结核分枝杆菌Rv1626的原核表达及其免疫功能研究(1)
摘要：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;②rRv1626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);③BCG+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rRv1626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 rRv1626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。
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关键词：结核分枝杆菌;Rv1626;原核表达;免疫原性;结核病
中图分类号：R378.91+1 文献标识码：A DOI：10.3969/j.issn.1006-1959.2019.01.022
文章編号：1006-1959(2019)01-0065-04
Prokaryotic Expression and Immune Function of Mycobacterium Tuberculosis Rv1626
YUAN Wei1，XU Li-fa1，WANG Xiao-chun1，ZHANG Jing-yan2，ZHU Xin-yi3
, 百拇医药
(1.School of Medicine，Anhui University of Science and Technology，Huainan 232001，Anhui，China;
2.Department of Clinical Laboratory，Affiliated Heping Hospital，Changzhi Medical College，Changzhi 046000，Shanxi，China)
Abstract：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-α and IL-2 secreted by spleen cells and antibody levels. Results ①The recombinant vector pPROEX-Rv1626 was successfully constructed and successfully expressed， purified and identified. ②rRv1626 protein induced IFN-γ levels 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-γ secreted 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 (F=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-γ and TNF-α were 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., http://www.100md.com(袁伟 许礼发 王晓春 张京燕 朱心怡)
, http://www.100md.com
关键词：结核分枝杆菌;Rv1626;原核表达;免疫原性;结核病
中图分类号：R378.91+1 文献标识码：A DOI：10.3969/j.issn.1006-1959.2019.01.022
文章編号：1006-1959(2019)01-0065-04
Prokaryotic Expression and Immune Function of Mycobacterium Tuberculosis Rv1626
YUAN Wei1，XU Li-fa1，WANG Xiao-chun1，ZHANG Jing-yan2，ZHU Xin-yi3
, 百拇医药
(1.School of Medicine，Anhui University of Science and Technology，Huainan 232001，Anhui，China;
2.Department of Clinical Laboratory，Affiliated Heping Hospital，Changzhi Medical College，Changzhi 046000，Shanxi，China)
Abstract：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-α and IL-2 secreted by spleen cells and antibody levels. Results ①The recombinant vector pPROEX-Rv1626 was successfully constructed and successfully expressed， purified and identified. ②rRv1626 protein induced IFN-γ levels 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-γ secreted 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 (F=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-γ and TNF-α were 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., http://www.100md.com(袁伟 许礼发 王晓春 张京燕 朱心怡)