河豚毒素DNA适配子的制备及应用

人工合成78个碱基的随机ss DNA文库,采用指数富集配基的系统进化技术与诱变聚合酶链式反应(polymerase chain reaction)结合的方法,通过筛选富集、克隆、测序,获得了与河豚毒素(tetrodotoxin,TTX)特异结合的单克隆DNA适配子A3。DNA适配子A3的二级结构主要为茎环结构,与TTX的亲和力为1.254。对适配子和TTX结合的磷酸盐缓冲液p H值、荧光染料结合时间进行优化,结果表明,最适p H值为7.5,最佳结合时间为10 min。建立的快速筛选检测TTX的DNA适配子-荧光染料法对TTX的检出限为10-6 mo1/L。     

河豚毒素DNA适配子核心区域的识别

用DNA folding form在线软件分析河豚毒素DNA适配子A3、G10的二级结构,根据二级结构,分别合成适配子A3、G10的各4个区域的5’端地高辛标记的序列。用丙烯酸-环氧乙烷珠子固定河豚毒素,测定抗地高辛碱性磷酸酶的适宜稀释倍数,用地高辛-抗地高辛碱性磷酸酶系统检测各区域与河豚毒素的亲和力,识别出适配子的核心区域。结果表明,抗地高辛碱性磷酸酶的适宜工作浓度为2×104倍稀释;合成的适配子A3的4个区域中,A3-2区域与河豚毒素的亲和力最强,即适配子A3的核心区域为A3-2区域,实际上含有47个核苷酸;合成的适配子G10的4个区域中,G10-4区域与河豚毒素的亲和力最强,即适配子G10的核心区域为G10-4区域,实际上含有56个核苷酸。     

全细胞SELEX法筛选单增李斯特菌的ssDNA适配体

以单增李斯特菌活细胞为靶标,采用指数富集配基的系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment,SELEX)从含40个随机碱基序列的单链DNA(single-stranded DNA,ss DNA)文库中筛选与之特异性结合的适配体。酶联配体吸附法(ELASA)测定筛选过程中次级文库与单增李斯特菌结合力的变化。对筛选得到的适配体进行序列测定,RNA structure软件预测二级结构,ELASA测定适配体与靶标的结合能力和特异性。结果显示,每轮筛选后次级文库与靶标的亲和力增强,特异性适配体逐步得到富集。测序获得23条适配体,其序列同源性不高,但预测的二级结构有些相似,根据二级结构将其分为3个群,分析结果表明二级结构与适配体的亲和力有关。挑选亲和力较强的21号(Lma-21)和35号(Lma-35)适配体能特异性地识别单增李斯特菌。本研究为开发食源性病原菌单增李斯特菌的新型检测试剂提供了基础材料。     

应用SELEX技术筛选沙门氏菌抗原的适配子

目的:应用指数富集配体系统进化(SELEX)技术筛选沙门氏菌抗原的高亲和性适配子。方法:首先合成一个全长78个核苷酸中间含35个随机序列的随机单链寡核苷酸序列(ssDNA)文库,再以环氧乙烷丙烯酸珠子作为筛选介质,利用生物素-抗地高辛碱性磷酸酶显色系统检测DNA适配子与沙门氏菌抗原的亲和力,以获得沙门氏菌抗原的高亲和性适配子。结果:随着筛选轮数的增加,DNA适配子与沙门氏菌抗原结合后显色,吸光度逐渐增加,初步获得了沙门氏菌抗原的高亲和性适配子。结论:实验所用的筛选流程是适当的,可以考虑推广用于类似靶目标的筛选。     

甲胺磷的核酸适体筛选

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RNA structure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。     

适配体功能化的DNA水凝胶的制备及其在食品安全小分子快速检测中的应用

DNA水凝胶是具有三维聚合物网络的高保水性材料。研究人员设计了多种DNA水凝胶交联制备方法, 并通过向其中引入其他功能分子或与其他类型的功能材料相互结合, 构建了具有优异性能的DNA水凝胶, 受到了广泛关注。适配体是基于指数富集的配体系统进化(systematic evolution of ligands by exponential enrichment, SELEX)技术从随机寡核苷酸文库中筛选获得的对目标物质具有良好特异性与亲和力的寡核苷酸序列。适配体功能化的DNA水凝胶具有靶向范围广、稳定性好、易于修饰、操作简单和成本低等优点, 得到了广泛应用。本文概述了构建适配体功能化的DNA水凝胶的基本设计原则与分类, 重点介绍了适配体功能化的DNA水凝胶在食品安全检测中的最新策略, 最后, 讨论了适配体功能化的DNA水凝胶面临的挑战以及对未来的展望, 旨在为其在食品安全领域的应用提供参考。     

基于适配体拉曼光谱技术快速检测肠炎沙门氏菌方法

目的:应用SELEX技术筛选高亲和力、高特异性适配体,利用该适配体结合拉曼光谱技术建立肠炎沙门氏菌快速检测方法。方法:采用全细菌指数富集的配体系统进化技术(whole-bacteria systematic evolution of ligands by exponential enrichment,whole-bacteria-SELEX)筛选肠炎沙门氏菌特异性核酸适配体,并采用酶联免疫吸附实验(enzyme-linked immuno sorbent assay,ELISA)与SERS技术对筛选出的适配体亲和力及特异性进行评价,建立肠炎沙门氏菌检测方法。结果:本研究通过对肠炎沙门氏菌进行十五轮SELEX筛选,并通过ELISA对其亲和力进行评价,筛选出Aptamer₄、Aptamer₁₀、Aptamer₁₂三条候选适配体,并通过SERS技术确认Aptamer₄为亲和力最佳适配体;将Aptamer₄与肠炎沙门氏菌、肺炎克雷伯氏菌等5种混合菌结合,结果表明,通过SERS技术可特异的检测出肠炎沙门氏菌,且该方法重复性较好,其最低检测限的细菌浓度为102 CFU/mL。且在猪肉样品的检测中,肠炎沙门氏菌的回收率为93.37%~100.18%。结论:应用SELEX方法成功筛选出与肠炎沙门氏菌高特异性、高亲和力适配体,并建立基于表面增强拉曼光谱技术快速检测肠炎沙门氏菌的方法,该方法特异性强、灵敏度高、成本低、快速简便,可应用于食品加工过程中肠炎沙门氏菌的快速检测。     

肠致病性大肠杆菌适配体筛选研究

目的食源性致病菌引起的食源性疾病已成为全球食品安全面临的巨大威胁,其中由大肠杆菌引起的食源性疾病最为常见。因此发展快速、准确检测大肠杆菌的方法对于保障食品安全、保护国民健康具有重要意义。方法应用Whole bacteria-SELEX(指数富集的配体系统进化技术),以肠致病性大肠杆菌为靶标,经过15轮的筛选富集,将所得产物进行克隆测序,并结合荧光分析考察所得序列的亲和力和特异性,根据解离常数值分析比较序列的亲和力和特异性,最终得到2条(Seq.1和Seq.28)与肠致病性大肠杆菌高特异性结合的适配体。结果适配体Seq.1和Seq.28均对肠致病性大肠杆菌表现出了相对良好的亲和力和特异性(对其他菌的相对亲和力均低于15%),解离常数分别为45.06±6.797和32.31±6.002 nmol/L。结论本文利用SELEX技术筛选特异性识别肠致病性大肠杆菌适配体,具有稳定性高、合成方便、易标记等特点,可进一步应用于食品中肠致病性大肠杆菌的快速检测。     

基于适配体的食源性致病菌检测方法研究进展

食源性致病菌是危害食品安全的重要因素,对人类健康造成了巨大的危害,越来越受到世界范围的广泛关注,因此建立简单高效的检测方法是食品卫生安全检测中的重要组成部分。适配体(Aptamer)是一段寡核苷酸序列,通过指数富集配体的系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX)从构建的随机文库中筛选得到。适配体对靶物质具有高度亲和力与选择性,多种用于食品中致病菌检测的适配体已被成功筛选和应用。介绍了基于适配体的食源性致病菌检测方法,讨论了适配体的发展及应用前景。     

氧化石墨烯指数富集法筛选食品中的组胺适配体

组胺(HA)是影响人类健康和食品质量的重要生化物质。对其进行快速、准确检测和鉴定至关重要。本试验利用氧化石墨烯对单链寡核苷酸(ssDNA)的强吸附作用,分离与靶标结合和未结合的ssDNA,对组胺适配体进行11轮的体外指数富集筛选,最终得到15条高亲和力和特异性的适配体。通过后续的适配体二级结构、亲和性和特异性分析,HAA-4和HAA-7被确定为最佳候选适配体,可被作为将来关于组胺的新型检测方法构建的候选适配体。本文首次利用氧化石墨烯指数富集(GO-SELEX)技术筛选出对组胺具有较高亲和特异性的适配体,为后续利用适配体对食品中组胺快速检测奠定了基础。     

Novel system for detecting SARS coronavirus nucleocapsid protein using an ssDNA aptamer

The outbreak of severe acute respiratory syndrome (SARS) in 2002 affected thousands of people and an efficient diagnostic system is needed for accurate detection of SARS coronavirus (SARS CoV) to prevent or limit future outbreaks. Of the several SARS CoV structural proteins, the nucleocapsid protein has been shown to be a good diagnostic marker. In this study, an ssDNA aptamer that specifically binds to SARS CoV nucleocapsid protein was isolated from a DNA library containing 45-nuceotide random sequences in the middle of an 88mer single-stranded DNA. After twelve cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure, 15 ssDNA aptamers were identified. Enzyme-linked immunosorbent assay (ELISA) analysis was then used to identify the aptamer with the highest binding affinity to the SARS CoV nucleocapsid protein. Using this approach, an ssDNA aptamer that binds to the nucleocapsid protein with a K_d of 4.93 ± 0.30 nM was identified. Western blot analysis further demonstrated that this ssDNA aptamer could be used to efficiently detect the SARS CoV nucleocapsid protein when compared with a nucleocapsid antibody. Therefore, we believe that the selected ssDNA aptamer may be a good alternative detection probe for the rapid and sensitive detection of SARS.     

Capture-SELEX技术筛选β-苯乙胺核酸适配体

利用指数富集配体的系统进化技术(SELEX)中基于固定ssDNA文库的捕获SELEX(Capture-SELEX)技术,筛选能特异性识别β-苯乙胺的核酸适配体。通过在Capture-SELEX技术中结合熔解曲线监测ssDNA文库的富集,并经过氧化石墨烯(GO)荧光法验证候选适配体的亲和力和特异性,证实获得一条特异性识别β-苯乙胺的适配体(PHE-2),解离常数(Kd)为(71.64±11.47)nmol/L。该适配体为β-苯乙胺的分析检测提供了新的分子识别元件。     

Selection,identification, and application of Aflatoxin B1 aptamer

Aflatoxins represent an important class of mycotoxins that are known to be mutagenic, carcinogenic, and teratogenic. Here, we report the use of the systematic evolution of ligands by exponential enrichment technology to screen for a DNA aptamer that recognizes Aflatoxin B1 (AFB1) with high affinity and specificity. AFB1 was first attached to magnetic nanoparticles and then incubated with an ssDNA library. After ten rounds of screening and amplification, 30 aptamer sequences were obtained following enrichment. Combined with a homological and structural analysis and affinity and specificity experiments, aptamer sequence 1, possessing the best affinity and specificity toward AFB1, was finally obtained. The dissociation constants value for aptamer sequence 1 was 11.39 nM. And, the specificity experiment results showed the binding between AFB1 aptamer with five other toxins was very week (did not exceed 15 % compared with AFB1). To demonstrate the potential use of this aptamer for quantitative analysis, a fluorescent bioassay with aptamer 1 was developed. The assay showed a wide linear range, with the AFB1 concentration ranging from 50 to 1,500 ng/L and a detection limit of 35 ng/L. Additionally, the spiked recovery experiment of AFB1 in peanut oil sample exhibited a recovery ratio between 94.2 and 101.2 % which showed good accuracy of the proposed aptamer-based bioassay. This fluorescent method represents a powerful tool for use in the detection of AFB1 without complex sample treatments.     

基于适配体的沙门氏菌检测方法研究进展

沙门氏菌(Salmonella)是重要的食源性致病菌,能引起人和动物中毒并引起多种严重的疾病,对人类健康造成了巨大危害,因此建立简单高效的检测方法是预防和控制沙门氏菌疾病的关键。适配体(aptamer)是通过指数富集配体的系统进化技术(systematic evolution of ligands by exponential enrichment, SELEX)从构建的随机文库中筛选得到一段对靶标物质具有高度亲和力和特异性的寡核苷酸序列,多种食品致病菌的适配体已被成功筛选并广泛应用于食品中致病菌的检测。本文对当前沙门氏菌检测技术进行简单论述,重点介绍了基于适配体的沙门氏菌检测方法,对各种方法的优势和缺点进行了比较和总结,并论述了适配体的发展及应用。     

噬菌体基因Ⅴ蛋白基因的合成、重组表达及功能分析

目的:研究丝状噬菌体基因V蛋白(gene V protein,GVP)基因的合成、重组表达及其功能。方法:根据GVP的基因序列,选择大肠杆菌偏爱的密码子,设计合成了8个寡核苷酸片段,利用重叠延伸PCR合成GVP基因序列,将其与原核表达载体pET-28a-c(+)质粒重组,转化大肠杆菌,获得GVP蛋白阳性表达菌株,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,产物经Ni+-NTA琼脂糖凝胶层析纯化,获得目的蛋白GVP,DNA结合实验检测其功能。结果:成功合成出GVP基因,重组体在大肠杆菌BL21(DE3)中诱导获得高效表达,DNA结合实验表明GVP与单链DNA间解离平衡常数Kd=7.27×10-5mol/L。结论:重组构建并高效表达的GVP蛋白具有较高单链DNA结合能力,可用于食品病原微生物特定单链DNA分子的浓缩和分离。     

In vitro selection of bacteriophage promoters employing a terminally capped template DNA and a streptavidin-binding aptamer

In vitro selection system for the isolation of bacteriophage promoters was developed. Employing the system, one round of the selection achieved ～20-30 fold enrichment of an active promoter from mixtures with an inactive one. In?addition, active T7 promoter variants were successfully selected from a library with six randomized bases.     

Screening of peptides with a high affinity to bile acids using peptide arrays and a computational analysis

Bile acid binding peptides have attracted attention for the improvement and prevention of hypercholesterolemia. In this study, screening of bile acid high affinity peptides was investigated using computationally-assisted peptide array analysis. Starting with the screening data obtained from a limited, random 6-mer library (2212 sequences), the peptides with a high affinity to bile acid were characterized by comparison of high- and low-affinity peptides using fuzzy neural network (FNN) analysis. The physical properties of amino acids at specific positions that contribute to bile acid binding activity were extracted as the structural rule; optimization was carried out using three repeated screening cycles of the rule extraction. The extracted structural rule indicates that Trp, Tyr, Phe, Leu, Ile and Val are enriched in bile acid binding peptides. The yields of bile acid binding peptides with an affinity of above the VAWWMY peptide (soystatin, control sequence) were significantly higher in the optimized structural rule (32.5%) compared to that of the random library (3.1%), and 6 peptides were obtained with above 2.0-fold increased binding activity.