重组人血管生成素制备和生物活性鉴定  

Preparation and Functional Characterization of Recombinant Human Angiogenin

作　　者：孙德森 盛静浩 

机构地区：[1]浙江大学医学院环境医学研究所 [2]浙江大学医学院分子医学研究中心,杭州310058

出　　处：《中国生物化学与分子生物学报》2015年第12期1315-1321,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.31170721;No.31400648)资助~~

摘　　要：血管生成素(angiogenin,ANG)是一种很强的促血管生成因子,与肿瘤的发生发展有着密切的关系,拮抗ANG被认为是抗肿瘤血管靶向治疗的一个有效途径.同时,ANG具有促进伤口愈合、保护神经元以及抗细菌感染等活性.但是,开展相关研究所需的最基本的天然ANG蛋白来源非常有限,大规模表达和纯化有生物活性的重组血管生成素蛋白(r ANG)具有广泛的应用前景.我们可以在大肠杆菌中表达r ANG,但表达产物在胞内聚集形成不溶性的包含体,需经变性、复性、阳离子交换层析和反向C18的HPLC方法纯化后,才能获得高纯度的r ANG.经SDS-PAGE鉴定,纯化蛋白质为单一条带,质谱鉴定蛋白分子量与理论分子量一致.经体外活性检测,证实纯化的蛋白质具有核糖核酸酶活性、促内皮细胞管腔形成和细胞核转位等生物学活性.本文详细描述了r ANG的表达、纯化、活性鉴定等过程和所使用的方法与技术.Angiogenin （ANG） is a key pro-angiogenic factor to associate with tumor occurrence and development. ANG antagonists are considered for effective vascular-targeted therapies. ANG also accelerate wound healing, protect neurons, and against bacterial infection. The natural source of ANG is limited, thus recombinant human angiogenin （rANG） with biological activity will be largely in need. We expressed rANG in E. coli, but mainly in insoluble forms in the inclusion bodies. Denaturation and renaturation processes were required. We used cation exchange chromatography and reverse C18 HPLC for preparing high purity of rANG. The purified protein was identified as a single band in SDS-PAGE with correct molecular weight, then confirmed by mass spectrometry. From in vitro assay, it showed that purified rANG possessed expected ribonuclease activity. The treatment with rANG of a nuclear translocation activity was observed to promote endothelial cell tube formation.

关 键 词：血管生成素 原核表达 核糖核酸酶活性 管腔形成 细胞核转位 

分 类 号：Q51[生物学—生物化学] Q78