Objective To develop a DNA vaccine against canine parvovirus(CPV) infection.Methods The VP2 gene of CPV with or without CpG motif was cloned by PCR.The eukaryotic expression vector pcDNA3 was modified by restriction digestion to remove its neo~r gene and to replace its amp~r with kan~r to conform gene therapy requirements.The recombinant plasmids were constructed by subcloning the VP2 gene with or without CpG motif into the modified vector pcDNAK and injected into BALB/c mice and Beagle dogs.Both primary and secondary antibody responses were detected by hemagglutination inhibition(HI) assay.Results Injection of the recombinant vector pcDNA3-VP2C1 containing one copy of the consensus CpG motif into BALB/c mice lead to more elevated antibody response than the empty vector pcDNAK and the recombinant vector pcDNAK-VP2 without CpG motif.Gene immunization experiments in dogs preimmunized with an inactivated vaccine showed that the pcDNAK-VP2C2 vector containing two copies of canine-specific CpG induced higher secondary immune response than that of the pcDNA3-VP2C1 vector.Conclusion These data indicate that the pcDNAK-VP2C2 vector containing canine-specific CpG is highly antigenic and could be used for further studies for development of gene vaccines against canine parvovirus disease.