蝉花蛋白提取方法比较

Comparison of the Extraction Methods of the Protein in Cordyceps Cicadae

目的：比较不同方法所提蝉花蛋白的浓度、数量和体外抗肿瘤活性,筛选最适蝉花蛋白提取方法。方法：采用六种方法提取蝉花中的总蛋白质,分别为Tris-HCl/饱和酚法（A法）、TCA/丙酮沉淀法（B法）、两步法（C法）、蜗牛酶酶解法（D法）、Tris-HCl提取法（E法）、磷酸盐缓冲液（PBS）提取法（F法）,通过蛋白质浓度检测、聚丙烯酰胺凝胶电泳（SDS-PAGE）分析及体外抗人乳腺癌细胞（MCF-7）肿瘤活性检测,比较不同提取方法对蛋白提取效果的影响。结果：从提取浓度来看,A法最优;从SDS-PAGE结果看,A法和B法即蛋白质沉淀法效果较好,条带丰度好且较清晰,C法条带丰度较好,但较模糊;从蛋白体外抗肿瘤活性结果看,C法、E法、F法的蛋白活性较高。结论：方法 A、B、C均适用于蝉花总蛋白的提取,但方法 A、B在制备过程中无法保持蝉花蛋白的生物活性,而方法 C即两步法提取的蝉花蛋白浓度较高、种类较多,体外抗MCF-7肿瘤活性也较强,为蝉花蛋白提取最适方法。

Objective：The concentration,quantity and in vitro anti-tumor activity of Cordyceps cicadae protein extracted by different methods were compared to screen out the optimal extracting technology of the protein in Cordyceps cicadae. Methods：Six extraction methods,such as Tris-saturated phenol extraction（method A）,TCA/acetone precipitation（method B）,two-step method（method C）,snailase digestion（method D）,Tris-HCl extraction（method E）,and phosphate buffer extraction（method F） were used to extract the total protein in Cordyceps cicadae. The effects of different extraction methods on the extraction of protein were compared by the protein concentration detection,SDS-PAGE electrophoresis analysis and in vitro anti-tumor examination on MCF-7 cells. Results：In terms of concentration,method A was the best,in terms of the results of SDS-PAGE electrophoresis,method A and B with protein precipitation had better effects with abundant and clear strips while method C with abundant but blurred strips;in terms of protein in vitro anti-tumor results,method C,E,F had higher protein activity. Conclusion：Method A,B and C are all indicated for the extraction of protein in Cordyceps cicadae,but method A and B could not maintain the biological activity of Cordyceps cicadae in the preparation while method C,i.e. two-step method extracts the protein in Cordyceps cicadae with high concentration,more categories and high in vitro anti-tumor activity which should be the optimal extraction method of protein in Cordyceps cicadae.