Development and identification of fully human scFv-Fcs against Staphylococcus aureus. (December 2016)

• Record Type: Journal Article
• Authors: Nian, Siji; Wu, Tong; Ye, Yingchun; Wang, Xu; Xu, Wenfeng; Yuan, Qing
• Is Part Of: BMC immunology. Volume 17:Number 1(2016)
• Page Start: 1
• Page End: 9
• Publication Date: 2016-12
• Subjects: Staphylococcus aureus -- Single-chain variable fragment -- Fragment crystallizable regions -- scFv-Fc -- Phage display; Immunology -- Periodicals; Immune System -- Periodicals; Immunity -- Periodicals; Immune System Diseases -- Periodicals; Immunologic Techniques -- Periodicals; 616.07905
• DOI: 10.1186/s12865-016-0146-z
• Languages: English
• ISSNs: 1471-2172

Abstract

Abstract Background Staphylococcus aureus, a gram-positive pathogen, causes many human infections. Methicillin-resistantS. 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. Results A single-chain variable fragment (scFv) library was constructed using mRNA from the peripheral blood mononuclear cells ofS. aureus infected volunteers. After the scFv library DNA was transformed intoEscherichia coli TG1, ~1.7 × 107 independent clones with full-length scFv inserts. The scFv library was screened by phage display for three rounds usingS. 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 toS. aureus . To improve the stability of scFvs, scFv-fragment crystallizable regions (-Fcs) were constructed and expressed inE. 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 withS. aureus . Conclusion scFv-Fcs againstS. aureus were successfully constructed and are good candidates for the development of future adjunctive therapy for severeS. aureus infections.