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人工合成编码ｃＡＭＰ依赖蛋白激酶（ｃＡＰＫ）的热稳定抑制剂第５～２４位氨基酸［ＰＫＩ（５～２４）］的ＤＮＡ片段，并将之克隆到ｐｈａｇｅｄｉｓｐｌａｙ载体ｆｄ－ｔｅｔ－ＤＯＧ１中，使ＰＫＩ（５～２４）以融合于ｇ３ｐ蛋白的形式展示于噬菌体表面，从而得到ＰＫＩ噬菌体。蛋白激酶抑制剂活性测定表明ＰＫＩ噬菌体可有效地抑制ｃＡＰＫ的活性。结合实验结果显示ＰＫＩ噬菌体能与固相化小鼠ｃＡＰＫ催化亚基α（Ｈｉｓ６－ｍＣα）高亲和结合，并在酸性条件下被定量洗脱。模型亲和筛选实验结果表明，利用固相化Ｈｉｓ６－ｍＣα亲和层析，可以从１０８倍的野生型噬菌体背景中选择性富集专一ＰＫＩ噬菌体。本工作为构建用于蛋白激酶结构功能等研究的特殊ＰｈａｇｅＤｉｓｐｌａｙ随机肽库成功地探索了可行途径。
A DNA fragment encoding amino acids 5 to 24 [PKI (5 to 24)] of the cAMP-dependent thermostable inhibitor of cAMP was artificially synthesized and cloned into the phagedisplay vector fd-tet-DOG1 such that PKI ( 5 ~ 24) displayed in the form of g3p protein displayed on the phage surface, resulting in PKI phage. Protein kinase inhibitor activity assay showed that PKI phage can effectively inhibit the activity of cAPK. The results of the experiments showed that PKI phage can bind with high affinity to the catalytic subunit α (His6-mCα) of the immobilized mouse and elute quantitatively under acidic conditions. The results of model affinity screening showed that the specific PKI phage can be selectively enriched from the 108-fold wild-type bacteriophage background using immobilized His6-mCα affinity chromatography. This work has successfully explored feasible ways to construct a special PhageDisplay random peptide library for the study of protein kinase structural function and so on.