结核分枝杆菌Rv3671c蛋白诱导人巨噬细胞死亡及TNF-α和IL-1β水平检测

Cell death of THP-1 induced by puried Rv3671c protein of tuberculosis and the detection of TNF-α and IL-1β in Mycobacterium tuberculosis

目的评价MTBRv3671c蛋白对人巨噬细胞（THP-1）的影响及机制。方法将编码MTBRv3671c蛋白的基因克隆到pET-28a质粒，并在大肠埃希菌中进行表达，采用镍亲和层析和离子层析法纯化重组Rv3671c蛋白，Lowry法测定蛋白浓度，并用纯化蛋白刺激分化成熟巨噬细胞，采用Hochest染色法观察细胞的转归（凋亡及坏死）情况，同时取上清液用ELISA法检测TNF-α和IL-1β的浓度。结果MTBRv3671c蛋白在大肠埃希菌中成功表达，层析纯化获得纯度为95．0％的重组Rv36710蛋白，蛋白浓度可达0．4mg／ml。Rv3671c蛋白刺激下经Hochest染色可见巨噬细胞核以坏死状多见，上清液中TNF-α和IL-1B水平最高分别可达19000、16500pg／ml；而未加蛋白干预组TNF-α和IL-1β水平最高仅为2100、3800pg／ml，均明显低于干预组。结论MTBRv36710蛋白可诱导人THP-1坏死，而该死亡可能与细胞因子TNF-α和IL-1β高表达有关。

[Abstract] Objective To assess the response in THP-1 treated with Rv3671c protein in Mycobacterium tuberculosis （M. tuberculosis ）. Methods The gene encoding Rv3671c protein of M. tuberculosis was cloned into pET-28a vector and then expressed in Escherichia coll. The Rv3671c was purified with Ni-NTA affinity and ion exchange chromatography. The detection of protein concentration was by Lowry method. THP-1 cell was stimulated with Rv3671c protein and cells were analyzed by Hochest staining under fluorescence microscopy to assay cell death （apoptosis and necrosis）. TNF-a and IL-1 ~ were detected by ELISA at each stimulating time. Results The Rv3671c protein of M. tuberculosis was successfully expressed in Escherichia coll. The purity of recombinant Rv3671c protein was 95% ,and the protein concentration was up to O. 4 mg/ml. The nucleus of THP-1 was isolated and necrosis-like under fluorescence when ceils were stimulated by Rv3671c protein. The levels of TNF-α and IL-1β in supernatant were 19 000 and 16 500 pg/ml respectively, and were significantly higher than control cells with the levels of 2100 and 3800 pg/ml separately. Conclusion The necrosis of THP-1 cells could be stimulated by Rv3671c protein of M. tuberculosis and it was probably associated with high cytokines TNF-α and IL-1βlevels.