Wang Xuan-Ang, Li Hong-Xuan, Zheng Lan-Lan, Ma Shi-Jie, Wang Ping-Li, Zhao Li, Chen Hong-Ying
Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, PR China.
Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, PR China; Key Laboratory for Animal-derived Food Safety of Henan Province, Zhengzhou 450000, PR China.
Vet Immunol Immunopathol. 2025 Jul;285:110951. doi: 10.1016/j.vetimm.2025.110951. Epub 2025 May 24.
Porcine epidemic diarrhea virus (PEDV) is a swine enteropathogenic coronavirus causing severe diarrhea and high mortality in neonatal piglets. Pigs of all ages are susceptible to PEDV, and the humoral immune response plays an important role in preventing PEDV infection. However, there is little information on monoclonal antibodies (mAbs) against PEDV derived from single B cells of pigs. In this study, we aimed to develop mAbs using antigen-specific single B cells from peripheral blood mononuclear cells (PBMCs) of pigs via fluorescence-activated cell sorting (FACS). Subsequently, the variable region genes of pig-derived mAbs were amplified and cloned into the plasmid pcDNA3.4 bearing the constant region gene of porcine-derived antibody. Pig-derived mAbs were expressed by transfecting the resultant antibody plasmids into HEK293F cells and validated using indirect Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and Western blotting. The results showed 60 double-positive (antigen and IgG) single B cells were obtained by flow sorting, of which 36 were positive for PEDV and 24 were positive for the N protein of PEDV. A total of 21 mAbs were expressed and purified. Indirect ELISA results showed that 20 bound specifically to PEDV, 19 recognized the N protein, and none reacted with S1D protein. Seven mAbs reacted with PEDV HN2021, as revealed by IFA. Western blotting showed that three N protein-specific mAbs identified linear epitopes, while the remaining 16 N protein-specific mAbs may recognize conformational epitopes. This study laid a foundation for the structural analysis of PEDV and the development of diagnostic reagents and antiviral drug.
猪流行性腹泻病毒（PEDV）是一种引起新生仔猪严重腹泻和高死亡率的猪肠道致病性冠状病毒。所有年龄段的猪都易感染PEDV，体液免疫反应在预防PEDV感染中起重要作用。然而，关于源自猪单个B细胞的抗PEDV单克隆抗体（mAb）的信息很少。在本研究中，我们旨在通过荧光激活细胞分选（FACS），利用猪外周血单核细胞（PBMC）中的抗原特异性单个B细胞来开发mAb。随后，扩增猪源mAb的可变区基因，并将其克隆到携带猪源抗体恒定区基因的质粒pcDNA3.4中。通过将所得抗体质粒转染到HEK293F细胞中表达猪源mAb，并使用间接酶联免疫吸附测定（ELISA）、间接免疫荧光测定（IFA）和蛋白质印迹法进行验证。结果显示，通过流式分选获得了60个双阳性（抗原和IgG）单个B细胞，其中36个对PEDV呈阳性，24个对PEDV的N蛋白呈阳性。共表达并纯化了21种mAb。间接ELISA结果表明，20种mAb与PEDV特异性结合，19种识别N蛋白，无一与S1D蛋白反应。IFA显示，7种mAb与PEDV HN2021反应。蛋白质印迹法表明，3种N蛋白特异性mAb识别线性表位，而其余16种N蛋白特异性mAb可能识别构象表位。本研究为PEDV的结构分析以及诊断试剂和抗病毒药物的开发奠定了基础。
