【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。更多还原

【Abstract】 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 expressed 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.更多还原