Construction of recombinant pseudorabies virus co-expressing ORF2 gene of porcine circovirus type 2 and VP2 gene of porcine parvovirus.

The recombinant plasmid pPI-2.EGFP.VP2.ORF2 was constructed by inserting VP2 gene of porcine parvovrius(PPV) and ORF2 gene of porcine circovirus type 2(PCV2) including complete open reading frame(ORF) into the eukaryotic expression vector pPI-2.EGFP.At hour 20 after co-transfection of the recombinant plasmid with the pseudorabies virus(PRV) strain SA215 genomic DNA into Vero cells with liposome,green fluorescent protein could be observed in some plaque using fluorescence microscope,and plaque purification was employed to obtain the virus,subsequently.The recombinant PRV named PRV SA215(D1) was collected through plaque purification repeatedly and proved to be successfully constructed by PCR amplification.The characterization of virus multiplication showed the recombinant virus could propagated on different cell lines such as Vero,MDBK,ST and IBRS-2 and PFU of the recombinant virus on Vero cell line was 1×106PFU.The animal test showed that 28 day-old piglets inoculated the recombinant virus(1×105PFU per piglet and 1×104PFU per piglet each) could generate specific PPV and PCV2 antibodies,which turned to positive on day 14 post-inoculation at 1×105PFU per piglet group,and on day 21 post-inoculation at 1×104PFU per piglet group.The result certificated that the recombinant virus PRV SA215(D1) was constructed successfully.