CAJ | 학술논문

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.
목적 탐토뇌심기염병독(EMCV)적3C단백매대진핵세포전록급번역궤제적영향.방법 구건진핵표체재체pcDNA3.1-3C,장표체재체분별여모양의뢰적록색형광단백(GFP)표체재체pEGFP-N1화형화충형광소매(Fluc)재체pGL3재체공전염세포후,검측GFP화Fluc적표체.Western Blot검측EMCV병독화pcDNA3.1-3C대진핵생물번역기시인자4GI(eIF4GI)급다취A미결합단백(PABP)적영향.RT-PCR검측pcDNA3.1-3C대GFP mRNA전록수평적영향.결과 EMCV-3C가이억제모양의뢰적GFP화Fluc적표체.EMCV 3C화EMCV병독감염균도치세포내PABP발생절할,이대eIF4GI균무명현작용.EMCV-3C야가도치GFP기인mRNA전록수평하강.결론 EMCV적단백매3C통과mRNA전록수평적억제화PABP적절할이억제진핵세포모양결구의뢰도경적단백번역,종이억제단백질적합성.
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.