Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu 225009, China.
Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China.
Vet Microbiol. 2024 Jun;293:110098. doi: 10.1016/j.vetmic.2024.110098. Epub 2024 Apr 25.
The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294-314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8-63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV.
犬冠状病毒（CCoV）的感染会导致犬急性肠胃炎的高度传染性疾病。有效的血清学诊断对于控制 CCoV 引起的疾病至关重要。CCoV 的核衣壳（N）蛋白是开发血清学方法的重要靶标。然而，对于 CCoV N 蛋白中的抗原表位知之甚少。在本研究中，我们通过融合用纯化的重组 GST-N 蛋白免疫的小鼠的脾细胞与 sp2/0 细胞，生成了针对 CCoV N 蛋白的单克隆抗体（mAb），命名为 13E8。表位作图显示，mAb 13E8 通过 Western blot 和 ELISA 识别 N 蛋白中 294-314aa 处的新型线性 B 细胞表位（命名为 EP-13E8）。序列分析表明，EP-13E8 的序列在分析的不同 CCoV 株中高度保守（100%），但与同属的其他冠状病毒如 FCoV、PEDV 和 HCoV 的相应序列的相似性较低（31.8-63.6%），除了 TGEV（95.5%的同一性）。结构分析表明，EP-13E8 的表位位于 N 蛋白表面的近距离位置。总之，针对 N 蛋白生成的 mAb 13E8 及其鉴定的表位 EP-13E8 为进一步开发基于表位的 CCoV 血清学诊断方法铺平了道路。
