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目的应用噬菌体随机肽库展示技术筛选能与整合素αvβ3分子具特异性结合功能的小肽分子,并对其功能进行初步鉴定。方法根据整合素αvβ3分子细胞外配体结合区域的晶体结构及蛋白质序列设计①～⑤号5条短肽,并作为筛选配基,分别经过3轮“吸附-洗脱-扩增”后,ELISA鉴定阳性克隆,DNA测序和分析,粘附试验进一步分析化学合成多肽的功能。结果经3轮亲和筛选后,噬菌体克隆得到有效富集,以⑤号肽为代表,ELISA鉴定显示7个阳性克隆能与⑤号合成短肽有较强结合活性。进行DNA测序,多重序列分析获得2个多肽基序:****HRH,HWR****。粘附试验表明化学合成多肽FITC-HLPWHRH,FITC-HWRLHPH与表达整合素αvβ3的细胞株具有一定的亲和性,且结合能被αvβ3分子配体纤维连接蛋白所抑制。结论通过噬菌体随机展示肽库技术在体外对合成的短肽进行筛选,可以获得与整合素αvβ3分子具特异性结合能力的小肽分子。
OBJECTIVE: To screen small peptide molecules with specific binding function to integrin αvβ3 by phage display of random peptide library and to identify its function. Methods According to the crystal structure and protein sequence of the integrin αvβ3 binding region, five short peptides ① ~ ⑤ were designed and used as screening ligands. After 3 rounds of “adsorption-eluting-amplification” , Positive clones by ELISA, DNA sequencing and analysis, and adhesion assays to further analyze the function of chemically synthesized polypeptides. Results After 3 rounds of affinity screening, the phage clones were effectively enriched. The ⑤ peptide was used as the representative, and 7 positive clones showed strong binding activity with the ⑤ synthetic short peptide. DNA sequencing, multiple sequence analysis to obtain two polypeptide motifs: **** HRH, HWR ****. Adhesion tests showed that the chemical synthetic peptides FITC-HLPWHRH and FITC-HWRLHPH had some affinity with the cell line expressing integrin αvβ3, and the binding ability was inhibited by αvβ3 ligand fibronectin. Conclusion The peptide synthesized by phage display peptide library technology can be used to screen small peptide synthesized in vitro, and the small peptide molecule with specific binding ability to integrin αvβ3 can be obtained.