Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa

Abstract

Background - Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot. Methods and Results - The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly4Ser)3 was introduced by overlap PCR. After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the Bβ15-22 peptide of fibrin (β-peptide). Hirudin was genetically fused to the C-terminus of the variable..