摘要
目的:建立一种高效的筛选跨膜受体靶向肽的噬菌体展示新方法,据此筛选出组织因子(TF)高亲和力靶向肽。方法:分别以纯化TF蛋白和具有TF高特异性表达的结直肠癌细胞株HT-29为靶标,用噬菌体七肽库进行序贯筛选(受体-细胞序贯筛选法,STRCA法),ELISA初步鉴定噬菌体克隆对HT-29亲和力。对噬菌体克隆进行测序,利用竞争抑制ELISA比较各合成肽的TF结合力。重复实验检测筛选结果的可靠性。结果:经过5轮筛选,与TF结合的噬菌体得到了富集,回收率由(2.25×10-4)%增加到(1.32×10-2)%;ELISA结果显示30个克隆中,阳性率为76.7%。噬菌体测序结果中,4种合成肽(分别命名为A、B、C、D肽)中A肽序列重复率为23.3%(7/30),B肽为23.3%(7/30),C肽为26.7%(8/30),D肽为10.0%(3/30);E肽为A、B肽合成的复合靶向肽。经竞争抑制ELISA比较5种肽的TF亲和力,其IC50分别为3.25 nmol/L、6.72 mol/L、3.24×103 mol/L、2.08×102 mol/L和45.77 mol/L。重复实验所获得的阳性噬菌体克隆序列与第1次实验相比,重复率为33.3%。结论:STRCA法是一种理想的筛选跨膜受体靶向肽方法,采用该法获得的TF靶向肽对TF具有高度的亲和力。
AIM： To screen tissue factor（TF） targeting peptides by establishment of a new and effective phage display method to acquire the peptides specifically bound to the transmembrane receptor.METHODS： Five rounds of panning were alternately conducted by targeting TF and HT-29 cell line which showed the detectable TF expression（screen for targeting receptor and cell alternately with phage display,STRCA）.The 30 phage clones were assessed by enzyme-linked immunosorbent assay（ELISA）.DNA sequencing was performed for the phage clones.The affinity of synthetic peptides was verified with competitive inhibition ELISA.The repeated experiment was conducted to verify the reliability of the results.RESULTS： The phages were effectively enriched after 5 rounds of panning with the improvement of the recovery rate from（2.25×10-4）% to（1.32×10-2）%.In 30 individual phages,ELISA positive rate was 76.7%,and the repetition of A,B,C and D peptides showed 23.3%（7/30）,23.3%（7/30）,26.7%（8/30） and 10.0%（3/30）,respectively.E peptide constructively consisted of A and B.The five synthetic peptides were verified by ELISA,and the IC50 of each peptide showed 3.25 nmol/L,6.72 mol/L,3.24×103 mol/L,2.08×102 mol/L and 45.77 mol/L,respectively.The positive phages were selected again in the second experiment to compare the results of the first experiment and the repeatability was 33.3%.CONCLUSION： STRCA can select TF targeting peptides with high affinity,which has the potential to become a therapeutic screening of transmembrane receptor-binding peptides.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2011年第12期2429-2432,共4页
Chinese Journal of Pathophysiology
基金
中山大学新兴和交叉学科重点培育项目(No.10ykjc02)
关键词
组织因子
噬菌体展示技术
跨膜受体
筛选
靶向肽
Tissue factor
Phage display
Transmembrane receptor
Screen
Targeting peptide
