西尼罗病毒E蛋白结构域Ⅲ在昆虫S2细胞中的表达与鉴定  

Expression and identification of WNV E domainⅢ in drosophila S2 cell lines

作　　者：史利军[1,2] 吕茂民[2] 黎诚耀[3] 孙卫华[2] 章金刚[1,2] 

机构地区：[1]华中农业大学动物医学院,湖北武汉430070 [2]军事医学科学院野战输血研究所,北京100850 [3]南方医科大学生物技术学院,广东广州510515

出　　处：《中国兽医学报》2008年第11期1288-1291,共4页Chinese Journal of Veterinary Science

基　　金：全军医药卫生科研基金资助项目(06H056)

摘　　要：根据GenBank中发表的西尼罗病毒(WNV)E domainⅢ基因序列设计1对引物,用PCR方法扩增domainⅢ基因片段,回收目的基因片段定向克隆入果蝇表达载体pMT/B/V5,构建重组表达载体pMT-DⅢ。测序验证正确后,用脂质体转染法与辅助质粒pCoBlast共转染果蝇S2细胞,通过Blasticidin进行加压筛选,获得了抗性细胞S2-DⅢ。提取S2-DⅢ细胞的基因组DNA,用PCR方法检测目的基因在果蝇S2细胞中的整合与表达。用硫酸铜溶液诱导表达,收集无血清的细胞表达上清,样品浓缩后进行SDS-PAGE及Western blotting鉴定。结果表明,研究成功构建了重组表达载体pMT-DⅢ,转染细胞经12mg/L的Blasticidin筛选及鉴定后获得了稳定表达WNV E do-mainⅢ蛋白的果蝇S2细胞株。Based on the sequence of WNV E domainⅢ gene in GenBank,a pair of primers was designed to amplify domainⅢ gene with PCR. Then was cloned into Drosophila Expression System to get recombinant expressed vector（pMT-DⅢ ）. After co-transfection of the pMT-DⅢ and pCoBlast into S2 cells. The positive S2 cells were further screened with 12 mg/L Blasticidin. pMT-Ⅲ was transfected into $2 cells which was named S2-DⅢ. S2-DⅢ was induced by copper sulfate. S2-DⅢ cell which could permanently express recombinant DⅢ protein. The assessment of purity and specificity of WNV DⅢ was identified by SDS-PAGE and Western blotting.

关 键 词：西尼罗病毒 E蛋白 结构域Ⅲ S2细胞 

分 类 号：R535[医药卫生—内科学] S852.65[医药卫生—临床医学]