Immune Mechanism and Therapy of Major Diseases of Luzhou Key Laboratory, Public Center of Experimental Technology, School of Basic Medical Sciences, Southwest Medical University, Luzhou, China.
Clinical Pharmacy & GCP Center, the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, China.
APMIS. 2022 Sep;130(9):578-589. doi: 10.1111/apm.13258. Epub 2022 Jul 12.
Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to almost all β-lactam antibiotics. Hence, new ways to control MRSA infection, such as antibacterial antibodies, need to be explored. α-hemolysin is the most important virulence factor widely expressed in S. aureus. This study aimed to develop a new fully human antibody against α-hemolysin of S. aureus and research its neutralizing effect. The single-chain antibody fragments (scFvs) against S. aureus were screened from a fully human scFv library using phage display technology. The selected scFvs had good binding affinities to α-hemolysin and S. aureus. The IgG-like scFv-Fc inserted into the pcDNA3.1 or pMH3 vector was expressed in HEK293F suspension cells to extend the half-life and restore Fc function. The size of purified scFv-Fc was about 55 kDa. The functions of expressed scFv-Fcs against α-hemolysin were validated. The cytotoxicity assays showed that scFv555-Fc had better protective effects on A549 cells than other scFv-Fcs. The results of anti-rabbit erythrocyte lysis and A549 cell apoptosis assay confirmed that scFv555-Fc had a significant neutralizing effect on α-hemolysin. The scFv555-Fc was used to construct the docking model of antigen-antibody complexes using Discovery Studio software. It predicted that the key binding sites of α-hemolysin were TYR28, LYS37, PHE39, ARG56, and LYS58, which might be the key toxic sites of α-hemolysin. A novel fully human scFv-Fc antibody neutralizing the α-hemolysin toxin of S. aureus was successfully developed. The findings might provide a new theoretical basis and treatment method for preventing MRSA infection.
耐甲氧西林金黄色葡萄球菌（MRSA）几乎对所有β-内酰胺类抗生素都有耐药性。因此，需要探索控制 MRSA 感染的新方法，如抗菌抗体。α-溶血素是金黄色葡萄球菌广泛表达的最重要的毒力因子。本研究旨在开发一种针对金黄色葡萄球菌α-溶血素的新型全人源抗体，并研究其中和作用。使用噬菌体展示技术从全人源 scFv 文库中筛选针对金黄色葡萄球菌的单链抗体片段（scFv）。筛选出的 scFv 与α-溶血素和金黄色葡萄球菌具有良好的结合亲和力。将 IgG 样 scFv-Fc 插入 pcDNA3.1 或 pMH3 载体中，在 HEK293F 悬浮细胞中表达，以延长半衰期并恢复 Fc 功能。纯化的 scFv-Fc 的大小约为 55 kDa。验证了表达的 scFv-Fc 对 α-溶血素的功能。细胞毒性测定表明，scFv555-Fc 对 A549 细胞的保护作用优于其他 scFv-Fc。抗兔红细胞裂解和 A549 细胞凋亡试验的结果证实，scFv555-Fc 对 α-溶血素有显著的中和作用。使用 Discovery Studio 软件构建了抗原-抗体复合物的对接模型。预测 α-溶血素的关键结合位点为 TYR28、LYS37、PHE39、ARG56 和 LYS58，这可能是 α-溶血素的关键毒性位点。成功开发了一种新型全人源 scFv-Fc 抗体中和金黄色葡萄球菌的α-溶血素毒素。这些发现可能为预防 MRSA 感染提供新的理论依据和治疗方法。
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