Generation Of Monoclonal Antibodies And Epitope Identification Of Infectious Bronchitis Virus M Protein

Avian infectious bronchitis (IB) , caused by infectious bronchitis virus(IBV), is an acute and highly contagious disease in chichens. Because of the high mutation frequency of IBV and little cross-protection between serologically distinct viruses , IB is still a major health problem affecting the chicken industry in most countries of the world. Antigenic epitope information of avian infectious bronchitis virus (IBV) is not only useful for investigating the relationship between antigenic structure and function of the IBV, but also significant for diagnosis of IBV infection and developing safe and effective multi-epitope vaccines against the disease.To begin with, reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the 681bp membrane (M) glycoprotein gene of avian infectious bronchitis virus and the gene was cloned into the pMD18-T vector for sequencing. By digestion of restriction enzymes BamHⅠand HindⅢ, the C-terminal 387bp fragments of M gene were subcloned into pET-30c to construct recombinant plasmid pET-30c-M . Then the recombinant plasmid was transformed into E.coli BL21(DE3) and induced with IPTG. It was demonstrated by SDS-PAGE that a protein of 20 kDa (M) was expressed in E.coli. The recombinant M protein was subsequently purified by affinity chromatography. Western blotting assay indicated that the chicken antiserum against IBV could recognize this protein. The results showed that the recombinant M protein possessed a good antigenicity and laid foundation for the preparation of monoclonal antibody (McAb) agaist IBV M protein and other functional research of M protein.Subsequently, SPF BALB/c mice were immunized subcutaneously(S/C)and intraperitoneally(I/P) with the recombinant M protein as antigen for the preparation of McAbs against M protein.Two McAbs against IBV M protein ,designated as 13A3 and 15E2, were finally identified by the indirect ELISA and Western blot assays using recombinant M protein and IBV as antigens. The results of subtype analysis showed that McAbs 13A3 and 15E2 all belonged to IgG1 and the light chains of all McAbs were theκchain. The two McAbs had good specificity and could specifically recognize both the recombinant M protein and IBV M protein,which were significant for the studies of both the function of M protein and epitope identification.In order to accurately study epitope structures of IBV M protein, 13 partially overlapping or consecutive peptides (MP3-1—MP12) spanning 387bp C-terminus of M gene were expressed for epitopes screening by Pepscan technology. One linear epitope of IBV M protein,199FATFVYAK206, was finally identified. The results of Western blot and homologous analysis indicated that the epitope was a highly conserved linear B-cell epitope of IBV M protein.To conclude, this study is not only significant for further understanding the epitope structure and immunological function of IBV M protein, but also useful for designing the diagnostic reagent and epitope vaccines.In addition, this study will lay foundation for the genetic and variant studies of the avian infectious bronchitis virus.