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
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)
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.