基于亲和层析方法研究栀子苷的药物靶标  

Study on Target Proteins of Geniposide based on Affinity Chromatography

作　　者：张琦[1] 张子剑[2] 谭琰[1] 王旭[1] 赵洁[1] 张傲哲 李亚楠[1] 万亮琴[1] 华茜[1] 

机构地区：[1]北京中医药大学基础医学院,北京100029 [2]北京中医药大学科研实验中心,北京100029

出　　处：《现代生物医学进展》2016年第15期2811-2815,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金面上项目(81473546)

摘　　要：目的:运用亲和层析方法初步探索栀子苷的特异结合蛋白。方法:采用环氧氯丙烷(Epichlorohydrin,ECH)作为活化试剂对琼脂糖凝胶进行环氧基修饰,通过偶联栀子苷与环氧活化的琼脂糖凝胶(epoxy activated Sepharose CL-6B,EAS6B),制备以栀子苷为配基的亲和介质,以高效液相色谱法验证其偶联。利用栀子苷-EAS6B亲和介质,从小鼠脑组织总蛋白中筛选特异结合蛋白。结果:优化偶联条件后,得到最佳的活化反应条件:15%ECH、0.8 mol/L Na OH于37℃反应3 h,环氧基密度达到122μmol/m L,琼脂糖凝胶亲和介质的偶联量为19.53%,偶联率为17.08μmol/m L。从小鼠脑组织总蛋白中筛选得到3个栀子苷特异结合蛋白的条带,经质谱鉴定,发现主要是由热休克蛋白、脑酸溶性蛋白和谷氨酰胺合成酶等组成。结论:确立了琼脂糖凝胶环氧活化的最佳条件,制备了栀子苷-EAS6B亲和介质并应用其筛选出栀子苷的结合蛋白。Objective： To preliminary identify the specific binding protein of geniposide using affinity chromatography. Methods：ECH, the abbreviation for Epichlorohydrin, was adopted as activating reagent for surface modification of agarose gel（epoxy activated Sepharose CL-6B, EAS6B）. Geniposide was coupled with EAS6 B by hydroxyl-epoxy interaction, and high performance liquid chromatography was put into use to verify the efficiency of coupling, and then geniposide-EAS6 B was applied to screening the binding protein of geniposide in mouse brain. Results： The optimal condition of epoxy activated Sepharose CL-6B was as followed： 15 % ECH, 0.8 mol/L Na OH was reacted at 37 ℃ for 3 h. After optimized the conditions, the epoxy group density was up to 122 μmol/m L, the conjugation ratio and amount were 19.53 % and 17.08 μmol/m L, respectively. Three bands on SDS-PAGE were obtained from total protein in mouse brain using affinity chromatography. Conclusions： We established the optimal condition of epoxy activated Sepharose CL-6B, and prepared the geniposide-EAS6 B affinity resin that was applied to screening the binding proteins of geniposide.

关 键 词：栀子苷 亲和层析 环氧活化琼脂糖凝胶 高效液相色谱 

分 类 号：Q503[生物学—生物化学] R284[医药卫生—中药学]