论文部分内容阅读
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 DIII region of the Japanese Encephalitis virus envelope protein (E protein) domain and understand its potential as a subunit vaccine, two primers were designed according to the sequence of SA14-14-2 strain (GenBank Accession No. D90195) The full-length JEV infectious clone pBR-JTF was used as a template to amplify the cDNA of JEVE protein DⅢ by PCR. The prokaryotic expression vector pET-JEDⅢ was constructed and transformed into E.coli Rosetta (DE3) for fusion expression. The fusion protein is soluble, and the expression amount is about 75% of the bacterial protein. The purified protein was used to immunize New Zealand rabbits and BALB / C mice, and the antigenicity and immunogenicity of JEDⅢwere verified by ELISA, Western blotting, plaque reduction assay and sucking mice. Western blotting and ELISA showed that the expressed product had good antigenicity. Immunized New Zealand rabbits with purified JEDⅢ protein could obtain anti-JEV specific antibody with a titer of 1: 7 × 105; BALB / C mice immunized with JEDⅢ protein, An anti-JEV-specific antibody titer of 1: 8.2 x 104 was obtained. And 1: 256 titers of neutralizing antibodies were obtained, and the protective effect of 75% was obtained in sucking mice. The above results show that the recombinant JEDIII protein, immunized mice and rabbits can produce anti-JEV specific antibodies and neutralizing antibodies, which can protect some of the mice from virulent challenge, antigenicity and immunogen Sex is better, there is the potential to further develop subunit vaccines.