構(gòu)建胰島素基因工程菌課件

構(gòu)建胰島素工程菌構(gòu)建胰島素工程菌1前言：胰島素的發(fā)現(xiàn)胰島素的結(jié)構(gòu)重組人胰島素及類型重組人胰島素基因工程菌的構(gòu)建：Constructionoftheecotin(大腸桿菌素)–proinsulinexpressionplasmidConstructionofpEGP1plasmid前言：重組人胰島素基因工程菌的構(gòu)建：2胰島素的發(fā)現(xiàn)1922FrederickBantingCharlesBest胰島素的發(fā)現(xiàn)1922FrederickBanting3人胰島素一級結(jié)構(gòu)CHAINA:21aminoacidsCHAINB:30aminoacids人胰島素一級結(jié)構(gòu)CHAINA:21aminoacid4胰島素立體結(jié)構(gòu)胰島素立體結(jié)構(gòu)51翻譯后不需要糖基化修飾，在細菌內(nèi)合成的胰島素具有完整的生物活性。2胰島素是一個相對較小的分子，由兩條多肽鏈組成，一條含21個氨基酸（A鏈）和另一條含30個氨基酸（B鏈）。胰島素具有兩個利于通過DNA重組技術生產(chǎn)的特點：

胰島素具有兩個利于通過DNA重組技術生產(chǎn)的特點：

6重組人胰島素1982年10月在美國上市，全球第一個商業(yè)化基因重組人胰島素。1.Humulin（優(yōu)泌林）

2.Novolin（諾和靈）

EliLillyandCompanyNovoNordisk重組人胰島素1982年10月在美國上市，全球第一1.Humu7歐洲市場的重組胰島素類似物歐洲市場的重組胰島素類似物8重組人胰島素類型1.重組前胰島素ChainBChainCChainA2.A,B鏈分開表達+ChainBChainA重組人胰島素類型1.重組前胰島素ChainBChainC93.B-miniC-A重組表達ChainBChainAMiniChainC4.B-XXX-A重組表達

X為氨基酸ChainBChainAX1X2Xn

N≤33.B-miniC-A重組表達ChainBChainA10重組人胰島素基因工程菌的構(gòu)建重組前胰島素ChainBChainCChainA重組人胰島素基因工程菌的構(gòu)建重組前胰島素ChainBCha11人胰島素原表達法基因工程菌的構(gòu)建戰(zhàn)略：tacb-GalCpeptideoriAprMetMetMMNCBpeptideApeptide人胰島素原的cDNA重組人胰島素原轉(zhuǎn)化分離純化人胰島素原表達法基因工程菌的構(gòu)建戰(zhàn)略：tacb-GalCp12人胰島素原表達法表達產(chǎn)物的后處理路線：SSSSCN胰蛋白酶CpeptideApeptideBpeptideMRRKRCNBrRSSSSCNNCSSSSR羧肽酶BB鏈中第22位上的Arg和第29位上的Lys由于良好折疊的原因，對胰蛋白酶不敏感人胰島素原表達法表達產(chǎn)物的后處理路線：SSSSCN胰蛋白酶C13人胰島素原表達法獲取人胰島素cDNA，克隆在含有Ptac和-gal基因的表達型載體上，兩基因通過Met的密碼子連接通過CNBr裂解法回收胰島素原蛋白體外折疊

胰蛋白酶去除C鏈，余下完整的A鏈和C末端帶一Arg的B鏈

羧肽酶獲得人胰島素結(jié)果：由于有C鏈的存在，胰島素原在體外能形成天然的空間構(gòu)象，使得正確折疊率達80％，成本為$50/g人胰島素原表達法14Materials1.RestrictionenzymesfromNEBandMBIFermentas2.PCRpurificationandgelextractionkitsfromQiagen3.pCR-BluntII-TOPOkitandfromInvitrogenTop10competentcells4.E.ColiBL21(DE3)GoldfromStratagene5.E.coliGM2163(dam-anddcm-)fromNEB

6.E.ColiSF120fromProf.GeorgeGeorgiou’slaboratory7.BovinetrypsinogenandthrombinfromSigma8.HisTrapFFcrudeandfromAmershamHiTrapNHS-activatedHPcolumns9.AntibodiesusedforELISAfromRoche10.StandardHumanproinsulinfromNIBSC11.MaxisorpRELISAplatesfromNunc,Copenhagen.12.ProteinmassstandardPeqGoldusedforSDS–PAGEfromPeqlab13.Mark12TMmarkerfromInvitrogen14.NuPAGE(4–12%)Bis–TrisgelsfromInvitrogen15.ZipTipC18fromMillipore16.MicroBCATMproteinassaykitfromPierce17.RP-HPLCNucleosilC18columnfromMachereyNagel,GermanyFrompage11to13and15

AjamaluddinMalik,MarcoJenzsch,AndreasLubbert,RainerRudolph,BrigitteSohlingProteinExpressionandPurification55(2007)100–111Materials1.Restrictionenzymes15Constructionoftheecotin–proinsulinexpressionplasmidpET20b-proinsulinThesourceofproinsulingeneforwardprimerreverseprimerPCRpCR-BluntII-TOPOkitsequencetransformE.ColiGM2163(dam-anddcm-)Constructionoftheecotin–pro16pEGP1BspEISalI(containingtheecotin-pepsinogenfusionproteingene)

(胃蛋白酶原)thepepsinogenfragmentwasremovedencodedE.coliecotintransformE.coliGM2163(dam-anddcm-)pEG-PIdigestBspEISalIdigestdephosphorylateandpurify.purify.ligateE.coliBL21(DE3)GoldpEGP1BspEISalI(containingth17pET-20bFrompET-20bFrom18forwardprimer5’-GGT

TCC

GGA

TCT

GGT

TCT

GGT

TCT

CTGGTCCCC

GlySerGlySerGlySer

GlySer

LeuVal

Pro

CGCGGTAGTCACCACCACCACCACCACCGT

TTTGTG

ArgGlySerHis

HisHisHisHisHis

Arg

AACCAACACCTGTGC

GGC-3’

Proinsulinreverseprimer5’-AGTGTCGACTTAGTTGCAGTAGTTCTCCAGCTG

GTA-3’

Ter

ProinsulinTCC

GGAGTCGACBspEISalIsequenceofprimerforwardprimer5’-GGTTCCGGAT19Schematicpresentationoftheecotin-proinsulinfusionproteinclonedinpTrc99a1-21：E.coliecotinsignalsequence22-162：E.coliecotin163-174：(GlySer)6

linker175-180:LVPRGSthrombin(凝血酶)cleavagesite181-186:(His)6187:Argtrypsin(胰蛋白酶)cleavagesite188-273:ProinsulinSchematicpresentationofthe20pCR?-BluntIITOPO?pCR?-BluntII21構(gòu)建胰島素基因工程菌ppt課件22RestrictionenzymesBspEI5'...T^CCGGA...3'

3'...AGGCC^T...5'

SalI5'...G^TCGAC...3'

3'...CAGCT^G...5'

EcoRI5'...G^AATTC...3'

3'...CTTAA^G...5'

RestrictionenzymesBspEI23E.coliGM2163(dam-anddcm-)

damdcm:甲基轉(zhuǎn)移酶保護DNA序列不被甲基化，提高限制性內(nèi)切酶正確識別酶切位點的機率E.coliBL21(DE3)GoldMoreLikeaMammalproduceanincreasedamountofrareE.colitRNAsthatcorrespondtocodonsusedmorefrequentlybyotherorganisms.Thismodificationovercomesexpressionproblemsduetocodonbias[1].提高E.colitRNAs結(jié)合在其他生物體里廣泛使用但在E.coli中很少使用的密碼子的機率?？朔嗣艽a子偏愛性造成的表達問題。E.coliGM2163(dam-anddcm-24pEGP1pTrc99a[2]pTrc-eco-peps(abbreviatedaspEGP1)ExpresspepsinogenfusedtoE.coliecotin[1][1]:vorgelegtderAnovelstrategyfortheperiplasmicproductionofheterologousproteinsinE.coliT7表達系統(tǒng)IPTG誘導pEGP1pTrc99a[2]pTrc-eco-peps(25CurrentProtocolsinMolecularBiologyJohnWileyandSons,Inc.Chapter1Escherichiacoli,Plasmids,andBacteriophagesSectionIIVectorsDerivedfromPlasmidsUnit1.5IntroductiontoPlasmidBiologyFigure1.5.11CurrentProtocolsinMolecular26ConstructionofpEGP1plasmidFrompage23topage26E.coliJM83ThesourceofecotingenePCRpHQPEX-30-5[1]5’end(Gly-Ser)3Primerforwardprimer3’endofpepsinogen(His)6primerreverseprimerPCRforwardprimerThesourceofpepsinogenConstructionofpEGP1pla27pCR-BluntII-TOPOkitEcoRI-BspEIBspEI-SalIpurifiedbyagarosegelelectrophoresis(瓊脂糖凝膠電泳)pTrc99aEcoRI-SalIdephosphorylatedligate

pEGP1pTrc-eco-pepspCR-BluntII-TOPOkitEcoRI-B28(A)Structuralmodeloftheecotindimer.(B)Schematicdrawoftheecotin-pepsinogenfusionproteinaspresentinpEGP1.1–20:ecotinsignalpeptide21-162:matureecotin163-174:GS6linker175-546:maturepepsinogen546-552:His6(A)Structuralmodeloftheec295’-TTCTAAGAATTCGAAGGAGATATACATAAT

GAAGACCATTCTACCTGCA-3’

ecotinsignalpeptidesequenceofprimer3’endofecotin-(Gly-Ser)3

primerreverseprimerforwordprimer5’-ATCTTATCCGGAACCAGAACCAGA

Gly

SerGly

Ser

GlySerACCACGAACTACCGCATTGTCAATTT-3’Glyecotin5’end(Gly-Ser)3primerforwordprimer5’-GTATATGTCGAC

TTAGTGGTGGTGGTG

Ter

HisHisHisHisGTGGTGAGCCACGGGGGCCAGACC-3’

HisHispepsinogen5’-GTTATATCCGGATCTGGTTCTGGTTCTGGT

Ser

GlySerGlySerGlySerGly

TACAAGGTCCCCCTCA-3’

pepsinogen3’endofpepsinogen(His)6primerreverseprimerGAATTCTCCGGAGTCGACBspEIEcoRISalI5’-TTCTAAGAATTCGAAGGAGAT30E.coliecotingeneGENBANKACCESSION:M60876J05757

1cgtagaggatcaaaagagtagcgggaagcgtggcaaaaacgggcttttgctcacatttca61aattggttataaatatatttatatagcgattgattcaccagagatatttctgctggtttg121ctctctcattagaatttaacactaaaagagcaggtaaaattgtctgaatgttctttaagt181tattcataaagcaaattaataaatctgatgaatatgttaaccttcagcgacatcatcggt241gaaaacctataaatgaagaaggaaagcaaa

aaaatgaaga

ccattctacctgcagtattg301tttgccgctttcgctaccac

ttccgcctgg

gcggcagaaagcgtccagccactggaaaaa

361atcgcgccttatccacaagctgaaaaagggatgaagcgtcaggtgattcagttaaccccg

421caagaagatgaatctaccctgaaagtagaactgttaatcggtcagacgctggaagtcgat481tgcaatttgcatcgtctcggcgggaagctggaaaacaaaacgctggaaggctggggctat

541gattattatgtctttgataaagtcagttccccggtttcaacgatgatggcctgcccggat

601ggcaagaaagagaagaaatttgtcaccgcgtatctgggcgatgctggaatgctgcgttac

661aacagcaagctgccgatcgtggtgtatacgccagacaatgtagatgtgaagtaccgcgtc

721tggaaggcggaagagaaaattgacaacgcg

gtagttcgctaaactgccgtgaagtgcggc781accccgtaggtcagacaaggcggtcagcgatccgacatccaacgcccgagccggttgcct841gatgcgacgctggccgtcttatcaggcctacaccgctgtgaagtgcggcaccccgtaggt901cagacaaggcggtcacgcgcatccgacatccaacgcccgagccggttgcctgatgcgacg961ctggcctcttatcaggcctacaccgctgtgaagtgctccaccccgtaggtcggataaggc1021ggtagcgatccgacatctaacgcaagccgttgcecotinsignalpeptidemature

ecotinE.coliecotingene1cgt31E.coliecotin1.Ecotinhasnoroleinthehostmetabolism.Duetoitsrelativelysmallsize,themetabolicburdenislow[1].2.Prokaryoticsignalpeptidetransportthefusionproteins

totheperiplasm(周質(zhì))[2].3.Playanimportantroleintranslocation[2]

.4.Ecotinisabroad-rangeserine-proteaseinhibitor，whenrattrypsinogen