Abstract: Using positive plasmid pMDT-18-N which contained the gene encoding nucleocapsid protein of porcine epidemic diarrhea virus strain CV777 as template, the N gene was amplified by the upper/lower primers each with the internal sites of Kpn Ⅰ and Xho Ⅰ, respectively. By digestion with restriction enzymes Kpn Ⅰ and Xho Ⅰ, the PCR product was then subcloned into eukaryotic expression vector pcDNA3.1(+) that digested with the same enzymes. After restriction enzyme digestion and DNA sequencing identification, the constructed recombinant plasmid was named pcDNA3.1(+)-N, deletion and insertion were not found in its sequence. The pcDNA3.1(+)-N was transiently transfected into Vero E6 cells, and the expression of N gene was detected by Western blot and indirect immunofluorescence assay with mouse antiserum against N protein. N protein subcellular localization were analyzed by confocal microscopy. Results verified that N gene could be expressed successfully in Vero E6 cells, and the N protein localizes both in the cytoplasm and the nucleus.