西方马脑炎核酸疫苗表达载体构建及免疫原性研究

Construction and Immunogenicity of Recombinant Expression Vectors of Western Equine Encephalomyelitis Virus

为构建西方马脑炎病毒(western equine encephalomyelitis virus,WEEV)结构基因C-E3-E2-6k-E1重组真核表达载体,并研究其作为核酸疫苗的免疫原性。采用PCR方法扩增目的基因,酶切之后连接到pcDNA3.1上构建真核表达载体pcDNA3.1-C-E,用酶切和测序分析方法鉴定正确后,重组质粒被转染到293T细胞,经电镜检测和间接免疫荧光方法证明基因可以表达后,用该重组质粒免疫小鼠,免疫后检测实验组小鼠外周血中CD4+T细胞/CD8+T细胞比例和血清中细胞因子IL-2、IL-4及IFN-γ浓度,以上实验组各项免疫指标与对照组相比差异均显著(P<0.05);ELISA方法检测实验组小鼠血清中WEEV的IgG抗体效价是1∶16。研究结果表明重组质粒pcDNA3.1-C-E可在细胞中获得瞬时表达,并且重组质粒作为核酸疫苗能够刺激小鼠产生免疫反应,具有较强免疫原性,为今后WEEV核酸疫苗研制奠定了良好基础。

In order to construct the expression vectors of all protein encoding genes C-E3-E2-6k-EI of Western Equine Encephalomyelitis Virus （WEEV） and confirm the immunogenicity of the vectors as the nucleic acid vaccine, the genes were amplified from the recombinant vector pVL1393-C-E3-E2-6k-E1 by PCR technique, the PCR products digested by enzyme were linked into pcDNA3.1 vectors, and the recombinant plasmids were designated as pcDNA3.1-C-E vectors. The identified recombinant plasmids were transfected into 293T cells. After the goal genes were expressed in 293T cells by the identification of the electron microscopy and indirect immunofluorescence （IIF）. The BALB/c mice were immu- nized by the recombinant plasmids. After the secondary immune, the ratio of CD4 ＋ T cell / CD8 ＋ T cell from the periph- era] blood of tile experimental mice and the concentration of IL-2,IL-4 and IFN-γ from the serum of experimental mice was significantly different compared with the control mice （ P 〈 0.05 ）. WEEV IgG antibody titers from the serum of ex- perimental mice was 1：16 by ELISA test after three times of immunization. The results showed that the experimental mice can produce strong immune reaction and the pcDNA3.1-C-E vectors are highly immunogenic in mice, which could pro- vide favorable foundation for DNA vaccine of WEEV in the future.