用于快速检测猪瘟抗体双功能分子的构建及表达-预防兽医学专业论文.docx

达产物大部分是以不溶的包涵体形式存在；在37℃，IPTG浓度为0．3mmol／L，诱导时间为3h的条件下重组蛋白可获得最佳表达。分别用6×His单克隆抗体和CSFV高免血清对表达的重组蛋白进行Western．blot检测，结果显示：重组蛋白与6xHis单克隆抗体及CSFV阳性血清均呈阳性反应，呈现清晰的特异性反应条带，而空载体诱导菌的菌体蛋白无上述反应条带。表明所表达的重组蛋白是特异的。3、2E8mE2重组蛋白包涵体的复性及检测超声波破碎处理重组菌，收集重组蛋白包涵体，用0．5％TritonX-100和lmol／LNaCI洗涤包涵体后用尿素溶解，利用Ni．NTA树脂对溶解的蛋白进行亲和层析纯化，然后利用谷胱甘肽再氧化法对纯化的变性溶解蛋白进行复性，最后利用间接ELISA方法测定复性后的重组蛋白与CSFV高免血清的反应性，结果显示：用洗涤的方法初步纯化后，目的蛋白纯度已经能达到86％，再通过亲和层析法的进一步纯化，蛋白纯度高达92％，纯化后的重组蛋白用谷胱甘肽再氧化法进行复性，复性后的重组蛋白与CSFV高免血清的反应性明显高于复性前的变性蛋白，提高了约3-4倍，表明复性是成功的。．关键词：猪瘟单链抗体双功能分子原核表达包涵体复性ⅡCONSTRUCTIONANDEXPRESSIONOFBIFUNCTIONAILMOLECULEOFDETECTINGCSFANTIBODYQUICKLYClassicalswinefever(CSF)causedbyClassicalswinefevervirus(CSFV)isakindofurgentfebricityandfataldisease．Ithasmadeagreatlossinworld’Sswineindustry．monitoringregulaurlyCSFantibodyhasimportantsignificancewithcontrolofCSF,atpresent，becausethereisstillnoaconvenientandfastandstabledetectivemethodofCSFantibodytorefertoprimarylevel，thestudyofbifunctionalreagentofdetectingCSFantibodyquicklyperformedaccordingtotheprincipleofhaemag百utination．Themainresultofstudyasfollow：1．Splicingofbifunctionalmoleculeandconstructionofprokaryoticvector2E8ScFv(ScFvgeneagainstHantigenofhumanerythrocyte)andmE2(mainantigenregionofE2geneofCSDweresplicedbyoverlapextension(SOE)PCRandassembledbifunctionalmoleculenamed2E8mE2，thenitwasdonedintoPMDl8-TSimplevector,thesequencedrecombinantbifunctionalmoleculewassub—clonedintoprokaryoticvectorPET-DsbAandindentifiedbyPCRandenzymedigest．Result showedthatPCRandenzymeHIdigestdetecteda1125bptargetband．TheresultindicatedconstructionofprokaryoticvectorPET-DsbA-2E8mE2hadbeensuccessful．2．Expressionofthe2E8mE2recombinantproteinRecombinantvectorPE卫DsbA-2E8mE2wastransformedintotheBL21(DE3)PlysSEcolicell andexpressedbycommonmethod，sonicationofthebacterialcellsandsolubilityanalysisofrecombinantproteinbySDS—PAGE，thenchoiceofexpressionconditionsbygradsoftemperatureandIPTGconcentrationandinducetime．Resultindicatedtherecombinantproteinwas gainedhighexpressionintheformofinclusionbodyatthele