乙酰胆碱酯酶核酸适体快速筛选的新方法研究

项目名称： 乙酰胆碱酯酶核酸适体快速筛选的新方法研究

项目编号： No.21307064

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 环境科学、安全科学

项目作者： 马翠萍

作者单位： 青岛科技大学

项目金额： 25万元

中文摘要： 目前，基于核酸适体的分析方法成为分析化学研究的热点领域。但有效的核酸适体太少成为制约核酸适体应用发展的瓶颈问题，而寡核苷酸-靶分子复合物与游离寡核苷酸难以快速有效分离是适体筛选过程中的关键问题。 在本课题中，以水体环境污染的生物标志物乙酰胆碱酯酶（Acetylcholinesterase）作为靶标蛋白，利用结合乙酰胆碱酯酶的核酸适体和游离寡核苷酸链在杂交时的Tm值的差异，结合靶标的核酸适体有效互补延伸形成模板，随后被PCR复制放大，实现与游离随机寡核苷酸链的快速分离。为了增强筛选的有效性，借鉴分子信标的分子结构，设计随机寡核苷酸链的自身互补结构开关，以加强溶液中开关反应的效率，建立一种新的结合乙酰胆碱酯酶的核酸适体筛选方法。 此方法的建立将对核酸适体的快速有效筛选提供方法学支持，并为建立基于核酸适体的环境分析检测方法奠定分子识别基础。

中文关键词： 分子开关；核酸适体筛选；环境污染检测；乙酰胆碱酯酶；

英文摘要： At present, the aptamer-based analytical methods have become hot areas of analytical chemistry. However, aptamer screening has been a bottleneck of the application of aptamer. It is the most important problem in aptamer screening that the complex of oligonucleotide and target and free oligonucleotide are very difficult to separate. Acetylcholinesterase （AChE）is often used as target in environmental contamination detection. So we select AChE as target protein in our research. When AChE combines with its aptamer, the Tm value is different with that of the free oligoucleotide. Thus, the oligonucleotides modified by AChE are extended to form the template of PCR and are amplified by PCR. Therefore, free oligonucleotides are successfully seperated. In addition, we further design oligonucleotides to be a self-complementary structure based on molecular beacon to improve aptamer screening. Finally, we will construct a novel method of AChE aptamer screening. The novel method will be fast and effective for aptamer screening of target protein, and greatly promote the development of analytical detection based on aptamer.

英文关键词： Molecular switch；Selection of aptamer；Detection of environmental contaminant；Acetylcholinesterase；