EMCV3C蛋白酶抑制真核细胞蛋白质合成的研究

摘要

Objective To study the impact on eukaryotic cell protein synthesis by EMCV 3C protease.Methods After encephalomyocarditis virus 3C gene was linked in eukaryotic expressing vector to form pcDNA3.1-3C vector,both pcDNA3.1-3C and pEGFP-N1 wihch express cap-dependent green fluoresce protein (GFP) protein co-transfected into BHK21,GFP protein were detected by Western Blotting and GFP mRNA was detected by RT-PCR.Then pcDNA3.1-3C and pGL3 vector expressing cap-dependent fire luciferase protein were also co-transfected into BHK21 cells,fluc were detected.The change of eukaryotic translation initiation factor 4G (eIF4G) and the poly (A)-binding protein (PABP) after EMCV virus infection or EMCV 3C transfection were detected by Western Blotting.Results EMCV 3C inhibited cap-dependent translation of both GFP and flucprotein expression.Cleavage of PABP was detected after both EMCV infection and EMCV 3C protease transfection but change of eIF4G was not observed.Conclusions EMCV-3C protease inhibited synthesis of host cell protein by inhibition of mRNA transcription and capdependent protein translation.%目的 探讨脑心肌炎病毒(EMCV)的3C蛋白酶对真核细胞转录及翻译机制的影响.方法 构建真核表达载体pcDNA3.1-3C,将表达载体分别与帽样依赖的绿色荧光蛋白(GFP)表达载体pEGFP-N1和萤火虫荧光素酶(Fluc)载体pGL3载体共转染细胞后,检测GFP和Fluc的表达.Western Blot检测EMCV病毒和pcDNA3.1-3C对真核生物翻译起始因子4GI(eIF4GI)及多聚A尾结合蛋白(PABP)的影响.RT-PCR检测pcDNA3.1-3C对GFP mRNA转录水平的影响.结果 EMCV-3C可以抑制帽样依赖的GFP和Fluc的表达.EMCV 3C和EMCV病毒感染均导致细胞内PABP发生切割,而对eIF4GI均无明显作用.EMCV-3C也可导致GFP基因mRNA转录水平下降.结论 EMCV的蛋白酶3C通过mRNA转录水平的抑制和PABP的切割而抑制真核细胞帽样结构依赖途径的蛋白翻译,从而抑制蛋白质的合成.