SELEX技术筛选HCV非结构蛋白NS3的DNA适配子  

Selection of High-Affinity DNA aptamer binding to HCV NS3 protein by SELEX

作　　者：徐国胜[1,2] 骆晓枫[1] 黄小荣[1] 林昌盛[1] 谢金卫[1] 周俊宜[1] 

机构地区：[1]中山大学中山医学院,广东广州510080 [2]广州血液中心,广东广州510095

出　　处：《热带医学杂志》2015年第5期600-602,609,共4页Journal of Tropical Medicine

基　　金：广东省科技计划项目(2010B031200013)

摘　　要：目的用配基指数富集的系统进化技术(SELEX)技术筛选丙型肝炎病毒(HCV)非结构蛋白NS3的DNA适配子。方法构建随机ss DNA文库,两端为固定序列,中间为39 nt的随机序列,总长度为85 nt,将NS3蛋白包被在96孔板上,ss DNA与NS3蛋白进行亲和反应,洗去未结合的ss DNA,洗脱回收与NS3蛋白结合的DNA,PCR扩增后,磁珠法分离成单链。重复以上筛选步骤,直至筛选出高特异性和亲和力的核酸适配子。将核酸适配子连接质粒载体克隆转入大肠杆菌,挑选阳性克隆提取质粒外送到公司进行测序。结果通过8轮筛选,成功筛选出DNA适配子,并获得其中6条克隆的DNA一级序列。6条适配子序列长度相同,均为85 nt,两端固定序列与设计相符,中间为随机序列,随机序列无共同保守系列。所有寡核苷酸适配子的二级结构以茎环和凸环为主。结论以NS3蛋白作为药物研究的靶点,结合SELEX筛选技术研究其核酸适配子,具有巨大的潜力和研究价值。Objective Given many advantages over antibodies, the aptamer has shown its potential in therapeatic and commercial applications. This paper was focused on the screening of the HCV-NS3 protein in vitro. Methods The Sytematic Evolution of Ligands by Exponential enrichment（SELEX） is a novel in vitro approach used in this study that allow rapid screening of aptamers that bind to target protein with high affinity. Results eight rounds of selection were performed and we obtained the NS3 specific aptamer that could bind the purified NS3 protein with high specificity.Conclusion These aptamers are of import and value for further studies on the basis of their abilities to bind the HCV NS3 protein and inhibit polymerase activity.

关 键 词：配基指数富集的系统进化技术 丙型肝炎病毒 NS3蛋白 适配子 

分 类 号：Q503[生物学—生物化学]