三斑海马蛋白质组学双向电泳技术的建立与优化  

Establishment and Optimization of Two-dimensional Electrophoresis Technique for Proteomics of Hippocampus trimaculatus

作　　者：王清蓉 万德光[1] 国锦琳[1] 陈璐[1] 

机构地区：[1]成都中医药大学中药材标准化教育部重点实验室四川省中药资源系统研究与开发利用省部共建国家重点实验室培育基地

出　　处：《中国实验方剂学杂志》2018年第5期50-54,共5页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81403056);四川省中医药管理局青年中医药研究项目(2014K083)

摘　　要：目的：建立适于三斑海马干药材蛋白质组研究的双向电泳技术,为后期进行深入的中药材海马蛋白组学研究奠定基础。方法：对影响蛋白质双向电泳的几个关键环节——提取方法、裂解液、纯化方法、上样量进行优化,筛选最佳方法流程。结果：优化出的海马蛋白质提取方法为液氮研磨法,最佳蛋白质提取裂解液为7 mol·L^（-1）尿素,2 mol·L^（-1）硫脲,含4%Chaps,1 mmol·L^（-1）PMSF,65 mmol·L^（-1）DTT和0.2%Bio-Lyte,最佳蛋白质纯化方法为超滤＋TCA丙酮沉淀两步纯化法,双向电泳最佳上样量为300μg。Image master 7.0软件对电泳图谱分析共检测出三斑海马蛋白质斑点236个,相对分子质量在14.4~97.4 k Da均有分布,分布差异不明显;蛋白质等电点主要分布在p H 4~9,其中以碱性蛋白质最为丰富。结论：利用上述方法所得的双向电泳图谱斑点清晰、分离均匀,图谱质量佳,该方法流程适合三斑海马药材蛋白质组学分析。该研究能为后期进行深入的海马蛋白质组学研究,筛选特征性蛋白质奠定基础。Objective： To establish a two-dimensional gel electrophoresis（ 2-DE） protocol for theproteomic study of Hippocampus trimaculatus,in order to lay the foundation for further research on Hippocampus in the late stage. Method： The methods of protein extraction,lysis,purification,and sample loading quantity were optimized. Result： The optimized protein extraction method was the liquid nitrogen grinding method. The best lysate contained 7 mol·L^（-1） Urea,2 mol·L^（-1） Thiourea,4% Chaps,1 mmol·L^（-1） PMSF,65 mmol·L^（-1） DTT and0. 2% Bio-Lyte. The best protein purification method was ultrafiltration ＋ TCA/acetone precipitation. The best sample loading quantity was 300 μg. A total of 236 protein spots were detected by image analysis,all of which were distributed within the range between 14. 4-97. 4 kDa, with unobvious differences. Protein isoelectric pointswere mainly distributed between pH 4-9,among which alkaline protein was the most abundant. Conclusion：The two-dimensional electrophoretic map obtained by the method mentioned above shows clear spots,uniform separation and a good quality. The method is suitable for the proteomic analysis of H. trimaculatus. This study can lay the foundation for further studying the proteomics of H. trimaculatus and screening characteristic proteins.

关 键 词：三斑海马 双向电泳 蛋白质组学 动物药 

分 类 号：R284.1[医药卫生—中药学] R931.74[医药卫生—中医学]