HCV E2蛋白诱导的体液免疫及CTL应答研究

The Humoral and Cellular (CTL) Immunological Property of HCV E2 Protein

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 E2 gene of Hepatitis C Virus was amplified by PCR, and cloned into the pQE30 at the downstream of LacZ promoter .The recombinant vector DNA was introduced into the E.coli JM109 cell to create the JM109 pQE2. The HCV E2 protein fused to 6 His peptides with molecular weight of 35kDa was produced in JM109 pQE2 cell. To study the immunological property of E2 protein, the rabbit and BALB/c mice were vaccinated with E2 protein purified by Ni-NTA-Superflow. The antibody against E2 was detected by ELISA, the results showed that the HCV E2 specific antibodies could be observed at the 14th day after immune, then the level of antibodies rose and reached the highest peak at the 55th day, then the antibodies had maintained at this high level with titer of 1:3200 in this study.The specific CTLs were gotten from the mice spleens, after simulated and augment in vitro,they were used as the effect cells to kill the target cells, which were called P815 transfected wtih pCE2. By LDH assay,we can fred that more than 30% of the target cells were killed at the ration E:T=200:1. and there are different effects in different immunity injection means. The results show that the E2 protein can provoke antibody in immune rabbits and specific CTLs in the mice. From this study, we suggest that the E2 protein is a good candidate for developing a vaccine to prevent HCV infection.