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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 explore the specific binding protein of geniposide by affinity chromatography. METHODS: Epichlorohydrin (ECH) was used as an activating reagent to modify the agarose gel with epoxy groups. The conjugates of geniposide and epoxy activated Sepharose CL-6B (EAS6B ) To prepare geniposide ligand for the affinity medium to verify its coupling by high performance liquid chromatography. Geniposide-EAS6B affinity media was used to screen specific binding proteins from mouse brain total protein. Results: The optimized activation conditions were as follows: 15% ECH and 0.8 mol / L NaOH were reacted at 37 ℃ for 3 h, the density of epoxy groups reached 122 μmol / m L, the affinity of agarose gel Of the coupling amount of 19.53%, the coupling rate of 17.08μmol / m L. Three bands of jasminoidin-specific binding proteins were screened from the total brain proteins of mice and identified by mass spectrometry. The bands were mainly composed of heat shock protein, brain acid-soluble protein and glutamine synthetase. CONCLUSION: The optimum condition of activation of agarose gel by epoxy was established. Geniposide-EAS6B affinity medium was prepared and applied to screen out the binding protein of geniposide.