日本脑炎病毒囊膜蛋白的基因原核表达及抗原性分析

Prokaryotic Expression of Japanese Encephalitis Virus Envelope Protein and Antigenic Analysis

参照GenBank中的日本脑炎病毒序列设计一对特异性引物,采用RT-PCR法扩增E基因得到cDNA,克隆至pMD-18T载体,经测序证实后亚克隆至原核表达载体pET-28a中,转化入BL21(DE3),IPTG诱导表达,对表达产物进行SDS-PAGE电泳分析。结果表明,成功构建了重组质粒pET-28a/JEV E,目的蛋白主要以包涵体形式表达。Western blot检测表明,表达产物与兔抗JEV多克隆抗体呈阳性反应,在ELISA试验中,用表达出的E蛋白包被,能够很明显地区分出猪日本脑炎阴性、阳性血清。

In order to construct and express E protein of Japanese encephalitis virus （JEV）, and analyze an （DE3）. The E protein expressed in BL21 （ED3） was determined by SDS-PAGE. The result showed that the recombinant plasmid pET-28a/JEV E was successfully constructed and the expressed product was mainly in the form of inclusion body. Western blot analysis showed that the recombinant protein had a positive reaction with polyclonal antibody of rabbit against JEV, in the ELISA tests the positive serum of swine should be identified from negative ones obviously. This research made a foundation for further research on the serological diagnosis and the construction and function analysis of E protein.