细胞间粘附分子1特异结合肽的筛选及其生物功能

采用两种方法对噬菌体展示随机十五肽库进行亲和淘选 .ELISA法筛选特异结合高亲和力的阳性噬菌体单克隆 ,测序 ,得到 6个与人细胞间粘附分子 1(ICAM 1)高亲和力的噬菌体展示十五肽单克隆 .再经ELISA法从这 6个噬菌体单克隆中选择与ICAM 1亲和力最高的单克隆 ,同时利用蛋白空间结构位象模拟技术对小肽与ICAM 1的亲和力进行模拟研究 .最终获取目的小肽的氨基酸序列为GRGEFRGRDNSVSVV .目的单克隆噬菌体与ICAM - 1的亲和常数Ka 为 7 87× 10 7L mol .体外合成、纯化并标记目的小肽 .ELISA法验证目的小肽与人ICAM 1的结合呈浓度依赖性 ,抗ICAM 1多抗不能拮抗目的小肽与ICAM 1的结合 .采用免疫组化方法证实 ,此目的小肽具有与炎症组织中高表达的ICAM 1特异性结合的功能 .在动物体内 ,荧光标记的目的小肽具有向高表达ICAM 1的炎症部位特异性聚集的功能 .说明此目的肽可尝试作为以ICAM 1为靶的“肽导向药物”的前导肽 .

Screening of ICAM-1-binding Peptide and Their Biological Functions

Two methods were used to screen intercellular adhesion molecule-1 (ICAM-1) binding peptides from phage-displayed random 15-peptide library. After 4-5 rounds affinity panning and ELISA analysis, six positive recombinant phage clones that could bind to ICAM-1 specifically were chosen. The DNA sequences of these positive clones were determined and amino acid sequences of 15-peptide were deduced. The affinity of the six positive recombinant phage clones was determined by ELISA. The space conformations of the peptides that had the consensus sequence were analyzed by computer-guided homologous modeling technique. The results showed that the No.4 peptide had the best affinity to ICAM-1. The amino acid sequence of the target peptide was GRGEFRGRDNSVSVV. The K a of No.4 displayed phage clone was 7.87×107 L/mol. The target peptide was synthesized and purified in vitro. It was also labeled with fluorescence for future study. The combination of target peptide with ICAM-1 depended upon concentration. The synthetic peptide could combine with ICAM-1 on inflammatory tissues by immunohistochemical staining. It also could accumulate toward ICAM-1 in vivo. It suggested that the target peptide might be used as the guiding peptide in “peptide guided drug" to inflammatory site with ICAM-1 expression in the future.