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应用PCR方法扩增出HCVE2基因编码417a.a～750a.a的DNA片段,克隆到原核表达载体pQE30 LacZ启动子下游,转化JM109菌株。在JM109菌株中诱导表达出N端含6个组氨酸的E2融合蛋白,用Ni-NTA-Superflow亲和层析柱纯化作为抗原免疫实验兔和BALB/c鼠。定期取免血,采用间接ELISA方法检测兔子体内针对E2的抗体水平和维持规律。结果显示,距初次免疫14d兔子体内已有抗体产生,直至免疫第55d抗体水平持续上升,之后抗体水平保持稳定,抗体滴度达到1:3200。六周后,取鼠脾脏制备淋巴细胞,定向刺激扩增后与经过重组真核表达质粒pCE2转染的P815细胞作用,利用LDH释放试验检测作用效果。在E:T=200:1的情况下,杀伤率超过30％。这些结果表明工程菌株表达的HCV E2蛋白具有良好的免疫原性,可以诱发免疫实验动物机体产生较高滴度的抗体及特异性CTL应答。由此我们认为E2蛋白是发展HCV预防工程蛋白疫苗的合适候选者。
The DNA fragment encoding 417a.a ~ 750a.a of HCVE2 gene was amplified by PCR and cloned into the downstream of pZE30 LacZ promoter of prokaryotic expression vector to transform JM109 strain. The E2 fusion protein containing 6 histidines at the N-terminus was induced in the JM109 strain and purified by Ni-NTA-Superflow affinity chromatography as antigen immunoassay rabbits and BALB / c mice. Blood was taken regularly, and the level of antibody against E2 in rabbits was detected by indirect ELISA and maintained. The results showed that there was antibody production in rabbits 14 days after the first immunization, and the antibody level continued to increase until the 55th day after immunization. After that, the antibody level remained stable with an antibody titer of 1: 3200. Six weeks later, the spleen of the mice was used to prepare lymphocytes. The cells were stimulated with PCE2 after transfected with the recombinant eukaryotic expression plasmid pCE2 by directional stimulation. The effect of LDH release test was tested. With E: T = 200: 1, the kill rate is over 30%. These results indicate that the HCV E2 protein expressed by the engineered strains has good immunogenicity and can induce high titers of antibodies and specific CTL responses in the body of the immunized experimental animals. From this we propose that E2 protein is a suitable candidate for the development of HCV prophylactic engineering protein vaccines.