表达小反刍兽疫病毒H基因的山羊痘病毒通用转移载体的构建及鉴定  

Construction and Identification of a Goat pox Virus Transfer Vector to Express Peste des Petits Ruminants H gene

作　　者：邵长春[1,2] 张强[2] 吴国华[2] 颜新敏[2] 李健[2] 王建科[2] 卢晓丽[2] 赵志荀[2] 崔丽凡[2] 高世功 

机构地区：[1]甘肃农业大学,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室,甘肃兰州730046 [3]中农威特生物科技股份有限公司

出　　处：《安徽农业科学》2009年第20期9384-9386,9404,共4页Journal of Anhui Agricultural Sciences

摘　　要：[目的]研制小反刍兽疫羊痘活载体疫苗。[方法]利用PCR技术扩增小反刍兽疫病毒H基因,克隆到pGEM-Teasy载体,NheⅠ和HindⅢ双酶切重组质粒,将目的片段插入到真核表达载体pEGFP-N1-P7.5中,得到重组载体pEGFP-N1-P7.5-H,重组载体HindⅢ、NheⅠ双酶切片段EGFP-P7.5-H平末端连接到KpnⅠ酶切后的载体pUC119-TK中,得到通用转移载体pUC119-TK-EGFP-P7.5-H。[结果]经酶切鉴定及PCR扩增检测,表明构建栽体正确。pUC119-TK-EGFP-P7.5-H转染羊痘病毒感染的BHK21细胞,48 h后报告基因表达。[结论]该研究为研制小反刍兽疫基因工程活载体疫苗奠定基础。[ Objective] This study was to develop a live vector goat pox virus vaccine of Peste des Petits Ruminants（PPR）. [ Method] Using PCR amplification tecbnique,PPR H gene was obtained, then ligated into pGEM-T easy vector; the reeombinants were digested by Nhe I and Hind Ⅲ, and ligated into pEGFP-NI-P7.5, yielding the recombinant vector pEGFP-N1- P 7.5-H; next the expression cassette EGFP-NI- P 7.5-H was first released from recombinant vector pEGFP-NI- P7.5-H by double digestion of Hind Ⅲ and Nhe Ⅰ and ligated into pUC119-TK that was digested by Kpn Ⅰ , yielding the transfer vector pUC119-TK-EGFP-PT. 5-H. [ Result] Identification and double enzyme digestion showed that the transfer vector pUC119-TK-EGFP-P7, 5-H was correctly constructed. From the transfer vector transfected BHK-21 cells which infected GTPV AV41, specific fluorescence was observed at 48th h after transfection. [ Conclusion] The construction of goat poxvirus live vector laid a foundation for the live vector vaccine of PPR vaccine.

关 键 词：羊痘病毒 H基因 转移载体 构建 鉴定 

分 类 号：S858.27[农业科学—临床兽医学]