Figure 1.

Schematic outline of the approach used for library construction. A library of V_H and genes was generated from rearranged human V-genes and cloned into the plasmid pCITE3A. The V_L genes used for scFv assembly were derived from a previously constructed scFv library contained in the plasmid pHEN1 (12). The vector containing the V_L repertoire also contained the scFv linker DNA 5′ to the V_L genes. Primers for reamplification of the V-gene repertoires were derived from sequences several hundred bp 5′ (the V_H genes) or 3′ (the V_L genes) of the scFv gene cloning sites. This approach facilitated the efficiency of PCR assembling a new scFv repertoire and increasing the efficiency of cutting assembled scFv genes with restriction enzymes. (A) V_H and linker-V_L gene repertoires were generated by PCR from the plasmid DNA of the separate libraries. The V_H genes wereamplified by using a plasmid specific primer    and an equimolar mixture of HuJ_H primers    . The linker DNA and V_L genes were amplified by using a plasmid specific primer   and an equimolar mixture of RHuJ_H primers     . The RHuJH primers are complementary to the HuJ_H primers. (B) The V_H and linker DNA-V_L gene repertoires were PCR assembled into a scFv gene repertoire. (C) The assembled scFv gene repertoire was cut with the restriction enzymes NcoI and NotI and cloned into the plasmid pHEN1 (17) for phage display.