应用噬菌体展示技术筛选缺氧诱导因子-1α相关肽  

The Screen of Relative Peptide of Hypoxia Inducible Factor-Ice from a Phage Displayed Library

作　　者：张爱霞[1] 王斌[1] 肖继皋[1] 

机构地区：[1]南京医科大学药理学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2003年第3期193-195,202,共4页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学青年基金(30000207);教育部留学回国人员基金;江苏省教育厅高校高新技术产业化基金(JH01-50);江苏省教育厅自然科学基金(KJB310001)重点资助项目

摘　　要：目的:应用噬菌体展示12肽库筛选低氧诱导因子-1α(HIF-1α)相关肽。方法:以抗HIF-1α单克隆抗体(HIF-1αmAb)为配基,采用亲和免疫法从随机12肽库中筛选与抗体特异结合的噬菌体。在筛选过程中,逐轮提高HIF-1α mAb的稀释度并增强洗脱强度。3轮筛选后,随机挑取10个克隆测序。通过膜斑点印迹(Dot-ELISA)进行特异性结合鉴定。结果:经过3轮筛选后,特异性结合的噬菌体得到有效的富集,并且Dot-ELISA反应阳性逐轮增强。测序后发现有4个克隆的序列完全相同,其12肽氨基酸序列为GPHHYWYHLRLP。结论:噬菌体展示肽库技术可以用于筛选HIF-1相关肽,并为后续的活性鉴定打下基础。Objective: To screen relative peptide of HIF-1α from 12 mer phage display library. Methods: The HIF-1α mAb was used to react with a randomly sequenced peptide library of 12 amino acides. The dilutions of HIF-la mAb were 1: 50, 1: 100, and 1: 200 respectively and the concentrations of Tween-20 in the eluent were increased from 0. 1% (V/V) to 0. 5% (V/V) namely from the first to the third round of selection. After the third round of biopanning, 10 clones of phages were randomly picked and their DNA inserts were sequenced. Dot-ELISA was employed to test the competency of clones bound to specific HIF-la mAb. Results: The libraries were enriched about 200-fold through 3 rounds of biopanning. The results of Dot-ELISA showed that the competency of phages bound to HIF-1α mAb increased gradually within three rounds of selection. Four of ten clones have the same DNA sequence, and their corresponding peptide sequences are: GPHHYWYHLRLP. Conclusion: Phage display library may be useful in selecting the relative peptide of HIF-1.

关 键 词：缺氧诱导因子-1Α 噬菌体展示肽库 相关肽 亲和筛选 测序 

分 类 号：R965[医药卫生—药理学] Q781[医药卫生—药学]