Infectious bronchitis (IB) is highly contagious disease of great economic importance to the poultry industry. IB Virus has three major structural proteins spike (5) glycoprotein, membrane (M) glycoprotein and nucleocapsid (N) phosphoprotein. As an potent immunogen of coronavirus, N protein is thought to be an appropriate candidate for development of diagnostics and recombinant vaccine due to its highly conserved and immunogenic nature. Cloning and sequencing of N gene of local isolate of IBV has already been done in M.P., so in present study we assessed its immunogenicity with the aim to further develop recombinant vaccine by exploiting it. N gene was cloned in pQE30 vector and highlevel expression of recombinant N protein was achieved in E. coli. SDS-PAGE analysis showed 2 bands of 52 kDa and 45 kDa. Recombinant N protein was purified using Ni-NTA affinity chromatography. Purified recombinant N protein was used to immunize rabbits. Characterization of polyclonal antiserum raised against the purified recombinant N protein was done by DID, WB, dot blot assay and indirect N-ELISA (using recombinant N protein as coating antigen). Polyclonal antisera reacted against the purified recombinant N protein in all the assays. Significant difference in OD450 was found between preimmune and hyperimmune sera in N-ELISA, which indicated potential immunogenecity of recombinant N protein. Purified recombinant N protein also reacted with chicken anti IBV sera collected from vaccinated poultry flocks in WB, N ELISA and dot blot assays which indicated its antigenicity and potential to develop diagnostics in future.
IB virus, N-ELISA, Nucleocapsid protein
