犬瘟熱病毒石河子株的初步分離及核衣殼蛋白基因原核表達.pdf

1、石河子大學碩士學位論文犬瘟熱病毒石河子株的初步分離及核衣殼蛋白基因原核表達姓名：楊井泉申請學位級別：碩士專業(yè)：預防獸醫(yī)學指導教師：薄新文201106IIAbstractCaninedistemperisacaninedistempervirus(CaninedistempervirusCDV)infectioncausedbyanacutefebrilehighlycontagiousdiseasestherefeitiscurrent

2、lyoneofthemostdamaginganimaldiseasesonthedogindustrythefurindustryanimalbreedingwildlifetrade.InthisstudyaRTnestedPCRassayfdetectingCaninedistempervirus(CDV)wasestablishedusing2pairsofprimers.Theprimersweredesignedusingn

3、ucleocapsidprotein(NP)geneinfmationofCDVOnderstepotstraininGenBank.TheassayamplifiedonlyaNPgenefromCDV：butnotfromothervirussuchasRabiesvirus(RV)Canineparvovirus(CPV)Verocell.Serial10folddilutionsof106TCID50CDV3strainwere

4、usedfbothPCRRTnestedPCR.ThelowerdetectionlimitfCDVRNA106103dilutionsftheRT–nestedPCRthetraditionsRTPCRrespectively.TheRTnestedPCRmethodwassuccessfullyusedtodetectfsuspectedCDVinfectioninclinicalsamplesfromShihezi，thuscou

5、ldbeusefulftheclinicalsamplesfromdifferenttissues.ItaslocanbeusedfaquickdiagnosisofCDtheepidemiologicalinvestigation.InthisstudythesupernatantofthetissuefromthedogsinfectedCDVwasinvestigatedonVerothecytopathicwasappeared

6、after5generationsthroughblindpassages.RTnestedPCRwasusedtodetectthesupernatantofcelllysisharvestedfrozen.TheCytopathicwerealongstretchinthecellnumerousroundcellsshrinkageofcellularmembranecellaggregatedattachedtothesurfa

7、ceofothercells.ThespecificbofPCRproductwasappearedbyelectrophesis.TheresultsshowedthatThespecificbofPCRproductistheCDVsequencespecificnucleicacidsequences.CompoundwiththesupernatantofthetissuefromthedogsinfectedCDVthecoi

8、ncidenceratewas100%indicatingthatthesupernatantofthetissuefromthedogsdidnotresultinDNAmutationstherefethesupernatantofCDVShiheziinfectedfrozencellwasharvested.Inthisstudytheprimersofthefulllengthnucleocapsidproteinthehem

9、agglutiningenesequencesofCDVShiheziOnderstepotattenuatedstraininGenBankweredesigenedtoamplifytheNPgeneofCDVShihezistrainanalysisthesequence.TheresultsshowedthattheNPgeneofCDVShihezistrainwassuccessfullyclonedtheproteinen

10、codedepitopeswereanalyzed.Theresultsshowthatamplificationofthenucleocapsidproteingenewas1688bpacompleteopenreadingframe(F)was1572bpencoding523aa.NucleicacidsequencehomologyphylogeicanalysisshowedthatCDVstrainsHM596313Shi

11、hezistrainshared100%identityattheaminoacidlevelonlytwobasedifferencesofthenucleicacidsequences.Hemagglutiningenewas607bpencoding202aasharedwithTNstrain(AY390347)aminoacidhomologywas98.02%withJapanKDK1strain(AB025271)was9