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.coli TG1,构建了噬菌体表面呈现的十肽库,实际库容为3.53×107,插入率为66.7%.经辅助噬菌体M13KO7超感染后,获得滴度为4.8 ×1011 pfu/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26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12个阳性噬菌体克隆都能够与血管形成素特异性结合.
A random decapeptide library was constructed by using phage-surface display. The oligonucleotide sequence (NNK) was digested with SfiⅠ and NotⅠ and ligated into the phagemid pCANTAB5E. The recombinant DNA was introduced into E.coli TG1 by electroporation, and 5.3×107 phage was harvested. The insert was present in 66.7% of phage,thus the random deca-peptide library had a complexity of 3.53×107.The titer of phage supernatant was 4.8×1011 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.
杨辉,张英起,颜真,韩苇,药立波,苏成芝.随机十肽库的构建及血管形成素结合肽的筛选[J].生物化学与生物物理进展,2000,27(6):641-645
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