SELEX法体外筛选胃癌细胞适配子方法的建立  

In vitro Selection of Aptamers to Gastric Cancer Cells by SELEX

作　　者：李真真[1] 韩跃武[1] 刘玲玲[1] 韩亚萍[1] 路艳[1] 王春霞[1] 

机构地区：[1]兰州大学基础医学院医学生物化学与分子生物学研究所,甘肃兰州730000

出　　处：《生物技术》2009年第3期42-46,共5页Biotechnology

基　　金：国家"863"计划重大项目子课题(2006AA10A208-1-4);甘肃省科技攻关项目(2GS064-A43-020-21)资助

摘　　要：目的:建立SELEX技术筛选胃癌细胞SGC-7901适配子的方法,并初步鉴定获得的SGC-7901细胞适配子。方法:体外合成全长88bp中间含52bp随机序列的ssDNA文库,通过优化PCR扩增条件,利用地高辛-抗地高辛抗体-碱性磷酸酶系统测定亲和力,经SELEX反复筛选获得胃癌SGC-7901细胞的特异性适配子。将最后一轮筛选产物克隆、测序并用相关软件分析适配子序列的一级结构和二级结构。结果:经12轮SELEX筛选,ssDNA文库与SGC-7901细胞的亲和力由0.16上升至1.14,表明特异性适配子得到逐步富集。22个克隆子测序,有4个序列完全一致,二级结构预测茎环可能是适配子与胃癌细胞作用的结构基础。结论:成功建立了SELEX技术体外筛选胃癌细胞SGC-7901高亲和性适配子的方法。Objective：To establish a SELEX screening method and characterize the aptamers against SGC- 7901 gastric cancer cells. Method：A single stranded library with 88 bp in length containing a random sequence of 52 bp was in vitro synthesized, the PCR conditions for ssDNA amplification were optimized, and the binding affinity between ssDNA library of each round and gastric cancer cells was determined by Digoxigenin - anti - digoxigennin - AP system. After repeated screenings, the aptamers that specially bind to SC, C - 7901 gastric cancer cells were obtained by SELEX. PCR products of the last round selection were cloned and sequenced. Relative software was employed to analyze the primary structure and prediction secondary structure of the aptamers. Result： After 12 rounds of SELEX screening, the binding affinity between the aptamers and target cells inceeased from 0.16 to 1.14,indicating that specific aptamers were enriched progressively.There were 4 aptamers have the absolute identical sequence after cloning and sequencing, the secondary structure prediction revealed possible stem - loop was the binding sites between the aptamers and gastric cancer cells. Conclusion：The method of in vitro selection of aptamers to gastric cancer cells by SELEX was successfully established.

关 键 词：胃癌细胞 SELEX 适配子 随机ssDNA文库 

分 类 号：R735.2[医药卫生—肿瘤] Q789[医药卫生—临床医学]