SARS冠状病毒E蛋白DNA疫苗的构建及其实验免疫研究  

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

作　　者：甘燕[1] 吴少庭[1] 秦莉[2] 潘晖榕[2] 戴五星[2] 袁仕善[1] 黄达娜[1] 

机构地区：[1]深圳市疾病预防控制中心,深圳518020 [2]华中科技大学同济医学院,武汉430030

出　　处：《中国人兽共患病学报》2006年第12期1139-1142,共4页Chinese Journal of Zoonoses

基　　金：深圳市科技计划项目资助(2003-k-07)

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。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.

关 键 词：SARS冠状病毒 小囊膜蛋白 DNA疫苗 

分 类 号：R373[医药卫生—病原生物学]