鸟分枝杆菌毒力相关因子EsxH对巨噬细胞凋亡的作用

Effects of Virulence-Related Factors EsxH of Mycobacterium avium on Macrophage Apoptosis

摘要: 目的：通过原核表达系统制备鸟分枝杆菌EsxH蛋白，并将其作用于U937细胞，初步探讨其对巨噬细胞凋亡的影响。方法：利用PCR扩增出EsxH编码序列，与表达载体pGEX-4T-3连接，构建重组载体pGEX-EsxH，将重组表达载体pGEX-EsxH转化入E. coli BL21 (DE3)，在IPTG作用下诱导表达目的蛋白。用超声破碎仪对菌体进行破碎，取上清通过谷胱甘肽琼脂糖凝胶柱纯化GST-EsxH融合蛋白。将纯化后的蛋白作用于U937细胞，用流式细胞仪检测细胞凋亡率。结果：扩增出基因片段大小为294 bp的EsxH基因，克隆入表达载体pGEX-4T-3，经IPTG诱导后成功表达一个分子量大小为36.5 kDα的融合蛋白GST-EsxH。将不同浓度的EsxH蛋白作用于U937细胞，经流式细胞术检测细胞凋亡率，发现处理组细胞凋亡率高于对照组，其差异具有统计学意义(P < 0.05)。结论：EsxH蛋白能够促进U937细胞的凋亡，为进一步研究鸟分枝杆菌的致病机制奠定了基础。

Abstract: Objective: In order to study the role of EsxH in Mycobacterium avium, EsxH protein was expressed in prokaryotic expression system and its effects on U937 cells apoptosis were investigated. Methods: The EsxH coding sequences were amplified by PCR and connected with the expression vector pGEX-4T-3 to construct the recombinant vector pGEX-EsxH. The recombinant expression vector pGEX-EsxH was transformed into E. coli BL21(DE3), and the target proteins were induced by IPTG. The GST-EsxH fusion proteins were purified by glutathione-agarose gel column after the cells were disrupted by ultrasonic disruption. The purified EsxH proteins were applied to U937 cells and the apoptotic rates were measured by flow cytometry. Results: A 294bp EsxH gene was amplified and cloned into the expression vector pGEX-4T-3, after induced by IPTG, a fusion protein GST-EsxH with a molecular weight of 36.5 kd was successfully expressed. Different concentrations of EsxH protein were applied to U937 cells, and the flow cytometry results showed that the apoptotic rate of the treated group was higher than that of the control group, with statistical significance (P < 0.05). Conclusion: EsxH protein could promote the apoptosis of U937 cells, laying the foundation for further study on the pathogenic mechanism of Mycobacterium avium.