重组登革病毒1-4型包膜蛋白EDIII的融合表达和免疫学特性研究

目的：构建含登革病毒（DENV）1-4型包膜蛋白（E蛋白）EDIII区的融合蛋白，并通过动物实验分析该融合蛋白的免疫机制。方法：通过生物信息学分析获得具有广泛代表性的DENV 1-4型EDIII区的氨基酸序列；将编码DENV 1-4型EDIII区的基因序列通过GS linker进行串联后插入原核表达载体pColdIII的多克隆位点，并将构建的重组质粒pColdIII-EDIII转化至大肠杆菌BL21（DE3）；通过SDS-PAGE及Western blot法鉴定目的蛋白表达，采用镍柱亲和层析法纯化目的蛋白。将纯化的蛋白免疫BABL/c小鼠，初次免疫之后再加强免疫2次。通过ELISA法检测血清中的抗体效价和细胞因子的分泌情况来评价其免疫效应。结果：重组蛋白EDIII免疫小鼠后可以诱导产生高滴度的特异性抗体，抗体效价为1:40 000。抗原刺激小鼠脾淋巴细胞可以诱导Th1和Th2细胞免疫应答。结论：成功表达了含DENV 1-4型E蛋白EDIII的融合蛋白，并通过动物实验证实该融合蛋白能诱导小鼠产生特异性的体液和细胞免疫应答，为DENV四价重组疫苗的研制奠定了基础。

Fusion expression and immune investigation of recombinant dengue virus EDIII tetravalent protein#br#

Objective: To construct dengue virus envelope protein EDIII tetravalent recombinant vaccines and to analyze its immunogenicity. Methods: Firstly, the gene sequences encoding dengue virus type 1-4 envelope protein EDIII were analyzed by bioinformatics methods, and the representative DENV1-4 EDIII was selected and connected by GS linker to obtain chimeric EDIII; Then, the chimeric gene coding EDIII was inserted into the multiple cloning site of the expression vector pColdIII to constructe the recombinant vector pColdIII-EDIII. The constructed pColdIII-EDIII plasmid was transformed into E.coli BL21 cells and induced by IPTG. The recombinant protein EDIII was purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis. The purified protein was used to immune BABL/c mice. After boosting twice, to detect the antibody titers in serum and the secretion of cytokines by ELISA to evaluate the immune response. Results: The mice immunized with EDIII could induce specific antibodies and the antibody titers was 1:40 000. The mouse spleen lymphocytes stimulated by antigen could produce cytokine including IFN-r, IL-4, IL-10, which were significantly higher in the experimental group. Conclusion: Our studies indicates that dengue virus envelope protein EDIII tetravalent vaccine can induce specific humoral and cellular immune response, and give priority to with the humoral immune response, which laid the foundation for the research of a new type of chimeric tetravalent vaccine in the future.