FluMag-SELEX技术体外筛选识别氯胺酮的DNA适体  

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

作　　者：孙美琪[1,2] 曹芳琦 胡小龙[2] 张玉荣[2] 陆鑫蔚 曾立波[2] 

机构地区：[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

出　　处：《法医学杂志》2014年第5期346-349,共4页Journal of Forensic Medicine

基　　金：国家重点基础研究发展计划(973计划)(2012CB722309);上海市科委项目(11231203200)

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78bp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0.66μmol/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。Objective To select specific DNA aptamer for determining ketamine by FluMag-SELEX. Meth- ods 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, speci- ficity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity. Re- suits 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 p, mol/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.

关 键 词：分子生物学 氯胺酮 SELEX适体技术 

分 类 号：DF795.2[医药卫生—法医学]