法医学杂志

法医学杂志 ›› 2014, Vol. 30 ›› Issue (5): 346-349.DOI: 10.3969/j.issn.1004-5619.2014.05.007

• 论著 • 上一篇    下一篇

FluMag-SELEX技术体外筛选识别氯胺酮的DNA适体

孙美琪1，2，曹芳琦3，胡小龙2，张玉荣2，陆鑫蔚1，2，曾立波2   

  1. （1. 中国医药工业研究总院上海医药工业研究院 创新药物与制药工艺国家重点实验室，上海 200437； 2. 上海市公安局物证鉴定中心 法医物证学现场应用技术公安部重点实验室 上海市现场物证重点实验室，上海 200083； 3. 上海市刑事科学技术研究院，上海 200083）
  • 发布日期:2014-10-25 出版日期:2014-10-28
  • 通讯作者: 曾立波，男，研究员，主要从事分子免疫学研究和法医毒物、毒品分析研究；E-mail：merry.magin@163.com
  • 作者简介:孙美琪（1987—），女，山东枣庄人，硕士研究生，主要从事毒物分析与分子免疫学研究；E-mail：mqsun1216@163.com
  • 基金资助:

    国家重点基础研究发展计划（973计划）（2012CB722 309）；上海市科委项目（11231203200）

DNA Aptamer Selection in vitro for Determining Ketamine by FluMag-SELEX

SUN MEI-QI1,2, CAO FANG-QI3, HU XIAO-LONG2, ZHANG YU-RONG2, LU XIN-WEI1,2, ZENG LI-BO2   

  1. （1. State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 200437, China; 2. Shanghai Key Laboratory of Crime Scene Evidence, Key Laboratory of Forensic Evidence and Science Technology, Ministry of Public Security, Institute of Forensic Science, Shanghai Public Security Bureau, Shanghai 200083, China; 3. Shanghai Research Institute of Criminal Science and Technology, Shanghai 200083, China）
  • Online:2014-10-25 Published:2014-10-28

摘要： 目的 利用FluMag-SELEX技术筛选出能特异性识别氯胺酮的DNA适体。 方法 体外合成78 bp的随机ssDNA文库，氯胺酮作为靶分子，表面修饰甲苯磺酰基的磁珠作为固相载体筛选特异性识别氯胺酮的适体。经过13轮筛选后进行DNA克隆测序，并进行一、二级结构分析。通过对荧光强度的分析检测适体的亲和性、特异性和Kd值。 结果 获得2条ssDNA适体Apt#4和Apt#8，能够特异性地与靶分子氯胺酮结合，Kd值分别为0.59和0.66 μmol/L。二级结构预测以茎环和G-四聚体为主，茎结构可能是适体结构稳定的基础，环和G-四聚体结构可能是与氯胺酮特异性结合的关键。 结论 FluMag-SELEX技术可以较好地提高适体的筛选效率，得到能特异性识别氯胺酮的DNA适体，有望用于氯胺酮的快速检测。

关键词: 分子生物学, 氯胺酮, SELEX适体技术

Abstract: Objective To select specific DNA aptamer for determining ketamine by FluMag-SELEX. Methods Based on magnetic beads with tosyl surface modification as solid carrier and ketamine as target, a random ssDNA library with total length of 78 bp in vitro was compounded. After 13 rounds screening, DNA cloning and sequencing were done. Primary and secondary structures were analyzed. The affinity, specificity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity. Results Two ssDNA aptamers （Apt#4 and Apt#8） were successfully selected with high and specific abilities to bind ketamine as target with Kd value of 0.59 and 0.66 μmol/L. The prediction of secondary structure was main stem-loop and G-tetramer. The stem was the basis of stability of aptamer’s structure. And loop and G-tetramer was the key of specific binding of ketamine. Conclusion FluMag-SELEX can greatly improve the selection efficiency of the aptamer, obtain the ketamine-binding DNA aptamer, and develop a new method for rapid detection of ketamine.

Key words: molecular biology, ketamine, SELEX aptamer technique

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