Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Cellular and Molecular Biology, Faculty of Science Faculty, Azarbaijan Shahid Madani University, Tabriz, Iran.
BMC Infect Dis. 2021 Mar 24;21(1):300. doi: 10.1186/s12879-021-05969-0.
Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library.
The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot.
Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A.
The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.
铜绿假单胞菌是医院感染的主要原因，尤其是在免疫系统受损的人群中。通过中和抗体靶向毒力因子是治疗抗生素耐药性铜绿假单胞菌感染的一种新方法。在这方面，外毒素 A 是铜绿假单胞菌中最有效的毒力因子之一。本研究旨在从人源 scFv 噬菌体文库中鉴定一种针对铜绿假单胞菌外毒素 A 结构域 I（ExoA-DI）的新型人 scFv 抗体。
在大肠杆菌中表达重组铜绿假单胞菌 ExoA-DI，通过 Ni-NTA 柱纯化，并用于筛选人抗体噬菌体文库。采用新的筛选程序防止稀有特异性克隆被消除。通过 ELISA 和 Western blot 评估高反应性的噬菌体克隆。
基于多克隆噬菌体 ELISA 的结果，第五轮生物淘选导致分离出几个 ExoA-DI 反应性克隆。通过单克隆噬菌体 ELISA 筛选出一个具有高亲和力的阳性克隆，并用于抗体表达。纯化的 scFv 与重组结构域 I 和全长天然外毒素 A 具有高反应性。
纯化的抗外毒素 A scFv 对外毒素 A 具有高度特异性。本研究中鉴定的人源 scFv 可为开发新型治疗剂以控制铜绿假单胞菌感染奠定基础。
