【摘要】 目的建立一种动态检测活细胞内泛素-蛋白酶体系统活性的方法。方法将表达绿色荧光蛋白（GFP）或红色荧光蛋白（DsRed₂）的质粒分别改建为表达带有内泛素-蛋白酶体系统降解信号CL1的GFP或DsRed₂的pGFP^u或pDsRed₂^u质粒,然后转染HEK293细胞,通过G418筛选得到稳定表达GFP^u或DsRed₂^u的细胞系。在蛋白酶体抑制剂N-Acetyl-Leu-Leu-Norleu-al（ALLN）处理GFP^u或DsRed₂^u细胞后,应用免疫印记技术检测细胞内GFP或DsRed₂含量的变化,应用荧光显微镜和激光扫描共聚焦显微镜技术观察GFP或DsRed₂荧光强度的变化。结果ALLN处理能使GFP^u和DsRed₂^u细胞内GFP和DsRed₂含量明显增加,荧光强度显著增强,并呈现明显的剂量/时间-效应关系。结论本文成功地建立了检测内泛素-蛋白酶体系统活性的方法,该方法能有效地对活细胞的内泛素-蛋白酶体系统活性进行实时动态检测。更多还原

【Abstract】 Objective To establish a new method for dynamically detecting activity of ubiquitin-proteasome in living cells.Methods By adding a sequence coding CL1-a degradation signal for the ubiquitin-proteasome system,the plasmids pGFP expressing GFP and pDsRed₂ expressing DsRed₂ were reconstructed into pGFP^u expressing GFP^u（GFP fused with CL1）,and pDsRed₂^u expressing DsRed₂^u（DsRed₂ fused with CL1）.HEK293 cells were transfected with pGFP^u and pDsRed₂^u,respectively,and then the stable cell lines expressing GFP^u or DsRed₂^u were obtained by G418 selection. After treatment with the proteasome inhibitor N-Aceryl-Leu-Leu-Norleu-al（ALLN）,the level of GFP or DsRed₂ in GFP^u and DsRed₂^u cells was analyzed by immunoblotting,and the fluorescent intensity of GFP or DsRed₂ was examined under fluorescent microscope and laser scanning confocal microscope.Results It was found that ALLN treatment obviously elevated the level and fluorescent intensity of GFP^u or DsRed₂^u in GFP^u or DsRed₂^u cells,in a dose- and time-dependent manner.Conclusion A new method for detecting activity of ubiquitin-proteasome is successfully established,and this method can be used for real-time and dynamic examination of activity of ubiquitin-proteasome system in living cells.更多还原