Development and identification of fully human scFv-Fcs against Staphylococcus aureus

BMC Immunology

Research Article

Wenfeng Xu¹  Yingchun Ye¹  Xu Wang¹  Qing Yuan¹  Tong Wu¹  Siji Nian¹ 

[1] The School of Basic Medical Sciences, Sichuan medical university, Room 218, Hanguang building, No 319, Zhongshan road, 646000, Luzhou, Sichuan, China;

关键词: Staphylococcus aureus;    Single-chain variable fragment;    Fragment crystallizable regions;    scFv-Fc;    Phage display;   

DOI  :  10.1186/s12865-016-0146-z

 received in 2015-01-02, accepted in 2016-04-19,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundStaphylococcus 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.ResultsA 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 × 107 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.ConclusionscFv-Fcs against S. aureus were successfully constructed and are good candidates for the development of future adjunctive therapy for severe S. aureus infections.

【 授权许可】

CC BY   
© Nian et al. 2016