利用重组酵母菌生产人胰岛素.doc

利用重组酵母菌生产人胰岛素

摘要利用套叠PCR技术合成重组人胰岛素基因，并在A链和B链之间加入Kex2酶切位点。将合成的重组人胰岛素基因以正确的阅读框插入到组成型分泌表达载体pGAPZα-A的α-factor信号肽序列的下游，构建成pGAPZα-A/Insulin重组分泌表达载体。表达载体用BlnI线性化处理后电击转化毕赤酵母GS115感受态细胞，构建成分泌型表达Insulin的工程菌。SDS2PAGE和Native2PAGE的分析表明：蛋白在分泌表达过程中，加入Insulin的A链（2.4k）和B链（3.4k）之间的Kex2酶切位点发挥了作用，Kex2蛋白酶将A链和B链切开，在α-factor信号肽的作用下通过分泌途径将A链和B链分泌到胞外，同时A链和B链又以二硫键形成了高级结构。WesternBlot结果表明：重组蛋白能够与鼠抗人Insulin抗体发生特异性反应，具有与天然Insulin相同的免疫原性。通过单因素分析和正交实验法对巴斯德毕赤酵母的高密度发酵培养基进行了筛选和优化；对高效表达人生长激素的培养条件进行了优化；根据优化的摇瓶发酵结果进行了补料分批发酵；对表达产物进行了初步鉴定。

关键词：重组人胰岛素，毕赤酵母，分泌表达，培养基优化，晶体收集

Abstract：UsenestedPCRsynthesisofrecombinanthumaninsulingene,joinKex2cleavagesitesbetweentheAandBchains.ThesyntheticrecombinanthumaninsulingeneinthecorrectreadingframewasinsertedintotheconstitutivesecretoryexpressionvectorpGAPZα-Aα-factorsignalpeptidesequencePreparationtheconstructpGAPZα-A/Insulinrecombinantsecretoryexpressionvector.TheexpressionvectorlinearizationBlnItreatedelectroporationcompetentcellsofPichiapastorisGS115constructedexpressionofsecretoryInsulinengineeredbacteria.TheanalysisSDS2PAGEandNative2PAGEshowedthat:theproteinintheprocessofsecretionexpressionaddedInsulinAchain(2.4k)andKex2cleavagesitesbetweentheB-chain(3.4k)playedarole,Kex2proteaseoftheAandBchainscutthroughthesecretorypathwayintheroleoftheα-factorsignalpeptideoftheaandBchainssecretedintotheextracellulardomain,whilethea-chainandB-chaindisulfidebondformationYouyiadvancedstructure.WesternBlot:Theresultsshowthattherecombinantproteinwasabletospecificallyreactwithmouseanti-humanInsulinAntibodieshavingthesamenaturalInsulinimmunogenicity.BysinglefactoranalysisandorthogonalexperimentmethodofhighdensityfermentationofPichiapastorismediumscreeningandoptimization;optimizedcultureconditionsofthehigh-levelexpressionofhumangrowthhormone;accordingflaskfermentationoptimizationresul