褐色橘蚜全蛋白双向电泳体系条件优化

Optimization of two-dimensional gel electrophoresis technology system for total protein from brown citrus aphid Toxoptera citricida

为优化有效分离褐色橘蚜Toxoptera citricida全蛋白的双向电泳体系,比较了饱和酚抽提法、三氯乙酸(trichloroacetic acid,TCA)/丙酮沉淀法、直接裂解液法和Tris-HCl法4种蛋白提取方法,以及蛋白上样量、聚焦条件和固相p H梯度(immobilized p H gradient,IPG)胶条对褐色橘蚜全蛋白双向电泳的影响。结果表明,TCA/丙酮沉淀法和直接裂解液法获得的全蛋白浓度分别为4.25、4.03μg/μL,显著高于饱和酚抽提法和Tris-HCl法,且TCA/丙酮沉淀法所提取的蛋白种类丰富度最高。采用TCA/丙酮沉淀法获得全蛋白,当上样量为100μg时,所得蛋白质点多、分辨率高、背景清晰,除盐3 h,等电聚焦量45 000 V·h,并选用p H 3~10非线性IPG胶条时,双向电泳图谱中横向拖尾现象少,蛋白点相对清晰,分布均匀。表明所建立的双向电泳体系,结合银染,可从褐色橘蚜全蛋白中识别出1 264个蛋白点,且重复性研究匹配率高,可用于褐色橘蚜蛋白质组学研究。

In order to optimize a two-dimensional electrophoresis（2-DE） system for studying total protein of brown citrus aphid Toxoptera citricida, four different methods of protein extraction including saturated phenol extraction, trichloroacetic acid（TCA）/acetone precipitation, lysate extraction and trihydroxymethyl aminomethane（Tris-HCl） buffer extraction methods were compared. Moreover, the factors including loading amount of protein samples, isoelectric focusing protocols and p H of immobilized p H gradient（IPG） strips were optimized. The results showed that TCA/acetone precipitation and lysate extraction yielded much higher amounts of proteins than other extraction methods, and the protein extraction rate was 4.25 and 4.03 μg/μL, respectively. Furthermore, TCA/acetone precipitation yielded the best richness of proteins. For T. citricida 2-DE, the optimized system included a loading amount of100 μg protein, desalinated for three hours, with an isoelectric focusing of 45 000 V · h, p H 3-10 nonlinear IPG strip and silver staining, which could produce good background, clear protein spots and few slight streak. This method had detected a total of 1 264 protein spots with a highly reproducible result in the proteomic study of T. citricida.