运用mRNA体外展示技术筛选胸苷酸合成酶RNA亲和肽  

Identification of Peptides That Bind With Thymidylate Synthase RNA Using mRNA Display Technique

作　　者：阎松[1,2] 牛荣丽[1] 张培军[1] 林秀坤[1,3] 

机构地区：[1]中国科学院海洋研究所,实验海洋生物学重点实验室 [2]中国科学院研究生院,北京100039 [3]Department of Medicine and Pharmacology,Yale University School of Medicine,New Haven CT 06520,USA

出　　处：《生物化学与生物物理进展》2005年第11期1081-1087,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30472043).~~

摘　　要：以体外选择方法筛选不同功能的核酸、肽和蛋白质是近年的研究热点,mRNA体外展示是一种新兴的高效多肽选择技术,其基本原理是通过含嘌呤霉素寡核苷酸的Linker使mRNA与它编码的肽或蛋白质共价结合,形成mRNA-蛋白质融合体,这一方法已用于多种功能肽的鉴定.以mRNA体外展示技术进行了由大容量多肽库中(>1013)筛选胸苷酸合成酶(thymidylatesynthase,TS)RNA亲和肽的研究,通过精密的实验设计,建立了一套完整有效的筛选方法,并对实验条件进行了优化.已进行了8轮筛选,结果表明,以mRNA体外展示技术获得的多肽分子,可以与TSmRNA亲和.将测序结果与初始肽库进行比较,发现亲和肽中碱性氨基酸及芳香族氨基酸含量明显增加,说明其在与RNA结合中具有重要作用.mRNA展示技术作为一种大容量文库的体外筛选方法,将广泛应用于与固定化靶物质具高度亲和性及特异性的多肽和蛋白质的筛选.Using in vitro selection method to isolate nucleic acids, peptides and proteins has been studied intensively in recent years. In vitro mRNA display is a new and effective technique for peptides selection, and the rationale of this technique is that a synthetic mRNA with puromycin could covalently link with the protein that it encodes, thus an mRNA-protein fusion is formed. This approach has been used in identification of many functional peptides. The peptides binding with thymidylate synthase RNA were isolated using mRNA display technique from a large peptide library （〉10^13 different sequences） . The selection scheme was constructed, and the experimental conditions, including library synthesis, formation of RNA-peptide fusion and RNA immobilization were optimized. Eight cycles have been processed and the results confirmed that the selected peptides could bind with thymidylate synthase mRNA specifically. Compared the amino acid sequences of the selected peptides with those from the initial random library, the basic and aromatic residues in selected peptides were enriched significantly, suggesting these peptide regions may be important in the peptide-TS mRNA interaction. As a novel in vitro selection approach, mRNA display technique would be developed as a powerful tool for isolation of functional peptides and proteins that could interact with immobilized targets with high affinity and specificity.

关 键 词：mRNA体外展示 筛选 胸苷酸合成酶 亲和肽 

分 类 号：Q78[生物学—分子生物学]