摘要
目的研究癌蛋白TRE17单泛素化的位点,为深入探讨其单泛素化的机制及鉴定其泛素连接酶E3提供研究基础。方法使用截短体逐步逼近的方法构建了一系列TRE17变异体,包括HA-TRE17(375)和HA-TRE17(303);用重叠延伸PCR方法构建了TRE17的中间缺失变异体HA-TRE17(447,Δ340-361)。随后,变异体质粒被分别转染HEK 293细胞,用Western blot方法鉴定各变异体及其单泛素化条带在HEK 293细胞内的表达。结果 HA-TRE17(447)、HA-TRE17(375)有单泛素化蛋白条带,而HA-TRE17(303)无单泛素化蛋白条带;与HA-TRE17(303)变异体一样,HA-TRE17(447,Δ340-361)也无单泛素化蛋白条带。结论 TRE17单泛素化位点可能位于340-361之间或附近的氨基酸残基。
Objective To explore the mono-ubiquitination site in TRE17 oncoprotein for providing a basis in the study of mechanism underlying the mono-ubiquitination and identification of ubiquitin ligase（E3）.Methods A series of nested deletion mutants of TRE17 was constructed,which included the first 375 and 303amino acids of TRE17,respectively.Subsequently,the mutants were transfected into HEK 293 cells and the mono-ubiquitilated and non-ubiquitilated proteins of TRE17 in HEK293cells were detected by Western blot.Results Cells transfected with HA-TRE17（447）or HA-TRE17（375）expressed a mono-ubiquitilated protein band besides their non-ubiquitilated form,while no mono-ubiquitilated band appeared in the cells transfected with HA-TRE17（303）.Consistent to HA-TRE17（303）,HA-TRE17（447,Δ340-361）was not mono-ubiquitilated in the cells.Conclusion The mono-ubiquitination site in TRE17 resides in or near the amino acids 340-361.
出处
《江苏医药》
CAS
2016年第9期993-996,共4页
Jiangsu Medical Journal
基金
国家自然科学基金(81272261)
