从噬菌体展示随机肽库中筛选豌豆凝集素的结合肽

目的:从噬菌体展示随机肽库中筛选能与豌豆凝集素(PSA)特异结合的短肽.方法:①用豌豆凝集素(PSA)作为靶蛋白,对噬菌体展示的随机六肽库进行亲和筛选;②α-甲基-D-甘露糖苷对筛选出的噬菌体和PSA结合的影响实验(点印迹法); ③选择性地合成了3条6肽ARMWSF、RYDYSY、LRLRQL,用不同浓度的6肽对PSA、ConA与HRP的结合进行竞争抑制实验.结果:经过三轮筛选后,这些噬菌体展示肽有明显的富集,从第三轮挑选的22个克隆的插入氨基酸序列可分为三大类;点印迹结果表明,这些噬菌体展示肽能与PSA特异结合,而α-甲基-D-甘露糖苷不同程度地抑制这种结合; LRLRQL不溶于水,ARMWSF和RYDYSY对PSA和HRP的结合有抑制,而对ConA和HRP的结合没有明显抑制.结论:人工合成的2条6肽和PSA的结合部位与α-甲基-D-甘露糖苷和PSA结合的部位并非相同.

Identification of peptides binding to Pisum sativum agglutinin from a phage-displayed random peptide library

OBJECTIVE: To obtain peptides binding specifically to Pisum sativum agglutinin (PSA) from a phage-displayed random peptide library. METHODS: (1) A phage-displayed random hexapeptide library was screened with PSA as target. (2) Dot blot was used to analyze the influence of the alpha-Met-D-mannoside on binding between PSA and phage-displayed peptides. (3) Three peptides (RMWSF, RYDYSY, LRLRQL) were selectively synthesized, and different concentrations were used to inhibit PSA and ConA binding to the HRP. RESULTS: The enrichment occurred obviously after three rounds of screening. The insert sequences of amino acids, displayed on 22 phage DNAs from the third round of screening, were divided into three groups. The binding of phage-displayed peptides to PSA was specific as shown by dot blot and could be inhibited by alpha-Met-D-mannoside. LRLRQL was not dissolved in water. ARMWSF and RYDYSY inhibited binding of PSA to HRP, but failed to inhibit binding ConA to HRP. CONCLUSION: The binding site of peptides ARMWSF and RYDYSY is different to that of alpha-Met-D-mannoside.