鹿茸蛋白2种不同提取方法的比较研究

Comparison of two protein extraction methods from velvetantler

目的探讨从鹿茸中获得促进神经细胞损伤修复蛋白的最佳提取方法。方法将新鲜鹿茸分别采用匀浆法和超微粉碎法获取鹿茸蛋白混合物(velvet antler proteins,VAPs);用Bradford法及SDS-PAGE检测并比较蛋白含量及蛋白组分差别;用扫描电镜观察不同提取方法超微结构不同。建立1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP+)MPP+诱导神经母细胞瘤细胞SH-SY5Y损伤模型,用该模型观察不同提取方法所得蛋白对损伤恢复的影响。结果超微粉碎法提取的鹿茸蛋白得率更高,得到的蛋白质条带更多,图谱清晰;电镜结果表明超微粉碎法得到的鹿茸蛋白干粉均无细胞结构,且颗粒较细;活性检测中,匀浆法提取的鹿茸蛋白从125μg/m L浓度开始可显著提高1m M MPP+作用下SH-SY5Y细胞的增殖率,而超微粉碎法提取的鹿茸蛋白的最低有效浓度为62.5μg/m L,促增殖率可达到25.45%。结论超微粉碎法既能大量提取鹿茸蛋白又能最大限度保证蛋白促细胞增殖活性,是较为理想的蛋白提取方式。

Objective To compare optimal protein extraction method of velvet antler（ VA） and explore the repairmen activity of velvet antler proteins（ VAPs） on 1-methyl-4-phenylpyridinium（ MPP＋） damaged nerve cell. Methods VAPs was obtained from fresh velvet antler by homogenization and micronization method respectively. The protein content of VAPs was measured by Bradford and the molecular weight was identified by SDS-PAGE. Scanning electron microscope（ SEM） was perfomed to observe the ultrastructure of VAPs. Neuroblastoma cell line（ SH-SY5Y） damage was induced by MPP＋,and activity of each compound was compared by the proliferation of damaged cell detected by MTT method. Results The yield of micronization method of VAPs was higher than homogenization method and with better characters both in SDS-PAGE and SEM. Activity detection indicated that VAPs at the concentration of 125 μg / m L by homogenate method could significantly increase the proliferation rate of damaged SH-SY5 Y cells. By contrast, VAPs by micronization method had more effective effect at 62. 5μg / m L, and proliferation rate could reach 25. 45%.Conclusion Micronization method does not only acquire more protein but also has cell proliferation activity,is a relatively ideal way of protein extraction of velvet antler.