Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences, Beijing, People's Republic of China, 100097.
College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, Jiangxi, People's Republic of China, 330045.
Appl Microbiol Biotechnol. 2017 Dec;101(23-24):8331-8344. doi: 10.1007/s00253-017-8566-0. Epub 2017 Oct 23.
Glycoprotein D (gD) of bovine herpesvirus-1 (BoHV-1) is essential for attachment and penetration of cells during infection and is a major target for neutralizing antibodies during an adaptive immune response. Currently there are no recombinant antibodies capable of binding gD epitopes for use in treating BoHV-1 infection. In this study, a bovine scFv gene derived from a hybridoma secreting monoclonal antibodies (McAbs) against the amino acid motif MEESKGYEPP of gD was expressed in E. coli. Molecular modeling, western blot and ELISA analysis showed that this scFv had a high affinity for BoHV-1 gD, with a Kd of 161.2 ± 37.58 nM and for whole BoHV-1 virus, with a Kd of 67.44 ± 16.99 nM. In addition, this scFv displayed a high affinity for BoHV-1 antigen in an ELISA and competed with BoHV-1 anti-serum in a competitive ELISA. Immunofluorescence assay (IFA) and laser confocal microscopy showed that this scFv could efficiently bind to and be internalized by BoHV-1 infected Madin-Darby bovine kidney (MDBK) cells. Importantly, this scFv was shown to inhibit BoHV-1 infectivity and to reduce the number of viral plaques by blocking viral attachment to MDBK cells. Our study suggests that this bovine single-chain antibody could be developed for use as a diagnostic and therapeutic agent against BoHV-1 infection in cattle.
牛疱疹病毒 1 型（BoHV-1）的糖蛋白 D（gD）对于感染期间的细胞附着和穿透是必不可少的，并且是适应性免疫反应期间中和抗体的主要靶标。目前，尚无能够结合 gD 表位用于治疗 BoHV-1 感染的重组抗体。在这项研究中，从分泌针对 gD 的氨基酸基序 MEESKGYEPP 的单克隆抗体（McAbs）的杂交瘤中表达了牛 scFv 基因。分子建模、western blot 和 ELISA 分析表明，该 scFv 对 BoHV-1 gD 具有高亲和力，Kd 为 161.2 ± 37.58 nM，对整个 BoHV-1 病毒的 Kd 为 67.44 ± 16.99 nM。此外，该 scFv 在 ELISA 中显示出对 BoHV-1 抗原的高亲和力，并在竞争 ELISA 中与 BoHV-1 抗血清竞争。免疫荧光分析（IFA）和激光共聚焦显微镜显示，该 scFv 可以有效地结合和内化感染 BoHV-1 的 Madin-Darby 牛肾（MDBK）细胞。重要的是，该 scFv 被证明可以通过阻止病毒附着到 MDBK 细胞来抑制 BoHV-1 的感染性并减少病毒斑的数量。我们的研究表明，这种牛单链抗体可开发用于预防和治疗牛的 BoHV-1 感染。
