论文部分内容阅读
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。
Objective To clone and express the fusion antigen ESAT-6 + CFP-10 (EC) of Mycobacterium tuberculosis (Mtb) and evaluate the secretion of specific cytokines in culture supernatant of THP-1 cells stimulated by EC. Methods The prokaryotic expression vector of fusion antigen EC was constructed. The purified THP-1 cells were stimulated with different concentrations of fusion antigen EC (10, 20μg / ml) and the supernatants were harvested at 12 and 24 hours respectively. Bio-Plex ProTMAssays cytokines The kit was used to detect the secretion of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-α and IFN-γ in the cell culture supernatant. Results The EC fusion antigen was successfully cloned and expressed. The secretion of IL-6, IL-8 and TNF-α in culture supernatant of THP-1 cells stimulated by fusion antigen EC was significantly increased, the difference was statistically significant (P <0.05 ), The secretion of other cytokines did not change significantly before and after stimulation. Conclusion The cloned fusion antigen EC has the biological activity, which can increase the levels of IL-6, IL-8 and TNF-α secreted by THP-1 cells.