IgA亲和体随机组合噬菌体文库的构建和体外分子进化

目的构建IgA亲和体随机组合文库并进行体外分子进化,研究IgA亲和体分子结构与功能的关系。方法基因合成两个IgA亲和体片段。3′端引入3个随机连接肽序列随机连接,克隆于噬菌粒展示载体pCANTAB5S,构建噬菌体展示随机组合分子文库。以人IgA为靶分子对该文库进行4轮亲和筛选。制备阳性筛选克隆的单克隆噬菌体,用ELISA鉴定IgA结合活性。结果成功构建噬菌体展示IgA亲和体随机组合分子文库,库容量为3.4×107,滴度为1.6×1012TU/L,阳性克隆占79%以上,序列分析IgA亲和体随机连接,随机连接肽序列呈随机分布。在人IgA分子诱导的分子进化过程中,展示多个IgA亲和体串联体的噬菌体比例明显增加,获得3种新型IgA亲和体组合分子结构形式:IgA亲和体二联体、三联体和四联体。ELISA结合实验表明IgA亲和体三联体和四联体的IgA结合能力远大于单体和二联体。结论通过构建IgA亲和体噬菌体展示随机组合文库和体外分子进化的方法,获得多种新型组合分子,其中IgA亲和体三联体和四联体的IgA结合能力明显增加,提示通过有效的分子进化成功地获得了高亲和力的IgA结合分子。

Construction of phage-displayed random combinatorial library of IgA affibodies and its directed in vitro evolution

Objective To construct a phage-displayed random combinatorial library of IgA affibodies and to analyze its directed in vitro evolution,so as to study the relationship of IgA affibody structure with its function.Methods The coding sequences of two affibodies,ZA1 and ZA2,were generated by overlapping PCR.The affibodies with a random linking peptide coding sequence in the 3′ terminal were randomly ligated and cloned into the KpnⅠsite of the phagemid pCANTAB5S to construct a combinatorial phage library.Totally four rounds of in vitro human IgA directed evolution were conducted,and selected phage clones were prepared individually to test the IgA binding activity by ELISA technology.Results The combinatorial phage library was successfully constructed;it contained about 3.4×10~7 clones with a titer of 1.6×10~(12) TU/L,and the positive clones accounted for more than 79%.Sequence analysis showed that the two single affibodies were randomly linked by random linking peptides.The composition of the phage clones displaying three or four affibodies in tandem increased remarkably along with the rounds of selection,which indicated the successful IgA directed evolution.Three new arrangements of two,three or four affibodies in tandem were obtained.ELISA results demonstrated a significantly enhanced IgA binding activity of three or four affibodies in tandem.Conclusion A series of new IgA binding recombinants have been obtained by directed in vitro evolution of combinatorial phage library displaying randomly linked IgA affibodies.The three or four affibodies in tandem have much higher IgA binding activity than others,indicating that the in vitro molecular evolution is an effective way to produce IgA binding proteins with high activity.