小反刍兽疫病毒H糖蛋白基因原核表达载体的构建及表达  

Construction of prokaryotic vector and expression of H glycoprotein gene of peste des petits ruminants virus

作　　者：刘玉洪[1] 花群义[2] 徐自忠[2] 杨云庆[2] 周晓黎[2] 董俊[2] 尹尚莲[2] 许靖逸[1] 高洪[1] 

机构地区：[1]云南农业大学,云南昆明650201 [2]云南出入境检验检疫局技术中心,云南昆明650228

出　　处：《中国兽医科学》2006年第9期692-695,共4页Chinese Veterinary Science

基　　金：国家"十五"重大科技攻关项目(2001BA804A48)

摘　　要：参照GenBank中小反刍兽疫病毒(PPRV)H抗原基因序列,人工合成了PPRV H基因,将其克隆至pUC18-T质粒中,转化E.coliJM109感受态细胞,构建并选择PPRV H基因克隆重组质粒,经核苷酸序列分析正确,将其克隆至pBAD/Thio-TOPO载体中,转化E.coliTOP10感受态细胞,核苷酸序列分析证实,成功构建了PPRV H基因重组表达载体。经不同浓度L-阿拉伯糖诱导,可稳定、高效地表达PPRV H抗原。SDS-PAGE分析结果表明,用终浓度为0.2 g/L的L-阿拉伯糖诱导5 h的表达量最高,表达蛋白为分子质量约83 ku的融合蛋白;经薄层扫描分析,其表达产量约占菌体总蛋白的10%。Western-blotting检测表明,诱导的蛋白能与PPRV H蛋白单抗发生特异性反应,说明表达的融合蛋白中含有PPRV H糖蛋白抗原。The H glycoprotein gene of peste des petits ruminants virus（PPRV）,which was synthesized artificially according to the Indian vaccine, was sub-cloned from pUC18-H, and inserted into pBAD/Thio- TOPO vector. The recombinant plasmid was identified by PCR. Sequence analysis confirmed that the H glycoprotein gene was inserted correctly. SDS-PAGE analysis revealed that the H protein gene was ex- pressed at high level in Escherichia coli TOP10. The expressed fusion protein, which made up 10% of total bacteria protein after being induced with 0.2 g/L of L-arabinose for 5 hours,was approximately 83 ku in molecular weight. In Western-blotting test, the protein can specifically react with the PPRV H monoclonal antibody,indicating that prokaryotic expression of PPRV H gene can produce H glycoprotein.

关 键 词：小反刍兽疫病毒 H糖蛋白基因 表达 

分 类 号：S852.659.5[农业科学—基础兽医学]