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Construction and characterization of single-chain variable fragment antibodies directed against the Bordetella pertussis surface adhesins filamentous hemagglutinin and pertactin.

ABSTRACT: A single-chain variable fragment (scFv) antibody library against Bordetella pertussis was constructed using M13 phage display. The library was enriched for phages surface displaying functional scFv by biopanning against B. pertussis immobilized on polystyrene plates. Two hundred eighty-eight individual clones from the enriched library were screened for binding to B. pertussis cells, filamentous hemagglutinin (FHA), and pertactin (PRN) in enzyme-linked immunosorbent assays (ELISAs). Based on the binding ability, the clones were put into eight groups. The scFv DNA inserts from the 288 clones were digested with BstOI, and 18 unique restriction patterns, named types 1 to 18, were found. Eight clones (types 1 to 7 and 18) were selected for further testing against FHA, PRN, and B. pertussis by ELISA. The results showed that types 1, 5, 7, and 18 bound strongly to B. pertussis cells as well as FHA and PRN. Type 3 bound strongly to the cells and FHA but weakly to PRN. Types 4 and 6 bound FHA only, and type 2 did not bind to the cells or antigens. The ability of the eight clones to inhibit B. pertussis from binding to HEp-2 cells was assayed. Types 1, 5, and 7, but not the remaining clones, inhibited the adherence of B. pertussis to HEp-2 cells. The scFvs were sequenced, and the deduced amino acid sequence showed that the scFvs were different antibodies. Maltose-binding protein (MBP) fusion proteins composed of three different regions of FHA (heparin-binding domain, carbohydrate recognition domain, and the RGD triplet motif) were constructed. The three fusion proteins and Mal85 (MBP-FHA type I domain) were used to map the binding sites for scFvs of types 1, 5, and 7 by ELISA. The results showed that all three scFvs bound to the heparin-binding domain fusion protein but not the other fusion proteins. BALB/c mice who received recombinant phage-treated B. pertussis had reduced bacterial counts in the nasal cavity, trachea, and lungs compared to the control groups.


PROVIDER: S-EPMC2168280 | BioStudies | 2007-01-01


REPOSITORIES: biostudies

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