豬圓環(huán)病毒2型ORF2基因在巴斯德畢赤酵母中的表達及初步應用.pdf

1、原創(chuàng)性聲明本人鄭重聲明:所呈交的學位論文，是本人在導師的指導下，獨立進行研究工作所取得的成果.除文中已經注明引用的內容外，本論文不包含任何其他個人或集體己經發(fā)表或撰寫過的作品成果。對本文的研究做出重要貢獻的個人和集體，均已在文中以明確方式標明。本人完全意識到本聲明的法律結果由本人承擔.學位論文作者(需親筆)簽名:Ct，、6A7!日學位論文版權使用授權書本學位論文作者完全了解學校有關保留、使用學位論文的規(guī)定，同意學校保留并向國家有關部門或

2、機構送交論文的復印件和電子版，允許論文被查閱和借閱。本人授權南京農業(yè)大學可以將本學位論文的全部或部分內容編入有關數據庫進行檢索，可以采用影印、縮印或掃描等復制手段保存和匯編本學位論文。保密口，在_年解密后適用本授權書。本學位論文所不保密嗎(請在以上方框內打“V日引勺學位論文作、(需親筆)簽名邪，爾龜:、、、月(需親筆)簽一jV7rA6月州日EXPRESSIONOFTHEF2GENEOFPCV2INPICHIAPASTORISPRELIM

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