CN116814638A - 一种四环素类抗生素广谱适配体 - Google Patents
一种四环素类抗生素广谱适配体 Download PDFInfo
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- CN116814638A CN116814638A CN202310863061.XA CN202310863061A CN116814638A CN 116814638 A CN116814638 A CN 116814638A CN 202310863061 A CN202310863061 A CN 202310863061A CN 116814638 A CN116814638 A CN 116814638A
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- C—CHEMISTRY; METALLURGY
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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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- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1048—SELEX
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- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
- C12N2320/13—Applications; Uses in screening processes in a process of directed evolution, e.g. SELEX, acquiring a new function
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
本发明公开了一种四环素类抗生素广谱适配体,属于分子生物学领域。本发明以四环素、土霉素、金霉素和强力霉素为混合靶标,采用基于氧化石墨烯的指数富集配体系统进化技术技术(GO‑SELEX)筛选获得了四环素类抗生素广谱适配体,并用荧光法验证了其高亲和力和高特异性,之后通过分子对接模拟进行序列分析和截短优化,明确了保守序列TTCAAGCG是四环素类抗生素广谱适配体的关键识别区域,获得了比原始适体具有更高亲和力和特异性的适体,解离常数为4.27~7.20 nM。该优化适配体为四环素类抗生素同步检测提供新的识别元件,具有广泛的应用前景。
Description
技术领域
本发明涉及一种四环素类抗生素广谱适配体,属于分子生物学领域。
背景技术
四环素类抗生素在奶牛养殖场中的的大量使用导致牛奶中存在兽药残留,这些残留物最终会通过食物链在人体内积累,并进一步引发一系列不良影响,比如影响牙齿发育、损害肝脏和肾脏、降低人体免疫力。因此,建立一种同时快速检测牛奶中4种四环素类抗生素残留的检测方法是保障乳品行业安全和人类健康的重要途经。
目前,牛奶中四环素类抗生素残留的检测方法主要分为两大类:一类是以质谱和液相色谱为主的仪器检测,操作复杂、仪器昂贵、需专门的技术人员进行操作;另一类是快速检测方法,仅检测单种抗生素或者几种抗生素,已经不能满足日益增多的大量样本快速、高效检测的要求。因此,迫切需要开发新的传感策略,为食品安全领域提供简单、高灵敏度和方便准确的四环素类抗生素残留同步检测方法。
适配体是通过指数富集配体系统进化技术(SELEX)从人工构建的随机寡核苷酸文库中筛选和扩增获得的单链短DNA或RNA序列,通常由20-80个碱基组成,库中寡核苷酸序列数可达 1013-1016个。适配体包含四个相似的亚基,通过链内某些互补碱基配对、静电作用等发生自身适应性折叠,形成复杂的二级、三级结构,为靶标提供核心结合区域,因此可与小分子、微生物、蛋白质等多种靶标相结合,且经多次筛选获得的核酸适配体与靶分子有很高的特异性和亲和力。综上,基于适配体的传感分析方法已成为抗生素残留检测的重要手段。
广谱性适配体的特性决定着分析检测的灵敏度和广谱特异性,是目前制约抗生素多残留分析技术发展的主要瓶颈。而目前有关抗生素广谱适配体筛选的研究报道有限,一定程度上制约了基于广谱适配体识别的抗生素多残留分析技术的发展。因此,有必要筛选出高特异性和亲和力的四环素类抗生素广谱适配体,利用适配体探针来增强化学稳定性。
发明内容
本发明的目的在于解决上述问题,以四环素、土霉素、金霉素和强力霉素为混合靶标,采用氧化石墨烯(GO)-SELEX进行四环素类抗生素广谱适配体的筛选,以获得四环素类抗生素广谱适配体,实现四环素类抗生素同步快速检测。
为实现上述技术目的,本发明采取的技术方案:一种四环素类抗生素广谱适配体,包括以下步骤。
(1)通过筛选获得四环素类抗生素广谱适配体,其序列为TCGTCGACGGATCCATGGCACATGGATTACTAGCGAACGCATGAGCCGGCTGCGGCGCATGCGT。
(2)四环素类抗生素广谱适配体序列分析。
(3)四环素类抗生素广谱适配体亲和力分析和特异性验证。
(4)四环素类抗生素广谱适配体截短优化。
优选的是,步骤(1)中四环素类抗生素广谱适配体的筛选,所采用的随机文库为79 nt的单链寡核苷酸,由生物公司合成,包括上、下游引物和中间35个随机核苷酸序列。
优选的是,步骤(1)四环素类抗生素广谱适配体正筛选以含有并四苯基本骨架的四环素、土霉素、金霉素和强力霉素作为混合靶标,与初始文库孵育后加入氧化石墨烯,经离心纯化PCR扩增后进行凝胶电泳验证,接着制备单链,重复以上筛选过程,随着筛选进行,与靶标亲和力高的ssDNA不断得到富集。 优选的是,步骤(1)四环素类抗生素广谱适配体反筛选,以四环素类抗生以外的抗生素如青霉素、氨苄青霉素三水、羟氨苄青霉素、苯唑西林钠作为反筛靶标,与上一轮产物混合孵育,加入氧化石墨烯吸附未与反筛靶标结合的单链寡核苷酸,离心去除能与反筛靶标结合的适配体,以提高所筛选适配体的特异性。
优选的是,步骤(1)筛选第一轮,将1 μL DNA 初始文库 (100 μM)加入到199μL结合缓冲液中,与混合靶标孵育后,加入500 μL 氧化石墨烯溶液 (2 mg/mL),以吸附未与靶标结合的ssDNA序列,混合2 h后,通过离心去除氧化石墨烯。
优选的是,步骤(1)PCR下游引物用生物素标记,PCR产物用提取试剂盒去除酶和引物。随后用3%琼脂糖凝胶进行电泳验证,其中DNA染料GelRed与纯化的PCR产物在loadingbuffer中混合。对生成的DNA条带进行成像和观察,以评估SELEX的效率。
优选的是,步骤(1)制备核苷酸单链,PCR产物与链霉亲和素修饰的磁珠混合孵育,DNA 通过生物素与链霉亲和素的作用连接在链霉亲和素磁珠上,通过NaOH溶液解离DNA双链,收集洗脱液作为下一轮的次级文库。
优选的是,步骤(1)最后一轮筛选PCR产物通过琼脂糖凝胶电泳验证,对PCR产物纯化回收,并与pMD-19T载体连接,通过热激转化转移到大肠杆菌感受态细胞,挑选阳性的菌落培养后提取质粒进行测序,获得适配体序列。
优选的是,步骤(2)通过M-fold分析适配体二级结构,结合利用DNAMAN8分析的一级结构的同源性和二级结构的相似性,以及自由能等因素,获得5条候选序列。
优选的是,步骤(3)利用荧光法,通过荧光分光光度计测量最大荧光值,用Origin8.0软件进行非线性拟合,测定筛选序列的解离常数以表征获得的适配体核酸序列亲和力和特异性。
优选的是,步骤(4)采用分子对接模拟研究亲和力最高的适配体与四环素类抗生素的主要结合位点,适配体能够形成结合口袋,在氢键作用下,与四环素类抗生素稳定结合,其保守序列TTCAAGCG是四环素类抗生素广谱适配体识别的关键区域。依据此结果,结合适配体二级结构分析,对此适配体序列进行截短优化。
优选的是,步骤(4)在保留发夹和环的基础上去除掉两端不参与结合口袋形成的多余碱基,采用三种截短方法对四环素类抗生素广谱适配体进行截短优化,获得亲和力和特异性更高的适配体序列TCGTCGACGGATCCATGGCACATGGATTACTAGCGAACGCATGAGCCGGCTGCGGCGCATGCGT。利用荧光法验证该序列的亲和力和特异性。
本发明具有以下有益效果。
本发明采用GO-SELEX,以含有并四苯基本骨架的四环素、土霉素、金霉素和强力霉素作为混合靶标,筛选获得了一种四环素类抗生素广谱适配体,此广谱适配体具有良好的亲和力和特异性,以筛选的适配体为识别元件构建生物传感器,可实现四环素类抗生素同步检测,为监管部门的现场快速灵敏监测提供重要基础。
附图说明
图1适配体筛选原理图。
图2适配体筛选过程回收率。
图3适配体与四环素分子对接模拟,其中A为四环素,B为土霉素,C为金霉素,D为强力霉素。
图4适配体序列剪切优化,其中A、B、C为三种不同的截短方法。
图5适配体亲和力分析,其中TET为四环素、AUR为金霉素、OXY为土霉素、DOX为强力霉素。
图6适配体特异性验证,其中AMP为羟氨苄青霉素、CEP为头孢氨苄、GEN为庆大霉素、CHL为氯霉素。
实施方式
下面结合附图和实施例对本发明作进一步详细说明,但实施例并不对本发明做任何形式的限定。
实施例1:一种四环素类抗生素广谱适配体,具体步骤为。
(1)适配体的筛选过程如图1所示,首先,取1 μL ssDNA随机文库(100 μM)加入到199 μL结合缓冲液(20 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mMCaCl2, 0.02% Tween 20, pH 7.6))中,混合均匀,于90℃加热变性10 min,迅速冰浴10min,室温放置10 min。接着加入等物质量的TET、AUR、OXY和DOX的混合物,混合均匀后室温轻微震荡 1 h,ssDNA 自适应形成特定的三维结构,一部分 ssDNA 形成的三维结构可以与TCs结合,形成复合物。然后加入500 μL氧化石墨烯溶液(2 mg/mL)吸附未与TCs结合的ssDNA,室温下轻微震荡 2 h。离心弃掉沉淀,上清液中含有能与TCs结合的 ssDNA,用提取试剂盒纯化后保存。
(2)PCR扩增,以1 μL ssDNA 为模板,1 μL不带生物素的上游引物、1 μL带生物素的下游引物和12.5 μL Taq PCR Master Mix按一定反应条件进行 PCR 扩增。在最佳循环下,PCR进行如下处理:预变性(95℃、5 min)、变性(95℃、30 s)、退火(55℃、30 s)、延伸(72℃、15 s),在72℃下最终延伸10 min。
(3)电泳分析,PCR产物经纯化后,用提取试剂盒去除酶和引物。随后用3%琼脂糖凝胶进行电泳验证,其中DNA染料GelRed与纯化的PCR产物在loading buffer中混合。对生成的DNA条带进行成像和观察,以评估筛选的效率。
(4)制备核苷酸单链,取 15 μL 的链霉亲和素磁珠,用 100 μL洗涤(B&W)缓冲液(10 mM Tris-HCl,1 mM EDTA,2 M氯化钠,pH 7.5)冲洗 5遍,用 80 μL 的 B&W 缓冲液悬浮链霉亲和素磁珠,加入 20 μL 第一轮 PCR 扩增产物,室温结合轻微震荡1 h。DNA 通过生物素与链霉亲和素的作用连接在链霉亲和素磁珠上,磁力架分离,除去未结合在磁珠上的 DNA,将沉淀物置于50 μL的氢氧化钠(0.1 M)中,在37◦C下洗脱30 min,接着用20μL氢氧化钠分开清洗沉淀两次,一并收集洗脱液作为下一轮的次级文库。
(5)反筛选,以四环素类抗生以外的抗生素如青霉素、氨苄青霉素三水、羟氨苄青霉素和苯唑西林钠作为反筛靶标,与上一轮产物混合孵育,加入氧化石墨烯吸附未与反筛靶标结合的单链寡核苷酸,往含有氧化石墨烯沉淀的离心管中加入结合缓冲液,离心弃掉上清液,重复操作 3 次,彻底去除能与反筛靶标结合的适配体。以提高所筛选适配体的特异性。
(6)筛选过程回收率如图2所示,通过核酸蛋白定量仪测定纯化回收的适配体浓度,计算适配体的回收率。第 1轮到第4轮的筛选中,回收率呈直线上升,表明能与四环素类抗生素结合的适配体得到富集。当第5轮和第8轮用反筛选进行时,因为结合反向靶标的适配体部分被消除以改善所需适配体的选择性,回收率降低,随着筛选过程的继续,回收率趋于稳定,表明能与四环素类抗生素结合的适配体趋于饱和,能与靶标特异性结合的适配体得到富集,筛选过程结束。
实施例2:四环素类抗生素广谱适配体的序列分析,具体步骤为。
(1)测序,经过13轮筛选后,用未经修饰的引物对最后一轮富集文库进行 PCR扩增并纯化,将纯化的PCR产物与pMD-19T载体连接,通过热激转化转移到大肠杆菌感受态细胞,挑选阳性的菌落培养后提取质粒进行测序。
(2)序列分析,采用DNAMAN8软件对所得序列进行同源性分析,并通过Mfold在线分析软件预测这些序列的二级结构和自由能(△G),综合分析选择5条不同家族的代表性序列作为适配体候选序列,通过模拟分析这5条序列的二级结构,发现二级结构中均含有发夹、茎环等结构,这些结构区域可能提供与靶标结合的关键位点。
实施例3:四环素类抗生素广谱适配体亲和力分析和特异性验证,具体步骤为。
(1)亲和力分析,利用荧光法测定获得的适配体与四环素类抗生素包括四环素、土霉素、金霉素和强力霉素的亲和力,发现解离常数在5.03-7.09 nM,亲和性较好。
(2)特异性验证,发现所获得的适配体对羟氨苄青霉素、头孢氨苄、庆大霉素和氯霉素等四环素类抗生素以外其他类抗生素不具有亲和性,特异性较好。
实施例4:四环素类抗生素广谱适配体截短优化,具体步骤为。
(1)利用分子对接模拟研究上述亲和力最高的适配体与四环素类抗生素的主要结合位点,如图3所示,适配体能够形成结合口袋,在氢键作用下,与四环素类抗生素稳定结合,其保守序列TTCAAGCG是四环素类抗生素广谱适配体识别的关键区域。
(2)采用三种截短方法对四环素类抗生素广谱适配体进行截短优化,如图4所示,利用上述荧光法验证三条序列的亲和力和特异性,根据解离常数和最大荧光值挑选亲和力和特异性最高的适配体序列,如图5和图6所示,该序列为 TCGTCGACGGATCCATGGCACATGGATTACTAGCGAACGCATGAGCCGGCTGCGGCGCATGCGT。
Claims (8)
1.一种四环素类抗生素广谱适配体,其特征在于,包括如下步骤:
(1)通过筛选获得四环素类抗生素广谱适配体,其序列为TCGTCGACGGATCCATGGCACATGGATTACTAGCGAACGCATGAGCCGGCTGCGGCGCATGCGT;
(2)四环素类抗生素广谱适配体序列分析;
(3)四环素类抗生素广谱适配体亲和力分析和特异性验证;
(4)四环素类抗生素广谱适配体截短优化。
2. 根据权利要求1所述的一种四环素类抗生素广谱适配体,其特征在于,步骤(1)所述四环素类抗生素广谱适配体的筛选,初始ssDNA文库含有79 nt 单链寡核苷酸,包括上、下游引物和中间35个随机核苷酸序列。
3.根据权利要求1所述的一种四环素类抗生素广谱适配体,其特征在于,步骤(1)四环素类抗生素广谱适配体正筛选以含有并四苯基本骨架的四环素、土霉素、金霉素和强力霉素作为混合靶标,与初始文库孵育后加入氧化石墨烯,经离心纯化PCR扩增后进行凝胶电泳验证,接着制备单链,重复以上筛选过程,随着筛选进行,与靶标亲和力高的ssDNA不断得到富集。
4.根据权利要求1所述的一种四环素类抗生素广谱适配体,其特征在于,步骤(1)四环素类抗生素广谱适配体反筛选以四环素类抗生以外的抗生素如青霉素、氨苄青霉素三水、羟氨苄青霉素、苯唑西林钠作为反筛靶标,与上一轮产物混合孵育,去除能与反筛靶标结合的适配体,以提高所筛选适配体的特异性。
5.根据权利要求3和4所述的一种四环素类抗生素广谱适配体,其特征在于,利用荧光法对筛选得到的适配体进行亲和力分析和特异性验证。
6.根据权利要求5所述的一种四环素类抗生素广谱适配体,其特征在于,基于分子对接模拟结果,判断适配体与四环素类抗生素的主要结合位点,发现其保守序列TTCAAGCG是四环素类抗生素广谱适配体识别的关键区域,适配体能够形成结合口袋,在氢键作用下,与四环素类抗生素稳定结合。
7.根据权利要求6所述的四环素类抗生素广谱适配体,其特征在于,依据分子对接模拟结果,结合适配体二级结构分析,对适配体序列进行截短优化,去除两端不参与结合口袋形成的多余碱基,获得亲和力和特异性更高的适配体序列TCGTCGACGGATCCATGGCACATGGATTACTAGCGAACGCATGAGCCGGCTGCGGCGCATGCGT。
8.根据权利要求7所述的四环素类抗生素广谱适配体,其特征在于,步骤(4)获得的适配体,采用通过荧光法测定获得的适配体亲和性和特异性。
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