摘要
目的利用原核表达系统表达、纯化结核分枝杆菌分泌蛋白PknG,并探讨PknG在炎症免疫应答中的作用。方法从NCBI数据库中获取结核分枝杆菌PknG全基因组序列,高保真PCR扩增结核分枝杆菌PknG全基因序列,构建PknG-pET28a融合表达质粒,IPTG诱导表达PknG蛋白,镍柱及不同浓度咪唑纯化及透析PknG蛋白,SDS-PAGE及western blot验证PknG蛋白的表达,脂多糖(LPS)处理巨噬细胞0、12和24 h,通过Real-time PCR及ELISA方法检测促炎细胞因子TNF-α及IL-6的表达。结果成功诱导表达及纯化结核分枝杆菌PknG蛋白,PknG抑制LPS处理的巨噬细胞促炎细胞因子TNF-α及IL-6的表达。结论结核分枝杆菌PknG蛋白抑制LPS诱导巨噬细胞促炎细胞因子TNF-α及IL-6的表达,可作为结核分枝杆菌感染合并脓毒血症过程中治疗的一个潜在靶点。
Objective To express and purify the secreted protein PknG of Mycobacterium tuberculosis through prokaryotic expression system and further investigate its role in the inflammatory immune response.Methods The whole genome sequence of Mycobacterium tuberculosis PknG was obtained from the NCBI database.After the PknG was amplified by high fidelity PCR and the PknG-pET28 a plasmid was constructed,the PknG protein expression was induced by IPTG,the PknG protein was purified by nickel columns and imidazole at different concentrations and finally the expression of PknG protein was verified by SDS-PAGE and western blot.After the macrophages were treated with LPS at 0 h,12 h and 24 h,the expression of pro-inflammatory cytokines TNF-αand IL-6 was detected by Real-time PCR and ELISA.Results PknG protein was successfully induced and purified.PknG inhibited the expression of pro-inflammatory cytokines TNF-αand IL-6 in LPS-induced macrophages.Conclusion PknG of Mycobacterium tuberculosiscan inhibit the expression of pro-inflammatory cytokines TNF-αand IL-6 in macrophages.Therefore,it may be related to the potential target for the treatment of Mycobacterium tuberculosis infection in the course of sepsis.
作者
彭章丽
沈瑶
付雪峰
PENG Zhangli;SHEN Yao;FU Xuefeng(Tuberculosis Division,Second Department of Respiratory and Critical Care Medicine,the Affiliated Hospital of Zunyi Medical University,Zunyi 563000,China)
出处
《实用医学杂志》
CAS
北大核心
2022年第1期45-49,共5页
The Journal of Practical Medicine
基金
国家自然科学基金地区基金(编号:81960004)
贵州省科技计划项目课题(编号:黔科合基础-ZK[2021]一般348)
遵义市科技局课题(编号:遵市科合HZ字2019(70)号)
遵义医学院硕士启动基金(编号:2015(009))。
