Construction of an scFv library by enzymatic assembly of VL and VH genes

The single-chain Fv fragment (scFv) is the most frequently used form of recombinant antibody. It is possible to establish clones specific to a certain target by displaying the scFv library on phages followed by biopanning against the target. For the construction of superior scFv libraries, the light-chain variable region (VL) and the heavy-chain variable region (VH) fragments should be assembled into the scFv without loss of diversity. We have provided an efficient method for constructing scFvs by enzymatic assembly of the VL and VH domains using the concerted action of λ-exonuclease and Bst DNA polymerase. First, we amplified the chicken VL and VH fragments using a phosphorylated primer with a 21-nucleotide overlap in the linker region. Then we recessed the overlapping parts of the VL and VH fragments with λ-exonuclease, which yielded single-stranded overhangs that specifically annealed between the VL and VH fragments; the complete double-stranded scFv was formed using Bst DNA polymerase. Complete scFvs were obtained using this method, whereby a library of scFvs was constructed from the immune library of chicken IgG. The diversity of this scFv library was analyzed by DNA fingerprinting method. The scFvs specific to the antigen could be isolated from this library after 5 rounds of panning.