茨城病病毒S7基因的克隆表達(dá)及其抗體間接ELISA檢測(cè)方法的建立.pdf

1、東北農(nóng)業(yè)大學(xué)碩士學(xué)位論文茨城病病毒S7基因的克隆表達(dá)及其抗體間接ELISA檢測(cè)方法的建立姓名：張文龍申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：預(yù)防獸醫(yī)學(xué)指導(dǎo)教師：王君偉20080620AbstractCloningandExpressionof$7GeneofIbarakiVirusandtheEstablishmentofanIndirectELISAforDetectionofAntibodyagainsttheVirusAbstractIbarak

2、idisease(IBAD)isallinfectiousdiseasecausedbyIbarakivirus(IBAV)manifestingitselfasacutefever1barakiviruswasamemberofgenusOrbivirusfamilyReoviridaeItprevailedinmanycountriesandareasinsoutheastAsiaAustraliaandAmericaandwasd

3、esignatedaslistBinfectiousdiseaseinListAandListBDiseasesfortheAnimalsImportedfornlotherCountriesintothePeople’sRepublicofChinaNeutralizationtestcomplementfixationtestandagardiffusionreactionwereusedworld—widelytodetectan

4、tibodiesagainstlbarakivirusinserumfordiagnosisofIbarakidiseaseIncurrentstudythefull—lengthS7geneofIbarakivirusencodingthemajorcapsidprotein。VP7，oflbarakivirusNo2strainwasamplifiedbyreversetranscriptpolymerasechainreactio

5、n(RTPCR)TheS7genewastheninsertedintopET28a()prokaryoticexpressionvectorandpFastBacHTatranspositionvectorrespectivelyMeanwhile，theantigenicityandhydrophobicityofVP7wereanalysedbysoftware，andthepartofS7geneencodingtheparto

6、fVP7withbestantigenicityandhydrophilicitywassubclonedintopET32a()prokaryoticexpressionvectorThepositivecloneofrecombinantprokaryoticexpressionvectorswereidentifiedandusedtotransformtheEscherichiacobBL21(DE3)competentcell

7、Therecombinantfull—lengthortruncatedVP7wereexpressedafterinductionThecorrectiverecombinanttranspositionvectoridentifiedwasusedtotransf01“111theEscherichiacoliDH10BaccompetentcellThepositiverecombinantshuttlevector(Bacmid

8、)involvingfulllengthS7genewasusedtotransfecttheSt9cells，andtherecombinantbaculoviruseswasharvestedThen，VP7proteinwasexpressedbySf9ceilsinfectedbyrecombinantbaculovirusesTheexpressionoftherecombinantVP7proteinswasidentifi

9、edbySDSpolyaerylamidegelelectrophoresisandWesternblotTheexpressionprotocolswereoptimized，andtherecombinantproteinswerepurifiedbyNi2chelatingaffinitychromatographyTherecombinantVP7proteinsexpressedbyEscherichiacoliwereuse

10、dtoestablishindirectELISATheresultsdemonstratedthatthetruncatedVP7wasbetterthanfull1engthVP7proteinascoatingantigenThenweoptimizedindirectELISAprotocol。Aseriesoftestswerecardedouttodeterminetheoptimalcoatingsituation，con