Construction and Identification of a Recombinant Canine Type 2 Adenovirus Expressing VP2 Protein of Canine Parvovirus

The E3-region-deleted vector,named pVAXΔE3,was constructed by removing the E3 Ssp I segment.The CPV VP2 expression cassette released from pVCPV-VP2 plasmid was ligated into Ssp I site of pVAXΔE3 plasmid,and the resulted recombinant plasmid was named pΔECPV-VP2.pΔECPV-VP2 and pPoly2-CAV-2 were digested with Nru I/Sal I,respectively.The purified NruI/SalI DNA fragment containing the CPV VP2 expression cassette was cloned into pPoly2-CAV-2 plasmid to generate pCAV-2/CPV-VP2.The recombinant genome,CAV-2/CPV-VP2,was released from pCAV-2/CPV-VP2 plasmid by digestion with Asc(I/Cla I.) CAV-2/CPV-VP2 was cotransfected into DK cells with CAV-2 genome without the Nru I/Sal I segment by Lipofectamine.The recombinant virus,named CAV-2/CPV,was gained and it produced classical cell cytopathic effects in DK cells.The morphologic character of CAV-2/CPV is similar to CAV-2.Expression of CPV VP2 gene by CAV-2/CPV in infected DK cells was confirmed by RT-PCR.CAV-2/CPV propagated slower than wild CAV-2.Hereditary feature of CAV2/CPV is stable.