乙型脑炎病毒贵州分离株E基因原核表达及其免疫原性的研究

收集某猪场1例发病种公猪的睾丸病料,处理后采用细胞培养与乳鼠脑内接毒相结合的方法盲传并结合RT-PCR方法分离鉴定,命名为乙型脑炎病毒(JEV)贵州分离株(GZ株),然后根据GenBank登录的日本乙型脑炎病毒SA14和SA14-14-2株全基因序列设计并合成1对扩增E基因的特异性引物,用RT-PCR方法扩增出JEV贵州分离株E基因,并将E基因克隆到pMD19-T载体上,构建重组质粒pMD19-T-E,经双酶切鉴定、测序及序列分析鉴定后,再将E基因亚克隆到pET-32a(+)原核表达载体上,成功构建乙脑病毒E基因原核表达质粒pET-32a-E,将构建好的原核表达质粒pET-32a-E转化至宿主菌BL21(DH3)中,诱导表达目的蛋白,表达产物经SDS-PAGE电泳和Western blot分析,得到了约59ku条带,与预期大小相符,进一步提取、纯化和浓缩目的蛋白,并将其加入弗氏佐剂后免疫小鼠,免疫后采血检测抗体。结果显示:乙型脑炎病毒贵州分离株E基因原核表达目的蛋白能诱导小鼠产生一定抗体水平,该研究为乙型脑炎亚单位疫苗的研究奠定了基础。

The Study of Prokaryotic Expression and Immunogenicity of E Gene of Japanese Encephalitis Virus Guizhou Isolation Strain

Japanese Encephalitis Virus(JEV) was isolated from a porcine’s intumescent testis.After specimens were made into homogenate and filtrated,it was inoculated to passage cells and injected into rat brain to isolate JEV,then JEV was identified by RT-PCR.According to published cDNA sequence of E gene in GenBank of JEV SA14 and SA14-14-2 strain,the synthesized specific primers were designed.E gene fragments of JEV Guizhou strain were amplified by using RT-PCR.The PCR products were sequenced and comparatively analyzed.The products of RT-PCR were cloned into pMD19-T vector to form recombinants pMD19-T-E,then were subcloned into Prokaryotic Expressing Vector pET-32a(+) after double enzyme digestion and sequence analysis.The pET-32a-E was transformed into the E.coli strain BL21(DE3) and the expression of the E protein was induced.After analysis by SDS-PAGE electrophoresis of the induced expressed protein and Western blot,we got about 59 kDa expressed protein in line with the expected size.Through further extracting and purifying the target protein,the expressed protein was used to immunize mice by adding adjuvant,and then the antibody titers of mice were detected.The results showed that the target protein induced anti-JE antibody in mice.This study laid the foundation of the research of subunit vaccine of Japanese Encephalitis.