2型猪链球菌中国强毒株及其covR基因突变株的蛋白质组学研究

目的 通过比较2型猪链球菌中国强毒株05ZYH33及其covR基因突变株△covR蛋白表达谱差异,质谱鉴定差异表达蛋白,分析CovR在蛋白表达调控中的作用。方法 首先将05ZYH33和△covR在THB培养基中培养至对数期,然后裂解细菌制备蛋白样品。第一向等电聚焦电泳在pH3~10的IPG胶条上完成后进行SDS-PAGE电泳,电泳胶经扫描分析后选取蛋白点进行质谱鉴定。结果 05ZYH33和△covR全菌蛋白裂解液经二维电泳分别得到559和491个蛋白点,经比对发现两菌株蛋白表达量差异3倍以上蛋白点40个,经质谱鉴定出15个蛋白,主要涉及细胞代谢的酶类如谷氨酸脱氢酶、腺苷酸激酶、PTS系统成分等,以及分子伴侣蛋白如GroEL和Dnak等;电泳分离得到△covR特异蛋白点124个,质谱鉴定出15个,主要为参与细胞糖代谢过程中的酶,如磷酸甘油酸激酶、甘油醛-3-磷酸脱氢酶、丙酮酸激酶等;质谱鉴定05ZYH33特异表达蛋白5个。结论 鉴定05ZYH33和△covR差异表达蛋白35个,部分蛋白涉及细菌毒力、宿主细胞粘附、细胞分裂等生命过程,同时蛋白分子伴侣在△covR中的表达变化说明CovR的调控可能发生在蛋白修饰水平,为研究CovR在调控细菌毒力中的作用和分子机制奠定基础。

Comparative proteomic research between the Streptococcus suis serotype 2Chinese highly virulent strain and the covR isogenic mutant

In order to search for the virulence factor regulated by CovR, the proteomics of the whole-cell protein were compared between Streptococcus suis serotype 2 wild strain 05ZYH33 and an isogenic mutant strain △covR by two-dimensional gel electrophoresis. The 05ZYH33 and △covR were cultured in Todd-Hewitt Broth medium then the whole-cell proteins sample were extracted. The 2-DE gel was conducted using the pH3-10 IPG strip for the first dimension IEF and followed by SDS-PAGE. After electrophoresis, the gels were stained and analyzed. Results showed that the 05ZYH33 and △covR had 559 and 491 protein spots respectively. Compared with the 05ZYH33, the mutant strain had 40 proteins more than 3 folds changed which identified 15 proteins by mass spectrum. Those proteins major involved in cell metabolic enzymes, such as adenylate kinase, glutamate dehydrogenase and PTS system components, etc., as well as molecular chaperone proteins GroEL and Dnak. The △covR had about 124 specific protein spots in which 15 proteins were identified by mass spectrum. Those proteins were participate in the cell process of sugar metabolism, such as phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase, etc. Beside of those 5 specific proteins of 05ZYH33 was identified by mass spectrum. Appraisement of 35 differentially expressed proteins involved in bacterial virulence, host cell adhesion and cell division, etc., and the molecular chaperone up-regulated expression showed that the regulation may be occurred in the profile of protein modification. These results provided better understanding on pathogenic mechanisms of Streptococcus suis type 2 at the level of protein expression.