牛Ⅰ型疱疹病毒感染性细菌人工染色体的构建和gN基因缺失病毒的细胞繁殖特性  

Bovine herpesvirus type 1 genome cloned as infectious bacterial artificial chromosome and replication of gN gene deletion mutant in vitro

作　　者：张存[1] 叶伟成[1] 王一成[1] 袁秀芳[1] 尹龙勃[1,2] 尹文玲[1,2] 刘蔓雯[1] 

机构地区：[1]浙江省农业科学院畜牧兽医研究所,杭州310021 [2]西北农业大学动物医学院,杨凌712100

出　　处：《科学通报》2009年第24期3823-3829,共7页Chinese Science Bulletin

基　　金：浙江省农业科学院国际合作专项资助项目

摘　　要：通过同源重组将pHA2质粒插入到牛Ⅰ型疱疹病毒ZJ分离株基因组的UL15和UL18基因之间,构建了重组病毒rBHV1-HA;提取重组病毒的环状基因组DNA,转化大肠杆菌DH10B,获得了含有BHV1全基因组的感染性细菌人工染色体克隆pBHV1.pBHV1转染MDBK细胞可以拯救出病毒,该病毒与野生毒株在细胞上的繁殖特性未见差异.通过两步Red E/T重组,构建了gN基因跨膜区缺失的pBHV1突变体,并转染获得了重组病毒rBHV1-△gN.病毒繁殖动态曲线显示,rBHV1-△gN的滴度比野生毒株低9%～20%.BHV1感染性克隆的成功构建,将为研究新型BHV-1基因缺失疫苗和牛的病毒载体疫苗提供技术平台.The recombinant bovine herpesvirus type 1 （rBHV1-HA） was constructed by inserting pHA2 plasmid into the viral genome between UL15 and UL18 cassettes. BHV1 infectious bacterial artificial chromosome had been confirmed after rBHV1-HA circular genome was extracted and transformed into E. coil strain DH10B. The resulting Bac clone, pBHV-1, was transfected into bovine kidney cells （MDBK） and the virus was rescued. The rescued virus, BHVl-res, have almost the same titres as the wildtype in replication in vitro. The transmembrane domain in gN open reading fram was deleted from pBHV-1 by Red E/T mutagenesis in E.coli. BHV-1 with partial deletion of gN, rBHV1-AgN, was reconstituted from the mutated Bac. rBHV1-AgN had lower titres by from 9% to 20% than rBHV-res in growth property in MDBK. This construction of infectious BHV1 clone should contribute greatly to the recombinant BHV1 vaccine with genes deletion and bovine universal viral vector.

关 键 词：牛Ⅰ型疱疹病毒 感染性克隆 细菌人工染色体 囊膜蛋白gN UL49.5 

分 类 号：S852.65[农业科学—基础兽医学]