肝癌特异性结合肽的筛选及鉴定  

Preliminary screening and identification of the hepatocarcinoma cell-binding peptide

作　　者：朱小华[1] 吴华[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院核医学科,武汉430030

出　　处：《中华核医学杂志》2004年第6期340-343,i002,共5页Chinese Journal of Nuclear Medicine

基　　金：国家自然科学基金资助项目 (30 1 71 0 62 )

摘　　要：目的　探讨利用噬菌体随机肽库筛选肝癌细胞特异性结合多肽或肝癌细胞优势结合多肽的可行性。方法　①利用噬菌体随机十二肽库 ,对人肝癌细胞系HepG2和人正常肝细胞系L0 2进行 4轮差异筛选、富集 ,获得与肝癌细胞亲和的噬菌体克隆。②用酶联免疫吸附测定 (ELISA)法测定其与肝癌细胞、正常肝细胞的亲和力 ,进一步筛选出高度亲和肝癌细胞的特异性阳性噬菌体克隆。比较其与其他肿瘤细胞的亲和力。③提取阳性噬菌体DNA并进行DNA序列测定 ,获得与肝癌细胞亲和的多肽序列。④用免疫荧光共聚焦成像进一步确定展示阳性多肽序列的噬菌体与肝癌细胞的结合部位。⑤根据筛选的多肽序列合成十六肽WH16 ,利用竞争抑制实验鉴定其与肝癌细胞的结合。结果　筛选及ELISA结果示得到与肝癌细胞特异性结合并内化入肝癌细胞的噬菌体克隆 ,测序结果示 5 6 6 7%的被检噬菌体展示相同的多肽序列FLLEPHLMDTSM ,推测FLEP是肝癌细胞特异性结合肽的基序。免疫荧光共聚焦成像进一步确定展示序列FLLEPHLMDTSM的噬菌体与肝癌细胞特异性结合并内化入细胞内。竞争抑制实验示WH16能有效抑制展示序列FLLEPHLMDTSM的噬菌体克隆与肝癌细胞的结合。结论　利用噬菌体随机肽库获得能与肝癌细胞特异性结合的多肽序列FLLE PHLMDTSM 。Objective To explore the feasibility of screening and isolating homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide library and to develop a new peptide which may be potentially used as targeting delivery carrier in the biological targeted diagnosis or therapy for liver cancer. Methods A 12-mer peptide phage display library was used to screen and isolate peptides that bind to human hepatocarcinoma cells, and four rounds of subtractive panning were carried out with the human hepatocarcinoma cell line HepG2 as the target. The affinities of selected phage clones for human hepatocarcinoma cells were determined with enzyme-linked immunosorbent assay (ELISA) and compared with that to human liver cell and other tumor cells of different tissue origins, respectively. In addition, the binding site in the tumor cells was observed with immunofluorescence analysis under confocal light microscopy. The amino acid sequences of phages that bind HepG2 specifically were deduced through DNA sequencing. Based on the results of DNA sequence, a 16-mer peptide (WH16) was designed and synthesized. Binding ability of the new peptide, WH16, was determined with competitive inhibition test. Results After four rounds of panning, the phages that were bound to and internalized in human hepatocarcinoma cells were isolated. ELISA and immunofluorescence analysis confirmed the affinity of these phages for hepatocarcinoma cells. 56.67%(17/30) of the isolated phages displayed repeated sequence FLLEPHLMDTSM, and FLEP was defined as conservative motif. Binding of the selected phage to HepG2 cells was inhibited by synthesized peptide WH16, that strongly support that cellular binding of the phage is mediated through its displayed peptide, and WH16 can also bind to HepG2. Conclusions It is feasible to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide libraries. The sequence of peptide that can bind

关 键 词：肝癌细胞 噬菌体 体克 多肽序列 特异性结合肽 阳性 筛选 克隆 竞争抑制 DNA序列 

分 类 号：R735.7[医药卫生—肿瘤] R373[医药卫生—临床医学]