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目的 :研究从噬菌体肽库中筛选到的与血管内皮生长因子受体Ⅱ (KDR)有特异结合活性的小肽 ,做为KDR靶向药物的先导物质的应用。方法 :从噬菌体肽库中筛选能特异结合KDR的噬菌体克隆 ,挑选结合力最强的克隆测序并化学合成小肽P5 ,梯度ELISA、阻断实验和竞争结合实验测定小肽在体外与KDR的结合活性。将P5与生物素 (NHS d Biotin)、BSA化学偶联 ,ELISA法和细胞免疫组化实验检测偶联物与KDR的结合活性。结果 :化学合成的小肽P5在体外能特异结合KDR ,Kd =16 8.6nmol/L ,约是KDR与配体血管内皮生长因子 (VEGF165)亲和力的 1/30 ,P5能阻断VEGF165与KDR的结合活性 ,但不能竞争VEGF165与KDR的结合活性。将P5作为导向物质化学合成的P5 BSA Biotin ,在体外同样具有与KDR和细胞表面KDR分子结合的特性。结论 :化学合成的小肽P5有望作为先导分子 ,在以KDR为靶点的肿瘤靶向治疗中得到应用。
Objective: To study the small peptides that specifically bind to vascular endothelial growth factor receptor II (KDR) screened from phage peptide libraries and use them as the lead substance for KDR targeted drugs. Methods: The phage peptide library was screened for phage clones that specifically bind to KDR. The strongest clones were selected for sequencing and chemical synthesis of the small peptide P5. Gradient ELISA, blocking experiments, and competition binding assays were used to determine the binding of small peptides to KDR in vitro. active. P5 was chemically coupled with biotin (NHS d Biotin) and BSA. ELISA and cellular immunohistochemistry experiments were performed to determine the binding activity of the conjugate to KDR. RESULTS: The chemically synthesized small peptide P5 could specifically bind to KDR in vitro, with Kd = 16 8.6 nmol/L, approximately 1/30 of that of KDR and ligand vascular endothelial growth factor (VEGF165). P5 can block VEGF165 and KDR. Binding activity, but not the binding activity of VEGF165 to KDR. P5 BSA Biotin, which is chemically synthesized using P5 as a directing substance, also has the property of binding to KDR and cell surface KDR molecules in vitro. Conclusion : The chemically synthesized small peptide P5 is expected to be a precursor molecule and is used in the targeted therapy of KDR as a target.