筛选鉴定CD44v9特异性结合肽并用于胃癌检测的研究  

Screening and identification of CD44v9 specific peptide ligand for gastric cancer detection

作　　者：张丹[1] 冯云 刘亚萍[1] 赵倩[1] 黄晋[2] 和水祥[1] ZHANG Dan;FENG Yun;LIU Yaping;ZHAO Qian;HUANG Jin;HE Shuixiang(Department of Gastroenterology,The First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061;Department of Photonics,School of Electronic Information and Artificial Intelligence,Shaanxi University of Science&Technology,Xi’an 710021,China)

机构地区：[1]西安交通大学第一附属医院消化内科,陕西西安710061 [2]陕西科技大学电子信息与人工智能学院光电系,陕西西安710021

出　　处：《西安交通大学学报（医学版）》2023年第3期383-388,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金青年项目(No.81602611)。

摘　　要：目的筛选并验证CD44v9特异性多肽配体。方法使用噬菌体展示12肽库筛选固相包被的CD44v9。测序发现待选序列,使用ELISA法进行验证,选择最佳序列合成探针。使用免疫荧光法验证C9-3多肽与CD44v过表达HEK-293细胞的结合,免疫组化法验证多肽探针与胃癌组织的特异性结合力。结果噬菌体筛选过程中出现明确的富集效应,测序发现30个克隆包含9条序列,其中C9-3序列重复次数最高。ELISA显示9个待选序列中,C9-3序列具有最高的吸光度绝对值及选择力,因此选定为合成探针序列。C9-3探针结合于CD44v过表达HEK-293细胞,而不结合空质粒转染HEK-293细胞。C9-3探针与胃癌组织及癌旁组织结合的免疫组化评分之间存在统计学差异(t=3.953,P<0.01)。胃癌组织中,C9-3探针与CD44v9的组化评分存在线性正相关关系(r=0.823,P<0.01)。结论本研究成功筛选出CD44v9配体多肽,可以通过CD44v9结合于细胞及胃癌组织,用于胃癌检测的探针。Objective To screen and verify a peptide ligand specific for CD44v9.Methods A 12-mer phage peptide library was screened on CD44v9 coated on solid phase.Candidate sequences emerged after sequencing.Candidate phages were selected using enzyme-linked immunosorbent assay.The best sequence was chosen for further study.Binding of C9-3 to CD44v overexpressed HEK-293 cells was determined using immunofluorescence.Binding affinity and specificity were verified on gastric cancer tissues with immunohistochemistry.Results Phages significantly were enriched during panning process.After sequencing,nine individual sequences occurred in 30 selected clones.Among the 9 candidate sequences,C9-3 exhibited the highest frequency.Results of ELISA showed that C9-3 had the highest OD value and selectivity.Thus,C9-3 was chosen for peptide probe synthesis.C9-3 probe stained CD44v overexpressed HEK-293 cells,but not empty vector transfected HEK-293 cells.Immunohistochemistry scores of C9-3 were significantly different between gastric cancer and paracancer tissues(t=3.953,P<0.01).A linear positive correlation was observed between C9-3 binding and CD44v9 expression(r=0.823,P<0.01).Conclusion In this study,peptide ligand of CD44v9 was successfully screened.The peptide can bind to cells and cancer tissues via CD44v9.It has potential for gastric targeting probes.

关 键 词：多肽探针 CD44V9 噬菌体展示 

分 类 号：R730.4[医药卫生—肿瘤]