摘要
目的:探究结核分枝杆菌RD区ESPJ和ESPK蛋白在巨噬细胞极化中的作用。方法:将结核分枝杆菌RD区Rv3878和Rv3879c基因分别构建至原核表达载体,体外诱导表达并纯化重组蛋白GST-ESPJ和HIS-ESPK,用重组蛋白刺激巨噬细胞,Western Blot和ELISA法检测重组蛋白对巨噬细胞极化的影响;构建重组菌BCG-Rv3878和BCG-Rv3879c,用重组菌感染巨噬细胞,RT-qPCR、Western Blot和ELISA法检测重组BCG对巨噬细胞极化的影响,并采用菌落计数法检测重组BCG在巨噬细胞内的存活。结果:重组ESPJ和ESPK蛋白刺激巨噬细胞均可诱导其向M2型极化,转录因子c-Maf表达增加;成功构建重组菌BCG-Rv3878和BCG-Rv3879c,重组BCG感染巨噬细胞也能诱导M2型巨噬细胞极化,巨噬细胞内c-Maf转录因子表达明显升高;菌落计数结果显示重组BCG胞内存活增加。结论:结核分枝杆菌ESPJ和ESPK蛋白可诱导M2型巨噬细胞极化并促进细菌的胞内存活,这可能与结核分枝杆菌的免疫逃逸和潜伏感染有关,转录因子c-Maf在其中可能起到至关重要的作用。
Objective:To explore the function of RD proteins ESPJ and ESPK in macrophage polarization induced by Mycobacterium tuberculosis.Method:Recombinant GST⁃ESPJ and HIS⁃ESPK proteins were purified.The recombinant proteins were used to stimulate macrophages,and macrophage polar⁃ization was detected by Western Blot and ELISA.Then recombinant Mycobacterium tuberculosis BCG⁃Rv3878 and BCG⁃Rv3879c were constructed.The effect of recombinant BCG infection on mac⁃rophage polarization was detected by RT⁃qPCR,Western Blot,and ELISA assays.The survival of recombinant BCG in macrophages was also examined.Results:Recombinant Mycobacterium tubercu⁃losis BCG⁃Rv3878 and BCG⁃Rv3879c were constructed successfully.Recombinant proteins,ESPJ and ESPK,and recombinant BCG induced M2 macrophage polarization,along with the increased expression of the transcription factor c⁃Maf.Bacterial colony⁃forming unit(CFU)counting results also showed increased intracellular survival of the recombinant BCG.Conclusion:The RD proteins ESPJ and ESPK can induce M2 macrophage polarization and promote bacterial intracellular survival,which may be related to the immune escape and latent infection of Mycobacterium tuberculosis,and the tran⁃scription factor c⁃Maf plays a crucial role in this process.
作者
田晴
钟燕霄
刘丽莎
章晓联
罗凤玲
TIAN Qing;ZHONG Yanxiao;LIU Lisha;ZHANG Xiaolian;LUO Fengling(Dept.of Immunology,Taikang Medical School(School of Basic Medical Sciences),Wuhan University,Wuhan 430071,Hubei,China)
出处
《武汉大学学报（医学版）》
CAS
2023年第7期800-807,共8页
Medical Journal of Wuhan University
基金
国家自然科学基金资助项目(编号:81000714)。
