蒙古黄芪中核糖核酸酶活性蛋白AmPR-10的纯化和性质研究  

Research on purification and activity characterization of AmPR-10 ribonuclease from Astragalus membranaceus

作　　者：任晋宏 王永辉 薛慧清 刘晔 Divid Adelson 陈薛静 李敏 高丽 

机构地区：[1]山西中医药大学实验中心,山西晋中030619 [2]阿德莱德大学生物信息与计算遗传学中心

出　　处：《中草药》2017年第19期3945-3953,共9页Chinese Traditional and Herbal Drugs

基　　金：国家国际合作专项项目(2013DFA30700);山西省黄芪资源产业化及产业国际化协同创新中心项目(HQXTCXZX-007)

摘　　要：目的采用全自动智能蛋白纯化系统AKTA Avant25建立蒙古黄芪病程相关蛋白(Astragalus membranaceus pathogenesis-related protein-10,AmPR-10)的稳定快速分离纯化方法,并分析其理化性质和生物学活性。方法蒙古黄芪经Tris-HCl缓冲液浸提后阴离子交换色谱捕获目标蛋白,疏水色谱精细分离,凝胶滤过色谱进一步精细分离得到AmPR-10。采用MALDI-TOF/TOF质谱法测定相对分子质量,质谱检测后MS/MS Ion Search检索鉴定蛋白,高碘酸-Schiff法判断是否为糖蛋白,琼脂糖凝胶电泳分析核糖核酸酶活性及不同影响因素对核糖核酸酶活性的影响。结果蒙古黄芪粗提液经Q Sepharose Fast Flow、Butyl Sepharose High Performance和SuperdexTM 75 10/300 GL 3步纯化后可得到电泳纯AmPR-10;质谱测定其相对分子质量为16 801;蛋白鉴定其属于PR-10蛋白家族;糖蛋白染色显示其不含糖链。AmPR-10具有显著的核糖核酸酶活性,且其活性对Na Cl浓度、pH值和金属离子的敏感度低,仅0.5 mol/L NaCl、pH 9.0、Mg^(2+)和Co^(2+)对其有微弱的抑制作用,EDTA对AmPR-10核糖核酸酶活性无影响。结论离子交换色谱-疏水色谱-凝胶过滤色谱3步法纯化AmPR-10快速稳定,可应用于其他中药蛋白质的分离纯化。AmPR-10可能在蒙古黄芪抵御RNA病毒侵染中发挥重要作用。Objective To establish a stable and rapid separation and purification method for Astragalus membranaceus（Am） pathogenesis-related protein-10（AmPR-10） using an automatic intelligent protein purification system AKTA Avant 25, and analyze its physiochemical and biological activity. Methods Amwas extracted by Tris-HCl buffer. The crude extract was captured by anion exchange chromatography, and finely separated by hydrophobic chromatography and gel filtration chromatography. The relative molecular weight of AmPR-10 was measured by MALDI-TOF/TOF mass spectrometry, the protein identification was determined by mass spectrometry and MS/MS Ion Search, the glycoprotein identification was estimated by periodic acid-Schiff method, and the ribonuclease activity and effect factors were analyzed by agarose gel electrophoresis. Results The electrophoretically pure AmPR-10 was obtained by three-step purification of Q Sepharose Fast Flow, Butyl Sepharose High Performance and SuperdexTM 75 10/300 GL from the crude extraction. The relative molecular weight of AmPR-10 was 16 801. AmPR-10 was highly homologous to PR-10 and has no carbohydrate chains. Incubated at 56 ℃ for 30 min, AmPR-10 exhibited significant ribonuclease activity to total RNA of mammalian cells. The activity was insensitive to Na Cl, p H value and mental ions, and weekly inhibited by 0.5 mol/L Na Cl, p H 9.0, Mg～（2＋） and Co～（2＋）. The activity was the same at EDTA as high as 20 mmol/L. Conclusion The three-step method of exchange chromatography-hydrophobic chromatography-gel filtration chromatography, a stable and rapid separation and purification method of AmPR-10, can be applied for other Chinese herbs. AmPR-10 might play an important role in resistance against virus.

关 键 词：蒙古黄芪 AmPR-10 AKTA Avant25 纯化 核糖核酸酶 活性蛋白 生物学活性 MALDI-TOF/TOF质谱法 高碘酸-Schiff法 

分 类 号：R284.2[医药卫生—中药学]