Expression of VP2 gene of canine parvovirus virus and development of a recombinant protein-based indirect ELISA for antibodies detection.

We developed an indirect ELISA method for detecting canine parvovirus virus(CPV) antibodies based on the recombinant pertactin protein expressed in Escherichia coli(DE3) strain.Base on analysis of CPV VP2 gene,we design a pair of primers to amplify the gene fragment except 5' and 3' rare codons in bacteria system,and constuct prokaryotic expression vector pET28a-VP2 and pET32a-VP2.The recombinant plasmids were transformed into E.coli BL21,and expressd after induced with IPTG.The expressed products were purified by His Tag of pETs vector,and identified by SDS-PAGE assay and Western-blot.The recombinant protein fragment pET28a-VP2 showed a great activity of immunity.The pET28a-VP2-based indirect ELISA was developed for detecting antibodies against CPV named iVP2-ELISA.The result showed that the opimal concentration of coated antigen was 17.8 mg/L and the best dilution of of serum was 1∶20.The positive criterion of this ELISA assay was D the tested serum 0.367 and D the tested serum/D the negtive serum 2.0.The method result was consistent with HA/HI assay and iVP2-ELISA,but not suggest a direct proportion relation.The assay showed excellent specificity,sensitivity and reduplication,and can be useful for epidemiological survey and clinical diagnosis of canine parvorivus.