猪脑心肌炎病毒gxlc株结构蛋白vp1的原核表达与间接elisa方法的建立

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1、的防控技术奠定了基础。关键词：脑心肌炎病毒；猪；结构蛋白VPl；原核表达；间接ELISA；血清学调查IlPROKARYOTICE)

2、omyocarditisVirus(EMCV)belongstot11egenus国以汤v妒凇ofthe‰ily尸勿D磁口V护姚8，谢lich

3、cancauseaninfectiousdiseaSeCharacterizedbyacuteencephalitisandmyocarditisinpigletsandseVerereproductiVefailureinsows．ApairofprimerswasdesignedaccordingtothestructureproteinVplgenesequenceofEMCVto锄pli分an831bp丹agmentbyreVersetraJlscriptionpolymerasechainreaCtion(

4、Rrr-PCR)．Theamplified矗阻gmentwasclonedin垃theexpressionVectorpE■32a(+)垃aCquiretherecombinantplasmidpET-32a—VP1．ARerident涵cationbyrestrictionanalysisandsequencing，therecombinantplasmidpET-32a—VPlwastransferredtoEcD疗BL21(DH3)competentcellsandthee)中ressionof如sionpro

5、teinVP1wasinducedbyIPTGTheresultofSDS-PIAGEanalysisshowedthatthe血sionproteinVPlwithabout52Kuwassuccessmllyexpressedinthefo册ofinclusionbody．Theresultofwestem-blotshowedthatt11erecombinamproteinVP1couldbespecificallyreactedwiththeEMCVpositiVesemmofpigs．Anindirect

6、ELISAassayfordetectionofEMCVantibodywasIlldevelopedwithtllepurifiedrecombinantproteinVP1asantigen．7I’heoptimalconditionsformeindirectELISAwereobtainedbyoptimization．Thecoatingliquidwascarbonatebu舵rofpH9．6withO．05mol／Lconcentration；theconcentrationofcoatingantig

7、enwasO．625ug／mLlayingat4℃ovemightaRer2hat37℃；thedilutionofsemms锄pleswas1：40andthereactivetimewas60minutes；t11edilutionoftllerabbit—anti—swineantibody(thesecondantibody)labeledw胁m冲was1：6000w沛workingconditionat37℃for3Ominutes；mecolorationtimeof圣Y堕Q!丝曼was15minutes

8、；thethresoldvaluebetweennegatiVeandpositiVeserumsamplewasO．33．Thespecifici够analysisshowedthatt11erecombin锄tproteinVPlcouldnotcross—reactedwiththepositiVeserumtoclassicalswinefeVervims(CSFV)，porcinecircoVims够pe2(PCV2)，porcinereproduCtiVeandrespirato巧syndromeVill

9、ls(PItRSV)，pseudorabiesVirus(PRV)orfoot．and—mouthdiseaseVirus(FMDV)．Atotalof2228semmsamplesofpigs矗’omGuangxiregioniIl20lO．20l1weredetectedforEMCVantibodybytheestablishedindi