脑心肌炎病毒（EMCV）3C真核表达载体的构建及其功能鉴定

Construction and functional identification of the eukaryotic expression plasmid EMCV 3C protease

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48h后，Western—Blot检测3c蛋白酶的表达，以及对PABPl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49×10^（3），可切割PABPl，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。

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 Confoeal Microscope. Results The recombinant plasmid EMCV-pEGFP-3C was confirmed by restriction enzyme analysis and sequencing. After EMCV-pEGFP-3 C 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 x 103. In the mean time, the GFP fusion protein was observed in the nucleus. Conclusions Eukaryotie expression plasmid of EMCV 3C gene was successfully constructed into plasmid EMCV-pEGFP- 3C. The expressed GFP fusion protein from the plasmid poses the character of EMCV 3C protease.