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A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

Marek’s disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek’s disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cance...

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Autores principales: Teng, Man, Zhou, Zi-Yu, Yao, Yongxiu, Nair, Venugopal, Zhang, Gai-Ping, Luo, Jun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505574/
https://www.ncbi.nlm.nih.gov/pubmed/36146851
http://dx.doi.org/10.3390/v14092045
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author Teng, Man
Zhou, Zi-Yu
Yao, Yongxiu
Nair, Venugopal
Zhang, Gai-Ping
Luo, Jun
author_facet Teng, Man
Zhou, Zi-Yu
Yao, Yongxiu
Nair, Venugopal
Zhang, Gai-Ping
Luo, Jun
author_sort Teng, Man
collection PubMed
description Marek’s disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek’s disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant—GX0101Δpp38—and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas—namely, 4F9, 31G7, 34F2, and 35G9—were demonstrated to secrete specific antibodies against MDV-1’s pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography–tandem mass spectrometry (LC–MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses—especially for large DNA viruses such as herpesviruses.
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spelling pubmed-95055742022-09-24 A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology Teng, Man Zhou, Zi-Yu Yao, Yongxiu Nair, Venugopal Zhang, Gai-Ping Luo, Jun Viruses Article Marek’s disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek’s disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant—GX0101Δpp38—and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas—namely, 4F9, 31G7, 34F2, and 35G9—were demonstrated to secrete specific antibodies against MDV-1’s pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography–tandem mass spectrometry (LC–MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses—especially for large DNA viruses such as herpesviruses. MDPI 2022-09-14 /pmc/articles/PMC9505574/ /pubmed/36146851 http://dx.doi.org/10.3390/v14092045 Text en © 2022 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
Teng, Man
Zhou, Zi-Yu
Yao, Yongxiu
Nair, Venugopal
Zhang, Gai-Ping
Luo, Jun
A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology
title A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology
title_full A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology
title_fullStr A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology
title_full_unstemmed A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology
title_short A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology
title_sort new strategy for efficient screening and identification of monoclonal antibodies against oncogenic avian herpesvirus utilizing crispr/cas9-based gene-editing technology
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505574/
https://www.ncbi.nlm.nih.gov/pubmed/36146851
http://dx.doi.org/10.3390/v14092045
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