表达牛呼吸道合胞体病毒G蛋白基因的重组Ⅰ型牛疱疹病毒的构建与鉴定  

Construction and identification of a recombinant BHV-1 expressing the G protein gene of bovine respiratory syncytial virus

作　　者：冯军科[1] 朱远茂[1] 任宪刚[1] 祖立闯[1] 李娇[1] 史鸿飞[1] 高欲燃[1] 童光志[1] 薛飞[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室／大动物传染病研究室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2010年第2期81-85,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省十一五科技攻关项目(GA06B202-3)

摘　　要：为构建表达牛呼吸道合胞体病毒(BRSV)G蛋白基因的牛疱疹病毒Ⅰ型(BHV-1)重组病毒,本研究将人工合成的BRSV全长G蛋白基因编码序列插入到巨细胞病毒(CMV)启动子之下构建TK基因缺失转移载体。利用磷酸钙-DNA沉淀法将该转移载体与亲本病毒BHV-1/TK-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过反向蚀斑筛选,得到重组病毒BHV-1/TK-/G+。PCR检测结果证实G蛋白基因已经插入到了亲本病毒BHV-1/TK-/LacZ+的基因组中,间接免疫荧光试验和western blot证实BHV-1/TK-/G+中的G蛋白基因在感染的细胞中获得了表达。本研究为研制BRSV及其他重要牛传染病的BHV-1病毒活载体疫苗奠定了基础。To construct a recombinant bovine herpesvirus 1 （BHV-1） expressing the G protein gene of bovine respiratory syncytial virus （BRSV）, a BHV-1 transfer vector with flanking BHV-1 TK gene was constructed by inserting a synthetic G protein gene sequence of BRSV under the control of CMV promoter. The transfer vector and the parental recombinant virus （BHV-1/TK^-/Lac Z^＋） DNA were co-transfected into BT cells using calcium phosphate-mediated transfection. The recombinant BHV-1 （designated as BHV-1/TK^-/G^＋） was obtained by selection for white virus plaques in presence of X-gal. PCR results showed that G gene was successfully inserted into the genome of BHV-1/TK^-/Lac Z^＋ and replaced the LacZ gene. The expression of G protein in infected cells was confirmed by indirect immunofluorescence assay and western blot. This work provided a basis for development of BHV-1 vector vaccines for BRSV and other important bovine infectious diseases.

关 键 词：牛呼吸道合胞体病毒 G蛋白基因 牛疱疹病毒Ⅰ型 磷酸钙-DNA沉淀法 

分 类 号：S852.65[农业科学—基础兽医学]