豬腦心肌炎病毒VP1基因的原核表達(dá)、血清學(xué)調(diào)查及分離毒株的鑒定.pdf

1、Y773591分類號(hào)：密級(jí)；嚏蛾差t夭等博士學(xué)位論文單位代碼：10019學(xué)號(hào)lB02207豬腦心肌炎病毒_：vPll基因的原核表達(dá)、血清7學(xué)調(diào)鷹及分離毒株的鑒定“，t?！踦姆hry06cexpressionofVPlgeneofswine：i衲phalomyocarditisvirus，serologicalsurveyandiidentificationofEMCVisolates研究生：指導(dǎo)教師：合作指導(dǎo)教師：申請(qǐng)學(xué)位門類級(jí)別：董塹

2、煎塹墊盎筮捶專業(yè)名稱：亟醴簦堡堂研究方向：盤塹金至照盎鱟璺盤瘞塋所在學(xué)院：盤塹匿塋毽2005年5月AbstractPorcineencephalomyocarditisisanacuteinfectiousdiseaseofswinecausedbyencephalomyocarditisvirus(EMCV)Thisdiseaseisclinicallycharacterizedbyencephalitis。myocarditisor

3、inflammationaroundcardiacmusclesInthisstudythestructuralproteinVPlgeneofEMCVwassuccessfullyclonedandexpressedAndtheexpressedrecombinantproteinVPlwasusedastheantigentodevelopanindirectELISAfordetectionofantibodyagainstEMC

4、VAserologicalsurveywascarriedoutforpigfarmsfromsomeareasofChinabyusingtheestablishedassayFiveencephalomyocarditisvirus(EMCV)strainsinpigswereisolatedandidentifiedfromclinicaltissuesamplesThecDNAofVPlandpartial3Dgenesfora

5、lltheisolatesweresequencedandanalysedThecompleteVPIgeneofEMCVwasamplifiedbyRTPCRandclonedintoprokaryotieexpressionvectorpGEX6P1TherecombinantproteinVPIcouldamountto271％ofthetotalmassofbacterialproteinsafterinductionwith0

6、1mmol／LIPTGAnalysisbySDSPAGEshowedthatthemolecularweightoftheexpressedproductwas600kDaAndWesternBlottinganalysisindicatedthatthepurifiedrecombinantproteincouldberecognizedbypolyclonalantiserumofswinepreparedwithEMCVUsing

7、purifiedrecombinantproteinVPlascoatingantigenallindirectELISAwasdevelopedfordetectionofantibndyinserumtoEMCVTheoptimalreactionconditionsfortheassayweredeterminedTheresultsindicatedthattheoptimalcoatingconcentrationofreco

8、mbinantproteinVPlwas0625Ixg／mL，thedilutionofserumsamplewas1：50，theworkingconcentrationofHRPlabeledrabbitantiporcineIgGwas1：800，serunlsamplefordetectingandHRPlabeledrabbitantiporcineIgGshouldbeincubatedat37“Cfor45min，thet

9、hresholdforserumsamplewas03TheindirectELISAhad900dspecificityandsensitivitycomparedwitllIFA1786serumsamplescollectedfromintensivepigfarmsin13regionsofChinawereexaminedusingtheELISAshowingthattheaveragepositiverateofEMCVa

10、ntibodywas7584％ItwasconcludedthatEMCVinfectionhasprevailedinChinaFiveEMCVstrainswereisolatedfromtheclinicaltissuesamplegorignatedfrompigfarmsinBeijing，Tianjin，Hubei，HebeiandLiaoningbyusingBHK21cellsTheseisolatesweredesig

11、natedEMCVBJC3，EMCVHBI，EMCVHuBI，EMCVLNIandEMCVTJIrespectivelyandidentifiedusingIFAThevirusparticleswithdiameterof27mllwereobservedbyimmuneelectronmicroscopy(IEM)ResultsofphysicochemicalcharacteristictestsindicatedthatEMCV

12、BJC3andEMCVHBlwerenotabletoendureacidandnotsensitivetochloroformeitherHoweverthetwoisolateshaddifferentsensitivitytotrypsinExposedunder60“Cforlh，thevirusescouldbeinactivatedandprotectionofbivalentcationattemperatureof50“

13、CwasnogligibleTheVPlgenesandpartial3DgenesoftheisolateswerasequencedSequenceanalysisrevealedthatthenucleotideidentityofVPlgeneamongtheisolateswasabove99％andthepredictedaminoacididentitywasabove975％Thenucleotideidentityof