噬菌体随机12肽库中VEGF模拟表位的筛选及初步鉴定

Screening and primary identification of antigen epitopes of vascular endothelial growth factor from phage display 12 peptide library

目的:从噬菌体随机12肽库中筛选能与抗血管内皮生长因子(VEGF)mAb阿瓦斯汀(Avastin)特异性结合的多肽.方法:用Avastin为靶分子,对噬菌体12肽库进行3轮生物淘洗,随机挑取80个克隆,ELISA法鉴定其亲和性,将亲和性高的阳性克隆进行DNA序列测定,推导与噬菌体外壳融合的12肽氨基酸序列.Fmoc固相法合成12肽,戊二醛交联12肽与血蓝蛋白(KLH),打点印迹(Dot blot)检测偶联物能否与Avastin结合.结果:ELISA显示,42个噬菌体克隆与Avastin有较强的亲和性,测序发现41个阳性克隆表达的融合12肽的序列一致,1个不同;化学合成的12肽能与Avastin特异性结合,12肽-KLH偶联物也能与Avastin特异性结合.结论:初步证明12肽模拟了VEGF部分表位.

AIM： To screen antigen epitopes of vascular endothelial growth factor（VEGF） by avastin in phage display 12 peptide library. METHODS： The avastin was used as target protein for biopanning of phage displayed 12 peptide library. After three round of blopanning, 80 phage clones were randomly selected and identified by ELISA. The positive phage clones were sequenced, and the fuse peptides were deduced by the DNA sequence. The 12 peptide was synthetized by Fmoc solid phase method, and conjugated with KLH by glutaral. Conformational accuracy was verified with Avastin in a dot blot assay. RESULTS： Forty-two of eighty phage clones were identified as positive clones by ELISA which bound to Avastin. Forty-one phage cones had the identical sequences. The 12 peptide bound with Avastin, and the conformation of peptide-KLH was right, it also bound with Avastin. CONCLUSION： 12 peptide can mimic the epitope of VEGF.