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- Title
Construction of a Fully Synthetic Human scFv Antibody Library with CDR3 Regions Randomized by a Split–Mix–Split Method and Its Application.
- Authors
Chang-Cheng Yin; Li-Li Ren; Lin-Lin Zhu; Xiang-Bin Wang; Zhong Zhang; Hua-Liang Huang; Xi-Yun Yan
- Abstract
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.
- Subjects
NUCLEIC acids; AMINO acids; DEOXYRIBOSE; TERATOGENESIS; RADIOGENETICS; SYNTHETIC antibodies
- Publication
Journal of Biochemistry, 2008, Vol 144, Issue 5, p591
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/jb/mvn103