1、原創(chuàng)性聲明本人鄭重聲明:所呈交的學(xué)位論文，是本人在導(dǎo)師的指導(dǎo)下，獨(dú)立進(jìn)行研究工作所取得的成果.除文中已經(jīng)注明引用的內(nèi)容外，本論文不包含任何其他個(gè)人或集體己經(jīng)發(fā)表或撰寫過的作品成果。對(duì)本文的研究做出重要貢獻(xiàn)的個(gè)人和集體，均已在文中以明確方式標(biāo)明。本人完全意識(shí)到本聲明的法律結(jié)果由本人承擔(dān).學(xué)位論文作者(需親筆)簽名:Ct，、6A7!日學(xué)位論文版權(quán)使用授權(quán)書本學(xué)位論文作者完全了解學(xué)校有關(guān)保留、使用學(xué)位論文的規(guī)定，同意學(xué)校保留并向國(guó)家有關(guān)部門或

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3、INARYAPPLICATIONAbstractSomekindsofdiseasesarerelatedwithporcinecircovirustype2inporcinegroupsincludingPMWSPDNSCTetal.EspeciallyPMWSisthemostseriousandhasmadegreateconomiclossesintheworld.Chinacannotescapebysheerluck.Atp

4、resentthereisnoefficientdiagnostickitinthedomesticmarket.Therapidandspecificdiagnosticmethodismuchimportanttocontrolthesediseases.ThenucleocapsidproteinofPCV2encodedbyORF2isamajorstructuralproteinofvirusandhasspecificimm

5、unogenicity.TheCapgeneisaconservedsequenceintheviralgenome.Theexpressedproductwouldbeusedindiagnosis.SoasfollowsinthisresearcharetosetupthediagnosticmethodonthebasisofCapproteinandprotectagainstthediseasesassociatedwithP

6、CV2.l.wedesignedonepairsof州mersandamplifiedtheORF2genesfromthevectorpeDNA3ORFwhichwasconstructedbyTanandthePCRproductswereinsertedintosecretoryPPICZ.AP.pastorisexpressionvectors.RecombinantstrainswithORF2genewereacquired

7、andinducedwithmethanol.SDSPAGEandWesternblotanalysisshowedthatthegenecouldbeexpressedinP.pastorisandtheproductshadgoodreactingability.2.Westudiedtherelationsbetweenexpressionyieldandgrowthconditionsandfoundtheoptimalexpr

8、essionconditionsoftherecombinantORF2proteinwere:2830C225rpmwhenBMGYwithOD60pbeing3therecombinantPichaPastoriswasaddtotheBMMYmediumofpH7.5whichwithOD60obeing1.5.Thenthesolutionwasincubated72handmethanolwasaddedtokeepitsco

9、ncentrationupto0.5%between24h.AfterinductionunderoptimalconditionstheyieldofORF2proteincouldbeaddedupto2781tgmL.Itwasfoundthatthesupernatantshouldbeconservedin20Caftercomparingefectsofdiferentconservationtemperatureonlys

10、isofproteinORF2.3.AnindirectELISAusingproteinORF2ascoatingantigenwasestablishedtomonitorantibodieslevelofpigsagainstPCV2anditsoperationmethodswereoptimized.Theoptimaloperationmethodswere:Aproteinconcentrationof42.51tgmLw

11、ascoatedovernightat4Con96wellmicrotiterplatesandtheplatesweresubsequentlyblocked3hwith1%BSAandthenincubatedfor45minwithseradilutedby1%BSA廠afterwashingtheplatestheantiswineimmunoglobulinconjuatewasaddedandincubatedfor30mi

12、nat37Cor45minatRTandthentheindicatorOPDwasaddedandincubatedfor15minat37Cor20minatRT0.12%HFwasaddedbeforeODwasmeasuredat630nm.200piecesofserafrompigsinfectedbyPCV2wereexaminedbythismethodandcomparedwiththemethodoflatexagg