Laboratory of Molecular Virology and Immunology, College of Veterinary Medicine, Hebei Agricultural University, Hebei Veterinary Biotechnology Innovation Center, Baoding, 071000, China; Rinpu (Baoding) Biological Pharmaceutical Co., LTD, Baoding, 071004, China.
Laboratory of Molecular Virology and Immunology, College of Veterinary Medicine, Hebei Agricultural University, Hebei Veterinary Biotechnology Innovation Center, Baoding, 071000, China.
Protein Expr Purif. 2020 Oct;174:105682. doi: 10.1016/j.pep.2020.105682. Epub 2020 Jun 2.
Canine parvovirus (CPV) non-structural protein-1 (NS1) plays crucial roles in CPV replication and transcription, as well as pathogenic effects to the host. However, the mechanism was not fully understood. Lack of NS1 antibody is one of the restricting factors for NS1 function investigation. To prepare NS1 monoclonal antibody (mAb), the NS1 epitope (AA461 ~ AA650) gene was amplified by PCR, and inserted into pGEX-4T-1vector to construct the prokaryotic expression vector of GST-tag-fused NS1 epitope gene. The NS1 fusion protein was expressed in E. coli, and purified with GSH-magnetic beads, and then used to immunize BALB/c mice. The mouse splenic lymphocytes were isolated and fused with myeloma cells (SP 2/0) to generate hybridoma cells. After several rounds of screening by ELISA, a hybridoma cell clone (1B8) stably expressing NS1 mAb was developed. A large amount of NS1 mAb was prepared from mouse ascites fluid. The isotype of NS1 mAb was identified as IgG1, which can specifically bind NS1 protein in either CPV-infected cells or NS1 vector-transfected cells, indicating the NS1 mAb is effective in detecting NS1 protein. Meanwhile, we used the NS1 mAb to investigate NS1 dynamic changes by qRT-PCR and location by confocal imaging in CPV-infected host cells and showed that NS1 began to appear in the cells at 12 h after CPV infection and reached the highest level at 42 h, NS1 protein was mainly located in nucleus of the cells. This study provided a necessary condition for further investigation on molecular mechanism of NS1 function and pathogenicity.
犬细小病毒 (CPV) 非结构蛋白-1 (NS1) 在 CPV 复制和转录以及对宿主的致病作用中发挥着关键作用。然而，其机制尚未完全阐明。缺乏 NS1 抗体是 NS1 功能研究的限制因素之一。为了制备 NS1 单克隆抗体 (mAb)，通过 PCR 扩增 NS1 表位 (AA461 ～ AA650) 基因，插入 pGEX-4T-1 载体构建 GST 标签融合 NS1 表位基因的原核表达载体。在大肠杆菌中表达 NS1 融合蛋白，并用 GSH-磁珠纯化，然后用于免疫 BALB/c 小鼠。分离小鼠脾淋巴细胞并与骨髓瘤细胞 (SP 2/0) 融合，以产生杂交瘤细胞。通过 ELISA 进行几轮筛选后，开发出稳定表达 NS1 mAb 的杂交瘤细胞克隆 (1B8)。从鼠腹水大量制备 NS1 mAb。NS1 mAb 的同种型鉴定为 IgG1，可特异性结合 CPV 感染细胞或 NS1 载体转染细胞中的 NS1 蛋白，表明 NS1 mAb 可有效检测 NS1 蛋白。同时，我们使用 NS1 mAb 通过 qRT-PCR 研究 CPV 感染宿主细胞中 NS1 的动态变化和通过共聚焦成像定位，并显示 NS1 在 CPV 感染后 12 小时开始出现在细胞中，在 42 小时达到最高水平，NS1 蛋白主要位于细胞核中。本研究为进一步研究 NS1 功能和致病性的分子机制提供了必要条件。
