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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
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)
关键词
小反刍兽疫病毒
H糖蛋白基因
表达
peste des petits ruminants virus（PPRV）
H glycoprotein gene
expression
