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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 kDa均有分布,分布差异不明显;蛋白质等电点主要分布在pH 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 technique suitable for studying the proteome of dry matter of three-spot hippocampus, which lays the foundation for the further study of hippocampal proteomics of traditional Chinese medicine. Methods: Several key steps affecting two - dimensional electrophoresis of protein - extraction method, lysis solution, purification method and sample loading were optimized and the best method was selected. Results: The optimized extraction method for hippocampal protein was liquid nitrogen grinding method. The optimum extraction solution of protein was 7 mol·L -1 urea, 2 mol·L -1 thiourea, 4% Chaps, 1 mmol·L -1 PMSF, 65 mmol·L -1 DTT and 0.2% Bio-Lyte. The best method of protein purification was ultrafiltration + TCA acetone precipitation two-step purification method, The sample volume is 300μg. Image master 7.0 software for the analysis of electropherograms detected a total of 236 spots of the three-spot hippocampus proteins, the relative molecular mass of 14.4 ~ 97.4 kDa are distributed, the distribution was not significant; isoelectric point of the protein mainly in the pH 4-9, of which The most abundant basic protein. Conclusion: The two-dimensional electrophoresis pattern obtained by the above method has clear spots, uniform separation and good map quality. The method is suitable for the proteomics analysis of the three-spotted hippocampus. This study can lay the foundation for further research on hippocampal proteomics and screening of characteristic proteins.