【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CD4+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。更多还原

【Abstract】 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.更多还原