噬菌体表面展示文库筛选人干细胞因子模拟肽  

Bio-panning of Human Stem Cell Factor Mimetic Peptides from Phage-displayed Random Peptide Library

作　　者：苏林[1] 孔燕[2,3,4] 刘长征[1] 杨克恭[1] 陈松森[1] 

机构地区：[1]中国医学科学院北京协和医学院基础医学研究所医学分子生物学国家重点实验室,北京100005 [2]北京大学临床肿瘤学院 [3]北京肿瘤医院 [4]北京市肿瘤防治研究所肾癌和黑色素瘤内科,北京100036

出　　处：《中国医学科学院学报》2011年第4期351-356,共6页Acta Academiae Medicinae Sinicae

基　　金：国家高技术研究发展计划项目(863项目)(2003AAZ3360)~~

摘　　要：目的用噬菌体表面展示文库筛选具有人干细胞因子(hSCF)生物学活性的模拟肽。方法采用酶联免疫吸附测定法(ELISA)从噬菌体环七肽库和十二肽库筛选与重组hSCF受体(rc-kit/Ig1-3)有较高亲和力的噬菌体克隆,提取噬菌体单链DNA进行序列测定,根据序列测定结果合成阳性小肽,四甲基偶氮噻唑盐(MTT)法检测合成小肽刺激UT-7细胞增殖的活性。结果经3轮筛选获得11个来源于环七肽库和8个来源于十二肽库与rc-kit/Ig1-3结合活性较高的噬菌体克隆,DNA测序结果显示环七肽库筛选到1个共有序列DPSPHTH,十二肽库没有筛选到共有序列。序列比对分析显示,这些小肽与hSCF没有同源序列。MTT法测定结果表明,合成的4个小肽均能促进UT-7细胞增殖,其中CE16和LE20刺激效果明显。结论获得4个具有较高hSCF生物学活性的模拟肽。Objective To screen human stem cell factor（hSCF） mimetic peptides in vitro with a phage-display random peptide library.Methods Phage clones with high hSCF receptor（rc-kit/Ig1-3）-binding activity was screened from phage-displayed random hepta/dodeca peptide library by phage enzyme-linked immunosorbent assay（ELISA）.Phage single DNA was extracted and sequenced.Four kinds of peptide with higher c-Kit/Ig1-3 binding activity were chosen for synthesis and characterized by using cell proliferation assay with 3-（4,5-dimethylthiazol-2-yl）2,5-diphenyltetrazolium bromide（MTT） method in UT-7 cells.Results Eleven Ph.D.-C7C clones and eight Ph.D-12 phage clones with high hSCF receptor-binding activity were selected from phage-displayed random hepta/dodeca peptide library,respectively.Sequence analysis showed there were no homologous sequence between hSCF and these screened mimetic peptides,except one homologous sequence DPSPHTH found in heptapeptide library.All these four synthesized peptides（CE3,CE16,LE4,and LE20）,particularly CE16 and LE20,stimulated UT-7 cell proliferation.Conclusion Four hSCF mimetic peptides were successfully isolated from phage-displayed random peptide library.

关 键 词：噬菌体表面展示文库 人干细胞因子 模拟肽 筛选 酶联免疫吸附测定法 

分 类 号：R542.220.5[医药卫生—心血管疾病]