Abstract:

To provide a candidate strain for the development of an efficient and safe ORFV genetic engineering vaccine, the deleted ORF112 gene recombinant ORFV strain was constructed using the homologous recombination technique. Taking the ORF 112 gene as the targeted gene, the left and right homology arms and the enhanced green fluorescent protein gene (EGFP) expression frame were in-fused by the overlapping PCR method, which was connected with the pUC19T vector to construct the pUC19T-ORFV-ΔORF112-EGFP transfer vector. Subsequently, the pUC19T-ORFV-ΔORF112-EGFP transfer vector was transfected into the Vero cells to cause the homologous recombination events with the wild-type ORFV genome. The positive ORFV-ΔORF112-EGFP strain was screened and purified by limited dilution and picking a single-cell clone method. The genetic stability and replication ability of the deleted ORF112 ORFV recombinant strain were further investigated. The positive recombinant ORFV strain was purified using limited dilution and picking a single-cell clone method. After the PCR verification and sequencing identification, the recombinant ORFV-ΔORF112-EGFP strain was successfully obtained. The expression of EGFP in the recombinant virus genome was stable, with no attenuating characteristics during the serial passages. The replication and growth characteristics of this recombinant ORFV stain are the same as wild-type ORFV. The ORFV-ΔORF112-EGFP recombinant strain has been successfully constructed, purified, and screened, which maintains good genetic stability and replication ability, providing the candidate strain for the development of a genetic engineering ORFV vaccine.

Key words: orf virus, ORF112 gene, homologous recombination, gene deletion