Cloning,Prokaryotic Expression and Immunogenicity Analysis of S Gene of Porcine Epidemic Diarrhea Virus HB/FN Strain

According to the S gene sequence of porcine epidemic diarrhea virus classic strain in GenBank and PMD-19T vector map,a pairs of primers were designed which were inserted into the restriction endonuclease BamHⅠand XhoⅠsites.Using PCR technology,a 1 080 bp fragment in suspected porcine epidemic diarrhea disease HB/FN strain was obtained and connected into PMD19-T vectors.By enzyme digestion and DNA sequencing,the results showed that its homology with S gene in GenBank was up to 100 %.Based on the antigenicity analysis,655 bp fragment of higher antigenicity was inserted into pET-28a(+),pET-32a(+) and pGEX-6p-1 to construct prokaryotic expression vector,and the first has the highest expression level.Induced by IPTG,SDS-PAGE analysis showed that S gene could be expressed by these vectors,the product is about 25 ku,the same with expected results.After purification,Western-blot assay indicated that the target protein could react with PEDV immune serum.Antibodies of the mice immunized with purified proteins showed good antigencity.