高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (4): 638.doi: 10.7503/cjcu20140951

• 分析化学 • 上一篇    下一篇

基于胶体金检测核酸适配子与蛋白相互作用的新方法

刘中成1,2(), 赵丽健1,2, 刘利鹏1,2, 张艳芬3, 王南南1,2, 陈瑶1,2, 王向欢1,2   

  1. 1. 河北大学药学院
    2. 河北省药物质量分析控制重点实验室
    3. 科学技术处, 保定 071002
  • 收稿日期:2014-10-27 出版日期:2015-04-10 发布日期:2015-03-19
  • 作者简介:联系人简介: 刘中成, 男, 博士, 副教授, 主要从事生物药学及分子药理学研究. E-mail: liuzc@hbu.edu.cn
  • 基金资助:
    国家自然科学基金(批准号: 81202338)、 河北省自然科学基金(批准号: H2013201128)和中国博士后科学基金(批准号: 2013M530885)

Novel Method Based on Colloidal Gold for Detection of Oligonucleotide Aptamer with Protein Interacting

LIU Zhongcheng1,2,*(), ZHAO Lijian1,2, LIU Lipeng1,2, ZHANG Yanfen3, WANG Nannan1,2, CHEN Yao1,2, WANG Xianghuan1,2   

  1. 1. College of Pharmaceutical Sciences
    2. Pharmaceutical Quality Control of Hebei Province
    3. Science and Technology Office, Hebei University, Baoding 071002, China
  • Received:2014-10-27 Online:2015-04-10 Published:2015-03-19
  • Contact: LIU Zhongcheng E-mail:liuzc@hbu.edu.cn
  • Supported by:
    † Supported by the National Natural Science Foundation of China(No.81202338), the Natural Science Foundation of Hebei Province, China(No.H2013201128) and the Postdoctoral Science Foundation of China(No.2013M530885)

摘要:

基于核酸适配子对靶蛋白的高特异性及胶体金比色法的高度灵敏性, 建立了一种分析核酸适配子与靶蛋白亲和力以及简便快速检测蛋白质的新方法. 核酸适配子保护的胶体金在高盐条件下可保持稳定, 靶蛋白与胶体金竞争结合适配子, 使胶体金发生聚沉, 呈现颜色变化, 可通过检测A520值分析适配子与靶蛋白的亲和力以及蛋白浓度. 通过对适配子浓度、 靶蛋白浓度、 共孵育时间、 竞争反应时间和氯化钠浓度等关键因素进行优化, 确定了最佳检测条件.

关键词: 核酸适配子, 靶蛋白, 胶体金, 亲和力

Abstract:

A novel method was developed for assaying affinity of aptamer with target protein and detecting the protein, which was based on the specific recognition of aptamers toward targets and high sensitivity of colloidal gold colorimetric. Oligonucleotide aptamer can protect colloid gold from coagulation in high salt conditions. When the aptamer bound to the target protein, the naked gold nanoparticles aggregated and the color of the solution changed. Then the affinity of target protein with aptamer and the concentration of target protein could be detected by monitoring A520 value. The best detection system was established, while the principal influence factors, such as the amount of aptamer and target protein, incubation time, competitive reaction time and the concentration of sodium chloride, were optimized.

Key words: Oligonucleotide aptamer, Target protein, Colloidal gold, Affinity

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