期刊
JOURNAL OF BIOCHEMISTRY
卷 144, 期 5, 页码 591-598出版社
OXFORD UNIV PRESS
DOI: 10.1093/jb/mvn103
关键词
antibody library; biopanning; phage display; randomization; single-chain antibody
资金
- Chinese Ministry of Sciences 863 Project [2006AA 02A245, 2006AA02Z457]
- National Protein Project [2006CB910901]
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 x 10(9) 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 x 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.
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