目的 探讨聚乙二醇修饰苦瓜籽核糖体失活蛋白(MAP30)的反应条件以及修饰产物的纯化方法。方法 在不同条件下,将聚乙二醇活性酯(mPEG)₂-NHS与MAP30反应,用SDS-PAGE分析不同反应条件对产物成分的影响;采用Sephacryl S-300HR分子筛柱色谱法对修饰产物进行分离纯化,TNBS法测定目标产物PEG-MAP30的氨基修饰度。结果 聚乙二醇修饰MAP30的最优反应条件为4℃,pH8.5硼酸硼砂缓冲液,MAP30质量浓度1mg/mL,MAP30与(mPEG)₂-NHS质量比1:10为最佳修饰条件,5min内反应完全;反应产物进一步纯化,经SDS-PAGE检测目标产物PEG-MAP30中不含未修饰的MAP30;TNBS法测定MAP30的氨基修饰度为44.9%。结论 初步确定了聚乙二醇修饰MAP30的反应条件和修饰产物的纯化方法。

Objective To explore conditions for the modification of MAP30 from Semen Momordica Charantiae with polyethylene glycol (PEG) and the method for the purification of PEG-MAP30. Methods To analyze the influence of various conditions on the components of PEG-MAP30 by SDS-PAGE,purify PEG-MAP30 by Sephacryl S-300 HR molecular sieve column chromatography,and detect the modification rate of amino by TNBS under the optimal reaction condition with the reaction of (mPEG)₂-NHS and MAP30. Results Temperature 4℃,boric acid-borax buffer with pH 8.5,concentration of MAP30 1 mg/mL,the mass ratio of MAP30 to (mPEG)₂-NHS (1:10),and,5 min for reaction time were selected as the optimal reaction condition. Molecular sieve column showed a good purification effect. The modification rate of amino was 44.9% tested by TNBS under the optimal reaction condition. Conclusion The factors influencing the modification of MAP30 with PEG are determined,and a purification method for PEG-MAP30 is developed.

国家自然科学基金资助项目(C01020404)