唾液酸化路易斯抗原X ssDNA适配子的筛选与鉴定  

Selection and verification of ssDNA aptamers to Sialyl LewisX by SELEX technique

作　　者：吴红艳[1] 谢伟[1] 冯茂辉[1] 段丽[1] 余佳莉[1] 陈家宽[1] 吴洲清[1] 谢辉[1] 汪付兵[1] 

机构地区：[1]武汉大学中南医院肿瘤外科肿瘤生物学行为湖北省重点实验室,430071

出　　处：《中华实验外科杂志》2009年第11期1546-1548,共3页Chinese Journal of Experimental Surgery

基　　金：武汉市科技攻关计划重点资助项目（200860423230）;湖北省自然科学基金资助项目（2004ABA159）;湖北省科技攻关计划资助项目（2005AA304B05、2005AA304B04）

摘　　要：目的建立体外获得唾液酸化路易斯抗原X（SLeX）ssDNA适配子（aptamers）的方法。方法构建长度为70m的初始随机单链DNA库，其中含30个随机序列，以溴化氰活化的琼脂糖小球为筛选介质，运用指数富集配体的系统进化技术（SELEX技术）获得SLeX的ssDNA适配子。将适配子库克隆、测序，采用DNAMAN软件对其结构进行分析，通过生物素-亲和素-辣根过氧化物酶显色系统测定适配子与SLeX的亲和力。结果经逐轮筛选，所得富集库与SLeX的亲和力逐步提高（A值从0．145增加到了0．460），大部分适配子克隆测序后与预期相符。结论经9轮筛选获得的ssDNA适配子与SLeX的体外结合有高度的特异性及亲和力。Objective To set up a method to obtain single-stranded DNA aptamers of Sialyl LewisX in vitro by SELEX technique and lay a foundation of application of these aptamers to metastasis of carcinoma in early diagnosis and targeted therapy. Methods A single-stranded DNA library containing 30 random sequences was constructed, at a length of 70nt. Chost cyanogen bromide （CNBr）-activated agarose beads served as screening medium, and the ssDNA aptamers of slex were obtained by SELEX technique. The aptamers were cloned and sequenced, and DNAMAN package was employed to analyze the conserved equences and the second structure of the aptamers. The affinity of aptamers to slex was determined by chromatic biotin-streptavidin-horseradish peroxides system. Results Through screening aptamers round by round, the affinity of aptamers to slex was increased gradually （ the absorbance values were increased from 0. 145 to 0.560）. Most aptamers had the correct length and fix-sequence after cloning and sequencing. Conclusion Mter selection of 9 rounds, the ssDNA aptamers we obtained had high specificity and affinity to slex in vitro.

关 键 词：唾液酸化路易斯抗原X 适配子 克隆 

分 类 号：R392.1[医药卫生—免疫学]