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目的采用两步层析法快速制备天花粉蛋白(TCS),并验证其SOD活性。方法粉碎栝楼块茎并抽提,经硫酸铵分级沉淀,再经Blue-Sepharose CL 6B和SP-Sepharose两步层析,可获得TCS纯制剂;采用SDS-PAGE和PAGE鉴定其均一性;用MTT法检测TCS对Hela细胞的抑制作用;采用PAGE-NBT活性染色法与邻苯三酚自氧化法相结合检测了TCS的SOD活性。结果用两步层析法可获得高纯度和高收率的TCS;经SDS-PAGE和PAGE鉴定,均为单一蛋白质着色带;TCS对Hela细胞增殖具有明显抑制作用,呈时间和浓度依赖关系;TCS不具有SOD活性。结论所用TCS纯化方法简单、快速、收率高,可大量制备具有生物活性的TCS,同时也首次证明了TCS不具有SOD活性。
Objective To rapidly prepare TCS by two-step chromatography and verify its SOD activity. Methods The tuberous stems of Rhizoma Paridis were smashed and extracted by ammonium sulfate fractionation, and purified by two-step chromatography using Blue-Sepharose CL 6B and SP-Sepharose to obtain pure TCS preparations. The homogeneity was determined by SDS-PAGE and PAGE. TCS was used to detect the inhibitory effect of TCS on Hela cells. SOD activity of TCS was detected by PAGE-NBT staining and pyrogallol autoxidation. Results TCS with high purity and high yield were obtained by two-step chromatography. SDS-PAGE and PAGE showed that the TCS were single protein bands. TCS had a significant inhibitory effect on the proliferation of Hela cells in a time and concentration-dependent manner. TCS does not have SOD activity. Conclusion The TCS purification method used is simple, rapid, high yield, can be prepared in large quantities with biological activity of TCS, but also for the first time that TCS does not have SOD activity.