马动脉炎病毒基因组全长cDNA克隆的构建  

Construction of full-length cDNA of equine arteritis virus

作　　者：韦祖樟[1] 袁世山[1] 

机构地区：[1]中国农业科学院上海兽医研究所中国动物卫生与流行病学中心上海分中心动物传染病防治研究室农业部动物寄生虫病重点实验室,上海200232

出　　处：《中国兽医学报》2009年第2期139-142,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30530580)

摘　　要：根据马动脉炎病毒(Equine arteritis virus,EAV)Bucyrus株全基因组序列设计并合成EAV特异引物,进而应用RT-PCR技术分6段扩增了EAV全基因组cDNA。将扩增的各个cDNA重叠片段PE124、PE631、PE1854、PE5191、PE61107、PE97Q分别克隆到载体PCR BluntⅡ-TOPO中,建立了EAV初级cDNA克隆。在扩增5′末端时,引入NotⅠ酶切位点和T3启动子序列;在基因组3′末段PolyA尾引入XhoⅠ酶切位点,后者供cDNA模板的线性化之用。将扩增片段在PCR BluntⅡ-TOPO中依次连接,最后亚克隆到质粒pBluescriptⅡKS(+)中,获得了EAV全长基因组cDNA克隆pWEAV。核酸序列分析表明:该毒株基因组全长为12 704个核苷酸,与EAVBucyrus分离株的同源性为99.1%;全长基因组中有104个核苷酸发生突变,相应地导致编码区内34个氨基酸的改变。To develop a reverse genetics system of equine arteritis virus （EAV）, six pairs of oligonueleotides were designed based on the full-length genomie sequence of EAV Bucyrus strain. Using RT-PCR technique,six overlapping eDNA fragments, designated as PE124, PE631, PE1854, PE5191, PE61107 and PE97Q, respectively, were amplified, followed by cloning into pCR Blunt Ⅱ-TOPO vector. A Not Ⅰ site and a T3 promoter were introduced immediately upstream of 5′-end,while a Xho Ⅰ Site was engineered downstream of 3′end of EAV poly（A） tail. Using pBluescript Ⅱ KS（＋） as a plasmid vector,the full length eDNA clone pWEAV of Bucyrus strain was obtained by connecting the six eDNA fragments utilizing single restriction endonuclase site. The construction of a full length genomic cDNA clone of EAV is a crucial step to obtain the infectious clone,which may facilitate further dissecting of structure and function relationship of EAV genome.

关 键 词：马动脉炎病毒 全长CDNA克隆 感染性克隆 

分 类 号：S852.652[农业科学—基础兽医学]