共表达鸡传染性贫血病毒VP1、VP2基因和鸡γ干扰素基因的重组鸡痘病毒的构建与鉴定OA

To construct a recombinant fowlpox virus transfer vector co-expressing the VP1 and VP2 genes of chicken infectious anemia virus(CIAV)and chicken interferon-gamma(ChIFN-γ),a homologous recombination strategy was employed.The VP1 and VP2 genes of CIAV,the ChIFN-γ gene,and the reporter gene lacZ were integrated into a non-essential region for viral replication within the genome of the fowlpox virus(FPV)S-FPV-017 strain.The VP1,VP2,and ChIFN-γ gene expression cassettes controlled by FPV early/late promoter LP2EP2 and lacZ reporter gene expression cassette controlled by P11 promoter(late promoter)were inserted into the non-essential region of FPV genome(S-FPV-017).The recombinant transfer plasmid pSY681-VP1-VP2-ChIFN-γ was transfected into chicken embryo fibroblasts(CEF)pre-infected with parental FPV strain S-FPV-017 for homologous recombination.After 8 rounds of plaque screening and purification,the recombinant virus rFPV-VP1-VP2-ChIFN-γ expressing CIAV VP1,VP2,ChIFN-γ and lacZ reporter gene was successfully obtained.PCR identification confirmed that CIAV VP1,VP2,and ChIFN-γ genes were integrated into the recombinant virus genome.Indirect immunofluorescence assay and Western-blot results demonstrated that the corresponding proteins were effectively ex-pressed in CEF cells infected with the recombinant virus.Compared with the parental virus,the recombi-nant virus showed no significant differences in replication characteristics and cytopathic effects on CEF cells.These findings demonstrate that multiple exogenous genes from avian pathogens can be simul-taneously inserted and expressed in a non-essential region of FPV genome,providing important founda-tion for development of multivalent recombinant viral vector vaccines.