GVHD : PGS.

TS Nguyn Thy Hng SVTH : Phm Ngc Xun Phm Ngc Thin Trn c Thin ng Minh Chu H Thanh Trc Lp : HC07SH2 Nm hc : 2010-2011

Ni dung chnh
I . Li m u i nt v bnh tiu ng II. Mt s loi insulin trn th trng III. C s khoa hc ca cng ngh sn xut insulin 1. Cu trc phn t insulin 2. Tng hp insulin trong c th 3. Vai tr sinh hc ca insulin IV. Cng ngh sn xut insulin. 1.Sn xut insulin tch chit t ng vt 2.Sn xut insulin t thc vt chuyn gen 3.Sn xut insulin t i tng vi sinh vt chuyn gen 4. Kt lun

I. Li m u
Bnh tiu ng l mt trong nhng cn bnh e da nghim trng ti sc kho con ngi.Trn th gii s ngi mc bnh tiu ng ngy cng tng, s gia tng s ngi mc bnh tiu ng s ko theo s gia tng cc bin chng ca cn bnh ny nh thn kinh, x va ng mch S ngi t vong trn th gii do bnh tiu ng lin tc gia tng trong nhng nm gn y. iu i hi phi tm ra nhng hng tip cn mi cho vic ngn nga v iu tr cn bnh ny.

Best v Banting- nhng ngi u tin chit sut c insulin nm 1921

Collip v MacLeod- nhng ngi u tin dng insulin chit sut iu tr bnh tiu ng type 1 cho Leonard Thomson

Vi nt v bnh tiu ng

1. Khi nim 2. Phn loi 3. Tnh hnh thc t v bnh tiu ng hin nay

1. Khi nim
Bnh i tho ng, cn gi l Bnh tiu ng, l mt bnh do ri lon chuyn ha cacbohydrat khi hoc mn insulin ca ty b thiu hay gim tc ng trong c th, biu hin bng mc ng trong mu lun cao Bnh tiu ng l mt trong nhng nguyn nhn chnh ca nhiu bnh him ngho, in hnh l bnh tim mch vnh, tai bin mch mu no, m mt, suy thn, lit dng, hoi th, v.v

2. Phn loi
Tiu ng loi 1: (tiu ng ph thuc insulin hay tiu ng ngi tr ) chim khong 5-10% tng s bnh nhn Nguyn nhn bnh: c ch t min (tuyn ty b tn cng v ph hy bi chnh c th, lm cho tuyn ty khng cn kh nng sn xut insulin na) Nhng triu chng in hnh: tiu nhiu, ung nhiu, i khi n nhiu, m mt, d cm v st cn, tr em chm pht trin v d b nhim trng.

 Tiu ng loi 2: ( tiu ng khng ph thuc insulin hay tiu ng ngi trng thnh) chim khong 90-95% trong tng s bnh nhn bnh tiu ng, thng gp la tui trn 40, nhng gn y xut hin ngy cng nhiu la tui 30, thm ch c la tui thanh thiu nin. Ty ngi bnh vn cn kh nng sn xut insulin, nhng khng . t c triu chng v thng ch c pht hin bi cc triu chng ca bin chng(nhi mu c tim, tai bin mch mu no), hoc ch c pht hin tnh c khi i xt nghim mu trc khi m. T l tiu ng tng theo tui, yu t di truyn cng ng vai tr quan trng. C mi lin quan trc tip gia bo ph v tiu ng loi 2.

Loi insulin

Mu sc

Ngun gc

Thi gian bt u c tc dng

Thi gian ht tc dng

Tc dng nhanh - Insulin Actrapid HM. - Insulinum maxirapid.

Ngi trong Ln

30 pht (tim di da)

8 gi

Tc dng bn chm - Insulatard HM. - Insulin Lente.

Ngi c Ln 1 gi

20 gi 18 gi

Loi pha trn - Mixtard HM 30/70.

Ngi

30 pht

20 gi

Tc dng rt chm - Utra - Lente

4 gi

30 gi

1. Cu trc phn t insulin

o Insulin ngi l mt polypeptide o Bao gm: Chui A 21 acid amin Chui B 30 acid amin C mt cu ni disulfur trong chui A v 2 cu ni disufur ni gia hai chui A v B. o Cng thc ha hc: C257H383N65O77S6 o Trng lng phn t: 5808
Cu trc ha hc ca insulin

2. Tng hp insulin trong c th

Preproinsulin: l chui peptid di 110 acid amine, tng hp trong cc t bo ca o Langerhans ca tuyn ty. Proinsulin: Khi Preproinsulin tin ti li ni cht, mt protease chuyn bit s ct b on peptide tn hiu to proinsulin. Proinsulin hnh thnh cu ni disulfur trong li ni cht, hnh thnh cu trc bc ba. Insulin: Hnh thnh qua qu trnh phn ct nh enzyme PC1/3 ti lin kt gia chui B v C v enzyme PC2 ngay v tr lin kt gia chui A v C.

3. Vai tr sinh hc ca insulin

 Insulin v trao i hydratcacbon Insulin v trao i lipit Nhng tc ng khc ca insulin

Gia tng tun hon insulin

T bo hp thu glucose

Nng lng trao i cht Tng hp cht bo Tng hp Glycogen

Kch thch tuyn ty tit ra insulin Tng lng Glucose trong mu

Lng Glucose cn li

Glucose trong mu

Gim lng Glucose trong mu

Hp thu Glucose vo mu

Kch thch tuyn ty tit ra Glucagon

Glycogen chuyn thnh Glucose gan

Gia tng tun hon Glucagon

S biu hin s iu ha hm lng glucose mu

Nhng tc ng khc ca insulin

- Insulin lm tng tnh hp th ca cc axitamin. - Insulin lm tng tnh thm cc ion kali, magie v photphat v c to iu kin cho qu trnh photphoryl ho v s dng glucoz.

Insulin lm tng tnh thm cc ion kali, magie v photphat v c

T ng vt

Insulin

T thc vt

T vi sinh vt

1.Sn xut insulin tch chit t ng vt

T nhng thp nin 1920 cho n nhng nm u ca thp nin 1980, insulin c to ra bng cch c lp t tuyn ty ca ng vt nh heo v b v c tinh sch Ngy nay loi insulin ny c tinh ch bng phng php sc k t tinh khit ha rt cao.

Chng loi Ngi Heo,ch,tinh dch c voi Th Gia sc(tru, b nga, d cu Nga Sei whale

Chui A cc v tr 8 9 10 Thr-Ser-ILe Thr-Ser-ILe Thr-Ser-ILe Ala-Ser- Val Ala-Gly- Val Thr-Gly-Leu Ala-Ser-Thr

Chui B cc v tr 30 Thr Ala Ser Ala Ala Ala Ala

S thay i chui cu trc amino acid ca cc loi ng vt so vi con ngi (Source W.F ganong, 2004)

Sn xut insulin t t bo gc tr bnh tiu ng

Nhng t bo gc t dy rn c kh nng sn xut mt hp cht c tn l C-peptide (mt cht protein tin thn ca insulin v ch hin din khi t bo sn xut ra insulin). S hin din C-pep c mt lng insulin nht nh c sn xut bi t bo gc c dng thay th cho t bo ty tng h hi hoc b ph hy.

Cc nh khoa hc Anh v M cho bit h SX c insulin t t bo gc ly t dy rn tr s sinh iu tr bnh tiu ng. (nh: www.neonet.ch)

Nhc im
Insulin V c cu trc khng hon ton ging insulin ngi Hot ng chc nng trong c th km hn insulin ngi Kh nng hp th km C th gy ra cc phn ng min dch trong c th Trong qu trnh tch chit khng th loi b c ht cc tc nhn gy bnh V Qui trnh tch chit i hi k thut cao Chi ph t (cn lng ln ty SX) gi thnh cao Kh SX lng ln vi quy m cng nghip

Sn xut Insulin tr bnh tiu ng t hoa rum

Cc nh khoa hc H Calgary cy mt gen Insulin nhn to vo cy hoa rum. Khi gen ny i vo hot ng, hoa rum s bt u sn sinh ra Insulin nhanh hn cc phng php truyn thng c tin hnh trn ln, b, men hay vi khun. Khong 4.000m2 hoa s c sn lng ln ti gn 1kg Insulin 6.475 ha hoa rum l c th chit xut Insulin cho ton b nhng ngi mc bnh tiu ng trn th gii.

Cy hoa rum dng chit sut insulin

Tr bnh tiu ng bng insulin t rau dip hoc cy thuc l

Nghin cu ny c thc hin bi gio s Henry Daniell, thuc Trng i hc Central Florida, v cc cng s. Nhm nghin cu a cc t bo thc vt ng kh c cha insulin di dng bt vo c th chut mc bnh tiu ng. Khi cc t bo ny tin vo rut chut, vi khun ang sng s phn hy cc thnh t bo v insulin thot ra s c a dn dn vo mu. Sau 8 tun l th nghim, cc chuyn gia nhn thy [glucose] trong mu v nc tiu chut tr li mc an ton, v cc t bo beta trong tuyn ty ca chut sn xut c insulin mc cn thit cho c th.

Gio s Henry Daniell (H Central Florida)

 Hin nay, c nhiu phng php sn xut insulin ti t hp da trn cc chng vi sinh vt bin i gen, ch yu l vi khun (E.coli, Bacillus brevis) hoc nm men (Saccharomyces cerevisiae , Pichia pastoris) C 2 nhm phng php sn xut Insulin ti t hp trong cc chng VSV bin i gen l : nhm phng php 2 chui v nhm phng php miniproinsulin.

Ph ng php 1: inh tng h p insulin ti t h p theo ph ng php 2 chui

Nhc im : Hiu sut to Insulin hot tnh khng cao.

Phng php 2: Sinh tng hp Insulin ti t hp theo phng php miniproinsulin (MPI)

Hng nghin cu mi:

Ni v cc h thng t bo dng biu hin insulin ti t hp, ngi ta s dng rt a dng t vi sinh vt ti t bo ng vt v c thc vt. Trong s , t bo vi sinh vt c s dng nhiu nht do chng d thao tc, d a vo p dng quy m sn xut cng nghip, nhiu nht l E. coli v nm men. Gn y, ngi ta a ra mt h thng biu hin khc cho cc loi protein ti t hp l Pichia pastoris.

 Hn 3 thp nin qua, E.coli c s dng rng ri nh t bo ch cho biu hin protein. Tuy nhin, vic ng dng E.coli trong qu trnh biu hin cc protein c ngun gc t eukaryote cn b hn ch do E.coli thiu cc b my ni bo thc hin cc qu trnh bin i sau dch m. T bo E.coli khi biu hin protein c khuynh hng gi li ui Met, ui amino acid ny c th nh hng ti s n nh ca protein v gy p ng min dch, sau khi biu hin, vic to ra iu kin gp cun cho protein rt kh khn thc hin, rt tn thi gian v qu trnh ny khng phi lc no cng thnh cng. Nhng iu ny lm tng gi thnh sn xut protein khi s dng h thng t bo E.coli

Nhng thun li ca h thng Pichia pastoris

Cng nh nm men S.cerevisiae, P.pastoris l vi sinh vt n bo d nui cy, d thao tc an ton i vi con ngi. P.pastoris l mt eukaryote, c cc qu trnh bin i sau dch m nh kh nng gp cun, hnh thnh cu ni disulfide, v glycosyl ha. Do , nhiu protein khi biu hin trong h thng vi khun b bt hot do hnh thnh di dng th vi nhng li c hot tnh sinh hc cao khi biu hin trong P.pastoris. C mt promoter mnh, s dng cht cm ng l methanol r tin hn so vi t bo E.coli C kh nng pht trin pH t 3z7 do c th iu chnh pH gim thiu ti a hot ng ca protease i vi protein tit ra

 C th pht trin vi mt t bo cao hn nhiu ln so vi S.cerevisiae C th biu hin protein vi hm lng t miligram ti gram c trong nghin cu phng th nghim ln trong sn xut quy m cng nghip Cc protein ca bn thn P.pastoris c tit ra mi trng vi hm lng rt thp nn qu trnh tinh sch v thu nhn protein mc tiu v sau s rt d dng. Thnh phn mi trng nui cy n gin, chi ph ln men thp, cc phng php s dng c thng mi ha, chng v cc vector biu hin c sn trn th trng Hn ch c s nhim trong qu trnh ln men Qu trnh ln men c th thc hin nhanh chng

a. Chng vi sinh vt v plasmid

Chng vi sinh vt: Pichia pastoris E.Coli

Plasmid (Invitrogen)  pPIC9K  TOPO pCR2.1

 Chng Pichia pastoris thng c s dng nhiu nht l GS115 (his4), c gene m ha cho histidinol dehydrogenease b t bin nn khng c kh nng t tng hp histidine; cha gene AOX1 v AOX2, pht trin c trn methanol mc ngang bng vi chng hoang di.  Chng E.coli DH5 hoang di (Nanogen Biopharma)

Vector nhn dng pCR2.1

Topo pCR2.1 3,931 bp

Kch thc 3,931 bp Trnh t sao chp f1 Gen khng ampicillin Gen khng kanamycin ..

Vector biu hin pPIC9K (invitrogen)

Thit k v tng hp gene m ha cho hMPI

Trong nghin cu ny, trnh t gene m ha cho insulin ngi c bin i mt s acid amin so vi trnh t cng b trn NCBI nhm lm tng kh nng biu hin protein, tng hiu qu gp cun, v tng hot tnh sinh hc.

 Trong nghin cu ny, hMPI c thit k bao gm 53 amino acid: chui B 29 amino acid; mini C 3 amino acid (Asp-Gly-Lys), chui A 21 amino acid. Sau qu trnh gp cun, 3 amino acid ni 2 chui A v B s c ct b bng trypsin to ra insulin trng thnh. Trong cu trc insulin, v tr LysB29 c th b phn ct khi x l bng tripsin nn chui B c thit k ch c 29 amino acid, ThrB30 c thm vo sau khi qu trnh x l tripsin hon tt. ProB28 c thay th bng AspB28, HisB10 c thay th bng AspB10

 Do c nhiu thay th trong trnh t amino acid ca insulin, nn thay v tng hp gene bng phng php PCR vi khun mu t mRNA ngi v vi cp mi c trng cho gene m ha cho insulin, sau tin hnh to t bin im ti nhng v tr cn thit, chng ti s dng phng php tng hp ha hc

 Hai chui oligonucleotide m ha cho hMPI c tng hp ha hc vi phn overlap di 20 nucleotide. Phn ng PCR c thc hin vi s hin din ca enzyme Klenow nhm ko di 2 on oligonucleotide t thnh trnh t DNA mch i hon chnh.

To dng t bo E.coli DH5 mang plasmid pCR2.1 cha gen m ha cho hMPI
Vi mc ch khuch i v lu tr gen hMPI cho cc th nghim tip theo, TA cloning kit c dng dng ha gen hMPI vo pCR2.1 (t l sn phm PCR:plasmid ~3:1) 160C qua m c s hin din ca T4 ligase. Qu trnh ni ny s to plasmid pCR2.1 ti t hp mang gen hMPI(pCR2.1/hMPI). Plasmid ny c in bin np vo E.coli DH5. Trn mi trng LB cha ampicillin (50 g/ml) c b sung X-gal (80 g/ml) v IPTG (1mM). Cc khun lc mang plasmid ti t hp c mu trng v kim tra li bng phng php PCR khun lc vi cp mi thng dng M13-F/R cho vector pCR2.1. Nhng khun lc cho kt qu dng tnh c tch chit plasmid, v khng nh li ln na bng phn ng ct gii hn vi EcoRI v NotI.

To dng t bo E.coli DH5 mang plasmid pCR 2.1 cha gene m ha cho hMPI

To dng t bo E.coli DH5 mang plasmid pPIC9k cha gen m ha cho hMPI
Plasmid ti t hp pCR2.1/hMPI v plasmid pPIC9K cng c ct bi EcoRI v Not I. gene mc tiu c tinh sch t sn phm ct plasmid ti t hp pCR2.1/hMPI v ni vo plasmid pPIC9K bng enzyme T4 ligase to plasmid ti t hp pPIC9K/hMPI. Plasmid ti t hp pPIC9K/hMPI c in bin np vo E.coli DH5 v tri trn mi trng thch LB cha 50g/ml ampicillin, 370C.cc khun lc c kim tra bng phng php PCR vi cp mi AOX1-F/R c trng cho vector pPIC9K ng thi vector ny c i gii trnh t chc chn gen m ha cho hMPI khng c g thay i so vi khi thit k

Plasmid ti t hp pCR2.1/hMPI

Plasmid pPIC9K

EcoRI v NotI pPIC9K/hMPI

E.Coli DH5 Mi trng thch LB Kim tra khun lc bng PCR Gii trnh t vi cp mi AOX1-F/R Khng nh gen m ha cho hMPI -50 g/ml Amp - 370C -AOX1-F: 5-GACTGGTTCCAATTGACAAGC-3
- AOX1-R:5-GCAAATGGCATTCTGACATCC-3

3.3 To dng t bo nm men P.pastoris mang gen m ha cho hMPI

Plasmid ti t hp pPIC9K/hMPI c ct bng Sal I ti v tr HIS4 to ra plasmid dng thng v in bin np vo t bo P.pastoris. Sau khi bin np, qu tnh ti t hp tng ng s din ra gia pPIC9K/hMPI v genome ca nm men. Nhng dng t bo c cha pPIC9K/hMPI ti t hp c sng lc trn mi trng MD. y l mi trng chn lc cho cc chng t bin khuyt dng hisdidine.

 Vector biu hin pPIC9K/hMPI c cha cc gene HIS4 c kh nng iu khin sinh tng hp histidine trong khi chng P.pastoris GS115 khng c kh nng ny, do , ch nhng dng t bo c s ti t hp tng ng gia pPIC9K/hMPI v genome ca P.pastoris mi c kh nng mc c trn mi trng ny. Cc khun lc mc ctrn mi trng MD c tin hnh tch chit genomic DNA kim tra s hin din ca gene hMPI bng PCR vi cp mi AOX1-F/R.

Plasmid pPIC9K/hMPI c ct bng SalI ti v tr HIS4

Plasmid dng thng in bin np Tb P.Pastoris Ti t hp tng ng gia pPIC9K v genone ca nm men

Sng lc trn mt MD

T bo P.Pastoris ti t hp c mang gen m ha cho hMPI Tch chit DNA v kim tra s hin din ca gen hMPI bng PCR

Cp mi AOX1-F/R

3.4 Sng lc dng t bo nm men P.pastoris c kh nng biu hin hMPI cao
Cc khun lc cho kt qu PCR dng tnh c nui cy lin tc trn mi trng YPD vi nng khng sinh G418 tng dn (1,2,4,6 mg/ml) nhm sng lc nhng chng P.pastoris ti t hp c mang nhiu bn sao ca gen m ha cho hMPI.

 Cc dng ny sau c nui cy trn mi trng MM. Mng nitrocellulose c p trc tip ln b mt nm men v tip tc nui cy qua m. Khi , hMPI c nm men tit ra s bm trn mng. Khng th c hiu khng hMPI(Abcam) c s dng pht tn hiu hMPI trn mng; ty thuc m nht ca tn hiu tng ng vi kh nng biu hin cao hay thp ca tng dng P.pasrstoris ti t hp !" chn lc c dng P.pastoris c mang nhiu bn sao gen mc tiu v c kh nng biu hin hMPI cao.

Kt qu kim tra kh nng biu hin mini-proinsulin ca cc dng t bo nm men Pichia pastoris bng p mng trn khun lc lai min dch vi khng th a dng khng insulin. Nhng mi tn ch cc dng t bo c kh nng biu hin mini-proinsulin cao.

3.5 Biu hin hMPI quy m nui cy lc

Chng P.pastoris ti t hp c cha gene m ha cho hMPI (RPPI02) c tng sinh qua m trong mi trng BMGY 280C, lc 250 vng/pht cho ti khi OD600 t 5-6. Sinh khi nm men c thu nhn bng ly tm 6000 vng/pht, v chuyn sang mi trng BMMY sao cho OD600 t 1. y l ch s OD ban u thch hp cho qu trnh cm ng MeOH v biu hin hMPI. Bnh nui cy c y bng khu trang y t ( qua kho st), lc 250 vng/pht tng ti a thong kh cho nm men trong sut qu trnh biu hin. Qu tnh biu hin c duy tr nht 200C nhm gim thiu kh nng biu hin cc protein tp ca chnh t bo nm men, tng % hMPI trong protein tng s. Sau 24h nui cy, MeOH 100% c thm vo mi trng nui cy n nng cui l 1% cho ti khi k thc qu trnh biu hin. mu c thu sau mi 24h kim tra mt t bo nm men trong mi trng nui cy.

Chng RPPI02 Mi trng BMGY T0 C=280 C

Lc 250 vng/pht

Tng sinh OD600 t 5-6 Thu nhn sinh khi

Ly tm 6000 vng/pht T=15 pht Mi trng BMMY T0 C=200 C Lc 250 vng/phut y bng khu trang y t Thm MeOH sau mi 24h nui cy Ly tm thu dch ni Kim tra bng pp Bradford v phn tch trn tricine SDS-PAGE 15%

Nui cy OD600 t 1 Cm ng v biu hin hMPI

Kim tra hm lng protein

3.6 Biu hin hMPI trn quy m fermentor

Chng RPPI02 c tng sinh bng nui cy lc vi mi trng YPD nhit 280C, lc 250 vng /pht trong khong 18-20h. sinh khi sau c chuyn vo 2L mi trng BM trong fermentoe 5L c b sung PTM1, tip tc tng sinh trn fermentor vi c cht l glycerol. Trong qu trnh ln men, tc tng trng ca P.pastoris c theo di thng qua s gia tng sinh khi t theo thi gian, lng O2 cung cp lin tc 6L/h.

3.7 Thu nhn v tinh sch hMPI

3.8 Chuyn ha hMPI thnh hI

hMPI sau khi c tinh sch s c ct bng tripsin loi b chui C-peptide; ng thi gn thm ThrB30 to thnh hI. Phng php: 20mg hMPI c ha trong 0.1 ml hn hp 34.3% acid acetic v 42.2% N,N dimethylformamide trong nc. 0.2ml 2M Thr-Obut trong N,N dimethylformamide c thm vo phn ng v duy tr 120C. 2mg trypsin pha trong 0.05ml 0.05M calcium acetate c b sung vo hn hp phn ng. Sau 96h 120C, protein c ta vi 4ml actetone lm lnh sn, cn protein c thu nhn bng ly tm 13000v/p trong 15p 40C.

1. Xy dng m hnh chut tiu ng type I bng STZ

C ch : Khi chut c tim i.p streptozotocin (STZ) kch thch qu trnh ph hy t bo tuyn ty. i tng : Chut nht trng ci 5 tun tui. Chia lm 6 nhm, 5 con/nhm.
Nhm1 Nhm 2-6

SCB

STZ + SCB

Xy dng m hnh chut tiu ng type I bng STZ

Tim STZ ln1
Ngy 0

B i

Tim STZ ln 2
Ngy 1 Ngy 2 Ngy 3 Ngy 4 Ngy 5

-4h
Kim tra nng glucose trong mu hng ngy

2. Th nghim hot tnh hI ti t hp in vivo

Th nghim trn 4 nhm: 1 nhm chut bnh thng, 3 nhm chut tiu ng (5con/nhm)

Chut khe PBS

Chut bnh PBS

Chut bnh Insulin (NOVO)

Chut bnh hI

Chng m

2. Th nghim hot tnh hI ti t hp in vivo

Tim insulin B i
0h N Kim tra nng glucose trong mu (1,5h/ln) 1,5h 3h 4,5h 6h 7,5h

S qu trnh theo di tc ng nhanh ca hI ln m hnh chut tiu ng typeI STZ

3. Kt qu th nghim
a) Tc ng nhanh ca hI :  Nhm i chng: c tim PBS c hm lng ng huyt tng theo thi gian trong sut 7,5h  Nhm tim insulin (NOVO) lng ng huyt gim mnh sau 1,5h. Sau , hm lng ng li tng ln, trong 4,5h sau khi tim insulin hm lng ng huyt vn duy tr di ngng 250mg/dl.  Nhm c tim hI tc ng ca insulin tng t nh insulin (NOVO). hI c tc dng lm gim lng ng huyt ca chut tiu ng Type I STZ tng t nh insuin(NOVO)

b) Tc dng lu di ca hI:  Nhm chng m (chut khe) vn duy tr hm lng ng huyt n nh, khi lng chut tng dn v 100% chut duy tr sng st.  Nhm chut tiu ng khng tim insulin c hm lng ng huyt tng dn theo ngy, sau 10 ngy lng ng huyt lun mc cao (" 500mg/dl) khi lng c th gim mnh. Sau 12 ngy 100% chut cht.  Nhm c tim hI v insulin (NOVO) cho kt qu din bin bnh tng t nhau. 100% chut sng sau 15 ngy quan st, hm lng ng huyt duy tr n nh. Bn cnh khi lng c th chut tng dn. hI c tc dng tng ng insulin (NOVO) trong qu trnh ci thin tnh trng bnh chut tiu ng TypeI STZ

4. Kt lun
Vi nhng kt qu t c thit lp thnh cng h thng t bo P.pastoris dng cho biu hin hMPI trn quy m nui cy lc v quy m fermenter vi hm lng protein t 150 g/l v 1g/l. Phng php ta protein sau ln men bng (NH4 )2SO4 v tinh sch protein bng sc k lc gel cng c xy dng vi hiu sut cao ln n 90%. hI th hin hoat tnh tt, tng ng vi sn phm insulin thng mi ca NOVO Chn Pichia pastoris l h thng ph hp biu hin protein ngi va cho nng sut cao, chi ph ln men thp, trong khi protein c biu hin c hot tnh tt khi th nghim in vivo.