pGEX┐e23scFvPE40融合基因的构建及其在大肠杆菌中的表达

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1、pGEX┐e23（scFv）PE40融合基因的构建及其在大肠杆菌中的表达作者：王慧，韩苇，颜真，张英起【关键词】单链抗体关键词:单链抗体，免疫毒素，基因表达摘要:目的在大肠杆菌中表达抗原癌基因c-erbB-2表达产物p185的单链抗体e23（scFv）/假单孢菌属外毒素活性片段PE40免疫毒素的融合蛋白，为乳腺癌、胃癌等多种c-erbB-2呈过量表达的恶性肿瘤的免疫治疗奠定基础.方法去除克隆在真核表达载体pLNCX中的e23（scFv）PE40基因5′端编码信号肽的核苷酸序列，并将改建后的融合基因克隆到原核融合表达载体pGEX-4T中表达.结果序列测定表明.改建

2、后的抗p185e23（scFv）/PE40序列正确.融合基因经IPTG诱导表达4h后，经SDS-聚丙烯酰胺凝胶电泳分析，在Mr90000处出现一条新生蛋白带，表达量约占菌体总蛋白的15%.结论成功改建并在原核中表达了抗p185e23（scFv）/PE40融合蛋白.INTRODUCTIONThec-erbB-2oncogeneislocatedin17q21，anden-codes185ku（p185）transmembranereceptor（HER2）thatbelongstotheepidermalgrowthfac-torreceptorfamilyand

3、hasintrinsictyrosinekinaseactivity7　　［1-4］.HER-2geneisactivatedbyamplifica-tionandoverexpressionofitsproteinproduct，whichleadstoitsconstitutiveactivationresultinginunregulatedcellgrowth，thenprocessestumorigenicortransformingactivity　　［5-6］.IthasbeenreportedthatHER2isoverexpressedin25

4、%～30%ofbreastcancers，urinarybladdercancerandgastriccarcino-ma.Thestudyofbreastcancersuggeststhatc-erbB-2hasadirectroleinthepathogenesisandclini-calaggressivenessofc-erbB-2overexpressingtu-mors　　［7，8］.Thepatientswithc-erbB-2positivehavehigherrisksofmetastasisandrecurrence，shorternon-t

5、umorlifespanandtotallifespan　　［9-13］.Theanti-p185humanizedmonoclonalantibodyHerceptincaneffectivelykillseveralc-erbB-2positivecelllinesanddramaticallyinhibitthegrowthofxenograftoverexpressingc-erbB-2innudemouse.Asare-sult，HerceptinhadbeenapprovedbyFDAofUSAtobeusedinthefirstlinechemot

6、herapyasanad-junctiveagentinc-erbB-2positivemetastasisbreastcancersinSeptember，19987　　［14］.Inourstudy，were-constructedasingle-chainchimericimmunotoxintermede23（scFv）PE40.Ine23（scFv）PE40，thevariabledomainofthelightchainofmAbe23wasattachedthroughapeptidelinkertothevariabledo-mainoftheh

7、eavychain，whichinturnwasfusedtodomainⅡandⅢofPE.ThenweexpressedthisimmunotoxinsuccessfullyinaprokaryoticfusionproteinexpressionvectorpGEX-4T.　　MATERIALSANDMETHODS　　MaterialspLNCX-e23（scFv）PE40thatconsistedofe23，ananti-p185singlechainvariablefragment（scFv）andPE40waspresentedbyProfessor

8、YangAn-Gang.