结核分枝杆菌Rv1626的原核表达及其免疫功能研究

Prokaryotic Expression and Immune Function of Mycobacterium Tuberculosis Rv1626

目的拟靶向结核分枝杆菌(M.tb)感染后的分泌期抗原Rv1626,构建其原核表达质粒pPROEX-Rv1626并表达纯化,通过人群和动物实验评价其免疫原性。方法构建重组载体pPROEX-Rv1626,以全血干扰素释放分析技术(WBIA)检测其是否能被山西省长治市M.tb感染者的T细胞特异性识别;同时免疫小鼠,检测其特异性诱导脾细胞分泌的IFN-γ、TNF-α和IL-2水平及抗体水平。结果 (1)成功构建重组载体pPROEX-Rv1626,并成功诱导表达、纯化和鉴定;(2)rRv1626蛋白诱导M.tb感染者外周血中淋巴细胞产生的IFN-γ水平均显著高于健康者对照者(P<0.001),ATB患者外周血中淋巴细胞分泌IFN-γ水平显著高于LTBI人群(P<0.001);(3)BCG+Rv1626/DMT组产生的特异性相关抗体滴度显著高于Rv1626/DMT组及BCG组(P<0.01);Rv1626/DMT组和BCG+Rv1626/DMT组的IgG2a/IgG1比值显著高于DMT组和BCG组(F=33.69),且前两组IgG2a/IgG1>1,倾向于Th1型细胞免疫应答;(4)不同免疫组小鼠无论是PPD或r Rv1626蛋白刺激,BCG+Rv1626/DMT组均分泌最高水平的IL-2、IFN-γ和TNF-α,其次为BCG组、Rv1626/DMT组,PBS组为最低。同时Rv1626/DMT组显著高于DMT组(P<0.01)。结论 r Rv1626能被M.tb感染者T细胞所识别,免疫小鼠能诱导抗原特异性Th1型细胞免疫应答,可能与其提供的免疫保护力密切相关。

Objective To target the secretory antigen Rv1626 after Mycobacterium tuberculosis(M.tb)infection,construct its prokaryotic expression plasmid pPROEX-Rv1626 and express it for purification.The immunogenicity was evaluated by human and animal experiments.Methods The recombinant vector pPROEX-Rv1626 was constructed and tested by whole blood interferon release assay(WBIA)to detect T cells specifically recognized by M.tb infected patients in Changzhi City,Shanxi Province.Simultaneous immunization of mice was used to detect their specific induction.The levels of IFN-γ,TNF-αand IL-2 secreted by spleen cells and antibody levels.Results①The recombinant vector pPROEX-Rv1626 was successfully constructed and successfully expressed,purified and identified.②rRv1626 protein induced IFN-γlevels in peripheral blood of M.tb infected patients was significantly higher than healthy controls(P<0.0001).At the same time,the level of IFN-γsecreted by lymphocytes in peripheral blood of ATB patients was significantly higher than that of LTBI(P<0.0005);③The specific antibody titers produced by BCG+Rv1626/DMT group were significantly higher than those of Rv1626/DMT group and BCG group(P<0.01).The ratio of IgG2a/IgG1 in Rv1626/DMT group and BCG+Rv1626/DMT group was significantly higher than that in DMT group and BCG group(=33.69),and the former two groups of IgG2a/IgG1>1 tended to Th1 type cellular immune response;④Different The mice in the immunized group were stimulated with PPD or rRv1626 protein,and the highest levels of IL-2,IFN-γand TNF-αwere secreted in the BCG+Rv1626/DMT group,followed by the BCG group and the Rv1626/DMT group,and the PBS group was the lowest.At the same time,the Rv1626/DMT group was significantly higher than the DMT group(P<0.005).Conclusion rRv1626 can be recognized by T cells infected by M.tb.Immunized mice can induce antigen-specific Th1-type cellular immune responses,which may be closely related to the immune protection provided by them.