结核分枝杆菌MycP1蛋白的原核表达及其对巨噬细胞的毒性作用

Prokaryotic expression of MycP1 of Mycobacterium tuberculosis and its cytotoxicity to macrophages

目的:构建结核分枝杆菌MycP1蛋白原核表达系统。方法:构建pGEX-4T-1-Rv3883c重组质粒,转化大肠杆菌,经IPTG诱导表达,采用SDS-PAGE和Westernblot法检测目标蛋白的表达情况。目标蛋白经亲和层析纯化后,以不同质量浓度MycP1作用于小鼠巨噬细胞ANA-1,48h后XTT法检测活细胞数,同时测定细胞上清液中乳酸脱氢酶(LDH)活力,以评价其细胞毒性。结果:成功构建pGEX-4T-1-Rv3883c质粒,该质粒能在大肠杆菌中表达,经鉴定,表达的融合蛋白为目标蛋白MycP1。该融合蛋白作用后可使ANA-1活细胞数下降(F=34.327,P<0.001),培养上清液中LDH活力升高(F=336.720,P<0.001)。结论:成功构建了MycP1原核表达系统,目标蛋白对巨噬细胞有一定的毒性效应。

To establish the prokaryotic expression system of MycP1 of Mycobacterium tuberculosis. Methods ：Therecombinant plasmid of pGEX-4T-1-Rv3883c was constructed and expressed in E. coli BL21 （ DE3 ） , and then induced by IPTG. The product was identified by SDS-PAGE and Western blot and purified with GST-affinity chromatography. The puri- fied protein was applied to mice maerophages ANA-1 cells,and the cytotoxicity was evaluated by detecting LDH activity in culture medium and XTT absorption. Result8 ：The recombinant plasmid pGEX-4T-1-Rv3883e was constructed successfully, and the purified product could decrease XTT absorption（ F = 34. 327,P 〈 0. 001 ） ,increase the level of LDH activity in cul- ture medium of ANA-1 cells （ F = 336. 720, t9 〈 0. 001 ）. Conclusion ： The prokaryotic expression system of MycP1 has beensuccessfully constructed,and the targeted protein has a toxic effect on macrophages.