第44卷第4期第406页2015年8月华中科技大学学报(医学版)ActaMedUnivSciTechnolHuazhongV0I．44No．4P．406Aug．2015结核分枝杆菌抗原HBHA在耻垢分枝杆菌中的重组表达和鉴定*余菲，寻阳，田茂鹏，李华秀，朱可橙，袁野，滕新栋，范雄林华中科技大学同济医学院附属协和医院，第一临床学院，武汉430022。华中科技大学同济医学院基础医学院病原生物学系，武汉430030摘要：目的构建高效表达结核分枝杆菌抗原肝素结合血凝素(HBHA)的重组耻垢分枝杆菌。方法针对结核分枝杆菌H37Rv株HBHA的编码基因Rv0475设计特异引物，利用PCR技术从细菌基因组中获取目的基因，并克隆人pET一30b(+)载体构建重组原核表达质粒pETHBHA，经IPTG诱导pETHBHA转化的重组E．coliBL21(DE3)细菌，Ni柱纯化携带C一端His标签的HBHA蛋白，并制备鼠源多克隆抗体。PCR扩增含自身启动子和调节序列的Rv0475基因并克隆人大肠埃希菌一分枝杆菌穿梭载体pMV261，构建重组分枝杆菌表达质粒pMVHBHA，并经酶切鉴定证实后，电转化人耻垢分枝杆菌，涂布含卡那霉素的7Hll平板，挑选抗性克隆并扩大培养，15SDS~PAGE和Westernblot鉴定重组耻垢分枝杆菌的表达。结果分别成功构建重组原核表达质粒pETHBHA和重组分枝杆菌表达质粒pMVHB—HA；重组蛋白HBHA在大肠埃希菌中成功表达并纯化后，BCA蛋白浓度测定试剂盒测定浓度为1．9125btg／gL，用其制备鼠源多抗；证实HBHA利用其自身的启动子和调节序列在重组耻垢分枝杆菌中表达。结论成功构建过表达HBHA的重组耻垢分枝杆菌，为研究该蛋白参与结核病的致病机制和研发新型疫苗和诊断试剂奠定了基础。关键词：肝素结合血凝素(HBHA)；耻垢分枝杆菌；结核病；表达中图分类号：R378．91DOI：10．3870／j．issn．1672—0741．2015．04．007RecombinantExpressionandIdentificationofAntigenHeparin—bindingHemagglutinin(HBHA)ofMycobacteriumTuberculosisinMycobacteriumSmegmatisYuFei．XunYang．TianMaopengetal1UnionHospital，TongjiMedicalCollege，HuazhongUniversityofScienceandTechnology，Wuhan430022，ChinaDepartmentofPathogenyMicrobiology，TongjiMedicalCollege，HuazhongUniversityofScienceandTechnology，Ⅱ430030，ChinaAbstractObjectiveToestablishrecombinantMycobacteriumsmegmatis(Msmegrnatis)strainhighlyexpressingantigenheparimbindinghemagglutinin(HBHA)ofMycobacteriumtuberculosis(M．tuberculosis)．MethodsThegeneRv0475，encodingHBHAof^tuberculosisH37RvstrainwasobtainedbvPCRandclonedintothevectorpET一30b(+)，toconstructtherecom—binantprokaryoticexpressionplasmidpETHBHA．RecombinantproteinHBHAwaspreparedasC—terminalHistaggedfusionproteinfrompETHBHAtransformedE．colBL21(DE3)strainafterinductionwithIPTGandwasusedtopreparemouseorig—inalpolyclonalantibodies．Rv0475containingitspromoterandregulatorysequencewasamplifiedbyPCRandclonedintoEcoli—I~ycobacteriumshuttlevectorpMV261．namelypMVHBHA．pMVHBHAwastransformedintosmegrnatisafteri-dentificationbyrestrictionenzymeanalysis．andrecombinantM．smegmatisstrainwascultivatedon7H11plate．Thekanamycinresistantcloneswereidentifiedwith15SDS-PAGEandWesternblotting．ResultsRecombinantprokaryoticexpressionplas—midpETHBHAandrecombinantmycobacteriaexpressionplasmidpMVHBHAwereconstructedsuccessfully．TherecombinantproteinHBHAwassuccessfullyexpressedandpurifiedinrecombinantE．colistrain．Theproteinconcentrationwas1．9125g／“LdeterminedbyamethodcalledBCAProteinAssayKit，andtheproteinwasusedtopreparemouseoriginalpolyclonalanti—bodies．RecombinantproteinHBHAwasexpressedinrecombinantMsrnegmatisusingitsownpromoterandregulatoryse—quences．ConclusionRecombinantMsrnegmatisstrainexpressingHBHAwasestablishedsuccessfully，whichprovidesfounda—tiontostudypathogenesisoftuberculosisanddevelopmentofnewvaccinesanddia...
