摘要
Antibodies selected from phage displayed libraries, especially nave ones, tend to have low affinity for their antigen, thereby limiting their use for certain applications Chain shuffling is a useful strategy to increase affinity of antibodies; itwould allow recombination a heavy or light chain repertoire in one library with a complementary variable domain of a specific antibody.In this work, two single-chain variable fragments(scFvs) against polyclonal antibodies of Bacillus thuringiensis Cry2 A toxins were shuffled with variable heavy and light chain repertoires of unpanned Tomlinson I library. The purified heavy chain and light chain PCR products were connected to form a full-length sc Fv gene in a strand overlap extension PCR reaction. The sizes of light chain and heavy chain shuffling libraries were 1.4×106 cfu/ml and 1.2×106 cfu/ml, respectively. Before panning, the affinity of primary light chain and heavy chain libraries were determined by polyclonal phage ELISA, and the absorbance of light chain shuffling library were 4.2 times higher than heavy chain library. After one round panning, 7 mutants were rapidly isolated from chain shuffling libraries. These mutants were ranked by a competitive phage ELISA, and the ratios of IC50 of templates to their mutants were between 0.51-1.26. This works showed an efficient way to rapidly obtain mutants with similar or slightly improved affinities by chain shuffling from the same na?ve library where the antibody was originally isolated.
Antibodies selected from phage displayed libraries, especially nave ones, tend to have low affinity for their antigen, thereby limiting their use for certain applications Chain shuffling is a useful strategy to increase affinity of antibodies; itwould allow recombination a heavy or light chain repertoire in one library with a complementary variable domain of a specific antibody.In this work, two single-chain variable fragments(scFvs) against polyclonal antibodies of Bacillus thuringiensis Cry2 A toxins were shuffled with variable heavy and light chain repertoires of unpanned Tomlinson I library. The purified heavy chain and light chain PCR products were connected to form a full-length sc Fv gene in a strand overlap extension PCR reaction. The sizes of light chain and heavy chain shuffling libraries were 1.4×106 cfu/ml and 1.2×106 cfu/ml, respectively. Before panning, the affinity of primary light chain and heavy chain libraries were determined by polyclonal phage ELISA, and the absorbance of light chain shuffling library were 4.2 times higher than heavy chain library. After one round panning, 7 mutants were rapidly isolated from chain shuffling libraries. These mutants were ranked by a competitive phage ELISA, and the ratios of IC50 of templates to their mutants were between 0.51-1.26. This works showed an efficient way to rapidly obtain mutants with similar or slightly improved affinities by chain shuffling from the same na?ve library where the antibody was originally isolated.
引文