Cloning and expression of fusion gene of Newcastle disease virus in a eukaryotic expression system

Newcastle disease is highly contagious devastating viral disease, which affects most of avian species worldwide. The Newcastle disease virus contains six structural and two non-structural proteins. The structural proteins hemagglutinin-neuraminidase (HN) and fusion (F) are responsible for inducing protective antibodies in birds. In the present study, complete coding region of F gene encoding 550 amino acid long fusion protein was amplified by RT-PCR and cloned in mammalian expression vector, pcDNA 3.1(+) at KpnI and ApaI site. The insert in recombinant clone was confirmed by digestion with same restriction enzymes. The orientation of insert was verified by digestion with NotI and EcoRI enzymes which resulted in release of a DNA fragment of approximately 900 bp, as seen in 1% agarose gel. The recombinant plasmid containing F gene in correct orientation (pcDNA.nd.f.lasota) was purified by QIAquick midi columns and used to transfect Vero cells for in vitro expression. The transfected Vero cell lysate showed a fusion protein band of approx 58.8 kDa at 48 h and 72 h post tranfection in 10% polyacrylamide gel in SDS-PAGE, which was not present in mock transfected cells. This protein reacted with anti NDV rabbit serum in western blot experiment confirming it to be NDV specific. Use of this clone as DNA vaccine against ND has been discussed.