An insertion plasmid called pKA4BPF was engineered by cloning an expression cassette composed of gB promoter, F protein gene and transcription termination factor into the commercially available plasmid vector pUC119 and used for the development of the recombinant virus.

The gB promoter was used with the aim of effectively expressing the NDV-F protein gene (see below) in the cells infected with MDV1. It is known that the homologous UL28 of HSV1 functions by incorporating the virus DNA into viral particles. However, it is not known how the protein encoded by UL28 functions in MDV1.

Homologous recombination was conducted by transferring the insertion vector plasmid pKA4BPF into a chicken embryo primary cell line infected with the MDV1 CVI988 C17 strain, the recipient organism virus, based on electroporation.

The gB (glycoprotein B) promoter region was cloned from the CVI988 C17 strain of the Gallid herpesvirus 2. It is a 0.5kb fragment amplified through PCR, with the EcoRI site added at each 5' end. The gB promoter sequence is configured mostly with the 3'-terminal of UL28 gene, containing 20% of its ORFs.

NDV-F gene derived from the avirulent Newcastle disease virus (NDV) D26 strain.

Transcription termination factor is a 0.25kb fragment derived from the commercially available expression plasmid pSVL. It contains the polyA addition signal and also the 3'-terminal sequence (77 bases) of large T antigen ORF of SV40 and the 3'-terminal sequence (61 bases) of VP1, the major virus capsid.