噬菌体展示IgA亲和体随机组合文库的构建

Construction of phage-displayed random combinatorial library of IgA affibodies

目的构建噬菌体展示IgA亲和体随机组合文库,研究IgA结合分子结构与功能的关系。方法基因合成IgA亲和体ZA1、ZA2片段。片段两端引入Kpn I酶切位点,3′端引入随机连接肽序列,Kpn I酶切,随机连接,克隆于噬菌粒展示载体pCANTAB5S的Kpn I克隆位点上,转化大肠杆菌TGI,辅助噬菌体M13K07拯救,构建噬菌体展示随机组合文库。结果基因合成IgA亲和体;构建噬菌体展示IgA亲和体随机组合文库,容量为3·4×107,滴度为1·6×1012TU/L;文库中含79%以上的阳性克隆;序列分析显示组合文库由ZA1、ZA2随机组合而成,连接方式符合随机连接的特点,各亲和体之间连接肽序列也呈随机分布。结论合成IgA亲和体,构建噬菌体展示IgA亲和体随机组合文库,该文库库容量、多样性和随机性可满足体外分子进化研究的要求。

Objective To construct a phage-displayed random combinatorial library of IgA affibodies, for studying the relationship between its structure and function. Methods The coding sequences of two affibodies, ZA1 and ZA2,were generated by Overlap PCR. The affibodies with the Kpn I recognition site in both ends and a random linking peptide in 3＇ terminal were synthesized by PCR and digested by Kpn I and ligated into the Kpn I site of the phagemid pCANTABSS. The recombinants were transformed into E. coli TGI, and the phage-displayed random combinatorial library was constructed by the rescue of help virus M13K07. Results Both coding sequences of ZA1 and ZA2 affibodies were synthesized . The phage-displayed random combinatorial library were constructed. The library consisted of about 3.4 × 10^7 clones with the titer of 1.6 × 10^12 TU/L. More than 79% clones in the library were positive for insertion. Sequence analysis showed that inserted fragments of clones in the library were composed of the two single affibodies ligated randomly. The nucleotide acids encoding random linking peptide also distributed randomlv. Conclusion The coding sequences of ZA1 and ZA2 affibodies were synthesized. The phage-displayed random combinatorial library of IgA affibodies was constructed. The capacity, variety and random of the library meet the requirements for in vitro molecular evolution study.