犬細小病毒HL-1株VP2基因真核表達及抗體ELISA檢測方法的建立.pdf

1、Y7853‘z目自目書升婁々：目％目丹婁：學位論文F缸。蓽犬細小病毒HL一1株VP2基因真核表達及抗體ELlsA檢測方法的建立導師姓名：李一經教授申請學位級別：農學碩上專業(yè)名稱：積防獸醫(yī)學論文提交日期2005年4月25日答辯日期2005年6月10日所在單位東北農業(yè)大學學位授予單位東北霹業(yè)犬學學位授予日期：答辯委員會主席教授2005年6月20日EukaryoticExpressionofCanineParvovirusHL1VP2Gene

2、andDevelopmentofanIndirectELISAfortheDetectionofViralAntibodieswiththeExpressionProduceABSTRACT疊Canineparvovirus(cvv)belongstothefamii；ParvovirusItisoneofthemajorpathogensthatcausebleedingenteritisandmyocarditisindogsCPV

3、2onlyinfectdogsandishighlycontagiousItsprevalenceis20％o100％，anditsmortalityindogsless12weeksis10％50％CPVenteritisischaracterizingbyseveresickness，diarrheaandexcretingrottendejeetaAccuratediagnosisandeffectivevaccinationfi

4、remainmeansforthepreventionofthediseaseResearchshowedthattheoutercapsidglycoproteinVP2ofCPVparticlesismajorprotectiveantigenandexcellentdiagnosisantigenSoVP2codingisthetargetproteinforthepreparationofthenewtypevaccineand

5、specificdiagnosisantigenInthisstudyCPVstrainHLIWaSpropagatedbnF81cellsApairofprimersWasdesignedtoampli母VP2genebyPCRThePCRproductisapproximate18kbinlengthTheVP2genewasclonedintothevectorpMD18Tvectorandtherecombinantplasmi

6、dWaSnamedpMD—VP2IdentificationsbyrestrictionenzymePCRandsequencingindicatedthattheVP2genehasbeenclonedsuccessfullyTheVP2sequencewascomparedwiththoseoftheotherCPVstrainVP2genesHL一1VP2shared9897％、9875％and9869％nucleotidewit

7、hthoseofCPVd(CPV2)，CPV15(CPV2a)andCPV39(CPV2b)respectivelyThededucedaminoacidsequenceswere9812％，9760％and9760％respectivelyVP2wasinsertedintoHindlIIandSalIsitesbfthevectorpMelBacCcreatingpMelVP2PurifiedpMelBacCVP2andBaeNBl

8、ueDNAgenmicwerecontransfectedinsectSf9cellsIdentificationwithPCRshowedVP2genehasbeenrecombinatedcorrectlywithbaculovirusDNARecombinantpurificatedbaculoviruswasscreenedbyblueplaquepurificationforthreetimesSDSPAGEandWester

9、nblotanalysesshowedthatHL—lVP2geneWasexpressedinrecombinantbaculovirusRecombinantfusionproteinWasusedasdetectingantigeninindirectenzymelinkedimmunosorbentassay(ELISA)todetectanti—CPVserBmantibodyTheoptimalELISAparameters