Rapid selection of recombinant orf virus expression vectors using green fluorescent protein / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct a universal, highly attenuated orf virus expression vector for exogenous genes using green fluorescent protein (GFP) as the reporter gene.</p><p><b>METHODS</b>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 IA82Delta 121 by homologous recombination. The recombinant IA82Delta121-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.</p><p><b>RESULTS</b>The recombinant orf virus IA82Delta121-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.</p><p><b>CONCLUSION</b>Green fluorescent protein is a selectable marker for rapid, convenient and stable selection of the recombinant viruses. Highly attenuated recombinant orf virus IA82Delta121-V can serve as a new expression vector for exogenous genes.</p>