应用噬菌体随机肽库筛选肿瘤MUC1/Y黏蛋白结合肽

MUC1/Y-Binding Peptides Generated by Biopanning a Phage Display Library

目的 :探索用噬菌体随机肽库筛选可用于肿瘤导向治疗的新型小分子载体。方法 :以MUC1/Y黏蛋白的胞外段蛋白(MUC1/Yex)为靶分子 ,用凝胶亲和法和酶联板法分别筛选十二肽噬菌体随机肽库 ,ELISA鉴定阳性克隆 ,DNA序列测定后确定MUC1/Yex结合肽的氨基酸序列 ;免疫组化鉴定阳性噬菌体克隆与正常及肿瘤细胞的结合能力及特异性。结果 :通过 4轮筛选 ,共获得 3种MUC1/Yex的结合肽 ,分别为HHWHSRSQLSWF ,HLKHKNYLPPTP和GNWYRHPHYLQP ,其中HXXHS表位可能在与MUC1/Yex的结合中起重要作用。阳性噬菌体克隆可与肿瘤细胞系MCF7,OVCA3,而不与正常外周血淋巴细胞结合。结论 :筛选获得的MUC1/Yex结合肽具有一定的亲合力和肿瘤特异性 ,可进一步用于肿瘤导向治疗的研究。

Objective: Using phage display randem peptide library to generate peptides as small molecular weight vec tors for tumor-targeting therapy. Methods: MUC1/Y extracellular domain was used as a target molecule to biopan Ph. D. 12 TM phage randem peptide library. Two protocols using affinity gel and cell culture plates respectively were carried out. Positive phage clones were identified by ELISA. ssDNA sequencing was done on 16 positive phage clones to get the amino acid sequences of MUC1/Y-binding peptides. Immunohistochemistry was done to show the capacity and specificity of positive phage clones to bind the tumor cell lines. Results: After 4 rounds biopanning, three MUC1/Y-binding peptides were generated including HHWHSRSQLSWF, HLKHUKNYLPPTP and GNWYRHPHYLQP. HXXHS may be the key motif for binding to MUCI/Y. The positive phage clones tested showed the ability to bind MCF, OVCA3 cell lines while no binding to normal peripheral blood lymphocytes( PBLs) was observed. Conclusion: The peptides generated by biopanning phage library showed some affinity and tumor-specificity and may be potential candidates as ligands for tumor-targeting therapy.