Cargando…
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...
Autores principales: | , , , , , |
---|---|
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 |
_version_ | 1784796506537066496 |
---|---|
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. |
format | Online Article Text |
id | pubmed-9505574 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT tengman anewstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT zhouziyu anewstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT yaoyongxiu anewstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT nairvenugopal anewstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT zhanggaiping anewstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT luojun anewstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT tengman newstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT zhouziyu newstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT yaoyongxiu newstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT nairvenugopal newstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT zhanggaiping newstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology AT luojun newstrategyforefficientscreeningandidentificationofmonoclonalantibodiesagainstoncogenicavianherpesvirusutilizingcrisprcas9basedgeneeditingtechnology |