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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.
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
Ngi trong Ln
8 gi
Ngi c Ln 1 gi
20 gi 18 gi
Ngi
30 pht
20 gi
4 gi
30 gi
T bo hp thu glucose
Lng Glucose cn li
Glucose trong mu
Hp thu Glucose vo mu
- 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.
T ng vt
Insulin
T thc vt
T vi sinh 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
S thay i chui cu trc amino acid ca cc loi ng vt so vi con ngi (Source W.F ganong, 2004)
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
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.
Phng php 2: Sinh tng hp Insulin ti t hp theo phng php miniproinsulin (MPI)
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
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
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)
Kch thc 3,931 bp Trnh t sao chp f1 Gen khng ampicillin Gen khng kanamycin ..
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
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
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 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.
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%
SCB
STZ + SCB
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
Chut bnh hI
Chng m
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.