Identification of catalytically active domain epitopes in neuraminidase protein of H9N2 subtype of avian influenza virus

ABSTRACTH9N2 subtype of avian influenza virus (AIV) is primarily a bird virus, which is widespread in clinical avian disease, and reported in cases of human infection. As one of the surface proteins of AIV, the neuraminidase (NA) protein plays an important role mainly in viral budding. However, vaccine development and detection methods for NA of H9N2 AIVs are in urgent clinical need. In this study, a truncated NA gene (205–900 bp) was cloned from the NA sequence of H9N2 strain, and then expressed using pET-28a (+) vector. This purified recombinant NA protein was used to immunize BALB/c mice, and the monoclonal antibodies were screened through the indirect enzyme-linked immunosorbent assay (ELISA). Next, eight prokaryotic expression vectors were constructed for epitope identification. After cell fusion, three hybridoma cell lines producing the antibodies special to NA protein were screened by ELISA, western blotting, and indirect immunofluorescence; these were named 1B10, 2B6, and 5B2, respectively. Epitope scanning techniques were used to identify three B-cell epitopes recognized by these three monoclonal antibodies, 196KNATASIIYDGMLVD210, 210DSIGSWSKNIL220 and 221RTQESECVCI230. The subsequent homology analysis revealed the three epitopes were highly conserved in H9N2 AIV strains. The structural predictions of the antigenic epitopes indicated that all three epitopes were located in the catalytic region of NA. These results provide a basis for studying the function of the NA protein of H9N2 AIV and technical support for the development of a universal detection method based on anti-NA monoclonal antibodies.KEYWORDS: H9N2 avian influenza virusNA proteinmonoclonal antibodyantigen epitopecatalytically active domainepitope scanning technique Disclosure statementNo potential conflict of interest was reported by the authors.Author contributionsXF designed the experiments. XH, YC, GY, ZC, and FX carried out the experiments. XH, JH, ZG, and XG analysed the data. XH, FX, and XF provided constructive suggestions. XH and XF wrote the paper. XH and XF checked and finalized the manuscript. All authors read and approved the final manuscript.Additional informationFundingThis research was funded by the Key Laboratory for prevention and control of Avian Influenza and Other Major Poultry Diseases, Ministry of Agriculture and Rural Affairs of the People Republic of China [grant number: YDWS202211], National Natural Science Foundation of China [grant number: 31872458] and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funding sources did not influence the work.