To screen the best genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens, the prMEIII gene, the polytope gene and the prMEIII-polytope fusion gene of the GenotypeⅠJapanese encephalitis virus GS strain were cloned into prokaryotic expression vector pET-30a. The recombinant proteins were obtained after the induction and purification. The prepared recombinant proteins were immunized to mice, and the immunogenicity of the subunit vaccine candidate antigens was evaluated through monitoring the humoral immune response by ELISA, detecting the neutralizing antibody titer by plaque reduction neutralization test, and testing the cell-mediated immune response by lymphocyte proliferation assay and cytokine profiling. The recombinant proteins with the molecular weights of 35 (prMEIII), 28 (polytope antigen) and 57 kDa (prMEIII-polytope) induced strong humoral and cellular immune responses in mice. Compared with prMEIII-polytope and polytope proteins, the prMEIII protein induced a significant expression of IL-2 and IFN-γ (P<0.05) and the significant lymphoproliferation of splenocytes (P<0.05). The neutralizing antibody titer induced by the prMEIII protein was close to that induced by the commercial attenuated vaccine SA14-14-2 (P>0.05). The study suggests that the prMEIII protein can be used for the development of the Japanese encephalitis virus subunit vaccine.
为了筛选出免疫原性最佳的基因Ⅰ型乙型脑炎病毒亚单位疫苗候选抗原，将基因Ⅰ型JEV GS 株的prMEIII 融合基因、polytope 复合表位基因和prMEIII-polytope 融合基因分别克隆构建到原核表达载体pET-30a 上，经诱导表达纯化获得重组蛋白。将制备的重组蛋白免疫小鼠，通过ELISA 监测体液免疫反应、通过噬斑减少中和试验滴定中和抗体滴度、通过细胞因子表达丰度和淋巴细胞增殖实验分析细胞介导的免疫反应，比较分析制备的乙型脑炎病毒亚单位疫苗候选抗原的免疫原性。结果表明：获得的分子量分别为35 kDa (prMEIII)、28 kDa(polytope 复合表位抗原) 和57 kDa (prMEIII-polytope) 的重组蛋白均能诱导免疫小鼠产生较强的体液免疫和细胞免疫反应。与prMEIII-polytope 和polytope 重组蛋白免疫组相比，prMEIII 蛋白可诱导免疫小鼠产生更高的IL-2 和IFN- γ 表达丰度和淋巴细胞增殖水平 (P<0.05)。prMEIII 蛋白免疫小鼠诱导产生的中和抗体滴度接近于商品化乙脑减毒疫苗SA14-14-2 (P>0.05)。上述研究结果表明，prMEIII 重组蛋白可以作为乙型脑炎病毒亚单位疫苗的备选蛋白。.
Keywords: Japanese encephalitis; immunogenicity; polytope; subunit vaccine.