表达外源绿荧光蛋白重组山羊痘病毒的构建及纯化  

Construction and Purification of Recombinant Goatpox virus Expressing Exotic Green Fluorescence Protein

作　　者：程小文[1] 胡森[1] 王喜军[1] 鲍恩东[2] 步志高[1] 

机构地区：[1]中国农科院哈尔滨兽医研究所兽医生物技术国家重点实验室,哈尔滨150001 [2]南京农业大学动物医学院,南京210095

出　　处：《农业生物技术学报》2006年第5期798-802,共5页Journal of Agricultural Biotechnology

基　　金：国家攻关项目(No.2004BA519A19和No.2005BA711A10);国家重点基础研究发展规划(973)项目(No.2005CB523200)资助

摘　　要：以我国自行培养并得到广泛应用的山羊痘病毒(Goatpoxvirus,GPV)疫苗株为亲本,以复制非必需胸苷激酶(tk)基因作为外源基因插入靶点及同源重组臂。利用转移载体pTK-lacZ-gfp及其与GPV发生同源重组获得的病毒(rGPV-lacZ-gfp)验证外源基因β-半乳糖苷酶(lacZ)和绿荧光蛋白(gfp)基因在背靠背启动子p11和p7.5启动下成功稳定表达后,将转移载体lacZ基因更换为gpt(黄嘌呤鸟嘌呤磷酸转移酶)基因(pTK-gpt-gfp),利用MPA阻断核酸代谢途径筛选并获得了遗传稳定表达的单一纯化病毒克隆(rGPV-gpt-gfp)。研究建立了表达外源基因重组GPV构建系统及其克隆纯化方法,为进一步开展GPV活载体疫苗研制及相关基础研究提供了技术平台。Vaccine strain of Goatpox virus （GPV） cultivated and widely used in domestic was employed as the mother virus. The tk gene of GPV, the nonessential region during virus replication, was chosen as target site and homologous arm when inserting foreign gene. The stability of lacZ and gfp genes expression in recombinant rGPV-lacZ-gfp downstream of back to back promoter 11 and promoter 7.5 was testified after recombination of the transfer vector pTK-lacZ-gfp with the mother GPV, then lacZ gene in pTK-lacZ-gfp was replaced by gpt gene in order to obtain a purified recombinant GPV expressing foreign gene stably （rGPV-gpt-gfp） by using MPA blocking off the nucleic acid metabolize. Further more recombinant GPV construction system and optimized the purification methods were established, which supply a technique platform for investigation of live vector vaccine and relative basal research.

关 键 词：重组山羊痘病毒 Β-半乳糖苷酶 绿荧光蛋白 黄嘌呤鸟嘌呤磷酸转移酶 

分 类 号：S188[农业科学—农业基础科学]