Construction of two-dimensional (2-D) polyacrylamide gel electrophoresis and characterization of whole-cell proteins produced by the Aeromonas hydrophila from fish
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摘要: 为建立鱼源嗜水气单胞菌全菌蛋白图谱并进行质谱分析,利用双向电泳(2-DE)结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/TOF-MS)技术对嗜水气单胞菌的蛋白质组学进行鉴定研究。结果显示,嗜水气单胞菌28 ℃培养12 h、三氯醋酸(TCA)/丙酮沉淀法提取全菌蛋白、等电聚焦20 000 Vh的2-DE图谱匹配率达81%;采用18 cm、pH 3-10 IPG胶条进行2-DE分析获得146个蛋白点,随机选取部分蛋白点进行肽质量指纹图谱(PMF)分析,共鉴定23个蛋白点包括膜脂蛋白、分子伴侣、30S核糖体蛋白S1、延伸因子、细胞色素C等,其中发现烯醇化酶、甘油醛-3-磷酸脱氢酶、二氢硫辛酰胺脱氢酶、精氨酸脱亚氨酶、ATP合酶α亚基、脱氧核糖核酸聚合酶亚基α、氨基甲酸激酶、腺苷酸激酶、尿苷磷酸化酶、硝基还原酶、肽酰脯氨酰异构酶共计11个代谢相关蛋白酶;基于分子功能进行蛋白分类(GO)分析,发现主要为结合(17.1%)、催化(14.3%)和转移酶(11.4%)等生物学过程。建立鱼源嗜水气单胞菌全菌蛋白图谱与部分蛋白鉴定,有助于理解嗜水气单胞菌的蛋白质组学及其分子机制。Abstract: In order to investigate the whole-cell proteomics and two-dimensional (2-D) gel maps of Aeromonas hydrophila isolated from the fish in this study, the proteomics analysis of A. hydrophila strain Zf-1 was performed using two-dimensional gel electrophoresis (2-DE) combined with matrix-assisted laser desorption/ionization timeof- flight mass spectrometry (MALDI-TOF/TOF-MS) techniques. A. hydrophila strain was prepared by shaking culture at 28 ℃ for 12 h, and the proteins of whole cell were extracted by using trichloroacetic acid (TCA)/acetone precipitation method. The results showed that 146 protein spots were observed in 2-DE gels performed with 18 cm pH3-10 IPG strips, and it shared matching profiles of about 81% protein expression spots while the 60 μg loading dose, and 7 cm pH3-10 IPG strips were used for isoelectric focusing (IEF) to 20 000 Vh. A total of 23 protein spots were further identified by the peptide mass fingerprinting (PMF) analysis, including Membrane lipoprotein, Molecular chaperones, 30S ribosomal protein S1, Elongation factor, Cytochrome C, etc; Furthermore, 11 enzymes of metabolism related proteins were found to be ATP F0F1 synthase subunit alpha, Dihydrolipoamide dehydrogenase, Enolase, Arginine deiminase, DNA-directed RNA polymerase subunit alpha, Glyceraldehyde-3- phosphate dehydrogenase, Carbamate kinase, Adenylate kinase, Uridine phosphorylase, Nitroreductase, and Peptidylprolyl isomerase. Analysis of protein functional classification with Gene Ontology (GO) of A. hydrophila based on molecular function showed that binding (17.1%), catalytic activity (14.3%), and transferase activity (11.4%) were responsible for the main biological functions. In conclusion, the proteomics analysis and 2-D map were constructed for A. hydrophila strain Zf-1 from the fish in this study, and the results will help to understanding the pathogenic mechanism and treatment of diseases caused by A. hydrophila from fish.
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