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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 study the monoubiquitination site of oncoprotein TRE17 and provide the basis for further exploration of its mechanism of mono-ubiquitination and identification of its ubiquitin ligase E3. Methods A series of TRE17 variants including HA-TRE17 (375) and HA-TRE17 (303) were constructed by truncated stepwise approach. The middle deletion mutant HA-TRE17 of TRE17 was constructed by overlap extension PCR , Δ340-361). Subsequently, the variant plasmids were separately transfected into HEK 293 cells and the expression of each variant and its mono-ubiquitinated bands in HEK 293 cells was identified by Western blot. Results HA-TRE17 (447), HA-TRE17 (375) had a single ubiquitinated protein band and HA-TRE17 (303) had no single ubiquitinated protein band; HA-TRE17 (447, Δ340-361) also has no mono-ubiquitinated protein band. Conclusion The TRE17 single ubiquitination site may be located between or near 340-361 amino acid residues.