WO2016011845A1 - 特异识别t-2毒素的寡核苷酸适配体 - Google Patents

特异识别t-2毒素的寡核苷酸适配体 Download PDF

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WO2016011845A1
WO2016011845A1 PCT/CN2015/079797 CN2015079797W WO2016011845A1 WO 2016011845 A1 WO2016011845 A1 WO 2016011845A1 CN 2015079797 W CN2015079797 W CN 2015079797W WO 2016011845 A1 WO2016011845 A1 WO 2016011845A1
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toxin
aptamer
seq
oligonucleotide
oligonucleotide aptamer
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French (fr)
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王周平
陈秀娟
顾华杰
夏雨
吴世嘉
段诺
马小媛
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江南大学
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Priority to EP15825274.2A priority Critical patent/EP3018212B1/en
Priority to US14/909,743 priority patent/US9856482B2/en
Publication of WO2016011845A1 publication Critical patent/WO2016011845A1/zh

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    • 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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    • G01N33/50—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
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/00—Applications; Uses
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    • C12N2320/13—Applications; Uses in screening processes in a process of directed evolution, e.g. SELEX, acquiring a new function
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a high affinity specific T-2 toxin-binding oligonucleotide aptamer prepared by SELEX technology (exponentially enriched ligand system evolution technology), It lays the foundation for rapid separation and analysis and analysis of T-2 toxin.
  • T-2 toxin is the most toxic of the class A trichothecene mycotoxins and is a sesquiterpene compound.
  • T-2 toxin can be produced by a variety of fungi, mainly from Fusarium, such as Fusarium oxysporum, Fusarium oxysporum and Fusarium oxysporum. It is widely distributed and seriously endangers human and animal health.
  • Toxicity of T-2 toxins includes acute toxicity, subacute toxicity, chronic toxicity, triad action, immunotoxicity, and damage to the blood and soft tissues.
  • the toxicity of T-2 toxin is relatively stable. It is kept at room temperature for 6 to 7 years or heated at 100-120 °C for 1 h, and its toxicity is still not reduced.
  • T-2 toxin Due to its high toxicity, the international concern about the harm of T-2 toxins to humans and animals. As early as 1973, FAO/WHO listed trichothecene T-2 toxin as one of the most dangerous natural food sources of food contamination. After the "Yellow Rain Poisoning" incident, the research on T-2 toxin was more extensive and in-depth.
  • T-2 toxin is mainly determined by thin layer chromatography (TLC), gas chromatography (GC), liquid chromatography (LC), liquid chromatography-mass spectrometry (LC-MS) and enzymes.
  • Immunological assays such as immunoassay (ELISA).
  • TLC thin layer chromatography
  • GC gas chromatography
  • LC liquid chromatography
  • LC-MS liquid chromatography-mass spectrometry
  • ELISA immunoassay
  • the derivatization of the GC method is mainly based on trimethylsilylation and fluoroacylation, and the derivatization reagent used in the LC method is cyanic acid.
  • the LC-MS method has high sensitivity, and the sample preparation is relatively simple, and no derivatization treatment is required, but the cost is high.
  • the antibody-dependent immunoassay is simple and sensitive, and is suitable for detecting a large number of samples without the advantages of large expensive instruments, but the false positive rate is relatively high and the quantification is not accurate; and T-2 is a molecular weight of 466 g/mol.
  • the small molecule hapten which is not immunogenic, needs to be combined with a macromolecular carrier protein to prepare a complete antigen to stimulate the animal to secrete antibodies. This process is not only cumbersome and expensive, but also results in stable batches of antibodies. Not as good as oligonucleotides, The storage conditions of the antibodies are also harsher than those of the oligonucleotides.
  • Oligonucleotide aptamers have been attracting attention as promising substitutes for antibody molecules. Oligonucleotide aptamers are passed through SELEX (Systematic Evolution of Ligands by
  • Exponential Enrichment an exponentially enriched ligand system evolution
  • SELEX technology is a new combinatorial chemistry technology developed in the 1990s. It is economical, simple, fast, and has a wide range of applications.
  • SELEX technology uses molecular biology techniques to construct a synthetic random oligonucleotide library, in which the random sequence length is generally around 20-40 nt, and the primer sequences at both ends are generally 20
  • the library capacity is approximately in the range of 1013-1015. Due to the random sequence of single-stranded oligonucleotides, it is easy to bind to proteins, peptides, drugs, amino acids, organic compounds, and even metal ions by secondary structures such as convex rings, hairpins, pseudo-knocks, and G-tetramers. , forming a strong binding complex.
  • the mycotoxin aptamers reported so far are mainly prepared by SELEX screening by immobilizing the target on an affinity chromatography column or magnetic beads, the former having ochratoxin A and the latter having fumonisin Bl, and also It was reported that zearalenone aptamers were obtained from different laboratories by chromatography and magnetic bead screening. However, there are no reports on T-2 toxin oligonucleotide aptamers and their preparation methods.
  • the object of the present invention is to provide an oligonucleotide aptamer capable of specifically binding to a T-2 toxin, which lays a good foundation for a novel separation and enrichment or analytical detection tool for T-2 toxin.
  • Another object of the present invention is to provide a method for preparing a T-2 toxin oligonucleotide aptamer, which can conveniently and accurately obtain a high affinity single-stranded DNA aptamer of T-2 toxin, and the effect Significant.
  • the method of the present invention utilizes SELEX technology based on graphene oxide separation, targets T-2 toxin, does not need to immobilize a small molecule target on a carrier, and performs cloning and sequencing of an enriched library after repeated screening by 10 rounds of SELEX. And analyze the affinity and specificity of the representative sequence to finally obtain the optimal oligonucleotide aptamer that specifically binds to the T-2 toxin with high affinity.
  • the present invention targets T-2 toxins commonly found in foods or feeds, and obtains oligonucleotides having high affinity for specific binding to T-2 toxins by using SELEX technology based on graphene oxide (GO) separation.
  • Ligand The oligonucleotide aptamer can be used to separate trace T-2 toxins from enriched samples, and can also be used for analysis and detection of T-2 toxins by special group modification, enriching laboratory detection methods, and can be used for ⁇ Test strips or portable small instruments for rapid detection in homes, farms, and factories, so the invention can be widely used in the field of mycotoxin detection.
  • 1 is a simulated secondary structure diagram of the full length sequence Seq. 2, Seq. 6, Seq. 16 and truncated sequences Tc. 2, Tc. 6, Tc.
  • Seq. 2 is a saturation binding curve of Seq. 2, Seq. 6, Seq. 16 and Tc. 6 oligonucleotide aptamers.
  • Example 1 GO-SELEX Screening of T-2 Toxin-Specific Binding Oligonucleotide aptamers
  • N 40 random nucleotides
  • Downstream primer 5'- CAGATGCACGACTTCGAGTC-3'
  • the random ssDNA library and the primers were all prepared in TE buffer and stored in -20 ° C for use.
  • ssDNA synthetic random single-stranded library
  • the PCR reaction system is: diluted random library as template DNA 1 (100 ng), upstream primer and phosphorylation downstream primer (20 ⁇ ) 1 ⁇ , dNTPmix (each 25 mM) 1 ⁇ , 10 x PCR amplification buffer 5 ⁇ , sterilized ultrapure water 40 ⁇ , Taq enzyme 1 ⁇ , total volume 50 ⁇ .
  • PCR amplification procedure predenaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s; annealing at 53 °C for 30 s; extension at 72 °C 30
  • the electrophoresis band is correctly positioned and a single PCR amplification product is purified by PCR product purification kit (Genemy Biotech. Co., Ltd.), redissolved in an appropriate volume of sterile ultrapure water, and passed through Thermo NanoDrop
  • the dsDNA concentration was determined by a 2000 ultra-micro spectrophotometer. Take a certain concentration of purified PCR product solution, add 1/10 volume of lOx reaction solution and mix well. Add the required exonuclease (5 U/ ⁇ according to the instructions, react at 37 °C for 30 min ⁇ 60 min, 75° The reaction was terminated by inactivation of the enzyme in a water bath for 10 min.
  • the electrophoresis was carried out by electrophoresis on an 8% polyacrylamide gel denatured with 7 M urea, and the optimal digestion conditions were determined, followed by amplification.
  • the first round of screening was performed in a binding buffer (BB, 10 mM Tris-HCl, 150 mM NaCl, 10 mM KC1, and 2.5 mM MgC12, pH 7.4).
  • BB binding buffer
  • Nmol random ssDNA library (in the 2nd-10th round of ssDNA library input reduction to 200, 100
  • the ssDNA library was evenly mixed with the free target T-2 toxin, and incubated for 2 h in a special binding buffer containing 1.0% methanol (total volume of 500 ⁇ M), which was gently shaken during the incubation period. After the end of the incubation, the mixture was transferred to a layer of GO that had been previously centrifuged and incubated at 25 ° C for 40 min. The ssDNA sequence bound to the T-2 target is retained in the supernatant, while the unbound ssDNA sequence is adsorbed by GO. Centrifuge at 13000 rpm/min 10
  • the supernatant was collected to obtain a ssDNA aptamer library bound to the T-2 toxin.
  • the supernatant was subjected to PCR amplification as an amplification template, and the purified product was digested with lambda exonuclease to prepare a single-stranded secondary library.
  • the purified ssDNA concentration was determined by a Thermo NanoDrop 2000 ultra-micro spectrophotometer to calculate the next round of library input volume.
  • the supernatant was collected by centrifugation and PCR amplified. After purification of the PCR product, the phosphorylated antisense strand was digested with lambda exonuclease to prepare the next round of single-stranded library.
  • the oligonucleotide aptamer-enriched library obtained by the 10th round screening was amplified with an unmodified primer, and the PCR product was purified and used with pMD-19T Cloning Vector (Takara Biotech. Co., Ltd.) Cloned and transformed into calcium chloride competent cells E. coli DH5ot. After 18 hours of culture on a solid medium containing 50 g/mL ampicillin, 30 white positive clones were randomly selected and cultured in LB liquid medium by "blue-white spot screening", and the plasmid MiniPrep kit (Generay Biotech) was used. Co., Ltd.) The plasmid was extracted and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.
  • T-2 toxin a fixed concentration of T-2 toxin ( ⁇ ) was adapted to a range of different concentrations (5, 10, 25, 50, 100, 200 ⁇ ).
  • the body sequence was incubated for a total volume of 500 ⁇ and incubated for 2 h in the dark. Then, GO was added in proportion to the aptamer concentration and incubation was continued for 40 min in the dark. After centrifugation, the fluorescence value at 490 nm excitation was measured with a Hitachi F-7000 fluorescence spectrometer. At the same time, a negative control without T-2 toxin was added to rule out non-specific binding.
  • the dissociation constant Kd value of each oligonucleotide aptamer was calculated using GmphPad Prism 5 software (see Table 1).
  • oligonucleotide aptamers of Seq. 2, Seq. 6 and Seq. 16 were selected for specific analysis.
  • Each 5'FAM-labeled oligonucleotide aptamer was mixed with target T-2 or FB1, ZEN, AFB1, OTA, respectively, and then incubated with GO solution (final concentration 0.3 mg/mL) at room temperature in the dark. 2 h.
  • GO solution final concentration 0.3 mg/mL

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Abstract

提供了一种特异结合T2毒素的小分子寡核苷酸适配体，序列为SEQ ID NO: 1。该单链寡核苷酸适配体是通过基于氧化石墨烯分离的指数富集配体系统在体外筛选获得的，能够高亲和力且特异的识别T2毒素，可用于分离富集样品中的痕量T2毒素，也可以转化为报告适配体用于分析检测食品中的T2毒素。

Description

说明书 t明名称： 特异识别 T-2毒素的寡核苷酸适配体
技术领域
[0001] 本发明涉及生物技术领域， 具体涉及一种经 SELEX技术 （指数富集的配体系统 进化技术） 制备而得的高亲和力特异性结合 T-2毒素的寡核苷酸适配体， 为快速 分离富集及分析检测 T-2毒素奠定了基础。
背景技术
[0002] T-2毒素 （T-2 toxin) 是 A类单端孢霉烯族真菌毒素中毒性最强的一种， 是一 种倍半萜烯化合物。 T-2毒素可由多种真菌产生， 主要产自如三线镰刀菌、 枝孢 镰刀菌和梨孢镰刀菌等镰刀菌属， 分布极广， 严重危害人畜健康。 T-2毒素毒性 作用包括急性毒性作用、 亚急性毒性、 慢性毒性、 三致作用、 免疫毒性以及对 血液系统和软组织的损害作用。 T-2毒素的毒性较为稳定， 室温放置 6〜7年或在 1 00-120°C加热 l h， 其毒性依然不减。 由于其毒性剧烈， 国际上非常重视 T-2毒素 对人畜的危害， 早在 1973年， FAO/WHO就将单端孢霉烯族 T-2毒素列为最危险 的天然存在的食品污染源之一； "黄雨中毒"事件发生之后， 关于 T-2毒素的研究 更为广泛和深入。
[0003] 目前， T-2毒素的测定方法主要有薄层色谱法 （TLC) 、 气相色谱法 （GC) 、 液相色谱法 （LC) 、 液相色谱质谱联用法 (LC-MS)和酶联免疫法 （ELISA) 等 免疫学检测法。 TLC法虽然分析成本较低， 但操作过程烦琐， 重复性差。 GC法 和 LC法都不能直接测定样品中的 T-2毒素， 通常需要衍生， GC法的衍生主要基 于三甲硅烷基化作用和氟酰基化作用， LC法使用的衍生化试剂为氰酸蒽， 在测 定过程中使用了有毒化学试剂。 LC-MS法灵敏度高， 样品制备相对简便， 不需 要衍生化处理， 但成本较高。 依赖抗体的免疫学检测法操作简单、 灵敏度高， 适用于检测大量样品， 无需大型昂贵仪器设备的优点， 但假阳性率比较高， 定 量不够准确； 且 T-2作为一种分子量 466 g/mol的小分子半抗原， 不具备免疫原性 ， 需将其与大分子载体蛋白结合制备完全抗原后才能刺激动物分泌抗体， 这个 过程不仅繁琐耗吋而且成本昂贵， 且制得的抗体批次间稳定性不如寡核苷酸， 抗体的储存条件也较寡核苷酸严苛。
近些年来， 寡核苷酸适配体作为抗体分子的前景性替代分子， 其研究较为引人 注目。 寡核苷酸适配体是通过 SELEX (Systematic Evolution of Ligands by
Exponential Enrichment, 指数富集的配体系统进化） 技术筛选的与靶物质特异性 结合的一簇小分子 DNA或 RNA片段。 SELEX技术是 20世纪 90年代发展起来的一 种新的组合化学技术， 具有经济、 简便、 快速、 适用范围广等特点。 SELEX技 术利用分子生物学技术， 构建人工合成的随机寡核苷酸文库， 其中随机序列长 度一般在 20-40 nt左右， 两端引物序列一般在 20
nt左右， 文库容量大约在 1013-1015范围内。 由于单链寡核苷酸随机序列， 容易 凸环、 发卡、 假节、 G-四聚体等二级结构， 故能与蛋白质、 肽段、 药物、 氨基 酸、 有机化合物， 甚至是金属离子相结合， 形成很强结合力的复合体。
技术问题
[0005] 至今报道的真菌毒素适配体， 主要是通过将靶标固定在亲和层析柱或磁珠上进 行 SELEX筛选制备的， 前者有赭曲霉毒素 A， 后者有伏马毒素 Bl， 也有报道不 同实验室分别用层析柱及磁珠筛选获得了玉米赤霉烯酮适配体。 但目前尚无有 关 T-2毒素寡核苷酸适配体及其制备方法的研究报道。
问题的解决方案
技术解决方案
[0006] 本发明的目的在于提供一种能与 T-2毒素特异性结合的寡核苷酸适配体， 为幵 发 T-2毒素的新型分离富集或分析检测工具奠定良好基础。
[0007] 本发明的另一目的是提供一种制备 T-2毒素寡核苷酸适配体的方法， 它可以方 便、 准确地获取 T-2毒素的高亲和力单链 DNA适配体， 效果显著。
[0008] 本发明方法利用基于氧化石墨烯分离的 SELEX技术， 以 T-2毒素为靶标， 无需 将小分子靶标固定在载体上， 通过 10轮的 SELEX反复筛选后， 对富集文库进行 克隆测序， 并分析代表序列的亲和力和特异性， 最终获得与 T-2毒素高亲和性特 异结合的最佳寡核苷酸适配体。
发明的有益效果
有益效果 [0009] 本发明以食品或饲料中常见的 T-2毒素为靶标， 利用基于氧化石墨烯 （GO) 分 离的 SELEX技术制备获得了与 T-2毒素高亲和力特异性结合的寡核苷酸适配体。 该寡核苷酸适配体可用于分离富集样品中的痕量 T-2毒素， 也可经特殊基团修饰 后用于分析检测 T-2毒素以丰富实验室检测手段， 并可用于幵发试纸条或便携式 小型仪器以实现家庭、 农田、 工厂快速检测， 因此该发明可以在真菌毒素检测 领域得到广泛应用。
对附图的简要说明
附图说明
[0010] 图 1是全长序列 Seq. 2、 Seq. 6、 Seq. 16及截短序列 Tc. 2、 Tc. 6、 Tc. 16的模拟 二级结构图。
[0011] 图 2是 Seq. 2、 Seq. 6、 Seq. 16及 Tc. 6寡核苷酸适配体的饱和结合曲线图。
[0012] 图 3是 Seq. 2、 Seq. 6及 Seq. 16寡核苷酸适配体的特异性试验结果。
本发明的实施方式
[0013] 以下结合说明书附图和实施例对本发明作进一步的说明， 但不是限制本发明。
[0014] 实施例 1 : T-2毒素特异性结合寡核苷酸适配体的 GO-SELEX筛选
[0015] 1、 体外化学合成初始随机单链 DNA (ssDNA) 文库及引物 （由美国 Integrated
DNA Technologies公司完成） ， 序列如下：
[0016] 5'-CAGCTCAGAAGCTTGATCCT-N40-GACTCGAAGTCGTGCATCTG-3' (40
N代表 40个随机核苷酸） ；
[0017] 上游引物： 5'-CAGCTCAGAAGCTTGATCCT-3'
[0018] 下游引物： 5'- CAGATGCACGACTTCGAGTC-3'
[0019] 5'磷酸化下游引物： 5'-P-CAGATGCACGACTTCGAGTC-3'
[0020] 将随机 ssDNA文库和引物均用 TE缓冲液配制成 ΙΟΟ μΜ贮存液存于 -20°C备用。
[0021] 2、 PCR扩增条件及 Lambda核酸外切酶消化制备单链次库的条件
[0022] 将合成的随机单链文库 （ssDNA) 稀释作为 PCR模板扩增出磷酸化的双链 DNA
(dsDNA) 产物， 研究 Lambda核酸外切酶消化磷酸化反义链制备单链次库的影 响因素， 最终确定制备单链次库的最佳条件。 [0023] PCR反应体系为： 稀释随机文库作为模板 DNA 1 (lOO ng) ， 上游引物及磷 酸化下游引物 （20 μΜ) 各 1 μί， dNTPmix (each 25 mM) 1 μί, 10 x PCR扩增 缓冲液 5 μί， 灭菌超纯水 40 μί， Taq酶 1 μί， 总体积为 50 μί。 PCR扩增程序： 94 °C预变性 5 min; 94°C变性 30 s; 53°C退火 30 s; 72°C延伸 30
s; 循环 20次； 最后 72°C延伸 5 min。 通过 8%非变性聚丙烯酰胺凝胶电泳验证扩 增效果。
[0024] 将电泳条带位置正确且单一的 PCR扩增产物用 PCR产物纯化试剂盒 （Genemy Biotech. Co., Ltd.) 纯化， 重溶于适当体积的灭菌超纯水中， 通过 Thermo NanoDrop 2000超微量分光光度计测定 dsDNA浓度。 取确定浓度的纯化 PCR产物 溶液， 加入 1/10体积的 lOx反应液混合均匀， 按说明书加入所需核酸外切酶 （5 U/μϋ ， 在 37°C下反应 30 min~60 min， 75°C水浴 10 min使酶失活终止反应。 通 过 7 M尿素变性的 8%聚丙烯酰胺凝胶进行电泳， 确定最佳酶切条件后再进行放 大酶切。 将酶切产物汇集在一个 1.5 mL离心管中， 加入等体积的酚： 氯仿： 异戊 醇 （V: V： V = 25 :24 :1) ， 旋涡混匀管内容物使呈乳状， 12000 rpm， 4°C离心 15 s， 将上层液体小心移入另一离心管， 弃去两相界面和有机相。 重复操作一次， 直至两相界面上见不到蛋白质为止。 向含样品的离心管中加入 1/10体积的 3 M醋 酸钠 （pH 5.2) 溶液及 2倍体积的无水乙醇， 充分混匀后放 -20°C冰箱内过夜。 取 出平衡， 12000 rpm， 4。C离心 15 min。 吸弃上清， 用 4。C预冷的 70%乙醇 0.5-1 mL 上下颠倒洗涤白色固体沉淀， 12000 rpm， 4°C离心 15 min。 弃上清， 风干沉淀后 ， 重溶于适当体积的缓冲液中， 通过 Thermo NanoDrop 2000超微量分光光度计测 定 ssDNA浓度。
[0025] 3、 体外 GO-SELEX筛选：
[0026] 第一轮筛选吋， 取溶解在结合缓冲液（BB, 10 mM Tris-HCl, 150 mM NaCl, 10 mM KC1, and 2.5 mM MgC12, pH 7.4)中的 1
nmol随机 ssDNA文库 （第 2-10轮 ssDNA文库投入量减少为 200， 100
pmol) ， 经 94°C水浴加热 5 min， 立即冰浴 15 min后室温放置 10
min。 然后， 将此 ssDNA文库与游离靶标 T-2毒素混合均匀， 并使其在含 1.0%甲 醇的特殊结合缓冲液 （总体积为 500 μϋ 中孵育 2 h， 期间不停地轻柔晃动。 孵 育结束后， 将混合液转移到加入事先离心洗涤好的 GO片层中， 25oC孵育 40 min 。 与 T-2靶标结合的 ssDNA序列被保留在上清液中， 而不结合的 ssDNA序列被 GO 吸附。 通过 13000 rpm/min离心 10
min， 收集上清液， 得到了与 T-2毒素结合的 ssDNA适配体库。 将此上清液作为扩 增模板进行 PCR扩增， 其纯化产物用 λ核酸外切酶消化磷酸化反义链制备获得单 链次级文库。 经 Thermo NanoDrop 2000超微量分光光度计测定纯化后的 ssDNA浓 度， 以此计算下一轮文库投入体积。
[0027] 为了提高适配体的特异性， 第 6-10轮采用了 Counter GO-SELEX。 首先将 ssDNA 次库与 FBI、 ZEN、 AFB1及 OTA的混合物孵育 60 min， 然后加入 GO片层混合均 匀孵育 40 min。 在这个过程中， 不与反筛靶标结合的寡核苷酸通过 π-π作用被吸 附到 GO表面， 而与反筛靶标结合的潜在适配体序列被保留在溶液中。 离心分离 ， 弃上清， 将得到的 GO/ssDNA沉淀物用 BB离心洗涤多次。 为了从 GO表面解离 恢复 ssDNA序列， 往重悬于特殊结合缓冲液的 GO/ssDNA复合物中加入靶标 T-2 毒素， 室温孵育 2
h。 孵育结束后， 离心收集上清液并 PCR扩增。 经纯化后的 PCR产物， 用 λ核酸外 切酶消化磷酸化反义链制备下一轮的单链文库。
[0028] 4、 克隆测序及序列分析
[0029] 将第 10轮筛选得到的寡核苷酸适配体富集库用无修饰的弓 I物扩增， PCR产物经 纯化后用 pMD-19T Cloning Vector (Takara Biotech.Co., Ltd)克隆并转化到氯化钙 感受态细胞 E.Coli DH5ot中。 在含 50 g/mL氨苄青霉素的固体培养基上培养 18小 吋后， 通过"蓝-白斑筛选"， 随机挑选 30个白色阳性克隆于 LB液体培养基中培养 ， 并用质粒 MiniPrep试剂盒 （Generay Biotech. Co., Ltd.) 提取质粒， 送至上海生 工生物技术公司测序。 对测序成功获得的 27条寡核苷酸适配体序列， 采用 DNA MAN软件对寡核苷酸适配体序列进行一级结构分析， 获得多条序列的同源性信 息； 并用 RNA Structure 4.6软件对寡核苷酸适配体序列的二级结构进行分析。 根 据 T-2毒素寡核苷酸适配体的一、 二级结构特征， 将 27条序列分为 3个家族。 从每 个家族中选出 1条能级较低、 结构稳定的序列为代表， 图 1所示为 3条代表序列 Se q. 2、 Seq. 6、 Seq. 16以及截短序列 Tc. 2、 Tc. 6、 Tc. 16的二级结构模拟图， 由上 海生工合成并标记 5'FAM基团以作进一步的亲和力和特异性分析。
[0030] 5、 T-2毒素适配体的亲和力和特异性分析
[0031] 5.1亲和力分析
[0032] 为了分析三条代表序列及其截短序列的亲和力， 将固定浓度的 T-2毒素 （Ι μΜ ) 与一系列不同浓度 （5， 10， 25， 50， 100， 200 ηΜ) 的适配体序列进行孵育 ， 总体积为 500 μί， 避光孵育 2 h。 然后， 加入与适配体浓度成比例的 GO， 继续 避光孵育 40 min。 离心后， 用日立 F-7000荧光光谱仪检测 490 nm激发下的荧光值 。 同吋， 设置不加入 T-2毒素的阴性对照以排除非特异性结合。 利用 GmphPad Prism 5软件计算各寡核苷酸适配体的解离常数 Kd值 （见表 1)
[0033] 表 1.全长序列及截短序列的解离常数 Kd值
[]
' :幸 稱 ' 解离常数
.4 ^.
Χ Ι ' '.·ΐ & Kd值 CnM)
Seq. 2 62.4±12J
Seq, 6 37.2±8„4
Seq, 16 20,8士 3.1
2 n.d.
To.. 6 :813士 21.7
T , 16 n.d.
[0034] 图 2为序列 Seq. 2、 Seq. 6、 Seq. 16及 Tc. 6的饱和结合曲线图。 分析可知， 与 T-2 毒素亲和力最强即 Kd值最小的寡核苷酸适配体序列为 Seq. 2、 Seq. 6及 Seq. 16。
[0035] 5.2特异性分析
[0036] 根据 5.1的分析结果， 挑选 Seq. 2、 Seq. 6和 Seq. 16三条寡核苷酸适配体进行特异 性分析。 将每一条 5'FAM-标记的寡核苷酸适配体， 分别与靶标 T-2或 FB1、 ZEN 、 AFB1、 OTA混合， 随后与 GO溶液 （终浓度 0.3 mg/mL) 室温下避光孵育 2 h。 另外设置每条适配体的阴性对照， 以 BB代替任一种真菌毒素。 随后， 将孵育混 合物在 13000 rpm/min转速下离心 10 min， 收集上清液， 用日立 F-7000荧光光谱 仪测定 490 nm激发下的荧光值。 结果显示 T-2毒素寡核苷酸适配体结合 T-2毒素靶 标的能力均强于其他四种真菌毒素， 特异性试验结果如图 3所示。
本发明包括但不限于以上实施例， 凡是在本发明的精神和原则下进行的任何等 同替换或局部该进， 都将视为在本发明的保护范围之内。
[0038] [0039]序列表
[0039] 〈110〉 江南大学
[0040] 〈120〉 一种特异识别 T-2毒素的寡核苷酸适配体
[0041] 〈130〉
[0042] 〈160〉 1
[0043] 〈170〉 Patentln version 3.5
[0044]
[0045] 〈210〉 1
[0046] 〈211〉 80
[0047] 〈212〉 DNA
[0048] 〈213〉 人工序列
[0049] 〈400〉 1
[0050] cagctcagaa gcttgatcct gtatatcaag catcgcgtgt ttacacatgc
[0051] cagatgcacg acttcgagtc 80

Claims

权利要求书
[权利要求 1] 一种特异识别 T-2毒素的寡核苷酸适配体， 其特征在于该寡核苷酸适 配体的序列如 SEQ ID NO: 1所示。
[权利要求 2] 如权利要求 1中所述的寡核苷酸适配体， 其特征在于其 5端或 端可 以进行 FITC、 氨基、 生物素或地高辛化学修饰。
[权利要求 3] 如权利要求 1中所述的寡核苷酸适配体在分离富集及分析检测 T-2毒素 中的应用。
PCT/CN2015/079797 2014-07-24 2015-05-26 特异识别t-2毒素的寡核苷酸适配体 WO2016011845A1 (zh)

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