肺炎链球菌糖代谢蛋白CcpA对荚膜多糖的调控作用

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

摘要:

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。

关键词:关键词:肺炎链球菌，ccpA基因，荚膜多糖，转录调控

Abstract:

Abstract:［Objective］We studied the regulation effect of glycometabolic protein，catabolite control protein A (CcpA)，on the biosynthesis of capsular polysaccharide (CPS) in Streptococcus pneumonia．［Methods］His-tagged CcpA protein was expressed in E．coli BL21(DE3) and purified by Ni2 + affinity chromatography．The anti-CcpA serum was obtained from immunized mice and the antibody titer was determined by ELISA．The conservation of CcpA was determined by Western blotting． In addition，binding of CcpA 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，ccpA mutant and the complement strains．［Results］CcpA protein was conserved in different pneuococcal serotypes included in this study．EMSA assay revealed that CcpA protein could bind the promoter region of the cps locus in a dose-dependent manner．The absence of ccpA gene led to an increased expression of capsular polysaccharide，and complement expression of CcpA protein significantly reduced the amount of capsular polysaccharide．［Conclusion］ CcpA is conserved in Streptococcus pneumonia，which plays a role in regulation of the expression of the capsular polysaccharide.

Key words:Keywords: Streptococcus pneumoniae，gene ccpA，capsular polysaccharide，transcription regulation