Identification of B-cell epitopes located on the surface in the PB2 protein of the H9N2 subtype avian influenza virus

ABSTRACT

Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was ⁴⁷⁵LRGVRVSK⁴⁸² and ⁵²⁸TITYSSPMMW⁵³⁷. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.

KEYWORDS:

• H9N2 avian influenza virus
• PB2 protein
• prokaryotic expression
• monoclonal antibody
• overlapping fragments
• PB2 antigenic determinants
• B-cell epitope

Ethical statement

The study was conducted in strict accordance with the recommendations in the Guidelines on “Ethical Treatment of Experimental Animals” (2006) No. 398 published by the Ministry of Science and Technology, China and the Regulation regarding the Management published by the Jiangsu Provincial People’s Government, and approved by Animal Ethics Committee at Nanjing Agricultural University (NJAU.No20211111167, 11 Nov. 2021).

Acknowledgements

Authors’ contributions: XF designed the experiments. YC, GY, XH, and JH carried out the experiments. YC, ZG, XG, YQ, and HS analysed the data. YC, GY, XH, and XF provided constructive suggestions. YC and XF wrote the paper. YC and XF checked and finalized the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was funded by 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.