A Single-Chain Antibody Using LoxP511 as the Linker Enables Large-Content Phage Library Construction via Cre/LoxP Recombination

摘要

To obtain natural or "me-better" antibodies (e.g., affinity-maturated antibodies), phage display libraries are widely used. However, the likelihood of obtaining satisfactory antibodies depends on the library content. Here, we used computeraided design to model the use of the LoxP511 site as a linker between the heavy and light variable domains of an antibody for construction of a large single-chain fragment (scFv) antibody phage library by using the Cre/LoxP recombinant system. Then, we constructed two novel scFvs based on 2C4, namely, AH_scFv15 (15 amino acid [aa] linker; common [SG_4]_3 sequence) and AH_scFv21 (21-aa linker; LoxP511 sequence), to verify the use of the LoxP511 site as a linker. Our results indicate that LoxP511 could be used effectively for the construction of a large (e.g., 5 × 10~(12)) phage display library of scFv antibodies from which it was possible to isolate an antibody with the same epitope as 2C4 but with higher affinity.