Construction of recombinant fowlpox virus transfer vector expressing VP3 gene of muscovy duck parvovirus

In order to construct the recombinant fowlpox virus vaccine against muscovy duck parvovirus(MDPV),2 pairs of primers were designed amplifying two arms of the thymidine kinase(TK) gene(TK left and TK right),according to genome of fowlpox virus FM strain on Genbank.Segment of TK left and TK right were linked by overlapPCR,inserted a segment of multiple clone site(MCS).The TK left-MCS-TK right segment were inserted to vector pBluescript II SK(+),and the abtained positive plasmid was named as pTK.The synthetized reverse tandem expression cassettes p7.5-MCS 1-SV40-MCS 2-P11 were inserted to MCS of plasmid pTK,and the abtained positive plasmid was named as pTKS.VP3 gene of MDPV was cloned into downstream of early promoter P7.5 and reporter gene EGFP was cloned into downstream of late promoter P11 on plasmid pTKS.The recombinant fowlpox virus transfer vector pTKS-VP3-EGFP were constructed and confirmed by digestion and sequencing.Plasmid pTKS-VP3-EGFP was used to co-transfect into CEF with fowlpox virus vaccine strain.The expression of VP3 in the recombinant fowlpox virus was confirmed by RT-PCR and expression of green fluorescent protein.This study provided useful information for development of a safe and effective recombinant FPV vaccine against MDPV.