艰难梭菌二元毒素A链适配体的筛选和鉴定  

Screening and identification of Clostridium difficile binary toxin A chain aptamer

作　　者：苏恒 刘红菊 吴爱武 SU Heng;LIU Hong-ju;WU Ai-wu(KingMed School of Laboratory Medicine of Guangzhou Medical University,Guangzhou,Guangdong 510182;Clinical Laboratory Department of Foshan Women's and Children's Hospital,Foshan,Guangdong 528000,China)

机构地区：[1]广州医科大学金域检验学院,广东广州510182 [2]佛山市妇幼保健院检验科,广东佛山528000

出　　处：《热带医学杂志》2021年第11期1396-1399,共4页Journal of Tropical Medicine

基　　金：广州市科技计划项目(201510010241)。

摘　　要：目的筛选和鉴定艰难梭菌(CD)二元毒素A链适配体(Apt)。方法利用指数富集配体系统进化(SELEX)技术筛选CDTa适配体,并使用非竞争ELISA法测定其亲和常数(KA)。结果成功构建了pET-28b-CDTa原核表达载体,并诱导表达出CDTa蛋白。经过11轮筛选,文库中随机ssDNA与二元毒素A链的结合率从3.2%增加到42.3%,筛选出来39条适配体,二级结构最稳定的是Apt-A53,其亲和常数为3.75×10-6。结论利用SELEX技术可成功筛选出高亲和力的艰难梭菌二元毒素A链适配体,为临床上高毒力艰难梭菌感染的实验室快速诊断提供一定的参考工具。Objective To screen and identify the aptamer(Apt) of Clostridium difficile(CD) binary toxins A chain.Methods The CDTa aptamers were screened by systematic evolution of ligands by exponential enrichment(SELEX)technique and their affinity constant(KA) was determined by non competitive ELISA.Results The pET-28 b-CDTa prokaryotic expression vector was successfully constructed and the CDTa protein was induced to express.After 11 cycles of screening,the binding rate of random ssDNA to the binary toxin A chain in the library increased from 3.2% to 42.3%,39 aptamers were selected randomly and sequenced,and the aptamer with the most stable secondary structure was the AptA53,whose affinity constant was 3.75×10-6.Conclusion The aptamers against CDTa was successfully obtained by SELEX,which might provide reference for the laboratory diagnosis of Clostridium difficile infection.

关 键 词：艰难梭菌二元毒素A链 SELEX 适配体 亲和常数 

分 类 号：R446.5[医药卫生—诊断学]