CAJ | 학술논문

目的 EMCV的pEGFP-3C真核表达载体的构建及功能鉴定.方法 RT-PCR扩增EMCV功能蛋白3C基因并克隆至pEGFP-C3载体中,经双酶切和测序鉴定.将重组质粒分别转染到BHK-21细胞和293T细胞48 h后,Western-Blot检测3C蛋白酶的表达,以及对PABP1的切割,共聚焦显微镜观测3C荧光表达位置.结果 EMCV的pEGFP-3C重组质粒经双酶切及测序鉴定构建正确.表达的GFP融合蛋白相对分子质量约为49×103,可切割PABP1,并表达于细胞核内.结论成功构建了EMCV的3C蛋白酶的真核表达载体pEGFP-3C,为进一步研究该蛋白酶的功能及作用奠定了基础.
목적 EMCV적pEGFP-3C진핵표체재체적구건급공능감정.방법 RT-PCR확증EMCV공능단백3C기인병극륭지pEGFP-C3재체중,경쌍매절화측서감정.장중조질립분별전염도BHK-21세포화293T세포48 h후,Western-Blot검측3C단백매적표체,이급대PABP1적절할,공취초현미경관측3C형광표체위치.결과 EMCV적pEGFP-3C중조질립경쌍매절급측서감정구건정학.표체적GFP융합단백상대분자질량약위49×103,가절할PABP1,병표체우세포핵내.결론 성공구건료EMCV적3C단백매적진핵표체재체pEGFP-3C,위진일보연구해단백매적공능급작용전정료기출.
Objective To construction of the eukaryotic expressing plasmid EMCV 3C proteinase and identify the function.Methods The EMCV 3C gene was clone into vector pEGFP-C3.Recombinant plasmid EMCV-pEGFP-3C was identified by restriction enzyme analysis and sequencing.After the plasmid was transfected into the both 293T cell and BHK-21 cell.3C protease was identified with Western Blotting.The cellular location of the expressed protein was observed by Confocal Microscope.Results The recombinant plasmid EMCV-pEGFP-3C was confirmed by restriction enzyme analysis and sequencing.After EMCV-pEGFP-3C was transfected into cell,both and the cleavage of the poly(A)-binding protein (PABP) were detected by Western Blotting,as well as the expressed GFP fusion protein with a relative molecular mass of about 49 × 103.In the mean time,the GFP fusion protein was observed in the nucleus.Conclusions Eukaryotic expression plasmid of EMCV 3C gene was successfully constructed into plasmid EMCV-pEGFP3C.The expressed GFP fusion protein from the plasmid poses the character of EMCV 3C protease.