H1N1亚型猪流感病毒HA基因的表达及单克隆抗体的制备  

Expression of HA gene of H1N1 subtype swine influenza virus and preparation of a monoclonal antibody against HA protein

作　　者：张祥斌[1] 谢青梅[1] 马静云[1] 曹永长[2] 冀君[1] 李少璃[1] 毕英佐[1] 

机构地区：[1]华南农业大学动物科学学院,广东广州510642 [2]中山大学生命科学学院,广东广州510275

出　　处：《中国兽医科学》2008年第11期962-967,共6页Chinese Veterinary Science

基　　金：广东省重大科技专项(2004A2090102、2005A20901004)

摘　　要：采用RT-PCR方法扩增了H1N1亚型猪流感病毒(SIV)广东株的血凝素(HA)基因,测序后对HA基因进行了生物信息学分析。将HA基因克隆到高效可溶表达载体pBCX上,成功构建重组表达质粒pBCX-HA。将其转化到大肠杆菌BL21(DE3)感受态细胞中,用IPTG进行诱导表达。诱导表达的HA融合蛋白分子质量约为94.0 ku,表达产物占菌体总蛋白的29.5%。经Western-blot分析证实可溶表达的融合蛋白与H1N1亚型猪流感病毒阳性血清具有良好的免疫反应性。用纯化的HA融合蛋白作包被抗原用于猪流感病毒单克隆抗体的筛选,成功筛选到2株针对HA蛋白抗原表位的单克隆抗体;ELISA结果表明,制备的SIV单克隆抗体ⅢF6A4C10与H1N1亚型SIV抗原具有特异的免疫反应性,为H1N1 SIV的鉴别诊断奠定了基础。The hemagglutinin （HA） gene of swine influenza virus （SIV） H1N1 subtype isolated from Guangdong Province of China was amplified by RT PCR, and then analyzed on its biological information after sequencing. The HA gene was inserted into the vector pBCX to construct recombinant plasmid pBCXHA. The pBCX HA was transformed into Escherichia coli BL21（DE3） to induce HA gene expression with IPTG. The molecular weight expressed fusion protein was 94.0 ku in SDS PAGE test,and the expression product accounted for 29.5 % of the total E. coti proteins. Western blot analysis showed that the expressed fusion protein could react favourably with positive blood serum against SIV H1N1. The expressed HA protein was used as coating antigen to select monoclonal antibodies against SIV. The EI.ISA results indicated that the prepared monoclonal antibody Ⅲ F6A4C10 reacted well with SIV H1N1,which was useful for diagnosis of SIV.

关 键 词：H1N1亚型猪流感病毒 血凝素 高效可溶表达 单克隆抗体 

分 类 号：S852.659.5[农业科学—基础兽医学] Q786[农业科学—兽医学]