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目的:用大肠杆菌表达系统表达人巨细胞病毒(HCMV)pp65蛋白和gB蛋白的优势抗原表位基因,制成HCMV亚单位疫苗,探究其在Balb/c小鼠体内的体液免疫和细胞免疫活性。方法:选取pp65蛋白的490~508aa和gB蛋白的607~621aa的基因片段,经PCR扩增目的片段,连接表达载体pET-32a(+),转化BL21(DE3)plys菌株,十二烷基硫酸钠-聚丙烯酰胺凝胶(SDSPAGE)分析蛋白质表达,金属螯合亲和镍柱法纯化目的蛋白。重组蛋白免疫Balb/c小鼠,Western blotting法和间接ELISA法检测重组蛋白的体液免疫活性;通过流式细胞仪和双抗夹心ELISA法检测重组蛋白的细胞免疫活性。结果:获得分子质量约为22kDa的融合蛋白,Western blotting检测显示抗体有特异性,间接ELISA法检测其效价为1∶102 400。与对照组相比,实验组小鼠外周血中CD4~+T细胞和CD8~+T细胞的数量,以及IFN-γ、IL-2、IL-12的含量有显著提高(P<0.01)。结论:制备的具有免疫优势抗原表位的重组蛋白能诱导小鼠产生较强的体液免疫和细胞免疫反应。
OBJECTIVE: To express HCMV pp65 and gB protein epitope genes by using E. coli expression system to make HCMV subunit vaccine and investigate its humoral and cellular immune activities in Balb / c mice . Methods: The gene fragments of 490 ~ 508aa of PP65 and 607 ~ 621aa of gB were selected. The target fragment was amplified by PCR and ligated with the expression vector pET-32a (+). The recombinant plasmid was transformed into BL21 (DE3) The protein expression was analyzed by sodium-polyacrylamide gel (SDSPAGE) and the target protein was purified by metal chelate affinity column method. The recombinant protein was used to immunize Balb / c mice. The humoral immune activity of the recombinant protein was detected by Western blotting and indirect ELISA. The cellular immunoreactivity of the recombinant protein was detected by flow cytometry and double antibody sandwich ELISA. Results: The fusion protein with a molecular mass of about 22 kDa was obtained. Western blotting showed that the antibody was specific. The titer of the fusion protein was 1:102 400 by indirect ELISA. Compared with the control group, the number of CD4 ~ + T cells and CD8 ~ + T cells in peripheral blood and the levels of IFN-γ, IL-2 and IL-12 in the experimental group were significantly increased (P <0.01). Conclusion: The prepared recombinant protein with immunodominant epitopes can induce strong humoral and cellular immune responses in mice.