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

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 t...

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Autores principales: Liu, Geyan, Wang, Kang, Yang, Zhen, Tang, Xiaoyu, Chang, Yungfu, Dai, Ke, Tang, Xinwei, Hu, Bangdi, Zhang, Yiwen, Cao, Sanjie, Huang, Xiaobo, Yan, Qigui, Wu, Rui, Zhao, Qin, Du, Senyan, Wen, Xintian, Wen, Yiping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10218323/
https://www.ncbi.nlm.nih.gov/pubmed/37239984
http://dx.doi.org/10.3390/ijms24108638
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author Liu, Geyan
Wang, Kang
Yang, Zhen
Tang, Xiaoyu
Chang, Yungfu
Dai, Ke
Tang, Xinwei
Hu, Bangdi
Zhang, Yiwen
Cao, Sanjie
Huang, Xiaobo
Yan, Qigui
Wu, Rui
Zhao, Qin
Du, Senyan
Wen, Xintian
Wen, Yiping
author_facet Liu, Geyan
Wang, Kang
Yang, Zhen
Tang, Xiaoyu
Chang, Yungfu
Dai, Ke
Tang, Xinwei
Hu, Bangdi
Zhang, Yiwen
Cao, Sanjie
Huang, Xiaobo
Yan, Qigui
Wu, Rui
Zhao, Qin
Du, Senyan
Wen, Xintian
Wen, Yiping
author_sort Liu, Geyan
collection PubMed
description 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.
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spelling pubmed-102183232023-05-27 Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody Liu, Geyan Wang, Kang Yang, Zhen Tang, Xiaoyu Chang, Yungfu Dai, Ke Tang, Xinwei Hu, Bangdi Zhang, Yiwen Cao, Sanjie Huang, Xiaobo Yan, Qigui Wu, Rui Zhao, Qin Du, Senyan Wen, Xintian Wen, Yiping Int J Mol Sci Article 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. MDPI 2023-05-12 /pmc/articles/PMC10218323/ /pubmed/37239984 http://dx.doi.org/10.3390/ijms24108638 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Liu, Geyan
Wang, Kang
Yang, Zhen
Tang, Xiaoyu
Chang, Yungfu
Dai, Ke
Tang, Xinwei
Hu, Bangdi
Zhang, Yiwen
Cao, Sanjie
Huang, Xiaobo
Yan, Qigui
Wu, Rui
Zhao, Qin
Du, Senyan
Wen, Xintian
Wen, Yiping
Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody
title Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody
title_full Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody
title_fullStr Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody
title_full_unstemmed Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody
title_short Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody
title_sort identification of a novel linear b-cell epitope of hbpa protein from glaesserella parasuis using monoclonal antibody
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10218323/
https://www.ncbi.nlm.nih.gov/pubmed/37239984
http://dx.doi.org/10.3390/ijms24108638
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