人参总皂苷亲和"垂钓"脑内蛋白质靶点的研究  

Research of ginseng total saponins pull down the proteins in brain

作　　者：陈飞燕[1] 欧阳柳凤 江玉翠 顾玲[1] 陈翠花[1] 徐雁[1] 赵玉男[1] 

机构地区：[1]南京中医药大学基础医学院实验研究中心,南京210023

出　　处：《中药药理与临床》2016年第3期41-45,共5页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学青年基金(81303246);江苏省"青蓝工程"优秀骨干教师和江苏省中西医结合优势学科项目资助;南京中医药大学基础医学院青年科技创新基金(14JCQN03)

摘　　要：目的:预测人参总皂苷在脑内的蛋白质作用靶点。方法:采用直接亲和法亲和"垂钓"出脑内可能与人参总皂苷相互作用的蛋白质;然后采用SDS-PAGE凝胶电泳对"垂钓"出的蛋白质进行分离、染色、挖取蛋白质斑点胶块,并采用MALDI-TOF/TOF串联二级质谱对未知蛋白质进行鉴定;采用生物膜层干涉技术(BLI)测定人参皂苷和疑似蛋白14-3-3之间的相互作用,来进一步确认疑似蛋白14-3-3是否与人参皂苷有直接的分子间相互作用,以及哪种人参皂苷单体与14-3-3蛋白的亲和力最强。结果:亲和"垂钓"和质谱共鉴定获得5个预测蛋白,分别为:14-3-3蛋白、肌动蛋白、肌酸激酶B型、ATP合成酶、未知蛋白质;动力学分析显示14-3-3蛋白与原人参二醇、原人参三醇存在分子间相互作用,并且KD值分别为7.8×10-4和5.62×10-4。结论:14-3-3蛋白是人参总皂苷脑内的作用靶点之一,人参皂苷体内代谢产物原人参二醇、原人参三醇可能是14-3-3蛋白的激活剂。Objective ：To predict the targets of ginseng total saponin in brain. Methods： In this study, We adopt direct affinity pull down the pro- teins in brain which may interact with ginseng saponins. Then, the unknown proteins were identified using tl;e MALDI-TOF/TOF after the SDS-PAGE gel electrophoresis separation. And the direct affinity chromatography using ginseng total saponins as ligands was adopted to dis- cover the protein targets of ginseng saponin in brain, on the other hand to explore whether the direct affinity chromatography is feasible for the finding of small molecular targets. Using the BLI technique, the affinity between 14-3-3 protein and ginsenosides was determined to further confirm whether 14-3-3 protein has the direct interaction with ginsenosides, and what kind of ginseng saponin individual has the strongest af- finity with 14-3-3 protein. Results：It showed that five predict proteins were acquired by SDS-PAGE and mass spectrometry. Respectively： the 14-3-3 protein, actin, creatinc kinase B, ATP syntbetase and unknown protein. Kinetics analysis showed that the affinity （KD） between protopanoxadiol and 14-3-3 protein is 7.8 × 104 M. Meanwhile, the affinity （KD） between panaxatriol and 14-3-3 protein is 5.62 × 104M. Conclusion ：The 14-3-3 protein is one of the targets for brain of ginseng total sapenins, and that protopanoxadiol, panaxatfiol could be activa- tors of 14-3-3 protein.

关 键 词：人参总皂苷 亲和"垂钓" 质谱 14-3-3蛋白 原人参二醇 原人参三醇 

分 类 号：R285[医药卫生—中药学]