Two genes originating from ILTV, namely UL32 and gB, were introduced into a FPV vector. The genes were ligated into a pUC18 transformation vector in addition to a lacZ marker gene from E. coli, using two synthetic promoters and an FPV homologous recombination site.

The vector was constructed by cloning a 3 kb fragment of FPV genomic DNA into the pUC18 vector. An internal fragment of 175bp was removed and replaced by a construct containing the UL32 coding sequenece with a synthetic PS promoter, followed by a lacZ gene directed by the synthetic promoter P17. This was followed by the coding sequence of the gB gene which was also directed by the synthetic promoter PS.

The synthetic promoters PS and P17 emulate the consensus early/late promoter and the early poxvirus promoter, respectively. Genes gB and UL32 code for the ILTV antigens, while the gene lacZ operates as a reporter gene to facilitate recombinant virus selection.

This construct and the the parental FPV strain were co-transformed into chicken embryo fibroblasts and incubated under conditions that favour homologous recombination.

Homologous recombination and integration of the genetic construct was confirmed by Southern blot using probes that hybridized in both sides of the insertion site. DNA sequencing was also conducted. Expression of genes UL32 and gB was confirmed by Western blot.