刺参肠组织蛋白质双向电泳体系的建立及优化

Establishment of two-dimensional gel electrophoresis(2-DE)for intestine tissue of Apostichopus japonicus

以刺参肠组织为材料,通过蛋白质提取方法、上样前处理方法、上样量以及聚焦条件的优化,建立了刺参肠组织蛋白质双向电泳技术体系.试验结果表明,采用TCA-丙酮沉淀法制备刺参肠组织蛋白质,能够得到高纯度的蛋白质提取液.刺参样品上样前不经过处理,等电聚焦电压1 000V,电流7mA,聚焦时间3h,平衡时间30min,最佳上样量25μg,pH 4～6.5、8.5cm的IPG胶条,银染能获得较好的双向电泳图谱.通过蛋白质双向电泳体系的优化,获得了刺参肠组织的蛋白质表达图谱。

The purpose of this research was to establish an optimized system to analyze Apostichopus japonicus proteomics.The protein extraction method,loading methods,sample volume and focusing condition in two-dimensional gel electrophoresis （2-DE） were optimized to obtain best results.Several methods of protein preparation （TCA-acetone precipitation treatment,Tris-HC1 treatment,Tris-HC1 acetone treatment and pure water treatment） were applied to extract total protein from A.japonicus intestine.The most effective profiles with the most electrophoretic protein spots were obtained by the TCA-acetone precipitation treatment.The 2-DE electrophoretic analysis in intestine achieved an optimized map by loading without buffer solution treatment at the sample volume of 25 μg,the pH of ampholytes ranged from 4 to 6.5.The samples were electrophoresed for 1000 V and 3 hours,and the gels were immersed in 2.5 ml equilibration buffer and equilibrated for 30 min.The system optimization of protein two-dimensional electrophoresis would provide supports in sea cucumber proteomics study.