鼠疫菌、副溶血性弧菌及肺炎克雷伯菌CRP蛋白的表达及其DNA结合活性分析  

Expression and DNA-binding activity of the cyclic AMP receptor proteins in Yersinia pestis,Vibrio parahaemolyticus and Klebsiella pneumoniae

作　　者：杨琳[1,2] 高鹤[2,3] 张义全[2] 刘霞[1,2] 谭亚芳[2] 郭兆彪[2] 黄新祥[1] 杨瑞馥[2] 周冬生[2] 

机构地区：[1]江苏大学生命科学研究院,江苏镇江212013 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071 [3]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206

出　　处：《中国媒介生物学及控制杂志》2011年第3期251-256,共6页Chinese Journal of Vector Biology and Control

基　　金：国家自然科学基金(30930001;30900823;30771179);973项目(2009CB522600)~~

摘　　要：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 determine expression and DNA-binding activity of the recombinant CRP（cyclic AMP receptor protein） in Yersinia pestis,Vibrio parahaemolyticus and Klebsiella pneumoniae for the study of transcriptional interregulation. Methods The coding region of the crp gene of Y. pestis,V. parahaemolyticus and K. pneumoniae was amplified by PCR,and cloned into the BamHⅠ and HindⅢ sites of a pET28a vector. The recombinant plasmid pET28a-crp was inducted into BL21λDE3. Over-expression of His-CRP in the LB medium was induced by adding 1 mmol/L IPTG（isopropyl-b-D-thiogalactoside） . The over-expressed protein was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns（Amersham） . Electrophoretic mobility shift assay（EMSA） and DNase I footprinting experiments were carried out to analyze the DNA-binding activity of the three CRP proteins in vitro. Results All three purified His-CRP proteins were able to bind to the upstream DNA regions of Y. pestis（psaA） ,V. parahaemolyticus（toxR） ,and K. pneumoniae（kfuA） . Conclusion Recombinant CRP proteins of Y. pestis,V. parahaemolyticus and K. pneumoniae were expressed and purified,suggesting their inter-regulatory effect on the transcription of key virulence genes of the three pathogens.

关 键 词：鼠疫菌 副溶血性弧菌 肺炎克雷伯菌 cAMP受体蛋白 交互调控 

分 类 号：R378[医药卫生—病原生物学]