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.4No.2
2013年4月JournalofFoodSafetyandQualityApr.,2013

甲胺磷的核酸适体筛选

1,2111112*
张强,胡秋莲,张冠,邱琳,陈几文,邱洪斌,刘贤金

(1.佳木斯大学公共卫生学院,佳木斯154007;2.江苏省农业科学院江苏省食品质量安全重点实验室
—省部共建国家重点实验室培育基地,南京210014)

摘要:目的筛选识别甲胺磷的DNA适体。方法体外合成ssDNA文库,将其固相化于琼脂糖凝胶颗粒
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技术,最终获得识别甲
胺磷的适体,采用DNAMAN和RNAstructure软件对适体进行一、二级结构分析。结果经过10轮筛选,
ssDNA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个
家族,二级结构预测以茎环结构为主。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磷
的DNA适体。
关键词:甲胺磷;适体;食品安全;检测技术

Selectionofaptameragainstmethamidophos

ZHANGQiang1,2,HUQiu-Lian1,ZHANGGuan1,QIULin1,CHENJi-Wen1,
QIUHong-Bin1,LIUXian-Jin2*

(1.CollegeofPublicHealth,JiamusiUniversity,Jiamusi154007,China;
2.KeyLabofFoodQualityandSafetyofJiangsuProvince—StateKeyLaboratoryBreedingBase,MinistryofAgriculture,
JiangsuAcademyofAgriculturalScience,Nanjing210014,China)

ABSTRACT:ObjectiveToselectaptamersagainstmethamidophos.MethodsThessDNApoolwhichwas
synthesizedinvitrowasimmoboilizedonsurfaceofagarosegel.ThessDNAsthatboundmethamidophoswere
releasedfromsurfaceofagarosegel.Methamidophosaptamerswereselectedbynon-solidphaseSELEX.The
primaryandsecondarystructureofmethamidophosaptamerswereanalysedbyDNAMANandRNAstructure
software.ResultsAffinitybetweenssDNApoolandmethamidophoswasincreasedafter10selectionrounds.
The20ssDNAswhichwerepositiveclonesweredividedintofivegroupsbasedonprimarysequencehomology.
Hairpinloopwasthemainmotifinthepredictedsecondarystructureanditwassupposedtobethebindingpart
oftheaptamerstomethamidophos.ConclusionTheefficiencyofscreeningoftheaptamerwasgreatlyim-
provedusingthismethodandthemethamidophos-specificDNAaptamerswereselectedsuccessfully.
KEYWORDS:methamidophos;aptamer;foodsafety;detectiontechnology

基金项目:黑龙江省卫生厅项目(2011-352)、佳木斯大学重点项目(Sz2011-006)、佳木斯大学大学生创新创业训练计划项目(2012sj005)
Fund:SupportedbyProjectofHealthDepartmentofHeilongjiangProvince(2011-352),TheKeyProjectsofJiamusiUniversity(Sz2011-006)
andCollegeStudents'InnovativeEntrepreneurialTrainingProject,JiamusiUniversity(2012sj005)