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Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek's Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses

文献类型： 外文期刊

作者： Teng, Man 1 ; Liu, Jin-Ling 1 ; Luo, Qin 1 ; Zheng, Lu-Ping 1 ; Yao, Yongxiu 6 ; Nair, Venugopal 6 ; Zhang, Gai-Ping 8 ; Luo, Jun 1 ;

作者机构： 1.Henan Acad Agr Sci, Minist Agr & Rural Affairs China, Key Lab Anim Immunol, Zhengzhou 450002, Peoples R China

2.Henan Acad Agr Sci, Henan Prov Key Lab Anim Immunol, Zhengzhou 450002, Peoples R China

3.Henan Acad Agr Sci, China Ctr Excellence Res Avian Dis, Zhengzhou 450002, Peoples R China

4.Henan Univ Sci & Technol, Coll Anim Sci & Technol, Luoyang 471003, Peoples R China

5.Henan Univ Anim Husb & Econ, Coll Vet Med, Zhengzhou 450046, Peoples R China

6.Pirbright Inst, Ash Rd, Guildford GU24 0NF, England

7.UK China Ctr Excellence Res Avian Dis, Ash Rd, Guildford GU24 0NF, England

8.Henan Agr Univ, Coll Vet Med, Int Joint Res Ctr Natl Anim Immunol, Zhengzhou 450002, Peoples R China

9.Yangzhou Univ, Jiangsu Coinnovat Ctr Prevent & Control Important, Yangzhou 225009, Peoples R China

关键词： herpesvirus; MDV; Meq; CRISPR; Cas9; gene editing; monoclonal antibody

期刊名称：VIRUSES-BASEL （ 影响因子：4.7； 五年影响因子：4.8 ）

ISSN：

年卷期： 2023 年 15 卷 4 期

页码：

收录情况： SCI

摘要： 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.

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