State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin, 150069, China; Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Xixiangtang District, Youai North Road 51, Nanning, 530001, China.
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin, 150069, China.
Virus Res. 2022 May;313:198742. doi: 10.1016/j.virusres.2022.198742. Epub 2022 Mar 11.
Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric coronavirus that causes vomiting, severe diarrhea, dehydration and death in suckling piglets. NS7a is putative accessory protein that is predicted to be encoded by SADS-CoV, but still to be confirmed experimentally. In the present study, recombinant NS7a protein was expressed in a prokaryotic expression system and used as an antigen to prepare monoclonal antibodies (mAbs) specific to NS7a protein. We obtained two anti-NS7a mAbs, termed AH5 and EH3, that were shown by western blotting to react with the natural NS7a protein in Vero E6 cells infected with SADS-CoV. Using the produced mAbs, we observed by confocal microscopy that NS7a protein was expressed in the cytoplasm. Further studies revealed that the motif VNTWQEFA was the minimal unit of the linear B-cell epitope recognized by mAb AH5, and the motif FDLFERF was the minimal unit of the linear B-cell epitope recognized by mAb EH3. Alignment of amino acids showed that these two epitopes were highly conserved among different SADS-CoV strains and SADS-related coronaviruses from bats, but with one substitution in these two motifs in bat coronavirus HKU2. In summary, we generated and characterized two mAbs against SADS-CoV NS7a protein, and demonstrated NS7a expression in SADS-CoV-infected cells for the first time.
猪急性腹泻综合征冠状病毒（SADS-CoV）是一种新兴的猪肠道冠状病毒，可引起仔猪呕吐、严重腹泻、脱水和死亡。NS7a 是假定的辅助蛋白，据预测由 SADS-CoV 编码，但仍需实验证实。在本研究中，重组 NS7a 蛋白在原核表达系统中表达，并用作抗原制备针对 NS7a 蛋白的单克隆抗体（mAb）。我们获得了两种抗 NS7a mAb，分别称为 AH5 和 EH3，Western blot 结果表明它们与 SADS-CoV 感染的 Vero E6 细胞中的天然 NS7a 蛋白反应。使用产生的 mAb，我们通过共聚焦显微镜观察到 NS7a 蛋白在细胞质中表达。进一步的研究表明， motif VNTWQEFA 是 mAb AH5 识别的线性 B 细胞表位的最小单位，而 motif FDLFERF 是 mAb EH3 识别的线性 B 细胞表位的最小单位。氨基酸序列比对表明，这两个表位在不同的 SADS-CoV 株和来自蝙蝠的 SADS 相关冠状病毒中高度保守，但在蝙蝠冠状病毒 HKU2 中这两个基序中有一个取代。总之，我们生成并鉴定了针对 SADS-CoV NS7a 蛋白的两种 mAb，并首次证明了 SADS-CoV 感染细胞中 NS7a 的表达。
