CN111349631B - 与鳍藻毒素-1特异性结合的适配体及其应用 - Google Patents
与鳍藻毒素-1特异性结合的适配体及其应用 Download PDFInfo
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- Chemical Kinetics & Catalysis (AREA)
Abstract
本发明涉及生物医药工程技术领域,提供了与鳍藻毒素‑1特异性结合的适配体及其应用。适配体的序列通式为:5’‑GAGGCAGCACTTCACACGAT‑N27‑CTGCGTAATGACTGTAGTGATG‑3’;其中,N为A、T、G、C四种脱氧核糖核苷酸碱基中的任意一个,N27代表长度为27bp的随机序列。优选为如SEQ ID NO.1~SEQ ID NO.6中任一条所示的序列,最优选为如SEQ ID NO.7~SEQ ID NO.10中任一条所示的序列,可以制备成适配体传感器或检测试剂,并将其应用于海水体样品中DTX‑1的检测,还可以为预防或治疗DTX‑1中毒药物的制备以及水体或水产品中DTX‑1的去除奠定基础。
Description
技术领域
本发明涉及生物医药工程技术领域,具体涉及一条基于磁珠SELEX技术筛选获得的与鳍藻毒素-1特异性结合的高亲和力适配体,可用于临床样本中鳍藻毒素-1的快速检测诊断和水体、食品中鳍藻毒素-1的监测。
背景技术
腹泻型贝类毒素(Nodularin-R,DSTs)是一种全球分布最广的海洋毒素。研究表明,DSTs不仅会导致恶心、呕吐、腹泻等消化道症状,而且还会促进肿瘤的发生,其毒性机理为抑制多种丝氨酸/苏氨酸蛋白磷酸酶(PP)的活性,主要是PP1,PP2A,PP2B。自1978年起,研究者们共发现了三种腹泻型贝类毒素,其中,鳍藻毒素-1(DTX-1)的毒性和致癌能力最强,也是在贝类体内沉积率最高的。人们主要因饮用被污染的水和误食被污染的水产品而中毒,中毒症状包括恶心、呕吐和腹泻等,目前临床尚无特效解毒剂。国内外卫生组织都将饮用水中DTX-1的最高含量规定为1μg/L。近些年来,一些被认定为“无DST”的海域也相继发现了腹泻型贝类毒素的存在,水产相关产业每年为此蒙受大量经济损失。因此,亟需建立一种准确、灵敏的DTX-1检测方法,以便更好的监控水体和水产品中DTX-1的水平,进而保障人们和水生生物的安全。
目前,常用的DTX-1的检测方法主要分为三大类,即生物分析法、物理化学分析法和免疫化学法。其中,小鼠生物法(MBA)是检测DTX-1最传统的生物分析方法。然而,该方法不仅成本高、特异度低、重现性差,而且还存在伦理道德问题。化学分析法,如高效液相色谱法(HPLC)和液相色谱质谱联用(LC-MS)等也逐渐完善,其中液相色谱质谱联用技术已经被欧盟确定为首要的检测方法。然而,该方法需要进行复杂的样品前处理过程,相关仪器设备昂贵,对技术人员有较高的技术要求。免疫化学法并未出现针对DTX-1的试剂盒,虽然OA的试剂盒已在实验室中虽然已经进行广泛应用,但是由于其缺乏特异性、步骤繁琐,难以满足海洋毒素的实时检测。因此,亟需建立一种快速、灵敏的新型检测方法。
近年来,生物传感器检测法因能克服传统检测方法的众多缺点而备受研究者们的关注。生物传感器的核心部件是分子识别元件,抗体是一种发展完善且备受关注的分子识别元件,但是,抗体不仅价格昂贵,容易变性,还会出现批次间的差异。因此,需要寻找一种新的分子识别元件。
在1990年,Tuerk和Gold首次通过指数富集的配基系统进化技术(SystematicEvolution ofLigands by Exponential Enrichment,SELEX),成功筛选到了具有高亲和力、强特异性的T4 DNA聚合酶核酸适配体。核酸适配体,即具有功能的单链寡核苷酸,包括ssDNA和RNA,能够形成发夹、假结、i-motif和G-四链体等空间结构,并通过分子间的相互作用,如疏水作用、范德华力和氢键等特异性识别并结合靶分子。作为一种新型分子识别元件,适配体与抗体相似,但优于抗体。例如,适配体能够识别多种靶标,如蛋白质、氨基酸以及金属离子等,甚至细胞和病毒;适配体能够与多种基团连接,修饰方便;适配体化学性质稳定,不容易变性;适配体可以直接化学合成,成本低廉。
目前,已经有很多学者将适配体应用于海洋生物毒素检测领域,并与生物传感器平台(如电化学、荧光技术和表面等离子共振等)结合,研发了许多快速、新颖的检测方法。然而,还没有关于DTX-1适配体的相关报道。
发明内容
本发明是为解决上述技术问题进行的,目的在于提供一种与鳍藻毒素-1特异性结合的适配体及其应用。
本发明的目的在于提供多条能够与DTX-1进行高亲和力特异性结合的单链DNA适配体,并对这些适配体与DTX-1之间的亲和力进行测试,得到亲和力常数(KD)值最小的一条适配体N59。本发明的第二目的在于以截短的方式对该适配体N59进行优化,提供结合能力更强,而且长度更短的DTX-1适配体(N59a)。本发明的第三目的在于提供适配体的应用,如适配体在制备鳍藻毒素-1分离富集试剂中的应用,在制备鳍藻毒素-1检测试剂、试剂盒或传感器中的应用,在制备治疗鳍藻毒素-1中毒药物中的应用,以及在自来水样品中DTX-1的快速检测中的应用,并为预防或治疗DTX-1中毒药物的制备和水体或水产品中DTX-1的去除奠定基础。
本发明的主要技术方案是:通过磁珠-SELEX技术筛选并得到一条能够与DTX-1以高亲和力特异性结合的单链DNA适配体(N59)。根据在线工具the mfoldweb server对其二级结构的预测,对适配体N59进行截短优化,并得到优化后的适配体N59a。通过与生物传感器平台相结合,可以制备DTX-1适配体传感器,并将其用于DTX-1的快速检测。此外,DTX-1适配体还为预防或治疗DTX-1中毒药物的制备以及水体或水产品中DTX-1的去除奠定基础。
本发明的第一方面提供了与鳍藻毒素-1特异性结合的适配体,该适配体的序列通式为:5’-GAGGCAGCACTTCACACGAT-N27-CTGCGTAATGACTGTAGTGATG-3’;其中,N为A、T、G、C四种脱氧核糖核苷酸碱基中的任意一个,27代表随机碱基的数量。
通过磁珠-SELEX技术筛选,得到如下代表性序列:
N11:如SEQ ID NO.1所示;
N16:如SEQ ID NO.2所示;
N50:如SEQ ID NO.3所示;
N29:如SEQ ID NO.4所示;
N59:如SEQ ID NO.5所示;
N63:如SEQ ID NO.6所示。
本发明的第二方面是以截短的方式对适配体N59进行优化,提供结合能力相当或者更优,但是长度更短的四条DTX-1适配体,分别被命名为适配体N59a、适配体N59b、适配体N59c和适配体N59d。在这4条适配体中,除了适配体N59d,其余适配体都能和DTX-1结合,其中,适配体N59-a与DTX-1之间的亲和力出现了明显提高,达到了64nM。
适配体N59a~适配体N59d的序列如SEQ ID NO.7~SEQ ID NO.10所示,优选N59a序列。
优选的,上述适配体N59a,可以在其3’端或5’端进行生物素、FITC和巯基等化学修饰。
本发明的第三方面提供了适配体的应用,如在制备鳍藻毒素-1分离富集试剂中的应用;在制备鳍藻毒素-1检测试剂、试剂盒或传感器中的应用;在制备治疗鳍藻毒素-1中毒药物中的应用。
优选的,治疗鳍藻毒素-1中毒药物是以与鳍藻毒素-1特异性结合的适配体作为唯一活性成份或者是包含与鳍藻毒素-1特异性结合适配体的药物组合物。
本发明的第四方面,提供了一种与鳍藻毒素-1特异性结合的适配体的药物组合物,以与鳍藻毒素-1特异性结合的适配体作为活性成份,还包括医学上可接受的药物载体。
优选的,药物组合物为去除水体或水产品中鳍藻毒素-1制剂。
本发明的药物组合物和药学上可以接受的辅料一起组成药物制剂组合物,从而更稳定地发挥疗效,这些制剂可以保证本发明公开的适配体核心序列的构像完整性,同时还能保护蛋白质的多官能团,防止其降解(包括但不限于凝聚、脱氨或氧化)。
本发明的第五方面是提供了适配体N59-T-02在自来水样品中DTX-1的快速检测中的应用,防止人们主要因饮用被污染的水和误食被污染的水产品而中毒。
本发明的有益保障及效果:
本发明提供了一种与鳍藻毒素-1特异性结合的适配体及其应用,通过实验验证,本发明的适配体能够快速特异性地与鳍藻毒素-1结合,其中适配体N59a与DTX-1之间的亲和力最高,达到了64nM。因此,本发明筛选得到的适配体可以制备适配体传感器或检测试剂,并将其应用于饮用水、海水体样品中DTX-1的检测。另外,这些适配体还可以为预防或治疗DTX-1中毒药物的制备以及水体或水产品中DTX-1的去除奠定基础。
此外,本发明根据DTX-1分子的特点,设计了磁珠-SELEX,通过正向筛选和反向筛选,获得了能够与鳍藻毒素-1以很高的亲和力特异性结合的单链DNA适配体,具有操作简便、重复性高等特点,生产成本低、纯化周期短。适配体作为一种新型分子识别探针,具有成本低廉、性质稳定和方便修饰等优点,适宜在生物医药工业化生产中大规模应用。
附图说明
图1是DTX-1适配体筛选的流程图;
图2是筛选过程中每一轮的单链DNA回收率结果图;
图3是截短后适配体N59的二级结构预测图,其中A为N59、B为N59a、C为N59b、D为N59c、E为N59d;
图4是适配体N59a的亲和力和特异性鉴定图。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室指南(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1.随机ssDNA文库及其引物的设计
1.ssDNA文库的设计
DTX-1适配体文库由69个碱基组成,两端分别为含有20和22个碱基的固定区域,中间则为含有27个碱基的随机区域:5’-GAGGCAGCACTTCACACGAT-N27-CATCACTACAGTCATTACGCAG-3’(N为A、T、G、C四种脱氧核糖核苷酸碱基中的任意一种,27代表随机碱基的数量)。
2.引物的设计
上游引物:5’-GAGGCAGCACTTCACACGAT-3’(SEQ ID NO.11)
下游引物A:5’-CATCACTACAGTCATTACGCAG-3’(SEQ ID NO.12)
下游引物B:5’-poly(dA20)-Spacer18-CATCACTACAGTCATTACGCAG-3’(SEQ IDNO.13)。
其中,下游引物A主要用于筛选中ssDNA的扩增,下游引物B主要用于克隆测序。
实施例2.DTX-1适配体的筛选
根据DTX-1分子的特点,采用适配体筛选的经典方法——磁珠法来进行筛选。
如图1所示,筛选流程主要包括四个步骤,即孵育、分离、洗脱和扩增,表1给出了具体筛选方案,具体过程如下:
(1)孵育:取3nmol ss DNA文库(第二轮到第五轮文库量为200pmol,第六轮到第十二轮文库量为120pmol)进行变复性处理,即先在95℃水浴10分钟;然后,冰浴骤冷5分钟;最后,在常温下放置5分钟;同时,用筛选缓冲液洗涤DTX-1磁珠数次,并取20μL加入到经过上述处理的ssDNA文库中;加入一定量的筛选缓冲液补足体系,使总体积为600μL,并在室温下旋转孵育2h。
(2)分离:旋转孵育2h后,利用磁力架实现溶液中的ssDNA和结合在磁珠上的ssDNA的分离。为了去除非特异性吸附的ssDNA以及结合非常不牢靠的ssDNA,在磁性分离后,需要用筛选缓冲液洗涤磁珠3-5次。
(3)洗脱:洗涤结束后,向磁珠中加入80μL无酶水,并95℃水浴20分钟,回收能够与与DTX-1特异性结合的ssDNA(共计三次)。
(4)扩增:以特异性结合的ssDNA作为PCR模板对其进行扩增。PCR反应体系(50μL)为:模板2.5μL;Hot start premix(2×)25μL;上游引物(10μM)2.5μL;下游引物(10μM)2.5μL;无酶水17.5μL。PCR反应条件为:95℃,5分钟;95℃,30s;54℃,45s;72℃,30s;72℃,5分钟,共25个循环。每次PCR扩增时均设置空白对照。
为了提高筛选的效率,从第六轮开始进行反向筛选。与正向筛选的不同之处在于:在用DTX-1磁珠与ssDNA文库进行孵育以前,先将ssDNA文库与阴性磁珠在室温下旋转孵育,孵育结束以后,用磁力架分离溶液中的ssDNA和结合在空白磁珠上的ssDNA,回收得到的溶液中的ssDNA即为正向筛选的ssDNA文库。
表1 DTX-1适配体筛选方案
经上述PCR扩增得到的DNA是dsDNA,而适配体则是ssDNA。为了获得ssDNA次级文库,特意设计了含有20个鸟嘌呤的下游引物B,使PCR后的两条链相差20个碱基,然后通过尿素变性聚丙烯酰胺凝胶电泳对两条链进行分离,再对目标链进行切胶回收。
尿素聚丙烯酰胺凝胶电泳的操作步骤具体如下:(1)将等体积的尿素上样缓冲液(2×)和PCR产物混合,充分混匀后在95℃下变性10分钟。(2)将上述样品缓慢加入到凝胶上样孔中,接通电泳仪,并使样品在恒定电压(300V)下电泳至溴酚蓝迁移到凝胶底端。(3)电泳结束后,首先,用剥胶板将凝胶剥离,然后,将凝胶放入盛有核酸染料(Gel-red,10000×)的玻璃皿中,并在四维旋转混匀仪上染色20分钟。(4)染色完成后,将凝胶置于凝胶成像仪下显像并观察。如果空白对照中能观察到目的条带,则该轮筛选失败;如果没有,则可以对实验组中的目的条带进行切胶回收。
将回收的凝胶离心破碎后,加入相同体积的煮胶缓冲液,并在50℃水浴锅中孵育20分钟(重复2次)。孵育结束后,对样品进行离心(12000RPM,5分钟),收集上清。采用凝胶提取试剂盒对回收得到的ssDNA进行纯化。纯化完成后,用核酸定量荧光计2.0对ssDNA进行定量分析,至此,次级文库制备完毕。
如图2所示,当筛选到第12轮的时候,ssDNA的回收率不再上升,预示达到了筛选终点。于是,利用上游引物和下游引物A对第12轮洗脱下来的ssDNA进行PCR扩增,并用核酸回收试剂盒对经聚丙烯酰胺凝胶电泳回收的dsDNA进行纯化以后,委托上海杰李生物工程股份有限公司进行克隆测序,总共测得了80条序列。在用Clustal X 2.1软件对所有序列进行比对分析以后,从中挑选了10条具有代表性的序列进行亲和力测定(生物膜干涉技术),其中有6条出现了亲和力较高的特异性结合,具体如表2所示。
表2 MB-SELEX筛选出的代表性序列的信息及亲和力值
实施例3.适配体N59的优化
在选取的10条适配体中,适配体N59与DTX-1之间亲和力常数(KD)值最小,为0.17μM。为了得到适配体N59的核心结构并对该适配体加以优化,对该适配体进行了截短处理。
图3显示了在线工具the mfold web server对适配体N59截短产物的二级结构预测图。
当适配体N59两端的固定区域被去除以后(N59a),适配体依然能够与DTX-1结合,而且KD值明显上升。因此,可以推断:适配体N59两端的引物结合区域不仅不参与其与DTX-1之间的结合,而且有可能对核心结构核心结构与DTX-1的结合产生产生阻碍。
根据在线工具the mfoldweb server对适配体N59二级结构的预测结果,对其进行了截短,并获得了适配体N59b、适配体N59c和适配体N59d。在这3条适配体中,除了适配体N59d,其余适配体都能和DTX-1结合,但是亲和力却有不同程度的降低。通过比较适配体N59a、适配体N59b、适配体N59c和适配体N59d的二级结构,可以推断:适配体N59a中较大的茎环结构在结合时具有重要意义,极有可能为其核心结构。考虑到时间和成本问题,我们没有进行进一步优化,而是以亲和力最高的适配体N59a作为最佳适配体,并将该适配体作为今后研究的对象。
本实施例中,N59及其截短后的序列信息和亲和力信息值如表3所示:
表3 N59及其截短后的序列信息和亲和力值
实施例4.生物膜干涉技术分析分子间的相互作用
生物膜干涉技术是一种免标记的技术,可以提供实时、高通量的生物分子相互作用信息。生物膜干涉技术的基本原理是仪器发射白光到传感器表面,同时收集光学膜层两个表面的反射光。因为不同频率的反射光受到光学膜层厚度和质量密度的影响有差异,一些频率的反射光形成了相长干涉(蓝色),另一些则形成了相消干涉(红色)。光谱仪检测到这些干涉光以后形成了一幅干涉光谱,并以干涉光谱的相位位移强度(Δλ)显示。因此,当结合到传感器表面的分子有数量上发生变化的时候,这种干涉光谱的位移就会被光谱仪检测到。而这种位移能够直接的反映出传感器表面生物膜厚度和质量密度的变化,从而对待测分子间的相互作用过程进行精确的定量测定。作为一种新型的免标记技术,生物膜干涉技术已经在生物分子间的相互作用研究中占据着重要地位。
将核酸适配体的5’端用生物素进行修饰,通过生物素和链霉亲合素相互作用将适配体固定在超级链霉亲和素(SSA)传感器表面。固定前,首先要对适配体进行变复性处理(95℃水浴10分钟,冰浴骤冷5分钟,室温放置10分钟),以帮助适配体重新折叠成稳定的空间结构。在用生物膜干涉仪OctetRED 96进行检测前,依次将缓冲液、生物素标记的适配体溶液、缓冲液以及DTX-1溶液加入96孔板的不同列中,加样体积为200μL。Octet RED 96系统的程序设定为:(1)传感器平衡(2分钟);(2)适配体耦合(3分钟);(3)传感器再平衡(2分钟);(4)DTX-1结合(5分钟);(5)DTX-1解离(5分钟)。所有的步骤都是在室温下完成的。检测完成后,利用Octet数据分析软件CFR Part 11 Version 6.x将实验组传感器的响应值中扣除对照组传感器的响应值,得到校正后的实际响应值。另外,采用1:1的结合模式对响应数据进行拟合,得到适配体与DTX-1的结合-解离曲线以及各种动力学参数,包括结合速率常数Kon、解离速率常数Kdis和亲和常数KD值。
利用上述方法,对适配体N59a和DTX-1、OA、STX、NOD、PTX、GTX以及随机序列(Random sequence)之间的相互作用进行测试,结果如图4所示。根据图4可知,N59a能够和DTX-1特异性结合,结合速率常数为5.44E+041/Ms,解离速率常数为1.61E-031/s,同时与OA进行少量结合,但亲和力常数差异巨大,N59a在关于DSP类毒素检测方面具有巨大的实际应用的价值。
以上内容已对本发明的实例进行具体说明,但本发明并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
序列表
<110> 中国人民解放军海军军医大学
<120> 与鳍藻毒素-1特异性结合的适配体及其应用
<130> 权利要求书 说明书
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gaggcagcac ttcacacgat cggacccaaa ctttacctta cccccatctg cgtaatgact 60
gtagtgatg 69
<210> 2
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaggcagcac ttcacacgat atttggggat cagccaggtc agtgccactg cgtaatgact 60
gtagtgatg 69
<210> 3
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gaggcagcac ttcacacgat cgtccctgcc cctgcctcct ttctatgctg cgtaatgact 60
gtagtgatg 69
<210> 4
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gaggcagcac ttcacacgat cgctgaagtc aacctcccct acctgtgctg cgtaatgact 60
gtagtgatg 69
<210> 5
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaggcagcac ttcacacgat ccaccaggcc aaacacgacc ccaaacactg cgtaatgact 60
gtagtgatg 69
<210> 6
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gaggcagcac ttcacacgat tccctcctcc tttatatccg gtccgatctg cgtaatgact 60
gtagtgatg 69
<210> 7
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccaccaggcc aaacacgacc ccaaaca 27
<210> 8
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gaggcagcac ttcacacgat ccaccaggcc aaacacgacc ccaaaca 47
<210> 9
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccaggccaaa cacgacccc 19
<210> 10
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccaccaggcc aaaca 15
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaggcagcac ttcacacgat 20
<210> 12
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
catcactaca gtcattacgc ag 22
<210> 13
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
catcactaca gtcattacgc ag 22
Claims (4)
1.与鳍藻毒素-1特异性结合的适配体,其特征在于,所述适配体的序列如SEQ ID NO.1~SEQ ID NO.9中任一条所示。
2.权利要求1所述的与鳍藻毒素-1特异性结合的适配体在制备鳍藻毒素-1分离富集试剂中的应用。
3.权利要求1所述的与鳍藻毒素-1特异性结合的适配体在制备鳍藻毒素-1检测试剂、试剂盒或传感器中的应用。
4.权利要求1所述的与鳍藻毒素-1特异性结合的适配体在饮用水体样品中鳍藻毒素-1快速检测中的应用。
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EP2770058A1 (en) * | 2013-02-26 | 2014-08-27 | Université de Perpignan | Ligand and method for detection of okadaic acid |
CN104894135A (zh) * | 2015-04-28 | 2015-09-09 | 中国人民解放军第二军医大学 | 一条与石房蛤毒素特异性结合的高亲和力适配体及其应用 |
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