结核分枝杆菌EsxV/W融合蛋白对耻垢分枝杆菌表型及巨噬细胞功能的影响

Effects of Mycobacterium tuberculosis EsxV/W Fusion Protein on the Phenotype of Mycolicibacteriumsmegmatis and Macrophage Function

目的:构建表达结核分枝杆菌EsxV/W融合蛋白的重组耻垢分枝杆菌(MS),分析重组MS的表型及对巨噬细胞功能的影响。方法:原核表达获取EsxV/W融合蛋白,纯化后免疫小鼠制备多克隆抗体;电击转化法构建重组菌株MS-EsxV/W及空载菌株MS-vec,使用制备的多克隆抗体检测融合蛋白在MS中的表达;分析表达EsxV/W融合蛋白的重组MS的菌落形态、生物膜及生长速率;菌落计数法检测重组MS在溶菌酶、孔雀绿、低pH、SDS、H2O2、NaNO2等不同环境条件下的存活能力,分析EsxV/W融合蛋白对MS表型的影响;重组MS侵染ANA-1巨噬细胞,Griess法检测ANA-1细胞NO的释放量,酶联免疫吸附实验检测ANA-1细胞肿瘤坏死因子-α(TNF-α)及白细胞介素-6(IL-6)的释放量,菌落计数法检测重组MS在ANA-1细胞内的存活能力。结果:成功构建能表达EsxV/W融合蛋白的重组菌株MS-EsxV/W,表达EsxV/W融合蛋白对MS的菌落形态、生物膜形成及生长速率无显著影响;表达EsxV/W融合蛋白不改变MS对溶菌酶、孔雀绿与低pH的耐受力,但可降低对SDS的耐受力,增强对H2O2与NaNO2的耐受力;侵染ANA-1细胞一定时间后,与MS-vec比较,MS-EsxV/W侵染的ANA-1细胞NO、TNF-α、IL-6释放量明显增加,且MS-EsxV/W的细胞内存活能力高于MS-vec。结论:EsxV/W融合蛋白可改变MS的表型并调控ANA-1巨噬细胞的免疫应答反应,有助于后续对esxV/W基因功能的探究。

Objective:To construct recombinant Mycolicibacterium smegmatis(MS) expressing Mycobacterium tuberculosis EsxV/Wfusion protein and analyze the phenotype of the recombinant MS and its effect on the macrophage function. Methods: The EsxV/Wfusion protein was obtained by prokaryotic expression, and polyclonal antibody was prepared by immunizing mice with purified EsxV/Wfusion protein. The recombinant strain MS-EsxV/W and the control strain MS-vec were constructed by electroporation, and theexpression of the fusion protein in MS was detected by using the generated polyclonal antibody. The colony morphology, biofilmformation, and growth rate of the recombinant MS expressing EsxV/W fusion protein were analyzed. The survival ability of therecombinant MS under different conditions, such as lysozyme, malachite green, low pH, SDS, H2O2 and NaNO2, was determined usingcolony counting method to assess the impact of EsxV/W fusion protein on the phenotype of MS. The recombinant MS was used to infectANA-1 macrophages. The release of NO from ANA-1 cells was detected by Griess method. Enzyme-linked immunosorbent assay wasperformed to measure the release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from ANA-1 cells, and the survivalability of the recombinant MS in ANA-1 cells was determined using colony counting method. Results:The recombinant strain MSEsxV/W expressing EsxV/W fusion protein, was successfully constructed. The expression of EsxV/W fusion protein had no significanteffect on the colony morphology, biofilm formation and growth rate of MS. The expression of EsxV/W fusion protein did not change thetolerance of MS to lysozyme, malachite green or low pH, but decreased the tolerance to SDS and increased the tolerance to H2O2 andNaNO2. After infecting ANA-1 cells for a certain period of time, the release of NO, TNF-α and IL-6 in ANA-1 cells infected by MSEsxV/W was significantly increased compared with MS-vec, and the intracellular survival ability of MS-EsxV/W was higher than thatof MS-vec. Conclusion:The EsxV/W fusion protein can change the phenotype of MS and regulate the immune response of ANA-1macrophages, which is conducive to the subsequent investigation of the function of esxV/W gene.