Rapidly isolation of anti-idiotype single-chain variable fragments against Cry toxins from chain shuffling libraries
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- 英文论文题名：Rapidly isolation of anti-idiotype single-chain variable fragments against Cry toxins from chain shuffling libraries
- 论文作者：Liu Yuan ; Lin Manman ; Liu Xianjin
- 英文论文作者：Liu Yuan ; Lin Manman ; Liu Xianjin ; Key Laboratory of Food Quality and Safety of Jiangsu Province
- 年：2016
- 作者机构：Key Laboratory of Food Quality and Safety of Jiangsu Province;
- 英文论文关键词：Single-chain variable fragments(scFvs) ; chain shuffling ; affinity maturation
- 会议召开时间：2016-11-04
- 会议录名称：第十一届全国免疫学学术大会摘要汇编
- 英文会议录名称：Abstracts of 2016 CSI Congress on Immunology
- 语种：英文
- 分类号：S852.4
- 学会代码：IGMD
- 会议名称：第十一届全国免疫学学术大会
- 会议地点：中国安徽合肥
- 主办单位：中国免疫学会
- 学会名称：中国免疫学会
- 页数：2
- 文件大小：1334k
- 原文格式：D
- 会议级别：全国
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.
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