关键词: PEDV, N蛋白, 真核质粒, 表达质粒

Abstract: In order to investigate the effect of porcine epidemic diarrhea virus N genes encoding proteins in the PEDV infection early diagnosis, a pair of specific primers was designed to amplify N gene according to the full-length genome sequence of N gene of the PEDV CV777 strain in this experiment, then N gene was cloned into the eukaryotic expression vector pPM-C-His to construct a recombinant plasmid named pPM-C-His-N, the recombinant plasmid was extracted and the intramuscular injection for Kunming mice was carried out to detect the expression level in mice. At the same time, the plasmid was transfected into Vero cells, and PEDV N expression at gene level and protein level was detected, then N protein distribution in cells was detected in vitro by indirect immunofluorescence assay (IFA). The results showed that the recombinant eukaryotic plasmid expressed successfully at gene level and protein level, the serum antibody titer in immunized mice was 1:51 200. Western blot results showed that the immune serum can react with recombinant N protein particularity. The study constructed recombinant eukaryotic expression plasmid of pPM-C-His-N successfully, and detected green fluorescence in Vero cells, which laid the foundation for establishment of diagnostic method of PEDV and research for pathogenic mechanism.

Key words: PEDV, N protein, prokaryotic plasmid, expression plasmid