Construction of a phage displayed library based on a lipocalin scaffold and preliminary screening of anticalins with specificity for the FcεRI-α receptor

Lipocalin噬菌体文库的构建及与FcεRI-α受体结合分子的初步筛选（英文）

The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin library was constructed using phagemid expression vector.Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells,and the lipocalin secondary library binding to mast cells was obtained.Then the lipocalin secondary library was enriched and screened with FcεRI-αreceptor protein as target molecule,and specific binding phages were eluted.After three rounds of screening,eight recombinant phage clones were randomly selected from elution clones of the third round.ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-αreceptor of mast cells.These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-αreceptor,and also lay the foundation for the development of biological small molecule drugs to treat Ig E associated allergic diseases.

根据Lipocalin蛋白家族的基因序列设计引物,通过PCR重叠延伸得到含随机突变蛋白的基因序列,重组到噬菌粒载体构建随机突变Lipocalin文库。以NSF60细胞对Lipocalin文库进行差减筛选,再用肥大细胞进行亲和筛选,得到结合肥大细胞的Lipocalin次级文库。以FcεRI-α受体蛋白为靶分子对Lipocalin次级文库进行富集筛选,洗脱特异性结合的噬菌体。经过3轮筛选,从第3轮的洗脱克隆中随机挑取8个重组噬菌体克隆。ELISA法检测出3个Anticalins分子能与肥大细胞FcεRI-α受体特异性结合。这为研发肥大细胞FcεRI-α受体阻断药物提供了生物类候选分子,也为Ig E相关的变态反应疾病的生物类小分子药物研发奠定了基础。