尼罗罗非鱼肌肉蛋白质双向电泳体系的建立

为建立1种尼罗罗非鱼肌肉组织蛋白质的双向电泳体系,实验将尼罗罗非鱼肌肉组织蛋白质提取后,用双向电泳(2-DE)分离蛋白质,分别对蛋白质样品的制备方法、IPG胶条长度及p H范围、SDS-PAGE凝胶浓度及染色方法4个关键因素进行了探索和优化。结果显示,采用液氮研磨-丙酮沉淀法制备样品蛋白质,使用24 cm p H 4~7的IPG胶条进行第一向等电聚焦电泳,第二向SDS-PAGE电泳采用浓度为12.5%的凝胶进行,双向电泳后的凝胶采用硝酸银染色法进行染色处理,该条件下扫描所得尼罗罗非鱼肌肉蛋白质二维电泳图谱具有蛋白质分离程度好、斑点清晰、分辨率高及横纹少等优点。研究表明,技术体系适用于尼罗罗非鱼肌肉蛋白质的分离,可用于后续罗非鱼肌肉蛋白质组学研究。

Establishment of two-dimensional electrophoresis (2-DE) system in muscle proteome of Nile tilapia (Orechromis niloticus)

Nile tilapia (Oreochromis niloticus) is a tropical species that prefers to live in shallow waters, and it has the merits of fast growth and high reprodu-ctive rate, also has the characteristics of less bone spurs, thick fleshy, which has been widely cultured in southern China, and China is by far the largest producer of farmed Nile tilapia. In order to establish a two-dimensional electrophoresis (2-DE) system for proteomic analysis of Nile tilapia muscle, protein samples of Nile tilapia muscle were separated using 2-DE after extracting. By optimizing different preparation methods, different pH of immobilized pH gradients(IPG) gel strips and their lengths, as well as different concentration of SDS-PAGE gel and staining methods (silver staining and Coomassie blue staining), we obtained the best 2-DE experiment conditions. The results show that the optimum method for protein samples preparation of Nile tilapia muscle is combination of liquid nitrogen grinding and acetone precipitation. For 2-DE assay, using 24 cm pH 4-7 IPG gel strips for isoelectric focusing(IEF) and 12.5% gel for the second SDS-PAGE. Besides, dyeing the gels by silver staining could get the optimized 2-DE map of Nile tilapia muscle protein with excellent degree of separation, clear protein points, high resolution and less cross band. The 2-DE technical system was successfully established in this study, which could be used for the separation of proteins in the future Nile tilapia muscle proteomics research.