
n nay, nh cng ngh DNA ti t hp ngi ta sn xut c protein v ca mt s loi
virus nh virus bnh di v vim gan B. Sn xut vaccine k thut gen l mt lnh vc pht trin
mnh hin nay ca cng ngh DNA ti t hp. y l loi vaccine c bo ch t vi khun c
chuyn gen m ha tng hp mt protein khng nguyn ca mt loi virus hay mt loi vi khun gy
bnh no . Hin nay, cc loi vaccine k thut gen c s dng cho ngi bao gm vaccine vim
gan B, vaccine di kiu mi, vaccine t kiu mi, vaccine st rt v vaccine bnh phong.
Virus vim gan B c v ngoi lypoprotein. Khng nguyn b mt l protein ch yu ca v
ngoi, c pht hin trong mu ngi b nhim. Ngi ta bin np gen tng hp khng nguyn ca
virus vim gan B vo vi khun E. coli sau sn xut sinh khi quy m ln cc vi khun E. coli
mang gen ti t hp ny, bin E. coli thnh nh my sn xut khng nguyn lm vaccine.
Mt s sn phm thng mi ca khng nguyn b mt vim gan B (c sn xut t nm
men S. cerevisiae ti t hp) hin ang c s dng l: Ambirix (GlaxoSmithKline), Comvax
56
(Merck), HBVAXPRO (Aventis Pharma), Hexavac (Aventis Pasteur), Infanrix-Penta
(GlaxoSmithKline), Pediarix (GlaxoSmithKline), Procomvax (Aventis Pasteur), Twinrix
(GlaxoSmithKline).
5. Mt s loi thuc khc
Hin nay c gn 140 loi protein tr liu c cho php s dng M v chu u. Cc
protein tr liu c th c chia lm hai loi: 1) cc protein khng b bin i hu dch m, v 2) cc
protein cn qu trnh hu dch m (hu ht l kiu N-glycosylation
9
) c chc nng sinh hc y .
Cc protein khng c qu trnh glycosyl ha (glycosylation)
10
, hu dch m, biu hin c hiu
hoc trong vi khun E. coli hoc trong nm men v hin nay chng chim khong 40% th trng
protein tr liu. Tuy nhin, mt s trong chng biu hin c hai h thng, chng hn nh insulin
ti t hp c sn xut c trong E. coli (Humulin, Eli Lilly, Indianapolis, IN, USA) ln trong nm men
(Novolog, Novo Nordisk, Bagsvaerd, an Mch).
Sn xut cc protein ti t hp thng i hi qu trnh N-glycosylation trng thi t nhin
ca chng, trong hu ht trng hp chng cn cc vt ch biu hin l ng vt c v (h thng c
kh nng bt chc glycosylation ca ngi). Cc vt ch prokaryote nh E. coli khng glycosyl ha
protein c, v cc h thng biu hin thuc eukaryote bc thp nh nm men hoc cc t bo cn
trng thng t n nh thc hin glycosylation ca ng vt c v. Tuy nhin, vic nui cy cc t
bo ng vt c v gp nhiu kh khn nh: mi trng dinh dng t tin, thi gian nui cy di
ngy, t bo rt mn cm vi cc lc trt ca h ln men... (xem chng 6). Do , cc yu cu
sinh trng cho vt ch biu hin cc ch phm sinh-dc khng c ci thin buc cc cng ty
phi quay li vi cc h thng biu hin ca nm men v nm si (eukaryote) do chng c kh nng
cung cp hiu sut protein cao (> 1 g/L) trong cc qu trnh ln men ngn ngy, c th tng quy
m sn xut (ln ti 100 m
3
).
Tt c cc protein tr liu da trn c s nm men hin nay c sn xut trong loi nm men
S. cerevisiae (Bng 5.3), nhng cc loi nm men khc cng c pht trin sn xut protein
tr liu. Nm men Pichia pastoris trc y c cng ty Philips Petroleum (Bartlesville, OK, USA) s
dng nh l mt h thng sn xut protein n bo, nhng sau c bin i di truyn biu
hin cc protein ngoi lai. Hn 120 protein ti t hp c biu hin trong loi vt ch ny, kh
nhiu trong s c ngun gc t ngi v ng vt c v. Gn y hn, nm men P. pastoris cng
c dng biu hin cc protein tr liu (ang c th nghim lm sng) (Bng 5.3).
Cng ty Genencor (Palo Alto, CA USA) trc y s dng cc nm si Aspergillus niger v
Trichoderma reesei cho sn xut quy m ln cc enzyme cng nghip ti t hp. Nhng gn y h
cng c gng biu hin cc protein tr liu (v d: cc IgG hon chnh) A. niger. Khng th ny
c sn xut vi hm lng < 1 g/L, c lp rp chnh xc v lin kt vi khng nguyn. Thnh
cng tng t vi biu hin khng th nguyn vn cng thu c P. pastoris, tuy nhin hm
lng kh thp < 40 mg/L. Ngi ta cng chng minh khng th c sn xut trong A. niger c
tp tnh dc ng hc (pharmacokinetic behavior) v hot tnh ADCC
11
tng t vi cc khng th
c ngun gc t cc t bo ng vt c v.
Cng ty Berna Biotechnology (Bern, Switzerland) cng pht trin phng php biu hin
protein thch hp da trn c s nm men methylotrophic Hansenula polymorpha, y l loi ang
c nghin cu sn xut cc vaccine ti t hp.
57
Bng 5.3. Mt s protein tr liu khc c sn xut bng cng ngh DNA ti t hp
III. Sn xut enzyme
ng dng thng mi chnh ca cc enzyme vi sinh vt l trong cng nghip thc phm v sn
xut bia mc d enzyme c tha nhn trong cc ng dng chn on v iu tr bnh. Hu ht cc
loi enzyme c tng hp trong pha log ca nui cy m v c xem nh cc cht trao i s cp.
Tuy nhin, trong mt s trng hp amylase (Bacillus stearothermophillus) c sn xut bi nui cy
idiophase v th c th xem l tng ng vi cc cht trao i th cp. Cc enzyme c th c sn
xut t ng-thc vt cng nh cc ngun vi sinh vt, nhng sn xut bng ln men vi sinh vt l
phng php kinh t v thch hp nht. Hn na, hin nay nh cng ngh DNA ti t hp ngi ta c
th chuyn gen vo cc t bo vi sinh vt sn xut cc enzyme ca ng-thc vt. Cc tin b ca
cng ngh DNA ti t hp m rng phm vi cc sn phm ln men tim tng ca vi sinh vt. C kh
nng a cc gen t cc c th bc cao vo cc t bo vi sinh vt nh l cc t bo nhn tng hp cc
protein (bao gm enzyme) ngoi lai. Cc t bo vt ch dng trong nhng trng hp ny l E. coli, S.
cerevisiae v mt s loi nm men khc.
Trong qu trnh sinh trng ca vi sinh vt, enzyme c hnh thnh trong t bo v mt s
c tit ra mi trng xung quanh. Trong sn xut ch yu l sn phm ca enzyme ngoi bo,
58
cn nu mun tch enzyme ni bo th phi ph v t bo. Cc vi sinh vt c dng trong sn xut
enzyme gm c vi khun, nm mc, nm men v x khun. Cc ch phm enzyme c sn xut t
vi sinh vt c ng dng trong nhiu ngnh cng nghip khc nhau, ch yu l cc enzyme thy
phn dng trong cng nghip thc phm nh:
- Amylase dng trong sn xut ng mt ng v chocolate, trong sn xut bia, ch bin
dextrin vi dch ng sn xut thc n cho ngi gi v tr em, trong sn xut nc qu v
trong y hc.
- Protease thy phn protein ca sa ch bin nhng mn n king c bit, c dng
trong thuc da, sn xut bt git, phim nh, t si, len d v trong y hc. Protease vi sinh vt c th
s dng cng vi amylase trong ch bin thc n gia sc.
- Pectinase l nhm enzyme thy phn pectin to thnh galacturonic acid, glucose, galactose,
arabinose, methanol
- Cytolase l h enzyme c hot tnh cao c th phn hy hemicellulose, pentozan, lignin Cc
enzyme ny c gi chung l cytolase (bao gm cellulase, hemicelllulase, pentosinase).
- Invertase l enzyme ni bo c dng rng ri trong sn xut bnh ko, ru mi, kem, mt
ong nhn to. N lm tng v ngt khi thy phn ng saccharose thnh fructose v glucose, lm
tng ha tan ca saccharose trong sn phm.
Trong mt tng lai gn, enzyme s c s dng rng ri trong y hc lm u d (probe)
cho cc thit b phn tch y khoa v cha bnh. Hin nay, mt s enzyme nh: glucooxydase,
hexokinase, esterase, urease, cholesteroloxydase, alcoholdehydrogenase c s dng kh
ph bin trong y hc. in hnh nht l s dng glucooxydase c nh trn b mt in cc platinum
trong thit b phn tch hm lng glucose mu. Trong y hc, enzyme cn c s dng cha tr
mt s bnh lin quan n s thiu ht ca mt s enzyme trong c th ngi, hoc dng enzyme
loi b nhng cc tht, m d tha gy nguy him cho mt s b phn ca c th ngi. Chng
hn: dng enzyme streptokinase v urokinase lm tan mu ng lm tc nghn mch mu.
1. Trn Th Thanh. 2003. Cng ngh vi sinh. NXB Gio dc, H Ni.
2. Nguyn Vn Uyn v Nguyn Tin Thng. 1999. Nhng kin thc c bn v cng ngh
sinh hc. NXB Gio dc, TP H Ch Minh.
3. Babey F and Mujacic. 2004. Recombinant protein folding and misfolding in Escherichia coli.
Nature Biotech. 22: 1399-1408.
4. Cutler SJ and Cutler HG. 2000. Biologically Active Natural Products: Pharmaceuticals. CRC
Press LLC, USA.
5. Gerngross TU. 2004. Advances in the production of human therapeutic proteins in yeasts
and filamentous fungi. Nature Biotech. 22: 1409-1414.
6. Klefenz H. 2002. Industrial Pharmaceutical Biotechnology. Wiley-VCH Verlag GmbH,
Weinheim, Germany.
nd
ed. Prentice
Hall, Inc. NJ, USA.
59
1
Psychrophiles: vi khun a lnh (di -20oC).
2
Mesophiles: vi khun a nhit trung bnh.
3
Thermophiles: vi khun a nhit cao.
4
Nhn tht: cn gi l nhn chun.
5
Sinh vt n bo: cn gi l sinh vt nguyn sinh.
6
Tin nhn: cn gi l nhn s.
7
Mt n v (unit) tng ng 0,6 mg ca chun quc t cho hot tnh penicillin.
8
Downstream processing: l cc bc ca qu trnh sinh hc tip theo sau s ln men v/hoc nui
cy t bo, mt chui cc hot ng phn tch v tinh sch cn thit thu c sn phm thuc
theo yu cu mc tinh sch cn thit.
9
N-glycosylation: cc oligosaccharide lin kt cng ha tr vi protein nguyn t nitrogen.
10
Glycosyl ha: l qu trnh b sung mt hoc nhiu phn t carbohydrate (gc ng) vo mt phn
t protein (glycoprotein) sau khi n c tng hp nh ribosome.
11
ADCC (Antibody-dependent cell-mediated cytotoxicity): Kh nng gy c t bo qua trung gian t
bo ph thuc vo khng th.
12
GM-CSF (granolocyte-macrophage colony stimulating factor): yu t kch thch qun lc i thc
bo ca t bo bch cu ht.
13
Cht chng ng mu.
14
Cht chng ng mu.
60
Chng 6
Nui cy t bo ng vt
I. M u
T bo ng vt tch t m c th c nui cy trn cc loi mi trng dinh dng tng hp
bn ngoi c th, chng sinh trng bng cch tng s lng v kch thc t bo. K thut nui cy
t bo ng vt to c hi nghin cu cc t bo ung th, phn loi cc khi u c tnh, m hnh
thc nghim kho st tc ng ca ha cht, xc nh s tng hp ca m trong cy ghp v
nghin cu cc t bo c bit cng s tng tc ca chng.
K thut nui cy t bo ng vt c v c th c ng dng sn xut cc hp cht ha sinh
quan trng dng trong chn on nh cc hormone sinh trng ca ngi, interferon, hot t
plasminogen m, cc viral vaccine v cc khng th n dng. Theo phng php truyn thng cc
hp cht ha sinh ny c sn xut bng cch s dng cc ng vt sng hoc c tch chit t xc
ngi cht. Chng hn, cc khng th n dng c th c sn xut bng cch nui cy cc t bo
hybridoma trong cc khoang mng bng (peritoneal cavity) ca chut, hoc hormone sinh trng dng
cha bnh ci (dwarfism) c th c tch chit t xc ngi cht. Tuy nhin, s lng thu c t
cc phng php ny rt hn ch v th vic ng dng rng ri chng trong iu tr cn gp nhiu kh
khn.
Mt vi sn phm gen ca ng vt c v cng c th c sn xut bi h thng vi khun bng
cch dng cng ngh DNA ti t hp. Tc sinh trng nhanh, thnh phn mi trng n gin v
r tin ca nui cy t bo vi khun khin chng c nhiu u im hn so vi nui cy t bo ng vt
c v. Tuy nhin, vi khun li thiu kh nng sa i hu dch m (post-translational modifications)
bao gm vic phn gii protein, lin kt cc tiu n v (subunit), hoc nhiu phn ng kt hp khc
nhau nh glycosylation, methylation, carboxylation, amidation, hnh thnh cc cu ni disulfide hoc
phosphoryl ha (phosphorylation) cc gc amino acid. Nhng sa i ny rt quan trng nh hng
n hot tnh sinh hc ca sn phm. V d, qu trnh glycosyl ha (glycosylation) c th gip bo v
protein chng li s phn gii chng, duy tr kh nng n nh cu trc v bin i khng nguyn. V
th, nhiu cng ty phi quay li vi h thng vt ch biu hin cc protein ngoi lai l cc t bo
ng vt c v. Hin nay, khong 60% protein ti t hp dng lm dc phm c sn xut t cc h
thng t bo vt ch ny.
1. Cc u im ca nui cy t bo ng vt
- H thng t bo ng vt l cc nh my t bo thch hp cho vic sn xut cc phn t phc
tp v cc khng th dng lm thuc phng bnh, iu tr hoc chn on (Bng 6.1).
- Cc t bo ng vt p ng c qu trnh hu dch m chnh xc i vi cc sn phm
protein sinh-dc (biopharmaceutical).
- Sn xut cc viral vector dng trong liu php gen (bin np mt gen bnh thng vo trong t
bo soma mang gen tng ng b khim khuyt cha bnh do s khim khuyt gy ra). Cc mc
ch chnh ca liu php ny l cc bnh ung th, hi chng suy gim min dch (HIV), chng vim
khp, cc bnh tim mch v x ha u nang.
61
- Sn xut cc t bo ng vt dng lm c cht in vitro trong nghin cu c cht hc v
dc hc.
- Pht trin cng ngh m hoc pht sinh c quan sn xut cc c quan thay th nhn to-sinh
hc/cc dng c tr gip, chng hn:
+ Da nhn to cha bng.
+ M gan cha bnh vim gan.
+ o Langerhans cha bnh tiu ng.
2. Mt s hn ch ca nui cy t bo ng vt
Mc d tim nng ng dng ca nui cy t bo ng vt l rt ln, nhng vic nui cy mt s
lng ln t bo ng vt thng gp cc kh khn sau:
- Cc t bo ng vt c kch thc ln hn v cu trc phc tp hn cc t bo vi sinh vt.
Bng 6.1. Cc sn phm ca nui cy t bo ng vt.
- Tc sinh trng ca t bo ng vt rt chm so vi t bo vi sinh vt. V th, sn lng ca
chng kh thp v vic duy tr iu kin nui cy v trng trong mt thi gian di thng gp nhiu
kh khn hn.
- Cc t bo ng vt c bao bc bi mng huyt tng, mng hn nhiu so vi thnh t bo
dy chc thng thy vi sinh vt hoc t bo thc vt, v kt qu l chng rt d b bin dng v v.
- Nhu cu dinh dng ca t bo ng vt cha c xc nh mt cch y , v mi trng
nui cy thng i hi b sung huyt thanh mu rt t tin.
- T bo ng vt l mt phn ca m c t chc (phn ha) hn l mt c th n bo
62
ring bit nh vi sinh vt.
- Hu ht cc t bo ng vt ch sinh trng khi c gn trn mt b mt.
II. T bo ng vt
Cc t bo ng vt l t bo eukaryote, chng c lin kt vi nhau bi cc nguyn liu gian
bo to thnh m. M ng vt thng c phn chia theo bn nhm: biu m (epithelium), m
lin kt (connective tissue), m c (muscle) v m thn kinh (nerve). Biu m to thnh lp ph v
lp lt trn cc b mt t do ca c th, c bn trong v bn ngoi. m lin kt, cc t bo thng
c bao bc trong th gian bo rng (ko di), c th l cht lng, hi rn hoc rn. Cc t bo
m c thng thon di v c gn vi nhau thnh mt phin hoc mt b bi m lin kt. M c
chu trch nhim cho hu ht chuyn ng ng vt bc cao. Cc t bo m thn kinh gm c thn
bo cha nhn v mt hoc nhiu phn m rng di v mnh c gi l si. Cc t bo thn kinh
c kch thch d dng v truyn xung ng rt nhanh.
1. Cc t bo dch huyn ph
T bo hng cu v bch huyt l cc m lin kt khng in hnh dng th lng. Cc t bo
mu hoc dch bch huyt l cc t bo dch huyn ph (suspension cells), hoc khng dnh bm khi
chng sinh trng trong nui cy in vitro. Cc t bo khng dnh bm khng i hi b mt sinh
trng.
Chng hn, cc t bo bch huyt (lymphocytes) (Hnh 6.1a) bt ngun t m bch huyt l
cc t bo khng dnh bm v c hnh cu ng knh t 10-20 m. Chng c th c nui cy
trong mi trng dch lng theo phng thc tng t vi khun.
2. Cc t bo dnh bm
Hu ht cc t bo ng vt bnh thng l cc t bo dnh bm, v th chng cn c b mt
gn vo v sinh trng. Trong cc ng dng, ngi ta s dng rng ri cc loi t bo dnh bm
l t bo biu m v nguyn bo si (fibroblast) (Hnh 6.1b v c). Cc t bo dnh bm cn c mt b
mt m sinh trng nh l thy tinh hoc plastic. a petri hoc cc chai trc ln l cc loi c
s dng rng ri nht. Cc chai c t nm trn mt trc ln quay trn chm trong t m. Chai c
dung tch 1 L cha khong 100 mL mi trng l thch hp cho cc t bo va sinh trng trn
thnh chai va tip xc vi mi trng v khng kh. Tuy nhin, chai trc ln ch dng cho quy m
phng th nghim v din tch b mt trn mt n v th tch ca chai nui cy kh nh (500 cm
2
/L).
Hnh 6.1. Cc t bo ng vt thng c s dng trong nui cy.
63
(a) t bo bch huyt, (b) t bo biu m, (c) nguyn bo si
T l din tch/th tch c th c tng ln khi cc t bo sinh trng trn cc gi th l
polymer bt bin (spongy), th gm (ceramic), cc si rng, bao vi th (microcapsule), hoc trn cc
ht nh c kch thc hin vi gi l microcarrier.
III. Mi trng nui cy
Nhu cu dinh dng ca cc t bo ng vt c v ln hn vi sinh vt do, khng ging cc vi
sinh vt, ng vt khng trao i cht nitrogen v c. V th, nhiu amino acid v vitamin cn phi
c b sung vo mi trng. Mi trng c trng dng trong nui cy t bo ng vt bao gm
cc amino acid, cc vitamin, cc hormone, cc nhn t sinh trng, mui khong v glucose. Ngoi
ra, mi trng cn c cung cp t 2-20% (theo th tch) huyt tng ca ng vt c v. Mc d
huyt thanh c thnh phn cha c xc nh y , nhng nhiu nghin cu cho thy n rt
cn thit cho s pht trin v tn ti ca t bo trong nui cy. Bng 6.2 trnh by thnh phn v hm
lng ca cc cht trong mi trng Eagle (Eagle 1959), y l mt trong nhng mi trng c
s dng ph bin trong nui cy t bo ng vt.
Bng 6.2. Thnh phn mi trng Eagle (1959).
64
Huyt thanh dng trong mi trng nui cy khng ch t tin m cn l ngun nhim bn
virus v mycoplasma. Do bn cht ha hc ca huyt thanh cha c xc nh y nn trong
mt s trng hp c th nh hng xu n kt qu nui cy. S hin din ca nhiu protein khc
nhau trong huyt thanh cng c th lm phc tp cc qu trnh phn tch v tinh sch u ra. V l
do , nhiu nghin cu c thc hin xy dng cng thc mi trng khng c huyt
thanh. Nhng cng thc ny cha cc hormone v cc nhn t sinh trng c tinh sch thay
th cho huyt thanh.
Trc y, huyt thanh ca thai b (fetal bovine serum-FBS), c b sung nng 1-20%,
l rt cn thit cho s sinh sn ca cc t bo ng vt c v. Nhng ngy nay, nhiu qu trnh nui
cy t bo quy m ln bt u thc hin trong mi trng khng c huyt thanh.
IV. K thut nui cy t bo ng vt
Phng php chnh trong nui cy t bo ng vt c v sn xut cc sn phm sinh-dc
l da trn c s nui cy dch huyn ph trong h ln men. T lu, h ln men c s dng
trong nui cy vi khun v nm men. u tin, s ln men l thut ng dng cho sn xut cn. Sau
, cc nh vi sinh vt hc ng dng cc nguyn tc trn tch chit cc vitamin, cc acid hu c
v cc khng sinh Kt qu dn n s pht trin nhanh chng cc phng php v cc h thng
ln men khc nhau.
65
Cc nguyn l tng t sau c ng dng cho nui cy sinh khi t bo ng vt v thc
vt. Tuy nhin, nui cy cc t bo ng vt v thc vt kh khn hn nhiu so vi vi sinh vt, ci
chnh l do qu trnh trao i cht trong cc loi t bo ny din ra chm, iu ny cng phn nh tc
sinh trng chm ca t bo. Cc t bo ng vt c nhu cu dinh dng phc tp hn so vi vi
khun v nm men, chng khng c thnh t bo nh vi khun v th rt d bin dng v v. Do ,
cc h thng khuy v sc kh c thit k khc vi nui cy vi khun. Mc d c mt s im
khng thun li, nhng h thng ln men c s dng nui cy t bo ng vt t nht cng
vi chc nm trc y. Cc dng t bo khc nhau nh BHK-21, LS, cc t bo Namalwa
c sinh trng trong h ln men theo phng thc nui cy chm ngp trong mi trng sn
xut cc viral vaccine v cc sn phm khc.
c im d bin dng v d v ca t bo ng vt c khc phc bng cch a vo
cc cnh khuy c dng hnh mi cho. Vic cung cp kh trc tip c th to ra bt kh d lm v t
bo, v th cn cung cp kh bng cch khuch tn thng qua ng silicone. Mi trng cha nhiu
protein huyt thanh c kh nng gy ra hin tng to bt nn cn khuy chm v nh. i vi nui
cy mt cao, cn cung cp thm oxygen. Phng php dng ng silicone sc kh c nhiu u
im do khng to ra bt kh v tc truyn oxygen l tha ng.
Nh vy, cc h ln men vi sinh vt c ci tin thch hp c th dng nui cy sinh khi
cc t bo ng vt sinh trng trong dch huyn ph. Nu mun nui cy mt dng t bo dnh bm
th nn dng mt h thng cht mang nh l microcarrier.
Cc dng t bo ng vt c v thng c s dng trong nui cy l CHO
4
, NS0
5
, BHK
6
,
HEK-293
7
v t bo vng mc ca ngi.
1. H thng sn xut
Pht trin mt qu trnh sn xut cng nghip cho protein ti t hp ca t bo ng vt c v
thng da theo h thng c trnh by hnh 6.2. u tin, gen quan tm c ti t hp vi cc
nhn t iu ha phin m (promoter) cn thit trong plasmid vector chuyn vo t bo. ng
thi, gen th hai (gen chn lc-selector, hay cn gi l gen ch th chn lc-selectable marker) cng
c chuyn cho t bo nhn phn bit t bo c bin np v khng bin np. S hin din
ca tc nhn chn lc trn mi trng nui cy sau khi chuyn gen mt vi ngy cho php phn
lp cc t bo ti t hp sng st. Cc gen ch th chn lc c dng ph bin nht l dihydrofolate
reductase (DHFR), mt enzyme tham gia trong qu trnh chuyn ha nucleotide, v glutamine
synthetase (GS). Trong c hai trng hp, s chn lc xy ra khi thiu cht chuyn ha thch hp
trong mi trng (hypoxantine v thymidine, trong trng hp ca DHFR hoc glutamine trong
trng hp GS), do ngn cn s sinh trng ca cc t bo khng bin np.
Sau khi chn lc, cc t bo sng st (xem nh l cc t bo n) c chuyn vo bnh nui
cy th hai, v qu trnh nui cy c pht trin sn xut cc qun th v tnh (clonal
populations). Cui cng, cc dng ring bit c nh gi kh nng biu hin protein ti t hp
chn ra dng c kh nng sn xut cao nht. T nhng dng ny, mt dng t bo c tc sinh
trng thch hp v cc sn lng cao c s dng sn xut protein ti t hp. Qu trnh nui
cy sau s c thit lp v ti u ha cho sn xut.
66
Hnh 6.2. Sinh sn v pht trin dng t bo cho cc qu trnh nui cy sn xut protein ti
t hp mong mun (protein o.i.). Cc ng gn sng ch ra s ln cy chuyn ca cc dng t
bo ring bit sng lc t bo a vo sn xut. Cc l nh l ngn hng t bo c ng lnh
trong nitrogen lng. Cc bnh nui xoay (spinner flask) m t cc h thng nui cy quy m nh ti
u ha quy trnh, v cc h ln men m t cc qu trnh sn xut quy m ln.
2. Ti u ha mi trng dinh dng v t bo vt ch
Hin nay, mi trng thng mi dng cho nui cy t bo c cht lng cao c mt s
nh cung cp hng u sn xut. Tuy nhin, vic sn xut protein ti t hp cng cn phi c ti
u ha bng cch kho st trn nhiu cng thc mi trng dinh dng. Thng thng, mt qu
trnh sn xut ring bit i hi mt vi cng thc mi trng khc nhau, trong mi cng thc
c thit k cho mt phase c trng ca s sinh trng.
Cc mi trng cho tc sinh trng nhanh i hi cy chuyn 3-5 ngy/ln. Qu trnh sn
xut m (6-8 ngy) hoc m m rng (10-21 ngy) di hn nhiu so vi thi gian cy chuyn c
trng. Pht trin mi trng thch hp l v cng quan trng v phi c thit k trn mt c s
ring bit, i vi mi qu trnh v mi dng t bo.
Tng t mi trng, cc t bo vt ch cng phi c ci thin chng li cc nh hng
ca iu kin nui cy lm gim kh nng sng st v/hoc tin hnh cc bc chuyn gen kch
thch sinh trng. Kt qu nghin cu ca nhiu phng th nghim cho thy, cc t bo vt ch c
th c ci thin sinh trng, kh nng sng st v sn lng nh cng ngh DNA ti t hp. Cc
proto-oncogene
8
, cc gen iu chnh chu k t bo (cyclins), cc gen yu t sinh trng (v d yu t
sinh trng ging insulin) v cc gen antiapoptosis c a vo trong t bo to ra cc vt
ch siu sn xut (superior production hosts).
Ci thin s bin i v sn xut protein hu dch m l mt pht trin y ha hn khc.
Chng hn, ngi ta thy hiu lc ca cc khng th c th c ci thin bng cch tng cng
hiu lc ca c quan phn ng min dch t nhin ca chng. S biu hin d tha (over
expression) c n nh ca N-acetylglucosaminyl-transferase-III, mt enzyme khng c biu
hin t nhin trong cc t bo CHO v NS0, trong cc t bo sn xut khng th ti t hp kch
thch to cc IgG nng cao v cc oligosaccharide khng fucosyl ha (fucosylation) trong vng
Fc. Nhng bin i ca dng glyco (glycoform) lm tng t 5-10 ln cc c t t bo ph thuc
khng th.
67
V. Cc khng th n dng
Cc t bo bch huyt (lymphocytes) l cc t bo mu trng cn cho cc phn ng min dch.
Cc B-lymphocyte, loi sn xut khng th, hin din trong l lch, cc u bch huyt (lympho nodes)
v mu. Khi mt cht ngoi lai i vo trong c th ca ng vt c xng sng, th cc tuyn B-
lymphocyte sn xut nhanh v tit ra cc phn t protein gi l immunoglobulin hay cn gi l khng
th (antibody). Cc khng th c cc v tr kt hp c th nhn ra hnh dng ca yu t quyt nh
c hiu trn b mt ca cht ngoi lai, cn gi l khng nguyn (antigent). Kt qu l cc khng th
c th lin kt vi khng nguyn c hiu, trung ha v o thi cc cht ngoi lai. Do tnh c hiu
ca chng trong vic nhn dng cc t bo hoc phn t c bit nn khng th l nhng cng c
rt quan trng cc nghin cu vin v thy thuc lm sng pht hin s hin din v nng ca
thuc, cc sn phm ca virus v vi khun, hormone v cc khng th khc trong mu.
C 5 loi khng th l immunoglobulin G, A, M, D v E. Hnh 6.3. minh ha cu trc ca mt
loi khng th immunoglobulin G (IgG), c cu trc protein dng hnh ch Y bao gm mt cp chui
nng (heavy chain)
9
v mt cp chui nh (light chain)
10
c lin kt bi cc cu ni disulfide. Mi
chui c 2 vng: (1) vng c th thay i c, vng ny khc nhau ty thuc vo mi loi khng th
v chng cha cc v tr lin kt c hiu vi cc khng nguyn khc nhau, (2) vng khng th thay
i c, c trng cho cc khng th ca mt phn lp nht nh. Bn cht ca lin kt khng th-
khng nguyn tng t vi bn cht ca phc hp c cht-enzyme.
C nhiu dng khc nhau ca B-lymphocyte v mi dng sn xut ra cc khng th khc nhau
nhn ra cc yu t quyt nh khng nguyn c hiu. V th, khi mt ng vt c tim mt tc
nhn min dch, th n phn ng bng cch sn xut ra mt hn hp khng th a dng hu nh
khng th phn chia c. sn xut mt lng ln khng th ng nht (khng th n dng) ch
nhn ra mt cu trc ha hc, chng ta phi cho sinh trng c mt dng t bo c bit ca B-
lymphocyte. Tuy nhin, ngi ta nhn thy l cc t bo tit (to) ra khng th khng th duy tr
c trn mi trng nui cy.
Hnh 6.3. Cu trc khng th immunoglobulin G (IgG).
1. Dung hp t bo
Khng ging nh cc t bo tit ra khng th, cc t bo myeloma (u ty) l loi t bo khi u
68
c tnh ca h thng min dch, c th c nui cy lin tc. Khler v Milstein (1975) pht trin
mt phng php dung hp cc t bo B-lymphocyte ca l lch vi t bo myeloma ca chut
lai hai loi t bo ny vi nhau, t bo lai myeloma (hay hybridoma), c th c c im ca c hai
dng t bo: l sn xut cc khng th c hiu v bt t. V hybridoma c bt ngun t mt t
bo B-lymphocyte n, nn n ch sn xut mt loi khng th gi l khng th n dng.
Phng thc c trng dung hp t bo nh sau (Hnh 6.4):
- Tim khng nguyn c chn vo trong chut. H thng min dch trong chut p ng
bng cch sn xut cc t bo B-lymphocyte tit ra khng th.
- Ly l lch ca chut v tch cc t bo B-lymphocyte.
- Nui cc t bo myeloma thch hp thiu HPGRT (hypoxanthine guanine phosphoribosyl
transferase), t bin HPGRT
-
, mt marker di truyn chn cc t bo lai sau khi dung hp.
- Dung hp cc t bo B-lymphocyte vi cc t bo myeloma bng cch trn chng trong mi
trng cha 40-50% polyethylene glycol (PEG). Mi trng s cha cc hn hp ca B-lymphocyte,
myeloma, v cc t bo hybrid-myeloma. Cc B-lymphocyte cha HPGRT, nh vy cc hybridoma
cng cha HPGRT. V th, cc t bo myeloma c coi nh l HPGRT
-
, trong khi cc t bo B-
lymphocyte v hybridoma l HPGRT
+
.
- Chn lc cc t bo HPGRT
+
bng cch nui cy hn hp trn mi trng cha HAT
(hypoxanthine, aminopterin v thymidine) l cht c ch sinh trng cc t bo HPGRT
-
. Do , cc
t bo myeloma s cht trn mi trng ny, trong khi cc t bo hybridoma s phn chia. Cc B-
lymphocyte khng dung hp s cht do khong thi gian sng b hn ch ca chng.
Hnh 6.4. Phng thc dung hp cc t bo B-lymphocyte.
69
2. Th nghim khng th
Cc khng th n dng c th c phn tch bng k thut th nghim pha rn nh cc th
nghim hp th min dch lin kt enzyme (enzyme linked immunosorbent assays-ELISA) hoc cc
th nghim min dch phng x (radioimmuno assays-RIA). Cc phng thc th nghim c trng
nh sau (Hnh 6.5):
- a dung dch khng nguyn c hiu (Ag) vo cc ging ca a microtitre c kh nng ht
bm khng nguyn bng s tng tc k nc khng c hiu. B sung mt loi protein khng gy
cn tr lin kt khng nguyn-khng th sau ny, nh l bovine serum albumin (BSA) chim cc
v tr gn cn li trn ging.
- B sung dung dch khng th n dng th nht (Mab1) l khng th c hiu vo ging.
Khng th s lin kt vi khng nguyn trn b mt rn.
- B sung khng th n dng th hai (Mab2) l khng th c c tnh tng phn vi
immunoglobulin ca loi m t t bo hybridoma bt ngun. Mt enzyme (E) cho phng php
ELISA hoc nh du ng v phng x cho phng php RIA c gn ng ha tr vi khng th
th hai.
- RIA: o hot tnh phng x bng phng php phng x t ghi (autoradiography) hoc bng
phng php m nhp nhy ca h thng nng rn (solid scintillation counter).
- ELISA: B sung c cht thch hp v o mt quang (OD) sn phm ca n trn my quang
ph bc sng thch hp.
Hnh 6.5. S th nghim khng th ELISA. Enzyme s c thay th bng nh du hot
tnh phng x trong trng hp th nghim RIA.
VI. Sn xut thuc v DNA vaccine
1. Interferon
Interferon l mt loi cytokine (protein) do t bo ng vt sinh ra mi khi chng b virus xm
nhp, interferon khng c hiu v c tc dng c ch sinh sn ca virus, nh hng n sinh
trng v pht trin (s phn ha) ca cc t bo khi u v t bo bnh thng nht nh no .
Interferon c th c coi nh mt loi thuc hu hiu chng cc bnh nhim virus v k c mt vi
dng ung th.
70
C th ng vt sn xut hai loi interferon l interferon I v interferon II. Interferon I c hot
tnh khng virus bao gm interferon-o (IFN-o, khi lng phn t khong t 17-26 kDa, c bch
cu sn xut) v interferon- (IFN-, khi lng phn t khong 21 kDa, c nguyn bo si sn
xut). C hai loi IFN-o v IFN- c tc dng chng li s sinh sn ca t bo, c bit l IFN-o, kch
thch hot ng cc t bo git t nhin (NK) lm tng biu hin cc phn t HLA
11
lp I. IFN-o c
ng dng iu tr ung th c hiu qu (c bit l ung th mu c tnh, ung th biu m t bo
thn), iu tr bnh nhim virus vim gan B v C mn tnh (c th cha khi t 40-50% s bnh nhn).
Interferon- (IFN-, c khi lng phn t khong 25 kDa, c sn xut t cc lympho T CD4+
12
(h1), T CD8+ v cc t bo NK) hot ha cho s biu hin ca cc phn t HLA lp II, lm thc y
s bit ha cc t bo mono thnh cc i thc bo.
Cc interferon ring bit c cc phm vi hot ng khc nhau trong cc loi khc nhau. o-
interferon chng t hiu qu chng li bnh hairy-cell leukemia v vim gan C. Ngoi ra, n cn c
hot tnh chng vim gan B mn tnh, cng nh iu tr bu sinh dc v mt vi bnh ung th nh
ung th mu v ty xng. Thuc xt mi cha o-interferon cung cp mt s cht bo v chng bnh
cm lnh do cc rhinovirus gy ra.
Trong mt thi gian di, vic cung cp interferon ngi cho nghin cu b hn ch do k thut
tch chit rt tn km. n nm 1979, Klein v cng s xy dng h thng sn xut n nh cho
interferon ca lymphoid ngi bng k thut nui cy dch huyn ph ca African Burkitts lymphoma
c ngun gc t dng t bo Namalva vi s cm ng bng virus gy bnh New Castle. Nui cy t
bo c sinh trng trong h ln men 50 L trn mi trng khng c huyt thanh v c cung
cp oxygen ha tan nng cao ti u s sinh trng ca t bo. Trung bnh 3,35 units
interferon/mL c thu hi trong sn phm cui cng cha tinh sch.
Tuy nhin, ngy nay vic tch chit mt lng ln interferon tr nn thun li hn nhiu
nh cng ngh DNA ti t hp. Gen m ha interferon c bin np vo t bo nm men S.
cerevisiae (eukaryote) c kh nng sn xut interferon vi sn lng rt cao. Thnh cng ny lm
gim ng k gi thnh ca interferon, m ra c hi s dng loi thuc ny trong iu tr cc bnh
vim nhim virus v mt vi dng ung th.
2. Hot t plasminogen m
Hot t plasminogen m (t-PA) l mt v d v protein tr liu c tng hp trong nui cy cc
t bo ng vt c v chuyn gen. t-PA l mt protease c tc dng phn hy cc si t huyt fibrin
(dng hot ha ca protein gy ng mu fibrinogen
13
, protein khng tan ng kt thnh cc si
hnh thnh mt cc mu ng) c s dng cha bnh nghn mch mu. Bnh nhn b
nhng cn au tim thng c ung t-PA v n ph tan nhng cc mu nh c kh nng lm tc
mch.
Cc nghin cu gn y cho thy c th s dng cc tuyn sa ca ng vt chuyn gen
sn xut cc protein him ca ngi. Chng hn, gen t-PA c a vo phi chut bng cng ngh
DNA ti t hp, sau phi chut c cy vo chut ci to thnh chut chuyn gen c kh nng
tit t-PA ngi qua sa.
3. DNA vaccine
Cn gi l vaccine DNA ti t hp, y l loi nucleic acid vaccine, da trn nguyn l mt gen
m ha cho protein khng nguyn c hiu c tim vo vt ch (t bo ng vt hoc vi sinh vt)
sn xut cc khng nguyn ny v khi ng mt phn ng min dch. Nhiu vaccine phng virus
71
hin nay (si, t lit, di) u c sn xut t nui cy t bo ng vt m khng phi l t bo vi
sinh vt.
Hnh 6.6 minh ha m hnh sn xut v iu tr bng liu php vaccine DNA ti t hp: Phn
lp mt hoc nhiu gen t tc nhn gy bnh (pathogen), a cc gen ny vo trong vng DNA ca
plasmid v ng li (a). Cc vng DNA sau c a vo trong cc nhm t bo nh, thng
bng cch tim vo t bo c (b) hoc y vo da nh sng bn gen (c). Cc gen c chn la m
ha cho cc khng nguyn, cc cht c th gy ra mt p ng min dch, thng c sn xut bi
tc nhn gy bnh.
Hnh 6.6. M hnh sn xut v iu tr bng liu php DNA vaccine.
3.1. Phng thc hot ng ca cc DNA vaccine
Cc DNA vaccine kch thch phn ng min dch bo v chng li tc nhn ngoi nhim
hoc tc nhn gy bnh, ch yu bng hai cch ca h thng min dch: nhnh th dch (yu t tn
cng cc tc nhn gy bnh bn ngoi t bo), v nhnh t bo (yu t o thi cc t bo b xm
nhim bi tc nhn gy bnh). Kh nng min dch s c duy tr khi hot ng ny to ra cc t
bo ghi nh (memory cells) lu di sn sng ngn chn tc nhn gy bnh.
Cc vaccine cm ng min dch theo phng thc DNA vaccine i vo t bo ch (v d: m
c) v sau hot ng sn xut khng nguyn ca t bo thng c tm thy trn tc nhn gy
bnh.
- Trong phn ng th dch, cc t bo mu trng c gi l t bo B lin kt gii phng cc
bn sao ca cc protein khng nguyn v sau tng ln nhiu ln. Nhiu th h sau ca t bo tit
ra cc phn t khng th (trong sut qu trnh xm nhim) v chng s kt thnh cm trn tc nhn gy
bnh nh du n v sau l ph hy.
- S biu hin cc on protein khng nguyn (hoc peptide) trn cc t bo b xm nhim c
th khi ng mt p ng t bo. S lin kt cc phc hp khng nguyn s cm ng cc t bo
72
mu trng (cytotoxic T cells
14
) sinh si ny n git cht cc t bo c lin kt hoc nhng yu
t khc biu hin cc chui peptide tng t. Cc t bo c hot ha cng s tr thnh cc t bo
ghi nh, sn sng o thi cc t bo b xm nhim bi tc nhn gy bnh trong tng lai.
3.2. Th nghim cc DNA vaccine trn ngi
Bng 6.3 lit k mt s DNA vaccine c th nghim trn ngi. Tt c cc vaccine ng vin
trong cc th nghim giai on u kim tra an ton v p ng min dch c s dng
iu tr tt. Khng c th nghim no khng c kt qu trong vic ngn nga bnh. Hu ht cc
nghin cu ny vn ang tip tc.
VII. T bo ng vt s dng trong cy ghp
y l lnh vc ng dng mi ca nui cy t bo ng vt c v. Trc y, khc phc cc
sai hng ca c th, ngi ta s dng cc tc nhn nh thuc, m hay c quan ghp, b phn gi.
Ngy nay, cng vi s pht trin ca k thut nui cy t bo, xu hng cy ghp t bo tr nn
ngy cng ph bin hn. Chng hn, vic ghp cc t bo ty sn xut insulin ca ln vo ngi
mc bnh tiu ng c thc hin vo nhng nm 1990, tuy nhin kt qu vn cha r rng.
Nghin cu ny nu thnh cng s gip bnh nhn khng cn ph thuc vo vic tim thng xuyn
insulin, ng thi loi b ti a cc hu qu ca bnh. Nguyn tc tng t cng c p dng cho
trng hp cc tn thng thn kinh. Mt cng trnh gn y cho thy vic ghp cc t bo thn
kinh ly t th vn ca thai cho php phc hi cc chc nng ca no cng nh chc nng vn ng
kh Macaque trc c mang cc biu hin ging chng bnh ma git Huntington ca ngi.
Bn cnh , k thut ghp t bo t thn cng rt c quan tm. Ngi ta hy vng bng
cch ghp cho bnh nhn cc t bo ngun to mu s khc phc c tnh trng khng hot ng
ca ty xng (chng hn trng hp b chiu x liu cao do tai nn). Cc t bo ny trc c
thu nhn t chnh ngi bnh, cho sinh sn v bit ha trong mi trng nhn to c cha cc nhn
t sinh trng v sau c tim tr li cho ngi bnh vi s lng ln. Mt nghin cu gn y
thnh cng trong vic ghp t thn t bo sn. Trc y, cha cc bnh thng tn u gi,
ngi ta buc phi s dng cc b phn gi. Tuy nhin, gii php ny t ra khng hiu qu lm: cc
vt liu sinh hc dng ch to b phn gi (thng l polyethylene) rt km bn so vi i hi vn
ng nhiu ca khp gi nn c sau 15 nm li phi thay. Mt khc, do t bo sn khng c kh
nng sinh sn b p thng tn nn khng th da vo s ti sinh t nhin. Nhm nghin cu
trn thu nhn sinh thit t mt phn sn lnh ca ngi bnh, tch ri tng t bo nh enzyme
thy phn. Cc t bo ny c nui cy v tim tr li vo ni b tn thng. , chng sn sinh
v ti to mt lp sn lnh thay cho phn h hng. K thut ny c p dng thnh cng trn vi
trm ngi bnh v c thng mi ha rng ri. Theo mt s tc gi th ty xng c th tit
vo trong mu cc t bo c kh nng bit ha thnh si c. Trn c s ny, h ghp t bo ty
xng ly t chut chuyn gen ln chut bnh thng b tn thng c. Sau vi tun, cc chut sau
chy nhanh nh th, vi cc t bo c cha nhn mu xanh.
Bng 6.3. Mt s DNA vaccine c th nghim trn ngi.
73
VIII. To c quan t t bo ng vt nui cy
Trong y hc, iu tr chng x va ng mch, ngi ta cn thay th cc mch mu b
hng. Cc mch mu nhn to km bn trong khi khu ni v rt d b tc nghn. Gn y, mt
nhm nghin cu sn xut thnh cng mch mu t nhin bng cch nui cy t bo ng mch
ch ca b, gm t bo c v t bo ni m, trn mt gi th polymer t tiu. Cc nh nghin cu
s dng mt thit b hot ng nh qu tim tht v tc ng ln cc mch ang hnh thnh ging ht
nh trong iu kin t nhin. Th nghim tng t vi cc t bo c ngun gc t ln cho php
sn xut mch mu m khi em ghp tr li trn ln cho nhng kt qu rt kh quan.
Tuy nhin, loi t bo ang c trin vng nht trong lnh vc ny l t bo mm phi. T bo
mm phi chut c s dng t lu, nhng phi n cui th k 20 ngi ta mi thu nhn c
t bo mm phi ngi bng hai cch khc nhau: t t bo phi nang v t t bo mm nguyn
74
thy. Cc nh khoa hc cho rng cc t bo vn c tnh ton th ny (totipotency) c kh nng ti to
m v c quan mi, thm ch c th chn ng c qu trnh lo ha nu c th c cung cp
chng thng xuyn. Mt ng dng thc t hn l vic iu tr cho nhng cp v sinh khng c kh
nng sn sinh t bo sinh dc.
s dng t bo mm phi vo mc ch iu tr cn phi thu nhn c nhng dng t bo
c mang b gen ca ngi bnh. Hin nay, c ba cch tip cn chnh:
- To dng tr liu: thay th vt liu di truyn ca non ngi bng nhn ca mt t bo trng
thnh. Bin php ny, v nguyn tc tng t nh k thut to dng cu Dolly. Khc bit duy nht
ch thay v sau cy phi tr li t cung m, ngi ta s nui n trong iu kin in vitro n giai
on phi nang. Cc t bo phi thai c thu nhn t v em nui cy.
- Cy nhn t bo ngi vo non ca mt ng vt c v bt k loi nhn.
- Lp chng trnh mi cho nhn ca t bo trng thnh bng cch kt hp t bo mm phi
vi nhn ca mt t bo sinh dng.
Bn cnh nhng trin vng rt ln, vic s dng mm phi ngi cng gi ra nhiu vn k
thut, ch yu l: (1) phng php cho php cc t bo mm phi ngi bit ha theo hng mong
mun, v (2) cc nhn t ch th v phng php chn lc t bo ch (target cells).
Nhng hiu bit mi v c ch qu trnh pht sinh c th cng vi vic pht hin nhng protein
cm ng ca qu trnh ny c th s gii quyt c cc vn nu trn. Gn y, ngi ta
phn lp c mt protein (t tn l Cerberus), protein ny l tn hiu u tin trong chui hin
tng hnh thnh u phi cc (Xenopus laevis). Chnh n cm ng s bit ha cc t bo ln
cn ngoi b thnh no v mt, cc t bo trung b thnh tim v t bo ni b thnh gan. Pht hin
ny gp phn m ra mt trin vng mi: sn xut hng lot cc c quan thay th bng cch nui cy
cc t bo mm phi vi s hin din ca mt protein cm ng c hiu hng s bit ha ca
chng thnh loi m mong mun.
IX. M hnh thc nghim
Nh chng ta bit, mt loi thuc mun dng iu tr bnh trc khi c php lu hnh
phi tri qua nhiu th nghim m bo hiu qu tc dng cng nh mc an ton ca n,
trong giai on th nghim bc u trn cc ng vt m hnh (model animal) c mt ngha rt
quan trng. Thng thng, nhng th nghim ny i hi mt thi gian di v tn km, ng thi
khng phi lc no cng p dng c cho ngi V vy, hin nay xu hng s dng t bo nui
cy in vitro lm m hnh th nghim ngy cng pht trin, nht l khi ngi ta c kh nng nui cy
hu nh mi loi t bo nh vo cc k thut hin i.
M hnh th nghim trn t bo c s dng trong cc kho st v s chuyn ha cc
cht c ngun gc ngoi lai nh h enzyme ni bo. Loi t bo c s dng thng dng nht l t
bo gan, do n c mt s u th sau: (1) gan ng vai tr ch yu trong chuyn ha cc hp cht
ha hc, c bit l cht c, (2) t bo gan nui cy vn gi nguyn (trong vi gi n vi ngy) cc
chc nng c trng bao gm c enzyme chuyn ha hin din vi hm lng ln. Vic nui cy
chung t bo gan vi t bo biu m cho php duy tr chng sng trong nhiu tun l. c bit, cc
t bo gan c bin i di truyn c th sinh tng hp cc cytochrome P450, y l cc
enzyme then cht trong chuyn ha cc cht ngoi lai, rt c ch xc nh loi cytochrome chu
trch nhim bin i mt loi thuc nht nh.
Mt ng dng khc ca nui cy t bo ng vt l kho st cc c tnh i vi vt cht di
truyn do cc cht chuyn ha trong t bo gn ln DNA. Ngi ta c th to ra cc h hng c
trng trn t bo bng cc cht c, t pht hin bo quan b nh hng v xc nh cc c ch
ha sinh v phn t ca qu trnh chuyn ha cht c. Loi t bo thng dng nht c s
75
dng trong trng hp ny cng l t bo gan, nhng hin nay ngi ta bt u s dng t bo
thn v phi m rng nghin cu.
Nhn chung, trin vng ca cc m hnh t bo ng vt nui cy rt ln, c bit l vi s pht
trin ca nhiu k thut hin i cho php m bo kh nng sng v chc nng n nh cho tt c
cc loi t bo nui cy, kh nng phc hi v sa cha cc h hng ca t bo cng nh t ng
ha cc thao tc.
1. Pham Kim Ngc v H Hunh Thy Dng. 2001. Sinh hc ca s sinh sn. NXB Gio
dc, TP H Ch Minh.
2. Bronzino JD. 1995. The Biomedical Engineering Handbook. CRC Press and IEEF Press,
USA.
3. Fox SR, Patel UA, Yap MG and Wang DI. 2004. Maximizing interferon-gamma production
by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling.
Biotechnol Bioeng. 85(2):177-84.
5. Lindner-Olsson E, Chatzissavidou N and Lllau E. 2001. Animal Cell Technology: From
Target to Market. Proceedings of the 17
th
ESACT Meeting, Sweden. Kluwer Academic Publishers,
Dordrecht, The Netherland.
6. Pollard JW and Walker JM. 1997. Basic Cell Culture Protocols. Humana Press Inc. Totowa,
New Jersey, USA.
nd
ed. Prentice
Hall, Inc. NJ, USA.
8. Wurm FM. 2004. Production of recombinant protein therapeutics in cultivated mammalian
cells. Nature Biotech. 22: 1393-1398.
1
Tissue plasminogen activator (tPA): mt protein chuyn ha trong t bo ca ng vt c v c tnh
nng kch thch plasminogen l tin cht ca m dng khng hot ng chuyn sang trng thi hot
ng dng iu tr cc cn au tim.
2
Growth hormone (GH): (a) chui polypeptide hormone do min trc ca tuyn yn tit ra iu chnh
tng kch thc c th. (b) bt k cht ni tit no tham gia iu chnh sinh trng trong cc c th
ng vt hay thc vt.
3
Vaccine: khng nguyn lm mt kh nng gy bnh nhng cn gi kh nng sinh khng th
tim chng gy min dch phng bnh. Vaccine c th gm mt c t khng hot ng hoc mt
cht khng c bng cch s dng cc vi khun hoc virus cht hoc gim c. C nhiu cch
tim chng: di da, xuyn da, tim bp, gy so hoc ung qua ng ming.
4
Chinese hamster ovary: t bo bung trng chut ng Trung Quc.
5
Mouse myeloma: t bo u ty ca chut.
6
Baby hamster kidney: t bo thn ca chut ng s sinh.
7
Human embryo kidney: t bo thn ca phi ngi.
8
Proto-oncogene: gen tin ung th.
9
Heavy chain: mch polypeptide c trng lng phn t khong 55 kDa.
10
Light chain: mch polypeptide c trng lng phn t khong 23 kDa.
11
HLA (Human Leucocyte Antigen): khng nguyn t bo bch cu ngi, cn c ngha l MHC
(phc h tng hp m ch yu-Major Histocompatibility Complex) ca ngi.
76
12
CD (Clusters of Differentiation): cm bit ha khng nguyn. L nhng phn t bc l trn b mt
t bo, c nhn bit bi nhng khng th n dng c hiu vi chng. S lng CD hin nay
ca cc t bo h min dch c tm ra ln n hng trm loi (CD1CD247).
13
Fibrinogen: protein huyt tng do cc t bo nhu m gan tng hp, tin cht ca fibrin, hot ha
hnh thnh mt cc mu ng khi mch mu b chn thng.
14
Cytotoxic T cells: cc t bo T gy c, l nhng t bo tiu dit cc t bo khc. Tt c cc t bo
T gy c u l cc T-CD8 ch nhn bit MHC-lp I, nhng t bo T-CD4 cng c th dit cc t
bo khc mt s trng hp. Cc t bo T gy c c vai tr quan trng trong vic bo v vt ch
chng li cc tc nhn gy c ni bo.
77
Chng 7
Nui cy t bo thc vt
I. M u
Thc vt l ngun cung cp cc hp cht ha hc khc nhau rt c gi tr, chng hn cc cht
dng lm dc liu, cc cht to mi, cc cht dng lm gia v, cc sc t v cc ha cht dng
trong nng nghip (Bng 7.1). Nhng sn phm ny, c bit nh l cc cht trao i th cp
(secondary metabolites), thng c sn xut vi mt lng rt nh (dng vt) trong thc vt v
khng c chc nng trao i cht r rng
1
. Chng dng nh l sn phm ca cc phn ng ha
hc ca thc vt vi mi trng chung quanh, l s thch nghi vi stress ca mi trng hoc l s
bo v ha hc chng li vi sinh vt v ng vt.
Mc d, ha hc tng hp hu c t nhiu thnh tu quan trng nhng nhiu hp cht trao
i th cp (thng gi l cc cht th cp) vn cn kh tng hp hoc c th tng hp c nhng
chi ph rt t. Chng hn, mt s hn hp phc tp nh tinh du hoa hng l khng th tng hp
ha hc c. sn xut cc sn phm th cp t thc vt, cc m thc vt ngoi sinh (chng
hn t cy hon chnh) c th c s dng nui cy t bo dch huyn ph (cell suspension
culture) trong iu kin v trng. C s khoa hc ca k thut ny l da trn tnh ton th ha sinh
(biochemical totipotency) duy nht ca t bo thc vt.
Nui cy t bo thc vt trong iu kin in vitro sn xut cc cht th cp c mt s u
im sau:
- Cc t bo thc vt c th c nui cy trong cc iu kin nhn to m khng ph thuc
vo thi tit v a l. Khng cn thit vn chuyn v bo qun mt s lng ln cc nguyn liu
th.
Bng 7.1. Cc sn phm tim nng ca thc vt ang c quan tm.
78
- C th kim sot cht lng v hiu sut ca sn phm bng cch loi b cc tr ngi trong
qu trnh sn xut thc vt, nh l cht lng ca nguyn liu th, s ng nht gia cc l sn xut
v s h hng trong qu trnh vn chuyn v bo qun.
- Mt s sn phm trao i cht c sn xut t nui cy dch huyn ph c cht lng cao hn
trong cy hon chnh.
Thch thc ln nht i vi cng ngh t bo thc vt l s n nh cho php nui cy t bo
thc vt trn quy m ln v t hiu sut ti a cho s tch ly v sn xut cc cht th cp. iu
ny c th thc hin bng cch chn lc cc kiu gen thch hp v cc dng t bo c sn lng
cao, xy dng cc cng thc mi trng dinh dng hp l nui cy t bo, thit k v vn hnh
cc h thng nui cy t bo (bioreactor) hiu qu. Chng ta cng c th s dng kinh nghim v
kin thc c c t nui cy vi sinh vt p dng cho nui cy t bo thc vt. Tuy nhin, t bo
thc vt v vi sinh vt c mt s c im khc nhau, v th cn phi ci tin v iu chnh cc iu
kin nui cy cng nh cu hnh ca ni phn ng (reactor) tm c cc yu cu c th ca
nui cy t bo thc vt.
Mt s c im chnh khc nhau gia t bo thc vt v vi sinh vt:
- T bo thc vt ln hn t bo vi khun hoc vi nm t 10-100 ln.
- S trao i cht ca t bo thc vt chm hn v th i hi phi duy tr mt iu kin v
trng trong thi gian lu hn.
- T bo thc vt c khuynh hng kt thnh mt khi gy ra s lng ng, c kh nng ha
79
trn km.
- Cc t bo thc vt mn cm d bin dng hn t bo vi sinh vt.
- Qu trnh sn xut cc cht th cp t bo thc vt ph thuc cc c ch iu ha phc tp
hn so vi cc t bo vi sinh vt.
- Cc t bo thc vt c n nh di truyn thp hn t bo vi sinh vt.
II. T bo thc vt
Hnh nh t bo thc vt c minh ha hnh 7.1. Mt t bo n thc vt thng c ng
knh khong t 20-40 m v di 100-200 m. Cu trc chnh ca t bo thc vt tng t cc t bo
c trng ca eukaryote. Tuy nhin, t bo thc vt c mt s c im ring bit nh thnh t bo
dy, khng bo ln v c lc lp.
T bo thc vt c bc chung quanh bi thnh t bo. Lp ngoi ca thnh t bo cha
pectin gip n lin kt vi cc t bo bn cnh. Lp trong ca thnh t bo l mng t bo. Mng
t bo hon ton khc vi thnh t bo v hnh dng, thnh phn v chc nng. Trong khi thnh t
bo cng rn, c cu trc tng i dy th mng t bo cht li mng (khong 75 ) v mm do.
Mng t bo bao gm protein v lipid trong khi thnh t bo l carbohydrate t nhin. Thnh t bo
c chc nng nng cho cy trong khi mng t bo iu ha s vn chuyn cc cht i vo v ra
khi t bo.
Hnh 7.1. Minh ha mt t bo thc vt.
Khng bo (vacuole) c vai tr tip nhn cc cht thi ca s trao i cht hoc cc cht th
cp ca thc vt. cc t bo non khng bo thng nh v nhiu. Khi t bo ln dn v gi hn
th khng bo cng m rng ln v kt thnh mt khi. cc t bo thc vt trng thnh, khng
bo c th chim ti 90% th tch t bo. Khng bo c bc chung quanh bi mng huyt tng
(plasma). Thnh phn chnh ca cc khng bo ln l nc cha cc cht ha tan nh cc ion v
c, cc amino acid, cc acid hu c, cc sc t ha tan trong nc (anthocyanin) v cc cht khng
ha tan dng tinh th v hnh kim. Ngoi ra, khng bo cng cha cc protein nh cc
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hydrolyse, catalase v photphatase. Phn bo tan mun cp n lipid chung quanh tt c cc
cu trc ni gia nhn v mng t bo.
Lc lp (chloroplast) l v tr ca quang hp trong t bo thc vt, n cha chlorophyll l sc t
lc phn ng vi nh sng sn xut cc carbohydrate. Nhn (nuclear) l trung tm iu khin ca
t bo cha DNA phin m v dch m thnh protein. Cc protein tng hp c sp xp v ng
gi trong cc ti ca b my Golgi. Ni sinh cht (endoplasmic reticulum) l mng li ca cc ng
nh ni lin cc phn khc nhau ca t bo. Ribosome c tp trung trn b mt ca mng li ni
sinh cht v tham gia vo hot ng sinh tng hp protein. Ty th (mitochondrion) cha vt liu di
truyn v nhiu enzyme quan trng trong s trao i cht ca t bo.
III. Cc loi nui cy t bo v m thc vt
Khi nim nui cy t bo v m thc vt thng c dng nh l mt thut ng chung
m t tt c cc loi nui cy thc vt iu kin in vitro. Trong cc qu trnh nui cy ny thng
xut hin hai kiu sinh trng sau:
1. Sinh trng khng phn ha (undifferentiated growth)
Sinh trng khng phn ha xut hin thng xuyn khi mt mu m ca cy hon chnh
c nui cy in vitro. Mu m sau lm bin mt mi cu trc c th nhn bit c ca cy
nguyn vn ban u.
1.1. Nui cy callus
Nui cy callus cho php cc khi t bo khng c hnh dng nht nh tng ln t sinh trng
khng phn ha ca mu vt trn mi trng dinh dng rn v trng (Hnh 7.2). Mu vt thng l
cc c quan t nh hoc cc mu m. Cc khi t bo ny khng tng ng vi mi cu trc m
c trng ca cy hon chnh. Thut ng nui cy callus c s dng do s phn chia v t chc
ca t bo m lc u c ngh l n cm ng vi s tn thng thc th ca thc vt trong qu
trnh tch ra khi cy hon chnh. Tuy nhin, sau ngi ta nhn thy n c c cm ng bi
cc cht iu ha sinh trng thc vt (plant growth regulators) trong mi trng dinh dng rn.
Hnh 7.2. Nui cy callus.
1.2. Nui cy dch huyn ph t bo
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Nui cy dch huyn ph t bo cha cc t bo v cc khi t bo, sinh trng phn tn trong
mi trng lng (Hnh 7.3 v 7.4). Thng c khi u bng cch t cc khi m callus d v
vn trong mi trng lng chuyn ng (lc hoc khuy). Nui cy dch huyn ph v th l s tin
trin t thc vt n mu vt, ti callus, v cui cng ti dch huyn ph. Nui cy dch huyn ph
thch hp hn cho vic sn xut sinh khi ca t bo thc vt so vi nui cy callus, do nui cy dch
huyn ph c th duy tr v c thao tc tng t vi cc h thng ln men vi sinh vt c ngp
chm trong mi trng lng.
1.3. Nui cy t bo trn
Nui cy t bo trn (protoplast) i hi s sinh trng ca protoplast trn mi trng c hoc
lng. Protoplast c th c chun b bng phng php c hc hoc enzyme loi b thnh t
bo. Cc protoplast c phn lp c th c s dng : (1) bin i thng tin di truyn ca t
bo thc vt, (2) to ra cy lai v tnh thng qua dung hp protoplast (protoplast fusion), (3) nghin
cu s xm nhim ca virus thc vt v nhng vn khc. Mt ng dng y trin vng khc
ca nui cy protoplast l vi nhn ging thc vt. Sau khi phn chia protoplast, thnh t bo c ti
sinh tng s pht trin callus v tip theo l cy hon chnh nh thc vt c th c nhn ln
nhiu ln.
2. Sinh trng c phn ha (differentiated growth)
Sinh trng c phn ha xut hin khi cc b phn ca thc vt c chuyn ln mi trng
nui cy l ni chng c th tip tc sinh trng vi cu trc c duy tr t trc. Cc c quan
thc vt c phn ha c th sinh trng trong qu trnh nui cy m khng b mt s ton vn ca
mnh cn gi l nui cy c quan (organ culture).
2.1. Nui cy r t
Nui cy r t c th c thit lp t u r tch ra nhiu loi thc vt khc nhau. Cc nui
cy r sinh trng nhanh c th thu c t cc loi cy hai l mm bng cch gy nhim chng vi
vi khun t Agrobacterium rhizogenes. Cc dng r t (hairy root) c hnh thnh c th dng
trong nui cy sn xut cc cht th cp (Hnh 7.5).
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Hnh 7.3. Nui cy dch huyn ph t bo thc vt trn my lc. A: Nui trong bnh tam gic c
lc chun b t bo. B: Nui trong h ln men lc sn xut sinh khi.
Hnh 7.4. H ln men c cnh khuy (bnh nui 5 L) dng nui cy dch huyn ph t bo
thc vt quy m phng th nghim.
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Hnh 7.5. Nui cy r t.
2.2. Nui cy phi
Nui cy phi (embryo) c th c thit lp cho cc phi tch ra t cc ht v trng, cc non
hoc qu. Cc phi c sn xut t k thut nui cy t bo, c gi l phi v tnh (somatic
embryo), c th c phn lp v ny mm cung cp mt cy trn mt mu vt. Nui cy phi c th
c ng dng sn xut nhanh cy ging t cc ht c thi gian ng ngh di. Phng php ny
c nhiu u im hn cc h thng nhn ging truyn thng nh l qu trnh ng nht di truyn,
sn xut sinh khi v nhn ging cc cy trng sch bnh.
IV. Mi trng nui cy
Mc d nhu cu dinh dng ca cc loi m nui cy l rt khc nhau nhng mi trng nui
cy m thc vt c trng cha cc thnh phn sau:
- Cc nguyn t a lng. Bao gm cc loi mui ca nitrogen, potassium, calcium,
phosphorus, magnesium v sulfur. y l su nguyn t chnh cn thit cho sinh trng ca thc vt
bc cao.
- Cc nguyn t vi lng. Bao gm cc loi mui ca st, km, mangan, boron, copper,
molybdenum v cobalt dng vt.
- Cc ph gia hu c. Mt lng nh cc loi vitamin (myo-inositol, thiamine, nicotinic acid,
pyridoxine, riboflavin), cc amino acid (thng cho php b qua nhng trong mt s trng hp
c bit th c th dng), v cc ph gia hu c khng xc nh khc (malt, dch chit nm men, dch
thy phn casein, nc da).
- Cc cht kch thch sinh trng thc vt. Thnh phn ph gia quan trng nht quyt nh
kt qu nui cy l cc cht iu ha sinh trng. Cc auxin (IAA
2
v cc dng tng t c tng
hp nhn to nh 2,4-D
3
, NAA
4
, IBA
5
, NOA
6
...), v cc cytokinin (zeatin, 2i-P
7
v cc dng tng t
c tng hp nhn to nh kinetin, BA
8
...) l nhng nhm cht kch thch sinh trng v pht sinh
hnh thi ch yu trong nui cy m v c quan ca thc vt.
- Ngun carbon. Thng s dng sucrose lm ngun carbon thay cho ngun carbon c
thc vt c nh t kh quyn bng quang hp. Trong a s cc th nghim nui cy, t bo thc vt
mt kh nng quang hp. Glucose cng thng c a vo mi trng v cho hiu qu tng
ng sucrose, trong khi fructose cho hiu qu km hn.
- Cc tc nhn lm rn (to gel) mi trng. Thng s dng l agar, mt loi
polysaccharide thu c t mt s loi to thuc ngnh to (Rhodophyta). Mt s hp cht khc
cng c th nghim thnh cng nh alginate, phytagel, methacel v gel-rite.
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Murasghige v Skoog (1962) xy dng mi trng dinh dng c bn (gi l mi trng
MS) thch hp cho hu ht cc th nghim nui cy t bo thc vt. Thnh phn mi trng nui cy
c trnh by bng 7.2.
V. Sn xut cc cht th cp
Cc cht trao i th cp hay cn gi l cc cht th cp c th xp trong ba nhm chnh:
alkaloid, tinh du v glycoside.
Cc alkaloid c dng tinh th l cc hp cht cha nitrogen, c th c tch chit bng cch
dng dung dch acid
9
. Alkaloid c hot tnh sinh l trn tt c ng vt v c s dng trong cng
nghip dc. H alkaloid bao gm: codein, nicotine, caffeine v morphine. Cc tinh du cha hn
hp terpenoid v c s dng nh l cht mi, cht thm v dung mi. Glycoside bao gm cc
phenolic, tanin v flavonoid, saponin v cc cyanogenic glycoside, mt s trong chng c s dng
lm cht nhum, cc cht mi thc phm v dc phm.
Bng 7.2. Thnh phn mi trng Murashige v Skoog (1962).
Mt trong nhng nhn t quan trng nh hng n vic sn xut cc cht th cp t t bo
thc vt l s phn ha hnh thi. Nhiu cht th cp c sn xut trong sut qu trnh phn ha t
bo. V th, chng thng c tm thy trong cc m c tnh c trng cao nh l r, l v hoa. Do
s phn ha hnh thi v s trng thnh khng xut hin trong nui cy t bo, nn cc cht th
cp c khuynh hng ngng to thnh trong nui cy t bo thc vt. Ch mt s gii hn h thng
nui cy t bo thc vt l c th sn xut mt lng va phi cc cht th cp, cho d thc vt t
nhin m t cc t bo c thu thp, l c th sn xut chng.
Tuy nhin, cc t bo khng phn ha trong nui cy dch huyn ph thng to thnh mt
khi khong vi trm t bo do tnh cht dnh nht ca b mt t bo, t s tit ra ca cc
polysaccharide cng nh mt cao ca t bo (Hnh 7.6). Do gradient nng v s tng tc
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t bo, cc t bo gia khi s c tip xc vi mi trng, iu khc vi cc t bo bn
ngoi. Do , s phn ha s xut hin ti mt mc no trong khi cho php to thnh cc
cht th cp.
Hnh 7.6. S kt khi trong nui cy t bo thc vt.
Mt s kt qu nghin cu cho rng mt s nui cy dch huyn ph c kh nng tng hp cc
sn phm c bit c nng cao hn so vi cy m t chng bt ngun. Chng hn: Schulte v
cs (1987) thng bo s to thnh cc anthraquinone trong cc nui cy t bo (c ti u cc
iu kin) vt tri cc cy sinh trng trong iu kin t nhin (17/19 loi khc nhau) thuc cc
chi Asperula, Galium, Rubia v Sherardia. Hiu sut anthraquinone cao nht l trng hp ca loi
Galium verum (1,7 g/L) v nng cao nht l loi Rubia fruticosa (20% trng lng kh).
c nhng bng chng r rng cho thy c mi quan h ngc (feedback) gia tc sinh
trng v kh nng sn xut cc cht th cp. Khi tc sinh trng cao, cc qu trnh s cp ca
t bo l phn chia t bo v sn xut sinh khi t bo din ra mnh m. Ngc li, trong pha tnh
khi s sinh trng gim n mc ti thiu, th lc ny hot ng sn xut v tch ly cc cht th cp
tng ln.
Thnh phn mi trng cng c nh hng mt cch ngha n s lng cc cht th cp
c sn xut. Yu cu c bn khi thit k cc cng thc mi trng dinh dng l m bo hon
thnh s sinh trng ca t bo. Sau khi t bo t n mt qun lc nht nh, s thay i thnh
phn mi trng cng c th nh hng n s tch ly sn phm. Chng hn, ngi ta ci thin
sn lng ca shikonin c ngun gc t nui cy dch huyn ph t bo ca cy Lithospermum
erythrorhizon bng cch dng mi trng sn xut thay cho mi trng sinh trng. Mi trng
sn xut thng cha nhiu sucrose hn nhng t cc thnh phn v c v vitamin hn so vi mi
trng sinh trng.
Tch ly sn phm bng nui cy t bo thc vt c th c kch thch bi cc elicitor sng
hoc khng sng. Cc elicitor sng l cc hp cht hoc cc cht c ngun gc t vi sinh vt v cc
elicitor khng sng l cc tc nhn gy stress nh chiu x UV, sc thm thu, hoc cc ion kim loi
nng. Cc elicitor sng (biotic) thng c sn xut bng cch nghin ng th h si nm v v
trng dch thu c. nh hng ca cc biotic elicitor ln s tch ly ca cc cht th cp ty thuc
vo c trng v nng ca elicitor, thi gian tip xc elicitor, v giai on sinh trng ca t bo
thc vt.
1. Cc cht th cp dng trong thc phm
1.1. Cc cht mu
- Anthocyanin
Cc anthocyanin l cc sc t tiu biu c trong cc loi thc vt ht kn (angiosperms) v cc
86
loi thc vt c hoa (flowering plants) ca cc h Poaceae, Fabaceae, Rosaceae, Cruciferae,
Vitaceae v Solanaceae tp trung cc b phn khc nhau nh: r, l, hoa v qu. Anthocyanin
tch chit t nho l ngun tim tng nht trn th gii. Hin nay, ngi ta cng tin hnh nui cy
t bo ca cc loi Vitis vinifera, Daucus carota v Euphorbia millii sn xut anthocyanin. K thut
nui cy t bo l phng tin l tng sn xut anthocyanin vi sn lng cao t 10-20% trng
lng kh. Sn xut anthocyanin ni chung t cc i trong sut pha tnh. Stress thm thu c
to ra do sucrose v cc tc nhn khc cho thy c th iu ha qu trnh sn xut anthocyanin trong
nui cy t bo V. vinifera.
- Betalaine
Cc betalaine l cc sc t tiu biu c trong cc loi thc vt ht kn v cc loi thc vt c
hoa thuc cc h Chenopodiaceae, Amaranthaceae v Phytolacaceae. Mt s loi nm n thuc b
Agaricinales cng sn xut betalaine. Nui cy t bo thc vt chng minh c kh nng sn
xut betalaine. Ging nh nhiu cht th cp khc, betalaine trong nui cy t bo thc vt t nng
cc i trong pha tnh ca sinh trng t bo. Tuy nhin, nui cy t bo dch huyn ph ca
Phytolacca americana li cho thy betalaine t nng cao nht pha sinh trng hm m (pha
log). T bo cy Chenopodium rubrum nui cy 15 ngy tui c th sn xut c 35-45 mg
betalaine/L mi trng. Nui cy t bo v nui cy r t ca cy Beta vulgaris cng c tin hnh
sn xut betalaine.
- Crocin v crocetin
Crocus sativus (cy ngh ty) l ngun cung cp crocin ch yu, mt loi sc t mu ti
c tm thy trong u nhy ca n. u nhy cy ngh ty (saffron) cng sn xut cc crocetin.
Crocin l mt digentiobiocide ester ca crocetin. y l loi nguyn liu c gi tr cao, do thc t cy
ngh ty ch c th sinh trng nhng vng a l c bit trn th gii v i hi nhiu nhn cng
khi thu hoch. Mun thu hoch 1 kg u nhy ngh ty (mt loi gia v c gi tr) cn phi c khong
150.000 hoa. Hin nay, ngi ta pht trin cc phng php nui cy t bo ca u nhy cy
ngh ty trong iu kin in vitro sn xut cc gc c bn ca saffron. M nui cy trong trng
hp ny c cm ng sn xut cc callus c mu cha crocin v cc crocetin v cng l
safrana (gc c bn ca cht mu) ca saffron.
- Capsaicin v cc capsaicinoid
Capsaicin l gc cay ch yu ca t, cc capsaicinoid chu trch nhim cho v cay l
dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin v homodihydrocapsaicin. Capsaicinoid c
s dng trong cng nghip thc phm nh mt ph gia. Ngoi ra, ngi ta cn s dng capsaicin
tinh khit bo ch dc phm iu tr chng vim khp v lm thuc gim au. Capsaicin c th
c sn xut bng nui cy bt ng t bo ca Capsicum frutescens. Cc t bo Capsicum bt
ng s sn xut capsaicin cao gp vi ln so vi t bo dch huyn ph t do. M gi non
(placenta) ca Capsicum c bt ng s sn xut lng capsaicin cao hn cc t bo bt ng.
1.2. Cc cht mi
- Vanilla
y l nguyn liu to mi ph bin v c s dng rng ri nht. Vanilla t nhin l th
trng rt ha hn cho sn xut cht mi bng phng php cng ngh sinh hc. N l hn hp
phc tp ca cc thnh phn cht mi chit ra t ht ca Vanilla planifolia. y l ha cht mi ph
bin v c s dng rng ri cho nhiu loi thc phm. Khong 12.000 tn vanilla c tiu th
hng nm, nhng trong ch khong 20 tn c tch chit t ht, phn cn li c sn xut
bng phng php tng hp ha hc, thng l t cc sn phm ha du nh guaiacol v mt i
87
khi t lignin, mt sn phm ca bt giy. Sn xut cc hp cht mi vanilla bng phng php nui
cy t bo cy V. planifolia c chng minh thnh cng. Nui cy t bo cy V. planifolia c
khi u bng nui cy cc c quan khc nhau ca cy nh l hoc on thn bng cch dng cc
t hp cht kch thch sinh trng khc nhau. Thng thng, kinetin c dng khi ng s
tng hp vanillic acid trong nui cy dch huyn ph ca V. planifolia. Sn xut vanilla c ci thin
bng cch b sung thm than hot tnh vo mi trng nui cy. Sn lng vanilla trong nui cy t
bo thng l 2,2%. Mt s ci tin iu kin nui cy lm gim thi gian nui cy t bo t 160
gi cn 50 gi v sn lng vanilla tng t 100 mg/L ln n hn 1.000 mg/L, tng ng 8% hm
lng vanilla trn trng lng kh.
- Garlic v onion
Cc cht mi garlic v onion cng c sn xut trong nui cy t bo. Cc cht mi ny pht
trin t cc tin cht (precusor) ngoi sinh trong qu trnh x l sau thu hoch. Nui cy t bo cy
ti (Allium sativum) khng phn ha (hoc cc callus phn ha c mu xanh lc) thu c mt s
loi tin cht amino acid (methyl, propyl, allyl v cysteine sulphoxides) v hai loi hp cht ninhydrin-
positive khng xc nh. Cht mi tng s thu c trong callus hnh cu mu trng (globular white
callus) v callus mu xanh lc phn ha chm (semidifferentiated green callus) tng ng l 4% v
13% so vi cy trng trong t nhin. K thut nui cy t bo thc vt ca cy hnh (Allium cepa)
sn xut cc cht mi onion cng c pht trin. Prince (1991) chng minh s tng ln ca
cc hp cht mi onion trong nui cy r. Thng qua vic b sung cc amino acid nh cysteine,
methionine v glutathione, nng cui cng ca sn phm c th tng ln rt ln.
1.3. Cc cht ngt
- Stevioside
Stevioside t nhin ngt hn sucrose khong 300 ln. Dch chit t cy hon chnh cha
khong 41% stevioside. Cc kim nghim v c cht hc cho thy rng stevioside hon ton an
ton cho ngi dng. Hn na, cc nghin cu nha khoa gi rng cc sn phm ny c th c
ch thc s sinh trng ca cc vi sinh vt vng ming. Sn xut stevioside trong nui cy callus
ca Stevia rebaudiana c nghin cu chi tit. Nhng nui cy chi to r cho v ngt, chng
minh rng c hai: r v l cn thit cho s tng hp stevioside.
2. Cc cht th cp dng trong dc phm
2.1. Cc alkaloid
Ngi ta c th thu c cc cht nh caffein t nui cy t bo cy Coffea arabica, betalain
trong callus c ci ng, berberin t t bo cy Coptis japonica (loi cy ny phi trng t 4-6 nm
mi thu c hm lng ng k berberin trong r, trong khi hm lng ny c th thu c sau 4
tun bng phng php nui cy t bo) Nhng cht ny c s dng rng ri trong cng
nghip hng liu v trong y hc.
Cht reserpine c tc dng cha bnh cao huyt p v cc bnh ri lon tun hon cng c
sn xut bng phng php nui cy t bo cy Rauwolfia serpentina. Nui cy t bo ca cy ny
trong 30 ngy trong h ln men quy m ln c th sn xut c 3.500 kg reserpine, tng ng
vi lng hng nm ca c th gii thu c t r cy .
Cc nh nghin cu thuc t hp dc phm Gibageigy (Based, Thy S) sn xut c
loi alkaloid l scopolamine t t bo cy Hyoscyanus aegypticus nui cy trong h ln men khng
c cnh khuy. Bng cch chn lc cc dng t bo cao sn nh k thut t bin t bo trn, bin
d n dng (monoclonal variation) v k thut gen, ngi ta tng c sn lng scopolamine
88
ln gp hng ngn ln.
Nhiu nghin cu cho thy nui cy callus v t bo ca cy Catharanthus roseus c hm
lng serpentin ngang vi cy dc liu bnh thng. Mt s nghin cu phn lp c cc
dng t bo Cantharanthus sn xut serpentin v ajmalacine t nui cy in vitro. Bng loi mi
trng sn xut c bit ngi ta a c sn lng alkaloid ca hai dng t bo tt nht ln
mt mc cao hn na, trong mt dng to c 162 mg/L serpentin, cn dng kia to c 72
mg/L serpentin cng vi 264 mg/L ajmalacine. Mi y, ngi ta hon thin c cng ngh nui
cy t bo ca cy C. roseus sn xut viblastine v vincristine l hai cht chng ung th rt mnh,
hin ang c nhu cu rt cao v chng c s dng cha ung th mu.
Sikuli v cng s (1997) sau khi gy nhim cy Datura stramonium vi Agrobacterium
rhizogenes nhn thy hm lng hyoscyamine r t cc i sau 6 tun nui cy < 100 mg/L.
2.2. Cc steroid
Trong lnh vc steroid v chuyn ha steroid, cc dng t bo c nng sut cao c Kaul
v cs cp n t nm 1969. H nui cy thnh cng t bo ca cy Dioscorea deltoidea
sn xut diosgenin, l nguyn liu th ch yu sn xut cc steroid chng th thai v cc hormone
tuyn thng thn.
Qu trnh chuyn ha cc hp cht glycoside tim (cardiac) bng nui cy t bo ca cy
Digitalis lanata cng c nghin cu. Ngi ta nhn thy, mc d cc t bo Digitalis ngng sn
xut glycoside tim nhng chng vn c kh nng hydroxyd ha digitoxin nguyn t C
12
to ra
digoxin. Digoxin l mt hp cht c ngha y hc ln hn digitoxin. Qu trnh hydroxyd ha xy ra
trong nui cy t bo rt nhanh v rt hiu qu khi a vo mi trng nui cy cht |-methyl-
digitoxin. Sau 12 ngy, ngi ta thu c 4 g |-methyl-digitoxin trong mt bnh nui dung tch 20
L.
2.3. Mt s cht khc
Th d in hnh nht l cng ngh sn xut shikonin, mt loi sc t c kh nng dit
khun, c trong r ca cy Lithospermum erythrorhizon. Bnh thng shikonin tch ly khng nhiu
trong r. Tuy nhin, cc nh khoa hc Nht to c dng t bo r cy Lithospermum c kh
nng tch ly n 15% shikonin v hon chnh cng ngh nui cy t bo sn xut shikonin. Cng
ngh ny cho php trong mt chu k nui cy thu hoch ti 5 kg hot cht v gip gim rt nhiu gi
thnh ca shikonin.
Hm lng tng i cao ca ubiquinone-10 c tm thy trong t bo thuc l nui cy in
vitro v ca L-dopa trong mi trng nui cy t bo Mucuma pruriens. Nui cy t bo ca cy
Panax pseudoginseng cho hm lng saponin kh cao. Nui cy t bo ca cy Glycyrrhiza
glabra thu c hm lng glycyrrhizin t 3-4% trng lng kh.
Hm lng cht th cp cao nht c tm thy trong nui cy t bo ca cy Coleus blumei
l cht rosmarinic acid chim 13-15% trng lng kh trong chu k nui 13 ngy, ln gp 5 ln so vi
hm lng trong cy trng iu kin t nhin. Trong nhng nm 1980, ngi ta cng sn xut rt
c hiu qu ginsengoside l hot cht ch yu ca nhn sm Panax ginseng. Cc anthraquinone l mt
89
nhm cc sn phm t nhin quan trng c vi khun, nm, a y v thc vt bc cao c cc hot tnh
sinh hc nh: khng khun, khng nm, gim huyt p, gim au, chng st rt, chng oxy ha, khng
bnh bch cu v cc chc nng t bin. thc vt bc cao, chng c tm thy rt nhiu h
thc vt khc nhau, chng hn Rubiaceae, Rhamnaceae, Polygonaceae, Leguminosae... Nui cy t
bo cc loi ca h Rubiaceae cho php thu c mt lng ln anthraquinone thm ch trong mt
s trng hp vt qu hm lng anthraquinone cy b m.
Cng ty Escagenetics (California, M) thnh cng trong sn xut taxol bng nui cy r t
(hairy-root). Taxol l cht tch chit t v v l kim ca cy thy tng (Taxus brerifolia) ang c s
dng hiu qu trong iu tr nhiu loi ung th. Vic cung cp taxol gp kh khn v bn thn cy thy
tng khan him v hm lng taxol trong chng rt thp. Escagenetics c th sn xut taxol vi
nng cao hn nng t nhin thy trong v v l cy thy tng.
VI. Sn xut cc protein ti t hp
Nui cy t bo thc vt c s dng sn xut cc sn phm t nhin cch y hn 20
nm v gn y hn chng c dng sn xut cc protein ti t hp. Protein ti t hp (protein
ngoi lai) l protein t nhin c sa i bng cng ngh gen nhm nng cao hoc thay i hot
tnh ca chng. Tng t nh cc t bo vi sinh vt, cc t bo thc vt rt thch hp cho cc
nguyn liu ti t hp do chng c th sinh trng trn mi trng tng i n gin khng cn b
sung protein, nhng do chng l cc sinh vt eukaryote bc cao nn c th tin hnh cc bin i
hu dch m nh trong t bo ca ngi. Nu protein ngoi lai c sn xut trong nui cy t bo
v c tit ra trong mi trng, nhiu hn phn c tch ly trong t bo, th vic thu hi v tinh
sch sn phm c th c tin hnh m khng c nhiu protein nhim bn. Cc protein c ngun
gc thc vt an ton cho ngi hn cc protein c ngun gc t t bo ng vt bi v cc cht
nhim bn v virus thc vt khng phi l tc nhn gy bnh ngi. Ngoi ra, nui cy t bo thc
vt cng l mt cng c thc nghim thun li cho vic kho st s sn xut protein ngoi lai trong
cy hon chnh (whole plants) (Bng 7.3).
Thc vt chuyn gen hin nay c xem l h thng sn xut rt kinh t cho vic sn xut cc
protein ngoi lai nh khng th, enzyme v hormone. Sn xut thng mi mt s protein ca vi
khun v ng vt c tin hnh bng thc vt. Yu t quan trng nh hng n hiu qu kinh
t ca sn xut protein da trn c s thc vt l hiu sut ca protein ngoi lai hoc nng ca
sn phm c tch ly trong sinh khi. Theo , ngi ta ch ci thin s biu hin gen ngoi
lai trong cy chuyn gen thng qua vic pht trin cc promoter tt hn, chn lc cc dng chuyn
gen n nh, v c ch gen im lng (silence gene). Tuy nhin, mt yu t quan trng l s t gy
protein ngoi lai lm gim nng ca sn phm chc nng trong m thc vt sau khi cc phn
t c tng hp v lp rp. S t gy protein ngoi lai lm bn sn phm vi cc on protein
mt hot tnh, v ngi ta cng gp kh khn khi loi b cc protein t gy ny trong cc hot ng
thu hi protein chc nng sn xut quy m ln. Tm hiu chi tit v v tr v c ch ca s t gy
ni v ngoi bo l rt cn thit c th pht trin phng php sao cho gim thiu c s tn
tht protein hu sau dch m.
1. GM-CSF ngi
90
GM-CSF ngi (human granulocyte macrophage-colony stimulate factor), l mt trong bn
glycoprotein c bit kch thch qun lc i thc bo ca t bo bch cu ht t tin sn sinh ra cc
bch cu ht, i thc khun v hai loi t bo mu trng quan trng. GM-CSF ngi c ng
dng lm sng trong iu tr bnh gim bch cu trung tnh (neutropenia) v bnh thiu mu khng
ti to (aplastic anemia). S dng GM-CSF ngi trong cy ghp ty xng gim thiu ng k
nguy c nhim trng do chng kch thch tng tng s bch cu trung tnh. GM-CSF ngi c
biu hin trong nhiu c th vt ch khc nhau nh: E. coli, nm men, A. niger, t bo ng vt c v
v t bo thc vt, v hin nay c sn xut dng trong lm sng.
Bng 7.3. Mt s protein ti t hp c sn xut bng nui cy t bo thc vt.
2. Khng th IgG1 ca chut
91
c sn xut bng cch nui cy r t v t bo dch huyn ph ca cy thuc l
(Nicotiana tabacum) chuyn gen. thc hin lp rp khng th hon chnh, t 2 n 4 on khng
th nh c tch ly trong sinh khi t bo. Cc nhn t c ch protease, cc tc nhn n nh
protein, cc nhn t c ch N-glycosylation v s tit protein, cc tc nhn ti hot ng glycan
(polysaccharide) v cc mu d i lc c s dng nghin cu c im ca cc on ny,
kho st cc v tr ca chng v c ch hnh thnh. Tt c cc phn t khng th c tit ra
trong mi trng nui cy.
3. Interleukin
L thut ng chung cho cc cytokine c bch cu sn xut (lymphokine), trong
interleukin-2 l mt cytokine quan trng nht i vi s pht trin v p ng min dch thch ng,
c s dng iu tr mt s bnh vim nhim virus v bnh ung th.
Interleukin-2 (IL-2) v interleukin-4 (IL-4) ca ngi c sn xut v tit ra mi trng nui
cy dch huyn ph t bo cy N. tabacum bin i di truyn. S tit qua mng huyt tng v
thnh t bo vo mi trng c thun li nh trnh t leader t nhin ca ca ng vt c v.
Nng ca IL-2 v IL-4 trong mi trng nui cy tng ng l 0,10 v 0,18g/mL, mc d nng
ca chng bn trong cc lymphokine l cao hn (IL-2 khong 0,8 g/mL v IL-4 khong 0,28
g/mL).
Phn tch Western blot
10
cho thy IL-4 c tit ra mi trng nui cy t bo thc vt l hai
chui polypeptide nh vi trng lng phn t khong 18-20 kDa. Hot tnh sinh hc ca IL-2 c
xc nh bi s sinh sn t bo ca dng t bo CTLL-2 ca chut ph thuc IL-2 [CT.h4S] c
chuyn nhim n nh bng receptor IL-4 ca ngi. Nhng khm ph ny cho thy rng nui cy
dch huyn ph t bo thc vt c th c s dng sn xut v tit ra mi trng loi cc
protein ng vt c v c hot tnh sinh hc dng trong chn on v iu tr.
VII. Chn dng t bo bin d soma
Ngi ta c th tin hnh x l v chn lc t bo thc vt ba mc cu trc chnh: callus, t
bo n (single cell) v t bo trn.
Trong phm vi cng ngh (nui cy) t bo thc vt, ngi ta thng tp trung cc nghin cu
cho mc ch chn dng t bo sn xut d tha (over production) cc loi sn phm ch yu l cc
amino acid v cc hp cht t nhin
11
.
Nhn chung, hin tng bin d di truyn xut hin cc t bo khng phn ha
(undifferentiation), cc protoplast phn lp, cc callus v cc m nui cy in vitro. Nui cy t bo
thc vt c kh nng to bin d di truyn tng i nhanh v khng cn phi ng dng cc k thut
phc tp khc.
Cc bin d chn lc c trong nui cy in vitro c nhiu cch gi khc nhau nh: dng callus
(calliclones-t nui cy callus) hoc dng protoplast (protoclones-t nui cy protoplast)... Tuy nhin,
thut ng bin d dng soma (somaclonal variation) c s dng ph bin nht, hoc bin d dng
giao t (gameclonal variation) ch cc dng b bin i di truyn pht trin t cc t bo giao t
hoc th giao t. S a dng ca bin d cc dng soma lm ni bt mt thc t rng bin d dng
soma l mt cng c rt hu hiu cho vic ci thin cc c im di truyn ca t bo.
92
VIII. Dung hp protoplast hay lai v tnh t bo thc vt
c trng ca t bo thc vt l c thnh cellulose bao quanh, sau n mng nguyn sinh.
Thnh cellulose gi cho t bo thc vt c hnh dng nht nh, cn cc hp cht pectin nm trong
thnh c nhim v lin kt gn cc t bo vi nhau thnh m. Mng nguyn sinh cho php protoplast
(t bo c ph b thnh cellulose v ch cn li mng sinh cht) c th hp thu vo t bo cc i
phn t (nucleic acid, protein) thm ch c cc c quan t nh lc lp, ty th, nhn bo theo c ch ca
amip. Nu cc protoplast cnh nhau, chng c th d ha lm mt, l hin tng dung hp
(protoplast fusion) hay cn gi l lai v tnh t bo (cell somatic hybridization). K thut dung hp
protoplast cho php m rng ngun gen ca cc loi thc vt, to ra cc dng t bo sn xut mi
12
mang cc c tnh di truyn u vit ca c b v m.
Mt s k thut dung hp protoplast nh:
- Dung hp bng ha cht. Phng php ny dng NaNO
3
hoc polyethylene glycol (PEG)
kch thch dung hp ca hai protoplast.
- Dung hp bng in (electrofusion). Phng php ny n gin hn, nhanh hn v hiu qu
hn dung hp bng ha cht. iu quan trng hn c l dung hp bng in khng gy c cho t bo
nh thng thy cc protoplast hoc cc th d nhn c x l bng PEG. Cng theo hng ny v
gn y c chng minh, ngi ta dng cc xung in (electric pulses) a trc tip DNA
ngoi lai vo trong t bo thc vt. Trong dung hp bng in, u tin cc protoplast c a vo
trong ngn dung hp nh c hai dy kim loi song song vi nhau ng vai tr l cc in cc. Tip ,
s dng in p (voltage) thp v trng AC dao ng nhanh (rapidly oscillating AC field) kch
thch cc protoplast sp thnh tng chui t bo (chui ngc trai-pearl chains) gia cc in cc.
Phng thc ny cho php cc t bo tip xc hon ton vi nhau trong mt vi pht. Sau khi cc t
bo xp hng hon chnh, qu trnh dung hp c thc hin theo tng t ngn ca xung DC in p
cao (high-voltage DC pulses). Xung DC in p cao to ra s ph v thun nghch ca mng nguyn
sinh cht (plasma membrane) v tr tip xc ca cc t bo, to ra s dung hp v ti t chc li
mng mt cch hp l. Mt qu trnh hon chnh bt u t lc a cc protoplast vo bn trong ngn
v chuyn chng ln mi trng nui cy, c th c thc hin trong nm pht hoc t hn.
2
nd
st
Singapore.
3. Cutler SJ and Cutler HG. 2000. Biologically Active Natural Products: Pharmaceuticals. CRC
Press LLC, USA.
4. Fischer R, Emans N, Schuster F, Hellwig S and Drossard J. 1999. Towards molecular
farming in the future: using plant-cell-suspension cultures as bioreactors. Biotech Appl Biochem. 30:
109-112.
5. Hellwig S, Drossard J, Twyman RM and Fischer R. 2004. Plant cell cultures for the
production of recombinant proteins. Nature Biotech. 22: 1415-1422.
6. Kieran PM, MacLoughlin PF and Malone DM. 1997. Plant cell suspension cultures: some
engineering condiderations. J Biotech. 59: 39-52.
93
7. Klefenz H. 2002. Industrial Pharmaceutical Biotechnology. Wiley-VCH Verlag GmbH,
Weinheim, Germany.
9. Ramawat KG and Merillon JM. 1999. Biotechnology: Secondary Metabolites. Science
Publishers Inc. USA.
10. Roberts MF and Wink M. 1998. Alkaloids: Biochemistry, Ecology, and Medicinal
Applications. Plenum Press, New York, USA.
nd
ed. Prentice
Hall, Inc. NJ, USA.
1
Cc cht s cp c sn xut vi s lng ln hn cc cht th cp v c cc chc nng trao i
c bit. Cc cht s cp thu c t thc vt bc cao c s dng nh l thc phm, cc ph gia
thc phm, v cc nguyn liu th trong cng nghip nh l cc carbohydrate, du thc vt, protein
v cc acid bo.
2
IAA: indoleacetic acid
3
2,4-D: 2,4-dichlorophenoxyacetic acid
4
NAA: o-naphthaleneacetic acid
5
IBA: indole-3-butyric acid
6
NOA: 2-naphthoxyacetic acid
7
2-iP: 6-(,-dimethylallylamino)purine
8
BA: N
6
-benzyladenine hay 6-benzylaminopurine (BAP)
9
Tn alkaloid c ngha l ging nh kim (alkali)
10
Western blot: k thut phn tch protein da trn nguyn l lin kt khng nguyn-khng th pht
hin protein c hiu c bn cht khng nguyn (thng c thm tch ln mng nitrocellulose sau
khi chy in di SDS-PAGE, v c nh ). Sau khi protein trn mng lai gn vi khng th th
nht c hiu v tip n l khng th th hai c nh du enzyme (alkaline phosphatase hoc
horse-radish peroxidase) th phc hp ny s c lin kt vi c cht to mu. S hin din
ca protein ngoi lai (sn phm dch m ca gen ngoi lai c chuyn vo t bo vt ch) s c
pht hin nh s xut hin mu ca phn ng lai.
11
Trong cng tc ging cy trng, chn dng t bo bin d soma c th khi qut mt s ng dng
sau:
- Chn dng t bo chng chu cc iu kin bt li ca ngoi cnh, v d: chng chu nng, lnh,
phn, mn, kh-hn...
- Chn dng t bo khng cc c t: c t do nm bnh tit ra, cc loi khng sinh
Cc dng t bo mang cc c tnh mong mun sau khi chn lc c s c ti sinh thnh c th
thc vt hon chnh pht trin ngun cy ging mi, thch hp cho cc iu kin sn xut nng
nghip c th.
12
Hoc cc ging cy trng mi cho sn xut nng nghip.
94
Chng 8
Cng ngh DNA ti t hp
Cng c chnh trong cng ngh sinh hc hin i l cng ngh DNA ti t hp
1
hay cn gi l
k thut di truyn. Cng ngh ny cho php thao tc trc tip trn cc nguyn liu di truyn ca cc
t bo ring bit. Bng cch a cc thng tin di truyn ngoi lai vo trong cc c th vi sinh vt, cc
t bo ng vt v thc vt sinh trng nhanh, chng ta c th sn xut ra cc sn phm ca gen
ngoi lai (cc protein) vi cc tc v hiu sut cao hn m thng khng th thc hin cc h
thng t bo khc.
Chng ny trnh by nhng nt chnh ca cc nguyn l c bn trong k thut di truyn v
cc vn lin quan n nui cy t bo c chuyn gen ngoi lai.
I. DNA v RNA
Deoxyribonucleotide acid (DNA) l phn t quan trng nht trong cc t bo sng v cha tt
c thng tin c trng ca t bo. DNA v ribonucleotide acid (RNA) l cc i phn t
2
polymer
mch thng c xy dng trn cc tiu n v ring r l cc nucleotides. Mt n v monomer
(nucleotide) c ba thnh phn sau (Hnh 8.1):
- ng nm carbon mch vng (pentose): deoxyribose cho DNA v ribose cho RNA.
- Mt nitrogen base ca dn xut hoc purine hoc pyrimidine lin kt ng ha tr vi nguyn
t carbon th nht ca ng bng lin kt kt N-glycoside (Hnh 8.1).
+ Purine: adenine (A), guanine (G).
+ Pyrimidine: cytosine (C), thymine (T) cho DNA v uracil (U) ch cho RNA.
- Mt gc phosphate gn vo carbon v tr th 5 ca ng bng lin kt phosphoester.
95
Hnh 8.1. Cu trc ca nucleotide.
Cc nucleotide ca DNA c gi l deoxyribonucleotide, v chng cha ng deoxyribose,
trong khi nucleotide ca RNA c gi l ribonucleotide v chng cha ng ribose. Mi
nucleotide cha mt vng c trng v mt vng khng c trng. Cc nhm ng v phosphate l
phn khng c trng ca nucleotide, trong khi cc base purine v pyrimidine to nn phn c trng
ca nucleotide.
Mt nucleotide ny s c kt hp vi cc nucleotide khc bng mt lin kt ha hc gia
cc nguyn t trong cc vng khng c trng to ra cc polynucleotide (Hnh 8.2). S lin kt
(c gi l cc lin kt phosphodiester) xy ra gia mt nhm phosphate v mt nhm OH trn
thnh phn ng.
im c trng nht ca DNA l n thng bao gm hai si b sung xon gn nh song song
xung quanh mt trc chung to thnh mt xon kp (double helix) tng t nh mt cu thang xon
c (Hnh 8.3). Mi si l mt polynucleotide. ng knh ca xon (chiu ngang bc thang) khong
20 , Chiu cao ca mi vng xon l 34 , gm 10 bc thang ngha l mi vng xon c 10
nucleotide trn mi si.
96
Hnh 8.2. Mt phn ca DNA.
Hai si c kt hp bng lin kt hydrogen gia cc cp base purine vi pyrimidine
3
. Adenine
(purine) lun lun bt cp vi thymine (pyrimidine), v guanine (purine) vi cytosine (pyrimidine). Kt
qu phn tch ha hc v nng phn t ca cc base trong DNA cho thy rng lng adenine
lun bng thymine v lng guanine lun lun bng cytosine. S bt cp base ny c trng n ni
adenine ch lin kt vi thymine v guanine ch lin kt vi cytosine, nh to ra mt kh nng
n nh cao bi lin kt hydrogen gia cc base b sung.
Hn na, c trng ca s bt cp base ny cho php truyn thng tin di truyn t th h ny
sang th h khc. Khi s nhn i t bo xut hin, xon kp ca DNA tho ra, v hai si DNA mi
c to thnh b sung cho hai si gc. Nh vy, mi t bo mi cha mt si DNA gc v mt si
DNA mi c tng hp trong xon kp ca n.
97
Hnh 8.3. M hnh xon kp ca phn t DNA.
Trnh t ca cc base (A, G, T v C) trong mt si DNA c trng cho mt trnh t ca cc
amino acid s c lp rp to thnh mt phn t protein. M di truyn l tp hp cc trnh t
base tng ng cho mi amino acid (codon). V ch c 4 base trong DNA v 20 amino acid trong
protein, cho nn mi codon phi cha t nht 3 base
4
. Hai base khng th lm thnh mt codon bi v
ch c 4
2
cp hp l ca 4 base. Nhng 3 base th c th bi v s c 4
3
= 64 b ba hp l. V s
lng b ba hp l ln hn 20, cho nn mt vi codon ch nh cng mt amino acid. Trong mt
ngha khc, m di truyn l rt phc tp, v d: UCU, UCC, UCA, UCG, AGU v AGC u cng m
ha cho serine (Bng 8.1).
Bng 8.1. M di truyn chung.
98
II. To dng gen
To dng mt gen l cng vic trung tm ca cng ngh DNA ti t hp. Gen l n v c s
ca thng tin di truyn nm trn nhim sc th ca t bo. Cc nhim sc th l cc cu trc si di
v mnh nm trong nhn, bao gm ch yu l DNA. Thng tin di truyn c bo qun trong mt
chui trnh t ca cc base nucleotide, gm c mt on DNA. Mi gen ghi r cu trc ca mt sn
phm gen c bit, thng l mt protein.
1. Cc trnh t DNA
thao tc gen, cn phi bit v tt c cc t chc ca cc trnh t DNA v cc n v chc
nng ca DNA tng tc vi cc cc n v khc trong tt c cc n v di truyn ca c th
(genome) l nh th no. Vic pht hin ra cc enzyme hn ch (restriction endonucleases, RE) ca
vi khun ct DNA nhng trnh t c trng, gip cho vic thao tc d dng hn, v n c th
gim chiu di ca cc phn t DNA thnh mt tp hp bao gm cc on ngn hn.
Cc enzyme hn ch hin din trong hu ht cc t bo vi khun ngn cn DNA ngoi lai
tip qun b my tng hp protein ca t bo. DNA ca chnh chng s c bo v khi tc dng
ca enzyme hn ch, nh s c mt ca cc enzyme ni bo c th methyl ha (methylation) cc
nucleotide c bit, v th cc nucleotide ny khng th b nhn bit bi cc enzyme hn ch.
Mi enzyme hn ch nhn bit v ct mt trnh t DNA c bit thng cha bn hoc su cp
nucleotide. V d enzyme EcoRI chit t E. coli ct trnh t GAATTC, enzyme BalI ca
Brevibacterium albidum ct trnh t TGGCCA (Hnh 8.4). C hn 500 enzyme hn ch khc nhau
c tinh sch t khong 250 vi sinh vt khc nhau. Cc enzyme hn ch ct gy cc phn t DNA
si i theo hai cch khc nhau nh trnh by hnh 8.4:
99
- Ct trn mt ng thng i xng to ra cc phn t u bng.
- Ct trn nhng v tr nm i xng quanh mt ng thng i xng to ra nhng phn t
u so le (u kt dnh).
Hnh 8.4. Hai kiu ct ca enzyme hn ch.
(a) to ra u bng, v (b) to ra u so le
V mt enzyme hn ch nhn bit mt trnh t duy nht, cho nn s v tr ct trn mt phn t
DNA c bit thng l nh. Cc on DNA c ct bi enzyme hn ch c th c phn tch
theo kch thc bng in di agarose gel nghin cu. Do s tng t ca t chc phn t trong
tt c cc c th, cho nn DNA vi khun, DNA thc vt v DNA ng vt c v tng hp nhau v
cu trc. V th, mt on DNA t mt dng sng ny c th d dng c pha trn vi DNA ca
mt dng sng khc. S tng t ny cng ph hp i vi plasmid, nhn t di truyn ngoi nhn
c tm thy trong nhiu loi vi khun khc nhau. Chng l nhng phn t DNA mch vng ng
si i c dng lm vector mang cc on DNA ngoi lai dng trong cng ngh DNA ti t hp.
2. S kt hp ca cc phn t DNA
Cc phng thc c bn ca cng ngh DNA ti t hp l: (1) Kt hp mt on DNA vo mt
phn t DNA (nh l vector
5
) c th ti bn, v (2) Cung cp mt mi trng cho php sao chp
phn t DNA c kt hp.
C ba loi vector ph bin c dng to dng cc on DNA ngoi lai v ti bn (sao
chp) trong E. coli. l plasmid, bacteriophage v cosmid. Tt c nhng vector ny phi c mt
s tnh cht cn thit sau:
- C kh nng t ti bn trong E. coli.
- Cha cc gen ch th chn lc d dng phn bit v tinh sch vector ca th ti t hp vi
cc dng khc.
- C cc vng DNA khng cn thit cho s sinh sn trong vi khun, v th DNA ngoi lai c th
c a vo trong cc vng ny.
- C th bin np vo t bo vt ch mt cch d dng.
DNA plasmid c th c phn lp t nui cy vi khun cha plasmid bng cch b sung cht
ty (nh l sodium dodecyl sulfate
6
) v bng cch ly tm s sinh tan (lysate). Phc hp nhim sc
th vi khun, ln hn plasmid nhiu, s lng xung y ca tube ly tm, plasmid siu xon v cc
100
on nhim sc th mch thng gi li trong th ni. Plasmid siu xon mt ln na c phn tch
bng ly tm sau khi x l vi CsCl v EtBr.
Plasmid cha cc gen m ha cho cc enzyme thng c li cho vi khun vt ch. Cc
plasmid mang cc kiu hnh khc nhau nh: khng khng sinh, sn xut khng sinh, phn hy cc
hp cht hu c phc tp, sn xut cc enzyme hn ch v enzyme bin i (modification
enzymes).
Cc plasmid c th c chuyn vo trong vi khun sau khi vi khun c x l sao cho t bo
c th thm qua nht thi cc phn t DNA nh. Qu trnh ny c bit nh l s bin np
(transformation). Vi khun c bin np thnh cng c th c chn lc da trn kiu hnh mi
m chng nhn c t plasmid, chng hn kh nng khng cc khng sinh.
Mt s plasmid hin din trong t bo c s bn sao thp (mt hoc mt vi bn sao trn t
bo) do DNA plasmid ch sao chp mt hoc hai ln trc khi t bo phn chia. Tuy nhin, cc
plasmid khc tn ti mt s bn sao ln hn (10 ti 100 bn sao trn mt t bo) do DNA ti bn lp
li cho n khi t c s bn sao thch hp. Cc plasmid c s bn sao ln c gi l plasmid ti
bn dng lng lo (relaxed plasmid), v y l mt trong nhng tnh cht hu ch ca vector to
dng.
Hnh 8.5 trnh by mt trong cc vector plasmid dng lng lo, pBR322 (th h th hai), di
4.363 bp, vector ny cha hai gen khng khng sinh l Ampicillin (Amp) v Tetracycline (Tet). S th
t ca cc nucleotide trn vector c bt u vi v tr EcoRI duy nht: T u tin trong chui
GAATTC c quy c l nucleotide th nht. Cc s th t sau c tip tc quanh phn t
vector theo hng t gen khng Tet ti gen khng Amp.
Plasmid c th c ct mt s th t no trong cc v tr xc nh bng enzyme hn ch.
V th, cc on c to ra c th to vng bng cch kt hp cc u kt dnh b tr. Hn na,
cc on c to ra bi mt enzyme c bit hot ng trn mt phn t DNA s c cng u kt
dnh vi on c to ra bi cng mt enzyme hot ng trn mt phn t DNA khc. V th, cc
on t hai phn t DNA khc nhau t hai c th khc nhau c th kt hp bng cc lin kt
hydrogen thun nghch khi cc on ny c trn li.
Nu s kt hp c gn li sau khi bt cp, th cc on c kt hp c nh bn vng, s
kt hp ca cc on ny c thc hin nh enzyme ligase (cn gi l polynucleotide ligase) lin
kt ng ha tr cc phn t ti t hp bng cch to ra lin kt phosphodiester gia u 5-PO
4
ca
mt polynucleotide vi u 3-OH ca polynucleotide khc.
101
Hnh 8.5. Plasmid vector pBR 322. Amp
r
v Tet
r
: gen khng ampicillin v tetracycline, ori: trnh t
khi u sao chp, v mt s v tr nhn bit cho cc RE.
Hnh 8.6. trnh by ton b phng thc sn xut DNA ti t hp (to dng gen). Plasmid c
ct cc v tr xc nh bng enzyme hn ch. DNA ca mt genome ngoi lai c ct cng mt
loi enzyme, mt s on trong c th c gen quan tm. Plasmid v cc on ca genome c
phi trn v kt hp nh enzyme ligase. Cc plasmid ti t hp c bin np vo vi khun bng
ng nui cy plasmid v vi khun.
III. Kh nng n nh ca cc vi sinh vt ti t hp
Khi cc vi sinh vt ti t hp c nui cy sn xut protein ti t hp, th hiu sut ca sn
phm protein c th b gim do s tn tht ca plasmid trong c th khi chng tri qua nhiu ln sinh
sn trong qu trnh sinh trng ca vi sinh vt. Kh nng mt n nh c th do: (1) S phn chia
khim khuyt ca plasmid trong qu trnh phn chia ca t bo, hoc (2) Mt kh nng n nh cu
trc to ra cc t bin ca DNA plasmid.
n nh s phn chia ca plasmid, cc t bo cn c sao chp sao cho s lng trung
bnh ca cc bn sao plasmid trn mt t bo c nhn gp i ch trong mt th h, v cc bn
sao plasmid cn c phn phi bng nhau ti cc t bo con khi phn chia t bo. Mt s kt qu
nghin cu cho thy s khng n nh ca plasmid c pht hin trong cc h thng c th vt
ch nh: E. coli, Bacillus subtilis cng nh nm men.
102
Hnh 8.6. Phng thc c bn to dng gen.
Mt b phn cc t bo mang plasmid trong qun th tng s c xem nh l mt hm s
lng cc th h sinh trng. V th, khi th tch ca h ln men tng ln th s lng cc th h
sinh trng (generation of growth) cng tng, nhng mt b phn cc t bo mang plasmid v hiu
sut sn phm li gim. S khng n nh ca plasmid s tip tc nu h ln men quy m ln c
hot ng lin tc.
1. ng hc ln men ca cc nui cy ti t hp
Nu gi xc sut cc t bo mang plasmid (X
+
) sn sinh ra cc t bo khng mang plasmid
(X
-
) sau mt ln phn chia l p. Khi y vi N t bo mang plasmid, sau mt ln phn chia s sn sinh
ra N(1-p) t bo mang plasmid v Np t bo khng mang plasmid.
Tng s t bo (X
+
) s l N(2-p). Trong sut thi k sinh trng theo hm m, tc sinh
trng ca cc t bo mang plasmid s c biu din nh sau:
Trong :
+
l tc sinh trng c trng ca cc t bo mang plasmid, C
X
+
l s lng cc
t bo mang plasmid trn mt n v th tch. Nu khi lng t bo tng ng xp x vi s lng
103
t bo, th phng trnh tc sinh trng cho cng c th p dng c khi C
X
+
l khi lng t
bo trn mt n v th tch.
Mt khc, tc sinh trng ca cc t bo khng mang plasmid s l:
Trong :
l tc sinh trng c trng ca cc t bo khng c plasmid, v C

X
-
s lng
cc t bo khng mang plasmid trn mt n v th tch.
Nu chng ta chp nhn rng
+
v p l hng s, th tch phn ca phng trnh (8.1) s l:
Trong : l nng ban u ca cc t bo mang plasmid.
Trong trng hp cc t bo khng mang plasmid, thay th phng trnh (8.3) vo (8.2) ta
c:
Gii phng trnh vi phn tuyn tnh bc mt cho, ta c:
V vy, cc phng trnh (8.3) v (8.5) d bo s thay i theo thi gian ca C
X
+
v C
X
-
a
ra cc gi tr ca p,
+
v
-
.
B phn cc t bo mang plasmid trong qun th tng s (f) c th c nh ngha nh sau:
Thay phng trnh (8.3) v (8.5) vo phng trnh (8.6) cho kt qu:
y l phng trnh ch ra s thay i ca b phn cc t bo mang plasmid theo thi gian
trong sut thi k sinh trng hm m ca qu trnh ln men m. iu ny c quan tm xem f
gim nh th no theo s lng th h.
Trong sut thi k sinh trng theo hm m, s lng th h (n) ca cc t bo mang plasmid
c th c tnh ton t mi quan h sau:
Kt hp cc phng trnh (8.7) v (8.8) s c kt qu phng trnh ca f theo th h th n nh
104
sau:
Trong , l t l ca cc tc sinh trng c trng:
Phng trnh (8.9) c th c dng d bo s thay i ca f theo s th h cho mt lot
qu trnh ln men m, nu chng ta chp nhn rng cc t bo nhn ln theo hm m trong khi nui
cy tng m mt. Hnh 8.7 cho thy nh hng ca p v ln f
25
(f ca 25 th h), l thng s b
gim khi tng v p. Khi p s 0,01 v < 1, th f
25
gn bng 1, khi cc t bo mang plasmid rt n
nh. Tuy nhin, khi tin ti 2, f
25
cng tr thnh 0. Gi tr c trng ca giao ng t 1 n 2.
Hnh 8.7. B phn t bo mang plasmid sau 25 th h (f
25
) nh l mt hm ca v p.
Hnh 8.8 cho thy s thay i ca f nh l mt hm ca n v . Gi tr p c t khng i l
0,01. Gi tr ca f gim nhanh khi n v tng ln. Khi bng 1,4 tt c t bo mang plasmid mt
plasmid ca chng sau khong 33 th h.
Hnh 8.8. B phn t bo mang plasmid (f) theo s lng th h n (p 0,01).
105
2. Nui cy trong h thng ln men thng khuy lin tc (CSTF)
Ni chung, chng ta cn phi kim tra kh nng n nh ca cc t bo ti t hp trong h
thng ln men thng khuy lin tc. Cn bng nguyn liu cho cc t bo mang plasmid chung
quanh mt CSTF sn xut nh sau:
Tng t, cn bng nguyn liu cho cc t bo khng mang plasmid c a ra nh sau:
Cng hai phng trnh (8.11) v (8.12) s c phng trnh nng tng s ca t bo:
Nu CSTF c hot ng sao cho nng tng s ca t bo khng i theo thi gian, th:
Nu o = 1, th phng trnh (8.14) c n gin ha thnh:
V th, tc sinh trng c trng ca t bo trong h ln men l khng i v c xc nh
bng tc pha long. Thay phng trnh (8.15) vo cc phng trnh (8.11) v (8.12) ta c:
Nh vy, trong sut qu trnh ln men lin tc, nng ca cc t bo mang plasmid s b
gim, trong khi nng cc t bo khng mang plasmid s tng ln.
Nu o = 1, th
+
khng cn l hng s i vi tc pha long khng i trong sut s hot
ng trng thi n nh ca CSTF na nhng vn ph thuc vo nng t bo (C
X
+
v C
X
-
v o
theo phng trnh 8.14). Kt qu
+
cng thay i theo thi gian. Bng cch gii ng thi cc
phng trnh (8.11), (8.12) v (8.14), chng ta c th c lng C
X
+
v C
X
-
s thay i theo thi gian
nh th no. iu ny c th gip kho st xem b phn t bo mang plasmid s gim nh th no
theo thi gian. Thay phng trnh (8.14) vo phng trnh (8.11) v chia cho C
X
+
v C
X
-
ta c:
Gii phng trnh trn cho thy gi tr f thay i theo thi gian. Gi tr lc u ca f dng gii
phng trnh (8.18) c th thu c t phng trnh (8.9).
106
Hnh 8.9 m t s thay i theo thi gian ca b phn t bo mang plasmid trong sut s hot
ng trng thi n nh ca CSTF. B phn t bo mang plasmid b gim theo thi gian cng vi
vic tng tc pha long ln. Khi p v D thp, v o gn ti 1, th CSTF c th hot ng mt
cch hiu qu trong mt thi gian di.
Hnh 8.9. nh hng ca tc pha long (D) ln b phn t bo mang plasmid trong s hot
ng trng thi n nh ca CSTF. Gi tr ban u ca f c tnh ton bng cch tha nhn s
ln sinh sn cn thit cho bc tip mu v nui cy m ban u, v s ln men lin tc trng thi
khng n nh l 20.
Tuy nhin, nu o tng ln n 1,4 th CSTF s mt hu nh tt c cc t bo mang plasmid
sau 100 gi hot ng trng thi n nh (Hnh 8.10).
Hnh 8.10. nh hng ca ln b phn ca cc t bo mang plasmid trong s hot ng
trng thi n nh ca CSTF. Gi tr ban u ca f c tnh ton bng cch tha nhn s ln sinh
sn cn thit cho bc tip mu v nui cy m ban u, v s ln men lin tc trng thi khng
n nh l 20.
3. Cc phng php n nh
Mt s yu cu cn c lu m bo cho s n nh ca h thng ti t hp c khuynh
hng b mt cc plasmid ca chng nh sau:
107
- Xy dng cc cng thc mi trng thch hp cho s sinh trng ca cc t bo mang
plasmid hn qua cc t bo khng mang plasmid.
- t cc p lc chn lc ln cc t bo khng mang plasmid bng cch dng cc t bin
quang t dng (auxotrophic mutants) hoc cc plasmid khng khng sinh.
- Dng plasmid hoc chng t bin ph thuc nhit .
- Dng i chng biu hin gen ph thuc nhit .
- Dng cc plasmid khng cha cc nhn t chuyn v (transposon). Cc nhn t chuyn v
(cn gi l gen nhy) l cc on DNA c th c chn vo trong mt vi v tr trong b gen v c
th gy ra t bin.
- Dng chng ti t hp khng hon ton.
IV. Bin i di truyn t bo thc vt
Mc d li ch ca nui cy t bo thc vt quy m ln c tha nhn, k thut ny vn
cn gp mt vi kh khn khi ng dng cng nghip sn xut cc sn phm th cp v s cp.
Vic s dng khng ng mc cc k thut nui cy m thc vt ch yu l do tc sinh trng
chm ca cc t bo thc vt so vi t bo vi sinh vt v sn lng cng thng thp hn.
Nhng pht trin gn y ca cng ngh DNA ti t hp cho thy mt ha hn rt ln trong
vic gii quyt vn ny. Cc t bo nui cy sinh trng nhanh c th c chn lc v bin i
di truyn to ra cc sn phm c gi tr thng mi nng cao hn hiu sut bnh thng ca
t bo. Cc sn phm nhiu tim nng ca cng ngh DNA ti t hp l cc protein ngoi lai v cc
cht trao i th cp.
Sn xut cc cht trao i th cp t cc t bo thc vt bin i di truyn c th tng sn
lng mt cch su sc v thng mi ha nhanh chng cc nui cy thc vt quy m ln. Tuy
nhin, cc gen cn thit cho sinh tng hp cc cht trao i th cp c gi tr kinh t quan trng n
nay vn cha c phn lp. Bi v cc cht trao i th cp thng c sinh tng hp thng qua
hot ng kt hp ca nhiu sn phm gen, nhiu gen c yu cu cho mi phng thc sinh tng
hp dn ti sn xut cc cht trao i th cp.
Trong khi , cc gen m ha cho cc protein c gi tr kinh t quan trng c nhn dng v
bin np thnh cng vo cc vi sinh vt bi v cc protein l cc sn phm gen trc tip. K thut
bin np gen ngoi lai vo trong t bo thc vt cng c pht trin cho cc ng dng nng nghip
ca nui cy t bo thc vt. V th, sn xut protein ngoi lai t cc t bo thuc l c bin i di
truyn c th t c nhiu thnh qu.
Di y l cc u im tim tng ca vic ng dng t bo thc vt nh l cc t bo vt ch
cho cc sn phm ngoi lai:
- Cc sn phm protein t cc t bo thc vt ti t hp c th l cc dc phm c hiu lc
v chc nng hn cc sn phm c ngun gc t vi sinh vt bi v s bin i hu dch m l c th
xy ra trong cc t bo thc vt.
- Mi trng (ch yu l ngun carbon) nui cy t bo thc vt c xc nh tt v khng t
tin, trong khi cc t bo ng vt i hi b sung huyt thanh rt t tin trong mi trng dinh
dng ca chng.
- Cc t bo dch huyn ph thc vt c th t n mt t bo rt cao (60% trng lng
ti hoc 2,4% trng lng kh). Kt qu ny cao hn kt qu m cc kiu nui cy khc c th t
108
c.
- S nhim bn bi vi khun v nm d dng c kim sot trong nui cy m thc vt. Hn
na cc tc nhn nhim bn ny thng khng phi l cc tc nhn gy bnh cho ngi.
Hiatt v cng s (1989) cho thy tim nng rt ln trong vic sn xut cc protein ngoi lai
t thc vt bng cch chng minh s sn xut immunoglobulin v lp rp cc khng th chc nng
trong cy thuc l c bin i di truyn. H bin np vo mnh l thuc l cc DNA b sung
(cDNA) tng hp t mRNA ca hybridoma chut v ti sinh mnh l thnh cy hon chnh. Cc
thc vt biu hin cc chui immunoglobulin hoc k ring r c lai sn xut con chu trong
c hai chui c biu hin ng thi. Mt khng th chc nng tch ly ti 1,3% protein l tng s
trong cc thc vt biu hin cc cDNA hon chnh cha cc chui leader.
1. K thut gen
Mt gen ngoi lai (ca vi khun hoc ng vt) cn phi c sa i biu hin tnh cht
ca chng trong cc t bo thc vt. iu ny cho thy rng thng tin di truyn c yu cu cho s
biu hin gen, nh l sn xut mRNA t gen v dch m mRNA thnh protein, l hon ton khc gia
thc vt v cc c th sng khc. c kt qu tt nht, c hai vng ngc hng (promoter) v
cng hng (terminator) ca gen cu trc (gen m ho cho sn phm protein mong mun-coding
gene) phi c thay th bng mt chui DNA ca thc vt cha thng tin thch hp nh trnh by
hnh 8.11.
1.1. Promoter
Promoter thc vt c th phn chia thnh hai loi, cu trc v cm ng. Cc promoter cu trc
c chc nng trong hu ht tt c cc m. Cc promoter cm ng thng l im lng cho n khi
chng c cm ng nh cc nhn t mi trng hoc nhn t pht trin.
Chng hn, cc promoter cho quang hp, cc c ch bo v v pht trin hoa c cm ng
ch di mt iu kin nht nh. Chn mt promoter thc vt ty thuc vo bn cht ca sn phm
mong mun. i vi hu ht cc trng hp, mt promoter cu trc mnh l ngun tt nht sn
xut mt sn phm ngoi lai. Tuy nhin, nu sn phm ngoi lai l bt li i vi sinh trng ca
thc vt, th cn thit s dng mt promoter cm ng sao cho sn phm s c tng hp ch di
cc iu kin mong mun. Cc promoter cho quang hp c cm ng nhanh nh nh sng trng
v tt trong sut thi gian ti. Cc gen tiu n v nh ca ribulose-1,5-biphosphate carboxylase v
cc gen protein light-harvesting c biu hin nhiu nht trong m xanh. V th, promoter t cc
gen ny c kh nng l ngun tt nht cho cc biu hin cm ng vi nh sng. Cc promoter cho
quang hp c phn lp t cc ngun thc vt khc nhau v dng cho s biu hin ca mt gen vi
khun.
109
Hnh 8.11. S biu hin gen ngoi lai trong thc vt. P: promoter, T: terminator, pGA482:
vector to dng thc vt, Km: gen khng kanamycin, Tc: gen khng tetracycline.
1.2. Terminator
Ngi ta bit rt t v terminator ca thc vt v cha chng minh c l liu terminator ca
ng vt c chc nng trong thc vt hay khng. Cho ti khi iu ny c lm r, th an ton
hn nn s dng mt terminator thc vt biu hin cc i mt gen ngoi lai. Terminator nos
(nopaline synthase) c dng thng xuyn nht mc d mt s terminator khc ca thc vt cng
c phn lp.
1.3. Cc chui tn hiu
Khng nn c tn hiu khi u dch m (ATG) gia chui promoter v gen cu trc v trnh t
ATG ny s lm gim mt cch ngha hiu sut dch m. Khong cch gia cc vng iu ha
(promoter v terminator) v gen cu trc phi khng c qu di. Nu khng th mRNA c sn
xut s km n nh.
Mt trong nhng nhn t quan trng xt v thc t thao tc gen l v tr cui cng ca protein.
Cc protein ngoi lai c sn xut c th c duy tr trong t bo cht, c chuyn vo trong
mt c quan t, hoc c tit ra ngoi t bo. Hu ht protein thc vt phn b trong t bo cht,
n c chuyn vo trong c quan t ca t bo cht hoc ra ngoi mng t bo th mt tn hiu
c trng (polypeptide) phi c gn vo u tn cng amino ca protein mong mun. Chui
peptide tn hiu ny c tch ra trong giai on sm ca s vn chuyn v ch mt protein chc
nng hon chnh c gii phng ti ni n cui cng. iu ny cha c hiu y khng bit
liu ch mt mnh chui peptide tn hiu c cho s vn chuyn ng cch hay khng. Nhng
nghin cu gn y cho thy rng s vn chuyn ca protein ngoi lai vo trong chloroplast ch yu
cu mt peptide tn hiu. Tuy nhin, c ch vn chuyn vo trong cc c quan t khc hoc trong
mi trng ngoi bo l rt khc nhau. Nu thng tin b sung trong th protein ch yu cng cn
110
thit, th s cc k kh khn khi thit k cho mt protein ngoi lai c vn chuyn m khng c s
thay i v cu trc protein khi lm hng chc nng ca protein.
Nhiu protein c tng hp trong cc t bo c bin i di truyn. V d: carbohydrate
thng c gn vi cc protein tm thy trn mng t bo. Mt s protein c phosphoryl ha.
Nhng bin i nh th hoc gy hot ng hoc lm bt hot chc nng ca protein. V vy, mt
gen ngoi lai phi c thao tc thch hp cho mt bin i hu dch m chnh xc. V d, nu mun
gn carbohydrate vo protein, th c th thit k mt qu trnh tit protein ra ngoi mng t bo.
Trong phng thc ny protein c th c bin i cho ph hp. Tuy nhin, cc c ch bin i
nh th c th khc nhau trong mi c th sng. V th, chn lc cn thn dng t bo thch hp l
rt cn thit.
2. Bin np gen
C nhiu k thut khc nhau c dng bin np gen vo t bo thc vt, chng hn bin
np gen gin tip qua Agrobacterium tumefaciens, bin np gen trc tip bng vi n
(microprojectile) nh sng bn gen (gene gun) hoc h thng di bom (bombardement), xung in
(electroporation), vi tim (microinjection), sng siu m (ultrasonic), tinh th silicon carbide, PEG
(polyethylene glycol)...
Tuy nhin, k thut c s dng rng ri nht bin np mt chui DNA mi vo thc vt l
da trn c s Ti-plasmid ca A. tumefaciens. Trong thi gian t bo thc vt vi vi khun t, th
mt chui c bit c gi l DNA vn chuyn (T-DNA) ca Ti-plasmid c chuyn t vi khun
vo t bo thc vt. Nu gen ngoi lai c xm nhim c trong T-DNA, th gen c th c chuyn
vo trong nhim sc th thc vt cng vi T-DNA. T-DNA t nhin mang cc gen sn xut
phytohormone dn n to thnh khi u cc t bo b xm nhim. trnh hin tng to khi u
v to iu kin cho vic chn lc nhanh cc th bin np, cc marker (gen ch th) khng ca thc
vt c pht trin bng cch dung hp gen khng khng sinh ca vi khun vi cc vng iu
ha ca thc vt nh m t phn trc. Theo phng thc ny cc marker khng kanamycin
v marker khng chloramphenicol c xy dng v s dng thay th cc gen phytohormone
trn vng T-DNA. Do Ti-plasmid t nhin kh thao tc trc tip in vitro bng cc phng php DNA
ti t hp v kch thc ln ca chng (khong 200 kb), v th ngi ta pht trin cc h thng
n gin hn.
Phng php hiu qu nht c s dng l h thng binary vector. Mt trong cc
binary vector c trnh by hnh 8.11. H thng ny ph thuc vo mt vector nh mang cc yu
t c yu cu ti thiu trong dng cis. Cc chc nng khc cn cho c ch chuyn gen c giao
cho mt helper plasmid ring bit, Ti-plasmid. Nhng phng thc ny cho php a vo v duy tr
mt cch d dng DNA ngoi lai cha trong mt gen c thao tc di truyn.
Cc t bo thc vt c th bin np vi mt gen ngoi lai bng phng php ng nui cy.
Trong phng php ny, cc vi khun A. tumefaciens cha binary vector v mt helper Ti-plasmid
c trn li vi cc t bo nui cy c hot tnh sinh trng hoc mnh ct ca thc vt (mnh l).
Sau khi hn hp ny khong hai ngy. Cc t bo bin np c chn lc trn mi trng agar c
khng sinh thch hp. Th bin np c th c pht hin bng mt thng sau khi ng nui cy t
2-3 tun. Mt vi t bo trong mnh l c bin np s c ti sinh thnh cy. Cc cy chuyn
gen ny sau s c s dng to callus dng trong nui cy dch huyn ph t bo cc h
ln men quy m ln.
V. Bin i di truyn t bo ng vt
Bin i di truyn cc t bo ng vt c th c ng dng sn xut mt protein c trng
111
hoc ci thin c im ca mt dng t bo sn xut. Cng tng t nh thc vt, hin nay c
nhiu phng php a DNA ngoi lai vo t bo ng vt c v bin i di truyn, v d: chuyn
nhim (transfection) hay cn gi l ha bin np, lipofection (DNA c a vo thng qua
liposome), xung in, vi tim DNA trc tip vo t bo, bn gen, dung hp (fusion) t bo ng vt c
v vi protoplast ca vi khun mang DNA hoc cc h thng viral vector (k c cc tiu phn
phage)... Cc dng t bo chuyn nhim s biu hin DNA ngoi lai n nh ch khi n c hp
nht trong genome. Ngc vi c th vi sinh vt, s hp nht ca DNA ngoi lai hu ht l khng
tng ng (non-homologous). V th, gen m ha sn phm protein c th c hp nht trong cc
vng ca genome, m vng khng thun li cho vic biu hin hiu qu ca gen.
1. K thut gen
Mt s gen ch th chn lc (selectable marker) cho cc t bo ng vt c v l c sn. Cc
marker tri (c th c dng bt chp dng t bo vt ch) hu ht lin quan vi tnh khng thuc.
Cc marker ln (c s dng trong s kt hp vi c im di truyn ca t bo vt ch) c th
bao gm cc enzyme ca s chuyn ha purine v pyrimidine, tnh khng thuc hoc chuyn ha
amino acid. Hai h thng thnh cng nht l h thng glutamine synthetase (GS) v h thng
dihydrofolate reductase (DHFR).
Enzyme GS (c tng hp nh gen gs) xc tc cho s to thnh glutamine t glutamate v
cc ion ammonium. Gen gs c th c dng nh l mt marker chn lc trong cc t bo
hybridoma v myeloma hoc cc t bo khng c gs khc. Cc t bo c chuyn nhim n nh
s biu hin gen gs v sinh trng n nh trn mi trng khng c glutamine.
Enzyme DHFR (c tng hp nh gen dhfr) dng cho dng t bo CHO dhfr
-
. Dng t bo
dfhr
-
khng n nh tng hp tetrahydrofolate mt cofactor cn thit trong chuyn ha mt carbon
(one-carbon). Cc dng t bo dfhr
-
ch sinh trng n nh trn mi trng cha thymidine v
hypoxanthine v cc tin cht (precursors) cn thit vt qua s khim khuyt ny. Cc t bo
chuyn nhim biu hin gen dhfr c kh nng sinh trng n nh trn mi trng khng b sung
cc cht ni trn.
Bin i di truyn ca cc t bo ng vt c v ci thin dng t bo cha c pht trin
rng ri nhng cng tng ln ng k. Cc lnh vc ang c quan tm l ko di s sng ca
cc t bo sn xut, sinh trng trong mi trng khng c huyt thanh, gim s to thnh cc sn
phm ph v cc c tnh glycosylation. Apoptosis, yu t xut hin trong hu ht cc nui cy t bo
ng vt c v, c th b nh hng nh vic a vo gen bcl2, mt gen khng apoptosis. Phng
thc ny s ko di s sng ca t bo v lin quan pha sn xut ca qu trnh nui cy.
2. Bin np gen
Chn phng php bin np gen ph thuc vo mc biu hin gen c mong i, biu
hin trong thi gian ngn hoc biu hin n nh; loi t bo ch nh t bo dch huyn ph hoc t
bo dnh bm, cc dng t bo thch nghi hoc phn ha. Mi phng php u i hi s ti u
cao, bao gm cc yu t nh: s lng t bo, nng DNA v vector biu hin.
2.1. Phng php chuyn nhim
Dng calcium phosphate kt ta DNA, c phm vi hiu qu t 1-10
4
khun lc/10
6
t bo/g.
S hp nht DNA ngoi lai trong DNA t bo mang tnh ngu nhin. DNA chuyn nhim thng ti
t hp trc khi hp nht lm cho th hi nhp mang nhiu bn sao DNA trong t bo. Hiu qu
112
chuyn np c th tng mt vi dng t bo c x l dimethyl sulfoxide (DMSO) hoc glycerol
trong mt thi gian ngn (4-6 gi) sau khi chuyn nhim. X l sc sau chuyn nhim bng
chloroquin diphosphate gy c cao. Mc c thay i gia cc dng t bo, c bit cc t bo
nui cy dch huyn ph v cc t bo phn ha giai on cui. Khi thay calcium phosphate bng
DEAE-dextran th chuyn nhim DNA c th t c hn vi mi x l sc sau chuyn nhim t bo
nui cy dch huyn ph v t bo phn ha.
2.2. Phng php lipofection
Phng php ny s dng cc lipid trung tnh hoc mang cation to thnh liposome. Phc
lipid hp nht vi mng huyt tng s phng thch DNA dnh bm vo trong phn bo tan. Phng
php ny cho hiu sut chuyn nap cao hn ha bin np. Tnh ng nht ca thnh phn lipid gia
mng t bo vt ch v lipofectant lm tng hiu qu dung hp v tng kh nng xm nhp ca DNA
gn km. Cc liposome mang cation (lipofectin) thch hp cho chuyn gen in vitro vi hiu sut trn
90% mt s loi t bo nui cy. Lipofectin c s dng bc cc virus. Vic to ra cc
liposome cha protein virus trong lp lipid ci thin hiu qu gn ca vector vi cc t bo c
bit, nh l t bo ung th gan. Ch c mt s phng php thch hp chuyn DNA qua mng
huyt tng bng thc m bo (endocytosis) v y mnh cc qu trnh ni th gy thoi bin v
sp xp li DNA. Liposome c dng phn phi in vivo v ex vivo cc gen ngi ti t bo ch
thch hp.
2.3. Phng php chuyn gen bng xung in
Phng php ny yu cu nghim ngt cc thng s ca thit b in xung lin quan n hiu
sut xm nhp ca DNA. Dng in c s dng to ra cc t bo treo trong dung dch DNA
cc l thng trn mng huyt tng v thng qua DNA theo grandient mt chui vo trong phn
bo tan. Phng php ny c s dng rng ri trong nui cy dch huyn ph lymphocyte (lympho
bo). Chuyn np gen bng xung in c khuynh hng to ra cc th hi nhp mang bn sao DNA
n v thng c s dng chuyn gen vo cc t bo mm phi (embryonic stem-ES). Vic
chuyn np gen thng qua in trng cc t bo to mu (hematopoietic cells) v cc th h t
tin ca chng l phng thc thch hp cho cc virals vector i hi h thng ng gi phc tp.
Cc t bo ty xng t tin (bone marrow progenitor cells) c nui trong mi trng c cytokine,
interleukin 3, s tng tn s chuyn nhim v biu hin gen cc t bo to bch cu ht
(granulopoietic) v th hng cu (erythroid) t tin. Cc marker chn lc tri nh l pSVNeo ghi m
cho mt gen ca prokaryote, neomycin phosphotransferase (neo) mang gen khi ng (promoter) v
gen tng cng (enhancer) ca Simian Virus 40 (SV 40), cho php phn lp cc t bo khng
neomycin bng cch dng mt dng ng ng ca neomycin, G418, trong mi trng b sung.
Chuyn np gen pSVNeo bng xung in vo trong t bo mm ty xng cho kt qu tt, v c th
ng dng chuyn np cDNA ca yu t ng mu (factor IX) vo trong t bo m (stromal cells)
c ngun gc ty xng.
2.4. Phng php vi tim
L phng php chuyn DNA trc tip nht v c hiu qu cao. Tuy nhin, s lng t bo th
nghim b gii hn ch trong vi trm. K thut tinh xo v thit b t tin ca vi tim hn ch s
dng rng ri phng php ny. Tin b ln nht ca phng php vi tim l kh nng gim st biu
hin ca DNA ngoi lai cc t bo ring r. Hn na, trong khi chuyn nhim v chuyn gen bng
xung in c th v cng c, th vi tim phn phi DNA trc tip vo trong nhn t bo m khng
gy nguy him n s nguyn vn ca t bo. gim c tnh ti mc ti thiu, th tch DNA
113
phi c hn ch nh hn 10% th tch nhn. c trng ca vi tim l cung cp phng thc
to ra cc ng vt c chuyn gen, nh gi biu hin gen c to dng trong cc t bo phi,
cung cp phng php trc tip to cc t bin chn on (insertional mutants), xc nh cc
nhn t iu ha biu hin gen c trng m v c trng t bo, v phn lp cc dng provirus
nguyn vn ca cc retrovirus tin hnh phn tch bnh l hc.
2.5. Phng php dng sng bn gen
Phng php ny s dng cc ht kim loi nh l tungsten hoc vng lm vi n. Vi n c
bc bng DNA v c bn i vi mt vn tc thch hp xm nhp vo t bo ch. Hiu qu ca
phng php ny tng ng vi phng php chuyn nhim. S biu hin thnh cng ca DNA
ngoi lai trong t bo chut NIH 3T3, t bo kh COS 7 v mt dng i thc bo (macrophage)
c thng bo. Sng bn gen v phng php tim trc tip DNA trn ca plasmid b hn ch i
vi tim v cc t bo c xng ca ng vt. Ch c 1-3% t bo nhn DNA v sn xut mt lng
nh protein c ghi m. Cc phng tin hin hnh hu ht u thch hp cho cc chin lc
vaccine trong vi mt lng nh protein l to ra mt phn ng min dch.
2.6. Viral vector
Cc viral vector l cc cu trc ti t hp trong mt hoc nhiu gen virus c thay th bi
DNA to dng. Chuyn np gen c trng m hoc t bo c thc hin bng cch chn lc cc
im th cm ca t bo c hiu virus. Virus mang DNA ti t hp xm nhim vo t bo ch c
trng v phn phi ti trng di truyn vo trong chng. Mt c im hp dn ca viral vector l iu
ha biu hin gen khi c mt promoter v enhancer ca virus. Cc viral vector c ngun gc t hu
ht cc virus DNA, bao gm SV 40, cc virus to u dng nh (papilloma virus) ngi v b, nhm
virus DNA ca parvoviridae (adeno-associated virus, AAV), adenoviruses (cc virus mang DNA si
kp), cc virus bnh mn gip (herpes) v virus bnh u ma (vaccinia). Kch thc ca cc on
chn (insert DNA) thay i t 2-3 kb cc papovavirus ti > 50 kb vaccinia. Cc viral vector c th
cho php biu hin trong thi gian ngn v hu nh 100% iu kin in vitro. DNA ca provirus
c sn xut nh phin m ngc RNA ca retrovirus (nhm virus mang RNA si n) hp nht
nh l mt bn sao n trong nhim sc th vt ch, gim ti mc ti thiu s phin m gen. Cc
viral vector c bit c thit k phn phi DNA ngoi lai vo trong cc t bo phn ha, cc t
bo mm phi, v cc lymphocyte. Mc d cc retrovirus c s dng chuyn gen, nhng
cng c mt vi kh khn do provirus ca n thiu kh nng hp nht vo cc t bo th ng, bi v
DNA ch hp nht khi t bo tri qua thi k phn bo. iu ny dn ti tht bi khi biu hin mc
cao cc gen chuyn np. c kh nng ti t hp, kch thc ti a ca on chn DNA
khong 7 kb.
Tm li, chn la mt phng php chuyn np gen thch hp ph thuc vo mc ch th
nghim v cc t bo ch (t bo dnh bm hay t bo dch huyn ph), chng l nhng t bo s
cp hay thch nghi, phn ha hay c nhiu tim nng. Th nghim biu hin ca DNA chuyn
nhim c th l tm thi (trong thi gian ngn) hoc bn vng (n nh) vi cc mc biu hin c
bn hoc c th suy din. Cc dng t bo dnh bm d thao tc bng cc phng php chuyn gen
khc nhau vi th nghim thnh cng biu hin gen sau . Khi c tnh ca phng php chuyn
nhim loi tr kh nng biu hin gen, th mt trong cc phng php khc c th cho php thit k
th nghim thnh cng. Khi phng php chuyn nhim, xung in, v viral vector tht bi th
phng php vi tim c th s thch hp. Chuyn np gen trong cc t bo khng dnh bm kh thc
hin nhng s dng viral vector v chuyn nhim DEAE, cng nh liposome c th l gii php hp
l. C kh nng vi tim cc t bo khng dnh bm nhng cn s dnh kt ha hc ca t bo vi c
cht.
114
Nng DNA dng trong th nghim chuyn gen thay i t 1ng n 10 g cho 10
5
-10
6
t bo
nhn. Trong vi tim, mt vi trm t bo c tim trc tip DNA nng t 1 pg n 1 g/l tng
ng vi 1-10
2
bn sao ca cu trc ti t hp. S lng bn sao a vo trong cc t bo nhn
vt ch t l vi kch thc vector, on chn (insert DNA) v nng DNA. Cc cu trc mch
thng hp nht thng qua ti t hp cao hn khong 10 ln cc cu trc mch vng. Chuyn nhim
thng qua liposome tr thnh mt k thut ph bin nh c tnh thp hn v hiu qu chuyn np
cao. Trong khi phng php xung in i hi mt s lng ln DNA (10-40g) v trung bnh s git
cht mt na t bo nhn.
VI. Cc k hiu
C nng , s lng t bo/m
3
hoc kg/m
3
C
X
+
s lng cc t bo mang plasmid trn mt n v th tch
nng ban u ca cc t bo mang plasmid
C
X
-
s lng cc t bo khng mang plasmid trn mt n v th tch
nng ban u ca cc t bo khng mang plasmid D tc pha long, s
-1
f s t bo mang plasmid trong qun lc t bo tng s, thng s khng c th nguyn
f
n
s t bo mang plasmid trong qun lc t bo tng s sau n th h, thng s khng c th nguyn
n s th h, thng s khng c th nguyn
p xc sut xut hin s t bo khng c plasmid trong mt th h
t thi gian, s
o t l ca cc tc sinh trng c trng (
-
/
+
), thng s khng c th nguyn
+
tc sinh trng c trng ca cc t bo mang plasmid, s
-1
-
tc sinh trng c trng ca cc t bo khng c plasmid, s
-1
X
+
t bo mang plasmid
X
-
t bo khng mang plasmid
2
nd
2. Bains W. 2003. Biotechnology from A to Z. Oxford University Press, Inc. New York, USA.
st
Singapore.
5. Old RW and Primrose. 1989. Principles of Gene Manipulation. Blackwell Scientific
Publications, Osney Mead, Oxford, UK.
6. Ratledge C and Kristiansen B. 2002. Basic Biotechnology. Cambridge University Press,
UK.
nd
ed. Prentice
8. Singleton P and Sainsbury D. 2001. Dictionary of Microbiology and Molecular Biology. 3
rd
ed. John Wiley & Sons, Ltd. UK.
115
9. Walker JM and Rapley R. 2002. Molecular Biology and Biotechnology. 4
th
ed. The Royal
Society of Chemistry, Cambridge, UK.
1
Xem chng 1 gii thiu chung v cng ngh sinh hc.
2
i phn t (macromolecular): l mt polymer c to thnh t hn 100 monomers.
3
Mt lin kt hydrogen gia hai si l mt lc ht yu gia mt nguyn t hydrogen c lin kt
ng ha tr (H-N) v mt nguyn t ketonic oxygen in tch m (C=O).
4
Hai mi amino acid c tm thy trong cc phn t protein l: Alanine (Ala), Arginine (Arg),
Asparagine (Asn), Aspartic acid (Asp), Cysteine (Cys), Glutamic acid (Glu), Glutamine (Gln), Glycine
(Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine
(Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) v Valine (Val).
5
Vector l phn t DNA c s dng a DNA ngoi lai vo trong t bo vt ch.
6
Cht ty lm bin i b mt t bo gii phng cc thnh phn t bo ra mi trng bn ngoi.
116
Chng 9
Tit trng
Hu ht cc qu trnh ln men cng nghip c tin hnh nh cc nui cy thun khit trong
ch c cc chng chn lc c php sinh trng. Nu mt c th vi sinh vt ngoi lai hin din
trong mi trng hoc trong bt k mt b phn thit b no , th chng s lm nhim bn mi
trng, sn xut ra cc sn phm c hi c th hn ch sinh trng ca cc c th c sn xut.
V th, trc khi bt u qu trnh ln men, mi trng v cc thit b ln men phi c tit trng
loi b tt c cc nguy c gy nhim v cc iu kin v trng ny phi c duy tr trong sut qu
trnh ln men.
I. Cc phng php tit trng
Tit trng mi trng ln men hoc cc thit b c th thc hin bng cch ph hy tt c cc
c th sng hoc bng phng thc nhit (m hoc kh), hoc cc tc nhn ha hc, chiu x (tia
cc tm hoc tia X) v bng cc phng php c hc (siu m hoc sng m thanh). Mt hng
khc l loi cc c th sng bng phng php lc hoc ly tm tc cao.
1. Nhit
Nhit l phng thc c s dng rng ri nht tit trng, c th s dng cho c hai loi
mi trng c v lng. N c th c ng dng di dng nhit kh hoc m (hi nc). Nhit
m thng hiu qu hn nhit kh, do kh nng khng nhit bn trong ca cc t bo vi khun
c tng ln mnh trong trng thi kh hon ton. Kt qu l t l cht ca t bo kh thp hn
nhiu so vi t bo m. S dn nhit trong khng kh kh cng c tc km hn trong khng kh
m. V th, nhit kh ch c dng tit trng dng c thy tinh hoc cc vt liu rn chu nhit.
Bng cch tng p sut ln bnh nui cy, nhit hi nc c th tng ln mt cch ngha trn c
im si ca nc. Ni tit trng p sut (autoclave) phng th nghim thng c hot ng
p sut hi nc khong 15 psi tng ng vi 121
o
C, cc bo t vi khun b git nhanh 121
o
C.
2. Ha cht
Cc tc nhn ha hc c th c dng git vi sinh vt bng kh nng oxy ha hoc alkyl
ha. Tuy nhin, chng khng c dng tit trng mi trng bi v cc ha cht ny c th c
ch sinh trng ca cc c th ln men. Cc tc nhn ha hc c s dng thng xuyn cho vic
x l loi b hoc lm gim mc nguy hi ca cc tc nhn gy bnh. Mt s tc nhn ha hc
khng khun chnh l: phenol v cc hp cht phenol (phenol, cresol, orthophenylphenol), alcohol
(ethyl, methyl), cc halogen (iodine, hypochlorite, chloramine), cc cht ty, thuc nhum, cc hp
cht ammonium bc bn, cc acid, kim v cc tc nhn gy v sinh dng kh (ethylene oxide, -
propiolactone, formadehyde).
117
3. Tia cc tm
Nhiu nguyn liu t bo hp th nh sng cc tm, dn n gy nguy him cho gen v sau
git cht t bo. Bc sng khong 256 nm c hiu qu dit khun cao nht. Tuy nhin, tia cc tm
c rt t kh nng xuyn qua vt cht. V th, vic s dng chng b hn ch i vi vic lm gim
qun th vi sinh vt trong phng ni m iu kin v trng cn thit c duy tr thng xuyn,
chng hn nh cc phng m ca bnh vin hoc cc bung lm vic sch trong phng th nghim.
Tia X gy cht c th vi sinh vt v c kh nng xuyn qua vt cht. Tuy nhin, chng khng
thc t nh cc cng c tit trng khc do chi ph t cng nh s lo lng v an ton lao ng.
4. Sng siu m
Sng m thanh hoc siu m c cng mnh cng c th ph v v git cht t bo. K
thut ny thng c s dng ph v t bo nhm tch chit cc thnh phn ca ni bo
(protein, enzyme...) hn l tit trng.
5. Lc
L k thut c s dng hiu qu nht trong vic loi b cc vi sinh vt trong khng kh hoc
trong cc loi kh khc. Trong trng hp dung dch lng, n c dng cho cc sn phm hoc cc
loi mi trng khng bn nhit, d dng b ph hy nh cc huyt thanh ngi v ng vt, cc loi
enzyme.
Trong s cc k thut c gii thiu trn, nhit m c hiu qu v kinh t nht cho cc yu
cu tit trng ni chung ca h ln men. V th, cc phn sau y ch m t ng hc ca hin tng
cht t bo v cc hot ng tit trng bng nhit m.
II. ng hc ca hin tng cht do nhit
Hin tng cht do nhit ca vi sinh vt, mt nhit c trng, c th m t bng phng
trnh ng hc bc mt:
Trong : k
d
l tc cht c trng, gi tr ca n ph thuc khng ch vo loi m cn vo
dng sinh l ca t bo. V d: gi tr k
d
ca bo t vi khun 121
o
C l 1 pht
-1
, trong khi gi tr
ny ca cc t bo sinh dng khc nhau t 10
1
pht
-1
n 10
10
pht
-1
ty thuc vo tng c th c
bit.
Tch phn ca phng trnh (9.1) cho kt qu:
118
Phng trnh (9.3) cho thy s suy gim theo hm m ca qun lc t bo. S ph thuc vo
nhit ca tc cht c trng k
d
c th c tha nhn theo phng trnh Arrhenius:
Trong : k
do
l h s Arrhenius bng 5,710
39
gi
-1
, R l hng s kh, T l nhit tuyt i, E
d
l nng lng hot ng c th thu c t dc ca th ln(k
d
) theo 1/T. V d: nng lng hot
ng ca E. coli l 127 kcal/gmol v ca Bacillus stearothermophilus (chng Fs 7954) l 68,7
kcal/gmol.
III. Tiu chun thit k
T phng trnh (9.2) v (9.4) tiu chun thit k cho s tit trng (V) c th c nh ngha
nh sau (Deindoerfer v Humphrey 1959):
V cng c xem nh l yu t Del, l thc o quy m ca cng vic c hon thnh. Yu
t Del tng ln khi s t bo cui cng gim. V d: Khi gim s t bo trong h ln men t 10
10
c
th c th sinh trng c xung cn 1 th yu t Del s bng:
Vic gim s lng t bo t 10
10
xung cn 1 dng nh rt n tng. Tuy nhin, thm ch
nu 1 c th cn sng th ton b h ln men vn b nhim. V th, tt c cc vi sinh vt cn sng
phi c o thi. Khi gim s lng t bo ti 0 th yu t Del bng , iu c ngha l v mt
l thuyt khng c kh nng ph v tt c cu trc ca cc t bo sng. V th, s lng t bo cui
cng cn thit c biu hin nh l phn s ca 1, bng vi xc sut ca s nhim bn. V d: n =
0,001 ngha l c hi cho mt nhn t gy nhim bn sng st khi b tit trng l 1/1000. Nhn t Del
lm gim s lng t bo trong h ln men t 10
10
c th sng xung cn 0,001 l:
Da trn c s tiu chun tit trng c tnh ton, chng ta c th thit k mt thit b tit
119
trng ti u.
IV. Tit trng tng m
Tit trng mi trng trong h ln men c th tin hnh tng m bng cch phun hi nc
(steam sparging) trc tip, bng cc b phn un nng bng in, hoc bng p lc tun hon khng
i lm ngng t hi nc thng qua cun dy t. Cc chu k tit trng c sp xp theo th t
un nng, gi nng v lm lnh. V th, yu t Del ton phn (total) s bng tng s yu t Del un
nng (heat), gi nng (hold) v lm lnh (cool):
Gi tr ca V
heat
v V
cool
c xc nh bng cc phng php dng cho qu trnh un nng v
lm lnh. Gi tr ca V
hold
c xc nh bng chiu di ca thi gian gi nng. Phng thc thit k
nh gi thi gian gi nng nh sau:
- Tnh ton tiu chun tit trng ton phn.
- Xc nh profile ca nhit theo thi gian trong sut cc chu k un nng, gi nng v lm
lnh ca qu trnh tit trng. Nu cc php o thc nghim khng tin hnh c, th cc phng
trnh l thuyt cho vic lm nng v lnh c th c s dng, l nhng phng trnh ng
thng, hm m hoc hyperbolic ty thuc vo kiu lm nng v lnh. Cc phng trnh c gi
cho cc qu trnh lm nng v lnh khc nhau nh sau (Deindoerfer v Humphrey 1959):
+ un nng tng m bng cch phun hi nc trc tip vo mi trng, phng trnh dng
hyperbolic:
Trong : T l nhit tuyt i (
o
K), T
0
l nhit tuyt i ban u ca mi trng (
o
K), H l
enthapy ca hi nc lin quan vi nhit ca mi trng cha nu chn (J/kg), m
s
l tc dng
chy ca khi hi nc (kg/s), t l thi gian (s), c l nhit c trng ca mi trng (J/kg
o
K), v M l
khi lng ban u ca mi trng trong ni tit trng m (kg).
+ un nng tng m bng tc khng i ca dng nhit, nh un nng bng nhit, phng
trnh dng ng thng:
Trong : q l tc truyn nhit (J/s).
+ un nng tng m bng ngun ng nhit, nh tun hon hi nc thng qua cun dy t,
phng trnh dng hm m:
Trong : T
H
l nhit tuyt i ca ngun nhit (
o
K), U l h s chuyn nhit ton phn (J/s
m
2o
K), v A l din tch mt ct ca s chuyn nhit xut hin trong khi tit trng (m
2
).
+ Lm lnh tng m bng cch dng b khng ng nhit lin tc, nh cho nc lnh chy
qua nh ng lm lnh xon, phng trnh dng hm m:
120
Trong : T
Co
l nhit tuyt i ban u ca bn nhit (
o
K), m
c
l tc dng chy ca khi
cht lng lm ngui (kg/s).
- V gi tr ca k
d
nh l mt hm thi gian.
- Ly tch phn cc din tch di ng cong k
d
theo thi gian cho cc qu trnh lm lnh v
lm nng nh gi tng ng V
heat
v V
cool
. Nu s dng cc phng trnh l thuyt, th ly tch
phn phng trnh (9.5) sau khi thay th cc profile nhit thch hp. Sau , thi gian gi nng c
th c tnh ton t phng trnh:
V. Tit trng lin tc
Tit trng c tin hnh trong kiu lin tc hiu qu hn kiu tng m. Tit trng lin tc c
mt s u im sau:
- Lp k hoch sn xut n gin, cho php s dng ti a thit b v s gim thiu s chm
tr.
- Cung cp cc iu kin ti sn xut.
- C th hot ng nhit cao (140
o
C, chng hn 121
o
C trong tit trng tng m), v th
thi gian tit trng c th rt ngn (thi gian gi ch t 1-2 pht).
- Cn t hi nc bng cch thu hi nhit t mi trng c tit trng. Kt qu l n cng cn
t nc lm lnh.
- D dng hn trong t ng ha qu trnh, nh vy cng lao ng t hn.
Mt thit b tit trng lin tc bao gm ba b phn chnh: un nng, gi v lm lnh.
1. B phn un nng
Phng php un nng c th chia lm hai loi: phun hi nc trc tip v lm nng gin tip
trong cc thit b trao i nhit ng lng ng (shell-and-tube) hoc c khung a (plate-and-frame).
Lm nng trc tip hiu qu hn gin tip do khng c vt cn (barrier) gia mi trng v ngun
nhit. Dng c phun hi nc lm nng nhanh mi trng ti mt nhit tit trng ti a. V th, s
tit trng trong sut thi gian un nng l ng k.
i vi lm nng gin tip, b phn trao i nhit c khung a thng hiu qu hn loi truyn
nhit ng lng ng. Tuy nhin, b phn u b hn ch i vi cc p lc thp (thng di 20 atm)
do cng lc cu trc yu ca n so vi b phn sau. Loi c khung a cng thun li cho cc h
thng c nht cao.
S thay i nhit lin quan vi thi gian lu trung bnh (
heat
) khi mi trng i qua ngun
ng nhit c th c tnh gn ng nh sau (Deindoerfer v Humphrey 1959b):
121
Vi Wl khi lng mi trng trong h thng tit trng.
Trng hp un nng bng cch dng ngun nhit dng nc ngc c tc dng chy v
cng sut nhit tng ng, th ta c phng trnh sau:
2. B phn gi nng
Mi trng c un nng i qua b phn gi nhit bao gm mt ng di. B phn gi c
duy tr trong cc iu kin on nhit. Nu nhit mt trong b phn ny l khng ng k, th nhit
c th c tha nhn l hng s. Thi gian lu trung bnh trong b phn gi l:
Trong : L l chiu di b phn gi, l tc trung bnh.
T yu t Del c tnh nh sau:
Vi n
0
l s lng t bo thi im bt u ca b phn gi.
Nu mi trng trong b phn gi chy nh mt dng nt l tng (ideal plug flow), th thi
gian lu ca mi trng trong phn ny l ging vi tt c cc mi trng khc. V vy, mc tit
trng khng thay i. Tuy nhin, vic khng gi ng mc tiu do bn cht nht ca cht lng, s ma
st ca thnh ng, cc xoy nc bt thng ca cht lng chy gy ra s phn chia dng nt l
tng. Kt qu l profile tc c gi tr cc i gia ng ng v gi tr ti thiu vng ln cn
thnh ng.
S phn chia ca dng nt l tng do s trn ln quanh trc c th c m t bng m hnh
phn tn (Levenspiel 1972). Hnh 9.1 trnh by yu t vi sai vi dy dx trong ng gi. S cn bng
nguyn liu c bn cho cc t bo l lng trong mi trng l:
122
Hnh 9.1. Cc cn bng nguyn liu quanh b phn s cp trong ng gi.
trng thi n nh, gii hn tch ly bng khng. S i vo v i ra khi mi trng ca t
bo nh mt dng chy khi v mt iu kin khuch tn ( phn tn) quanh trc. S lng t bo
i vo tr cho nhng t bo ri i bng dng chy khi l:
Trong : C
n
l mt s lng t bo, S din tch mt ct ca ng.
Tng t vi s khuch tn phn t, dng chy hng trc x ca t bo l lng trong mi
trng do s phn tn quanh trc c th c biu din nh sau:
Trong : D l h s phn tn quanh trc c m t bng mc trn ngc (backmixing)
trong dng chy. Nu D bng 0, th s phn phi tc hng ti dng chy nt l tng. Nu D
bng , th dng chy trong ng s phi trn tt ging nh mt ci bnh c trn hon ton. i vi
dng chy xo trn hn lon, th h s phn tn tng quan nh l mt hm ca s Reynolds (Hnh
9.2).
S lng cc t bo i vo v i ra nh s phn tn quanh trc l:
123
S lng cc t bo b git khi tit trng l k
d
C
n
Sdx. V th, bng cch thay th phng trnh
(9.19), (9.21) vo trong phng trnh (9.18) v n gin ha, ta c:
i vi hng s D v , phng trnh (9.22) c th c bin i trong dng khng c th
nguyn:
Trong :
N
Pe
c bit nh l s Pclet. Khi N
Pe
= th n l dng nt l tng. Cc iu kin cho vic
gii phng trnh (9.23) l:
Gii phng trnh (9.23) ta c:
Trong :
3. B phn lm lnh
i vi b phn lm lnh, dng mt bung lm mt c kh nng loi nhit l rt hiu qu. Mt
k thut khc l a mi trng nng qua mt van m rng vo trong bung chn khng c xem
l qu trnh lm lnh nhanh (flash cooling). C hai k thut ny t tn thi gian, v th thi gian lm
124
lnh trong sut qu trnh tit trng c cho l khng ng k.
B phn trao i nhit ng lng ng v c khung a cng c th c dng lm lnh bng
cch dng bn ng nhit l:
Trong : T
C
l nhit tuyt i ca bn nhit.
Trng hp lm lnh bng cch dng bn nhit dng nc ngc c tc dng chy v kh
nng nhit bng nhau, th ta c phng trnh sau:
4. Tit trng khng kh
i vi ln men hiu kh (aerobic fermentations), th cn cung cp khng kh lin tc. Tc
sc kh c trng cho ln men hiu kh khong 0,5-1,0 vvm (th tch kh/th tch cht lng/pht). iu
ny i hi mt lng ln khng kh. V th, khng ch mi trng m khng kh cng phi v trng.
Tt c nhng k thut v trng dng cho mi trng cng c th p dng cho khng kh. Tuy nhin,
tit trng theo phng thc nhit khng thc t v mt kinh t v cng khng hiu qu do hiu sut
truyn nhit thp ca khng kh so vi cht lng. K thut tit trng c hiu qu nht cho khng kh l
phng php lc bng cch dng b lc mng (membrane filter) hoc b lc si (fibrous filter).
Nt bng, thng c dng nh l np y cho ng nghim hoc bnh tam gic ng dung
dch v trng, l mt v d tt loi vi sinh vt ra khi khng kh bng si lc. Mt b lc n gin
c lm bng cch nhi bng trong ct. Tuy nhin, vi cc b lc lm bng bng th s gim p ln
v s m t c th l iu kin thun li cho s nhim bn. V th, cc si thy tinh thch hp khi
lc mi trng do chng to ra mt s gim p thp hn v t c kh nng m t hoc chy. H
thng lc hin i bng si l cc ng hnh tr lm t cc vi si borosilicate lin kt, chng c bao
bc trong mng li gia c polypropylene. Loi thit k ny c th phn phi hn 3 m
3
/s khng kh
v trng s gim p sut 0,1 bar.
Vi cc b lc si, cc tiu th trn khng c thu thp bng cc c ch ng cht
(impaction), ngn chn (interception) v khuch tn (diffusion).
4.1. ng cht
Khi dng kh mang cc phn t chy quanh ng gp (collector), th cc phn t ny s theo
lung khng kh cho ti khi chng r ra quanh ng gp. Cc tiu th nh khi lng ca chng s c
ng lng (sc y ti) y tip tc chuyn ng hng ti ng hnh tr v chc thng dng
kh (Hnh 9.3). Hiu sut thu gom bng c ch ng cht (
imp)
theo qun tnh l mt hm ca s
Stokes v Reynolds nh sau:
Trong : N
St
l s Stokes, mt ,
p
mt cc phn t, d
p
ng knh phn t, D
c
ng
knh ng gp, v
0
tc cht lng ngc hng khng b xo trn, nht lu cht (nc v kh), C
f
l yu t hiu chnh Cunningham. Gi tr ca C
f
c th c c lng t s hiu chnh theo kinh
125
nghim c pht trin bi Davis (Strauss 1975):
Trong : l ng i t do trung bnh ca cc phn t kh da trn phng trnh Chapman-
Enskog:
Vi M
w
trng lng phn t ca cc phn t kh.
Hnh 9.3. Kiu lung kh quanh si hnh ng, cho thy hng i ca cc phn t c thu thp bi s ng cht
theo qun tnh.
Hiu sut
imp
c nh ngha l phn t nh tip cn vi ng gp ng cht.
Trong : l s Reynolds ca ng gp.
Mt tng quan khc c xut bi Friedlander (1967) l:
Nh vy, hiu sut tng ln vi vic tng ng knh phn t hoc tc dng kh.
4.2. Ngn chn
M hnh ng cht theo qun tnh tha nhn cc phn t c khi lng, v v th c qun tnh,
nhng khng c kch thc. Mt c ch ngn chn c xem nh l cc phn t c kch
thc, nhng khng c khi lng, v v th chng c th theo dng kh chuyn ng quanh ng
gp. Nu dng kh i qua gn b mt ca si, th cc phn t s tip xc vi si v b loi b
126
(Hnh 9.4). Hiu sut ngn chn (q
int
) ph thuc vo t l ca ng knh phn t vi ng knh
ca ng gp (k = d
p
/D
c
):
int
c pht trin bng cch dng phng trnh tc dng chy ca Langmuir (Strauss
1975). T l k c xem nh l thng s ngn cn. Hiu sut thu gom bng ngn chn tng ln cng
vi vic tng kch thc ca cc phn t.
4.3. Khuch tn
Cc phn t c ng knh nh hn khong 1 micron (m) biu l mt s chuyn ng Brown
c cng to ra s khuch tn. Nu dng chy cha cc phn t ny ti gn ng gp th
cc phn t ny s va trng ng gp v b loi b. Ngc vi hai c ch trc, hiu sut thu gom
bng khuch tn tng ln cng vi vic gim kch thc phn t hoc tc khng kh. Kch thc
c trng ca cc phn t c thu gom bng c ch ny l nh hn 0,5 m. Hiu sut thu gom
bng khuch tn (q
dif
) c th c lng bng mt phng trnh tng t phng trnh Langmuir nh
sau (Strauss 1975):
Trong : Z l thng s khuch tn, c nh ngha nh sau:
Vi D
Br
l s khuch tn do chuyn ng Brown.
Friedlander (1976) gi s tng quan sau:
Trong : N
Pe
l s Pclet, mt thng s khng c th nguyn quan trng trong l thuyt
khuch tn i lu. N c nh ngha nh sau:
127
Vi N
Sc
l s Schmidt, c nh ngha nh sau:
S khuch tn nh chuyn ng Brown cho cc phn t c kch thc nh hn micron
(submicron) c th c c lng t biu thc:
Trong : k l hng s Boltzmann (1,3805410
-23
J/
o
K).
4.4. C ch kt hp
Hiu sut thu gom tng s ca b lc si thu c t hiu qu phi hp ca ba c ch c
trc. Mt phng thc n gin phi hp hiu sut thu gom ca cc c ch khc nhau l b
sung chng cng vi nhau. Nhng iu ny gi l cc phn t c th c thu gom khng ch
mt ln. Mt hng tt hn l dng mi tng quan sau:
y l yu t ch cho php cc phn t khng c thu gom bng c ch ny th c thu
gom bng c ch khc. Thay th phng trnh (9.32), (9.34) v (9.35) vo phng trnh (9.41) s
cho kt qu trong mi tng quan i vi hiu sut thu gom bng cc c ch kt hp (combined
mechanism,
c
). Pasceri v Friedlander (1960) hiu chnh hiu sut thu gom kt hp nh sau:
Nh cp, vi vic tng tc dng kh b mt th
imp
v
int
tng trong khi
dif
gim. V
th, hiu sut thu gom phi hp thng gim ti mt im ti thiu v sau tng cng vi vic tng
tc dng kh b mt.
VI. Cc k hiu
A din tch mt ct ca s chuyn nhit xut hin trong khi tit trng, m
2
C
f
yu t hiu chnh Cunningham, khng c th nguyn
C
n
mt s lng t bo, s lng t bo/m
3
c nhit c trng ca mi trng, J/kg
o
K
D h s phn tn quanh trc
D
Br
s khuch tn do chuyn ng Brown, m
2
/s
D
c
ng knh ng gp, m
d
p
ng knh phn t, m
d
t
ng knh ng, m
128
E
d
nng lng hot ng cho s tiu dit t bo bng nhit trong phng trnh Arrhenius, J/kmol
H enthapy ca hi nc lin quan vi nhit ca mi trng cha nu chn, J/kg
J
n
lung t bo do s phn tn quanh trc, m
-2
s
-1
k hng s Boltzmann: 1,3805410
-23
J/
o
K hoc 1,3805410
-16
erg/
o
K
k
d
tc cht c trng, s
-1
hoc kg/m
3
/s
L chiu di ca b phn gi, m
M khi lng ban u ca mi trng trong ni tit trng m, kg
M
w
trng lng phn t ca cc phn t kh, kg/kmol
m
s
tc dng chy ca khi hi nc, kg/s
m
c
tc dng chy ca khi cht lng lm ngui, kg/s
N
Pe
s Pclet (L/D hoc
0
D
c
/D
Br
), khng th nguyn
N
Re
s Reynolds (d
t
u/
L
), khng th nguyn
s Reynolds ca ng gp (D
c
0
/), khng th nguyn
N
Sc
s Schmidt (/D
Br
), khng th nguyn
N
St
s Stokes (C
f
p
d
p
2
0
/18 Dc), khng th nguyn
n s t bo trong h thng
q tc truyn nhit, J/s
R hng s kh: 8,31410
3
J/kmol
o
K hoc 8,31410
7
erg/mol
o
K
S din tch mt ct ca ng, m
-2
T nhit tuyt i,
o
K
T
0
nhit tuyt i ban u ca mi trng,
o
K
T
C
nhit tuyt i ca bn nhit,
o
K
T
Co
nhit tuyt i ban u ca bn nhit,
o
K
T
H
nhit tuyt i ca ngun nhit,
o
K
t thi gian, s
U h s chuyn nhit ton phn, J/s m
2o
K
u tc , m/s
tc trung bnh, m/s
v tc cht lng trong phm vi khng gian trng ca b lc, m/s
v
0
tc cht lng ngc hng khng b xo trn, m/s
W khi lng mi trng trong h thng tit trng, kg
x khong cch nh hng-x, m
Z thng s khuch tn c nh ngha trong phng trnh (9.36), khng th nguyn
q hiu sut thu gom, khng th nguyn
k t l ca phn t v ng knh ng gp (d
p
/D
c
), khng th nguyn
ng i t do trung bnh ca cc phn t kh, m
nht lu cht (nc v kh), kg/m s
L
nht cht lng, kg/m s
129
mt , kg/m
3
p
mt cc phn t, kg/m
3
thi gian lu trung bnh, s
tiu chun thit k cho s tit trng, khng th nguyn
York, USA.
2
nd
st
Singapore.
nd
ed. Prentice
nd
130
Chng 10
Khuy trn v thng kh
I. M u
Mt trong nhng nhn t quan trng cn c lu khi thit k h ln men l kh nng
khuy trn thch hp cc thnh phn ca n. Cc vn chnh ca s khuy trn trong h ln men l
s phn tn ca cc bong bng kh, to huyn ph cc c th vi sinh vt (hoc t bo thc vt v
ng vt) v tng cng s chuyn nhit v chuyn khi trong mi trng.
Ni chung, hu ht cc cht dinh dng u c kh nng ha tan cao trong nc, do trong
thi gian ln men nu ch phn b u mi trng khi cc t bo tiu th cht dinh dng th s
khuy trn khng tht cn thit. Tuy nhin, trng hp oxygen ha tan th ngi ta li rt mong
mun c mt s khuy trn tt v kh nng ha tan ca n trong mi trng ln men l rt km, trong
khi yu cu oxygen cho s sinh trng ca cc vi sinh vt hiu kh (hoc t bo thc vt v ng vt)
li rt cao.
V d: khi oxygen c cung cp t khng kh, nng cc i c trng ca n trong dung
dch nc l t 6-8 mg/L. Nhu cu oxygen ca t bo, mc d c th ph thuc rt ln vo loi t
bo, thng l khong 1 g/L gi. Ngay c khi mi trng ln men c bo ha hon ton vi
oxygen, th oxygen ha tan s c c th tiu th t hn mt cht nu nh n khng c cung cp
lin tc.
quy m phng th nghim, s khuy trn c to ra nh my lc (shaker) l thch hp
nui cy t bo trong cc bnh thy tinh hoc ng nghim. Cc my lc vng hoc lc ngang to ra
mt s phi trn nh v trao i kh b mt rt hiu qu. Trng hp ln men quy m pilot hoc
quy m sn xut, s khuy trn thng c to ra bng cch khuy c hc c hoc khng c sc
kh. Ph bin nht l s dng loi cnh khuy (impeller) to ra dng chy ta trn vi su cnh khuy
mng c gn vo trong mt a, gi l turbine a c cnh khuy mng (flat-blade disk turbine)
hoc Rushton turbine (Hnh 10.1 v 10.2).
Cc cnh khuy dng ta trn (cc mi cho v turbine) to ra dng chy ta trn t cnh ca
turbine hng ti vch ngn ca bnh nui (vessel), trong dng chy chia ra theo hai hng: mt
hng i ln dc theo vch, ri i tr vo vng trung tm theo b mt cht lng, v i xung vng
cnh khuy dc theo trc khuy. Mt hng khc i xung dc theo vch v y, sau i vo vng
cnh khuy.
131
Hnh 10.1. S Rushton turbine.
Hnh 10.2. S bnh ln men c cnh khuy.
Mt khc, cc cnh khuy dng chy theo trc (cnh qut v cc mi cho khng bng phng)
to ra dng chy i xung y bnh, sau i ln dc theo vch v quay xung vng trung tm ca
cnh khuy. V th, cc turbine a c cnh khuy mng c u im hn ch on mch (short-
circuiting) ca kh dc theo trc truyn ng (drive shaft) nh s nn kh, a vo t pha di, dc
theo hng vo trong vi thot (discharge jet).
1. Con ng chuyn khi
Con ng ca cc cht kh t mt bong bng vo mt c quan t trong t bo c th c
phn chia trong mt vi bc nh sau:
a. Chuyn t kh nn (bulk gas) trong mt bong bng ti mt lp kh tng i nguyn cht
(relatively unmixed gas layer).
b. Khuch tn thng qua lp kh tng i nguyn cht.
c. Khuch tn thng qua lp cht lng tng i nguyn cht quanh bong bng.
d. Chuyn t lp cht lng tng i nguyn cht ti khi cht lng nn (bulk liquid).
132
e. Chuyn t khi cht lng nn ti mt lp cht lng tng i nguyn cht quanh mt t bo.
f. Khuch tn thng qua lp cht lng tng i nguyn cht.
g. Khuch tn t b mt ca mt t bo ti mt c quan t m trong oxygen b tiu hao.
Cc bc c v e l chm nht. S khuy trn v thng kh s tng cng tc chuyn khi
trong cc bc ny v tng din tch tng tc gia kh v cht lng.
Chng ny trnh by mt s mi tng quan khc nhau i vi s chuyn khi lng-kh, din
tch tng tc, kch thc bong bng, s tc nghn kh, s tiu th cng sut khuy v h s th tch
chuyn khi, l nhng cng c quan trng thit k v hot ng cc h ln men. S ti hn i
vi vic tng quy m sn xut v s khuy trn nhy cm vi lc trt cng c trnh by. u
tin, chng ta tm hiu cc khi nim c bn ca s chuyn khi m quan trng l hiu c s
chuyn khi lng-kh trong h ln men.
II. Cc khi nim c bn v chuyn khi
1. S khuch tn phn t trong cht lng
Khi nng ca mt thnh phn bin thin t mt im ny n mt im khc, th thnh phn
ny c xu hng chy theo hng lm gim nhng s khc bit cc b trong nng .
Dng phn t ca cu t A lin quan vi vn tc phn t trung bnh ca tt c cu t J
A
l t l
vi gradient nng dC
A
/dz khi:
Phng trnh (10.1) l nh lut th nht ca Fick c vit cho chiu z. K hiu D
AB
trong
phng trnh (10.1) biu din kh nng khuch tn cu t A vo B, tc l gi tr o chuyn ng
khuch tn ca n.
Dng phn t ca A lin quan vi ta c nh (stationary coordinate) N
A
l bng:
Trong : C l nng tng s ca cc cu t A v B, v N
B
l dng phn t ca B lin quan
vi ta c nh. i vi dung dch long ca cu t A th:
1.1. S khuch tn
L thuyt ng hc cht lng khng c nhiu u im so vi cht kh. V th, mi tng quan
cho kh nng khuch tn trong cht lng l khng r rt nh trong cc cht kh. Trong s nhng mi
tng quan c cp, th tng quan Wilke-Chang (1955) c s dng rng ri nht cho
cc dung dch long ca cc cht khng in phn:
133
Khi cc dung mi l nc, Skelland (1974) gii thiu s dng mi tng quan c pht
trin bi Othmer v Thakar (1953):
Hai mi tng quan cho trc khng ph hp v th nguyn, v th cc phng trnh s dng
n v SI nh sau:
kh nng khuch tn ca A trong B, trong mt dung dch rt long, m
2
/s
M
B
khi lng phn t ca cu t B, kg/kmol
T nhit ,
o
K
tc ha tan, kg/m/s
V
bA
th tch phn t ha tan im si bnh thng, m
3
/kmol (0,0256 m
3
/kmol cho oxygen)
yu t kt hp i vi dung mi: 2,26 i vi nc; 1,9 i vi methanol; 1,5 i vi ethanol;
1,0 cc dung mi khng kt hp nh benzene v ethyl ether.
2. H s chuyn khi
Dng chy khi (mass flux), tc chuyn khi q
G
trn n v din tch, t l vi s chnh lch
nng . Nu mt cht ha tan chuyn t pha kh vo pha lng, th dng chy khi ca n t pha kh
ti b mt chung N
G
l:
Trong : C
G
v C
Go
l nng kh mt bin (gas-side concentration) tng ng phn chnh
v vng phn gii (b mt chung) (Hnh 10.3). k
G
l h s chuyn khi ring r cho cho pha kh v A
l din tch vng phn gii.
Tng t, dng chy khi ca pha lng mt bin (liquid-side phase) N
L
l:
Trong : k
L
l h s chuyn khi ring r i vi pha lng, q
L
l tc hp th kh.
Do lng cht ha tan c chuyn t pha kh ti vng phn gii phi bng lng cht ha
tan t vng phn gii ti pha lng, nn:
134
Hnh 10.3. Profile nng gn vng phn gii kh-lng v mt ng cong trng thi cn
bng.
Thay phng trnh (10.6) v (10.7) vo trong phng trnh (10.8) ta c:
Phng trnh (10.9) c dc ca ng cong kt ni (C
L
, C
G
) v (C
Li
, C
Gi
) nh trnh by
hnh 10.3.
S dng phng trnh (10.6) hoc (10.7) xc nh h s chuyn khi gp nhiu kh khn do
chng ta khng th o nng ca vng phn gii C
Li
hoc C
Gi
. V th, thun li cho vic xc nh
ton b h s chuyn khi c th dng phng trnh sau:
Trong : l nng kh mt bin s cn bng vi nng kh hin din trong pha lng.
Tng t, l nng cht lng mt bin s cn bng vi nng cht lng hin din trong pha
kh. Nhng thng s ny d dng c t ng cong trng thi cn bng trnh by hnh 10.4. K
G
v K
L
c nh ngha li l cc h s chuyn khi ton b tng ng cho cc mt bin ca kh v
lng.
135
Hnh 10.4. ng cong trng thi cn bng gii thch ngha ca v
3. C ch ca chuyn khi
Mt vi c ch khc nhau c a ra cung cp c s cho l thuyt chuyn khi gian k
(interphase). Ba c ch tt nht c bit l: thuyt hai mng (two-film), thuyt thm qua
(penetration) v thuyt phc hi b mt (surface renewal).
3.1. Thuyt hai mng
Thuyt ny gi thit rng c tnh kh di chuyn hon ton c bao gm trong hai mng gi
bn ny hoc bn kia vng phn gii, trong s di chuyn xy ra nh khuch tn phn t. M hnh
ny dn n kt lun rng h s chuyn khi k
L
t l vi kh nng khuch tn D
AB
v t l nghch vi
dy ca mng z
f
nh sau:
3.2. Thuyt thm qua
Thuyt ny tha nhn rng xoy nc hn lon i t phn chnh ca pha ti vng phn gii,
chng duy tr mt thi gian phi khng i t
e
. Cht ha tan c tha nhn l thm vo trong
xoy nc c sn vng phn gii bi mt qu trnh khuch tn phn t trng thi khng n nh.
M hnh ny d bo rng h s chuyn khi t l trc tip vi cn bc hai ca kh nng khuch tn
phn t:
Trong : l p sut tuyt i.
3.3. Thuyt phc hi b mt
Thuyt ny xut rng c mt gii hn thi gian v tn cho cc nhn t b mt v hm phn
b tui b mt (surface age). L thuyt ny d bo mt ln na h s chuyn khi t l vi cn bc
hai ca kh nng khuch tn phn t:
Trong : s l tc phn on ca s phc hi b mt.
Tt c l thuyt ni trn i hi phi bit mt s thng s cha xc nh nh: dy mng c
tht z
f
, thi gian phi t
e
hoc tc phn on ca s phc hi b mt s. Ni chung, nhng tnh cht
ny t c bit n, n mc c ba l thuyt l khng hon chnh. Tuy nhin, nhng l thuyt ny
gip chng ta hnh dung c ch chuyn khi vng phn gii v cng bit s ph thuc hm m ca
kh nng khuch tn phn t trn h s chuyn khi.
III. Xc nh vng phn gii
tnh ton tc hp th kh q
L
ca phng trnh 10.7, chng ta cn bit din tch vng phn
gii kh-lng l thng s c th o c bng cch ng dng mt vi k thut nh l chp nh,
truyn sng v quang ph laser.
136
Din tch vng phn gii (a) trn mt n v th tch c th c tnh ton t ng knh trung
bnh Sauter D
32
(m) v phn on th tch ca pha kh H, nh sau:
ng knh trung bnh Sauter, mt gi tr trung bnh ca b mt th tch, c th c tnh ton
bng cch o cc kch thc git trc tip t cc hnh nh ca phn tn theo phng trnh sau:
Xc nh kch thc cc git bng hnh nh l phng php d lm trong s nhiu k thut xc
nh do n khng i hi s nh c trc (calibration). Tuy nhin, chp mt bc nh r rng c
th l rt kh khn v c cc bc nh ny l mt cng vic n iu t nht, tn nhiu thi gian.
Cc bc nh c th chp thng qua chn hoc thnh bn ca bnh ln men. loi b tnh trng
khng r rng do b mt b cong ca thnh bnh, bnh ln men c th c cho ngp chm trong mt
ci thng hnh ch nht hoc mt ti nc c gn trn thnh. Nhc im ca phng thc ny
l vic o kch thc git b hn ch i vi nhng vng gn thnh bnh, l ni khng th i din
cho ton b s phn tn trong h ln men.
S phn b kch thc git c th c o gin tip bng cch dng k thut truyn sng. Khi
mt chm sng i qua mt vng c phn tn kh-lng, th nh sng c ta ra bi cc bong bng
kh. Ngi ta nhn thy rng th ca t l dp tt (hm thun nghch ca truyn sng 1/T) da
theo din tch vng phn gii trn mt n v th tch ca phn tn a, to ra mt ng thng,
nh sau:
V l thuyt, m
1
l phn t n v, cn m
2
l mt hng s c lp ca s phn b kch thc
git vi iu kin l tt c cc bong bng kh gn nh hnh cu.
K thut truyn sng c s dng thng xuyn nht cho vic xc nh kch thc trung bnh
ca bong bng kh trong s phn tn kh-lng. K thut ny c mt s u im nh o nhanh v hot
ng trc tuyn.
IV. Tc nghn kh
Tc nghn kh l mt trong nhng thng s quan trng nht m t thy ng hc ca h ln
men. Tc nghn kh ty thuc ch yu vo vn tc b mt ca kh v s tiu th cng sut, v
thng l rt nhy cm vi cc tnh cht vt l ca cht lng. Tc nghn kh c th c xc nh d
dng bng cch o mc cht lng c thng kh trong sut thi gian hot ng (Z
F
) v mc
cht lng sch (Z
L
). Nh vy, vic tc nghn kh trung bnh tiu phn H c tnh theo cng thc
sau:
137
1. Phun kh (sparging) bng khuy trn khng c hc
i vi mt h thng hai pha, trong pha lin tc duy tr ch thch hp ca n, th s tc
nghn kh s lin quan vi vn tc kh b mt V
s
v vn tc tng bong bng kh V
t
:
Akita v Yoshida (1973) t mi tng quan tc nghn kh i vi vic hp th oxygen
cc dung dch nc khc nhau trong ct bong bng nh sau:
Trong : g l gia tc do trng lc, D
c
l ng knh ct bong bng, v o l p lc b mt, v
c
l
th tch cht lng ca pha lin tc, v
c
l mt ca pha lin tc.
2. Phun kh bng khuy trn c hc
Calderbank (1958) t mi tng quan tc nghn kh i vi vic phn tn lng-kh c
khuy trn bng turbine dng a c cnh khuy mng nh sau:
Trong 2,610
-4
c n v (m) v V
t
= 0,265 m/s khi kch thc bong bng trong khong 2-5
mm ng knh, P
m
l cng sut b tiu hao do cnh khuy trong s phn tn cht lng c thng
kh, v v l th tch cht lng.
Trng hp vn tc kh b mt cao (V
s
> 0,02 m/s), thay P
m
v V
t
ca phng trnh (10.20)
bng cch a vo cng sut hiu qu P
e
v V
t
+ V
s
tng ng.
V. Xc nh tc hp th oxygen
c lng cc thng s thit k a oxygen vo h ln men, chng ta c th s dng cc
mi tng quan c trnh by trong cc phn trc y, ng dng cho mt phm vi rng cc h
thng kh-lng b sung vo h thng nc-khng kh. Tuy nhin, phng thc tnh ton ny di dng
v cc gi tr d bo t nhng mi tng quan ny c th thay i rt nhiu.
Cng c trng hp chng ta cng khng th tm thy cc mi tng quan thch hp p
dng cho kiu v th tch ca h ln men mun s dng. Trong nhng trng hp nh th, chng ta
c th t o tc chuyn oxygen hoc dng cc mi tng quan da trn nhng th nghim ny.
Tc hp th oxygen trn mt n v th tch q
a
/v c th c c lng nh phng trnh:
138
Do oxygen l loi kh t ha tan, nn h s chuyn khi ton b K
L
bng h s chuyn khi
ring r k
L
. Mc tiu ca chng ta trong thit k h ln men l cc i ha tc chuyn oxygen vi
s tiu th cng sut ti thiu cn thit khuy trn cht lng v cng gim thiu lu tc kh.
cc i ha tc hp th oxygen, chng ta phi cc i ha k
L
, a, . Tuy nhin, s khc bit
nng c hn ch hon ton bi v gi tr c gii hn bi kh nng ha tan cc i rt
thp ca n. V th, cc thng s quan tm chnh trong thit k l h s chuyn khi v din tch vng
phn gii.
Bng 10.1 lit k kh nng ha tan ca oxygen 1 atm trong nc di cc nhit khc
nhau. Cc gi tr thu c l nng oxygen cc i trong nc khi n trong s cn bng vi
oxygen tinh khit. Kh nng ha tan ny gim khi c b sung acid hoc mui nh trnh by bng
10.2.
Bng 10.1. Kh nng ha tan oxygen trong nc 1 atm.
Bng 10.2. Kh nng ha tan ca oxygen trong dung dch mui hoc acid 25
o
C.
139
Thng thng, chng ta s dng khng kh cung cp nhu cu oxygen cho h ln men. Nng
cc i ca oxgen trong nc trong s cn bng vi khng kh p sut kh quyn l
khong mt phn mi lm ca kh nng ha tan c lit k, theo nh lut Henry:
Trong : l p sut tng phn (partial pressure) ca oxygen v (T) l hng s oxygen
ca nh lut Henry nhit T. Gi tr ca hng s nh lut Henry c th thu c t cc kh nng
ha tan c lit k bng 10.2. V d: 25
o
C, l 1,26 mmol/L v l 1 atm do n l oxygen
tinh khit. Bng cch thay th cc gi tr ny vo trong phng trnh (10.22), chng ta thu c
(T) l 0,793 atm L/mmol. V th, nng oxygen cn bng cho s tip xc nc-kh 25
o
C s l:
Theo iu kin l tng, tc chuyn oxygen phi c o trong h ln men cha mi
trng dinh dng v t bo trong sut qu trnh ln men thc t. Tuy nhin, iu ny kh tin hnh
do bn cht phc tp ca mi trng v s thay i lu bin hc (rheology) trong sut qu trnh sinh
trng ca t bo. Phng thc chung l s dng mt h thng tng hp xp x nh cc iu kin
ca qu trnh ln men.
1. Phng php oxy ha sodium sulfite
Phng php oxy ha sodium sulfite da trn nguyn tc oxy ha sodium sulfite thnh sodium
sulfate vi s c mt ca cht xc tc (Cu
2+
hoc Co
2+
) nh sau:
Phn ng ny c cc c im sau, p ng cho vic o tc chuyn oxygen:
- Tc phn ng c lp vi nng ca sodium sulfite trong khong 0,04 n 1 N.
- Tc phn ng nhanh hn nhiu so vi tc chuyn oxygen. V th tc oxy ha c
iu chnh ch bi tc chuyn khi.
o tc chuyn oxygen trong h ln men, lm y h ln men bng dung dch sodium
sulfite 1 N cha t nht 0,003 M Cu
2+
. M b phn sc v bt u bm gi khi bong bng kh u tin
xut hin trong h ln men t b phn sc kh. Cho php s oxy ha tip tc t 4-20 pht, sau
dng dng kh, b phn khuy trn v timer cng mt thi gian, ri ly mu. Trn mi mu vi mt
lng d thuc th iodine chun bng pipette sch. Chun bng dung dch sodium thiosulfate
chun (Na
2
S
2
O
3
) ti im cui ca cht ch th tinh bt. Mt khi oxygen a vo c o, th h s
th tch chuyn khi k
L
a c th c tnh ton bng cch dng phng trnh (10.21), trong C
L
l
bng 0 v l nng oxygen trng thi cn bng.
K thut oxy ha sodium sulfite c hn ch ca n v trong thc t dung dch khng th xp x
vi cc tnh cht vt l v ha hc ca mi trng ln men. Thm mt vn na l k thut ny i
hi cc nng ion cao (1-2 mol/L), s c mt ca cc ion ny c th nh hng n din tch vng
phn gii v, trong mt mc thp hn, n h s chuyn khi. Tuy nhin, k thut ny hu ch khi
140
so snh vi hiu sut ca cc h ln men v nghin cu nh hng ca s pht trin quy m sn
phm v cc iu kin hot ng.
2. K thut tch khng kh
K thut ny gim st s thay i nng oxygen trong mt cht lng giu oxygen c kh
oxygen bng cch cho nitrogen i qua n. in cc ca php o cc ph (polarography) thng
c dng o nng . Cn bng khi trong mt bnh nui cho ra:
Ly tch phn phng trnh cho gia t
1
v t
2
cho kt qu:
T phng trnh trn k
L
a c th c tnh ton da trn cc gi tr o c C
L
(t
1
) v C
L
(t
2
).
3. Xc nh trc tip
Trong k thut ny, chng ta o trc tip hm lng oxygen ca dng kh i vo v i ra khi
h ln men bng cch s dng thit b phn tch oxygen khng kh. S hp th oxygen c th c
tnh ton nh sau:
Trong : Q l tc dng kh.
Mt khi oxygen hp th c o, th k
L
a c th c tnh ton bng cch dng phng trnh
(10.21), trong C
L
l nng oxygen ca cht lng trong h ln men v l nng ca oxygen
s trng thi cn bng vi dng kh. Nng oxygen ca cht lng trong h ln men c th c
o bng mt b cm bin oxygen trc tuyn (on-line oxygen sensor).
Nu th tch ca h ln men l kh nh (< 50 L), th s bin thin ca trong h ln
men cng kh nh. Tuy nhin, nu kch thc ca h ln men l rt ln, th s bin thin c th c
ngha. Trong trng hp ny, gi tr trung bnh logarithm ca dng kh chy vo v chy ra
c th c s dng, khi :
4. K thut ng lc hc
Bng cch s dng k thut ng lc hc chng ta c th c lng gi tr k
L
a i vi s
141
chuyn oxygen trong sut qu trnh ln men thc t vi cc t bo v mi trng nui cy thc s.
K thut ny da trn nguyn tc ca s cn bng oxygen ca nguyn liu trong mt h ln men m
hiu kh trong lc cc t bo ang hot ng sinh trng khi:
Trong : l tc ca h hp t bo (g O
2
/g t bo gi).
Trong khi nng oxygen ha tan ca h ln men l n nh, nu t nhin chng ta ngt s
cung cp khng kh, th nng ca oxygen s b gim (Hnh 10.5) vi tc nh sau:
V k
L
a trong phng trnh (10.28) l bng 0. V th, bng cch o dc ca ng cong C
L
theo t, chng ta c th c lng C
X
. Nu chng ta m dng kh thm mt ln na, th nng
oxygen ha tan s c tng ln theo phng trnh (10.28), phng trnh ny c th c sp xp
li cho mt mi quan h tuyn tnh nh sau:
Hnh 10.5. K thut ng hc cho vic xc nh k
L
a.
VI. Cc k hiu
A din tch vng phn gii, m
2
a din tch vng phn gii kh-lng trn mt n v th tch ca s phn tn cho cc s Reynolds ca
cnh khuy thp, m
-1
C nng , kmol/m
3
D
AB
kh nng khuch tn ca cu t A vo B, tc l gi tr o ca chuyn ng khuch tn, m
2
/s
142
kh nng khuch tn ca cu t A trong mt dung dch B rt long m
2
/s
J
A
dng phn t ca cu t A lin quan vi vn tc phn t trung bnh ca tt c cu t, kmol/m
2
s
K h s chuyn khi ton phn, m/s
k h s chuyn khi ring r, m/s
k
L
a h s th tch chuyn khi
g gia tc do trng lc, m/s
2
H tiu phn th tch (phn on) ca pha kh trong s phn tn, khng c th nguyn
N
A
, N
B
dng khi ca A v B lin quan vi ta c nh, kmol/m
2
s
N
G
, N
L
dng khi t pha kh ti pha lng v t pha lng n pha kh, tng ng, kmol/m
2
s
P p sut tng s, N/m
2
P
e
cng sut hiu qu c a vo nh phun kh v khuy c hc, W
P
m
cng sut b tiu hao do cnh khuy trong s phn tn cht lng c thng kh, W
q tc chuyn khi, kmol/s
Q tc dng kh, m
3
/s
s tc phn on ca s phc hi b mt, s
-1
t
e
thi gian phi cho l thuyt thm qua, s
V
s
vn tc kh b mt, m/s
V
t
vn tc tng bong bng kh, m/s
v th tch cht lng, m
3
v
c
th tch cht lng ca pha lin tc
Z
F
, Z
L
mc cht lng c sc kh trong sut thi gian hot ng, m
z
f
dy ca mng trong l thuyt hai mng, m
nht, kg/m s
t p sut tuyt i, N/m
2
mt , kg/m
3
c
mt ca pha lin tc
o p lc b mt kg/s
2
York, USA.
2
nd
st
Singapore.
nd
ed. Prentice
143
nd
144
Ph lc
Mt s thut ng c bn
Agrobacterium tumefaciens. L loi vi khun t gy bnh cho thc vt hai l mm c s
dng nh cc vector t nhin mang cc gen ngoi lai (foreign gene) vo m v t bo thc vt. A.
tumefaciens c cha mt plasmid ln kch thc khong 200 kb gi l Ti-plasmid (tumor inducing
plasmid) chnh l tc nhn truyn bnh cho cy. Khi cy b nhim A. tumefaciens qua cc vt thng,
biu hin bnh r nht l cc khi u c hnh thnh ngay ch ly nhim. S hnh thnh khi u sau
c th tip tc m khng cn thit phi c s hin din ca vi khun. Kh nng ny c c do A.
tumefaciens chuyn mt on DNA ca Ti-plasmid (T-DNA), c cha cc gen sn xut ra auxin v
cytokinin, xm nhp vo h gen (genome) ca cy b bnh.
Agrobacterium rhizogenes. Cng l mt loi vi khun t gy bnh cho thc vt hai l mm.
C ch ly nhim ca A. rhizogenes i vi cy cng tng t nh A. tumefaciens, nhng trong
vng T-DNA ca A. rhizogenes ch c gen sn xut ra auxin, v th s thay i hnh thi chnh ca
thc vt l chng to ra rt nhiu r t (hairy roots) khi b nhim bnh.
Amino acid. L mt phn t nh c cha mt nhm carboxyl (-COOH) v mt nhm amine (-
NH
3
) cng ni vi mt nguyn t carbon. Amino acid l n v c s ca protein.
Apoptosis. Cht theo chng trnh l mt hnh thc cht t nhin ca t bo theo mt chng
trnh c kch hot ni ti. c im ca apoptosis l s phn hy nhn t bo v ngng t nhn,
trong cc mnh nhn c bao bc bi cc mng cha c t bo cht, sau c cc i thc
bo tiu ha
Bt ng t bo (cell immobilization). Cc k thut gn t bo vi cc phn t ln hoc trn
cc b mt c phn tch d dng khi dng sn phm. Phng php ny m bo hot ng lin tc
ca h ln men (fermenter/bioreactor) khng b nguy c ra tri t bo. S bt ng cng c th cung
cp cc iu kin c li cho s phn ha t bo v s truyn t thng tin gia cc t bo, bng cch y
thc y sn phm c sn lng cc cht trao i th cp cao. Mt s phng php bt ng t bo
thng c s dng l: gn ln b mt, to th xp, s dng bao vi th v t kt khi.
Bin d dng soma (somaclonal variation). Hin tng bin d di truyn xut hin cc t bo
soma khng phn ha (undifferentiation), cc protoplast phn lp, cc callus v cc m nui cy in
vitro. Nguyn nhn ca bin d ch yu l do nhng thay i v s lng v cu trc ca nhim sc
th.
Bin i hu dch m (post-translational modification). L nhng bin i sau qu trnh dch
m, thay i cc lin kt ha tr xy ra trong chui polypeptide, sau khi chui polypeptide tch khi
ribosome v trc khi tr thnh protein hot ng thc s.
Bin np (transformation). Qu trnh a vo v dung np mt cch chc chn DNA ngoi lai
trong t bo thc vt bt lun bng cch g c c mt bin i di truyn th u c coi l bin
np thnh cng.
Bin np gen gin tip qua Agrobacterium tumefaciens (Agrobacterium tumefaciens-
mediate transformation). K thut ny c thit k da theo phng thc gy bnh cy hai l
mm ca vi khun A. tumefaciens chuyn DNA ngoi lai vo m thc vt bng cch ng nui
cy (co-cultivation) vi khun ti t hp vi m nui cy ca cy hai l mm hoc cy mt l mm.
Bin np gen bng vi n (microprojectile). L k thut chuyn gen nh sng bn gen (gene
gun, bombardement). Nguyn tc ca phng php ny l s dng cc vin n vng hoc tungsten c
145
kch thc hin vi (1-1,5 m). Vi n c trn vi DNA theo mt t l thch hp cng vi cc cht
ph gia v sau khi kt ta DNA bao quanh vi n, hn hp c lm kh trn trn mt a kim loi
mng kch thc 0,5-0,8 cm. a kim loi c gn vo u mt vin n ln (macroprojectile) lm
bng nha hoc bng nn hay cc vt liu nh va kht vi nng sng. Khi bn, p sut hi y vin
n ln i vi tc cao. Ra n u nng, mt li thp mn cn vin n ln li, nhng cc vi n
vn tip tc qu o vi gia tc ln n ch v xuyn vo t bo. Mt t l nht nh DNA ngoi lai
hp nht vi DNA t bo v biu hin, thc hin qu trnh bin np gen.
Bin np gen bng vi tim (microinjection). L k thut s dng ph bin trong cng ngh t
bo ng vt (animal cell biotechnology). Trn hin vi trng, DNA plasmid c th c tim vo
protoplast v thc hin bin np gen thnh cng kh nhiu i tng thc vt. Tuy nhin, k thut
ny hin nay t c cc phng th nghim s dng, v thao tc vi tim di knh hin vi i hi thit
b vi thao tc (micromanipulator) cc nhy, thit b ko v mi kim tim t cc ng thy tinh (puller)
rt t tin. Ngoi ra, n cn i hi k nng thao tc v s kin nhn cao ca k thut vin.
Bin np gen nh xung in (electroporation). Thit b in xung (electroporator) l thit b
c kh nng to ra cc xung in trong thi gian rt ngn (5-6 phn nghn giy) v in th (pulse
strenght) chnh xc (500 V/cm) vi thi gian tt dn (decay time) 20 ms. Protoplast c t gia hai
tm kim loi cch nhau t 1-4 mm trong mt cuvette bng nha. in th cao, xung in to cc l
tm thi (c 30 nm) trn mng protoplast v DNA bn ngoi c th xm nhp vo cht nguyn sinh.
Bin np gen nh siu m (ultrasonic). Sau khi tch, protoplast c x l nh bng siu m
c s hin din ca DNA ngoi lai. Sng siu m s gip DNA i vo t bo v biu hin.
Bin np gen nh silicon carbide. Tinh th silicon carbide c cng rt cao, khi lc vi t bo
chng c tc dng nh cc mi kim nh m thng thnh t bo gip DNA ngoi lai xm nhp vo
bn trong t bo.
Biu hin gen (gene expression). Qu trnh phin m v dch m to ra sn phm protein
ca gen.
Cm ng (induction). Hormone gy to mt loi cu trc, b phn hay mt qu trnh no
trong iu kin in vitro.
Cp base (base pair, bp). Hai nucleotide hai chui khc nhau ca trn mt phn t DNA
mch kp b sung vi nhau bi nhng lin kt hydrogen: A-T hoc G-C v l n v o chiu di ca
mt phn t DNA.
Cu disulfide (disulfide bridge). Mt lin kt ng ha tr to thnh gia hai chui polypeptide
qua trung gian ca mt gc cystine.
Cy chuyn (passage hoc subculture). Chuyn t bo, m hay mu vt nui cy sang bnh
nui c cha mi trng mi pha kt hp vi tch nh hoc lm long mt nhn s lng.
cDNA (complementary DNA). Mt phn t DNA si n b sung cho mt phn t mRNA,
c tng hp t khun mu mRNA ny nh enzyme phin m ngc (reverse transcriptase). Sau
mRNA trong th lai mRNA-DNA b phn hy bng enzyme RNase H, cn si DNA s c dng
lm khun mu tng hp nn mt si DNA khc nh enzyme DNA polymerase. Hai si DNA ny
s bt cp vi nhau tr thnh phn t cDNA si i.
Cht trao i th cp (secondary metabolites). Dng nh y l sn phm ca cc phn
ng ha hc ca thc vt vi mi trng chung quanh, l s thch nghi vi stress ca mi trng
hoc l s bo v ha hc chng li vi sinh vt v ng vt. Cc cht ny c sn xut vi mt
lng rt nh (dng vt) trong thc vt v khng c chc nng trao i cht r rng.
Chng t bo (cell strain). Chng t bo gm nhng t bo c c im ring bit c chn t
nui cy khi sinh hay dng t bo c trc. Thng phi nu r ngun gc ca chng t bo trong
cc cng b khoa hc nht l khi cc chng c ngun gc t cc phng th nghim khc.
146
Chng ph (substrain). c tch v nhn t mt nhm t bo ca mt chng vi nhng tnh
trng m chng b m khng c.
Cosmid. L cc vector c bit c xy dng bi plasmid v u cos ca bacteriophage ,
dng to dng cc on DNA c kch thc ln (trn 45 kb) ca eukaryote. Cc thnh phn c
bn ca cc vector cosmid bao gm: mt marker khng khng sinh v mt trnh t khi u sao chp
ca plasmid (ori), on DNA mang u kt dnh (cos) ca phage .
Cng ngh DNA ti t hp (DNA recombinant technology). H thng cc phng php
phng th nghim cho php ct on DNA t mt sinh vt ghp ni vo DNA ca mt sinh vt
khc to ra phn t DNA ti t hp. Phn t ny c a vo cc sinh vt khc nhau to ra
nhng ging chng vi sinh vt, thc vt v ng vt mi, p ng nhu cu ngy cng cao ca sn
xut v i sng con ngi. Cng ngh ny c ng dng rng ri trong y hc, dc hc, nng
nghip v nhiu ngnh cng nghip.
DNA (deoxyribonucleic acid). Gm hai chui polynucleotide xon li vi nhau to nn chui
xon kp, c mt trnh t c trng ca cc deoxyribonucleic mang thng tin di truyn cho tt c cc
t bo v vi virus cha DNA.
DNA polymerase. Enzyme xc tc cho s tng hp si DNA mi t nhng
deoxyribonucleoside 5-triphosphate trn c s mt khun mu DNA.
DNA replicase. Ton b phc hp enzyme v protein c hiu cn thit cho s ti bn DNA
(DNA replication).
DNA siu xon (DNA supercoiling). DNA xon li trn bn thn n, thng l kt qu ca s
gp khc, m xon hoc xon li ca chui xon kp DNA.
Dch m (translation). Qu trnh tng hp phn t protein t khun mu mRNA.
Dng (clone). Tp hp cc phn t, t bo hoc c th ging ht nhau cng bt ngun t mt
phn t, t bo hay c th ban u.
Dng giao t (gametoclone). Nhng thc vt c to ra t giao t, bo t gim nhim hoc
th giao t.
Dng t bo (cell line). Khi nim ch s nui cy ca nhng t bo c ngun gc chung t
ln cy chuyn u tin.
Dung hp t bo (cell fusion). K thut lm cho hai hay nhiu t bo dung hp vi nhau thnh
mt t bo.
Dung hp protoplast (protoplast fusion). K thut lm cho hai hay nhiu t bo trn (t bo
thc vt ph b thnh cellulose) dung hp vi nhau thnh mt t bo.
in di (electrophoresis). in di l k thut phn chia cc phn t nh protein hoc cc on
nucleic acid trn c s khi lng v in tch thc ca chng bng s dch chuyn khc nhau thng
qua giy, hoc thng qua gel trong in trng. Ni cch khc, in di l s dch chuyn trong in
trng ca nhng phn t tch in trong dung dch. K thut in di trn gi rn (agarose gel v
polyacrylamide) c s dng rng ri cho DNA, RNA v protein.
hp th nh sng (absorbency). Hin tng mt chm nh sng khi i qua mt mi
trng c th b gim v cng do hp th v do hiu ng tn x.
ng hc enzyme (enzyme kinetics). L vn tc phn ng enzyme c biu din bng
lng c cht b chuyn ha (mol) hoc lng sn phm c to thnh (mol) trong mt n v thi
gian (giy).
ng hc t bo (cell kinetics). L kt qu ca h thng cc phn ng ha sinh v cc qu
trnh vn chuyn phc tp, bao gm nhiu pha v cc h thng nhiu thnh phn. Trong sut thi
gian sinh trng, hn hp khng ng nht ca cc t bo gi v non thay i lin tc v t thch
147
nghi vi mi trng dinh dng l yu t cng thay i lin tc trong cc iu kin vt l v ha hc.
E. coli (Escherichia coli). Vi khun thng c trong rut non ca ng vt c xng sng. E.
coli c coi nh sinh vt mu cho vic nghin cu hot ng ca t bo.
Enzyme gn DNA (DNA ligase). Mt enzyme to ra mt lin kt phosphodieste gia u 3-
OH ca mt on DNA v u 5-PO
4
ca mt on DNA khc. on ni lin c kt hp b sung
i base vi si DNA khun mu.
Enzyme hn ch (restriction enzyme, RE). Cc enzyme hn ch c phn lp t
prokaryote, chng c kh nng phn hy DNA phage, hn ch kh nng sinh trng ca phage trong
vi khun bng cch ct phn t DNA. Hin nay, c khong 500 loi RE khc nhau. Cc enzyme ny
ct DNA si i cc v tr nhn bit c bit ca chng gm t 4-6 cp nucleotide c trnh t i
xng o ngc nhau, cc on ngn ny gi l palindrome (on i xng: l on DNA c hai si
hon ton i xng ging ht nhau nu lt ngc u ui).
Exon. Cc on DNA trong gen c chc nng phin m. Exon tn ti c sinh vt prokaryote
ln eukaryote. eukaryote, cc exon nm xen k vi cc on intron chim ti 90% tng s DNA
ca t bo v khng c chc nng phin m.
Gen (gene) hay cistron. n v c bn ca di truyn, l mt on nhim sc th m ha mt
chui polypeptide hoc mt phn t RNA. Gen bao gm cc vng nm trc v sau vng m ha v
c nhng trnh t (intron) nm gia cc phn m ha (exon).
Gen khng apoptosis (antiapoptosis gene). L gen khng li hin tng cht t nhin
c chng trnh ha (apoptosis) to ra cc vt ch siu sn xut (superior production hosts).
Gen chn lc (selector) hay gen ch th chn lc (selectable marker). L cc gen ch th
c chuyn cho t bo nhn phn bit t bo c bin np v khng bin np. S hin din
ca tc nhn chn lc trn mi trng nui cy sau khi chuyn gen mt vi ngy cho php phn
lp cc t bo ti t hp sng st.
Gen iu ha (regulatory gene). Mt gen m sn phm ca n tham gia vo s iu ha biu
hin ca mt gen khc. V d: gen m ha mt protein km hm.
Gen khi ng (promoter). Mt trnh t trn phn t DNA m RNA polymerase c th
kt hp c khi u s phin m.
Gen nhy hay nhn t chuyn v (transposon). Mt on DNA nh c cu trc c bit nn c
kh nng di chuyn t mt v tr ny n mt v tr khc trn phn t DNA hay t mt nhim sc th
ny sang mt nhim sc th khc trong t bo lng bi. Gen nhy ch chuyn n nhng v tr xc
nh trong genome. Trong nhiu trng hp gen nhy c th gy t bin ti v tr n di chuyn n.
Gen nhy c th dng lm phng tin gy t bin nh hng.
Gen tng cng (enhancer). Mt trnh t dng cis, c kh nng y mnh vic s dng mt
s promoter eukaryote, c th hot ng theo c hai hng bt k v tr no so vi promoter
kch thch s phin m ca mt gen nht nh.
Gen tin ung th (proto-oncogene). Tm thy trong genome eukaryote, l thnh phn tng
ng ca cc gen ung th tm thy trong cc retrovirus.
Gen ung th (oncogene). Gen m ha cho nhng sn phm c kh nng bin t bo
eukaryote thnh t bo ung th.
Ghp ni (splicing). S loi b cc intron v ni lin cc exon mRNA trong qu trnh hon
thin sau phin m.
Glycosyl ha (glycosylation). L qu trnh b sung mt hoc nhiu phn t carbohydrate (gc
ng) vo mt phn t protein (glycoprotein) sau khi n c tng hp nh ribosome.
Helper plasmid. Plasmid tr gip mang vng vir (virulence) v ori c s dng trong giao
148
phi b ba (triparental matting): Agrobacterium khng c plasmid, E. coli c helper plasmid v E. coli
c binary vector mang gen ngoi lai to thnh mt Agrobacterium ti t hp c binary vector c
dng cho vic bin np vo t bo v m thc vt.
H gen (genome). Trnh t DNA ton phn ca mt sinh vt, cha ton b thng tin di truyn
ca c th.
H ln men (fermenter) hay ni phn ng sinh hc (bioreactor). L loi thit b m trong n
s bin i ha sinh c tin hnh bi cc t bo sng hoc cc thnh phn t bo in vivo (enzyme).
Ba k thut ln men c bn l: ln men m, ln men m c cung cp dinh dng v ln men lin tc.
Cc h ln men c thit k da trn c s ca ba k thut ny nhng c ci tin cho tng trng
hp c th tng hiu sut nui cy.
Hiu sut bm (attachment efficiency). Phn trm s t bo bm c ln b mt mi trng
nui cy trong mt thi gian nht nh.
Hiu sut nui tri (plating efficiency). Khi nim ny ng ngha vi hiu sut to dng, ni
ln phn trm s t bo pht trin thnh dng khi nui tri trn b mt mi trng.
Hiu sut to dng (cloning efficiency). Phn trm s t bo to c dng khi nui tri trn
b mt mi trng.
Hybridoma. Dng t bo c hnh thnh t s phi hp mt t bo ung th v mt t bo
bch cu lymphocyte B. Hybridoma c kh nng sn sinh cc khng th (immunoglobulin) mt cch
vnh vin.
Intron. L mt on DNA c phin m nhng b loi b trong qu trnh hon thin ca
mRNA, khng c mt phn t mRNA hon chnh.
In vitro. Dng ch mt qu trnh xy ra trong dch chit t bo khng cha t bo nguyn
vn, hay ch cc t bo nui cy trong mi trng nhn to.
In vivo. Dng ch cc hin tng xy ra trong t bo nguyn vn hay trong c th.
Khng nguyn (antigen). Phn t c kh nng kch thch sn sinh mt khng th khi xm
nhp vo mt c th sng.
Khng th (antiboby). Mt protein (immunoglobulin) do t bo bch cu lymphocyte B sn
sinh, c kh nng nhn bit mt khng nguyn l c trng v lc s khi u mt p ng min
dch.
Khng th n dng (monoclonal antibody). c sn sinh t mt dng hybridoma, v mi
dng hybridoma pht xut ch t mt t bo bch cu lymphocyte B nn ton b cc phn t khng
th sn sinh ra u y ht nhau.
Khng bo (vacuole). C vai tr tip nhn cc cht thi ca s trao i cht hoc cc cht th
cp ca thc vt. cc t bo non khng bo thng nh v nhiu, khi t bo ln dn v gi hn th
khng bo cng m rng ln v kt thnh mt khi. cc t bo thc vt trng thnh, khng bo
c th chim ti 90% th tch t bo. Khng bo c bc chung quanh bi mng huyt tng
(plasma). Thnh phn chnh ca cc khng bo ln l nc cha cc cht ha tan nh cc ion v
c, cc amino acid, cc acid hu c, cc sc t ha tan trong nc (anthocyanin) v cc cht khng
ha tan dng tinh th v hnh kim. Ngoi ra, khng bo cng cha cc protein nh cc hydrolyse,
catalase v photphatase. Phn bo tan mun cp n lipid chung quanh tt c cc cu trc ni
gia nhn v mng t bo.
Kilobase (Kb). Mt nghn cp base ca mt phn t DNA.
K thut v trng (aseptic technique). Qui trnh ngn nga s nhim nm, vi khun, siu vi
khun hoc cc loi vi sinh vt khc i vi nui cy m v t bo.
Lai t bo (cell hybridization). S dung hp hai hay nhiu t bo khng ging nhau to mt
149
th t bo hn hp.
Ln cy chuyn (passage number). S ln t bo, m hay mu vt nui cy c cy chuyn,
qua c th tnh tui v h s ng trng ca chng.
Lc lp (chloroplast). L v tr ca quang hp trong t bo thc vt, n cha chlorophyll l sc
t lc phn ng vi nh sng sn xut cc carbohydrate.
Li ni sinh cht (endoplasmic reticulum). Mt bo quan c trong t bo cht ca nhng
sinh vt eukaryote, l mt phc hp mng c hai mng, lin quan n qu trnh tng hp v vn
chuyn protein.
Mng t bo (cell membrrane). L lp trong ca thnh t bo. Mng t bo bao gm protein
v lipid, n c chc nng iu ha s vn chuyn cc cht i vo v ra khi t bo.
M di truyn (genetic code). Tt c nhng b ba nucleotide DNA (hoc mRNA) m ha c
hiu 20 amino acid khc nhau ca protein.
M kt thc (termination codon). UAA, UAG v UGA l nhng m kt thc hoc cn gi l
m dng (stop codon), l nhng tn hiu kt thc tng hp protein.
Mt qun lc (population density). S lng t bo trn n v din tch nui cy hay trn
n v th tch nui cy.
Mu vt (explant). M c tch t nguyn liu ban u dng duy tr hoc nui cy.
Methyl ha (methylation). Mc ch bo v DNA ca cc on palindrome, ngha l gn gc
methyl (CH
3
) v tr cn thit nn khng b enzyme hn ch ct. Khi c s methyl ha th enzyme
khng nhn bit c v tr ct hn ch v do DNA khng b ct, DNA ca phage khng c gn gc
methyl s b ct. Trong cng ngh DNA ti t hp, enzyme methyl ha c gi l methylase enzyme.
Methylase c dng bo v on DNA cn gn vo. Tt c cc chng E. coli u cha hai enzyme
methyl ha DNA l: dam-methylase v dcm-methylase.
Mi trng nhn to (chemically difined medium). Dung dch dinh dng dng nui cy
ch cha nhng thnh phn m cu trc ha hc c bit.
M so (callus). Khi m thc vt gm nhng t bo khng phn ha, c kh nng phn chia,
c pht sinh t cc t bo phn ha t nhiu. Khi thc vt b thng tn thng to loi m ny
trn vt so, v th c tn gi l m so.
Nhn (nuclear). L trung tm iu khin ca t bo cha DNA phin m v dch m thnh
protein. Cc protein tng hp c sp xp v ng gi trong cc ti ca b my Golgi.
Nhn dng (clonal propagation). Nhn ging v tnh nhng dng thc vt c ngun gc t mt
c th hay mt mnh ct duy nht, m bo hon ton ng nht v di truyn.
Nhn ging in vitro (in vitro propagation). Nhn ging mt loi thc vt trong ng nghim
(bnh thy tinh, bnh plastic, hp plastic...) trn mi trng nhn to v trong iu kin v trng. ng
ngha vi khi nim vi nhn ging (micropropagation).
Nucleic acid. Nhng polynucleotide sinh hc t nhin trong nhng n v nucleotide c
kt hp vi nhau bi nhng lin kt phosphodieste thnh trnh t ring bit: DNA v RNA.
Nui cy dch huyn ph t bo (cell suspension culture). Phng thc nui t bo n hay
cm nhiu t bo (cell aggregate) trng thi l lng trong mi trng lng trong bnh tam gic trn
my lc (shaker) hoc trong cc h ln men chm (fermenter/bioreactor) thu sinh khi t bo (cell
biomass) hoc dch nui cy (borth), phc v cho vic tch chit cc hp cht c hot tinh sinh hc t
t bo (i vi cc hp cht ni bo) hoc tinh sch chng t dch nui cy (i vi cc hp cht ngoi
bo).
Nui cy m (tissue culture). Duy tr v sinh trng cc loi m trong iu kin in vitro nhm
iu khin phn ha v hnh thi v chc nng ca chng.
150
Nui cy m thc vt (plant tissue culture). Duy tr v nui dng t bo, m, c quan hay
cy hon chnh ca thc vt trong iu kin in vitro.
Nui cy khi u hay nui cy s cp (primary culture). Nui cy u tin khi tch t bo,
m hoc mu vt t c th ban u tnh n ln cy chuyn u tin t s thu c dng t bo.
Nui cy phi (embryo culture). Duy tr v pht trin phi non hoc trng thnh c phn
lp t ht.
Nui cy t bo (cell culture). Khi nim ch nhng nui cy trong ng nghim (in vitro) ca
nhng t bo k c t bo n khng phn ha thnh m.
Pha lag (lag phase). L pha tnh khi u hoc tim tng. y l thi k khi u ca qu
trnh nui cy, trong sut thi k ny s thay i s lng t bo l bng khng hoc khng ng k.
Mc d s lng t bo khng tng ln, nhng t bo c th sinh trng bng cch tng kch thc
trong sut thi k ny.
Pha log (logarithm phase). L pha sinh trng theo hm m. cc c th n bo (vi sinh
vt) hoc t bo ng-thc vt, s nhn i tng dn s lng t bo cho kt qu tc sinh trng
tng ln lin tc trong qun th giai on ny.
Pha tnh (stationary phase). L giai on m s sinh trng ca qun th t bo thng b
hn ch hoc do s dng ht ton b cc cht dinh dng c sn hoc do s tch ly cc sn phm
c ca s trao i cht. Kt qu l tc sinh trng gim v s sinh trng cui cng dng li.
Pha cht (death phase). L giai on tip theo ca pha tnh m trong cc c th trong qun
th b cht. S cht xut hin hoc do s suy yu ca vic bo qun nng lng ca t bo, hoc do
s tch ly cc sn phm c t. Ging nh s sinh trng, s cht l mt hm m. Trong mt s
trng hp, c th khng ch cht m cn phn hy (qu trnh phn gii).
Phn ha hay bit ha (differentiation). Mt kha cnh ca s pht trin bao gm s hnh
thnh cc loi t bo, cc loi m, cc loi c quan khc t mt hp t ban u di s iu khin
c bit ca cc gen.
Phin m (transcription). S tng hp mRNA t khun mu DNA.
Phin m ngc (reverse transcription). S tng hp DNA t khun mu mRNA nh
enzyme phin m ngc (reverse transcriptase).
Phng x t ghi (autoradiography). K thut pht hin cc phn t c nh du phng x
thng qua hiu ng to nh ca cc phn t ny trn phim X-quang.
Phng trnh Monod (Monod equation). L mt trong nhng phng trnh c s dng
rng ri nht th hin nh hng ca nng c cht (cht dinh dng) ln tc sinh trng c
trng ca t bo ():
Trong : C
S
l nng ca c cht gii hn (limiting substrate) trong mi trng v K
S
l h
s h thng. Gi tr ca K
S
tng ng vi nng ca cht dinh dng khi tc sinh trng c
trng bng mt na gi tr cc i ca n (
max
)
Plasmid. Mt phn t DNA mch vng ngoi nhim sc th, sao chp c lp, khng ph
thuc vo DNA ca nhim sc th.
Protein. Mt phn t ln gm mt hoc nhiu chui polypeptide, mi chui c mt trnh t
amino acid nht nh, khi lng phn t ca protein t vi nghn n vi triu.max
Ribosome. c tp trung trn b mt ca mng li ni sinh cht v tham gia vo hot ng
151
sinh tng hp protein.
RNA thng tin (messenger RNA, mRNA). Mt trong ba loi RNA tham gia vo tng hp
protein. Loi ny c mang thng tin c m ha trong trnh t cc nucleotide xc nh trnh t
amino acid ca polypeptide.
RNA vn chuyn (transfer RNA, tRNA). Mt trong ba loi phn t RNA c sinh ra bi qu
trnh phin m v tham gia vo vic tng hp protein: RNA vn chuyn a amino acid n ribosome
vo chng ghp vi thng tin trn mRNA.
Sc k (chromatography). Thut ng sc k ch cc k thut phn tch v iu ch cho
php tch bit cc hp phn khc nhau ca mt hn hp. Php phn tch sc k da vo s di
chuyn khc nhau trong mt pha ng ca cc cht ha tan c gn trn mt pha tnh trng
thi rn. Ngi ta thng chn cc cht c kh nng kt gn c vi cc cht (ha tan) nh phn
tch lm pha tnh. Tng tc gia cht ha tan v pha tnh c th l tng tc hp ph, tng tc
ion (trao i ion), tng tc k nc, tng tc kiu ry phn t hoc tng tc c hiu sinh hc.
Sinh khi (biomass). Tng khi lng kh ca tt c cc cht sng trong mt n v din tch
hoc th tch.
Sinh trng cn bng (balanced growth). L s sinh trng m trong sut qu trnh s
nhn i sinh khi xy ra cng vi s nhn i ca tt c cc c tnh xc nh khc ca qun th
nh l protein, DNA, RNA v nc ni bo.
S ti bn hay sao chp (replication). S tng hp mt phn t DNA xon kp ging vi
phn t DNA m.
To dng gen (gene cloning). Cn gi l nhn dng hay tch dng gen, l s sn sinh nhiu
bn sao ca mt phn t DNA, thng l phn t DNA ti t hp, bng cch sao chp phn t
trong mt vt ch thch hp.
Ti t hp (recombination). Qu trnh trong nhim sc th hay phn t DNA t ra ri cc
phn t c ni li theo mt t hp mi. Qu trnh ny c th xy ra trong t bo sng (qua s
trao i cho trong phn bo gim nhim) hay trong ng nghim nh cc enzyme ct v ni DNA.
Tng nui dng (feeder layer) hay t bo nui dng (nurse cells). Lp t bo c th b
chiu x lm mt kh nng phn bo c tri bn di cung cp mt s cht cn thit cho lp t
bo khc nui bn trn.
T bo lai (hybrid cell). L t bo c mt nhn c hnh thnh sau khi dung hp hai t bo dn
n s hnh thnh mt nhn hn hp.
T bo mm phi (embryonic stem cell). T bo phi cha bit ha, c th c nui cy
trong mt thi gian di m vn gi c tnh a th (ngha l kh nng bit ha theo nhiu hng
khc nhau).
Thnh t bo (cell wall). c cu to bi cc carbohydrate t nhin (cellulose v
hemicellulose). Lp ngoi ca thnh t bo cha pectin gip n lin kt vi cc t bo bn cnh.
Thnh t bo c chc nng nng cho cy.
Th n nh ha tnh (chemostat). Trong h ln men lin tc tc dng chy dinh dng
c ci t mt gi tr c bit v tc sinh trng ca nui cy s iu chnh tc dng chy
ny, nh vy s duy tr c nng mi trng dinh dng thch hp vi mt t bo.
Th n nh c (turbidostat). c s dng khi h ln men lin tc tin hnh cc tc
pha long cao gn vi im ra tri (washout point), khi ta c th ngn cn s ra tri bng cch
iu ha tc dng chy trong trng hp tht thot t bo thng qua dng chy ra ngoi vt qu
s sinh trng t bo trong h ln men.
Thi gian gp i qun th (population doubling time). Thi gian m s lng t bo ca
152
dng hay chng nui cy tng n gp i k t khi bt u nui.
Thc khun th (bacteriophage). Mt virus c th ti bn trong mt t bo vi khun.
Tip mu (inoculation). Bc a mu vo trong nui cy khi u (initiation culture).
Tnh ton th (totipotency). Mt c tnh ca t bo l c kh nng pht trin thnh mi kiu t
bo c trong c th trng thnh m t n c tch ra, tc l c kh nng ti sinh thnh mt c th
hon chnh.
Tc phn chia t bo (cell division rate). S phn chia t bo trn mt n v thi gian.
Tc phn chia l hng s trong sut thi gian sinh trng (theo hm m) ca t bo.
Tc sinh trng t bo (cell growth rate). S thay i s lng t bo theo thi gian. Tc
sinh trng khng phi l mt hng s trong sut thi gian sinh trng (theo hm m) ca t bo.
Tc sinh trng c trng ca t bo (cell specific growth rate). S thay i theo
logarithm t nhin ca s lng t bo theo thi gian.
Trnh t cis. Trnh t trn mt phn t DNA c tc ng (iu ha) n cc trnh t khc trn
cng phn t DNA . Cc trnh t cis khng m ha cho protein.
Tui th h t bo (cell generation time). Thi gian gia hai ln phn chia ca t bo. Khi
nim ny khng ng ngha vi thi gian gp i qun th.
Ty th (mitochondrion). Cha vt liu di truyn v nhiu enzyme quan trng trong s trao i
cht ca t bo.
Vector. L cc phn t DNA c s dng trong to dng gen v nhn bn chng trong t bo
vt ch (E. coli hoc nm men). C ba nhm vector chnh gm: (1) Nhm plasmid, (2) Nhm
phage/phagemid, v (3) Nhm nhim sc th nhn to (artificial chromosome: BAC v YAC). tng
v vector chuyn gen bt ngun t cc plasmid ca vi khun. Vector chuyn gen l phn t DNA c
kh nng t ti sinh, tn ti c lp trong t bo v mang c cc gen cn chuyn.
Vector hai ngun (binary vector). L dng s dng hai hay nhiu loi plasmid v vi khun
cng lc, v d: vi khun E. coli v Agrobacterium, cc plasmid trong trng hp ny thch ng vi c
E. coli v Agrobacterium.
Virus. Phn t c mang nucleic acid (DNA hoc RNA) nm trong mt v bc protein, c kh
nng sao chp trong t bo ch v lan truyn t t bo n sang t bo kia.
Xon kp (double helix). Cu trc ba chiu t nhin ca hai chui DNA b sung, i song v
xon vi nhau.

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Giao Trinh Cong Nghe Te Bao

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1

) th phng trnh (4.7) s tr thnh:

l tc sinh trng c trng ca cc t bo khng c plasmid, v C

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