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| 刺参肠组织蛋白质双向电泳体系的建立及优化 |
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田燚1, 莫海波1, 常亚青1, 湛垚垚1, 陈百尧2,3, 伏光辉2,3, 安建2,3
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1.大连海洋大学 农业部北方海水增养殖重点开放实验室，116023;2.连云港市海洋与水产科学研究所，222044;3.连云港市海珍品增养殖试验场，222044
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| 摘要: |
| 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，为进一步研究刺参差异表达蛋白的筛选及蛋白质组学研究提供技术保障。 |
| 关键词: 刺参 肠组织 蛋白质双向电泳 |
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| 基金项目:国家自然科学基金项目(41106128)、国家“863”计划项目(2012AA10A42)和江苏省科技支撑计划（BE2012421） |
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| Establishment of two-dimensional gel electrophoresis (2-DE) for intestine tissue of Apostichopus japonicus |
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| Abstract: |
| 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-HCl treatment, Tris-HCl 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 range 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. |
| Key words: Apostichopus japonicus intestine Protein two-dimensional electrophoresis (2-DE) |