Whole-Genome Analysis Of Vero Cell-adapted Porcine Epidemic Diarrhea Virus And Preparation Of Hyperimmune Antiserum Against PEDV N-Protein

Porcine epidemic diarrhea(PED)is a highly pathogenic enteric disease caused by porcine epidemic diarrhea virus(PEDV).In recent years,PEDV has resulted in a severe economic losses on the swine industry in China and all over the world.The researchers from various countries have pay more attention on the prevention and control of PEDV.To further elucidate the molecular characterization of field strains of PEDV and preparation of hyperimmune serum against PEDV N protein,the following researches have been performed:1.The full-length genome sequence determination and molecular characterization of Vero cell-adapted PEDV strain circulating in Jiangxi: The 33 pairs of specific primers containing overlapping regions were used to amplify the complete genome sequence of passage 50 of PEDV CH/JX/01 strain isolated and stored in our laboratory.The genome has a full length of 28,054 nt,and the genome organization of the strain was 5'UTR-ORF1a/b-S-ORF3-EMN-3' UTR which was similar to classical strain of PEDV CV777.The nucleotides insertions and/or deletions were not observed in comparison with CH/JX/01 P0,P5,and P30.However,multiple nucleotide substitutions were identified in ORF1a/b,S,E,and M genes,and the number of S gene mutations showed an increasing trend when compared to CH/JX/01 P0,P5,and P30.The phylogenetic trees were generated based on the complete genome,S and N genes of 43 PEDV strain.The results indicated that the four strains of CH/JX/01 were clustered into subgroup 2a in PEDV group 2(G2),and did not fall in the same genotype with the classical PEDV strain of CV777.Phylogenetic analysis of the complete genome sequence indicated that CH/JX/01 had closest relationship with NW8(MF782687)isolated in Shaanxi in 2015.However,the phylogenetic analysis of the S gene showed PEDV CH/JX/01 strain had closer relationship with CH/GDZH02/1401(KR153325)in 2014 and CH/GX/2015/750A(KY793536)in 2015.The homology analysis showed that the N gene of PEDV CH/JX/01 strain shared higher nucleotide identity with 39 reference strains available in Genbank than the complete genome sequence and S gene,and the CH/JX/01 strain had 100% amino acid identities with SD2014(KX064280),XM1-2(KX812523),CH/HNZZ47/2016(KX981440)and CH/GDZH02 /1401(KR153325),respectively.2.The expression of PEDV N protein and preparation of hyperimmune antiserum against expressed N protein: The N gene of PEDV CH/JX/01 was amplified by specific primers and ligated with the expression vector pcold I to construct the recombinant plasmid pcold-PEDV-N and then transfected into competent cells of E.coli BL21(DE3).The conditions were optimized in order to induce the expression of recombinant N protein.SDS-PAGE and western blot results showed that recombinant N protein could be existed as soluble proteins and have good immunogenicity.The recombinant PEDV N protein purified by Ni-chromatographic column was used as an antigen to immunize mice in order to prepare hyperimmune sera.By utilizing expressed PEDV N protein as a coating antigen,an indirect ELISA was established.The result of the ELISA demonstrated that hyperimmue antiserum titer was as high as 1:25,600 and had a good specificity against PEDV verified by indirect ELISA,western blot and IFA,which revealed the antiserum can be used for PEDV detection.