线虫surface coat proteins提取方法的建立与双向电泳分析

Two Dimensional Electrophoresis and Extraction of Surface Coat Proteins of Caenorhabditis elegans

目的:建立一套适用于蛋白质双向电泳体系的线虫surface coat proteins(SCPs)样品制备技术,为今后研究线虫surfacecoat蛋白质组学及线虫病理生理学奠定基础。方法:以秀丽隐杆线虫(Caenorhabditis elegans)为研究材料,对比和分析不同的蛋白提取沉淀方法,进而采用SDS-PAGE电泳技术和双向电泳技术对所提蛋白进行评价。结果:通过35%乙醇结合TCA-丙酮沉淀法获得的质量较好的线虫SCPs,在12%的SDS-PAGE分析中该法提取的蛋白背景浅,蛋白条带多且清晰尖锐,含有丰富的蛋白信息量。通过双向电泳分析,可从提取的蛋白中鉴定出清晰蛋白点400多个。随机选择5个蛋白斑点,进行基质辅助激光解吸电离飞行时间质谱鉴定,鉴定得到高度匹配的已知线虫蛋白质2个。结论:所建立的方法可为今后研究线虫surface coat蛋白质组学及线虫病理生理学提供重要工具。

Objective：A new preparation protocol of protein extracts is established for 2-DE of nematode surface coat proteins,which will be provide a basis for studying nematode surface coat proteomics and pathophysiology.Method：With Caenorhabditis elegans as the study material,we compared several nematode surface coat proteins extraction methods to determine their efficacy in separating nematode SCPs by SDS-PAGE and 2D-PAGE.Result：The 12% SDS-PAGE analysis result suggest that 35% ethanol method combined with TCA/acetone precipitation was found to be the most effective step.By using the optimized method,more than 400 proteins were identified in the following 2-DE electrophoresis gel analysis.MALDI-TOF MS results demonstrated that 2 of 5 protein spots were identified to nematode proteins.Conclusion：The developed method in this study can become an important tool for studying nematode surface coat proteomics and pathophysiology in the future.