随机十肽库的构建及血管形成素结合肽的筛选

Construction of Random Decapeptide Library and Screening Identification of Angiogenin-binding Peptides.

将合成的含有随机序列 (NNK) 10 的寡核苷酸片段 ,克隆入噬菌体呈现载体噬菌粒pCANTAB5E的SfiⅠ ,NotⅠ位点 ,即cpⅢ蛋白信号肽与成熟肽之间 ,电转化E .coliTG1,构建了噬菌体表面呈现的十肽库 ,实际库容为 3 5 3× 10 7,插入率为 6 6 7% .经辅助噬菌体M13KO7超感染后 ,获得滴度为 4 8× 10 11pfu/ml的噬菌体上清 .经过两轮panning筛选和富集 ,从构建的随机十肽库中筛选到 2 6个具有血管形成素结合活性的重组噬菌体克隆 ,对其中 12个阳性噬菌体克隆的短肽序列进行了分析 ,ELISA检测结果显示

A random decapeptide library was constructed by using phage surface display. The oligonucleotide sequence (NNK) was digested with Sfi Ⅰ and Not Ⅰ and ligated into the phagemid pCANTAB5E. The recombinant DNA was introduced into E.coli TG1 by electroporation, and 5 3×10 7 phage was harvested. The insert was present in 66 7% of phage,thus the random deca peptide library had a complexity of 3 53×10 7.The titer of phage supernatant was 4 8×10 11 after the helper phage M13KO7 super infection. This library was screened using angiogenin protein. 26 ANG binding clones were indentified from 94 enriched individual phagemid clones after two rounds of panning, The nucleotide sequences encoding peptide recombined in 12 positive phagemid clones were determined. ELISA showed that all of them could specifically bind to ANG.