In order to reconstruct the system for identification of short antigenic peptides, the chicken BF2 gene of Chinese Sanhuang (SH) chicken line was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich peptide, by splicing overlap extension PCR (SOE-PCR). The MBP-BF2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/E. coli TB1 system. The purified MBP-BF2-(G4S)3-beta(2)m protein was cleaved by Factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the BF2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD). In addition, the refolded BF2-(G4S)3-beta2m protein was used to bind three predicted nonameric peptides derived from the hemagglutinins of the avian influenza virus (AIV) H5N1 and H9N2 subtypes. The BF2-(G4S)3-beta2m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and tandem mass spectrometry analysis, respectively. The results indicate that the secondary structures and predicted three-dimensional crystal structure of BF2-(G4S)3-beta(2)m are similar to those of the monomers of chicken BF2 and beta(2)m. The BF2-(G4S)3-beta(2)m protein could bind two of the three predicted nonamer peptides derived from AIV hemagglutinin. The experimental system demonstrated that the reconstructed BF2-(G4S)3-beta(2)m protein complex can be used to identify nonamer peptides, including T-cell epitopes in chicken.