表达抗原 Rv3478的重组卡介苗的实验研究  

Immunogenicity analysis of a recombinant BCG vaccine strain overexpressing Rv3478 protein

作　　者：孔聪[1] 朱琳[1] 粟海波[1] 黄琪[1] 李光华[1] 宋娜[1] 徐颖[1] 王洪海[1] 

机构地区：[1]复旦大学生命科学学院,上海200438

出　　处：《中华微生物学和免疫学杂志》2015年第5期359-366,共8页Chinese Journal of Microbiology and Immunology

摘　　要：目的：本研究选取结核杆菌优秀抗原Rv3478，构建重组疫苗rBCG：Rv3478并对其进行免疫原性评价。方法 PCR扩增基因Rv3478，构建重组质粒Rv3478-pMV261之后电转进入BCG感受态细胞中，另外将扩增的基因片段导入pET-28a中，构建表达载体Rv3478-pET-28a（ BL21）表达蛋白Rv3478，蛋白免疫小鼠获取多克隆抗体，蛋白印迹方法筛选得到表达Rv3478的重组卡介苗；用构建好的重组卡介苗刺激巨噬细胞，收集细胞培养上清 ELISA 检测细胞因子的分泌；重组疫苗rBCG：Rv3478（R3）免疫小鼠，PBS、BCG、BCG：pMV261（R0）作对照组。分别在免疫4周和12周后处死小鼠，用ELISPOT、ELISA、流式细胞术等方法检测疫苗细胞免疫和体液免疫水平。结果成功表达蛋白Rv3478，免疫小鼠后获得多克隆抗体；成功构建重组疫苗R3；免疫小鼠后，能够引发强烈的细胞免疫应答（强烈的IFN-γ反应，高水平的TNF-α的分泌），促进外周血CD4＋／CD8＋T细胞比例增加， CD44＋CD62L＋T细胞百分比增加。结论构建的重组疫苗R3能够引发强烈的免疫应答，显示出良好的免疫原性，具有良好的抗结核潜力，值得进一步研究。Objective To construct a recombinant Bacillus Calmette-Guerin （ BCG ） vaccine strain, rBCG：：Rv3478-pMV261, expressing the Rv3478 protein of Mycobacterium tuberculosis and to inves-tigate its immunogenicity.Methods The gene fragments encoding Rv3478 antigen were amplified by PCR and then respectively cloned into pMV261 and pET-28a vectors to construct the recombinant expression plas-mids （Rv3478-pMV261 and Rv3478-pET-28a）.The Rv3478-pMV261 plasmids were transformed into the BCG cells to construct the rBCG vaccine strains, while the Rv3478-pET-28a plasmids were transformed into Escherichia coli BL21 strains for the expression of Rv3478 protein.Polyclonal antibodies were induced in mice upon the immunization with Rv3478 protein.The rBCG vaccine strains overexpressing Rv3478 protein were screened out with Western blot assay.The C57BL/6 mice were divided into four groups including the PBS treated group, BCG treated group, rBCG：：pMV261 （ R0） treated group and rBCG：：Rv3478-pMV261 （ R3） treated group.All mice were sacrificed in 4 or 12 weeks after immunization.Enzyme-linked immunos-pot assay （ ELISPOT） , ELISA and flow cytometry analysis were performed to evaluate the induced humoral and cell-mediated immune responses in mice.Results The Rv3478 protein was successfully expressed and could induce polyclonal antibodies in mice.High levels of IFN-γand TNF-αwere detected in mice treated with R3, indicating that the immunization with R3 enhanced the cellular immunity.Moreover, the ratios of CD4＋to CD8＋T cells and the percentages of CD44＋CD62L＋T cells were increased in mice upon the immuni-zation with R3.Conclusion The recombinant BCG vaccine strain overexpressing Rv3478 protein could in-duce stronger cell-mediated immune responses in mice.It might be have a great significance as a new tuber-culosis（ TB） vaccine strain against TB infection in the future.

关 键 词：结核病 重组卡介苗 Rv3478 疫苗 

分 类 号：R392[医药卫生—免疫学]