副溶血弧菌总蛋白双向电泳体系的建立

Establishment of Two-Dimensional Gel Electrophoresis(2-DE) System for Analysis of Total Protein in Vibrio Parahaemolyticus

目的：建立并优化副溶血弧菌总蛋白质双向电泳技术体系,为后续差异蛋白质组学研究奠定基础。方法：以副溶血弧菌为研究对象,对比研究样品制备、裂解液配方、IPG胶条长度、IPG胶条pH范围、水化上样方式、等电聚焦程序、上样量和二向胶浓度等因素,优化双向电泳体系。结果：采用超声破碎与TCA沉淀相结合的样品制备方法,能够获得较高的蛋白提取率;使用17 cm pH4~7的IPG胶条,主动水化上样,上样量100μg,上样体积300μL,适当延长除盐时间（2.5 h）和等电聚焦总电压时间积（80 000 vhr）,采用12.5%的二向分离胶,能得到较高的电泳图谱分离度和分辨率。结论：本研究建立的副溶血弧菌总蛋白质双向电泳体系具有较高的可操作性和重现性。

Objective：To develop and optimize a two-dimensional gel electrophoresis（2-DE） system for analysis of total protein in Vibrio Parahaemolyticus which could provide a technical basis for the further study on differential pro-teomics.Methods：The total proteins extracted from V.parahaemolyticus were separated by carrierampholytes pH gradients based immobilized isoelectric focusing gel electrophoresis for first and the vertical SDS-polyacrylamide gel electrophoresis for second to the line of bidirectional gel electrophoresis two-dimensional gel electrophoresis（2-DE）.The gels were dyed and scanned to gain gel image,then PD-Quest software was used for gel image analysis.The 2-DE related techniques were constructed and optimized by comparative tests on some important factors including different extraction methods,ly-sis buffer components,length of IPG strips,pH range of IPG strips,isoelectric focusing programs,sample volume,and so on.Results：The results showed that the resolution and reproducibility of 2-DE profiles was significantly improved by adding Tris and TBP in lysis buffer,the combination of sonication and trichloroacetic acid/acetone（TCA） precipitation for sample preparation,active rehydration of 17 cm（pH 4~7） IPG gel strips,loading the sample 100 μg with sample volume of 300 μl,prolonging the time of desalting（2.5 h） and isoelectric focusing time（80 000 vhr）,preparing 12.5% SDS-PAGE gel,and dying the gels by the silver stained.Conclusion：The established two-dimensional gel electrophoresis（2-DE） system in this study was highly feasible and reproducible in application.