α-鹅膏[蕈]毒环肽核酸适配体的筛选和结构分析  

Screening and Structure Analysis of the Aptamers against α-Amanitin

作　　者：汪颖[1] 乔璞[1] 宋玉竹[1] 张金阳[1] 陈强[1] 韩芹芹[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500

出　　处：《中国生物化学与分子生物学报》2016年第12期1341-1346,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：云南省人才培养项目(No.KKSY201226103);云南省应用基础研究计划项目(No.KKS0201326002);云南省科技创新平台建设计划(No.2012DA002)资助~~

摘　　要：α-鹅膏[蕈]毒环肽(α-amanitin)是从致命鹅膏毒伞子实体中分离的多肽物质。本文采用配体指数富集系统进化(systemic evolution of ligand by exponential enrichment,SELEX)技术,以α-鹅膏[蕈]毒环肽为靶蛋白,以亲和填料epoxy-activated sepharose 6B为筛选介质,从体外合成的随机单链DNA文库中筛选其核酸适配体。经过12轮筛选,将第12轮筛选产物克隆测序,对获得的12条核酸适配体进行分析。二级结构预测分析表明,茎环和口袋结构为主要的结构形式,提示其可能是核酸适配体与α-鹅膏[蕈]毒环肽特异性结合的基础。对得到的核酸适配体进行特异性和灵敏度检测,其中E06核酸适配体的特异性最好,为核酸适配体检测蘑菇中α-鹅膏[蕈]毒环肽的残留奠定了基础。α-Amanitin is a kind of polypeptide isolated from the fruiting body of Amanita exitialis.According to systematic evolution of ligands by exponential enrichment（ SELEX） method,a ss DNA random library was subjected to 12 rounds of selection against α-amanitin. Affinity filler epoxy-activated Sepharose 6B was used as the screening medium. The twelfth round of screened product was cloned and sequenced. The secondary structure of the obtained 12 aptamers analysis revealed the stem loop and pocket were the main structures. We speculalted that they were the basic structure of specific binding of aptamer to α-amanitin. The results of aptamer specificity and sensitivity showed that E06 was the best one. Our results laid the foundation for detecting the α-amanitin in mushroom.

关 键 词：α-鹅膏毒肽 SELEX筛选核酸适配体技术 核酸适配体 核苷酸 

分 类 号：Q789[生物学—分子生物学]