clpE缺陷对肺炎球菌蛋白表达的影响

Effect of ClpE depletion on the bacterial protein expression profiles of Streptococcus pneumoniae

目的了解ClpE对肺炎球菌蛋白表达的影响。方法用长臂同源多聚酶链式反应(LFH-PCR)方法失活clpE基因,用PCR、测序鉴定缺陷菌株;通过固相pH梯度双向凝胶电泳(2D-PAGE)分别分离野生菌和clpE缺陷菌的总蛋白质;经凝胶银染色、PDQuest2DE软件分析获得2个菌株的蛋白质表达谱;选取差异蛋白质点进行MEDLI-TOF质谱分析,查询数据库,获取有意义的蛋白点。结果以肺炎链球菌D39的图谱为参考,△clpE与其匹配率为61%。挑选17个有明显差异表达的蛋白点进行MALDI-TOF-MS分析,获得了17张肽质量指纹图,经数据库查询后,4个蛋白点获得了有意义的结果,分别为:次黄嘌呤鸟嘌呤核糖核酸转移酶(hypoxanthine-guanine phosphoribosyltransferase),吡咯烷羧酸肽酶(pyrrolidone-carboxylate peptidase1),甲酸四氢叶酸连接酶(formate-tetrahydrofolate ligase)和双功能蛋白pyrR(bifunctional proteinpyrR)。结论野生菌表达蛋白的种类和数量均高于突变菌株,提示ClpE可通过影响某些特殊蛋白质的表达,帮助细菌适应宿主的不同环境,使宿主致病。

Objective To investigate the effect of ClpE on the protein expression profiles of Streptococcus pneumoniae. Methods clpE-deficient Streptococcus pneumoniae strain was constructed by long flanking homology-polymerase chain reaction (LFH-PCR) and identified by PCR and sequencing. The total bacterial proteins were analyzed by two-dimensional gel electrophoresis and imaging analysis, and the differentially expressed protein spots were excised by dot-gel digestion with trypsin. Peptide mass fingerprinting (PMF) ...