蚯蚓蛋白质组双向电泳技术体系的建立及条件优化

Establishment and optimization of two-dimensional gel electrophoresis technology system for earthworm Eisenia fetida epithelium proteome

对赤子爱胜蚓(Eisenia fetida)表皮组织的两种蛋白质提取方法进行了比较,并对双向电泳IPG胶条pH范围,上样量条件进行了优化,结果表明:裂解液提取法提取的蛋白质量浓度较低,蛋白种类缺失,采用TCA-丙酮沉淀法提取法,可显著提高提取液蛋白质提取效果,样品中蛋白质质量浓度可达12.49μg/μL.蚯蚓表皮组织蛋白质主要分布在pH范围4～7之间,适于选用24cmpH 4～7线性梯度IPG胶条,采用考马斯亮蓝染色法时最适上样量为600μg,所得电泳图谱可检测蛋白点达(629±20)个,且重复性实验匹配率较高.用TCA-丙酮沉淀法提取蚯蚓表皮组织蛋白质组,按优化的参数进行双向电泳分离,所得图谱分辨率高、重复性好,可用于蚯蚓蛋白质组学研究.

In order to establish and optimize two-dimensional electrophoresis（2-DE） technology system for earthworm Eisenia fetida epithelium proteome, sample preparation method, loading quantity of samples, appropriate IPG strips and system repetition were studied. The results showed that, compared with the lysis buffer extraction method, TCA-A extraction method could increase the protein extracting efficiency up to 12.49 μg/μ2L. While the 24 cm IPG strip（pH 4～ 7） and 600 μg loading sample quantity were considered to be the optimal condition for 2-DE analysis of earthworm Eisenia fetida epithelium proteome, up to （629±20） protein spots can be detected on the gel after coomassie brilliant blue R-250 staining, repeatability of the system also turned out to be good. Therefore, the high resolution and repeatable 2-DE technology system could be wildely used in proteome study of earthworm Eisenia fetida.