Molecular Characterisation Of Canine Parvovirus VP2 Gene

A pair of primers were designed and synthesized based on VP2 gene of CPV from Genebank. The genomic DNA samples of canine parvovirus strain isolated in Yangzhou were extracted and used as templates for polymerase chain reaction (PCR) to amplify the VP2 gene. The specific 1740 bp band was amplifed and cloned into pUC18 vector at the restriction endonuclease EcoRI and Sail sites. The recombinant palsmids were identified by restriction endonuclease analysis. The exogenous insert of recombinant plasmid was further confirmed by sequence analysis. After sequencing, VP2 gene of Yangzhou isolation was compared with that of American reference strains CPV-d (type 2) ,CPV-15 (type2a) , CPV-39(type 2b). The results showed 99.5%, 99.7%, 99,7% homology among these different strains.VP2 fragments from CPV Yangzhou strain were amplified with another two pairs of primers. PCR fragments were cloned to the downstream of GST gene according to the right Open Reading Frame in pGEX-6p-l vector. After inducing with 1.0mM IPTG and 37℃, the inserts were expressed as two fusion proteins containing the GST protein. According to SDS-PAGE analysis, two GST-VP2 fusion proteins were found as 54kD and 61kD bands, respectively. The expressed products were extracted from the gel and added as immunogen to immunize mice intraperitoneally, 5 doses with one-week intervals. The specificity of antibody was detected positively against CPV-infected FK81 cell in the indirect immunofluorescent assay (IFA). The results of the study showed that the in vitro expressed products of VP2 gene maintained the naive antigenicity of CPV.The sequence analysis of VP2 gene will contribute to the study of antigenic drift, and the fusion proteins will be useful for the generation of specific monoclonal antibodies and recombinant vaccines.