中文摘要：

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。

英文摘要：

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 CD4^＋/CD8^＋ 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 CD4^＋ T cells in the immunized group was significantly increased compared with that of the control ones. Whereas the number of CD8^＋ 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.