肺炎链球菌糖代谢蛋白CcpA对荚膜多糖的调控作用  

Regulation effect of Ccp A protein on the biosynthesis of capsular polysaccharide in Streptococcus pneumoniae

作　　者：王丽滨[1] 徐红梅[1] 吴凯峰[2] 郑玉强[1] 王建敏[1] 马峰[1] 张雪梅[1] 尹一兵[1] 张群[1] 

机构地区：[1]重庆医科大学检验医学院,临床检验诊断学教育部重点实验室,重庆400016 [2]遵义医学院第三附属医院检验科,贵州遵义563002

出　　处：《微生物学报》2015年第6期732-738,共7页Acta Microbiologica Sinica

基　　金：高等学校博士学科点专项科研基金联合资助课题--肺炎链球菌荚膜多糖cps基因座转录调控的分子机制研究(20135503110004)~~

摘　　要：【目的】研究肺炎链球菌糖代谢蛋白CcpA对肺炎链球菌荚膜多糖(CPS)的调控作用。【方法】利用大肠杆菌(Escherichia coli)BL21(DE3)工程菌原核表达CcpA蛋白,使用Ni2+亲和层析的方法纯化蛋白。利用纯化后的CcpA蛋白免疫昆明小鼠并制备多克隆抗体;采用ELISA法测定抗CcpA抗体效价。随后,利用Westertn blot方法分析CcpA蛋白在肺炎链球菌中的保守性。另外,利用EMSA方法分析CcpA与cps基因座启动子区域片段的结合。最后,构建ccpA基因缺失株和ccpA基因回复株;利用ELISA法测定野生D39菌株、ccpA基因缺失株和ccpA基因回复株的荚膜多糖含量。【结果】Western blot结果显示CcpA蛋白在多种血清型的肺炎链球菌均有表达,CcpA蛋白可与cps基因座启动子区域结合,且呈剂量依赖性;ccpA基因缺失时,细菌CPS含量升高,回复表达CcpA蛋白后,CPS含量显著降低。【结论】CcpA是肺炎链球菌中一种保守表达的蛋白,可通过调节cps基因座启动子负性调控肺炎链球菌荚膜多糖的表达。[Objective] We studied the regulation effect of glycometabolic protein,catabolite control protein A（Ccp A）,on the biosynthesis of capsular polysaccharide（CPS） in Streptococcus pneumonia.[Methods] His-tagged Ccp A protein was expressed in E.coli BL21（DE3） and purified by Ni2 ＋affinity chromatography.The anti-Ccp A serum was obtained from immunized mice and the antibody titer was determined by ELISA.The conservation of Ccp A was determined by Western blotting.In addition,binding of Ccp A protein to the promoter region of cps locus was verified by EMSA.The amount of capsular polysaccharide was determined by ELISA and compared among wild type D39 strain,ccp A mutant and the complement strains.[Results]Ccp A protein was conserved in different pneuococcal serotypes included in this study.EMSA assay revealed that Ccp A protein could bind the promoter region of the cps locus in a dose-dependent manner.The absence of ccp A gene led to an increased expression of capsular polysaccharide,and complement expression of Ccp A protein significantly reduced the amount of capsular polysaccharide.[Conclusion] Ccp A is conserved in Streptococcus pneumonia,which plays a role in regulation of the expression of the capsular polysaccharide.

关 键 词：肺炎链球菌 ccp A基因 荚膜多糖 转录调控 

分 类 号：R378[医药卫生—病原生物学]