噬菌体展示随机十五肽库的构建

为了满足药物筛选的需要，本实验以pCANTAB 5E噬菌粒为载体，构建表达于丝状噬菌体尾丝蛋白P3N末端的随机十五肽库。通过自行设计引物与模板，人工合成编码随机十五肽的寡核苷酸片段及含有酶切位点的引物。通过PCR技术进行模板扩增获得编码随机十五肽的基因。将扩增后的目的基因经过Sfi Ι，Not Ι两个限制性内切酶双酶切后，与经同样双酶切的5′去磷酸化的噬菌粒载体连接，将重组产物电转入大肠埃希菌TG1感受态细胞。集合菌落后进行库容和多样性检验。所建肽库库容可达5×10⁸。随机挑取20个克隆测序，其核苷酸序列及推断出的氨基酸序列均随机。成功构建了库容量与多样性都满足筛选要求的噬菌体展示随机十五肽库。

Construction of a phage display random fifteen-peptide library

A phage display random fifteen-peptide library expressed in the filamentous phage tail fiber protein P3N terminal was constructed using pCANTAB 5E vector. First, a random oligonucleotides fragment containing fifteen codons was designed and synthesized. The fragment was amplified by PCR. Then, the amplified target gene was digested by two restriction endonuclease of Sfi I and Not I, and connected with pCANTAB 5E phagemid vector. Finally, the recombinant vector was transformed into electro-competent cells of E. coli TG1. The capacity of peptide library was up to 5×10⁸. Twenty clones were randomly selected for sequencing; the nucleotide sequence and deduced amino acid sequences were randomized. The random fifteen-peptide library was successfully established for screening requirements.