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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。
Objective: To investigate the effect of Mycobacterium tuberculosis fusion protein Ag85B-Hsp16.3, Ag85B-ESAT6 and secretory protein Hsp16.3 on human hepatoma HepG-2 cells. Methods: The constructed expression vectors pProEXHTa-Ag85B-Hsp16.3, pProEXHTa-Ag85B-ESAT6 and pProEXHTb-Hsp16.3 containing the three genes of interest were respectively transformed into E. coli DH5α, and were induced to express Ag85B -Hsp16.3, Ag85B-ESAT6 and Hsp16.3 three proteins were purified by Ni2 + affinity chromatography, and dialysis method for the purpose of protein renaturation. Refolded protein was reacted with HepG-2 cells at different concentrations and time, respectively. Cell growth was detected by MTT assay. Results: Three proteins were successfully purified and refolded. Statistical analysis of MTT data showed that the three proteins at the final concentration of 10μg / ml had no significant effect on the growth of HepG-2 cells. When the final concentrations of the three proteins were 20, 40 and 80μg / ml, the three proteins could inhibit the growth of HepG-2 cells Growth, and the inhibitory effect increases with the increase of the final concentration of the protein and the prolongation of the action time. There was no significant difference between the different types of protein inhibition. Conclusion: Mycobacterium tuberculosis partially secreted protein can inhibit the growth of HepG-2 cells.