CN116769783B - 一种特异性识别志贺毒素ⅱ型b亚基的核酸适配体及其应用 - Google Patents
一种特异性识别志贺毒素ⅱ型b亚基的核酸适配体及其应用 Download PDFInfo
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- CN116769783B CN116769783B CN202310683332.3A CN202310683332A CN116769783B CN 116769783 B CN116769783 B CN 116769783B CN 202310683332 A CN202310683332 A CN 202310683332A CN 116769783 B CN116769783 B CN 116769783B
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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Abstract
本发明公开了一种特异性识别志贺毒素Ⅱ型B亚基的核酸适配体及其应用,属于生物检测技术领域。本发明通过磁性氧化石墨烯筛选法筛选出与Stx2B特异性结合的适配体,对候选序列进行亲和力和特异性表征,得到三条适配体,为降低成本、消除假阴性结果和提高灵敏度,通过计算机模拟引导的适配体结构设计与实验验证相结合的方式得到截短优化后的适配体单体,亲和力与截短之前相比有所提升,在检测中表现良好,具有方法简单、灵敏度和准确度高的优点。
Description
技术领域
本发明涉及一种特异性识别志贺毒素Ⅱ型B亚基的核酸适配体及其应用,属于生物检测技术领域。
背景技术
肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli,EHEC)属于致病性大肠杆菌之一,又称为产志贺毒素大肠杆菌(Shiga toxin-producing Escherichia coli,STEC),是一种人畜共患的食源性致病菌。EHEC的主要毒力因子是噬菌体编码的志贺毒素(Shiga toxin,Stx),也称为志贺样毒素(Shiga-like toxin,SLT)或Vero毒素。Stx主要有两种抗原形式:志贺毒素Ⅰ型(Stx1)和志贺毒素Ⅱ型(Stx2)。与Stx1相比,在产生Stx2的STEC感染中观察到的肾和神经后遗症更加严重。志贺毒素由一个A亚基(32kDa)单体和一个B亚基五聚体(每个单体7.7kDa)非共价连接。B五聚体(StxB)负责与靶细胞上表达的Gb3受体特异性结合,与Gb3结合后,毒素-受体复合物通过内吞作用转运到细胞中,A亚基具有针对28S rRNA的N-糖苷酶活性,导致真核细胞中蛋白质合成受到抑制,导致细胞死亡。
目前,检测肠出血性大肠杆菌志贺毒素通常有质谱法、PCR法、Vero细胞毒性实验、酶联免疫试剂盒。质谱法虽然准确性高,但需要昂贵的大型仪器,成本高昂。PCR法能准确得检测大肠杆菌所携带的Stx基因,但这个过程需要很长时间,因为细胞必须培养几个小时才能进行测试。虽然许多菌株可能携带stx基因,但志贺毒素的表达差异很大,且需要有专业的操作和严格的实验环境,不能用于快速的现场检测。Vero细胞毒性实验只能判断大肠杆菌菌株中志贺毒素的生物学活性和强弱,判断是否存在志贺毒素,但该方法还需要细胞培养,耗时长,对检验条件和检验人员要求高,且结果受检验人员个体差异的影响。酶联免疫试剂盒虽然可以做到快速简便,但是依赖于抗体,抗体的制备复杂且成本较高,且储存条件较高,批次间差异明显。
核酸适配体是一段功能性的单链DNA或RNA寡核苷酸,长度通常为20-100个核苷酸,是通过称为指数富集配体系统进化(Systematic Evolution of Ligands byExponential Enrichment,SELEX)的过程从大量随机核酸库中提取出来的。适配体通过形成各种高阶结构识别靶标(发夹环、凸起、G-四链体、双螺旋、三链体等),以结合多种靶标,包括金属离子、小分子化合物、蛋白质、细胞和整个生物体。自从1990年代发现适配体以来,适配体已成为生物分析、诊断和治疗应用中的高亲和力和特异性探针。适配体通常被称为化学抗体,与抗体相比,适配体具有结构稳定,免疫原性低,无需任何基于动物的合成,可最大限度地减少批次间差异且延长保质期,可以灵活地被其他功能部分修饰以增强稳定性和靶向亲和力等优势。但目前尚未有关于Stx2B特异性适配体筛选研究的报道。
发明内容
为解决上述问题,本发明提供了一种特异性识别肠出血性大肠杆菌志贺毒素Ⅱ型B亚基的核酸适配体,具有特异性高、合成方便、易标记功能基团等特点,并基于该核酸适配体开发了一种双模式探针,可用于志贺毒素Ⅱ型的检测。
本发明的第一个目的是提供一种特异性识别肠出血性大肠杆菌志贺毒素Ⅱ型B亚基的核酸适配体,所述核酸适配体选自以下任一种:
(1)SEQ ID NO.1-3所示的核苷酸序列之一;
(2)SEQ ID NO.1-3所示的核苷酸序列截短优化后的序列之一。
进一步地,SEQ ID NO.1-3所示的核苷酸序列截短优化后的序列分别如SEQ IDNO.4-6所示。
进一步地,所述核酸适配体的5′端或3′端修饰有功能基团或分子。
进一步地,所述功能基团或分子选自同位素、电化学标记物、酶标记物、荧光基团、生物素、亲和配基、巯基中的至少一种。
本发明的第二个目的是提供上述核酸适配体在检测志贺毒素Ⅱ型中的应用。
本发明的第三个目的是提供上述核酸适配体在制备活细胞成像制剂中的应用,如对适配体进行荧光标记或放射性同位素标记,通过其能够靶向Stx2B亚基的特性实现体内成像或定位。
本发明的第四个目的是提供一种用于检测志贺毒素Ⅱ型的产品,该产品中包括上述核酸适配体。
进一步地,所述用于检测志贺毒素Ⅱ型的产品为双模式检测传感器。
进一步地,所述双模式检测传感器包括:复合锰的铁基MOF材料与纳米金形成的复合物、上述任一核酸适配体、核酸适配体的部分互补序列和磁珠;其中,所述复合物与部分互补序列共价连接,所述核酸适配体与磁珠亲和连接。
进一步地,所述部分互补序列上修饰有巯基。
进一步地,所述部分互补序列的核苷酸序列如SEQ ID NO.7所示。
本发明的有益效果:
本发明通过磁性氧化石墨烯筛选法筛选出与Stx2B特异性结合的适配体,对候选序列进行亲和力和特异性表征,又在得到的三条序列的基础上通过计算机模拟引导的适配体结构设计与实验验证相结合的方式对其进行截短优化后,亲和力与截短之前相比有所提升。本发明为志贺毒素Ⅱ型的检测提供了稳定性良好、亲和力高、易制备、易修饰和标记的高特异性适配体序列,基于其所建立的检测方法灵敏度高,特异性强,成本低,不需要依赖大型仪器设备和专业技术人员。
附图说明
图1为不同适配体的特异性结果;
图2-4为亲本序列S4~S6预测的可能结合位点;
图5-7为截短后单体的二级结构预测;
图8-10为比色和SERS双模式的适配体传感器的检测性能。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1Stx2B的核酸适配体的筛选
1、筛选
选用磁性GO-SELEX筛选方法对Stx2B重组蛋白进行筛选,筛选基本步骤如下:(1)文库与靶标混合,在第一轮筛选中,将随机文库(1nM)与Stx2B蛋白(50μg/mL)混合,在BB溶液中37℃摇晃孵育2h,总体系为1mL,之后的筛选中总体系均为300μL。(2)加入磁性氧化石墨烯孵育。加入磁性氧化石墨烯(CO/ssDNA的质量比为300)吸附未与Stx2B蛋白结合的ssDNA,继续37℃孵育0.5h。(3)磁分离去掉磁性氧化石墨烯后,与Stx2B特异性结合的ssDNA形成ssDNA-靶标复合物分散在上清液中,收集上清液中的ssDNA-靶标复合物,以上清液为PCR扩增模板,以进行下一步PCR扩增。
(4)PCR扩增。(5)单链制备与纯化。
每轮筛选前,取出磁性氧化石墨烯溶液(10mg/mL),超声至均匀悬浮体系,用结合缓冲液(BB溶液)多次冲洗。将ssDNA文库用TE溶液(10mM Tris-HCl,1mM EDTA,pH 7.4)溶解,95℃加热10min,立即冰上冷却10min,并放置在室温下10min待用。
随机单链DNA文库:
5′-TGAGCCCAAGCCCTGGTATG-N40-GGCAGGTCTACTTTGGGATC-3′,其中,N代表碱基A、T、C、G中的任一个,N40代表随机片段长度为40个碱基。
2、PCR扩增
PCR总体系(50μL)如下:0.5μL FAM标记上游引物(10μM),0.5μL磷酸化标记下游引物(10μM),0.5μL Taq DNA聚合酶(5U/μL),1μL dNTP mix(5mM),5μL 10×PCR缓冲液,1μLssDNA模板,41.5μL灭菌水。PCR程序如下:95℃预变性5min;95℃变性30s;58℃退火30s;72℃延伸20s;循环10次;72℃延伸2min,最后4℃冷却。随后,利用8%非变性聚丙烯酰胺凝胶对PCR产物进行鉴定。PCR产物通过上海GENEray PCR产物纯化试剂盒纯化。
由于在SELEX过程中,每轮筛选后文库中寡核苷酸的数量大幅度减少,为了满足下一轮筛选所需寡核苷酸的浓度需求,并且富集对靶标具有高亲和力的序列,需要对含有ssDNA-靶标复合物的上清液进行PCR扩增。为了获得较多的PCR产物,避免产生非特异性扩增产物,提高扩增效率,需要使用非变性聚丙烯酰胺凝胶电泳对每一轮PCR扩增过程的退火温度和循环轮数进行优化验证,并在接下来的PCR扩增时选择58℃作为退火温度,选择第10轮作为循环轮数。
3、单链制备
采用Lambda核酸外切酶特异性识别磷酸化标记的下游引物进行酶切以制备次级文库。具体如下:向PCR纯化产物中加入Lambda酶及10×Lambda酶切反应缓冲液(670mmol/LGlycine-KOH,25mmol/L MgCl2,500μg/mL BSA,pH 9.4@25℃),混合均匀后置于37℃酶切反应,75℃10min终止反应。酶切产物使用含7mol/L尿素的8%变性聚丙烯酰胺凝胶进行鉴定。最后利用乙醇沉淀法纯化酶切后的ssDNA,收集后作为下一轮筛选的次级文库。后续筛选总体系为300μL,其余质量比不变。
PCR扩增后,采用PCR纯化试剂盒进行PCR扩增产物的纯化,去除PCR过程中引入的离子、dNTP、引物和酶等。接下来采用Lambda核酸外切酶特异性识别磷酸化标记的下游引物的酶切方式进行单链制备,但是酶切过程中,如果酶切时间过短,会导致PCR纯化产物酶切未完全,产物中仍存在双链;如果酶切时间过长,Lambda核酸外切酶会以较低速率切除正向引物。因此,需要对每一轮酶切时间进行优化,最后选择45min为酶切时间。最后,经过乙醇沉淀法纯化酶切产物得到次级文库。
4、循环筛选
第一轮的筛选条件为:文库量为1nmol,靶标浓度为50μg/mL,磁性氧化石墨烯质量为7.5mg,文库与靶标孵育时间为2h,磁性氧化石墨烯孵育时间0.5h。第二轮的筛选条件为:文库量为100pmol,靶标浓度为50μg/mL,磁性氧化石墨烯质量为0.75mg,文库与靶标孵育时间为2h,磁性氧化石墨烯孵育时间0.5h。
5、筛选进程的监测
随着筛选轮数的增加,利用FAM标记,将每一轮筛选得到的上清液中ssDNA的荧光强度与筛选前文库中ssDNA的荧光强度对比,计算得出每一轮筛选ssDNA的回收率(富集率=靶标孵育后上清液中荧光强度/初始ssDNA荧光强度)。当回收率趋于平稳后,停止筛选。
SELEX过程经过多次循环筛选,特异性适配体不断富集,因此需要测定每一轮的ssDNA回收率以监测与Stx2B具有强结合能力的ssDNA的富集程度。结果显示,随着筛选轮数的增加,富集率呈逐渐上升的趋势,说明与Stx2B亲和力高的ssDNA在不断富集。回收率第9轮开始趋于平稳,在第10轮保持稳定,表明Stx2B适配体富集程度达到饱和。因此,实验中对stx2B进行10轮的富集筛选。
6、高通量测序及序列分析
以第十轮筛选磁分离后的上清液为模板,采用无标记的正向引物和反向引物进行PCR扩增,经8%非变性聚丙烯酰胺凝胶电泳验证后,将PCR扩增产物送至上海生工生物工程技术服务有限公司进行高通量测序。利用Seqman软件读取测序结果,并初步比对。利用Mfold在线工具(http://www.unafold.org/mfold/applications/dna-folding-form.php)预测序列的二级结构。根据序列的二级结构、自由能(ΔG)及重复出现次数等因素,选出结构稳定,重复次数多且ΔG较低的代表性序列作为候选适配体序列,用于后续亲和力、特异性表征,以期获得与Stx2B结合的最优适配体。
将第10轮产物经过无修饰的引物PCR扩增纯化后,委托上海生工生物工程技术有限公司进行高通量测序,获得40000多条序列,根据高通量测序中出现的频次、二级结构、二级结构自由能级(ΔG)等挑选出前六条序列作为候选适配体,分别命名为S1、S2、S3、S4、S5、S6。侯选适配体的二级结构通过Mfold进行预测,并将这些候选适配体合成并进行亲和力、特异性分析。
7、亲和力特异性分析
用酶联寡核苷酸测定法(ELONA)检测适配体与Stx2B的结合能力。首先,将Stx2B蛋白稀释在包被缓冲液(pH值为9.6、0.05M的碳酸盐缓冲液)中,96孔板的每孔包被有1μg蛋白质,在4℃下培养过夜。用PBST(0.5% Tween20)缓冲液洗涤3次后,加入5%脱脂奶粉,在37℃下封闭1小时。然后,稀释在结合缓冲液(BB溶液)中不同浓度的生物素化适配体(10、25、50、100、150、200、400nM)添加到这些孔中,在37℃下摇晃1小时。用PBST洗涤3次后,将100μLHRP-共轭链霉亲和素(1:750)添加到孔中,并将平板在37℃下培养30min。用PBST洗涤3次后,将100μL 3,3',5,5'-四甲基联苯胺(TMB)添加到孔中,在37℃下培养15min。最后,加入50μL硫酸终止反应,在450nm处通过酶标仪测定微孔的吸光度。ELONA分析重复3次进行。
接下来,对Stx2同样用ELONA法检测亲和力,以此证明三条适配体同样以高亲和力结合Stx2。结果如表1所示,此六条适配体解离常数均在纳摩尔级别,对比得出S4、S5、S6的解离常数和另外三条相比较低,亲和力较好。
表1候选序列的亲和力分析
选用ELONA法对候选适配体进行特异性分析。操作与亲和力检测一致,但是在特异性分析实验中,使用BSA、Stx1B、Stx1、Stx2作为靶标的结构类似物或共存物代替Stx2B蛋白包被在96孔板中。结果如图1所示,与阳性对照Stx2B相比,S4、S5、S6对Stx2的信号响应更高,这说明S4、S5、S6同样对Stx2具有亲和力。相比之下,S1与Stx2B和Stx2结合力均比较弱,S2和S3虽然表现出较强结合Stx2B和Stx2的能力,但与BSA、Stx1、Stx1B也具有较强的交叉反应率,但是S4、S5、S6对BSA、Stx1、Stx1B的吸光度较S1、S2、S3低,说明S4、S5、S6是与Stx2B具有高亲和力和高特异性的适配体序列,因此,接下来的实验对S4、S5、S6三条适配体进行截短优化。
实施例2与Stx2B特异性结合的核酸适配体的截短优化
1、适配体与靶标的分子模拟对接研究
Stx2B蛋白的分子结构从RCSB PDB数据库获取。首先利用Mfold在线工具预测适配体的二级结构,随后预测该序列的三维结构模型。最后利用ZDOCK程序对DNA三维结构与Stx2B的结构模型进行分子对接模拟。选取每个对接的最佳姿态,并进行比较。通过对接100次的构象,并选取最低能量构象分析结果发现适配体与Stx2B对接后进行相互作用时,主要相互作用力为疏水、范德华、氢键及静电作用。
适配体S4与靶标Stx2的分子对接结果:蛋白SER31残基上的羰基氧与适配体G37的氢形成1对氢键,氢键键长为蛋白THR55残基氧原子与适配体A38上氢形成1对氢键,氢键键长为/>GLY61上的氨基氢与A38上的氮形成1对氢键,键长为/>其中蛋白的ASP17,THR18,TRP29,THR30,ARG32,TRP33,ASN34,SER53,SER54,THR55,GLY61,PHE62,ALA63与适配体结构的C35,C36,G37,A38,A39,G60,G61相互作用。
适配体S5与靶标Stx2的分子对接结果:蛋白TRP33残基上的氨基氢与适配体A44的五元环氧形成1对氢键,氢键键长为蛋白ASN34残基上的氢原子与适配体T46上的羰基氧形成1对氢键,氢键键长为/>其中蛋白的ASN14,GLU15,ASP16,ASP17,THR18,TRP29,THR30,SER31,ARG32,TRP33,ASN34与适配体结构的T30,A31,C32,T43,A44,C45,T46相互作用。
适配体S6与靶标Stx2的分子对接结果:蛋白THR55残基上的羟基上氢与适配体A53上的五元环氧形成1对氢键,氢键键长为蛋白GLY59残基上的羟基氢原子与适配体T33上的氧形成1对氢键,氢键键长为/>其中蛋白的LYS12,ASN14,GLU15,ASP16,ASP17,THR18,TRP29,THR30,SER31,ARG32,SER53,SER54,THR55,CYS56,GLY59,GLY61,PHE62与适配体结构的G31,G32,T33,C34,C52,A53,C54相互作用。
2、适配体结构优化
根据适配体的二级结构、自由能(ΔG)及分子对接结果进行适配体的截短,并利用酶联寡核苷酸测定法对截短后的序列进行Stx2B的亲和力分析,并对亲本序列、截短序列对Stx2的亲和力进行分析。
(1)剪裁
通过分子对接结果,预测得出适配体与靶标可能的结合位点,如图2-4所示,适配体与靶标可能的结合区域用涂笔画出,所在的茎环区域用方框圈出,可以发现结合位点都在茎环区域且大部分不在两端引物结合区,因此,根据分子对接结果,只保留结合位点所在的茎环结构,利用Mfold网站预测其二级结构和吉布斯自由能,综合考虑适配体的空间结构稳定性及序列长度,分别对S4、S5、S6进行剪裁优化,得到截短后的单体,分别命名为S4-34(34mer)、S5-35(35mer)、S6-29(29mer),如图5-7所示。
表2适配体亲本序列及剪裁后适配体单体的序列
(2)亲和力分析
使用酶联寡核苷酸测定法考察适配体剪裁后对Stx2B亚基和Stx2的亲和力,结果见表3。表1与表3对比,发现截短后的适配体S4-34、S5-35、S6-29的Kd值较亲本适配体都有所下降,即截短后亲和力提高,说明截短后的单体是适配体与靶标结合的关键基序,符合分子对接的预测。
表3候选序列的亲和力分析
实施例3比色和SERS双模式的适配体传感器的构建
1、合成Mn/Fe-MIL(53)@AuNSs复合物
首先通过FeCl3·6H2O、Mn(CH3COO)2·4H2O、DMF、H2BDC等原料的一步水热反应的简便有效的方法合成Mn/Fe-MIL(53)。其次,利用柠檬酸钠溶液、氯金酸溶液制备金种,利用HCl、HAuCl4、金种溶液、AgNO3、AA溶液合成AuNSs。最后,将PEI与Mn/Fe-MIL(53)混合合成Mn/Fe-MIL(53)@PEI。将AuNSs溶液加入Mn/Fe-MIL(53)@PEI溶液中,合成Mn/Fe-MIL(53)@AuNSs复合物。
2、合成Mn/Fe-MIL(53)@AuNSs-磁珠复合物
首先将互补链cDNA(5’-TAGCAAAGACCGT-SH-3’,SEQ ID NO.7)用TCEP激活,然后加入Mn/Fe-MIL(53)@AuNSs震荡反应,离心后获得Mn/Fe-MIL(53)@AuNSs-cDNA。接着将生物素标记的适配体S6-29(5’-ACGGTCTTTGCTATTTACGGCAACACCGC-Biotin-3’)与磁珠溶液在室温下轻轻摇晃反应。将溶液置于磁力架中分离并去除上清液。产品用BB洗涤得到磁珠-适配体。将Mn/Fe-MIL(53)@AuNSs-互补链与磁珠-适配体混合得到Mn/Fe-MIL(53)@AuNSs-磁珠复合物。
3、建立标准曲线
将不同浓度的Stx2加入到制备好的的磁珠-适配体与Mn/Fe-MIL(53)@AuNSs-互补链共轭物中孵育,磁分离收集上清液进行比色测量和SERS测量。
4、特异性实验
在特异性实验中,使用BSA、Stx1B、Stx1作为靶标的结构类似物或共存物进行实验,Stx2作为阳性对照进行SERS测量和比色测量。
5、结果
比色模式下的检测结果如图8所示,随着Stx2浓度的增加,650nm处吸光度不断增加,在0.05-500ng/mL的范围中,650nm处的吸光度差值与蛋白质浓度的对数之间呈线性关系,线性回归方程拟合为y=0.0457x+0.1609(R2=0.99455),LOD为3.6pg/mL(3σ/k,σ和k是空白测量的标准偏差(n=3)和校准曲线的斜率)。
SERS模式下的检测结果如图9所示,随着Stx2浓度的增加,1074和1584cm-1处的SERS强度不断增加,1074cm-1的SERS强度的增加较1584cm-1更为明显,因此选用1074cm-1建立标准曲线,在1-1000ng/mL的范围中,1074cm-1处的相对SERS强度与蛋白质浓度的对数之间呈线性关系,线性回归方程拟合为y=2008.3275x+921.16759(R2=0.97883),LOD为0.82ng/mL(3σ/k,σ和k是空白测量的标准偏差(n=3)和校准曲线的斜率)。
在特异性实验中,使用BSA、Stx1B、Stx1作为靶标的结构类似物或共存物进行实验,Stx2作为阳性对照进行实验,用酶标仪测定650nm处的吸光度,同时,将待测物与4-MBA混合后进行表面增强拉曼散射测定。如图10所示,与阳性对照相比,其他结构类似物或干扰物比色和SERS信号相对较低,说明该双模式传感器的特异性较好。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (8)
1.一种特异性识别肠出血性大肠杆菌志贺毒素Ⅱ型B亚基的核酸适配体,其特征在于,所述核酸适配体选自SEQ ID NO.1-6所示的核苷酸序列之一。
2.根据权利要求1所述的核酸适配体,其特征在于:所述核酸适配体的5′端或3′端修饰有功能基团或分子。
3.根据权利要求2所述的核酸适配体,其特征在于:所述功能基团或分子选自同位素、电化学标记物、酶标记物、荧光基团、生物素、亲和配基、巯基中的至少一种。
4.权利要求1-3任一项所述的核酸适配体在制备志贺毒素Ⅱ型检测产品中的应用。
5.一种用于检测志贺毒素Ⅱ型的产品,其特征在于:包括权利要求1-3任一项所述的核酸适配体。
6.根据权利要求5所述的产品,其特征在于:所述产品为双模式检测传感器。
7.根据权利要求6所述的产品,其特征在于:所述双模式检测传感器包括复合锰的铁基MOF材料与纳米金形成的复合物、权利要求1-3任一项所述的核酸适配体、核酸适配体的部分互补序列和磁珠;其中,所述复合物与部分互补序列共价连接,所述核酸适配体与磁珠亲和连接。
8.根据权利要求7所述的产品,其特征在于:所述部分互补序列上修饰有巯基。
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