猪巨细胞病毒gb基因的原核表达与重组蛋白间接elisa方法的建立(6113)

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1、扬州大学硕士学位论文2个表达载体(分别命名为pGEX—gBl和pGEX-gB2)，测序结果表明与在GenBank已收录的序列同源性分别为99．4％和100％。将重组质粒转化入大肠杆菌BL21(DE3)中，经IPTG诱导，由SDS-PAGE电泳分析证实gBl和gB2基因获得了表达，蛋白分子量分别约为39KDa和36KDa，分别命名为GST-gBl和GST-gB2。其中重组蛋白GST-gBl以包涵体的形式存在，GST-gB2以可溶性蛋白形式存在。经Western．blot分析证实表达的重组蛋白抗原性良好。GST-gBl

2、用切胶纯化法，GST-gB2通过GSH—Sefinose柱纯化。以纯化的重组蛋白GST-gB2作为诊断抗原建立间接ELISA检测方法。运用方阵试验法确定最适抗原包被浓度为2gg／nm，血清的最佳稀释度为1：100，阳性临界值为0．224，阴性临界值为O．189，建立的ELISA方法的灵敏度为1：3200。关键词：PCMVgB基因；同源性；原核表达；间接ELISA赵锋祥猪巨细胞病毒gB基因的原核表达及重组蛋白间接ELISA方法的建立3ProkaryoticexpressionofgBgeneofPorcineCyto

3、megalovirusandDevelopmentofindirectELISA●●l●‘0IrecomblnantproteinM．D．Candidate：FengxiangZhaoSupervisor：Prof．JianshengXuAbstractPorcinecytomegalovirusinfection(PCMVI)isanoppommisticinfectionineasyinfectedherds，itcancauseclinicalsymptomasfetusandyoungswinedied，r

4、hinitis，pneumonicfeverandretardedgrowthandSOon．Itsinfectedanimallimitedinswine．PCMVwasfirstdiscoveredisolatedbyDoneinUKinl955．Thediseaseisepidemicinallovertheworld，thediseaseincidenceisespeciallyhi曲inUK，AmericaandJapan．Ininfectedherds，morethan90％ofswinesareser

5、opositive．Itindicatedthatthediseaseinfectionisubiquitouswitllherds．AccordingtothegBgeneofPCMVfromGenBank，twopairsofprimersweredesignedbyselectingconservedistrictinPCMVgBsequence，theoneisdetectedprimer,theotheristotallengthofPCMVgBgene．Usingdetectedprimertodete

6、ctnasalswabspecimens丘Iom‰gzllou，Huaian，YanchengandHaianthroughPCR．Theresultsshowedthat23of53clinicalsampleswerepositive谢tllapositiverateof43．4％．Usingtotallengthprimeramplifiedtotalsequence，itslengthWaS2627bp．ThegenesequenceWasclonedintopUCm—Tvector．Thesequenci

7、ngresultshowedthatthenucleotidehomologywas97．8％-99．4％andaminoacidhomologyWas97．1％一99．3％comparedwiththePCMVgBfromGenBank．ThecladogramwasanalyzedbyDNAStars，The扬州大学硕士学位论文4resultshowedthatthesampleofthisstudywasinthesamecluster、析thUKstrain．GermanystrainandHunanstr

8、aininourcountry．ThebiologycharacteristicsandsignalpeptideofPCMVgBantigenproteinwereanalyzedbybioinformaticssoftwareandDNAStars．TheimmunityreactivityofPCMVgBantigenproteinwaspredict