Prokaryotic Expression Of The Nucleocapsid Protein Of Infectious Bronchitis Virus And The Analysis Of Its Antigenicity

Infectious bronchitis (Avian Infectious Bronchitis,IB) is an acute and highly contagious viral disease caused by avian infectious bronchitis virus (Avian Infectious Bronchitis Virus,IBV). As the characters of the major structural protein of IBV, the highly immunogenic property,the important immunogen of IBV humoral and cellular immune response and the highly conservative property,IBV nucleocapsid (N) protein is considered as a suitable antigen in detecting IBVspecific antibody.A pair of primers were designed and synthesized based on the sequence of the nucleocapsid(N)protein gene of Avian Infectious Bronchitis virus(IBV),and the N gene was amplified by RT-PCR. The fragment was cloned into pGEM-T vector and transformed into E. coli DH5αto get recombinant plasmid pGEM-T-N. After identification by digesting with EcoRⅠand XhoⅠenzyme, the cloned fragment was subcloned into prokaryotic expression vector pET-28a(+). Recombined expression plasmid pET-28a-N was transformed into BL21(DE3) and then induced with IPTG. N protein was purified by affinity chromatography taking advantage of the affinity of Ni2+ for His tag protein. It was demonstrated by SDS-PAGE and Western blotting that the fusion protein which was 50 kDa,was expressed in E. coli BL21(DE3) successfully.The main pattern of the expressive target protein in the cell is soluble.The Western blotting result showed that the protein could recognize the positive serum against IBV,indicating that the expressed product had highly immunogenic property.