鴨CD8α分子克隆與多克隆抗體制備及MHCⅠ和β2m的表達(dá).pdf

1、中國農(nóng)業(yè)大學(xué)碩士學(xué)位論文鴨CD8α分子克隆與多克隆抗體制備及MHCⅠ和β2m的表達(dá)姓名：廖定為申請學(xué)位級別：碩士專業(yè)：獸醫(yī)生物醫(yī)學(xué)指導(dǎo)教師：夏春20070501AbstractInordertogetproteincomplexesofMHCIB2mCD8forthefurtherstudiesofduckimmunerespone徹CTLrecognitionoffluevirustheproteinoftheduckCD8nchai

2、n(DuCD8a)andMHCI／IB2mWereexpressed，theantiDuCDSaployclonedantibodieswereproducedTheDuCDSawasamplifiedbyPCRmethodfromthecDNAPCRlibraryofPekingducLTheclonerecombinedvectorwereconslruclwiththePCRproductsandthepGEMTEasyVecto

3、rResultsshowthattheclonedDuCDSa正F205151)has645bpinI∞gIl，codingfora214aminoacidspeptideItsharesa927％aahomologywiththepublishedCD8asequenceofCairinamoschata(Muscovyduck)anda991％aahomologywitIIthatofotherPekingduckTheCD8ase

4、quencesofPekingDucktherearcthreepointmutationsitesAmongtheCD8agenesofPekingDucktherewerenlreepointmutationsites：A—123C，A一367_GT536_CThcmutationsitesofDucD8aarcmostlyinthepsheetsandCDRdistrictsTheexpressionplasmid(pQE30／D

5、uCDSa)containstheDuCD8aWasconstructedandtransformedintoEcolistrainJMl09，whichWasinducedbyIPTGtoexpressrecombinantproteinTheexpressedprotein(rDuCD8a)WaspurifiedandanalyzedbySDSPAGEInordertoproducepolyclonalantibodiestheBa

6、lb／cmicewasimmunizedbytherDuCD8aTheSDSPAGEanalysisshowedthatthemolecularweightis26kDaandthemountsoftheexpressedrDuCD8nis15O％intotalmassofbacterialproteinbythin—layerscanneranalysisThethreedimentional(3D)structuresoftheDu

7、CDgaproteinwereestimatedbyhomologymodelingThe3DstructureofDuCD8aproteinestimatedbyhomologymodelingshowstypicalIgVlikedomainconsistingofnineISsheetswithahelixThis3DstructureWaSsimilarashuman’sbutsomedifferenceagainstmice(

8、eleven15sheets)ELISAresultsrevealedthatthetiterofantiDuCD8aantibodiesWasabout1：I，280，000Atlasttheexpressionplasmid：pET21aIAnplMHCIBSPandpET21a／Anplp2mwasconstructedusingthesequenceofAnpIMHCI—BSPandAnplfjj2mTheSDSPAGEanal

9、ysisshowedthatthemolecularweightofthethreekindsAnpIMHCIBSPareall315kDathemolecularweightoftheAnplf32mis144kDaTheexperimentresultsprovidedapowerfultooltofurtherstudythetetramerictechnologytoidentifyTcellepitopes，moreoveri

10、tpavesthewayfortheestablishmentofthesystemthatdetectscellmediatedimmuneresponseofavianinfluenzavirusandthemolecularmechanismsofthewaterfowlantigenpresentingwithMHCIKeywords：Pekingduck，CD8n，MHCI，prokaryoticexpression，poly