应用细胞删减筛选法筛选整合素αvβ_3结合肽

Subtractive Screening of Targeted Short Binding Peptides of Integrinαv β_3

目的:应用新的噬菌体删减筛选技术筛选能与整合素αvβ_3具特异性结合功能的小肽分子,并简单分析其生物学功能。方法:以高表达整合素αvβ_3分子的M2黑色素瘤细胞株为靶细胞,以低/不表达整合素αvβ_3分子的C23黑色素瘤细胞株为吸附细胞,对噬菌体随机七肽库进行消减筛选,ELISA鉴定阳性克隆,DNA测序和分析。结果:经过4轮亲和筛选,3轮删减筛选,经固定ELISA鉴定,随机选取50个克隆得到2个能特异性与M2细胞结合而不与C23细胞结合的阳性克隆,进行DNA测序后,其多肽序列如下:HTPPYIL;MNFNAML。结论:通过噬菌体噬菌体删减筛选技术获得与整合素alpha v beta 3具特异性结合能力的小肽分子,为基于整合素alpha v beta 3的肿瘤血管生成的早期诊断及治疗的多肽药物前体设计提供实验依据。

Objective：Use the new subtraetive phage display technology to seek short peptides capable of binding integrinαvβ3 and detect their affinity function.Methods：Taking M2 melanoma cell line eells expressed integrinαvβ3 as target cells,C23 melanoma cell line cells do not expressed to do integrinαvβ3 as absorption cells,subtractive screening in phage displayed 7-peptide library,phage clones was identified by sandwich ELISA.The DNA sequence of positive phage clones was determined.Results：After four rounds of binding screening,three rounds of subtractive screening,We finally found 3 of 50 phage clones were identified positive by competitive ELISA,which had comparatively strong binding activity to M2 cells did not to C23 cells.Their sequences were obtained,Conclusions：The peptides with high affinity to integrinαvβ3 binding can be obtained through the subtractivc screening of phage random peptide library, which is beneficial to the further studies on the function of integrinαvβ3 in the early diagnosis of cancer and targeting angiogenesis inhibitors specific drug design.