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

Abstract

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.

Development and identification of a fully human single‐chain variable fragment 29 against TSLP

“…The fully human scFv library was constructed by referring to our previous reports . Eighty microliters of frozen cell suspensions (scFv library) under glycerol were incubated in 40 mL of LB, containing 100 µg/mL of ampicillin and 2.0% glucose, and shaken at 37°C until the optical density at a wavelength of 600 nm (OD 600 ) was 0.2.…”

“…The scFvs those revealed a different fingerprint mapping were sequenced again. The vector for soluble scFvs expression was designed based on pUC vector, which include lac promoter, PhoA leader, Flag tag, and His tag . The 10 scFvs with correct and different sequences were digested with Nco I and Not I, ligated with expression vector, and transformed into Escherichia coli DH5αF ’ .…”

Affinity improvement of the fully human anti‑TSLP recombinant antibody

“…Screening of mutated anti-TSLP-scFvs in the prokaryotic expression vector pLZ16. a TSlP-plZ16 plasmid was constructed in our previous study based on the puc vector (11). The mutated anti-scFv fragment and the plZ16 vector were digested with Noti and NocI restriction enzymes at 37˚C for 4 h. The products were ligated using 1 µl T4 ligase (Takara Bio, Inc.) at 16˚C for 12 h and transformed into DH5αF' competent cells (Tiangen Biotech co., ltd.).…”

“…The sp-scFv-Fc-84 (Pre-mutated) and sp-scFv-Fc-M4 (mutated) in pcDNA3.1 were amplified by PCR using 30 cycles at 98˚C for 10 sec, 55˚C for 30 sec and 72˚C for 90 sec with exTaq enzyme (Takara Bio, inc.) (11). Forward primers pcdna3.1-F (5'-cTa GaG aac cca cTG cTT ac-3') and pcdna3.1-r (5'-TaG aaG Gca caG TcG aGG-3') were employed.…”

Naïve Human Antibody Libraries for Infectious Diseases

“…The assembled fragment is then digested with restriction enzymes before cloning it upstream of the gene III coat protein in the phagemid [97]. Ligated products of antibody sequences and phagemids are transformed into bacterial cells carrying the F pilus such as XL1-Blue MRF' [97], ER2738 [122], TOP10F' [123], E. coli SS 320-M13cp [124,125] and TG1 [120,126]. The choice of cells that are used must have high transformation efficiencies.…”