Preparation Of The Monoclonal Antibody And Epitope Identification Of Infectious Bronchitis Virus N Protein

Avian infectious bronchitis (IB) caused by the avian infectious bronchitis virus (IBV), is an acute and highly contagious diseases which cause respiratory tract, genito-urinary system and digestive system damages in chickens. Since 1931, the disease was first reported by Schalk and Hawn, IBV have continually developed and more than 30 kinds of serotypes have emerged. Beacause the poor cross-protection of different serotypes, it brought great difficulties to the diagnosis and treatment of the IB, resulting in huge economic losses in poultry industry. IBV epitope studies by monoclonal antibody technology, not only help to understand the IBV antigenic structure in depth, but also facilitate to design scientific and rational epitope vaccines and diagnostic reagents. Therefore, it has an important significance for diagnosis and treatment of IB.In this study, BALB/c mice were immunized with IBV CK/CH/LDL/97Ⅰas antigen for the preparation of monoclonal antibodies (MAbs) of IBV N protein. By cell fusion technique, one hybridoma cell line designated as 6D10, which secreted anti-IBV N protein MAb, was obtained through Western blot and indirect ELISA assays. Subtype analysis showed that MAb 6D10 belonged to IgG1 and the light chain was theκchain. The MAb 6D10 has good specificity and could recognize IBV CK/CH/LDL/97Ⅰand IBV recombinant N protein specifically, which laid foundation for epitope location and research of function of IBV N protein.In order to identify MAb 6D10 corresponding epitope, the MAb 6D10 against IBV N protein was used as target molecular to screen for binding pepties from phage display random 12-peptide library and obtained a consensus sequence FGPRTK after three rounds of biopanning. The sequence was corresponded to the IBV CK/CH/LDL/97ⅠN protein 242FGPRTK247. The 6 peptides were expressed as GST fusion protein and were tested with MAb 6D10 by western blot and ELISA analysis. The fusion protein could be recognized specifically by MAb 6D10. The results showed that the 6 peptides—FGPRTK was a linear B cell epitope of IBV CK/CH/LDL/97ⅠN protein. To test the reactogenicity of the identified epitope, the fusion epitope protein was reacted with anti-IBV serum by ELISA and the fusion epitope protein has good reactivity with anti-IBV serum. Homology analysis showed that the epitope sequence FGPRTK was completely homologous to the corresponding regions of N protein of different serotype IBV strains tested.Thus, the epitope that we identified was a highly conserved linear B cell epitope.The results of this study will lay the foundation for further understanding of the relationship between IBV N protein antigen structure and function, and for the establishment of diagnostic methods.