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目的检测并分析幽门螺杆菌(Hp)尿素酶β亚基(UreB)与Hsp60之间的相互作用。方法克隆Hp 26695的尿素酶β亚基基因(UreB)融合GST标签和Hsp60基因融合His标签,分别在大肠埃希菌中进行异源表达。提取两种蛋白,采用pull-down方法检测两者间的相互作用。利用Modeller 9v2软件,以E.coli的GroEL为基础模建Hp Hsp60的三维结构,再与已知的UreB结构利用AutoDock 4.2软件进行分子共模拟,分析二者的相互作用面和关键氨基酸。结果构建了Hp Hsp60和尿素酶β亚基(UreB)的大肠埃希菌异源表达体系并获得纯化蛋白;Pull-down试验显示部分未融合GST标签的Hsp60出现在GST-UreB的洗脱液中,表明Hsp60与GST-UreB之间存在相互作用;以E.coli的GroEL为基础模建了Hp Hsp60的三维结构,利用docking技术构建UreB与Hp Hsp60的相互作用模型,发现其主要的相互作用面位于Hsp60的α9与UreB的α2之间,其可能形成氢键的关键位点为UreB的α2上的T147氨基酸与Hsp60的α9上的E237、K238。结论 Hp Hsp60能直接与尿素酶β亚基(UreB)相互作用,Hsp60可能通过这种相互作用在UreB成熟过程中发挥分子伴侣功能。这为阐明Hp尿素酶的组装与成熟机制奠定了基础。
Objective To detect and analyze the interaction between Helicobacter pylori (Hp) urease β subunit (UreB) and Hsp60. Methods The urease β subunit gene (UreB) fusion Hp 26695 fusion GST tag and Hsp60 gene fusion His tag were respectively expressed in Escherichia coli. Two proteins were extracted and the pull-down method was used to detect the interaction between the two proteins. Using Modeller 9v2 software, the three-dimensional structure of Hp Hsp60 was constructed based on GroEL of E.coli, and then co-molecularly mimicked with known UreB structure using AutoDock 4.2 software to analyze the interaction surface and key amino acids. Results The heterologous expression system of Escherichia coli Hp Hsp60 and urease β subunit (UreB) was constructed and the purified protein was obtained. The pull-down assay showed that some of the unlabeled GST-tagged Hsp60 appeared in the eluate of GST-UreB , Indicating the interaction between Hsp60 and GST-UreB. The three-dimensional structure of Hp Hsp60 was constructed on the basis of GroEL of E.coli. The interaction between UreB and Hp Hsp60 was constructed by using docking technique. The main interaction surface Located between α9 of Hsp60 and α2 of UreB, the key sites that may form hydrogen bonds are T147 amino acids on α2 of UreB and E237 and K238 on α9 of Hsp60. Conclusion Hp Hsp60 can directly interact with urease β subunit (UreB), and Hsp60 may play a molecular chaperone role in UreB maturation through this interaction. This laid the foundation for clarifying the mechanism of assembly and maturation of Hp urease.