日本脑炎病毒SA14-14-2E蛋白结构域Ⅲ的抗原性和免疫原性分析

Immunogenicity and antigenicity of Japanese encephalitis virus envelope protein domain Ⅲ

为了表达日本脑炎病毒囊膜蛋白(E蛋白)结构域DⅢ区,了解其作为亚单位疫苗的可能性,本研究根据SA14-14-2病毒株序列(GenBank Accession No.D90195)设计两条引物,以全长JEV感染性克隆pBR-JTF为模板,通过PCR扩增出JEVE蛋白DⅢ的cDNA片段,构建了原核表达载体pET-JEDⅢ,转化大肠杆菌Rosetta(DE3)进行融合表达。融合蛋白为可溶性表达,表达量约占菌体蛋白的75%。用纯化后蛋白免疫新西兰兔和BALB/C鼠,通过ELISA,Western blotting,噬斑减少实验,及乳鼠攻毒实验验证JEDⅢ的抗原性和免疫原性。Western blotting及ELISA结果表明纯化后的表达产物具有良好的抗原性,纯化的JEDⅢ蛋白免疫新西兰兔,可以获得高达1:7×105滴度的抗JEV特异性抗体;JEDⅢ蛋白免疫BALB/C鼠,可以获得1:8.2×104滴度的抗JEV特异性抗体。并且获得1:256滴度的中和抗体,乳鼠攻毒实验能达到75%的保护效果。以上结果说明本研究表达、纯化的重组JEDⅢ蛋白,免疫小鼠以及兔后,能产生抗JEV的特异性抗体,中和性抗体,能够保护部分乳鼠接受毒种的攻击,抗原性及免疫原性较好,有进一步开发研制成亚单位疫苗的潜能。

To express the domain Ⅲ gene of Japanese encephalitis virus （JEV） and to learn the possibility of developing the DⅢ protein as a subunit vaccine, we amplified the JEV DⅢ gene by PCR and constructed the expression plasmid pET-JE DⅢ by inserting JEV DⅢ gene into the prokaryofic expression vector pET-32a（＋）. The domain III protein of the attenuated strain SA14-14-2 was expressed as a thioredoxin （Trx） fusion protein, which was unique in forming a large fraction of the soluble recombinant protein. We immunized the rabbits and mice with the purified protein, tested the antigenicity and imrnunogenicity of JEV DⅢ protein by ELISA, Western blotting, plaque reduction test and observed the protective efficacy on challenged weanling mice with JEV. Rabbits immunized with the purified JEV Dill protein generated 1：7×10^5 anti-JEV specific antibody titers. BALB/c mice immunized with the purified JEV E DⅢ protein generated 1：8.2×10^4 anti-JEV specific antibody fiters. And the neutralized antibody titer can reach 1：256, the survival rate of the immunized weanling mice was approximately 75%. Overall, this study highlighted that recombinant JEV E DⅢ protein delivered in mice and rabbits can generate high antibody titers against JEV, and protect some mice challenged with JEV. These studies can provide useful information for further developing the domain Ⅲ recombinant protein as subunit vaccine against JEV.