【摘要】 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-Plex ProTMAssays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。更多还原

【Abstract】 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 p ET-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 and20 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-α and IFN-γ in THP-1 cell culture supernatants were detected using Bio-Plex ProTM Assays kit. Results The M. tuberculosis fusion antigen EC was cloned and expressed successfully. The secretion levels of IL-6,IL-8 and TNF-α in 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.更多还原