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ISG15由干扰素刺激基因15编码,是最早被发现的类泛素修饰分子.病毒感染以及干扰素刺激可以强烈诱导其表达.与泛素类似,ISG15可以共价连接到其他蛋白分子上进行修饰,但ISG15及其连接修饰的功能作用还有很多尚未知.最近的研究表明,ISG15及其修饰作用在先天免疫中起着重要的作用.将牛类ISG15基因克隆进入pET28a( +)原核表达载体,并且表达了可溶的融合有His-tag标签的bISG15融合蛋白.使用Ni-NTA葡聚糖进行纯化浓缩.纯化蛋白免疫Balb/c小鼠并获得抗血清.Western印迹实验显示,抗血清可以特异地识别在真核细胞中表达的bISG15 .浓缩的bISG15以及制备的抗血清用于建立bISG15的体外修饰系统.实验证明,使用该系统bISG15可以连接到细胞蛋白上进行修饰.
ISG15 is the first ubiquitin-modified molecule to be encoded by interferon stimulating gene 15. Similar to ubiquitin, ISG15 can be covalently linked to other protein molecules for modification, However, there are still many unknown functional roles of ISG15 and its linker modification.Recent studies have shown that ISG15 and its modification play an important role in innate immunity.IgG5 gene was cloned into pET28a (+) prokaryotic expression vector, And expressed a soluble His-tag fusion protein bISG15 fusion protein was purified using Ni-NTA dextran purified protein Balb / c mice were immunized and obtained antiserum Western blotting experiments showed that antisera can be specific To identify bISG15 expressed in eukaryotic cells.Concentrated bISG15 and the prepared antisera were used to establish the in vitro modification system of bISG15.It has been experimentally proved that bISG15 can be modified to connect to cellular proteins by using this system.