鱼源嗜水气单胞菌全菌蛋白双向电泳与质谱鉴定

Construction of two-dimensional(2-D) polyacrylamide gel electrophoresis and characterization of whole-cell proteins produced by the Aeromonas hydrophila from fish

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°C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、p H 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。

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 p H3-10 IPG strips, and it shared matching profiles of about 81% protein expression spots while the 60 μg loading dose, and 7 cm p H3-10 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, 30 S 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.