鸡传染性支气管炎病毒DB株核蛋白基因的克隆及在杆状病毒系统中的表达  

Expression of Avian Infectious Bronchitis Virus DB Strain Nucleoprotein by Recombinant Baculovirus System

作　　者：高磊[1] 王玮[1] 刘胜旺[1] 谷守林[1] 孔宪刚[1] 蔡红[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2002年第3期168-171,共4页Chinese Journal of Preventive Veterinary Medicine

摘　　要：利用PT_PCR方法扩增鸡传染性支气管炎病毒 (IBV)DB株的核蛋白基因 ,并对其序列进行了测定 ,DB株IBV的核蛋白基因长度为 12 30bp ,编码蛋白由 4 0 9个氨基酸残基组成。与已发表的参考毒株进行核苷酸同源性比较 ,发现DB株与澳大利亚群毒株的亲缘关系最为密切。同时构建了杆状病毒重组转移载体pBlue_DB_N ,将其与线性化的杆状病毒DNA共转染Sf9昆虫细胞 ,经过 3轮蚀斑纯化和聚合酶链式反应 (PCR)鉴定 ,获得重组杆状病毒rBac_DB_N。SDS_PAGE分析和Westernblot检测的结果表明DB株IBV核蛋白基因在重组杆状病毒感染的Sf9昆虫细胞内获得表达 ,融合蛋白最大表达量占细胞蛋白总量的19 .4With the specific RT_PCR,the nucleoprotein(NP)genes of DB strain was amplified and cloned into the plasmid vector pUC19 at SmaI site.The generated recombinant plasmid designated as pUC19_DB_N was identified harboring NP gene by PCR?restriction endonuclease digestion and sequencing.The complete coding region of NP gene of DB strain was then subcloned into baculovirus transfer vector pBlueBacHis B.The recombinant baculovirus transfer vector containing NP gene in correct orientation was designated as rBac_DB_N.The transfer vector was used to cotransfect insect cell Sf9 with linearized baculovirus DA mediated with Cell Fectin reagent.The recombinant baculoviruses were generated and by three cycle of plaquen cloning.The expression of NP in Sf9 cell was determined by Western blot with ployclonal antibodies against IBV.The infected cells were collected at different time post_infection,analyzed by SDS_PAGE,the result showed that the expressed product had a molecular mass of approximately 56KDa,the expression level of the recombinant proteins was up to 19.4% of the total cell protein.

关 键 词：传染性支气管炎 核蛋白 重组杆状病毒 病毒DB株 基因克隆 鸡 

分 类 号：S858.31[农业科学—临床兽医学] Q786[农业科学—兽医学]