豬瘟兔化弱毒疫苗株cDNA分子克隆與嵌合分子克隆的構(gòu)建.pdf

1、碩士學位論文Y87368S豬瘟免化弱毒疫苗株cDNA分子克隆與嵌合分子克隆的構(gòu)建COnstructiOnOfthecDNACloneandChimericClOnesofClassicalSwineFeverVirusC—strainvaccine研究生：指導(dǎo)教師：至塑態(tài)垡生查盟塞亟盤查良型煎撞新疆農(nóng)業(yè)大學中國農(nóng)業(yè)科學院哈爾濱獸醫(yī)研究所本研究得到國家973項目(2005CB52320)的資助。二00六年六月AbstractCl嬲sica

2、ls、^，inet’ever(cSF)，whichiscausedbyclassicalswinefeVerVims(CSFV)，isahighlycor婦百0us趾doftenfatalViraldiseaseofpigsnischaracterizedbyhighf曲er鋤dh鋤orrhages，舭dclassi：fiedasanOIElistAdiseaseGreatlossestopigindlls時worldwidchavcb

3、eenc棚edbymeprevalenceofCSF，especiallyofnontypicalfornlsHogcholeral印irli乃cdvims(HCLV)(alsoI【Ilown髂C—s艦岫，aVerysafe鋤de伍caciousVaccmedeVelopedinCllilHh嬲beenplayingakeymleint圮controlofmediseaseHoweVerpigsvaccinatedbythisvacci

4、necaIlIlmbedis血guished舶m訊l岣pecSFvin?！辴edpigsbyconvemiona王serologicalassaysDevelopmemofmarkerVaccincsmayhelptosolVetllisproblem11lisstudyaimstoconsmlct血e血ll1eng血cDNAcloneofHCLVtoestablisharcVersegeneticssystemaSpIatformfo

5、rstudyofCSFVNiIleprimerpairSweredesigncdaccordingt0mepublishedseq啪cesofHCLVUsingRTPCR，nineoVerlappingcDNA丘agments(Fl一9)covcringtllecompletegenomeofHCLV／HVIuwereanlplified，clonedandsequenced，andthenthecompletegenomesequen

6、cew嬲assembledHomologycomparisonbe錒ecnthcHCLV小VRIsequenceaIldotller7HCLVsequencesshowsmattheHCLVgenomeisrathcrgeneticaIlystableUsingone—steplig砒ionofmuIti如gmentme廿10d，twohalf_1engthcDNAclonespOKl2／F1—5a11dpOKl2／F69wereobt

7、aincdbycloIling行agmentsF1to5andF6to9imopOKl2111enmeMohalflengnlcDNAc10neswcreligated，resultinginme劬llengmcDNAclonepOKl2／Fl一9(p0KHCLV)Byinsertion，substitution，deletion血pOKHCLV，weobtainedaseriesofchimericclonespOKHCLV厄GFP(

8、inseningEGFPgeneintoHCLVgenome)，p0KHCLV甩IAVep(insertinganEIAVepitopeintoHCLVgenome)，pOKHcLV／3’UTR(substimting3’untr趾slatingregionofShimenfor血atofHCLV)，p0KHcLV／shimen(substituting3’halfpanofShimenstrainformatofHCLV)，pOKsl

9、limen，HCLV(substituting5’halfpanofshimenstrainforthatofHcLV)，pOⅪcLVdel4764(deleting4764bpinthecodingregionofHCLV)，aIldp0KCIHCLV(placingHCLVgenomeuIlderthccontmlofCMVpromoter)Themolecularcloneandchimericclonescanbeusedtos