动态检测活细胞内泛素-蛋白酶体系统活性方法的建立

ESTABLISHMENTOF A METHOD FOR DYNAMICALLY DETECTING ACTIVITY OF UBIQUITIN-PROTEASOME SYSTEM IN LIVING CELLS

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(DsRed_2)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DsRed_2的pGFP^u或pDsRed_2~u质粒,然后转染HEK293细胞,通过G418筛选得到稳定表达GFP^u或DsRed_2~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DsRed_2~u细胞后,应用免疫印记技术检测细胞内GFP或DsRed_2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DsRed_2荧光强度的变化。结果ALLN处理能使GFP^u和DsRed_2~u细胞内GFP和DsRed_2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。

Objective To establish a new method for dynamically detecting activity ofubiquitin-proteasome in living cells. Methods By adding a sequence coding CLl-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-Acetyl-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 DsRed^u 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 ofubiquitin-proteasome system in living cells.