Nian Siji, Wu Tong, Ye Yingchun, Wang Xu, Xu Wenfeng, Yuan Qing
The School of Basic Medical Sciences, Sichuan medical university, Room 218, Hanguang building, No 319, Zhongshan road, Luzhou, Sichuan, 646000, China.
BMC Immunol. 2016 Apr 29;17(1):8. doi: 10.1186/s12865-016-0146-z.
Staphylococcus aureus, a gram-positive pathogen, causes many human infections. Methicillin-resistant S. aureus (MRSA) is the most common drug-resistance bacteria. Nearly all MRSA bacteria are resistant to several drugs. Specific antibodies are the main components of the host's humoral immunity, and play a significant role in the process of the host's resistance to bacterial infection.
A single-chain variable fragment (scFv) library was constructed using mRNA from the peripheral blood mononuclear cells of S. aureus infected volunteers. After the scFv library DNA was transformed into Escherichia coli TG1, ~1.7 × 10(7) independent clones with full-length scFv inserts. The scFv library was screened by phage display for three rounds using S. aureus as an antigen. The single clones were chosen at random and the scFvs were expressed for enzyme-linked immunosorbent assay (ELISA) assessment. Approximately 50 % of the clones were positive with good binding activity to S. aureus. To improve the stability of scFvs, scFv-fragment crystallizable regions (-Fcs) were constructed and expressed in E. coli DH5α. The expressed scFv-Fcs were purified and identified by western blot. These antibodies were further characterized and analyzed for bioactivity. The results showed that the expression level and folding of scFv-Fcs induced at 25 °C without isopropyl β-D-1-thiogalactopyranoside (IPTG) were higher than that induced at 32 °C with 1.0 mmol/L IPTG. scFv-Fcs had good bioactivity and could specifically bind with S. aureus.
scFv-Fcs against S. aureus were successfully constructed and are good candidates for the development of future adjunctive therapy for severe S. aureus infections.
金黄色葡萄球菌是一种革兰氏阳性病原体，可引发多种人类感染。耐甲氧西林金黄色葡萄球菌（MRSA）是最常见的耐药菌。几乎所有的MRSA菌株都对多种药物耐药。特异性抗体是宿主体液免疫的主要组成部分，在宿主抵抗细菌感染的过程中发挥着重要作用。
利用金黄色葡萄球菌感染志愿者外周血单个核细胞的mRNA构建了单链可变片段（scFv）文库。将scFv文库DNA转化到大肠杆菌TG1中后，获得了约1.7×10⁷个带有全长scFv插入片段的独立克隆。以金黄色葡萄球菌为抗原，通过噬菌体展示对scFv文库进行三轮筛选。随机挑选单克隆并表达scFv用于酶联免疫吸附测定（ELISA）评估。约50%的克隆呈阳性，对金黄色葡萄球菌具有良好的结合活性。为提高scFv的稳定性，构建了scFv-可结晶片段（-Fc）并在大肠杆菌DH5α中表达。对表达的scFv-Fc进行纯化并通过蛋白质免疫印迹法进行鉴定。对这些抗体的生物活性进行了进一步表征和分析。结果表明，在25°C无异丙基-β-D-1-硫代半乳糖苷（IPTG）诱导条件下scFv-Fc的表达水平和折叠情况高于在32°C 1.0 mmol/L IPTG诱导条件下的情况。scFv-Fc具有良好的生物活性，能够与金黄色葡萄球菌特异性结合。
成功构建了抗金黄色葡萄球菌的scFv-Fc，它们是未来开发严重金黄色葡萄球菌感染辅助治疗药物的良好候选物。
