Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China.
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, New York, NY 14850, USA.
Int J Mol Sci. 2023 May 12;24(10):8638. doi: 10.3390/ijms24108638.
() is the etiological pathogen of Glässer's disease, which causes high economic losses to the pig industry. The heme-binding protein A precursor (HbpA) was a putative virulence-associated factor proposed to be potential subunit vaccine candidate in In this study, three monoclonal antibodies (mAb) 5D11, 2H81, and 4F2 against rHbpA of SH0165 (serotype 5) were generated by fusing SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with the rHbpA. Indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) demonstrated that the antibody designated 5D11 showed a strong binding affinity with the HbpA protein and was chosen for subsequent experiments. The subtypes of the 5D11 were IgG1/κ chains. Western blot analysis showed that mAb 5D11 could react with all 15 serotype reference strains of . None of the other bacteria tested reacted with 5D11. In addition, a linear B-cell epitope recognized by 5D11 was identified by serial truncations of HbpA protein and then a series of truncated peptides were synthesized to define the minimal region that was required for mAb 5D11 binding. The 5D11 epitope was located on amino acids 324-LPQYEFNLEKAKALLA-339 by testing the 5D11 monoclonal for reactivity with 14 truncations. The minimal epitope 325-PQYEFNLEKAKALLA-339 (designated EP-5D11) was pinpointed by testing the mAb 5D11 for reactivity with a series of synthetic peptides of this region. The epitope was highly conserved among strains, confirmed by alignment analysis. These results indicated that mAb 5D11 and EP-5D11 might potentially be used to develop serological diagnostic tools for . Three-dimensional structural analysis revealed that amino acids of EP-5D11 were in close proximity and may be exposed on the surface of the HbpA protein.
（）是格氏染色病的病原体，给养猪业造成了巨大的经济损失。血红素结合蛋白 A 前体（HbpA）被认为是一种潜在的与毒力相关的因子，可能是 血清型 5 菌株 SH0165 的亚单位疫苗候选物。在本研究中，通过融合 SP2/0-Ag14 骨髓瘤细胞和用重组 HbpA 免疫的 BALB/c 小鼠脾细胞，产生了针对 rHbpA 的 3 株单克隆抗体（mAb）5D11、2H81 和 4F2。间接酶联免疫吸附试验（ELISA）和间接免疫荧光试验（IFA）表明，抗体 5D11 与 HbpA 蛋白具有很强的结合亲和力，并被选为后续实验。5D11 的亚型为 IgG1/κ 链。Western blot 分析表明，mAb 5D11 可与所有 15 种血清型参考菌株的 反应。没有其他测试的细菌与 5D11 反应。此外，通过 HbpA 蛋白的串联截断鉴定出 5D11 识别的线性 B 细胞表位，然后合成一系列截断肽以定义 mAb 5D11 结合所需的最小区域。通过测试 5D11 单克隆抗体与 14 个截断物的反应性，确定 5D11 表位位于 324-LPQYEFNLEKAKALLA-339 氨基酸上。通过测试 mAb 5D11 与该区域的一系列合成肽的反应性，确定最小表位 325-PQYEFNLEKAKALLA-339（命名为 EP-5D11）。通过序列比对分析证实，该表位在 菌株中高度保守。这些结果表明，mAb 5D11 和 EP-5D11 可能被用于开发针对 的血清学诊断工具。三维结构分析表明，EP-5D11 的氨基酸非常接近，可能暴露在 HbpA 蛋白的表面。
