融合免疫佐剂Lipo多肽与包膜蛋白EDⅢ区的登革病毒四价疫苗的构建及免疫效应研究

Construction of fusion immunoadjuvants Lipo peptides and envelope protein EDⅢ dengue virus tetravalent vaccine and its immune effect

目的构建编码免疫佐剂Lipo多肽与登革病毒1-4型包膜蛋白EDIII区的重组甲病毒载体,并在小鼠体内研究其免疫效应,为研制新型的登革病毒四价疫苗奠定基础。方法先通过GS linker将编码免疫佐剂Lipo多肽的基因序列与编码登革病毒1-4型包膜蛋白EDIII区的基因序列进行串联获得LipoEDIII;然后将LipoEDIII基因插入甲病毒载体DREP的多克隆位点,构建重组甲病毒载体DREP-LipoEDIII;将DREP-LipoEDIII转染293细胞,Western blot检测融合蛋白的表达;随后将DREP-LipoEDIII免疫ICR小鼠,不加任何佐剂,再加强免疫2次后,通过ELISA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。结果成功构建了含有"融合免疫佐剂Lipo多肽与包膜蛋白EDIII区"的重组甲病毒质粒DREP-LipoEDIII。DREP-LipoEDIII免疫小鼠后可以诱导产生特异性抗体,抗体效价为1∶160。抗原刺激小鼠脾淋巴细胞可以产生IFN-r、IL-4、IL-10细胞因子,实验组的浓度明显高于对照组。结论在不需要加入任何外源佐剂的情况下,本研究构建的融合免疫佐剂Lipo多肽的登革四价疫苗能够诱导小鼠产生特异性的体液和细胞免疫应答,且以细胞免疫应答为主,为今后新型登革病毒四价疫苗的研究奠定了基础。

We constructed a recombinant tetravalent DENV alphavirus vaccine coding of immunoadjuvants Lipo peptides and dengue virus type 1-4envelope protein EDIII zone and studied its immune response in mice.Firstly,the gene sequence encoding immunoadjuvants Lipo and the gene sequence encoding dengue virus type 1-4envelope protein EDIII zone was connected by GS linker to obtain LipoEDIII.Then,the gene LipoEDIII was inserted into the multiple cloning site of the alphavirus vector DREP to construct the recombinant alphavirus vector DREP-LipoEDIII.Furthermore,the 293 cells were transfected with the recombinant alphavirus vector DREP-LipoEDIII,the fusion protein expression was detected by Western blot.At last,the ICR mice were immunized with DREP-LipoEDIII without any adjuvant.The antibody titers in serum and the secretion of cytokines were detected by ELISA after the twice booster to evaluate the immune response.The mice immunized with DREP-LipoEDIII could induce specific antibodies and the antibody titer was 1∶160.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.Our studies indicates that the tetravalent DENV alphavirus vaccine containing the fusion Lipo peptides can induce specific humoral and cellular immune responses in mice without adding any exogenous adjuvant,which laid the foundation for the research of novel tetravalent dengue virus vaccine in the future.