摘要
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 establish two step chromatography for the preparation of trichosanthin（TCS） and confirm if TCS possess the SOD activity. METHODS TCS was isolated and purified by a combination of ammonium sulphate fractionation and Blue - Sepha- rose CL - 6B and SP - Sepharosecolumn from the grinded and extracted root tuber of Trichosanthes kirilowii. The homogeneity of purified TCS was assessed by using SDS - PAGE and PAGE. The inhibition of TCS on Hela cells was detected by MTT assay. Pyrogallolautoxi - dation method and NBT staining were used to determine TCS＇ s SOD activity. RESULTS Through the process of ammonium sulphate precipitation of crude extract, and two ehromategraphies, Blue - Sepharose CL - 6B and SP - Sepharose, high - purity and high - yield TCS were obtained. It showed a single band on SDS - PAGE and PAGE. TCS obviously inhibited the proliferation of the protein on Hela cells,which displayed in time and dose dependent manners. SOD activity of TCS was not observed. CONCLUSION This method is simple, rapid and with high - yield, and can be used to prepare TCS with biological activity in a large scale. And that TCS possesses no SOD activity has been confirmed for the first time.
出处
《华西药学杂志》
CAS
CSCD
北大核心
2014年第5期510-512,共3页
West China Journal of Pharmaceutical Sciences
关键词
天花粉蛋白
SOD活性
核糖体失活蛋白
两步层析法
栝楼
TCS
SOD activity
Ribosome inactivating protein
Two step chromatography method
Trichosanthes kirilowii.
