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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 potent pro-angiogenic factor that is closely related to tumorigenesis and development, and antagonizing ANG is considered as an effective anti-tumor target therapy.At the same time, ANG has the potential to promote Wound healing, neuron protection and anti-bacterial activity, etc. However, the most basic sources of native ANG proteins required for carrying out the relevant studies are very limited in that large-scale expression and purification of bioactive recombinant angiogenic proteins (r ANG) A wide range of application prospects.We can express r ANG in E. coli, but the expression of the product aggregated within the formation of insoluble inclusion bodies, subject to denaturing, refolding, cation exchange chromatography and reverse C18 HPLC method to To obtain high purity r ANG.After identification by SDS-PAGE, the purified protein was a single band, and the molecular weight of the identified protein was the same as the theoretical molecular weight.It was confirmed by in vitro activity assay that the purified protein had ribonuclease activity and promoted the formation of endothelial lumen And nuclear translocation and other biological activity.This article described in detail the r ANG expression, purification, activity identification process and the use of technology and technology Surgery.