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作 者:史静华 李东硕 岑山[2] 陆宇[1] 徐建[1] Shi Jinghua;Li Dongshuo;Cen Shan;Lu Yu;Xu Jian(Beijing Chest Hospital Affiliated to Capital Medical University,Beijing Tuberculosis Chest Cancer Institute,Beijing 101149;Institute of Medical Biotechnology,Chinese Academy of Medical Sciences,Beijing 100050)
机构地区:[1]首都医科大学附属北京胸科医院北京市结核病胸部肿瘤研究所,北京101149 [2]中国医学科学院医药生物技术研究所,北京100050
出 处:《中国抗生素杂志》2024年第8期890-897,共8页Chinese Journal of Antibiotics
基 金:国家自然科学基金(No.81803588);北京市自然科学基金(No.7202029)。
摘 要:目的 表达纯化结核分枝杆菌MmpL5蛋白,从而进一步检测MmpL5蛋白与药物之间的相互作用,探究MmpL5外排系统所导致的耐药机制。方法 通过分子对接技术预测MmpL5蛋白与药物结合位点。基于分子对接结果,以结核分枝杆菌H37Rv基因组为模板,构建MmpL5-L1截短蛋白的表达质粒,并在大肠埃希菌中进行表达纯化。利用荧光淬灭技术和表面等离子体共振技术检测截短蛋白与药物之间的相互作用。结果 成功在大肠埃希菌中表达纯化结核分枝杆菌膜截短蛋白MmpL5-L1(Q52-R202),MmpL5-L1截短蛋白与氯法齐明相互作用使蛋白产生荧光淬灭,表面等离子体共振实验表明MmpL5-L1截短蛋白与贝达喹啉的亲和常数(KD)为4.033×10~(-5) mol/L,与氯法齐明的亲和常数(KD)为1.218×10~(-5) mol/L。结论 本文预测并确证了结核分枝杆菌MmpL5蛋白与贝达喹啉、氯法齐明结合的关键结合氨基酸区域,为贝达喹啉、氯法齐明与MmpL5蛋白结合进而通过MmpS5-MmpL5外排提供了直接证据,对进一步阐明贝达喹啉耐药以及氯法齐明交叉耐药机制奠定了一定基础。Objective To express and purify the MmpL5 protein of Mycobacterium tuberculosis so as to further investigate the interaction between MmpL5 protein and drugs and study the drug resistance mechanism caused by the MmpL5 efflux system.Methods The binding sites of the MmpL5 protein and drugs were predicted by molecular docking.Based on the results of molecular docking,the expression plasmid of the MmpL5-L1 truncated protein was constructed using the Mycobacterium tuberculosis H37Rv genome as a template.The MmpL5-L1 truncated protein was expressed and purified in Escherichia coli.The fluorescence quenching technique and the surface plasmon resonance technique were used to investigate the interaction between truncated proteins and drugs.Results MmpL5-L1(Q52-R202),a truncated protein of Mycobacterium tuberculosis,was successfully expressed and purified in Escherichia coli.The interaction between the truncated protein of MmpL5-L1 and clofazimine was validated by protein fluorescence quenching.The surface plasmon resonance experiment showed that the affinity constant(KD)of the truncated protein of MmpL5-L1 with bedaquiline was 4.033×10-5 and that with clofazimine was 1.218×10-5.Conclusion The key amino acid region of MmpL5 binding to bedaquiline and clofazimine,which provided direct evidence for the binding of bedaquiline and clofazimine to MmpL5 protein and then efflux through the MmpS5-MmpL5 system,was successfully predicted and confirmed.This work laid a certain foundation for further elucidating the mechanisms of bedaquiline resistance and clofazimine cross-resistance.
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