肠道病毒71型VP1蛋白原核表达及初步鉴定

Expression of VP1 protein of enterovirus 71 in E. coli and verification of the expressive product.

目的采用分子克隆技术,构建肠道病毒71型(EV71)VP1全长基因大肠杆菌原核系统表达载体,诱导重组VP1融合蛋白表达。方法自EV71感染患者血清中提取病毒总RNA,进行一步法RT-PCR,扩增编码VP1蛋白的全长基因片段(891 bp),以pET32(a)为表达载体,构建重组表达质粒pET32(a)-VP1,转化E.coli.Rosseta感受态细胞,获得重组工程菌株。经诱导培养,SDS-PAGE电泳,免疫印迹鉴定表达产物。结果获得了含重组表达质粒pET32(a)-VP1的正相阳性工程菌株,经IPTG诱导能高效表达VP1融合蛋白。结论重组工程菌可表达VP1融合蛋白,对研究EV71发病机制及疫苗研制具有重要意义。

Objective The aim of this study are to construct recombinant expression vector containing VP1 whole gene of enterovirus 71 （EV71）by DNA recombinant technology and to induce expression of VP1 fusion protein in E. coli. Methods The interested gene with total extracted from the serum of patients. These patients were infected by EV71 virus. We inserted it into pET32（a）expression vector to construct recombinant expression vector pET32（a）- VP1. The verified recombinants pET32（ a）- VP1 were transformed into E. coli. Rossena to produce bacteria strain with positive recombinants,then the strain were analyzed with SDS - PAGE and immunoblotting to verify the expression product -- VP1 fusion protein. Results The positive bacteria containing the recombinant expressive vector pET28（a） - VP1 were constructed successfully, and high level expression of VP1 fusion protein was performed by inducing with IPTG. Conclusion It played an important role in investigating the pathogenesis of EV71 and developing vaccine in the future that the VP1 fusion protein of the positive strain with recombinant were expressed efficiently in E. coli system.