靶向肿瘤干细胞标记CD133特异性结合肽的筛选和初步鉴定  

Selection and identification of specific-binding peptides for cancer stem cell surface marker CD133

作　　者：田平阁[1] 周春平 张超[1] 杨慧[1] 吴小金[1] 陆滟霞[1] 刘国炳[2] 李学农[1] 

机构地区：[1]南方医科大学基础医学院病理学系广东省分子肿瘤病理学重点实验室,广东广州510515 [2]南方医科大学南方医院妇产科,广东广州510515

出　　处：《南方医科大学学报》2011年第5期761-766,共6页Journal of Southern Medical University

基　　金：国家自然科学基金(30871156);广东省科技计划项目(2007B031001001;2009B030803041);广东省自然科学基金(8151051501000057)~~

摘　　要：目的筛选可与人肿瘤干细胞表面标记物CD133特异性结合的短肽并进行初步鉴定。方法首先合成人肿瘤干细胞表面标记物CD133细胞外片段的生物素化蛋白,以此为靶,利用噬菌体肽库技术,高通量液相淘选CD133的特异性短肽。应用夹心ELISA选择出结合较强的克隆。提取DNA测序,通过竞争性阻断实验验证其特异性,根据噬菌体基因序列推导出多肽序列。通过免疫荧光技术初步验证合成多肽和人大肠癌细胞的结合情况。结果 4轮液相筛选后,与CD133特异性结合的噬菌体得到有效富集,第4轮与第1轮相比,富集了388倍。经ELISA鉴定的20个噬菌体单克隆中,有13个与CD133有较高亲和力,具有阳性意义。经比对得到11条完全一致的氨基酸序列TISWPPR,2条完全一致的氨基酸序列STTKLAL,竞争性阻断ELISA实验表明第一条特异性较强。结论成功利用噬菌体肽库技术,淘选出1条可和人CD133特异性结合的高亲和力短肽,为后续CD133的研究奠定了基础,表明从噬菌体肽库中液相淘选生物素化小分子的高亲和力多肽的方法切实可行。Objective To select the peptides that specifically bind human cancer stem cell surface marker CD133 from the Ph.D.-7TM phage peptide library.Methods With a biotinated extracellular fragment of human cancer stem cell surface marker CD133 as the target protein,the CD133 high-affinity peptides were screened from the phage peptide library by liquid phase panning.The clones with high-binding force with human CD133 were then identified by sandwich ELISA and their single-stranded DNA was extracted to test the specificity by competitive ELISA.The amino acid sequences of the selected peptides derived from the phage DNA sequences were synthesized after sequence alignment analysis,and their capacity of binding with colorectal carcinoma cells was assessed by immunofluorescence technique.Results After 4 rounds of liquid phase selection,the phages capable of specific binding with human CD133 were effectively enriched,with an enrichment ratio of 388 times compared to that at the fourth and first rounds.Thirteen out of the 20 clones from the fourth round of panning were identified as positive clones,among which 11 had identical amino acid sequence of TISWPPR,and 2 had the sequence of STTKLAL,and the former sequence showed a stronger binding specificity to CD133.Conclusion We have successfully obtained a peptide that specifically binds human CD133 from the Ph.D.-7TM phage peptide library,demonstrating the feasibility of screening small molecule high-affinity polypeptides from phage peptide library by liquid-phase panning.

关 键 词：肿瘤干细胞 CD133 噬菌体展示 肽库 

分 类 号：R730.2[医药卫生—肿瘤]