Expression of Newcastle disease virus hemagglutinin-neuraminidase (HN) gene in mammalian expression vector

Newcastle disease (ND) is a highly contagious economically important viral disease of avian species. ND virus (NDV) genome contains six genes which encode for six structural and two non-structural proteins. The glycoprotein HN present on viral envelope is responsible for attachment of virus to cellular receptor and is a major host protective antigen. In the present study, complete coding sequence of HN gene was amplified by RT-PCR using primers designed from published sequence. The PCR product was cloned in pGEMT easy vector (Promega, USA) and insert was confirmed by digestion with restriction enzyme NotI. The HN gene insert was subcloned at NotI site in pcDNA3.1(+) mammalian expression vector against cytomegalovirus enhancer promoter. The right orientation of HN gene insert in recombinant pcDNA was confirmed by digestion with EcoRI. Vero cells transfected with recombinant plasmid pcDNA.ndhn using both calcium phosphate and Lipofectin reagent demonstrated expression of 74 kDa HN protein at 48 h, 72 h and 96 h post transfection in SDS-PAGE which was confirmed by western blotting using anti NDV rabbit serum. Intramuscular immunization of rabbit with recombinant pcDNA.ndhn induced NDV specific antibody which had a titre of 1:32 in hemagglutination inhibition test indicating recombinant clone to be expressing HN protein upon inoculation in animals. Potential of this gene construct as DNA vaccine has been discussed.