牛分枝杆菌TB9.8蛋白的表达及其诱导小鼠巨噬细胞RAW264.7细胞因子的分泌

The expression of Mycobacterium bovis antigen protein TB9.8 and its effects on the cytokine secretion in RAW264.7 macrophages

为研究牛分枝杆菌TB9.8蛋白对小鼠巨噬细胞RAW264.7的生长及分泌细胞因子的影响,本研究将编码牛分枝杆菌TB9.8蛋白的基因克隆于pET-30a质粒中并在E.coli中进行表达。经SDS-PAGE电泳和western blot分析表明,表达的重组蛋白(rTB9.8)约9.8 ku,具有良好抗原性。以不同剂量的纯化rTB9.8刺激巨噬细胞RAW264.7,采用动态实时细胞检测技术分析rTB9.8对RAW264.7生长影响的最佳时间点和最佳剂量,结果表明rTB9.8可以在8 h^24 h对RAW264.7细胞的增殖产生明显的抑制作用。此外,rTB9.8能够显著诱导RAW264.7细胞表达TNF-α、IL-6和IL-12。本实验为进一步研究牛分枝杆菌rTB9.8与宿主细胞相互作用提供了实验依据。

To evaluate the immune effects of Mycobacterium bovis TB9.8 protein on mouse macrophage RAW264.7, the tb9.8 gene was cloned into pET30a and expressed in E. coli BL21（DE3）. The target protein was purified with urea gradient elution, and verified by western blot. IFN-3~ release test showed that recombinant TB9.8 exhibits good antigenicity. During the following tests, RAW264.7 was treated with different concentrations of rTB9.8, and the RT-CES system was used to determine the optimal timing and dosage. The concentrations of TNF-ct, IL-6, and IL-12 in supernatants were titrated by enzyme-linked immunosorbent assay （ELISA）. Our study showed that rTB9.8 could strongly inhibit the growth of RAW264.7 between 8h and 24h aider treatment, and when exposed to rTB10.4, RAW264.7 produced significantly higher levels of TNF-α, IL-6, and IL-12. Our results provide the basis for further research on the interaction of M. boris TB9.8 and its host.