SARS冠状病毒E蛋白DNA疫苗的构建及其实验免疫研究

目的构建SARS冠状病毒（SARS-CoV）小囊膜蛋白（E蛋白）的DNA疫苗pVAC-E，观察其在小鼠中诱导的免疫应答。方法采用PCR方法体外扩增SARS冠状病毒E蛋白的基因片段，克隆入真核表达载体pVAC，构建pVAC-E重组质粒。通过脂质体介导瞬时转染非洲绿猴肾（Vero）细胞，Western-blot鉴定E蛋白在细胞中的表达。以基因枪方式免疫小鼠，ELISA检测小鼠血清特异性抗体，MTT法测定淋巴细胞转化率，流式细胞仪检测小鼠脾脏T淋巴细胞亚群分布。结果成功构建SARS-CoVDNA疫苗pVAC-E。Western-blot结果示，转染后的Vero细胞可表达一约9kD大小能被SARS病人血清特异识别的蛋白条带。免疫小鼠中未检测到明显的抗体滴度升高。淋巴细胞转化率在免疫组和对照组间无差别（P〉0．05）。脾脏T淋巴细胞亚群分析示免疫组小鼠CD才细胞显著升高（P〈0．05），CD8^＋细胞与对照组相比无显著差异。结论构建的真核表达质粒pVAC-E能在Vero细胞中表达，表达产物具免疫活性，免疫小鼠后能诱导一定的细胞免疫应答。

Construction of DNA vaccine encoding the small envelope protein of SARS coronavirus and the experimental immunization study in mice

To construct the eukaryotic expression plasmid of gene fragment of envelope protein of SARS coronavirus (E) and to observe its immune responses in Balb/c mice, the specific gene of E was amplified by PCR, and was subcloned to the eukaryotic expression plasmid pVAC. Then the recombinant plasmid was transfected into Vero cell line with lipofectamine 2000. The expression of E protein was detected by western blotting. The plasmid was inoculated into Balb/c mice by gene gun injection. The specific antibody to E protein was detected by ELISA, and the proliferation activity of T lymphocytes was tested by MTT assay. The percentage of spleen CD~ + _4/CD~ + _8 T cells were analyzed by flow cytometry. It was found that the recombinant plasmid pVAC-E was successfully constructed, and the expression of E protein was detected in vitro. Specific antibodies cannot be detected after immunization. The proliferate responses between control and immunized spleen cells showed no difference (P>0.05). The number of CD~ + _4 T cells in the immunized group was significantly increased compared with that of the control ones. Whereas the number of CD~ + _8 T cells was higher than that of the control, but with no significant difference(P>0.05). It demonstrated that the recombinant pVAC-E can be expressed in mammalian cell, and induce cellular immunity in mice.