WO2020014883A1 - 一种特异性识别妥布霉素的单链dna适配体及其应用 - Google Patents

一种特异性识别妥布霉素的单链dna适配体及其应用 Download PDF

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WO2020014883A1
WO2020014883A1 PCT/CN2018/096048 CN2018096048W WO2020014883A1 WO 2020014883 A1 WO2020014883 A1 WO 2020014883A1 CN 2018096048 W CN2018096048 W CN 2018096048W WO 2020014883 A1 WO2020014883 A1 WO 2020014883A1
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tobramycin
aptamer
stranded dna
detection
sequence
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周楠迪
聂晶晶
张玉红
韩旭艳
田亚平
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the invention relates to a single-stranded DNA aptamer that specifically recognizes tobramycin and its application, and belongs to the field of biotechnology.
  • Tobramycin is an aminoglycoside antibiotic. It is a broad-spectrum antibacterial drug with good antibacterial effect. It is suitable for treating various infection symptoms of the respiratory, digestive, urinary, and reproductive systems. It is widely used in animals and aquaculture. Because tobramycin is a medicine shared by humans and animals, in addition to its direct side effects on humans, tobramycin remaining in food and the environment will eventually affect human health and safety.
  • Traditional antibiotic residue detection methods include microbial detection and instrumental analysis, including gas chromatography (GC), high performance liquid chromatography (HPLC), liquid-mass spectrometry (HPLC-MS), and liquid chromatography-string Polar mass spectrometry (HPLC-MS / MS), etc. Microbial detection method is widely used.
  • the instrumental analysis method has high sensitivity and good accuracy, but often the equipment is expensive, and it also has high requirements for the laboratory personnel. It requires high sample pretreatment, and the analysis is time-consuming and difficult to popularize. It is difficult to meet the needs of field testing.
  • Immunoassay method uses antigen-antibody specific binding reaction to detect various substances, and has been widely used in the field of food antibiotic residue detection.
  • Enzyme-linked immunosorbent assay combines the immune response of the antigen and antibody with the highly efficient catalytic reaction of the enzyme to effectively ensure high selectivity and sensitivity in the analysis.
  • the most reported method is the immunoassay method.
  • ELISA kit products for the detection of antibiotic residues on the market, but they are expensive and the types of enzyme-labeled antibodies are limited. Only a few types of common antibiotic residues can be detected. Because antibiotics are small molecule compounds, they do not have immunogenicity, and there are many restrictions on production and application.
  • antibiotic haptens with weak immunogenicity in antigen preparation are difficult to produce high titer antibodies; they are produced in animals such as mice and rabbits
  • heterogeneous antibodies may produce non-specific reactions (false positives); the preparation of antibodies is expensive, time-consuming and laborious, and clones are not easy to save; the quality varies from batch to batch; the activity of antibodies is difficult to maintain for a long time and is sensitive to temperature. Prone to irreversibility.
  • SELEX technology is a combinatorial chemistry technology that uses random oligonucleotide libraries to screen in vitro oligonucleotide fragments that can specifically bind to various ligands.
  • This technology has no special requirements for target molecules and can be proteins or nucleic acids . Oligopeptides, small molecule organics and even metal ions, the selected oligonucleotides are called aptamers. Due to the advantages of aptamers in many aspects, it has been widely used as a target-specific recognition element in biosensor analysis.
  • the aptamer sequence obtained by SELEX screening is generally long, which increases the uncertainty of the conformation of the aptamer, which is not conducive to its stable binding to the target molecule in different solution environments, and also increases the cost of synthesis.
  • the rational design based on structural analysis can be used to truncate and optimize the aptamer sequence, retain its binding site to the target molecule, and delete a large number of redundant sequences, which can greatly reduce the cost of synthesis.
  • the secondary structure of the original sequence is often destroyed, and the affinity and sensitivity will be reduced.
  • 79-nucleotide tobramycin aptamer ap32 was obtained using magnetic beads SELEX screening. Starting from aptamer ap32, the ap32 sequence was truncated through secondary structure analysis and rational design. By optimization, a piece of aptamer ap32-34nt with a nucleotide length of 34 was obtained, but the affinity of aptamer ap32-34nt was reduced.
  • the present invention on the basis of ap32-34nt, removes part of the terminal base pairs and unpaired bases and shortens the length of the secondary structure stem region to obtain a sequence with greatly shortened sequence length and improved affinity.
  • the aptamer ap32-15nt.
  • the first object of the present invention is to provide a single-stranded DNA aptamer that specifically recognizes tobramycin, the nucleotide sequence of which is shown in SEQ ID NO.1.
  • the 3 'end or 5' end of the aptamer modifies a functional group or molecule.
  • the modified aptamer has the same function as the aptamer described above.
  • the functional group or molecule is used to improve the stability of the aptamer, provide a detection signal, or to connect the aptamer with other substances to form a composition.
  • the functional group or molecule is a fluorescent group, digoxin, isotope, electrochemical label, enzyme label, biotin, amino group, affinity ligand or thiol group.
  • the aptamer further includes the nucleotide sequence shown in SEQ ID No. 1 as a core sequence, and the sequence is extended on both sides or unilaterally, and is adapted to the adaption. Aptamers with the same function.
  • the second object of the present invention is to provide the application of the single-stranded DNA aptamer that specifically recognizes tobramycin for separation and enrichment or analysis and detection of tobramycin.
  • a third object of the present invention is to provide a composition for detecting tobramycin, comprising the aptamer.
  • a fourth object of the present invention is to provide a test paper for detecting tobramycin, including the aptamer.
  • a fifth object of the present invention is to provide a kit for detecting tobramycin, including the aptamer.
  • a sixth object of the present invention is to provide a chip for detecting tobramycin, including the aptamer.
  • the present invention removes part of the terminal base pairs and unpaired bases and shortens the length of the secondary structure stem region to obtain a sequence with a greatly shortened sequence length and improved affinity.
  • Aptamycin aptamer ap32-15nt, and the use of aptamer ap32-15nt for a variety of tobramycin detection methods and detection reagents, aptamers have the advantages of high affinity, high specificity, stable structure, etc.
  • the established detection method has the advantages of short detection period, high sensitivity, low cost, and strong specificity.
  • Figure 2 Simulation of the secondary structure of the truncated aptamer ap32-15nt and its docking with tobramycin molecules;
  • Figure 3 The standard curve of the relationship between the peak current value and the concentration of tobramycin in the DPV curve for electrochemical detection of tobramycin;
  • Figure 4 Standard curve of the relationship between the absorbance of AuNPs solution of tobramycin at 520 nM and the concentration of tobramycin by gold colloid colorimetry;
  • Figure 5 Schematic diagram of test strip detection of tobramycin.
  • the secondary structure of the aptamer ap32-34nt (Kd 58.92nmol ⁇ L -1) obtained from previous research in the laboratory, and its nucleotide sequence is shown in SEQ ID NO. 4 as 5'-CGTCGACGGATCCATGGCACGTTATAGGTCGACG-3 ' Based on the aptamer (ap32-34nt aptamer, see the large paper "Screening of Tobramycin-Specific Single-Stranded DNA aptamers and Sequence Optimization and Application Research", Zhang Yuhong, Jiangnan University). Aptamer sequences, as shown in Table 1:
  • the stem-loop structure is analyzed, and the dissociation constant (K d ) is determined by the fluorescence method.
  • the specific steps are as follows (1)-(11):
  • Epoxy-based magnetic microspheres a particle diameter of 1-2 ⁇ m and a concentration of 10 mg ⁇ mL -1 ;
  • Tobramycin solution configured to a concentration of 10 mmol ⁇ L -1 ;
  • step (1) The epoxy-based magnetic microspheres in step (1) and the tobramycin in step (3) are washed and combined under suitable conditions; the appropriate conditions refer to making the epoxy-based magnetic microspheres compatible with Conditions of specific binding of ofloxacin, including temperature of 37 ° C, action time of 12h, and binding buffer composition;
  • step (5) Blocking the ethanolamine solution in step (5) and step (4) under suitable conditions;
  • suitable conditions include a temperature of 37 ° C and a blocking time of 6 hours;
  • step (2) The steps (6) and the truncated aptamer in step (2) are combined under appropriate conditions; suitable conditions include a temperature of 25 ° C. and a binding time of 1 h;
  • step (8) The aptamer obtained in step (8) is eluted under appropriate conditions; suitable conditions include a temperature of 80 ° C and a binding time of 15 minutes;
  • step (10) collecting the aptamers eluted in step (9) and measuring the fluorescence intensity
  • the fitting curve and Kd value are shown in Figure 1.
  • the secondary structure of the aptamer ap32-15nt is shown in Figure 2.
  • Autodock4.0 software to perform molecular docking simulation with tobramycin. After a series of calculations and simulations, it was determined that the aptamer was mainly composed of tobramycin. When tobramycin interacts with the aptamer, it mainly interacts with bases 2-5, 7, and 9 on the aptamer ( Figure 2).
  • Example 2 aptamer ap32-15nt electrochemical detection of tobramycin
  • the designed hairpin structure sequence is (SEQ ID No. 5): 5'-AAAAAAGACTAGGCACTAGTCAAAAAACCCCGATCCTAGTCTTTCCC-3 '; where the italic portion is the truncated aptamer sequence.
  • the designed signal transduction probe sequence is (SEQ ID NO. 6): 5'-GCGAAAAAAGCG- (CH 2 ) 6 -HS-3 ', and the 3' end of the probe is modified with a thiol group to self-assemble to the surface of a gold electrode.
  • the designed primer sequence is (SQE ID NO.7): 5’-AAAGACTAGGA-3 ’
  • a hairpin structure of Hp
  • signal transduction probe the primers were designed; and formulated concentrations of 10 ⁇ mol ⁇ L -1, 1 ⁇ mol ⁇ L -1, 10 ⁇ mol ⁇ L -1 .
  • step (1) The hairpin structure (Hp) in step (1) is heated at 95 ° C for 5 minutes and placed in a hot water bath at 41 ° C for 2h to form a hairpin structure.
  • Pretreatment of gold electrode Carefully polish the gold electrode (diameter 3mm) with alumina powder (particle diameters 0.5 and 0.05 ⁇ m), and then immerse them in ethanol and ultrapure water for 5min, respectively. Scanning cycle in 0.5M H 2 SO 4 with scanning range: 0.35V to -1.5V and scanning speed of 100mV / s. Electrochemically activated polished electrodes were obtained until a stable CV map was obtained. Rinse with pure water and blow dry with nitrogen for later use.
  • step (4) Formation of triple-stranded structure: The single-stranded DNA obtained in step (4) is further modified on the electrode of step (6) to form a triple-stranded structure on the electrode.
  • step (7) The electrode obtained in step (7) is incubated with the [Ru (NH 3 ) 6 ] 3+ solution in step (2) for 1 h at room temperature for electrochemical detection.
  • the gold electrode obtained in step (8) is a working electrode, and is detected by differential pulse voltammetry (DPV).
  • DPV differential pulse voltammetry
  • Tobramycin of different concentrations is taken.
  • the measurement is performed.
  • DPV curves after reaction under different tobramycin conditions.
  • the relationship between the peak current value and tobramycin in the DPV curve was analyzed, and a linear fitting curve was drawn ( Figure 3).
  • Figure 3 With the increase of tobramycin concentration, the oxidation peak current signal also increases. In the range of tobramycin concentration from 10nmol ⁇ L -1 to 200nmol ⁇ L -1 , the response current is linear with the concentration of tobramycin.
  • the application of the method for detecting tobramycin based on the finally determined truncated aptamer detects the tobramycin in a milk sample.
  • the specific steps are as follows: Trichloroacetic acid is added dropwise to the milk sample. (20%) The pH was adjusted to 4.6, and then a 45 ° C water bath was used for 10 minutes to precipitate the protein, and the coagulated protein and fat were removed by centrifugation at 10,000 r ⁇ min -1 for 25 minutes to obtain a pretreated milk sample. The samples processed in this step are tested according to steps (1)-(8) in the above steps.
  • Solution A AuNPs were prepared using trisodium citrate reduction method and concentrated 5 times;
  • Solution B tobramycin aptamer ap32-15nt, formulated at a concentration of 150nmol ⁇ L -1 ;
  • Liquid C NaCl solution, formulated at a concentration of 120mmol ⁇ L -1 ;
  • step (4) sequentially add tobramycin of different concentrations, and protect from light for 50 minutes;
  • step (6) Add 50 ⁇ L of the C solution of step (3) to the solution obtained in step (5), observe the color change of each centrifuge tube, and perform spectral characterization with a spectrophotometer, and draw the relationship between the absorbance of the AuNPs solution and the concentration of tobramycin.
  • Example 4 Detection of tobramycin by a tobramycin test strip
  • DNA1 (SEQ ID NO.8): 5'-HS- (CH 2 ) 6 -TCAGGACTAGTGCCTGTCCAACGTCAGATCC-3 '
  • DNA2 (SEQ ID NO.9): 5’-Biotin-CCGATGGATCTGACGT-3 ’
  • the gold label pad is a special inert medium. After the synthesized gold label conjugate is dropped on the gold label pad, it will be adsorbed in the special inert medium to make the product. In order to make the gold label couple added on the gold label pad dropwise, The complex can be completely released after rehydration, and the gold label pad needs to be pretreated. First, cut the gold label pad and sample pad to the appropriate size, soak them in the sample pad and gold label pad pretreatment buffer for 30 minutes, and then place them in a 45 ° C constant temperature drying oven to dry. Drop on the treated gold label pad Add AuNPs-ap32-15nt and AgNPs-DNA1, dry in a 37 ° C incubator, and store at 4 ° C in the dark under dry conditions for future use;
  • NC membrane nitrocellulose membrane
  • biotinylated DNA2 was combined with streptavidin (SA). Mix 10 ⁇ L, 1mg ⁇ mL -1 streptavidin and 20 ⁇ L, 10 ⁇ mol ⁇ L -1 biotinylated DNA2 and mix for 2h at 4 °C. Due to the strong binding force between SA and biotin The SA and biotinylated DNA2 were fully coupled. Continue adding 10 ⁇ L of 5 mmol ⁇ L -1 biotin to the above solution to supplement unbound sites on SA.
  • the PVC base plate, sample pad, gold label pad, nitrocellulose membrane, and water absorption pad are the five components of the colloidal gold test strip.
  • the PVC base plate is a sticky base plate, and other film materials can be directly pasted and assembled on the base plate.
  • the treated sample pad and gold label pad are respectively adhered to the PVC bottom plate, of which the sample pad and gold label pad ,
  • the gold label pad and the NC film overlap each other by 2mm
  • the water absorption pad and the NC film are overlapped by 2mm and pasted on the PVC base plate, and the excess part is cut off.
  • Each part is firmly adhered, cut into test strips with a size of 60mm ⁇ 4mm, and placed in an aluminum foil bag with a desiccant, and stored at 4 ° C in a refrigerator for future use (Figure 5A);
  • the test strip First put the prepared test strip in the test strip cartridge. After the solution to be tested is added dropwise to the sample loading hole, the liquid moves to the water absorption pad with the siphon action of the capillary, and the test solution passes the test line on the NC membrane and The quality control line, react with it, let it stand for 10 minutes. After the test strip is completely colored, when the tobramycin is not present, the detection line can capture the AgNPs-DNA1-ap32-15nt-AuNPs complex, which makes AuNPs accumulate and display. color. The SA modified on the C line can directly capture AuNPs-ap32-15nt under the action of biotin-avidin, and make the C line develop color. At this time, both T and C lines showed negative results ( Figure 5B).

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Abstract

提供一种特异性识别妥布霉素的单链DNA适配体及其应用，属于生物技术领域。在ap32-34nt的基础上，通过去除部分末端碱基对和不配对碱基并缩短二级结构茎区长度的方式，得到序列长度大大缩短而亲和力有提高的妥布霉素适配体ap32-15nt，并将适配体ap32-15nt用于多种妥布霉素检测方法和检测试剂的构建，适配体具有亲和力高、特异性高、结构稳定等优点，所建立的检测方法具有检测周期短，灵敏度高，成本低，特异性强等优点。

Description

一种特异性识别妥布霉素的单链DNA适配体及其应用 技术领域
本发明涉及一种特异性识别妥布霉素的单链DNA适配体及其应用，属于生物技术领域。
背景技术
妥布霉素属于氨基糖苷类抗生素，是一类广谱抗菌药，其抗菌效果好，适于治疗呼吸、消化、泌尿、生殖系统的各种感染症状，广泛应用于动物和水产养殖业中。由于妥布霉素属于人兽共用药，其毒副作用除对人体有直接损害以外，食品和环境中残留的妥布霉素最终也会影响人类的健康安全。传统的抗生素残留检测方法有微生物检测法和仪器分析法，包括气相色谱法(GC)、高效液相色谱法(HPLC)、液-质联用仪法(HPLC-MS)、液相色谱-串极质谱法(HPLC-MS/MS)等。微生物检测法应用较为广泛，其优点是费用低，一般实验室都能操作，但测定时间长，结果误差较大，操作复杂，因此不能适应高通量高灵敏定量检测的需求。而仪器分析方法灵敏度高，准确性好，但往往设备昂贵，对实验人员也有较高的要求，对样品的预处理要求较高，分析费时而不易推广，难以满足现场检测的需要。免疫分析法是利用抗原抗体特异性结合反应检测各种物质，在食品抗生素残留检测领域已较为广泛地应用。酶联免疫吸附测定(ELISA)将抗原和抗体的免疫反应和酶的高效催化反应有机结合，有效保证分析中的高选择性和高灵敏度，报道最多的是免疫分析方法。目前市场上已有一些针对抗生素残留检测的ELISA试剂盒产品，不过价格昂贵，酶标抗体的种类有限，只能检测少数种类常见的抗生素残留。由于抗生素是小分子化合物，本身不具有免疫原性，生产与应用存在较多的限制，包括：抗原制备中免疫原性弱的抗生素半抗原难以产生高效价的抗体；产生于鼠、兔等动物的异源 抗体在使用中有可能产生非特异性反应(假阳性)；抗体的制备成本高，费时费力，克隆株不易保存；批间质量不一；抗体的活性难以长时间保持，对温度敏感，易发生不可逆变性。这些都在很大程度上限制了免疫分析方法在抗生素检测中的应用。
SELEX技术是一种利用随机寡核苷酸文库在体外筛选能与各种配体特异性结合的寡聚核苷酸片段的组合化学技术，该技术对靶分子无特殊要求，可以是蛋白质、核酸、寡肽、小分子有机物甚至是金属离子，筛选出的寡核苷酸被称为适配体。由于适配体在多个方面的优势，已被广泛用作生物传感分析中靶分子特异性的识别元件。但通过SELEX筛选得到的适配体序列一般比较长，增加了适配体构象的不确定性，不利于其在不同溶液环境中与靶分子的稳定结合，也提高了合成成本。利用基于结构分析的理性设计可以对适配体序列进行截短优化，保留其与靶分子结合的位点，删除大量冗余序列，能够大大降低合成成本。但是在截短过程中，往往会破坏原有序列的二级结构，亲和力、灵敏度等会降低。
本实验室前期研究利用磁珠SELEX筛选得到的长度为79个核苷酸的妥布霉素适配体ap32，从适配体ap32出发，通过二级结构分析和理性设计对ap32序列进行截短优化，得到一段核苷酸长度仅为34的适配体ap32-34nt，但是适配体ap32-34nt的亲和力有所降低。
发明内容
为解决上述问题，本发明在ap32-34nt的基础上，通过去除部分末端碱基对和不配对碱基并缩短二级结构茎区长度的方式，得到序列长度大大缩短而亲和力有提高的妥布霉素适配体ap32-15nt。
本发明的第一个目的是提供一种特异性识别妥布霉素的单链DNA适配体，其核苷酸序列如SEQ ID NO.1所示。
在本发明的一种实施方式中，所述适配体的3’端或5’端修饰功能基团或分子。
在本发明的一种实施方式中，修饰后的适配体与上述的适配体具有相同功能的适配体。
在本发明的一种实施方式中，所述功能基团或分子用于提高适配体的稳定性、提供检测信号，或者用于连接适配体与其他物质形成组合物。
在本发明的一种实施方式中，所述功能基团或分子为荧光基团、地高辛、同位素、电化学标记物、酶标记物、生物素、氨基、亲和配基或巯基。
在本发明的一种实施方式中，所述适配体还包括以如SEQ ID NO.1所示的核苷酸序列为核心序列，在两边或单边延伸序列，并与所述的适配体具有相同功能的适配体。
本发明的第二个目的是提供所述的特异性识别妥布霉素的单链DNA适配体在分离富集或分析检测妥布霉素中的应用。
本发明的第三个目的是提供一种用于检测妥布霉素的组合物，含有所述的适配体。
本发明的第四个目的是提供一种用于检测妥布霉素的试纸，包含所述的适配体。
本发明的第五个目的是提供一种用于检测妥布霉素的试剂盒，包含所述的适配体。
本发明的第六个目的是提供一种用于检测妥布霉素的芯片，包含所述的适配体。
本发明的有益效果：本发明在ap32-34nt的基础上，通过去除部分末端碱基对和不配对碱基并缩短二级结构茎区长度的方式，得到序列长度大大缩短而亲和力有提高的妥布霉素适配体ap32-15nt，并将适配体ap32-15nt用于多种妥布霉素检测方法和检测试剂的构建，适配体具有亲和力高、特异性高、结构稳定等优点，所建立的检测方法具有检测周期短，灵敏度高，成本低，特异性强等优点。
附图说明
图1：适配体ap32-15nt的K d值拟合曲线；
图2：截短适配体ap32-15nt二级结构和其与妥布霉素分子对接模拟图；
图3：电化学方法检测妥布霉素的DPV曲线中峰电流值与妥布霉素浓度关系标准曲线；
图4：金胶比色法检测妥布霉素的AuNPs溶液在520nM处的吸光值与妥布霉素浓度关系标准曲线；
图5：试纸条检测妥布霉素原理图。
具体实施方式
为了更好地理解发明的实质，下面通过实施例来详细说明发明的技术内容。
实施例1：妥布霉素ssDNA适配体截短
在实验室之前的研究得到的适配体ap32-34nt(Kd＝58.92nmol·L -1，其核苷酸序列如SEQ ID NO.4所示，为5’-CGTCGACGGATCCATGGCACGTTATAGGTCGACG-3’)二级结构的基础上(适配体ap32-34nt参见大论文《妥布霉素特异性单链DNA适配体的筛选及其序列优化和应用研究》张玉红，江南大学)，进行截短优化，得到几组适配体序列，如表1所示：
表1适配体与对应的核苷酸序列
适配体 核苷酸序列(SEQ ID NO.1～3)
ap32-15nt 5’-GACTAGGCACTAGTC-3’
ap32-13nt 5’-GACGGGCACCGTC-3’
ap32-15nt-2 5’-GACGTGGCACACGTC-3’
分析茎环结构，利用荧光法进行解离常数(K d)的测定，具体步骤如下(1)-(11)：
(1)环氧基磁性微球：粒径1-2μm，浓度为10mg·mL -1；
(2)截短适配体：用荧光基团(FAM)修饰上述三个截短的适配体；
(3)妥布霉素溶液：配置成浓度为10mmol·L -1；
(4)乙醇胺溶液：配置成浓度为0.5mol·L -1；
(5)使步骤(1)中的环氧基磁性微球和步骤(3)中的妥布霉素在适宜的条件下洗涤，结合；所述适宜条件是指使环氧基磁性微球能和氧氟沙星特异性 结合的条件，包括温度37℃、作用时间12h、结合缓冲液成分；
(6)将步骤(5)与步骤(4)中的乙醇胺溶液在适宜条件下封闭；适宜的条件包括温度37℃，封闭时间6h；
(7)使步骤(6)与步骤(2)中的截短适配体在适宜的条件下结合；适宜的条件包括温度25℃，结合时间1h；
(8)收集经步骤(7)处理得到的适配体；
(9)对步骤(8)中得到的适配体在适宜的条件下洗脱；适宜的条件包括温度80℃，结合时间15min；
(10)收集步骤(9)中洗脱得到的适配体进行荧光强度测定；
(11)以适配体浓度为横坐标，荧光强度为纵坐标，用Grapad Prism 5.0进行非线性拟合，可对适配体的解离常数K d值进行分析。
最终，适配体ap32-15nt的亲和力有所提高，而另外两个适配体的亲和力均大幅度下降，拟合曲线与Kd值参见图1，适配体ap32-15nt的Kd值为42.12nmol·L -1，与ap32-34nt(Kd＝58.92nmol·L -1)相比，亲和力具有较大的提升，并且与原始的适配体ap32(Kd＝52.32nmol·L -1)相比，亲和力也得到一定的提升。
适配体ap32-15nt的二级结构如图2所示，利用Autodock4.0软件与妥布霉素进行分子对接模拟，在经过一系列的计算模拟之后确定出适配体上主要与妥布霉素作用的碱基位点，妥布霉素与适配体作用时，主要与适配体上的2-5，7,9位碱基发生相互作用(图2)。
实施例2：适配体ap32-15nt电化学方法检测妥布霉素
设计发夹结构序列为(SEQ ID NO.5)：5’-AAAAAAGACTAGGCACTAGTCAAAAAACCCCGATCCTAGTCTTTCCC-3’；其中斜体部分为截短的适配体序列。
设计信号转导探针序列为(SEQ ID NO.6)：5’-GCGAAAAAAGCG-(CH 2) 6-HS-3’，探针的3’端修饰巯基以自组装至金电极表面。
设计引物序列为(SQE ID NO.7)：5’-AAAGACTAGGA-3’
(1)发夹结构(Hp)，信号转导探针，引物的序列设计；并配制浓度分别为10μmol·L -1，1μmol·L -1，10μmol·L -1。
(2)三氯六氨合钌([Ru(NH 3) 6] 3+)溶液：用10mmol·L -1的Tris-HCl配制浓度为10μmol·L -1。
(3)将步骤(1)发夹结构(Hp)在95℃加热5min并置于41℃的热水浴中2h以形成发夹结构。
(4)单链DNA的置换：在含有1μmol·L -1引物，5U phi29 DNA聚合酶，1mmol·L -1dNTP和5U Nt.AlwI切刻内切酶的15.6μL的1×CutSmart缓冲液中加入2.4μL步骤(3)中的发夹结构(Hp)。然后将含有妥布霉素的样品溶液(2μL)加入缓冲液中，在37℃温育120min以进行酶促反应。然后将所得混合物浸入65℃的热水浴中10min以使酶失活。最终得到大量单链DNA目标序列。
(5)金电极的预处理：用氧化铝粉末(粒径为0.5和0.05μm)分别小心地抛光金电极(直径3mm)，然后分别在乙醇和超纯水中浸泡5min。通过循环扫描在0.5M H 2SO 4中，扫描范围：0.35V至-1.5V，扫描速度100mV/s，进行电化学活化抛光电极，直至获得稳定的CV图为止，最后将处理好的电极用超纯水冲洗并用氮气吹干备用。
(6)信号转导探针的固定：将步骤(1)的信号转导探针用10mmol·L -1Tris-HCl(10mmol·L -1三(2-羧乙基)膦TCEP，100mmol·L -1NaCl，pH7.4)，孵育1h后，将步骤(5)得到的金电极浸于100μL孵育后的辅助探针中，37℃孵育13h，使捕获探针在金电极表面上形成自组装单分子层；用1mmol·L -1巯基己醇封闭电极1h，得到修饰捕获探针的金电极。
(7)三链结构的形成：步骤(4)得到的单链DNA继续修饰在步骤(6)的电极上，使其在电极上形成三链结构。
(8)将步骤(7)得到的电极在室温下与步骤(2)中的[Ru(NH 3) 6] 3+溶液孵育1h，用于电化学检测。
步骤(8)所得金电极为工作电极，用差分脉冲伏安法(DPV)进行检测；取不同浓度的妥布霉素，上述步骤(1)-(8)同样操作和试剂进行反应后，测 定在不同浓度的妥布霉素条件下反应后的DPV曲线图，分析DPV曲线中峰电流值与妥布霉素之间的关系，绘制线性拟合曲线(图3)。随着妥布霉素浓度的增加，氧化峰电流信号也随之增强，在妥布霉素浓度在10nmol·L -1到200nmol·L -1范围内，响应电流与妥布霉素浓度呈线性相关，拟合曲线方程y＝0.0005x+0.0788(x是妥布霉素浓度/nmol·L -1，y是峰电流值/μA)，线性相关系数R 2＝0.994。
所述基于最终确定的截短适配体检测妥布霉素的方法的应用，对牛奶样品中的妥布霉素的进行了检测，具体步骤如下：向牛奶样品中逐滴滴加三氯乙酸(20％)将pH调节到4.6，然后45℃水浴10min以沉淀蛋白，以10000r·min -1离心25min除去凝结的蛋白质和脂肪，得到经预处理后的牛奶样品。将步骤处理好的样品按照上述步骤中的的步骤(1)-(8)进行检测。
实施例3：金胶比色试剂盒对妥布霉素的检测
(1)A液：使用柠檬酸三钠还原法配制AuNPs并浓缩5倍；
(2)B液：妥布霉素适配体ap32-15nt，配制浓度为150nmol·L -1；
(3)C液：NaCl溶液，配制浓度为120mmol·L -1；
(4)取50μL的步骤(1)中的A液与100μL步骤(2)中的B液在室温下避光反应1h；
(5)向步骤(4)依次添加不同浓度的妥布霉素，避光反应50min；
(6)向步骤(5)所得溶液中添加50μL步骤(3)的C液，观察每个离心管颜色变化，并用分光光度计进行光谱表征，绘制AuNPs溶液吸光度与妥布霉素浓度关系图。
在不同浓度的妥布霉素存在下，随着妥布霉素浓度的增加，肉眼可观察到溶液颜色将会从原来的酒红色转变成蓝色(图4)；AuNPs溶液在520nm处的吸收值与妥布霉素浓度的关系，当妥布霉素的浓度在100-1400nmol·L -1范围内时，AuNPs溶液的在520nm处的吸光度随着妥布霉素浓度的增大而逐渐减小，呈现良好的线性关系(图4)。线性关系式为：y＝-2.599e-4x+1.02186(x是妥布霉素浓度/nmol·L -1，y是吸光度值)，线性相关系数R 2＝0.99047。
实施例4：妥布霉素检测试纸条对妥布霉素的检测
(1)修饰的妥布霉素适配体(ap32-15nt)：5’-HS-(CH 2) 6-GACTAGGCACTAGTC-Biotin-3’
DNA1(SEQ ID NO.8)：5’-HS-(CH 2) 6-TCAGGACTAGTGCCTGTCCAACGTCAGATCC-3’
DNA2(SEQ ID NO.9)：5’-Biotin-CCGATGGATCTGACGT-3’
(2)样品垫、金标垫的预处理
金标垫是特殊的惰性介质，将合成好的金标偶联物滴加到金标垫上后会吸附在特殊的惰性介质中才能制成产品，为了使滴加在金标垫上的金标偶联物再水化后能完全释放，需要对金标垫进行预处理。首先，将金标垫和样品垫裁剪成合适的大小后在样品垫、金标垫预处理缓冲液中浸泡30min，然后置于45℃恒温干燥箱中烘干，在处理好的金标垫上滴加AuNPs-ap32-15nt和AgNPs-DNA1，在37℃恒温培养箱中烘干，干燥条件下4℃避光保存备用；
(3)硝酸纤维膜(NC膜)的预处理
首先，将生物素(biotin)化的DNA2与链霉亲和素(SA)结合。将10μL，1mg·mL -1链霉亲和素和20μL，10μmol·L -1生物素化的DNA2混合均匀后在4℃的条件下混合2h，由于SA和生物素之间的强结合力会使SA和生物素化的DNA2充分偶联。继续向上述溶液中加入10μL，5mmol·L -1生物素补充SA上未结合的位点，孵育1h后用超滤离心管(30kDa)去除未结合的生物素和DNA，取1μL结合好的SA-biotin-DNA2在NC膜上靠近金标垫一端6mm处制备检测线(T线)。取1μLSA在靠近吸水垫一端6mm处划质控线(C线)。将制备好T、C线的硝酸纤维膜在37℃下烘干，使其充分固定在NC膜上，干燥条件下4℃保存备用；
(4)试纸条的组装
PVC底板、样品垫、金标垫、硝酸纤维膜、吸水垫是胶体金试纸条的五个组成部分，PVC底板是有粘性的底板，可以将其他膜材料在底板上直接粘贴组装。首先，将NC膜粘贴到PVC底板的中间位置，轻压使NC膜和底板紧密贴 合，然后，将处理过的样品垫和金标垫分别粘在PVC底板上，其中样品垫与金标垫，金标垫与NC膜各重叠2mm，最后将吸水垫与NC膜重叠2mm粘贴到PVC底板上，多余部分裁掉。各部分粘贴牢固，裁成规格为60mm×4mm的试纸条，与干燥剂一同放到铝箔袋中，4℃冰箱保存备用(图5A)；
(5)试纸条的检测
首先将制备好的试纸条置于试纸条卡盒中，在加样孔滴加待检测的溶液后，液体随毛细管的虹吸作用向吸水垫移动，检测液经过NC膜上的检测线和质控线，与其反应，静置10min，待试纸条显色完全后，当不存在妥布霉素时，检测线可以捕获AgNPs-DNA1-ap32-15nt-AuNPs复合物，使AuNPs积累而显色。在C线上修饰有SA可以在生物素-亲和素作用下直接捕获AuNPs-ap32-15nt，使C线显色。此时T、C线均显色呈阴性结果(图5B)；当存在妥布霉素时，由于ap32-15nt与妥布霉素的亲和力远高于其与互补序列的结合力，因此ap32-15nt与妥布霉素结合并在检测线上脱落下来，因此，T线颜色变浅或完全褪色，C线仍然可以捕获生物素化的ap32-15nt正常显色，此时呈现阳性结果(图5C)。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (9)

  1. 一种特异性识别妥布霉素的单链DNA适配体，其特征在于，其核苷酸序列如SEQ ID NO.1所示。
  2. 根据权利要求1所述的特异性识别妥布霉素的单链DNA适配体，其特征在于，所述适配体的3’端或5’端修饰功能基团或分子。
  3. 根据权利要求2所述的特异性识别妥布霉素的单链DNA适配体，其特征在于，所述功能基团或分子为荧光基团、地高辛、同位素、电化学标记物、酶标记物、生物素、氨基、亲和配基或巯基。
  4. 根据权利要求1所述的特异性识别妥布霉素的单链DNA适配体，其特征在于，所述适配体还包括以如SEQ ID NO.1所示的核苷酸序列为核心序列，在两边或单边延伸序列，并与权利要求1所述的适配体具有相同功能的适配体。
  5. 权利要求1～4任一项所述的特异性识别妥布霉素的单链DNA适配体在分离富集或分析检测妥布霉素中的应用。
  6. 一种用于检测妥布霉素的组合物，其特征在于，含有权利要求1～4任一项所述的适配体。
  7. 一种用于检测妥布霉素的试纸，其特征在于，包含权利要求1～4任一项所述的适配体。
  8. 一种用于检测妥布霉素的试剂盒，其特征在于，包含权利要求1～4任一项所述的适配体。
  9. 一种用于检测妥布霉素的芯片，其特征在于，包含权利要求1～4任一项所述的适配体。
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