| 张琦 张子剑 谭琰 王旭 赵洁 张傲哲 李亚楠 万亮琴 华茜.基于亲和层析方法研究栀子苷的药物靶标[J].,2016,16(15):2811-2815 |
| 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 |
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| DOI： |
| 中文关键词: 栀子苷 亲和层析 环氧活化琼脂糖凝胶 高效液相色谱 |
| 英文关键词: Geniposide Affinity chromatography ECH HPLC |
| 基金项目:国家自然科学基金面上项目（81473546） |
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| 中文摘要: |
| 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 NaOH 于37 ℃反应3 h，环氧基密度达到122 滋mol/mL，
琼脂糖凝胶亲和介质的偶联量为19.53 %，偶联率为17.08 umol/mL。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。 |
| 英文摘要: |
| 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 EAS6B by hydroxyl-epoxy interaction, and high performance liquid chromatography
was put into use to verify the efficiency of coupling, and then geniposide-EAS6B 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
NaOH was reacted at 37 ℃ for 3 h. After optimized the conditions, the epoxy group density was up to 122 umol/mL, the conjugation ratio
and amount were 19.53 % and 17.08 umol/mL, respectively. Three bands on SDS-PAGE were obtained from total protein in mouse
brain using affinity chromatography.Conclusion:We established the optimal condition of epoxy activated Sepharose CL-6B, and prepared
the geniposide-EAS6B affinity resin that was applied to screening the binding proteins of geniposide. |
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