Công nghệ sản xuất insulin tái tổ hợp

8/6/2019 Cng ngh sn xut insulin ti t hp

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GVHD : PGS.TS Nguyn Thy HngSVTH : Phm Ngc Xun

Phm Ngc ThinTrn c Thinng Minh ChuH Thanh Trc

Lp : HC07SH2 Nm hc : 2010-2011


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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 insulin1. 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


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I. Li m u

Bnh tiu ng l mt trong nhng cn bnhe da nghim trng ti sc kho con

ngi.Trn th gii s ngi mc bnh tiung ngy cng tng, s gia tng s ngi mcbnh tiu ng s ko theo s gia tng cc binchng ca cn bnh ny nh thn kinh, x va

ng mch S ngi t vong trn th gii dobnh tiu ng lin tc gia tng trong nhngnm gn y. iu i hi phi tm ra nhnghng tip cn mi cho vic ngn nga v iutr cn bnh ny.


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

Collip v MacLeod- nhng ngi u tindng insulin chit sut iu tr bnhtiu ng type 1 cho Leonard Thomson


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Vi nt v bnh tiu ng

1. Khi nim

2. Phn loi

3. Tnh hnh thc t v bnh tiu ng hin nay


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1. Khi nim

Bnh i tho ng, cn gi l Bnh tiu ng,l mt bnh do ri lon chuyn ha cacbohydratkhi hoc mn insulin ca ty b thiu hay gimtc ng trong c th, biu hin bng mc ngtrong mu lun cao

Bnh tiu ng l mt trong nhng nguyn nhnchnh ca nhiu bnh him ngho, in hnh lbnh tim mch vnh, tai bin mch mu no, mmt, suy thn, lit dng, hoi th, v.v


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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 insulinna) 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.


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Tiu ng loi 2: ( tiu ng khng ph thuc insulin hay tiung ngi trng thnh) chim khong 90-95% trong tng s

bnh nhn bnh tiu ng, thng gp la tui trn 40, nhnggn y xut hin ngy cng nhiu la tui 30, thm ch c latui 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 chngca bin chng(nhi mu c tim, tai bin mch mu no), hoc chc 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 trquan trng. C mi lin quan trc tip gia bo ph v tiu ngloi 2.


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Loi insulin Mu sc Ngun gc Thi gian bt u ctc dng Thi gian ht tcdng

Tc dng nhanh- Insulin Actrapid

HM.- Insulinum

maxirapid.

trongNgi

Ln

30 pht(tim di da)

8 gi

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

cNgi

Ln1 gi

20 gi18 gi

Loi pha trn- Mixtard HM 30/70.

c Ngi 30 pht 20 gi

Tc dng rt chm

- Utra - Lente c B 4 gi 30 gi


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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:

C257H383N65O77S6o Trng lng phn t: 5808

Cu trc ha hc ca insulin


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2. Tng hp insulin trong c th

Preproinsulin: l chui peptid di110 acid amine, tng hp trong cc t

bo ca o Langerhans ca tuynty.

Proinsulin: Khi Preproinsulin tin tili ni cht, mt protease chuyn

bit s ct b on peptide tn hiuto proinsulin. Proinsulin hnh thnh

cu ni disulfur trong li ni cht,hnh thnh cu trc bc ba.

Insulin: Hnh thnh qua qu trnhphn ct nh enzyme PC1/3 ti lin

kt gia chui B v C v enzyme PC2ngay v tr lin kt gia chui A v C.


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3. Vai tr sinh hc ca insulin

Insulin v trao i hydratcacbon

Insulin v trao i lipit

Nhng tc ng khc ca insulin


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S biu hin s iu ha hm lng glucose mu

Gia tng tun honinsulin

Glycogen chuyn thnhGlucose gan

Tng lngGlucose trong

mu

Kch thch tuyn ty titra insulin

Glucosetrong mu

Hp thu Glucose vo

mu

Lng Glucose cn li

T bo hp thu glucose Nng lng trao i chtTng hp cht boTng hp Glycogen

Gim lng Glucosetrong mu

Kch thch tuyn ty tit

ra Glucagon

Gia tng tun honGlucagon


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Nhng tc ng khc ca insulin

- Insulin lm tngtnh hp th ca cc

axitamin.- Insulin lm tngtnh thm cc ionkali, magie v

photphat v c toiu kin cho qutrnh photphorylho v s dngglucoz. Insulin lm tng tnh thm cc ion kali,

magie v photphat v c


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Insulin

T ng vt

T thc vt

T vi sinh vt


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1.Sn xut insulin tch chit t ng vt

T nhng thp nin 1920 cho n nhng nm u ca thpnin 1980, insulin c to ra bng cch c lp t tuyn tyca ng vt nh heo v b v c tinh sch

Ngy nay loi insulin ny c tinh ch bng phng php sck t tinh khit ha rt cao.


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Chng loi Chui A cc v tr

8 9 10

Chui B cc v tr

30

NgiHeo,ch,tinh dch c voi

Th

Gia sc(tru, b nga, d

cu

Nga

Sei whale

Thr-Ser-ILeThr-Ser-ILe

Thr-Ser-ILe

Ala-Ser- Val

Ala-Gly- Val

Thr-Gly-Leu

Ala-Ser-Thr

ThrAla

Ser

Ala

Ala

Ala

Ala

S thay i chui cu trc amino acid ca cc loi ng vt so vi conngi

(Source W.F ganong, 2004)


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Sn xut insulin t t bo gc tr bnhtiu ng

Nhng t bo gc t dy rn ckh nng sn xut mt hp chtc tn l C-peptide (mt cht

protein tin thn ca insulin vch hin din khi t bo sn xutra insulin).

S hin din C-pep c mt

lng insulin nht nh c snxut bi t bo gc c dngthay th cho t bo ty tng h hi hoc b ph hy.

Nhng t bo gc t dy rn ckh nng sn xut mt hp chtc tn l C-peptide (mt cht

protein tin thn ca insulin vch hin din khi t bo sn xutra insulin).

S hin din C-pep c mt

lng insulin nht nh c snxut bi t bo gc c dngthay 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 tdy rn tr s sinh iu tr bnh tiu

ng. (nh: www.neonet.ch)


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Nhc imNhc 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


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Sn xut Insulin tr bnh tiu ng thoa rum

Cc nh khoa hc H Calgary cy mtgen Insulin nhn to vo cy hoa rum. Khi

gen ny i vo hot ng, hoa rum s btu sn sinh ra Insulin nhanh hn cc

phng php truyn thng c tin hnhtrn ln, b, men hay vi khun.

Khong 4.000m2 hoa s c sn lng lnti gn 1kg Insulin 6.475 ha hoa rum lc th chit xut Insulin cho ton bnhng ngi mc bnh tiu ng trn thgii.

Cy hoa rum dng chit sut insulin


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Tr bnh tiu ng bng insulin trau dip hoc cy thuc l

Nghin cu ny c thc hin bi gio s HenryDaniell, thuc Trng i hc Central Florida, v

cc cng s. Nhm nghin cu a cc t bo thc vt ng khc cha insulin di dng bt vo c th chut mc

bnh tiu ng. Khi cc t bo ny tin vo rutchut, vi khun ang sng s phn hy ccthnh t bo v insulin thot ra s c a dn dnvo mu.

Sau 8 tun l th nghim, cc chuyn gia nhn thy[glucose] trong mu v nc tiu chut tr limc an ton, v cc t bo beta trong tuyn ty cachut sn xut c insulin mc cn thitcho c th.

Gio s Henry Daniell (HCentral Florida)


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Hin nay, c nhiu phng php sn xut insulin ti thp da trn cc chng vi sinh vt bin i gen, chyu 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 phngphp 2 chui v nhm phng php miniproinsulin.


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

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


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


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Hng nghin cu mi:

Ni v cc h thng t bo dng biu hininsulin ti t hp, ngi ta s dng rt a dngt vi sinh vt ti t bo ng vt v c thc

vt. Trong s , t bo vi sinh vt c sdng nhiu nht do chng d thao tc, d avo p dng quy m sn xut cng nghip,nhiu nht lE. coli v nm men. Gn y,

ngi ta a ra mt h thng biu hin khccho cc loi protein ti t hp lPichia

pastoris.


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Hn 3 thp nin qua, E.coli c s dng rng ri

nh t bo ch cho biu hin protein. Tuy nhin, vicng dng E.coli trong qu trnh biu hin cc proteinc ngun gc t eukaryote cn b hn ch do E.colithiu cc b my ni bo thc hin cc qu trnhbin i sau dch m.

T bo E.coli khi biu hin protein c khuynh hnggi li ui Met, ui amino acid ny c th nhhng ti s n nh ca protein v gy p ngmin dch, sau khi biu hin, vic to ra iu kin gp

cun cho protein rt kh khn thc hin, rt tnthi gian v qu trnh ny khng phi lc no cngthnh cng. Nhng iu ny lm tng gi thnh snxut protein khi s dng h thng t bo E.coli


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Nhng thun li ca h thng Pichia pastoris

Cng nh nm men S.cerevisiae, P.pastoris l vi sinhvt n bo d nui cy, d thao tc an ton i vicon ngi.

P.pastoris l mt eukaryote, c cc qu trnh bin isau dch m nh kh nng gp cun, hnh thnh cuni disulfide, v glycosyl ha. Do , nhiu proteinkhi biu hin trong h thng vi khun b bt hot dohnh thnh di dng th vi nhng li c hot tnhsinh hc cao khi biu hin trong P.pastoris.

C mt promoter mnh, s dng cht cm ng lmethanol 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


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C th pht trin vi mt t bo cao hn nhiu ln sovi S.cerevisiae

C th biu hin protein vi hm lng t miligram tigram c trong nghin cu phng th nghim ln trongsn 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 schv 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 miha, chng v cc vector biu hin c sn trn thtrng

Hn ch c s nhim trong qu trnh ln men Qu trnh ln men c th thc hin nhanh chng


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a. Chng vi sinh vt v plasmid

Chng vi sinh vt:Pichia pastorisE.Coli

Plasmid (Invitrogen)pPIC9K

TOPO pCR2.1


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Chng Pichia pastoris thng c s dng nhiu nht l

GS115 (his4), c gene m ha cho histidinol dehydrogeneaseb t bin nn khng c kh nng t tng hp histidine; chagene AOX1 v AOX2, pht trin c trn methanol mcngang bng vi chng hoang di.

Chng E.coli DH5 hoang di (Nanogen Biopharma)


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Vector nhn dng pCR2.1

Kch thc 3,931 bp

Trnh t sao chp f1 Gen khng ampicillin

Gen khng kanamycin

..

Topo pCR2.13,931 bp


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Vector biu hin pPIC9K(invitrogen)


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Thit k v tng hp gene m ha cho hMPI

Trong nghin cu ny, trnh t gene m hacho insulin ngi c bin i mt s acidamin so vi trnh t cng b trn NCBI nhmlm tng kh nng biu hin protein, tng hiuqu gp cun, v tng hot tnh sinh hc.


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Trong nghin cu ny, hMPI c thit k baogm 53 amino acid: chui B 29 amino acid; miniC 3 amino acid (Asp-Gly-Lys), chui A 21 aminoacid. Sau qu trnh gp cun, 3 amino acid ni 2chui A v B s c ct b bng trypsin to rainsulin trng thnh. Trong cu trc insulin, v trLysB29 c th b phn ct khi x l bng tripsinnn chui B c thit k ch c 29 amino acid,

ThrB30

c thm vo sau khi qu trnh x ltripsin hon tt. ProB28 c thay th bng AspB28,HisB10 c thay th bng AspB10


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Do c nhiu thay th trong trnh t amino acidca insulin, nn thay v tng hp gene bng

phng php PCR vi khun mu t mRNAngi v vi cp mi c trng cho gene mha cho insulin, sau tin hnh to t binim ti nhng v tr cn thit, chng ti s

dng phng php tng hp ha hc


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Hai chui oligonucleotide m ha cho hMPI

c tng hp ha hc vi phn overlap di 20nucleotide. Phn ng PCR c thc hin vis hin din ca enzyme Klenow nhm ko di2 on oligonucleotide t thnh trnh t DNA

mch i hon chnh.


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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 genhMPI vo pCR2.1 (t l sn phm PCR:plasmid ~3:1) 160Cqua m c s hin din ca T4 ligase. Qu trnh ni ny s toplasmid pCR2.1 ti t hp mang gen hMPI(pCR2.1/hMPI).

Plasmid ny c in bin np vo E.coli DH5. Trn mitrng LB cha ampicillin (50 g/ml) c b sung X-gal (80g/ml) v IPTG (1mM).

Cc khun lc mang plasmid ti t hp c mu trng v kim trali bng phng php PCR khun lc vi cp mi thng dngM13-F/R cho vector pCR2.1.

Nhng khun lc cho kt qu dng tnh c tch chitplasmid, v khng nh li ln na bng phn ng ct gii hn

vi EcoRI v NotI.


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To dng t bo E.coli DH5 mang plasmid pCR2.1 cha gene m ha cho hMPI


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To dng t bo E.coli DH5 mang plasmidpPIC9k cha gen m ha cho hMPI

Plasmid ti t hp pCR2.1/hMPI v plasmid pPIC9K cngc ct bi EcoRI v Not I. gene mc tiu c tinh scht sn phm ct plasmid ti t hp pCR2.1/hMPI v ni vo

plasmid pPIC9K bng enzyme T4 ligase to plasmid tit hp pPIC9K/hMPI. Plasmid ti t hp pPIC9K/hMPI c in bin np voE.coli DH5 v tri trn mi trng thch LB cha 50g/mlampicillin, 370C.cc khun lc c kim tra bng

phng php PCR vi cp mi AOX1-F/R c trng chovector 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


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Plasmid ti t hppCR2.1/hMPI

PlasmidpPIC9K

pPIC9K/hMPI

E.Coli DH5

Gii trnh t vi cpmi AOX1-F/R

Mi trng thchLB

-50 g/ml Amp- 370C

Kim tra khunlc bng PCR

-AOX1-F: 5-GACTGGTTCCAATTGACAAGC-3- AOX1-R:5-GCAAATGGCATTCTGACATCC-3

Khng nh gen m

ha cho hMPI

EcoRI v NotI


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

Plasmid ti t hp pPIC9K/hMPI c ct bng Sal Iti v tr HIS4 to ra plasmid dng thng v in bin

np vo t bo P.pastoris. Sau khi bin np, qu tnhti t hp tng ng s din ra gia pPIC9K/hMPIv genome ca nm men. Nhng dng t bo c chapPIC9K/hMPI ti t hp c sng lc trn mi

trng MD. y l mi trng chn lc cho ccchng t bin khuyt dng hisdidine.


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Vector biu hin pPIC9K/hMPI c cha cc geneHIS4 c kh nng iu khin sinh tng hphistidine trong khi chng P.pastoris GS115 khngc kh nng ny, do , ch nhng dng t bo cs ti t hp tng ng gia pPIC9K/hMPI v

genome ca P.pastoris mi c kh nng mc ctrn mi trng ny. Cc khun lc mc ctrn mi trng MD c

tin hnh tch chit genomic DNA kim tra shin din ca gene hMPI bng PCR vi cp miAOX1-F/R.


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Plasmid pPIC9K/hMPI c ct bng SalI ti vtr HIS4

Plasmid dng thng

in bin np

Tb P.Pastoris

Sng lc trn mt MD

T bo P.Pastoris ti thp c mang gen m ha

cho hMPI

Ti t hp tng nggia pPIC9K v genone

ca nm men

Tch chit DNA v kim tra s hin din ca

gen hMPI bng PCR

Cp mi AOX1-F/R


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3.4 Sng lc dng t bo nm menP.pastoris c kh nng biu hin hMPI cao

Cc khun lc cho kt quPCR dng tnh c nui

cy lin tc trn mitrng YPD vi nng khng sinh G418 tng dn(1,2,4,6 mg/ml) nhm

sng lc nhng chngP.pastoris ti t hp cmang nhiu bn sao cagen m ha cho hMPI.


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Cc dng ny sau c nui cy trn mi

trng MM. Mng nitrocellulose c p trc tipln b mt nm men v tip tc nui cy qua m.

Khi , hMPI c nm men tit ra s bm trnmng. Khng th c hiu khng hMPI(Abcam)c s dng pht tn hiu hMPI trn mng; tythuc m nht ca tn hiu tng ng vi khnng biu hin cao hay thp ca tng dng

P.pasrstoris ti t hp !" chn lc c dngP.pastoris c mang nhiu bn sao gen mc tiu v ckh nng biu hin hMPI cao.

K ki kh bi hi i i i li


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Kt qu kim tra kh nng biu hin mini-proinsulinca cc dng tbo nm men Pichia pastoris bng p

mng trn khun lc lai min dch vi khng th a

dng khng insulin. Nhngmi tn ch cc dng tbo c kh nng biu hin mini-proinsulin cao.


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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 250vng/pht cho ti khi OD

600 t 5-6. Sinh khi nm men c thu nhnbng ly tm 6000 vng/pht, v chuyn sang mi trng BMMY saocho OD

600 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 khutrang 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 biuhin c duy tr nht 200C nhm gim thiu kh nng biu hincc protein tp ca chnh t bo nm men, tng % hMPI trong proteintng s. Sau 24h nui cy, MeOH 100% c thm vo mi trngnui 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 trongmi trng nui cy.


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Chng RPPI02

Tng sinh

Thu nhn sinh khi

Nui cy

Cm ng v biu hin hMPI

Kim tra hm lng protein

Mi trng BMGYT0 C=280 C

Lc 250 vng/pht

Ly tm 6000 vng/phtT=15 pht

Mi trng BMMYT0 C=200 CLc 250 vng/phuty bng khu trang y t

Thm MeOH sau mi 24hnui cy

Ly tm thu dch niKim tra bng pp Bradfordv phn tch trn tricine

SDS-PAGE 15%

OD600 t 5-6

OD600 t 1


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3.6 Biu hin hMPI trn quy mfermentor

Chng RPPI02 c tng sinh bng nui cy lcvi mi trng YPD nhit 280C, lc 250

vng /pht trong khong 18-20h. sinh khi sau c chuyn vo 2L mi trng BM trongfermentoe 5L c b sung PTM1, tip tc tngsinh trn fermentor vi c cht l glycerol. Trong

qu trnh ln men, tc tng trng caP.pastoris c theo di thng qua s gia tngsinh khi t theo thi gian, lng O2 cung cplin tc 6L/h.


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3.7 Thu nhn v tinh sch hMPI


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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 hnhp 34.3% acid acetic v 42.2% N,Ndimethylformamide trong nc. 0.2ml 2M Thr-Obuttrong N,N dimethylformamide c thm vo phn

ng v duy tr 120

C. 2mg trypsin pha trong 0.05ml0.05M calcium acetate c b sung vo hn hp phnng. Sau 96h 120C, protein c ta vi 4mlactetone lm lnh sn, cn protein c thu nhnbng ly tm 13000v/p trong 15p 40C.


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1 Xy dng m hnh chut tiu ng type I


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1. Xy dng m hnh chut tiu ng type Ibng STZ

C ch : Khi chut c tim i.p streptozotocin(STZ) kch thch qu trnh ph hy t bo tuynty.

i tng : Chut nht trng ci 5 tun tui.

Chia lm 6 nhm, 5 con/nhm.

Nhm1 Nhm 2-6

SCB STZ + SCB


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Xy dng m hnh chut tiu ngtype I bng STZ

Ngy 0

Kim tra nng glucose trong mu hng ngy

B i

Ngy 1 Ngy 2 Ngy 3 Ngy 4 Ngy 5

Tim STZln 2

Tim STZln1

-4h


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

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

Chutkhe

PBS PBSInsulin(NOVO)

hI

Chutbnh

Chutbnh

Chutbnh

Chng m


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2. Th nghim hot tnh hI ti t hp

in vivoTim insulin

B i

N

0h 3h1,5h 4,5h 6h 7,5h

Kim tra nng glucose trong mu (1,5h/ln)

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


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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 gimmnh sau 1,5h. Sau , hm lng ng li tng ln,trong 4,5h sau khi tim insulin hm lng ng huytvn duy tr di ngng 250mg/dl.

Nhm c tim hI tc ng ca insulin tng t nhinsulin (NOVO).

hI c tc dng lm gim lng ng huyt ca chuttiu ng Type I STZ tng t nh insuin(NOVO)


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b) Tc dng lu di ca hI: Nhm chng m (chut khe) vn duy tr hmlng ng huyt n nh, khi lng chut tng dnv 100% chut duy tr sng st.Nhm chut tiu ng khng tim insulin c hmlng ng huyt tng dn theo ngy, sau 10 ngylng ng huyt lun mc cao (" 500mg/dl) khi

lng c th gim mnh. Sau 12 ngy 100% chutcht.Nhm c tim hI v insulin (NOVO) cho kt qudin bin bnh tng t nhau. 100% chut sng sau

15 ngy quan st, hm lng ng huyt duy tr nnh. Bn cnh khi lng c th chut tng dn. hI c tc dng tng ng insulin (NOVO) trongqu trnh ci thin tnh trng bnh chut tiu ngTypeI STZ

4 Kt lun


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4. Kt lun Vi nhng kt qu t c thit lp thnh cng h

thng t bo P.pastoris dng cho biu hin hMPI trnquy m nui cy lc v quy m fermenter vi hmlng protein t 150 g/l v 1g/l.

Phng php ta protein sau ln men bng (NH4 )2SO4v tinh sch protein bng sc k lc gel cng c xydng vi hiu sut cao ln n 90%.

hI th hin hoat tnh tt, tng ng vi sn phminsulin thng mi ca NOVO

Chn Pichia pastoris l h thng ph hp biu hinprotein ngi va cho nng sut cao, chi ph ln menthp, trong khi protein c biu hin c hot tnh ttkhi th nghim in vivo.


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Công nghệ sản xuất insulin tái tổ hợp