Zhang Li, Miao Wei, Zhou Mo, Lin Miao, Fu Changyao, Wu Zhi, Lei Xinnuo, Xu Jialong, Cao Shinuo, Zhu Shanyuan
Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Engineering Technology Research Center for Modern Animal Science and Novel Veterinary Pharmaceutic Development, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China.
College of Veterinary Pharmacy, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China.
Vet Sci. 2024 Nov 1;11(11):533. doi: 10.3390/vetsci11110533.
Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that infect pigs' intestinal epithelial cells, causing high morbidity and mortality. Due to the rapid mutation of PEDV, vaccine efficacy is uncertain, prompting exploration of alternative treatments. Nanobodies, also known as variable heavy chain domains of heavy chain-only antibodies (VHHs), offer significant potential in biomedical applications due to their small size and high specificity. In this study, yeast two-hybrid technology was employed to screen for eight specific VHH sequences targeting the PEDV S protein from a synthetically constructed nanobody yeast library. The VHH genes were then cloned into expression plasmids for recombinant protein production, and the resulting VHHs (termed PEDV S-VHHs) were purified. Indirect immunofluorescence assay (IFA) and Western blotting analysis confirmed that these VHHs specifically bind to both PEDV and its S protein. Neutralization assays demonstrated that seven PEDV S-VHHs exhibited potent neutralizing activity against PEDV. Additionally, a combination of these seven antibodies showed enhanced antiviral effects. Preliminary predictions were also made regarding the binding sites between these VHHs and PEDV. The PEDV S-VHHs described in this study hold potential as candidates for the prevention and treatment of PEDV infection.
猪流行性腹泻病毒（PEDV）是一种高度传染性的冠状病毒，可感染猪的肠道上皮细胞，导致高发病率和高死亡率。由于PEDV的快速突变，疫苗效果不确定，促使人们探索替代治疗方法。纳米抗体，也称为仅重链抗体的可变重链结构域（VHHs），因其尺寸小和特异性高而在生物医学应用中具有巨大潜力。在本研究中，利用酵母双杂交技术从合成构建的纳米抗体酵母文库中筛选出八个靶向PEDV S蛋白的特异性VHH序列。然后将VHH基因克隆到表达质粒中以生产重组蛋白，并对所得的VHH（称为PEDV S-VHHs）进行纯化。间接免疫荧光试验（IFA）和蛋白质印迹分析证实，这些VHHs能特异性结合PEDV及其S蛋白。中和试验表明，七种PEDV S-VHHs对PEDV表现出强大的中和活性。此外，这七种抗体的组合显示出增强的抗病毒效果。还对这些VHHs与PEDV之间的结合位点进行了初步预测。本研究中描述的PEDV S-VHHs有望作为预防和治疗PEDV感染的候选物。
