Exploration of the BF2*15 major histocompatibility complex class I binding motif and identification of cytotoxic T lymphocyte epitopes from the H5N1 influenza virus nucleoprotein in chickens.

The binding motif of BF2*15 major histocompatibility complex (MHC) class I was explored by analyzing the interaction between an infectious bronchitis virus octapeptide and BF2*15, and the cytotoxic T lymphocyte (CTL) epitope from the nucleoprotein (NP) of H5N1 virus was identified using experimental methods. Computational methods, including homology modeling, molecular dynamics simulation, and molecular docking analysis, were used. The recombinant plasmid pCAGGS-NP was constructed, and NP expression was confirmed by indirect immunofluorescence and Western blot in transfected 293T cells. Antibodies against NP in pCAGGS-NP-inoculated specific-pathogen-free chickens were detected by enzyme-linked immunosorbent assay (ELISA). Interferon γ (IFN-γ) mRNA was quantified, and IFN-γ production was evaluated using quantitative reverse transcription PCR and capture ELISA, respectively. CD8(+) T-lymphocyte proliferation was detected using flow cytometric analysis. The BF2*15 MHC class I binding motif "x-Arg/Lys-x-x-x-Arg/Lys" was explored. Quantification of chicken IFN-γ mRNA, evaluation of IFN-γ production, and measurement of CD8(+) T-lymphocyte proliferation confirmed that the peptide NP67-74 of H5N1 was the BF2*15 MHC-class-I-restricted CTL epitope.