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

Abstract

Background: 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.

Results: 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.

Conclusion: scFv-Fcs against S. aureus were successfully constructed and are good candidates for the development of future adjunctive therapy for severe S. aureus infections.

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

Fig. 1

Plasmid map of pLZ16. The vector was constructed based on the pUC vector and used for soluble single-chain variable fragment- fragment crystallizable region (scFv-Fc) production

Fig. 2

Amplification of V_H, V_κ, and V_λ gene repertoires. Lane M: DNA Marker II (Tiangen, CN). The expected size of the amplified V_H and V_L (including V_κ and V_λ) was ~350 bp. A part of amplified V_H, V_κ, and V_λ were shown in Fig. 2

Fig. 3

a Thirty clones were chosen randomly from the single-chain variable fragment (scFv) library and the positive clones inserted into full scFv genes were identified by polymerase chain reaction (PCR). Lane M: 2000 bp DNA Marker (TaKaRa, JP). Lanes 1–30: amplified scFvs from different clones randomly picked. b PCR products of scFvs were digested by BstN I. 2000 bp DNA Marker (TaKaRa, JP). Lane M: 2000 bp DNA Marker (TaKaRa, JP). Lanes 1–30: Fingerprints of scFvs digested by BstN I

Fig. 4

Amino acid sequences of 10 clones which were randomly picked from the library and analyzed by DNA sequence

Fig. 5

The specificity of selected single-chain variable fragment (scFvs) detected by enzyme-linked immunosorbent assay. Number 15 and 16 were negative controls

Fig. 6

a Constructed scFv-Fcs (S78, S128, S117, S182) were amplified and their sizes were ~1.5 kb. Lane M: 2000 bp DNA Marker (TaKaRa, JP). b Expression level of scFv-Fcs (S78, S128, S117, S182) expressed at 25 °C and 32 °C, respectively, were tested by enzyme-linked immunosorbent assay

Fig. 7

a The scFv-Fcs (S78, S117) expressed at 25 °C and 32 °C respectively were purified and run SDS-PAGE. Lane M: prestained protein ladder (Fermentas, USA); b The scFv-Fcs (S78, S128, S117, S182) were identified by western blot. Lane M: prestained protein ladder (Fermentas, USA)

Fig. 8

a Binding activity of scFv-Fcs (S78, S128, S117, S182) and S. aureus were detected by dot blot. The immunoreactions was developed by chemiluminscence (b). Detection of the specificity of scFv-Fcs (S78, S128, S117, S182) by enzyme-linked immunosorbent assay

Fig. 9

Analysis of the association and dissociation of S117 and S. aureus. The S117 was immobilized on AHC biosensor, the different concentration of S. aureus were tested. (run1: 1 × 10⁷ cfu/mL, run 2: 2 × 10⁷ cfu/mL, run 3: 4 × 10⁷ cfu/mL)