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目的:克隆、表达并纯化肠出血性大肠杆菌(EHEC)O157∶H7新预测分子伴侣蛋白Ecs4563,以进一步开展其结构与功能的研究。方法:利用PCR方法从EHEC O157∶H7基因组中扩增出分子伴侣基因ecs4563,构建重组表达质粒,在大肠杆菌BL21(DE3)中实现诱导表达,采用Western印迹法进行鉴定,通过Ni2+螯合柱纯化获得目的蛋白。结果:构建了pET-24a-ecs4563重组质粒,实现了目的蛋白的融合表达,Ni2+金属螯合法纯化了目的蛋白,通过Western印迹法验证了融合蛋白的特异性。结论:得到了新预测分子伴侣Ecs4563,为下一步研究蛋白功能,进一步研究EHEC O157∶H7的致病机制奠定了基础。
OBJECTIVE: To clone, express and purify EcH4573, a novel predicted molecular chaperone of enterohemorrhagic Escherichia coli (EHEC) O157: H7, to further study its structure and function. Methods: The molecular chaperone ecs4563 was amplified from the genome of EHEC O157:H7 by PCR. The recombinant plasmid was constructed and expressed in E. coli BL21 (DE3). The recombinant plasmid was identified by Western blotting and purified by Ni2 + chelating column Get the target protein. Results: The recombinant plasmid pET-24a-ecs4563 was constructed and the fusion protein was expressed. The target protein was purified by Ni2 + metal chelating method. The specificity of the fusion protein was verified by Western blotting. Conclusion: The new predicted molecular chaperone Ecs4563 was obtained, which laid the foundation for further study of protein function and further study on the pathogenesis of EHEC O157: H7.