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时间:2020-03-24
《重组人肝刺激物在原核细胞中的表达与纯化.doc》由会员上传分享,免费在线阅读,更多相关内容在工程资料-天天文库。
1、研究论文重组人肝刺激物在原核细胞中的表达与纯化杜海军.孙红柳.陈莉•安威•酋祁医科大学细熬生物呆.北京1M054m要,利用基因童组技术.构建成人肝刺激因子(hHSS)和谷胱甘肽转移RKGST)融合衷达直体.转化大肠杆菌BL-21(DE3)・以His-Tag亲和层析纯化衷达产物.FaciorXa切割分肉hHSS单体.并检测其生物学活性。结果显示・在pET-42a衷达体系中hHSS以可溶性蛋白和包涵体两种形式存在.GST-hHSS表达Received2001-06-22Accepted2001-08-18Thiaworkwassuppor
2、tedbytheNauonaiNaturalScienceFoundationofChina(GrantNo.39800275)Correspondrngauthor.Tel:丄86-10-63291?211Fax:-F86-10-63051130E-maiLanwei@cpums,edu.cn占菌体可溶性董白的3U?:}FaciorXa切割GST与hHSS之间肽IB,得到33和15kD两条蛋白带•经Western杂交证实33kD条带为GST・而15kD条带的分子燉与hHSS基因序列推测蛋白结果相符.经His・Tag再次纯化可获得hH
3、SS单体.初步证实重组hHSS具有促进肝癌细胞增殖活性。关■词:人肝剌激因子.融合蛋白.原核表达学科分类号:Q435:R333.4ProkaryoticexpressionandpurificationofhumanhepaticstimulatorsubstanceDUHai-Jun,SUNHong-Liu,CHENLi,ANWei*Depart?ntrntofCellBiologytCapitalUniversityofMedicalSciences4、ilityofprokaryoticexpressionofhumanhepaticstimulatorsubstancehHSSgenewasinsertedinthedownstreamofglutathionS-transferase(GST)inapET-42aexpressionvectorandrecombinantGST-hHSSfusionproteinwasexpressedunderIPTGinductioninBL-21(DE3)cells.TherecombinantHSSwaspurifiedw5、ithHis•Tagaffinitychromatographytanditsbioactivitywasanalyzed.TheresultsshowedthatGST-hHSSfusionproteinwasexpressedbothasasolubleorainclusivebodyinbacterialcytosol.ThesolubleGST-hHSSexpressionreachedupto30%ofthewholesolubleproteinofbacteriaasdeterminedbydensitometry.T6、hecleavageofGSThHSSfusionproteinwithFactorXaproducedtwofragmentsoftheprotein,whichsized33and15kD・respectively.Themolecularweightofre・combinanlUSSproteinwasidenticaltotheoreticaldeductionbasedontheDNAsequences.Theproteinhomologyof15kDhHSScouldbeefficientlyelutedoutafterF7、aciorXacleavage.ItisfurtherindicatedthattherecombinanthHSSisableioproliferatehepatomacellsofBEL-7402inthepreliminaryexperiments.Keywords:humanhepaticstimulatorsubstance;prokaryoticexpression;fusionprotein肝刺激因子(hepaticstimulatorsubstance^HSS)是广泛存在于初断乳动物肝脏或再生肝脏中的一种促肝细胞增殖因8、子D'2].有关HSS的研究虽取得了一定进展口“:,但由于它在肝细胞中含量极低,提取和纯化十分困难,至今仍未获纯品,故迄今许多实验仍采用部分纯化的HSS。这不仅严重制约了HSS作用机制的深入研究,也影响了其在临床的实际应
4、ilityofprokaryoticexpressionofhumanhepaticstimulatorsubstancehHSSgenewasinsertedinthedownstreamofglutathionS-transferase(GST)inapET-42aexpressionvectorandrecombinantGST-hHSSfusionproteinwasexpressedunderIPTGinductioninBL-21(DE3)cells.TherecombinantHSSwaspurifiedw
5、ithHis•Tagaffinitychromatographytanditsbioactivitywasanalyzed.TheresultsshowedthatGST-hHSSfusionproteinwasexpressedbothasasolubleorainclusivebodyinbacterialcytosol.ThesolubleGST-hHSSexpressionreachedupto30%ofthewholesolubleproteinofbacteriaasdeterminedbydensitometry.T
6、hecleavageofGSThHSSfusionproteinwithFactorXaproducedtwofragmentsoftheprotein,whichsized33and15kD・respectively.Themolecularweightofre・combinanlUSSproteinwasidenticaltotheoreticaldeductionbasedontheDNAsequences.Theproteinhomologyof15kDhHSScouldbeefficientlyelutedoutafterF
7、aciorXacleavage.ItisfurtherindicatedthattherecombinanthHSSisableioproliferatehepatomacellsofBEL-7402inthepreliminaryexperiments.Keywords:humanhepaticstimulatorsubstance;prokaryoticexpression;fusionprotein肝刺激因子(hepaticstimulatorsubstance^HSS)是广泛存在于初断乳动物肝脏或再生肝脏中的一种促肝细胞增殖因
8、子D'2].有关HSS的研究虽取得了一定进展口“:,但由于它在肝细胞中含量极低,提取和纯化十分困难,至今仍未获纯品,故迄今许多实验仍采用部分纯化的HSS。这不仅严重制约了HSS作用机制的深入研究,也影响了其在临床的实际应
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