基于氧化石墨烯-SELEX技术对氟苯尼考核酸适配体的筛选及其结构分析  

Screening and structure analysis of florfenicol based aptamers based on graphene oxide SELEX technologytitis

作　　者：肖海华 王君雁 李劼[1] XIAO Hai-hua;WANG Jun-yan;LI Jie(School of Animal Science and Technology,Shihezi University,Shihezi 832003,China)

机构地区：[1]新疆石河子大学动物科技学院,新疆石河子832003

出　　处：《中国预防兽医学报》2021年第5期468-476,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：新疆兵团科技局高新技术攻关项目(2016AB014)。

摘　　要：氟苯尼考(FFC)具有胚胎毒性,在食用动物组织中残留会严重危害消费者的健康与安全。为建立快速检测动物性食品中FFC残留的方法奠定基础,本研究根据引物设计原则,设计了上下游引物,根据引物序列构建长度为80 bp的随机单链寡核苷酸(ssDNA)文库,其3'端和5'端为固定的20 bp引物结合位点,中间为40 bp的随机序列。通过氧化石墨烯-富集配体系统进化技术(GO-SELEX)筛选,即将经过预处理的ssDNA文库与等摩尔量的FFC及氧化石墨烯(GO)沉淀共孵育。通过GO的π-π共轭键吸附未结合靶标FFC的ssDNA,已结合靶标的ssDNA(FFC-ssDNA)则留在上清液中,通过离心获得FFC-ssDNA复合物。将每一轮筛选的FFC-ssDNA复合物经离心、抽提、醇沉淀回收所得到的ssDNA做为模板进行PCR扩增,通过链霉亲和素将PCR产物的双链DNA(dsDNA)彻底裂解为单链,作为次级ssDNA文库进入下一轮筛选。每一轮的筛选均计算一次回收率,当回收率变化趋于稳定时,引入反筛选物氯霉素(CAP)、甲砜霉素(TAP),以去除与FFC特异性结合较差的ssDNA。当回收率达到饱和,筛选停止。最终,根据回收率的计算结果共进行了12轮核酸适配体的筛选,其中有10轮为正筛选,第7轮和第11轮为反筛选。PCR结果显示,经12轮筛选均得到了所需要的目的ssDNA片段。对第9轮和第12轮的筛选产物克隆并测序,共得到46条不同的核酸适配体序列。经对46条核苷酸序列的二级结构、自由能(△G)预测和亲缘性分析,最终得到A1、A26、A27、A31、B18、B28、B30、B37共8条候选核酸适配体。通过测定8条候选适配体的解离常数(Kd值)分析其与FFC的亲和性。结果显示,8条候选适配体的Kd值在11.811 nmol/L~54.097 nmol/L;利用相同浓度的FFC、CAP、TAP对其中亲和性较高的A26和B18进行特异性试验,结果表明两条核酸适配体均可以与FFC特异性结合,且B18与FFC的特异性结合作用更强,可作为FFC的特异性核酸�Florfenicol(FFC)is embryotoxicity toxic,and its residues in food animal tissues can seriously harm the health and safety of consumers.In order to establish a method for rapid detection of FFC residues in animal food,the upstream and downstream primers were designed according to the primer design principle.A random single stranded oligonucleotide(ssDNA)library with the length of 80bp was constructed according to the primer sequence.The 3'and 5'ends were fixed 20bp primer binding sites,with a random sequence of 40bp in the middle.Through GO-SELEX screening,the pretreated ssDNA library was coincubated with equal moles of FFC and GO.Through GOπ-πthe conjugated bonds adsorbed ssDNA that did not bind to the target FFC,while the ssDNA that had bound to the target(FFC ssDNA)was left in the supernatant,then the FFC ssDNA complex was obtained by centrifugation.After centrifugation,extraction and alcohol precipitation,the ssDNA obtained from FFC ssDNA complex in each round of the screening was used as template for PCR amplification.The double stranded DNA(dsDNA)of PCR product was completely split into single strand by streptavidin,which was used as secondary ssDNA library to enter the next round of screening.The recovery rate was calculated once after each round of screening.When the change of recovery rate tended to be stable,the antiscreening products of chloramphenicol(CAP)and thiamphenicol(TAP)were introduced to remove the ssDNA with the poor specificity for FFC.When the recovery reaches saturation,screening stops.Finally,12 rounds of aptamer screening were carried out according to the calculation results of recovery rate,of which 10 rounds were positive screening,and the seventh and eleventh rounds were reverse screening.PCR results of ssDNA obtained from 12 rounds of screening showed that the desired ssDNA fragments were obtained through 12 rounds screening.Forty-six different aptamer sequences were obtained by cloning and sequencing the products from the ninth and twelfth rounds.Based on the secondary structure,fre

关 键 词：氧化石墨烯 氟苯尼考 核酸适配体 指数富集配体系统进化技术 

分 类 号：S851.4[农业科学—预防兽医学]