甲胺磷的核酸适体筛选.pdf

第4卷第2期
食品安全质量检测学报
Vol 4 No. 2
2013年4月
Journal of Food Safety and Quality
Apr.,2013
甲胺磷的核酸适体筛选
张强2,胡秋莲,张冠,邱琳,陈几文,郵洪斌,刘贤金
(1.佳木斯人学公共卫生学院,佳木斯154007;2.江苏省农业科学院江苏省食品质量必全重点实验室
一省部共建国家重点实验室培育基地,南京210014)
摘要:目的筛选识别胺磷的DNA适体。方法体外合成sINA文库,将其固相化于琼脂糖凝胶粒
表面,液相中甲胺磷与其结合的DNA分子从固相表面分离,进而建立非固相化 SELEX找术,最终获得识别中
胺僯的适体,采用 DNAMAN和 RNA structure软件对适体进行一、二级结构分析。结果经过10轮筛选
sINA文库与甲胺磷的亲和力呈上升趋势,随机挑选的20个阳性克隆适体根据一级结构的同源性可分为5
家族,二级结构预测以茎坏结构为主。结论本方法大大提高了适体的筛选效率,最终筛选得到能识别甲胺僯
的DNA适体
关键词:甲胶磷;适体;食品父全;检测技术
Selection of aptamer against methamidophos
ZHANG Qiang"-, HU Qiu-lian, ZHANG Guan, QIU Lin, CHEN Ji-wen
QIU Hong-bin, LIU Xian-jin
(1. College o/Pub/ic dea/th, Jiamusi University, Jiamusi 154007, China
2. Key Lab of ood Quality and afety of iangsu Province State Key abrator Breeding Base, Ministry of griculture
liangs cademy o/4griczllaru/ Science, Najing 210014, China)
ABSTRACT: Objcctive To select aptamers against methamidophos. Methods The ss)NA pool which was
synthesized in vitro was immobilized on surface of agarose gel. The SSINAS that bound methamidophos were
released from surface of agarose gel. Methamidophos aptamers were selected by non-solid phase ST X. The
primary and sccondary structure of methamidophos aptamers were analysed by DNAMAN and RNA structure
software. Results Affinity betwecn SSDNA pool and methamidophos was incrcascd aftcr 10 sclcction rounds
Thc 20 SSDNAS which were positive clones were divided into five groups bascd on primary scqucnce homology
Hairpin loop was the main motif in the prcdicted sccondary structure and it was supposed to be the binding part
of the aptamers to methamidophos. Conclusion The cfficicncy of screening of the aptamer was greatly im-
proved using this method and the methamidophos-specific DNA aptamers were selected successfull
KEY WORDS: methamidophos, aptamer; food safcty, detection technology
基金项目:黑庀江省卫生厅项日(2011-352)、佳木斯大学重点项月(S72011-006)、佳木斯大学大学生创新创业训练计划项日(2012sj005)
Fund: Supported by Irojcct of Health Departmcnt of Heilongjiang Irovincc(2011-352) The Key Projects of Jiamusi University(Sz2011-006
and College Students' Innovative I ntrepreneurial Raining Project, Jiamusi University(20128j005)
通讯作者:刘贤金,研究员,主要研究方向为食品质量安全检测,E-Mail: jaasliu(ci jaas.ac.cn
Corresponding author LIU Xian-jin, Professor, Food Quality and Safcty and Detcction Institute, Jiangsu Acadcmy of Agricultural Scicncc
No. 50 Zhongling Strect, Nanjing 210014, China. E-mail: jaasliu(a jaasaccn