【摘要】 目的:构建丙型肝炎病毒包膜区基因(E1、E2)真核表达载体,命名为PCI-neo-E1和E2。方法:将真核重组体转染NIH-3T3细胞中,利用RT-PCR方法鉴定相应的基因片段,显示获得了相应的稳定转染的细胞克隆。提取转染细胞蛋白,进行WesternBlot分析。结果:HCVE1、E2基因在真核细胞中获得有效表达。将所构建的PCI-neo-E1、E2免疫Balb/C小鼠,获得特异的抗体反应。结论:通过HCV包膜区DNA免疫的研究表明能激发特异的免疫反应,为进一步进行HCV疫苗研究奠定基础。更多还原

【Abstract】 Objective To investigate the capacity of HCV core DNA vaccine to induce the humoral and cellular immune response. Methods Firstly, the core gene of HCV was cloned to PGEM-T vector verified by enzyme digestion and direct sequencing. Secondly, the cloned gene was inserted to mammalian cells expression vector PCI-neo and expressed in the stable transfection clones of SP2/0 cells, which was confirmed by the mRNA and protein analysis. Subsequently, the capacity of PCI-neo-C to induce the humoral and cellular response in the female BALB/c mice were determined by the antibody titer and the specific cytotoxic T lymphocyte (CTL) response against HCV core protein. Results The PCI-neo-C had exactly the target gene sequence and the correct manner of enzyme digestion. The HCV core expression in the stable cell lines were confirmed by the mRNA found by Northern blot assay and a 20 ku protein found by Western blot assay. The titer of antibody against HCV core protein increased up to 1:640; moreover, the specific CTL response against the HCV core protein was detectable. Conclusions A HCV core plasmid expression in the mammalian cells is constructed successfully. The DNA vaccine based on this plasmid could elicit a specific immune response in mice, including both humoral and cellular response. This plasmid might be useful in the development of new HCV vaccines.更多还原