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目的原核表达乙型脑炎病毒(JEV)NS1和E蛋白,并初步探讨其免疫原性。方法采用RT-PCR法扩增JEVSA14-14-2株NS1和E蛋白基因片段,分别克隆入原核表达载体pET-30a(+)和pET-32a(+),构建重组表达质粒pET-30NS1和pET-32Et,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析目的蛋白的表达形式及表达水平。两种蛋白经亲和层析纯化后,Western blot鉴定其反应原性。通过小鼠主动和被动保护力试验及豚鼠病毒血症试验,检测两种蛋白的免疫原性。结果酶切及测序证明重组表达质粒构建正确;表达的重组NS1和E蛋白的相对分子质量分别约为40000和47000,均以包涵体形式表达;纯化的重组NS1和E蛋白的浓度分别为160.7和380μg/ml,均具有良好的反应原性;小鼠主动和被动保护力试验表明两种蛋白均有保护活性;豚鼠病毒血症试验显示,NS1蛋白免疫豚鼠后能明显抑制病毒血症的产生。结论已成功表达并纯化了JEVSA14-14-2株NS1和E蛋白,两种蛋白均有较好的免疫保护活性。
Objective To prokaryotically express NSE and E proteins of Japanese encephalitis virus (JEV) and to investigate their immunogenicity. Methods The NS1 and E gene fragments of JEVSA14-14-2 strain were amplified by RT-PCR and cloned into the prokaryotic expression plasmids pET-30a (+) and pET-32a (+) respectively. The recombinant plasmid pET-30NS1 and pET- -32Et, transformed into E.coli BL21 (DE3) and induced by IPTG. The expression and expression of the target protein were analyzed by SDS-PAGE. After purified by affinity chromatography, the two proteins were identified by Western blot. The immunogenicity of the two proteins was tested by the active and passive protective mice and the guinea pig viremia test. Results Restriction analysis and sequencing proved correct recombinant plasmid; relative molecular mass of the recombinant E and NS1 protein expressed approximately 40,000 and 47,000, respectively, are expressed in the form of inclusion bodies; purified recombinant NS1 protein concentrations and E were 160.7 and 380μg / ml, both have good reactionogenicity; active and passive protective mice test showed that both proteins have protective activity; guinea pig viremia test showed that NS1 protein immunized guinea pigs can significantly inhibit viremia. Conclusion The NS1 and E proteins of JEVSA14-14-2 strain have been successfully expressed and purified. Both proteins have good immunoprotective activity.