Abstract: Objective To construct a universal, highly attenuated orf virus expression vector for exogenous genes using green
fluorescent protein (GFP) as the reporter gene. Methods The flanking regions of the ORFV132 of orf virus DNA were amplified
by PCR to construct the shuttle plasmid pSPV-132LF-EGFP-132RF. The shuttle plasmid was transfected into OFTu cells and
GFP was incorporated into orf virus IA82△121 by homologous recombination. The recombinant IA82△121-V was selected by
green fluorescent signal. The deletion gene was identified by PCR and sequencing. The effects of ORFV132 knockout were
evaluated by virus titration and by observing the proliferation of the infected vascular endothelial cells in vitro. Results The
recombinant orf virus IA82 △121-V was obtained successfully and quickly, and the deletion of ORFV132 did not affect the
replication of the virus in vitro but reduced its virulence. Conclusion Green fluorescent protein is a selectable marker for rapid,
convenient and stable selection of the recombinant viruses. Highly attenuated recombinant orf virus IA82△121-V can serve as
a new expression vector for exogenous genes.