犬瘟热病毒的分离、鉴定与单克隆抗体的研制(6109)

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1、华中农业大学2011届硕士学位论文AbstractCaninedistemper(CD)isahighlycontagiousandfataldiseaseofdogsorothersusceptibleanimalswhichinfectedbycaninedistempervirus(CDV)．Atpresent,caninedistemperispandeIIlicinChinaandbecomesolleofthemostseriousdiseasesfordogbreedingindustry．Accordingtothec

2、haracteristicsofcellculture，thestudyisolatedastrainofcaninedistempervirus(CDV)attenuatedstrainfromSecondUnitedattenuatedsampleinVerocellsusinghereroviusantiserumblockingassaying．TheCPEinVerocellfeaturinggatheredshedding,rounding,net-balloonanddeathappearedsteadilyafterg

3、enerationcultivation．ThediseasedcellsidentifiedbyRT-PCRshowedpositive．ThephysicsandChemistryexperimentsshowedthat,theviruswasinactivated56"Cfor30min，moresensitivetochangesofacidity．ThestrainhelditsvirlxLeneestabilityinpH7．O～8．0environmentbutsensitiveabovepill0．0andbelow

4、pH4．0．CDVisanenvelopedvires，losseditsinfectionactivityafter20minutes诚tllchloroform．TheHAtestshowthat，CDVcouldnotagglutinateredbloodcellofchicken，pig，sheepandrabbit．TheTCID50ofCDVattenuatedstrainis10-6．25／mL．The6to8weeksoldBALB／cfemalemicewereimmunizedsubcutaneously、析tlI

5、CDVOonderstepoortstrain,whichWaspurifiedandconcentratedwitllpolyeth，7leneglyc01．Afterbeingimmunizedthreetimes，themicewereboostedintraveinally、ⅣitIlequalthedoseforthefinalimmunization．Threedayslater，spleencellsfromimmunizedmicewerefusedwithSP2／0myelomacells．Afterrepeated

6、screeningandsubcloning，5hybridomacelllineswereobtainedwhichcouldsteadilysecretemonoclonalantibodyagainstCDV．Theywerenamed：1G5、2D7、386、4C7and4G5。Monoelonalantibodiesin5linesallhadhigherspecificitytoCDV,whiehdidnotreact、析tlladenovirustypelIandcanineparvovirus．TheIFAtestsh

7、owedthatallthe5mAnscanreactwithVerocellsinfectedbyCDV．Theascitestitersof5hybridomacelllineswere1G5>100x2lo、2D7>100x29、386>100x27、4C7>100×28、4G5>100x210．thetiterofcellcI．tLturesupematantWas2s"--29．Theheavychainsinmonoclonalantibodiesof5myelomacelllinesbelongedtoIgG1andli

8、ghtchainsbelongedtoKchains．Keyword：Caninedistempervirus；isolation,identification；MonoclonalantibodyⅡ犬瘟热病毒的分离、鉴