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  

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

作　　者：任守凤[1] 刘文权[1] 曹国梅[1] 梁韶晖[1] 潘长旺[1] 

机构地区：[1]温州医科大学基础医学院寄生虫学教研室,浙江温州325035

出　　处：《温州医科大学学报》2016年第4期235-240,244,共7页Journal of Wenzhou Medical University

基　　金：浙江省自然科学基金资助项目(LY13C080001)

摘　　要：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p Cold Ⅱ的多克隆位点,并将构建的重组质粒p Cold Ⅲ-EDⅡ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ELⅢSA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。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 p Cold Ⅲ to constructe the recombinant vector p Cold Ⅲ-EDⅢ. The constructed p Cold Ⅲ-EDⅢ plasmid was transformed into E.coli BL21 cells and induced by IPTG. The recombinant protein EDⅢ was purifi ed by Ni-NTA affi nity chromatography and confi rmed by SDS-PAGE and Western blot analysis. The purifi ed 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 specifi c 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 signifi cantly higher in the experimental group. Conclusion： Our studies indicates that dengue virus envelope protein EDⅢ tetravalent vaccine can induce specifi c 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.

关 键 词：登革病毒 包膜蛋白 融合表达 四价疫苗 

分 类 号：R373.33[医药卫生—病原生物学]