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© 2023 by the authors. 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/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

Marek’s disease (MD) caused by pathogenic Marek’s disease virus type 1 (MDV−1) is one of the most important neoplastic diseases of poultry. MDV−1-encoded unique Meq protein is the major oncoprotein and the availability of Meq-specific monoclonal antibodies (mAbs) is crucial for revealing MDV pathogenesis/oncogenesis. Using synthesized polypeptides from conserved hydrophilic regions of the Meq protein as immunogens, together with hybridoma technology and primary screening by cross immunofluorescence assay (IFA) on Meq-deleted MDV−1 viruses generated by CRISPR/Cas9-gene editing, a total of five positive hybridomas were generated. Four of these hybridomas, namely 2A9, 5A7, 7F9 and 8G11, were further confirmed to secrete specific antibodies against Meq as confirmed by the IFA staining of 293T cells overexpressing Meq. Confocal microscopic analysis of cells stained with these antibodies confirmed the nuclear localization of Meq in MDV-infected CEF cells and MDV-transformed MSB-1 cells. Furthermore, two mAb hybridoma clones, 2A9-B12 and 8G11-B2 derived from 2A9 and 8G11, respectively, displayed high specificity for Meq proteins of MDV−1 strains with diverse virulence. Our data presented here, using synthesized polypeptide immunization combined with cross IFA staining on CRISPR/Cas9 gene-edited viruses, has provided a new efficient approach for future generation of specific mAbs against viral proteins.

Details

Title
Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek’s Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses
Author
Teng, Man 1   VIAFID ORCID Logo  ; Jin-Ling, Liu 2 ; Luo, Qin 3 ; Lu-Ping, Zheng 2 ; Yao, Yongxiu 4   VIAFID ORCID Logo  ; Nair, Venugopal 4   VIAFID ORCID Logo  ; Gai-Ping Zhang 5 ; Luo, Jun 1   VIAFID ORCID Logo 

 Key Laboratory of Animal Immunology, Ministry of Agriculture and Rural Affairs of China and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China 
 Key Laboratory of Animal Immunology, Ministry of Agriculture and Rural Affairs of China and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China 
 Key Laboratory of Animal Immunology, Ministry of Agriculture and Rural Affairs of China and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; College of Veterinary Medicine, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China 
 The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford GU24 0NF, UK 
 International Joint Research Center of National Animal Immunology and College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou University, Yangzhou 225009, China 
First page
817
Publication year
2023
Publication date
2023
Publisher
MDPI AG
e-ISSN
19994915
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2806631943
Copyright
© 2023 by the authors. 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/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.