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目的通过噬菌体展示肽库技术筛选VPAC1高亲和力结合十二肽配体,并进行特异性和亲和力鉴定。方法建立稳定表达VPAC1的真核细胞系;利用噬菌体肽库展示技术进行全细胞差减筛选,随机挑取40个噬菌体克隆进行测序,初步鉴定确定阳性克隆,明确外源十二肽序列,固相合成该十二肽;利用体外竞争结合ELISA及流式细胞分析,鉴定目标多肽与VPAC1受体结合的特异性及亲和力。结果成功构建稳定表达VPAC1细胞系;经过4轮差减筛选,回收噬菌体逐轮得到富集,随机挑取40个克隆测序,共得到15条序列不同的多肽,经ELISA鉴定有7个克隆与细胞有特异性高结合能力;阳性噬菌体克隆与细胞结合通过多肽VP1介导;VP1能特异高亲和地与VPAC1受体结合。结论通过噬菌体展示肽库技术成功筛选得到与VPAC1高亲和力特异结合十二肽。
OBJECTIVE: To screen VPAC1 high affinity binding dodecapeptide ligand by phage display peptide library technology and to identify its specificity and affinity. Methods Eukaryotic cell lines stably expressing VPAC1 were established. Phage display peptide library screening was used to screen the whole cell subtraction. 40 phage clones were randomly selected and sequenced. The positive clones were initially identified and identified, and the sequence of exogenous dodecapeptide, Synthesis of the dodecapeptide; using competitive ELISA and flow cytometry analysis to identify the specificity and affinity of the target polypeptide binding to the VPAC1 receptor. Results The VPAC1 cell line was successfully constructed. After four rounds of subtractive screening, the recovered phage were enriched round by round. 40 clones were randomly selected and sequenced to obtain 15 different polypeptides. Seven clones and cells were identified by ELISA Specific binding ability; positive phage clones and cell binding through the polypeptide VP1 mediated; VP1 can be highly specific and VPAC1 receptor binding. Conclusion The peptide with high affinity and specificity to VPAC1 was successfully screened by phage display peptide library technology.