摘要
以O型FMDV重组质粒pMD18T-O-VP1为模板,利用PCR技术扩增得到O型FMDV-VP1基因片段,将此基因片段与原核表达载体pET32a连接构建重组表达载体pET32a-O-VP1,经PCR和测序鉴定后,用IPTG诱导VP1基因的表达,收集不同诱导时间的菌液,进行SDS-PAGE电泳,摸索掌握最佳诱导时间,切取最佳诱导时间电泳胶片做Western-blotting,分析检验表达产物与其抗血清的反应性。结果显示,分子量约为45 ku的蛋白条带反应显著,能被口蹄疫阳性血清识别,表明FMDV-VP1基因在大肠杆菌中得到高效表达,纯化复性的表达蛋白有望开发为诊断抗原和多肽疫苗。
With O-FMDV recombinant plasmid pMD18T-O-VP1 as a template, O-FMDV- VP1 gene fragments were obtained by using PCR amplification. This gene fragments wwas connected with prokaryotic expression vector pET32a to construct recombinant expression vector pEq32a-O-VP1. After PCR identification and sequencing, IP1G was used to induce the expression of VP1 gene. Different induction time of the bacterium were collected for SDS-PAGE electrophoresis. The best induction time was mastered. And the electrophoresis film with the best induction time was cut to do Western-blotting.The expression product testing with its anti-serum responsiveness was analyzed. The results showed that the protein with the molecular weight of about 45 ku had significant strip reaction and it c~.dd be identified by positive serum of feed-and-mouth disease. These results showed that the FMDV- VP1 genes were effficiently expressed in E. coll. The purified expression protein is expected to develop diagnostic antigen mad peptide vaccines.
出处
《安徽农业科学》
CAS
北大核心
2009年第15期6876-6878,共3页
Journal of Anhui Agricultural Sciences
关键词
口蹄疫病毒
VP1基因
原核表达
蛋白复性
Foot-and-mouth disease virus
VP1 gene
Prokaryotic expression
Protein renaturation
