肝脏去唾液酸糖蛋白受体特异性RNA适配子的SELEX筛选与鉴定  

Selection and Identification of RNA Aptamer Specific for Hepatic Asialoglycoprotein Receptor by SELEX

作　　者：刘嘉[1] 杨燕[2] 胡斌[1] 马智勇[1] 余源[2] 黄红平[2] 陆蒙吉[3] 冯新华 郭培宣[5] 杨东亮[1,2] 

机构地区：[1]华中科技大学同济医学院附属同济医院临床免疫研究室,湖北武汉市430030 [2]华中科技大学同济医学院附属同济医院实验医学研究中心,湖北武汉市430030 [3]华中科技大学同济医学院微生物学教研室,湖北武汉市430030 [4]贝勒医学院分子与细胞牛物学系TX77030,美国 [5]辛辛那提大学医学院生物医学工程系OH45267,美国

出　　处：《医学分子生物学杂志》2009年第3期197-202,共6页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30571646）,国家高技术研究发展计划资助项目（863计划）（No.2006AA02Z128）

摘　　要：目的获得能够特异性高亲和力结合肝脏特异性去唾液酸糖蛋白受体（asialoglycoprotein receptor，ASGPR）的RNA适配子，为开发诊断和治疗肝脏疾病的靶向性试剂和药物奠定基础。方法合成一个长度为115nt含有25个随机序列的单链DNA随机文库，通过体外转录构建出单链RNA适配子随机文库，以肝脏ASGPR大亚基为靶蛋白，采用SELEX（systematic evolution of ligands by exponential enrichment）技术筛选具有高亲和力的AsGPR特异性RNA适配子；通过膜结合测定实验、凝胶阻滞实验鉴定筛选适配子对靶蛋白的特异性和亲和力。结果经过12轮筛选获得了具有高亲和力的肝脏ASGPR特异性RNA适配子。结论成功地筛选出了具有离亲和力的肝脏ASGPR特异性RNA适配子库。Objective To obtain RNA aptamer with high affinity and specificity to human liver specific asialoglycoprotein receptor （ ASGPR）, with the aim of developing new reagents or drugs for diagnosis and targeting treatment of liver diseases. Methods A single-stranded 115 nt random DNA library containing 25 random oligonucleotides was synthesized in vitro. Random RNA aptamers library was constructed by in vitro transcription. Aptamers that can specifically bind to human liver asialoglycoprotein receptor （ASGPR） were isolated from the RNA random library by use of the SEL-EX（systematic evolution of ligands by exponential enrichment） procedure. Filter biding assay and gel shift assay were performed to determine the specificity and affinity of the selected aptamers. Results and conclusion RNA aptamer that can specifically bind to ASGPR with high affinity was successfully obtained after 12 rounds of selection.

关 键 词：适配子 SELEX技术 去唾液酸糖蛋白受体 

分 类 号：Q819[生物学—生物工程]