结核分枝杆菌ESAT-6＋CFP-10融合抗原克隆表达及其生物学活性

目的：克隆表达结核分枝杆菌（Mycobacterium tuberculosis，Mtb）融合抗原ESAT-6＋CFP-10（EC），评价融合抗原EC刺激THP-1细胞培养上清中特定细胞因子分泌水平的变化。方法构建融合抗原EC原核表达载体，用纯化后的不同浓度融合抗原EC （10，20μg／ml ）刺激THP-1细胞，分别收集刺激12和24 h细胞培养上清，采用Bio-PlexProTM Assays细胞因子测定试剂盒检测细胞培养上清中IL-2、IL-4、IL-6、IL-8、IL-10、GM-CSF、TNF-α和IFN-γ的分泌水平。结果成功克隆表达此菌EC融合抗原；经过融合抗原EC刺激THP-1细胞培养上清中IL-6、IL-8和TNF-α的分泌水平明显升高，差异具有统计学意义（ P＜0．05），其余细胞因子的分泌水平在刺激前后没有显著变化。结论克隆表达的融合抗原EC具有生物学活性，能使THP-1细胞分泌的IL-6、IL-8及TNF-α水平升高。

Cloning,expression and biological activity of Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10

Objective To clone and express Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10 ( EC) and to evaluate the biological activity of the fusion antigen EC in inducing specific cytokines secretion from THP -1 cells. Methods The fusion antigen EC gene was cloned into pET-30 a prokaryotic expression vector and expressed highly in E.coli BL21.Then, the THP-1 cells were stimulated with purified fusion antigen EC of different concentrations (10 and 20 μg/ml).Culture supernatants were collected after 12 h and 24 h, respectively.The secretion levels of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-αand IFN-γin THP-1 cell culture supernatants were detected using Bio-PlexProTM Assays kit.Results The M.tuberculosis fusion antigen EC was cloned and expressed successfully .The secretion levels of IL-6, IL-8 and TNF-αin EC infected THP-1 cells were significantly higher than those in THP-1 cells (P<0.05).The secretion levels of other cytokines did not change significantly .Conclusion The obtained M.tuberculosis fusion antigen EC has biological activity in inducing the THP-1 cells to secrete a higher level of IL-6,IL-8 and TNF-α.