摘要
目的研究香菇C91-3菌丝体发酵液中的粗蛋白LFP91-3的分离纯化方法,提取具有抗肿瘤活性的蛋白组分,并初步鉴定其药效及特性。方法 LFP91-3经盐析、透析、Sephadex G-100凝胶过滤层析分离。用考马斯亮蓝法测定各管收集液中的蛋白浓度,依此绘制洗脱曲线,按曲线对收集液分组;用MTT比色法分别测定各组蛋白收集液对肿瘤细胞的生长抑制作用。对抑瘤率最大的一组各管收集液分别做SDS-PAGE电泳银染,取电泳条带相对较单一的峰尖部数管收集液合并,即为本实验的提取蛋白,以MTT比色法测定其对肿瘤细胞的生长抑制作用。用考马斯亮蓝法和硫酸-苯酚定糖法测定提取蛋白中蛋白和糖的比例。流式细胞仪检测提取蛋白诱导肿瘤细胞的早期凋亡率和晚期凋亡率;透射电镜观察经该蛋白作用的肿瘤细胞的凋亡形态。结果 LFP91-3经分离纯化,可得Ⅰ～Ⅳ共4组蛋白峰,且第3组的收集液抑瘤率最高。SDS-PAGE电泳结果显示第3峰顶部23～26管收集液主要含有19 kDa左右的蛋白,其他分子量的蛋白含量极低。测得该蛋白中蛋白和糖比例约为3∶1。MTT法显示提取蛋白在体外能明显抑制鼠肉瘤细胞株S180细胞的生长。流式细胞仪检测结果表明提取蛋白能在体外诱导S180细胞凋亡。透射电镜下,经提取蛋白作用的S180细胞,呈明显的凋亡形态。结论粗蛋白LFP91-3经盐析、透析、Sephadex G-100凝胶过滤层析分离共得到4组蛋白,第3组对S180细胞的抑制作用最强,其中第23～26管收集液主要含有分子量为19 kDa左右的蛋白,而且含有糖成分,提示可能为糖蛋白。该提取蛋白对S180细胞的生长具有较强的抑制作用,此作用与诱导肿瘤细胞凋亡有关。
Objective To research the methods of isolating and purifying the composition of LFP91-3 antineoplastic effect in vitro. Methods Salt-out,dialyze and concentrate the fermentation liquid.Chromatography was taken by Sephadex G-100,concentration of protein in every tube was determined by Bradford method and the eluting curve was drawn.Collected liquid was grouped according to the curve.The restraining tumor rate was detected by MTT method.Purity and molecular weight of protein for 23-26 tubes of the third fraction were determined by SDS-PAGE electrophoresis.Protein to polysaccharide ratios in purified protein was determined by Bradford method and sulphuric acid-phenol method,respetively.Apoptotic rate of tumor cells induced by purified protein was determined by flow cytometry.Morphology of apoptosis cells was observed by TEM. Results（1）Compounds were divided into Ⅰ,Ⅱ,Ⅲ,Ⅳ groups of fractions.The third fraction of collected liquid had the highest rate of restraining tumor.（2）The results of SDS-PAGE electrophoresis demonstrated that molecular weights of protein in 23-26 tubes of the third fraction were mostly about 19kDa.Contents of other proteins were extremely low.Protein to polysaccharide ratios in purified protein was 3：1.（3）Results of MTT methods demonstrated that the purified protein could restrain growth of S180 cells significantly.Flow cytometric analysis showed that apoptotic rate of the tumor cells induced by purified protein was higher than intervention significantly,and the effect could be revealed by trans-mission electron microscope. Conclusion Four groups of protein can be obtained from the fermentation liquid containing crude proteins by centrifugation,salt-out,dialyzation,concentrate and gel permeation（Sephadex G-100） chromatography.The third fraction has the strongest inhibition to proliferation of S180 cells in vitro.The purified protein in 23-26 tubes of the third fraction,which is 19kDa approximately and contained a quarter of polysaccharide,has strong inhibition to S180 ce
出处
《时珍国医国药》
CAS
CSCD
北大核心
2012年第3期538-542,共5页
Lishizhen Medicine and Materia Medica Research
基金
国家自然科学基金(No.30540015)
关键词
发酵液
香菇
蛋白
凝胶过滤层析法
Fermentative Liquor
Lentinus edodes
Protein
Gel permeation
Chromatography
