Construction of a Fully Synthetic Human scFv Antibody Library with CDR3 Regions Randomized by a Split–Mix–Split Method and Its Application

摘要

The randomization scheme of hypervariable region takes crucial role in construction of a synthetic antibody library. The codon bias and inevitable ‘stop’ codon of conventional ‘NNK’ and ‘NNS’ codons limit their applications. Here we report a split–mix–split DNA synthesis method that can control over the amino acid composition and distribution of randomized sequences effectually. A fully synthetic human antibody library with a diversity of 1.56 × 109 was successfully generated with complementarity determining region 3 (CDR3) randomized by this strategy. Sequencing analysis indicated that 60% of colonies had completely correct scFv genes and the amino acid composition and distribution were designed well in accordance. The utility was demonstrated by screening of scFv clones against BHL (anti-CD3 × anti-ovarian carcinoma bispecific antibody). These results proved the feasibility of the split–mix–split DNA randomization strategy in library construction and site-directed mutagenesis.