表达抗原Rv1255c的重组卡介苗的免疫原性研究  

Immunogenicity of recombinant BCG vaccine expressing Rv1255c antigen

作　　者：张天然 翁术锋 章金怡 粟海波[1] 徐颖[1] ZHANG Tian-ran;WENG Shu-feng;ZHANG Jin-yi;SU Hai-bo;XU Ying(Department of Genetics,School of Life Sciences,Fudan University,Shanghai 200438,China)

机构地区：[1]复旦大学生命科学学院遗传系,上海200438

出　　处：《微生物学免疫学进展》2022年第2期9-16,共8页Progress In Microbiology and Immunology

基　　金：国家自然科学基金(81971900);广东省医学科学技术研究基金(B2017066)。

摘　　要：目的 选取结核分枝杆菌RD10区抗原Rv1255c,构建重组卡介苗(rBCG::Rv1255c),评价其诱导先天性免疫和适应性免疫应答的能力。方法 通过分子克隆构建重组质粒pMV261-Rv1255c,将其电转进入BCG感受态细胞,通过蛋白印迹方法筛选得到表达Rv1255c的重组卡介苗;rBCG::Rv1255c腹腔免疫C57BL/6小鼠,在免疫后24 h和7 d,用ELISA、流式细胞术等方法检测吞噬细胞和淋巴细胞的比例、NO和H;O;的含量;rBCG::Rv1255c皮下免疫小鼠,在免疫后6周,用ELISA检测脾淋巴细胞分泌TNF-α、IFN-γ和IL-2的水平,用流式细胞术检测CD4;和CD8;T细胞分泌IFN-γ、IL-2的水平。结果 成功构建重组卡介苗rBCG::Rv1255c;腹腔免疫C57BL/6小鼠后,与BCG相比,rBCG::Rv1255c能够激活更强的先天性免疫应答,包括中性粒细胞数升高(P=0.000 1);NO(P=0.007 6,P<0.01;P<0.000 1)和H;O;(P=0.000 2,P<0.001;P=0.035 5,P<0.05)含量增加;同时rBCG::Rv1255c皮下免疫6周后的小鼠脾淋巴细胞针对特异抗原分泌更高水平的TNF-α(P<0.000 1)、IFN-γ(P=0.009 8,P<0.01)和IL-2(P=0.000 7,P<0.001),CD4;和CD8;T细胞分泌更高水平的IFN-γ(P=0.002 7,P<0.01)、IL-2(P=0.000 2,P<0.001;P=0.025 9,P<0.05)。这表明rBCG::Rv1255c可以更好地激活适应性免疫应答。结论 与BCG相比,rBCG::Rv1255c可以促进吞噬细胞的激活,增强TH1型细胞因子分泌,显示出良好的免疫原性,可作为潜在的抗结核疫苗进一步研究。Objective In this research, we constructed a recombinant BCG which expressing the antigen Rv1255 c of RD10 region of Mycobacterium tuberculosis and evaluated its ability to induce innate immunity and acquired immune response. Methods Recombinant plasmid pMV261-Rv1255 c was constructed by molecular cloning and transferred into BCG competent cells. Recombinant BCG vaccine expressing Rv1255 c was screened by western blot. The mice immunized intraperitoneously with recombinant BCG vaccine(rBCG::Rv1255 c) were evaluated the proportion of phagocytes to lymphocytes by flow cytometry and measured the content of NO and H;O;by ELISA at 24 hours and 7 days after immunization. The mice immunized by subcutaneous injection with recombinant BCG vaccine(rBCG::Rv1255 c) were detected the levels of TNF-α, IFN-γ and IL-2 secreted by splenic lymphocytes ELISA, and the levels of IFN-γ and IL-2 secreted by CD4;and CD8;T cells by flow cytometry at 6 weeks after immunization. Results Recombinant BCG vaccine rBCG::Rv1255 c was constructed successfully. After peritoneal immunization of C57 BL/6 mice, rBCG::Rv1255 c activated a stronger innate immune response compared with BCG. In which, the neutrophil(P=0.000 1)count and the contents of NO(P=0.007 6, P<0.01;P<0.000 1) and H;O;(P=0.000 2, P<0.001;P=0.035 5, P<0.05) significantly increased. The spleen lymphocytes of mice immunized subcutaneously with rBCG::Rv1255 c secreted higher levels of TNF-α(P=0.002 7, P<0.01), IFN-γ(P=0.009 8, P<0.01) and IL-2(P=0.000 7, P<0.001) in response to specific antigens, CD4;and CD8;T cells of rBCG::Rv1255 c immunized mice secreted higher levels of IFN-γ(P=0.002 7, P<0.01), IL-2(P=0.000 2, P<0.001;P=0.025 9, P<0.05). These results indicated that rBCG::Rv1255 c could activate the adaptive immune response. Conclusion Compared with BCG, rBCG::Rv1255 c can promote the activation of phagocytes and enhance the secretion of TH1 cytokines, showing good immunogenicity. It could be used as a potential anti-tuberculosis vaccine for further study.

关 键 词：重组卡介苗 先天性免疫 适应性免疫 结核分枝杆菌 Rv1255c抗原 

分 类 号：R183[医药卫生—流行病学]