日本脑炎病毒E基因与流感病毒HA基因的串联表达及应用  

Coexpression for E Gene of Japanese Encephalitis Virus and HA Gene Epitopes of Human Influenza Virus and the Application for the Expression Product

作　　者：郑其升[1] 杨耀武[2] 张晓勇[1] 周斌[1] 曹瑞兵[1] 李鹏[1] 陈德胜[1] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,江苏南京210095 [2]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《中国病毒学》2005年第1期11-15,共5页Virologica Sinica

基　　金：国家"863"高技术发展计划资助项目(2001AA240912)

摘　　要：根据GenBank公布的日本脑炎病毒(Japanese Encephalitis Virus, JEV) SA14 14 2株的核酸序列和人流感病毒的血凝素基因(ha)序列, 设计一对特异性引物, 用 PCR方法扩增编码 JEV囊膜蛋白主要抗原域基因, 其中含ha基因主要核苷酸序列。将PCR产物定向克隆入原核表达载体 pET 32a(+), 构建原核表达载体 pET EHA。阳性质粒转化BL21(DE3)宿主菌, 经 IPTG诱导获得表达, 重组蛋白以包涵体的形式存在。Western blot分析表明表达产物具有良好的免疫学活性。利用纯化的表达产物与流感病毒血凝素单抗及乳胶建立了诊断日本脑炎病毒抗体水平的乳胶凝集试验。结果表明乳胶凝集方法具有简便快速、敏感性高、特异性强、价格低廉、可现场检测等优点, 是一种适合基层兽医单位用于日本脑炎病毒抗体水平检测的新方法。Using a pair of specific primers designed according to the nucleotide sequence of Japanese encephalitis virus and Hameoaggulatinin of Human influenza virus from GenBank, the gene of JEV E protein's main antigenic domain including the sequence of Human influenza virus ha gene was amplified with PCR method. The PCR product was successively cloned into pET-32a(+) vector to get a prokaryotic expression plasmid pET-EHA. After the positive plasmid was transformed into the host cell BL21(DE3) , the target gene was successfully expressed in the form of inclusion bodies when induced with IPTG. Western-blotting analysis proved the recombinant protein has good reactivity ability against JEV antibodies. The Latex Agglutination Test (LAT) method for the detection of JEV antibodies was established with the purified recombinant protein. The result indicated that LAT was a simple, rapid, sensitive, specific, inexpensive method which is suitable for detecting antibodies against JEV in serum and serological survey of JE .

关 键 词：日本脑炎病毒 囊膜蛋白 HA表位 乳胶凝集 

分 类 号：R346[医药卫生—基础医学]