传染性支气管炎病毒核衣壳蛋白基因克隆及在E.coli中的表达  

Cloning of Nucleocapsid Gene of Infectious Bronchitis Virus and Its Expression in E.coli

作　　者：周继勇[1] 丁红梅[1] 程丽琴[1] 沈行燕[1] 

机构地区：[1]浙江大学动物预防医学研究所,杭州310029

出　　处：《中国生物化学与分子生物学报》2002年第4期450-455,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金 (No.3 0 0 70 5 70 );浙江省自然科学基金 (No .3 99411);浙江省重点项目 (No .99110 2 0 3 0 )资助GenBankNo:AF3 5 2 3 0 8

摘　　要：核衣壳蛋白基因 (N基因 )是传染性支气管炎病毒的重要结构基因 .根据已报道的序列设计引物 ,利用RT PCR技术从病毒RNA中扩增和克隆到了N基因的cDNA ,并测定了核苷酸序列 .克隆的N基因片段ORF全长 12 30bp ,编码 4 0 9个氨基酸 .将该片段序列与其他IBV病毒株比较 ,核苷酸的同一性为 87 0 %～ 98 6 %,氨基酸的同一性为 91 0 %～ 98 1%.将该cDNA亚克隆到pBV2 2 0表达载体 ,转化大肠杆菌DH5α菌株 ,Western印迹检测 ,获得了分子量约 4Nucleocapsid(N) gene is one of the major structural genes of infectious bronchitis virus(IBV). A pair of specific primers were designed and synthesized according to the published sequence of N gene of IBV, cDNA of N gene was obtained by RT PCR from viral RNA of IBV ZJ971 strain, and further cloned into pBluescript SK(+) vector, and its nucleotide sequence was determined by the dideoxy mediated chain termination method. The results showed that the complete open read frame (ORF) of N gene encoding 409 amino acid was 1 230 bp in length. A comparison of the nucleotide and deduced amino acid sequence of N gene with that of other IBV strains showed thot the identity of nucleotide was between 87 01%～98 6%, and that of the deduced amino acid was between 91 0%～98 1%. cDNA of N gene was subcloned into prokaryotic expression vector pBV220, and the specific non fusion protein of molecular weight 45 kD (N protein) was expressed in E. coli DH5α, Western blotting assay indicated that the monoclonal antibody against N protein of IBV could recognize this protein.

关 键 词：禽病毒 传染性支气管炎病毒 核衣壳蛋白 基因克隆 E.COLI 表达 

分 类 号：S852.659.6[农业科学—基础兽医学] S858.315.3[农业科学—兽医学]