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《豬巨細胞病毒gb基因的原核表達與重組蛋白間接elisa方法的建立(6113)》由會員上傳分享，免費在線閱讀，更多相關(guān)內(nèi)容在學術(shù)論文-天天文庫。
1、揚州大學碩士學位論文2個表達載體(分別命名為pGEX—gBl和pGEX-gB2)，測序結(jié)果表明與在GenBank已收錄的序列同源性分別為99．4％和100％。將重組質(zhì)粒轉(zhuǎn)化入大腸桿菌BL21(DE3)中，經(jīng)IPTG誘導，由SDS-PAGE電泳分析證實gBl和gB2基因獲得了表達，蛋白分子量分別約為39KDa和36KDa，分別命名為GST-gBl和GST-gB2。其中重組蛋白GST-gBl以包涵體的形式存在，GST-gB2以可溶性蛋白形式存在。經(jīng)Western．blot分析證實表達的重組蛋白抗原性良好。GST-gBl
2、用切膠純化法，GST-gB2通過GSH—Sefinose柱純化。以純化的重組蛋白GST-gB2作為診斷抗原建立間接ELISA檢測方法。運用方陣試驗法確定最適抗原包被濃度為2gg／nm，血清的最佳稀釋度為1：100，陽性臨界值為0．224，陰性臨界值為O．189，建立的ELISA方法的靈敏度為1：3200。關(guān)鍵詞：PCMVgB基因；同源性；原核表達；間接ELISA趙鋒祥豬巨細胞病毒gB基因的原核表達及重組蛋白間接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謝tllapositiverateof43．4％．Usingtotallengthprimeramplifiedtotalsequence，itslengthWaS2627bp．ThegenesequenceWasclonedintopUCm—Tvector．Thesequenci
7、ngresultshowedthatthenucleotidehomologywas97．8％-99．4％andaminoacidhomologyWas97．1％一99．3％comparedwiththePCMVgBfromGenBank．ThecladogramwasanalyzedbyDNAStars，The揚州大學碩士學位論文4resultshowedthatthesampleofthisstudywasinthesamecluster、析thUKstrain．GermanystrainandHunanstr
8、aininourcountry．ThebiologycharacteristicsandsignalpeptideofPCMVgBantigenproteinwereanalyzedbybioinformaticssoftwareandDNAStars．TheimmunityreactivityofPCMVgBantigenproteinwaspredict
