State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.
Virol J. 2018 Aug 30;15(1):133. doi: 10.1186/s12985-018-1042-3.
Porcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry. Interaction of the viral spike (S) surface glycoprotein with the host cell receptor is recognized as the first step of infection and is the main determinant of virus tropism. The mechanisms by which neutralizing antibodies inhibit PEDV have not been defined. Isolating PEDV neutralizing antibodies are crucial to identifying the receptor-binding domains of the viral spike and elucidating the mechanism of protection against PEDV infection.
B cell hybridoma technique was used to generate hybridoma cells that secrete specific antibodies. E.coli prokaryotic expression system and Bac-to-Bac expression system were used to identify the target protein of each monoclonal antibody. qPCR was performed to analyze PEDV binding to Vero E6 cells with neutralizing antibody.
We identified 10 monoclonal antibodies using hybridoma technology. Remarkably, 4 mAbs (designed 2G8, 2B11, 3D9, 1E3) neutralized virus infection potently, of which 2B11 and 1E3 targeted the conformational epitope of the PEDV S protein. qPCR results showed that both 2B11 and 2G8 blocked virus entry into Vero cells.
The data suggested that PEDV neutralizing antibody inhibited virus infection by binding to infectious virions, which could work as a tool to find the receptor-binding domains.
猪流行性腹泻病毒（PEDV）是一种新兴的致病性冠状病毒，给养猪业带来了巨大的经济负担。病毒刺突（S）表面糖蛋白与宿主细胞受体的相互作用被认为是感染的第一步，也是病毒嗜性的主要决定因素。中和抗体抑制 PEDV 的机制尚未确定。分离 PEDV 中和抗体对于鉴定病毒刺突的受体结合域以及阐明针对 PEDV 感染的保护机制至关重要。
采用杂交瘤技术产生分泌特异性抗体的杂交瘤细胞。使用大肠杆菌原核表达系统和 Bac-to-Bac 表达系统鉴定每个单克隆抗体的靶蛋白。用 qPCR 分析中和抗体与 Vero E6 细胞结合的 PEDV。
我们使用杂交瘤技术鉴定了 10 种单克隆抗体。值得注意的是，4 种 mAb（设计为 2G8、2B11、3D9、1E3）能够有效中和病毒感染，其中 2B11 和 1E3 针对 PEDV S 蛋白的构象表位。qPCR 结果表明，2B11 和 2G8 均可阻止病毒进入 Vero 细胞。
数据表明，PEDV 中和抗体通过与感染性病毒颗粒结合来抑制病毒感染，可作为寻找受体结合域的工具。
