【摘要】 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。更多还原

【Abstract】 Objective To express the NS1 and E proteins of Japanese encephalitis virus(JEV)SA14-14-2 strain in prokaryotic cells and study its immunogenicity. Methods The gene fragments encoding NS1 and E proteins of JEV SA14-14-2 strain were amplified by RT-PCR and cloned into prokaryotic expression vectors pET-30a(+)and pET-32a(+)respectively. The constructed recombinant plasmids pET-30NS1 and pET-32Et were transformed to E. coli BL21(DE3)respectively for expression under induction of IPTG. The expressed product was analyzed for form and expression level by SDS-PAGE, purified by affinity chromatography, identified for reactogenicity by Western blot, and evaluated for immunogenicity by active and passive protection test in mice and virusemia test in guinea pigs. Results Both restriction analysis and sequencing proved that recombinant plasmids pET-30NS1 and pET-32Et were constructed correctly. Both expressed recombinant NS1 and E proteins, with relative molecular masses of about 40 000 and about 47 000 respectively, existed in forms of inclusion body, reached concentrations of 160. 7 and 380. 0 μg / ml respectively, showed good reactogenicity. Both active and passive protection tests in mice showed protective effect of the two proteins. Virusemia test proved that NS1 protein inhibited virusemia in guinea pigs significantly. Conclusion Both NS1 and E proteins of JEV SA14-14-2 strains were successfully expressed and purified, which showed high immune protective activities.更多还原