Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody

Author:

Liu Geyan1,Wang Kang1,Yang Zhen1,Tang Xiaoyu1,Chang Yung-Fu2ORCID,Dai Ke1,Tang Xinwei1,Hu Bangdi1,Zhang Yiwen1,Cao Sanjie1ORCID,Huang Xiaobo1,Yan Qigui1,Wu Rui1ORCID,Zhao Qin1,Du Senyan1,Wen Xintian1,Wen Yiping1

Affiliation:

1. Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China

2. Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, New York, NY 14850, USA

Abstract

Glaesserella parasuis (G. parasuis.) is the etiological pathogen of Glässer’s disease, which causes high economic losses to the pig industry. The heme-binding protein A precursor (HbpA) was a putative virulence-associated factor proposed to be potential subunit vaccine candidate in G. parasuis. In this study, three monoclonal antibodies (mAb) 5D11, 2H81, and 4F2 against recombinant HbpA (rHbpA) of G. parasuis SH0165 (serotype 5) were generated by fusing SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with the rHbpA. Indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) demonstrated that the antibody designated 5D11 showed a strong binding affinity with the HbpA protein and was chosen for subsequent experiments. The subtypes of the 5D11 were IgG1/κ chains. Western blot analysis showed that mAb 5D11 could react with all 15 serotype reference strains of G. parasuis. None of the other bacteria tested reacted with 5D11. In addition, a linear B-cell epitope recognized by 5D11 was identified by serial truncations of HbpA protein and then a series of truncated peptides were synthesized to define the minimal region that was required for mAb 5D11 binding. The 5D11 epitope was located on amino acids 324-LPQYEFNLEKAKALLA-339 by testing the 5D11 monoclonal for reactivity with 14 truncations. The minimal epitope 325-PQYEFNLEKAKALLA-339 (designated EP-5D11) was pinpointed by testing the mAb 5D11 for reactivity with a series of synthetic peptides of this region. The epitope was highly conserved among G. parasuis strains, confirmed by alignment analysis. These results indicated that mAb 5D11 and EP-5D11 might potentially be used to develop serological diagnostic tools for G. parasuis. Three-dimensional structural analysis revealed that amino acids of EP-5D11 were in close proximity and may be exposed on the surface of the HbpA protein.

Funder

Key R&D support plan of Chengdu Science and Technology Bureau

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

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