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目的表达有活性的鼠疫耶尔森菌(鼠疫菌)、副溶血性弧菌以及肺炎克雷伯菌的cAMP受体蛋白(CRP)并进行DNA体外结合活性分析,为深入研究3种CRP蛋白间的交互转录调控作用奠定基础。方法应用大肠埃希菌pET系统体外表达鼠疫菌201株、副溶血性弧菌5421株以及肺炎克雷伯菌tw518株的CRP蛋白;应用生物信息学对3种CRP蛋白进行同源性比较,并对CRP与靶DNA的结合基序(CRP consensus)进行分析预测;通过体外凝胶阻滞实验(EMSA)和DNaseⅠ足迹实验验证His?CRP与DNA的结合活性。结果成功表达出3种菌有活性的His?CRP融合蛋白,3种CRP蛋白对鼠疫菌pla、副溶血性弧菌toxR及肺炎克雷伯菌kfuA基因均有结合活性。结论 3种CRP蛋白都能直接结合到鼠疫菌pla、副溶血性弧菌toxR及肺炎克雷伯菌kfuA基因的启动子区,说明上述3种CRP蛋白对3种病原菌的重要毒力基因的转录可能具有交互调控作用。
Objective To express cAMP receptor protein (CRP) of Yersinia pestis (Yersinia pestis), Vibrio parahaemolyticus and Klebsiella pneumoniae and to analyze the in vitro DNA binding activity. In order to further investigate the relationship between the three CRP proteins The role of interactive transcriptional regulation lay the foundation. Methods The E. coli pET system was used to express CRP protein of 201 strains of Yersinia pestis, 5421 strains of Vibrio parahaemolyticus and 2.55 strains of Klebsiella pneumoniae in vitro. Homology comparison of three CRP proteins was performed by bioinformatics The binding motif (CRP consensus) between CRP and target DNA was predicted. The binding activity of His CRP to DNA was verified by EMSA and DNase I footprinting. Results The His? CRP fusion protein was successfully expressed in three kinds of bacteria. The three CRP proteins had binding activity to plague plague, toxR parahaemolyticus, and kfuA gene of Klebsiella pneumoniae. Conclusion All three CRP proteins can directly bind to the plague pla, toxR and K. pneumoniae kfuA gene promoter region, indicating that the three CRP proteins on the three pathogens important virulence gene transcription May have interactive regulation.