结核分枝杆菌融合蛋白Ag85B-Hsp16.3、Ag85B-ESAT6和分泌蛋白Hsp16.3体外对人肝癌细胞HepG-2的作用

Effect of Ag85B-Hsp16.3 Ag85B-ESAT6 and Hsp16.3 of Mycobacterium Tuberculosis on Human Hepatoma Carcinoma Cell HepG-2 in Vitro

目的:探讨结核分枝杆菌融合蛋白Ag85B-Hsp16.3、Ag85B-ESAT6及分泌蛋白Hsp16.3对人肝癌细胞HepG-2的作用。方法:将已构建的含3种目的基因的表达载体pProEXHTa-Ag85B-Hsp16.3、pProEXHTa-Ag85B-ESAT6和pProEXHTb-Hsp16.3,分别转入宿主菌E.coliDH5α中,诱导表达后分别获得Ag85B-Hsp16.3、Ag85B-ESAT6和Hsp16.3三种蛋白,采用Ni2+亲和层析柱进行纯化,并用透析方法进行目的蛋白的复性。复性的蛋白按照不同浓度和作用时间分别与肝癌细胞HepG-2反应,用MTT法检测细胞生长情况。结果:三种蛋白被成功纯化并复性。MTT数据统计分析显示,终浓度10μg/ml的三种蛋白对HepG-2细胞生长没有明显作用,当三种蛋白的终浓度分别为20、40、80μg/ml时均能够抑制HepG-2细胞的生长,并且抑制作用随着蛋白终浓度的增大以及作用时间的延长而增强。不同类别的蛋白抑制作用没有明显差别。结论:结核分枝杆菌的部分分泌蛋白能够抑制肝癌细胞HepG-2的生长。

To investigate the effect of Ag85B-Hsp16.3, Ag85B-ESAT6 and Hspl6.3 of Mycobacterium tuberculosis （MTB） on HepG-2 in vitro. Methods：The three recombinant plasmids （pProEXHTa-Ag85B-Hsp 16.3, pProEXHTa-Ag85B-ESAT6 and pProEXHTb-Hspl6.3） were transfered into E.coli DH5a respectively. The three proteins were induced by IPTG and purified by Ni-NTA purification system under denaturing condition. Following renaturation by dialysis and filtration, the three proteins were respectively added into HepG-2 cells of various concent ration （101xg/ml,201xg/ml,401μg/ml and 801μg/ml）. The cells were incubated for 24h or 48h, and then the inhibition rate was examined by MTT test assay. Results：The proteins were successfully purified and all had a moderate killing effect on Hepg-2 cells. The effect was dependent on the concent ration of the protein as well as the action time.But there was no statistical difference between different proteins. Conclusion： Some of the secreted proteins of Mycobacterium tuberculosis can inhibit cell growth of liver caricer cell HepG-2.