非洲马瘟病毒VP7基因的克隆与表达  

Cloning and Expression of VP7 Gene of African Horse Sickness Virus

作　　者：曾昭文[1] 花群义[2] 段纲[1] 周晓黎[2] 董俊[2] 杨云庆[2] 尹尚莲[2] 项勋[1] 常华[1] 

机构地区：[1]云南农业大学动物科技学院,云南昆明650201 [2]云南出入境检验检疫局,云南昆明650228

出　　处：《中国农学通报》2006年第10期49-53,共5页Chinese Agricultural Science Bulletin

基　　金：国家质检总局重点科技项目"非洲马瘟检验技术研究"(2002IK009-02)

摘　　要：为获得非洲马瘟病毒VP7蛋白,以用于制备AHS血清学诊断试剂并为AHS新型疫苗的构建奠定基础,笔者采用原核表达系统表达VP7蛋白。在GeneBank中查找非洲马瘟病毒VP7基因序列,人工合成VP7基因,将VP7基因片段克隆插入pBAD/Thio-TOPO表达载体,将重组质粒转入大肠杆菌TOP10。经测序鉴定,筛选获得VP7基因正向插入、有正确读码框的阳性克隆,经L-Arabinose诱导表达VP7融合蛋白抗原。SDS-PAGE分析表明以终浓度为0.2%的L-阿拉伯糖进行诱导,4h后表达可达到高峰,融合蛋白质量约54.72kDa。Westernblotting检测和间接ELISA表明诱导表达的融合蛋白能与非洲马瘟病毒VP7单克隆抗体发生特异性反应,说明蛋白有特异的免疫学活性。To obtain the African horse sickness virus （AHSV） VP7 protein, which is used to prepare serological diagnostic reagent and construct AHS new generation vaccine, the prokaryotic expression is used to express VP7 protein. The VP7 gene was synthesized from the database of GeneBank, then the VP7 gene was cloned into pBAD/Thio-TOPO vector, and the recombinant plasmid was transferred into Escherichia coli TOP10. The recombinant plasmid was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted VP7 gene. The results of SDS-PAGE revealed that the VP7 recombinant fusion protein was expressed in TOP10 in a high level and the optimal amount of the expressed fusion protein was being induced with L-arabinose at 0.2% concentration for 4 hours. It had a molecular mass of approximately 54.72 kDa. The results of Western blotting and indirect ELISA revealed that the fusion protein was able to react with ASHV VP7 monoclonal antibody and it suggested that the fusion protein had immunologically reactive activity.

关 键 词：非洲马瘟病毒 VP7基因 克隆 表达 

分 类 号：S852.652[农业科学—基础兽医学]