WO2020211878A1 - 纳豆激酶核酸适配体及其筛选方法 - Google Patents

纳豆激酶核酸适配体及其筛选方法 Download PDF

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WO2020211878A1
WO2020211878A1 PCT/CN2020/090634 CN2020090634W WO2020211878A1 WO 2020211878 A1 WO2020211878 A1 WO 2020211878A1 CN 2020090634 W CN2020090634 W CN 2020090634W WO 2020211878 A1 WO2020211878 A1 WO 2020211878A1
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nattokinase
nucleic acid
screening
seq
aptamer
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French (fr)
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法芸
赵海杰
管明阳
王�琦
刘会洲
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中国科学院青岛生物能源与过程研究所
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    • 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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    • 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
    • C12N15/1089—Design, preparation, screening or analysis of libraries using computer algorithms
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    • 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
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)
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    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the invention belongs to the technical field of bioengineering, and particularly relates to a nattokinase nucleic acid aptamer and a screening method thereof.
  • the magnetic bead method is commonly used in protein screening in this field.
  • the protein target is fixed on the magnetic beads.
  • the oligonucleotide molecules in the liquid phase diffuse freely, and the corresponding target molecules on the surface of the solid support can bind to it.
  • the oligonucleotide chain bound to the target molecule is eluted/separated, and then subjected to PCR amplification and the next cycle. This method requires multiple cycles, takes a long time, and is prone to non-specific adsorption.
  • NK Nattokinase as a thrombolytic agent has the characteristics of high fibrinolytic activity, safety and economy. Because NK activity is greatly affected by external factors, the quantitative determination method of nattokinase activity is still an important technical bottleneck restricting its efficient separation, purification, and thrombolytic mechanism research.
  • the present invention provides nattokinase nucleic acid aptamers and a screening method thereof, with the purpose of solving some of the problems in the prior art or at least alleviating some of the problems in the prior art.
  • a method for screening nattokinase nucleic acid aptamers includes the following steps:
  • the designed nucleotide library is mixed and incubated with nattokinase, separated by capillary electrophoresis, and the bound complexes are collected;
  • the candidate aptamers were tested for their structure, affinity and specificity with nattokinase to obtain high-affinity, high-specificity nattokinase nucleic acid aptamers.
  • capillary electrophoresis separation capillary temperature 25° C., 0.5 psi, 20 s injection, separation voltage 25 KV, capillary electrophoresis running buffer is pH 7.8, 50 mmol/L boric acid-borax buffer, and detection wavelength It is 280nm.
  • nattokinase peaked at 3.3 min
  • 69nt-ssDNA peaked at 9.9 min
  • the peak of NK-ssDNA complex appeared at 15.2 min
  • the collection time was about 18.76 min-24.56 min.
  • a 69 nt random sequence library is used to combine with nattokinase, the 69 nt random sequence is 5'-TTGAGCCTACGAGCGATACC-29N-GATGTCAGGTGTCTCGTCGT-3', where 29N represents 29 random sequences of T, G, A, C.
  • primer sequences involved in asymmetric PCR are shown in SEQ ID NO: 18 and SEQ ID NO: 19 respectively, wherein the concentration of the downstream primer is 0.5 ⁇ mol/L, and the ratio of the upstream and downstream primers is 30:1.
  • primer sequences involved in the PCR amplification of the complex obtained in the last round of screening are shown in SEQ ID NO: 23 and SEQ ID NO: 24.
  • nucleic acid purification method includes magnetic bead purification.
  • Nattokinase nucleic acid aptamer the nucleotide sequence of the aptamer is any one of SEQ ID NO: 1 to SEQ ID NO: 17.
  • nattokinase nucleic acid aptamer is obtained by the screening method of any one of claims 1-8.
  • a nattokinase protein molecular recognition kit comprising the nattokinase nucleic acid aptamer of claim 8.
  • the invention adopts capillary electrophoresis technology to screen aptamers, which greatly improves the screening efficiency of aptamers.
  • the present invention uses surface plasmon resonance technology to measure the dissociation constants of seven aptamers, and obtains their affinity, which is 8.7-87 nM, indicating that the affinity is relatively strong.
  • the nucleic acid aptamer provided by the present invention has precise specificity, high affinity, and convenient chemical modification, and can be used as an effective molecular recognition tool for the highly sensitive analysis of proteins.
  • the nucleic acid aptamer-based recognition technology is NK assay And provide a basis for the development direction of efficient separation and purification. There are no reports about nattokinase aptamers.
  • nattokinase aptamer to establish its concentration or activity determination method solves the analysis problem of high accuracy, high sensitivity and high specificity of NK.
  • the use of aptamers as molecular recognition tools provides a key basis for the separation and purification of NK.
  • the present invention uses surface plasmon resonance technology to initially explore the interaction between candidate aptamers and targets, understand the kinetic and thermodynamic characteristics of the interaction between molecules, and provide a key basis for the establishment of an aptamer evaluation system. It is helpful for nucleic acid aptamers to give full play to their unique advantages and promote the research progress of NK active sites, catalytic mechanism, separation and purification.
  • Figure 1 is the experimental result of NK wavelength determination in Example 1;
  • Figure 2 is a diagram showing the separation of NK and ssDNA by capillary electrophoresis in Example 1;
  • Figure 3 is a capillary electrophoresis separation diagram after mixing NK and ssDNA in Example 1;
  • Example 4 is an experiment for determining the optimal annealing temperature in the characterization experiment of the PCR process in Example 1;
  • Figure 5 is a CE electrophoresis diagram in the characterization experiment of the PCR process in Example 1;
  • Figure 6 is an electrophoresis diagram of an optimized asymmetric PCR primer ratio
  • Figure 7 is a CE electrophoresis diagram of the first round of screening of NK nucleic acid aptamers in Example 1;
  • Figure 8 is a CE electrophoresis diagram of the second round of screening of NK nucleic acid aptamers in Example 1;
  • Figure 9 is a gel electrophoresis diagram of the third round of screening of NK nucleic acid aptamers in Example 1;
  • Figure 10 is a graph showing the results of determination of the affinity of NK nucleic acid aptamers in Example 2.
  • Figure 11 is a diagram showing the specificity determination result of NK nucleic acid aptamer in Example 2.
  • Figure 12 is the CE electrophoresis analysis result of the affinity of nucleic acid aptamers in Example 2.
  • genes, proteins or fragments thereof involved in the present invention can be natural purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (for example, bacteria, yeast, plants) using recombinant technology.
  • the present invention screens out the aptamer that specifically binds to the nattokinase target as a new type of nucleic acid aptamer, adding new members to the aptamer family.
  • the use of capillary electrophoresis technology to screen aptamers greatly improves the efficiency of aptamer screening and solves the problems of cumbersome operation, time-consuming, low efficiency and poor repeatability of traditional screening methods.
  • Using surface plasmon resonance technology and capillary electrophoresis technology to initially explore the interaction between candidate aptamers and the target understand the dynamics and thermodynamics of molecular interactions, and provide a key basis for the establishment of an aptamer evaluation system. It is conducive to nucleic acid adaptation as a molecular recognition tool body to give full play to its unique advantages, and promote the research progress of NK active sites, catalytic mechanism, separation and purification.
  • the present invention discloses a nattokinase nucleic acid aptamer and a screening method thereof, and the specific examples are as follows.
  • the experimental conditions involved in the screening process are first explored.
  • the experimental conditions such as the running buffer and the optimal detection wavelength are optimized to determine the 50mmol/L boric acid-borax buffer (pH 7.8)
  • the detection wavelength is 280nm. details as follows:
  • Capillary activation Before the capillary is used for the first time, rinse it with methanol, 0.1mol/L hydrochloric acid solution, regenerant (0.1mol/L NaOH solution) and buffer in sequence.
  • the rinse condition is: 20psi, 3.5min.
  • When changing different solutions use it Rinse with ultrapure water for 2.0min under the condition of 20psi.
  • Capillary cleaning before each injection, flush with ultrapure water and running buffer in sequence, flushing conditions: 15psi, 3.0min.
  • the capillary needs to be reactivated after several loadings.
  • the reagents used in the experiment were sonicated and filtered through a 0.22 ⁇ m pore filter membrane and used on the machine.
  • the electrophoresis pattern is shown in Figure 6, where bands: M.DL2000DNA Marker; 1.ssDNA; 2. Blank control; 3-6 are PCR products under primer ratios: 3.40:1, 4.30:1, 5.20:1, 6.10: 1.
  • the first round of screening is the first round of screening.
  • nattokinase was added to the random oligonucleotide library solution that was reduced to room temperature after denaturation, so that the concentration in the solution was about 0.0025 mg/mL (1.0 ⁇ mol/L), and incubated at 30°C for no less than 30 minutes. Slowly mix upside down every 20min. At this time, the amount of nattokinase is sufficient to fully bind to the oligonucleotide chain capable of binding to nattokinase in the random oligonucleotide library. After being placed at room temperature for 10 minutes, the sample was injected and separated.
  • the collected complex was used to replace the random nucleic acid library, and the second round of nattokinase aptamer screening was performed.
  • the experimental conditions were consistent with the first round of screening.
  • the previous round of screening was used to obtain NK-ssDNA complexes instead of random libraries. The screening was repeated several times to obtain an aptamer with sufficient affinity for nattokinase.
  • the first round of collection was supplemented with nattokinase and incubated, and then collected in sections after sample injection analysis.
  • the electrophoresis pattern of the aptamer collection is shown in Figure 8.
  • NK-ssDNA complex collected in the second round of screening and use this as a template for PCR amplification.
  • the PCR system and procedures are the same as those in Table 2 and Table 3 below, and 69nt-ssDNA (sequence as shown in Table 1 Show) and ultrapure water as a template control experiment, the gel electrophoresis results of PCR products are shown in Figure 9, where 1: identification nucleic acid; 2: 69nt-ssDNA as the template PCR product; 3: NK-ssDNA as the template Template PCR product; 4: Blank control; 5: 69nt-ssDNA.
  • the bands of the products amplified using the 69nt-ssDNA library as the template are basically the same as those of the collected NK-ssDNA complex samples. There are almost no diffuse bands, and the bands are located near the 100bp Marker. No product was produced in the blank control PCR experiment group with pure water as the template. It shows that the generated band is the correct band, the PCR amplification is successful, the collection contains NK-ssDNA complex, and has a certain concentration, the concentration is not higher than 1.0nmol/Lol/mL. PCR successfully amplified the ssDNA in the collected NK-ssDNA complex, and the third round of NK-ssDNA collection was effective.
  • the purchased nattokinase was purified by chromatography, identified, and the purified protein and the experimental conditions determined in Example 1 were used for aptamer screening.
  • the screening method is as follows:
  • nucleic acid primer design software Primer to design the sequence required for the experiment, and use the NUPACK software and The Mfold Web Server to evaluate the secondary structure of the designed nucleic acid strand.
  • the concentration of the fixed fluorescently labeled oligonucleotide library is 0.5 ⁇ mol/L, and the concentration of nattokinase is 0.125 ⁇ mol/L. Mix the two, incubate at 30°C under dark conditions, take out and shake gently every 10min. After 30 min, the sample was injected and separated.
  • the separation conditions are: capillary temperature 25°C, 0.5psi, 20s injection, separation voltage 25kV; before each injection for the first time, use methanol, ultrapure water, 0.1mol/L hydrochloric acid solution, ultrapure water, regenerant A in sequence (0.1mol/L NaOH solution), ultrapure water, running buffer flushing, flushing conditions: 20psi, 5.0min; flush with ultrapure water and running buffer between every two injections, flushing conditions: 15psi, 3.0 min.
  • the random library is combined with NK, and the secondary library is prepared by asymmetric PCR.
  • nucleic acid sequences of primers and templates used in the PCR system are shown in Table 1;
  • the secondary library obtained by asymmetric PCR is used as a template to perform amplification.
  • the specific operation is as follows: the Qubit2.0 DNA detection kit is used to accurately quantify the genomic DNA to determine the amount of DNA that should be added to the PCR reaction.
  • the primers used in PCR are introduced into Illumina bridge PCR compatible primers:
  • 190294-F primer AGCAGCACAGAGGTCAGATG, SEQ ID NO: 23.
  • 190294-R primer TTCACGGTAGCACGCATAGG, SEQ ID NO: 24.
  • the PCR system proceeds as follows:
  • the configured PCR system performs PCR amplification according to the following reaction conditions, the procedure is the same as the asymmetric PCR procedure:
  • NK-APT-01 CGCCTGAGGGATGACCTGTTCATGTAGGG SEQ ID NO: 1.
  • NK-APT-02 GACTACCGCGGAGATCTCCAATCGGGATG SEQ ID NO: 2.
  • NK-APT-03 AAATGAGGACCTCATTATCTTAGGAAGGT SEQ ID NO: 3.
  • NK-APT-04 GCCTTAAAAACGTCCTTAGTGACGTTTAC SEQ ID NO: 4.
  • NK-APT-05 GCTCACGTAGATTAAACTATGCTAAGAGC SEQ ID NO: 5.
  • NK-APT-06 CATTGCAATGTAACTGTCTAGCTTGACTG SEQ ID NO: 6.
  • NK-APT-07 CCGGTCGCCATGGGGGAGCTGCGTACCGC SEQ ID NO: 7.
  • NK-APT-08 CCATTCCAACTCGCTTTAAACATAAATCG SEQ ID NO: 8.
  • NK-APT-10 GGGTTACGATCAGTCTCATGGAAACGACC SEQ ID NO: 10.
  • NK-APT-11 GTTGCAAGGAAAGCTATGCCTATGCCCCA SEQ ID NO: 11.
  • NK-APT-12 CTCGTGTAAAACGATGGTAGCTGTACTTG SEQ ID NO: 12.
  • NK-APT-13 AAAGGCGCTGTGCACAGTGTCCCCGCGGG SEQ ID NO: 13.
  • NK-APT-14 CCGAGGGGAATTCACCACGAATCGCCTCT SEQ ID NO: 14.
  • NK-APT-15 TCGATAGTTGTACTGGAGTGAATCACTGA SEQ ID NO: 15.
  • NK-APT-16 AAGACTCCAGTCGCGGCCTCTATCCGGGA SEQ ID NO: 16.
  • NK-APT-17 ACCCAAGTAGGCGAGTTCAGAACTGTGTG SEQ ID NO: 17.
  • the specific operation is: select nucleic acid aptamers with representative secondary structure, and perform 5'6-FAM (FITC) fluorescent label synthesis. Dissolve the nucleic acid aptamer to 0.5 ⁇ mol/L with 50mmol/L pH7.8 boric acid buffer, put the easily soluble random oligonucleotide library at 95°C for denaturation not less than 10min, and slowly cool to room temperature.
  • FITC 5'6-FAM
  • Figure 12 is a capillary electrophoresis diagram of seven aptamers and complexes provided by an embodiment of the present invention.
  • the surface binds to nattokinase with a strong affinity.

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Abstract

本发明提供了一种纳豆激酶核酸适配体的筛选方法，包括步骤：设计构建两端带有固定序列的随机寡聚核苷酸文库；将核苷酸文库与纳豆激酶混合孵育，并进行毛细管电泳分离，收集结合的复合物；以收集的复合物作为模板进行不对称PCR扩增，并制备次级文库；对次级文库进行多轮筛选，以最后一轮筛选得到的复合物进行PCR扩增，得到与纳豆激酶结合的核苷酸文库；对核苷酸文库纯化后测序，获得候选适配体。还提供了一组纳豆激酶核酸适配体。

Description

纳豆激酶核酸适配体及其筛选方法 技术领域
本发明属于生物工程技术领域，尤其涉及纳豆激酶核酸适配体及其筛选方法。
背景技术
目前本领域内进行蛋白筛选时常用磁珠法筛选，将蛋白靶标固定到磁珠上，液相中的寡核苷酸分子自由扩散，遇到固体支持物表面的相应靶分子可以与其结合，以除去未结合或结合的弱寡核苷酸分子。洗脱/分离与靶分子结合的寡核苷酸链，然后进行PCR扩增和下一个循环。此方法需要多次循环，耗时长，容易产生非特异性吸附。
纳豆激酶(Nattokinase，NK)作为溶栓剂具有纤溶活性高、安全、经济的特点。由于NK活性受外界因素的影响大，纳豆激酶活性的定量测定方法仍是限制其进行高效分离纯化、溶栓机理研究的重要技术瓶颈。
在测定NK酶活性的几种方法中，科学家们使用最多的是纤维蛋白平板法，通过表观的溶解圈的面积大小定量纤溶活性，检测误差大，灵敏度低。此外该方法受时间和温度及产品纯度影响较大，另因纤维蛋白源和凝血酶价格高导致检测成本难以降低。中国、日本、印度、韩国和加拿大的科学家都使用这种纤维蛋白降解法。有日本科学家在使用酶联免疫吸附法。NK测定方法虽然很多，但没有完全规范统一的标准。
上述各活性检测方法的局限性常导致相关研究结果之间存在差异，难以进行比对。在其生产优化和分离提纯的研究中，不同菌种、不同发酵工艺所得分子量等理化性质也有差别，使得其准确测定和分离较困难。
在实际应用中，酶活性的测定有时并不直接反映NK的真实产量。这些都限制了其作为溶栓剂的规模生产和开发应用。
综上所述，现有技术存在的问题是：
(1)现有技术中，各活性检测方法的局限性导致相关研究结果之间存在差异，难以进行比对；在其生产优化和分离提纯的研究中，对于不同菌种、不同发酵工艺所得的分子量很难进行准确测定和分离。
(2)纳豆激酶的性质不够稳定，难以得到纯品，其活性测定的方法仍然采用传统方法。
(3)传统筛选方法操作繁琐、费时、效率低、重复性差。
发明内容
针对现有技术存在的问题，本发明提供了纳豆激酶核酸适配体及其筛选方法，目的在于解决现有技术中的一部分问题或至少缓解现有技术中的一部分问题。
本发明是这样实现的，一种纳豆激酶核酸适配体的筛选方法，包括以下步骤：
设计构建两端带有固定序列的随机寡聚核苷酸文库；
所设计核苷酸文库与纳豆激酶混合孵育，并进行毛细管电泳分离，收集结合的复合物；
以收集的复合物作为模板进行不对称PCR扩增，并制备次级文库；
对次级文库进行多轮筛选，以最后一轮筛选得到的复合物进行PCR扩增，得到与纳豆激酶结合的核苷酸文库；
对核苷酸文库纯化后测序，获得候选适配体；
对候选适配体进行结构、与纳豆激酶亲和力以及特异性检测，获得高亲和力、高特异性纳豆激酶核酸适配体。
进一步地，所述毛细管电泳分离的条件为，毛细管温度25℃，0.5psi，20s进样，分离电压25KV，毛细管电泳的运行缓冲液为pH7.8、50mmol/L硼酸-硼砂缓冲液，检测波长为280nm。
进一步地，所述毛细管电泳分离过程中，纳豆激酶在3.3min出峰，69nt-ssDNA在9.9min出峰，NK-ssDNA复合物的峰出现15.2min，收集时间约为18.76min～24.56min。
进一步地，使用69nt随机序列文库与纳豆激酶结合，所述69nt随机序列为5′-TTGAGCCTACGAGCGATACC-29N-GATGTCAGGTGTCTCGTCGT-3′，其中29N表示有29个T,G,A,C随机的序列。
进一步地，不对称PCR涉及的引物序列分别见SEQ ID NO：18和SEQ ID NO：19所示，其中下游引物的浓度为0.5μmol/L，上、下游引物比例为30:1。
进一步地，所述以最后一轮筛选得到的复合物进行PCR扩增涉及的引物序列见SEQ ID NO：23和SEQ ID NO：24所示。
进一步地，所述核酸纯化的方法包括磁珠法纯化。
纳豆激酶核酸适配体，所述适配体的核苷酸序列为SEQ ID NO：1～SEQ ID NO：17中的任一条。
进一步地，所述纳豆激酶核酸适配体利用权利要求1-8任一所述的筛选方法获得。
一种纳豆激酶蛋白质分子识别试剂盒，包括权利要求8所述的纳豆激酶核酸适配体。
综上所述，本发明的优点及积极效果为：
本发明采用毛细管电泳技术进行适配体的筛选，极大地提高了适配体的筛选效率。
本发明利用表面等离子共振技术测定七条适配体的解离常数，得到其亲合力，为8.7-87nM，表明其亲合力较强。本发明提供的核酸适配体具有精准的特异性、高亲合力、便于化学修饰，可作为一种有效的分子识别工具用于蛋白质的高灵敏分析，基于核酸适配体的识别技术为NK测定和高效分离纯化的发展方向提供依据。目前未见有纳豆激酶适配体的相关报道。
利用纳豆激酶适配体建立其浓度或活性测定方法，解决了NK的高准确性、高灵敏度、高特异性的分析问题。将适配体作为分子识别工具为NK分离纯化提供了关键依据。
本发明采用表面等离子共振技术初步探索了候选适配体与靶标的相互作用，了解分子间相互作用的动力学和热力学等特性，为适配体评价体系的建立提供关键依据。有利于核酸适配体充分发挥其独特优势，促进NK活性位点、催化机理、分离纯化等领域的研究进展。
附图说明
图1是实施例1中NK波长确定实验结果；
图2是实施例1中NK和ssDNA毛细管电泳分离图；
图3是实施例1中NK和ssDNA混合后毛细管电泳分离图；
图4是实施例1中PCR过程的表征实验中最佳退火温度确定实验；
图5是实施例1中PCR过程的表征实验中CE电泳图；
图6是不对称PCR引物比例优化电泳图；
图7是实施例1中NK核酸适配体的筛选的第一轮筛选的CE电泳图；
图8是实施例1中NK核酸适配体的筛选的第二轮筛选的CE电泳图；
图9是实施例1中NK核酸适配体的筛选的第三轮筛选的凝胶电泳图；
图10是实施例2中NK核酸适配体亲和力测定结果图；
图11是实施例2中NK核酸适配体特异性测定结果图；
图12是实施例2中核酸适配体亲和力CE电泳分析结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例对本发明进行进一步详细说明，各实施例及试验例中所用的设备和试剂如无特殊说明，均可从商业途径得到。此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
本发明中涉及的基因、蛋白或其片段可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物)中产生。
本发明筛选出与纳豆激酶靶标特异性结合的适配体为一种新型核酸适配体，为适配体家 族增加了新成员。采用毛细管电泳技术进行适配体的筛选，极大地提高了适配体的筛选效率，解决了传统筛选方法操作繁琐、费时、效率低、重复性差的难题。采用表面等离子共振技术和毛细管电泳技术初步探索了候选适配体与靶标的相互作用，了解分子间相互作用的动力学和热力学等特性，为适配体评价体系的建立提供关键依据。有利于核酸适配作为分子识别工具体充分发挥其独特优势，促进NK活性位点、催化机理、分离纯化等领域的研究进展。
本发明披露了纳豆激酶核酸适配体及其筛选方法，具体如下各实施例。
实施例1 实验条件的建立
本发明本实施例中首先对筛选过程中涉及的实验条件进行摸索，本实施例中优化了运行缓冲液、最佳检测波长等实验条件，确定50mmol/L硼酸-硼砂缓冲液(pH7.8)作为毛细管电泳的运行缓冲液和纳豆激酶样品的溶剂，检测波长为280nm。具体如下：
1、适用于纳豆激酶适配体筛选的毛细管电泳分析方法的建立
毛细管电泳的分析条件：柱温25℃，0.5psi，20s进样，25kV电压下分离。
毛细管活化：毛细管首次使用前，依次用甲醇、0.1mol/L盐酸溶液、再生液(0.1mol/L NaOH溶液)和缓冲液冲洗，冲洗条件为：20psi，3.5min，更换不同溶液时，需用超纯水在20psi条件下冲洗2.0min。
毛细管清洗：每次进样前，依次用超纯水、运行缓冲液冲洗，冲洗条件：15psi，3.0min。毛细管经多次上样后需重新活化。
实验所用试剂超声后经0.22μm孔径滤膜过滤后上机使用。
2、NK波长确定实验
以50mmol/L硼酸-硼砂缓冲液(pH7.8)作为运行缓冲液，考察浓度为1.00mg/mL样品在214、254、280nm三个波长下的响应值，以获得最佳检测波长。
实验结果如图1，由图可知，214nm处的样品响应值最强，其次为280nm，254nm处的样品响应值最弱。核酸的紫外吸收在260nmol/L处。由于214nm处的干扰严重，因此选择280nm作为毛细管电泳分析的检测波长。
3、NK、NK-ssDNA、ssDNA三者分离实验
使用50mmol/L硼酸-硼砂缓冲液(pH7.8)溶解纳豆激酶和ssDNA(即下文中的69nt随机序列文库)，使NK浓度为2mg/mL，ssDNA浓度5μmol/L，在相同条件下分别进行毛细管电泳分析，分析条件为前述优化后的结构。
实验结果如图2，从毛细管电泳谱图上可以看出，纳豆激酶在3.40min～3.67min出峰，而ssDNA出峰时间为12.5min～16.5min。纳豆激酶样品峰与ssDNA样品峰间隔较大，分离 很好。
准确配制0.005mg/mL NK和100μmol/L ssDNA的混合溶液，进行毛细管电泳分析，平行测定三次，实验结果如图3。结果表明NK与ssDNA在混合状态下仍可以得到完全分离。
4、PCR过程的表征
最优扩增条件下实验：实验选取55℃、57℃、59℃、61℃、63℃、65℃作为退火温度，以随机序列为模板进行PCR。确定最佳退火温度在61-63℃之间后，进一步选取61℃、62℃、63℃作为退火温度进行实验，退火温度为61℃时，溶解曲线在62℃有峰出现，82℃有峰存在，但峰很小(如图4所示)。CE电泳时无杂峰出现(如图5所示)，说明在61℃时，反应的特异性好。
5、通过不对称PCR制备次级文库条件优化
在最佳的PCR条件下，不断改变引物浓度和两种引物的比例(引物序列见表1所示)，最终确定在引物P2的浓度为0.5μmol/L，引物比例为30:1时，不对称PCR的产物量较大，虽然有非特异性扩增产物存在，但在凝胶电泳中特异性扩增产物可以与非特异性产物很好的分开，可以用于后期胶回收制备单链次级文库。
电泳图谱见图6，其中，条带：M.DL2000DNA Marker；1.ssDNA；2.空白对照；3-6为引物比例下的PCR产物：3.40:1，4.30:1，5.20:1，6.10:1。
6、NK核酸适配体的筛选
第一轮筛选：
筛选开始前，将易溶解的随机寡核苷酸文库放至95℃环境下变性不低于10min，缓慢冷却至室温。随后将在变性后降至室温的随机寡核苷酸文库溶液中加入纳豆激酶，使之在溶液中的浓度约为0.0025mg/mL(1.0μmol/L)，30℃孵育不低于30min，每20min缓慢的上下颠倒混匀。此时，纳豆激酶的用量足够与随机寡核苷酸文库中能够与纳豆激酶结合的寡核苷酸链充分结合。放置室温10min后，进样分离。
对图7中收集窗口1和收集窗口2部分进行收集，并存放至内有10μL运行缓冲液的样品瓶中。为使产物中核酸浓度足够高，连续收集10次。
从图中可以看出，随着随机寡聚ssDNA文库的浓度加大，在靶标纳豆激酶与游离的核酸文库峰之间，基线有向上漂移的倾向。这应该是电泳过程中ssDNA-NK复合物持续不断的解离产生“毛细管涂层”所致，实验中同时对“基线漂移”部分和NK-ssDNA复合物出峰位置进行收集。
第二轮筛选：
经过第一轮筛选收集获得NK-ssDNA复合物后，用收集所得复合物替代随机核酸文库，进行第二轮纳豆激酶的适配体筛选，实验条件与第一轮筛选一致。在随后的筛选轮次中，均使用上一轮筛选收集获得NK-ssDNA复合物替代随机文库。重复进行多次筛选，以便获得与纳豆激酶有足够亲合力的适配体。
在第二轮次的适配体筛选试验中，对第一轮次的收集物补加纳豆激酶后孵育，进样分析后分段收集，适配体收集的电泳图谱如图8。
第三轮筛选：
取第二轮次筛选所收集的NK-ssDNA复合物，以此为模板进行PCR扩增，PCR体系及程序同下文中的表2和表3，并设置以69nt-ssDNA(序列如表1所示)和超纯水为模板的对照试验，PCR生成物的凝胶电泳结果如图9所示，其中1：标识核酸；2：69nt-ssDNA为模板的PCR产物；3：以NK-ssDNA为模板的PCR产物；4：空白对照；5：69nt-ssDNA。
从图中可以看出，以69nt-ssDNA文库为模板扩增的产物和以收集的NK-ssDNA复合物样品条带基本一致，几乎没有弥散条带产生，且条带位于100bp Marker附近，以超纯水为模板的空白对照PCR实验组中无产物生成。说明产生的条带是正确的条带，PCR扩增成功，收集物中含有NK-ssDNA复合物，且具有一定的浓度，浓度不高于1.0nmol/Lol/mL。PCR对所收集的NK-ssDNA复合物中的ssDNA成功的进行了扩增，第三轮次NK-ssDNA收集有效。
实施例2 纳豆激酶核酸适配体的筛选
将购置的纳豆激酶进行层析纯化，鉴定，应用纯化的蛋白及实施例1中确定的实验条件进行适配体筛选。筛选方法如下：
1、核酸文库及引物设计
利用核酸引物设计软件Primer设计实验所需序列，并利用NUPACK软件、The mfold Web Server对所设计的核酸链进行二级结构评估。
2、毛细管电泳分离纳豆激酶、核酸和复合物
固定荧光标记的寡聚核苷酸文库的浓度为0.5μmol/L，纳豆激酶的浓度为0.125μmol/L，将两者混合，避光条件下30℃孵育，每10min取出轻轻摇动一次，30min后进样分离。分离条件为：毛细管温度25℃，0.5psi，20s进样，分离电压25k V；首次每次进样前，依次用甲醇、超纯水、0.1mol/L盐酸溶液、超纯水、再生液A(0.1mol/L NaOH溶液)、超纯水、运行缓冲液冲洗，冲洗条件：20psi，5.0min；每两次进样之间分别用超纯水和运行缓冲液冲洗，冲洗条件：15psi，3.0min。
纳豆激酶在3.3min出峰，69nt-ssDNA在9.9min出峰。在3.96min～11.88min对“基线漂 移”部分进行收集。NK-ssDNA复合物的峰出现15.2min，收集时间约为18.76min～24.56min，对复合物进行收集。
3、选用随机文库与NK结合，通过不对称PCR制备次级文库。
PCR体系所用引物、模板等的核酸序列见表1；
表1 PCR体系中所用的序列及长度
Figure PCTCN2020090634-appb-000001
PCR体系所用各物质及其浓度见表2。
表2 PCR反应体系
Figure PCTCN2020090634-appb-000002
PCR体系的基本反应程序见表3
表3 PCR反应基本程序
Figure PCTCN2020090634-appb-000003
不对称PCR体系及程序见表4和表5。
表4 不对称PCR反应体系
Figure PCTCN2020090634-appb-000004
Figure PCTCN2020090634-appb-000005
表5 不对称PCR程序
Figure PCTCN2020090634-appb-000006
4、以前述筛选得到复合物或次级文库进行PCR扩增(次级文库也可以按照优化的条件再进行多轮次筛选)，得到与纳豆激酶结合的纯核酸。本实施例中以通过不对称PCR获取的次级文库为模板，进行扩增，具体操作如下：利用Qubit2.0DNA检测试剂盒对基因组DNA精确定量，以确定PCR反应应加入的DNA量。PCR所用的引物引入Illumina桥式PCR兼容引物：
190294-F引物：AGCAGCACAGAGGTCAGATG，SEQ ID NO：23。
190294-R引物：TTCACGGTAGCACGCATAGG，SEQ ID NO：24。
PCR体系按照如下进行：
Figure PCTCN2020090634-appb-000007
配置好的PCR体系按照如下反应条件进行PCR扩增，程序同不对称PCR程序：
不对称PCR程序
Figure PCTCN2020090634-appb-000008
5、将扩增得到的核酸纯化。
具体操作如下：
1)在40ul PCR产物中加入体积0.6倍-0.8倍的磁珠(参照生工生物工程磁珠法测序产物纯化试剂盒)，震荡充分悬浮后放在磁力架上吸附5min，小心的用移液枪吸出上清。
2)加入50ul 0.6倍-0.8倍的磁珠洗涤液，震荡充分悬浮后放在磁力架上吸附5min，小心吸出上清。
3)加入90ul WashBuffer(或者70％乙醇)，反向放置在磁力架上，使磁珠吸附到PCR管的另外一面，充分吸附后吸出上清。
4)将PCR管或8联管放在55℃烘箱5min，使里面的酒精完全挥发。
5)加入30ul Elution Buffer洗脱。
6)将PCR管放在吸附架上5min，充分吸附，移出上清到干净的1.5mL离心管中，定量备用。
7)将纯化后的核酸利用Qubit2.0DNA检测试剂盒精确定量，以方便按照1：1的等量混合后测序。等量混合时，每个样品DNA量取10ng，最终上机测序浓度为20pmol。
8)测序得到核酸的序列，最终获得17条候选适配体，如下：
NK-APT-01 CGCCTGAGGGATGACCTGTTCATGTAGGG SEQ ID NO：1。
NK-APT-02 GACTACCGCGGAGATCTCCAATCGGGATG SEQ ID NO：2。
NK-APT-03 AAATGAGGACCTCATTATCTTAGGAAGGT SEQ ID NO：3。
NK-APT-04 GCCTTAAAAACGTCCTTAGTGACGTTTAC SEQ ID NO：4。
NK-APT-05 GCTCACGTAGATTAAACTATGCTAAGAGC SEQ ID NO：5。
NK-APT-06 CATTGCAATGTAACTGTCTAGCTTGACTG SEQ ID NO：6。
NK-APT-07 CCGGTCGCCATGGGGGAGCTGCGTACCGC SEQ ID NO：7。
NK-APT-08 CCATTCCAACTCGCTTTAAACATAAATCG SEQ ID NO：8。
NK-APT-09 CTGTGAACCAAGGATTCATGCCGGTAAAC SEQ ID NO：9。
NK-APT-10 GGGTTACGATCAGTCTCATGGAAACGACC SEQ ID NO：10。
NK-APT-11 GTTGCAAGGAAAGCTATGCCTATGCCCCA SEQ ID NO：11。
NK-APT-12 CTCGTGTAAAACGATGGTAGCTGTACTTG SEQ ID NO：12。
NK-APT-13 AAAGGCGCTGTGCACAGTGTCCCCGCGGG SEQ ID NO：13。
NK-APT-14 CCGAGGGGAATTCACCACGAATCGCCTCT SEQ ID NO：14。
NK-APT-15 TCGATAGTTGTACTGGAGTGAATCACTGA SEQ ID NO：15。
NK-APT-16 AAGACTCCAGTCGCGGCCTCTATCCGGGA SEQ ID NO：16。
NK-APT-17 ACCCAAGTAGGCGAGTTCAGAACTGTGTG SEQ ID NO：17。
6、通过Mfold软件，在25℃、Na +为50mmol/L条件下，对所得序列进行二级结构分析可知，所得序列普遍存在颈环结构。选取亲合力较大的七条候选适配体，用CE-荧光检测技术对亲合力进行了初步鉴定。
首先对未加入纳豆激酶的核酸文库进行CE分析；加入纳豆激酶，孵育前后再一次进行 CE电泳分析。亲合力用峰面积变化百分比S＝(A 0-A 1)/A 0表示，其中A 0为核酸文库的峰面积，A 1为与靶标纳豆激酶混合孵育后，核酸文库中未与靶标结合部分的峰面积。比例越大，说明纳豆激酶与核酸文库间亲合力越大。
具体操作为：选取二级结构具有代表性的核酸适配体，进行5`6-FAM(FITC)荧光标记合成。将核酸适配体用50mmol/L pH7.8硼酸缓冲液溶至0.5μmol/L，将易溶解的随机寡核苷酸文库放至95℃环境下变性不低于10min，缓慢冷却至室温。取50μL，加入浓度为0.05mg/mL纳豆激酶溶液10μL，加入50mM pH7.8硼酸缓冲液溶补足至100μL，获得aptamer浓度为0.25μmol/L、NK浓度为0.005mg/mL的混合液，将混合液30℃孵育30min后上样分析。
图12是本发明实施例提供的七条适配体与复合物的毛细管电泳图。表面与纳豆激酶相互结合的亲合力较强。图中：(a)、SEQ ID NO：2；(b)、SEQ ID NO：3；(c)、SEQ ID NO：4；(d)、SEQ ID NO：12(e)、SEQ ID NO：13；(f)、SEQ ID NO：14；(g)、SEQ ID NO：15。
7、对候选适配体与纳豆激酶亲合力进行鉴定，具体如下：
稳态亲合力测定条件：温度：4℃，流速30μl/min，结合时间100s，解离时间100s。将0-220nmol/纯化的NK依次流经芯片，得到明显的SPR稳态响应图，平衡态得到的响应值对不同蛋白浓度进行非线性拟合，得到芯片上蛋白-适配体的亲合力K C为5.4nmol/Lol/L。
配制相当2KC浓度的NK，在这个蛋白浓度条件下加入不同浓度的十条竞争适配体，在同一个芯片上得到不同的SPR响应值，对其进行非线性拟合得到其他九条适配体在溶液中的KS。竞争适配体的浓度梯度范围为0-200nmol/L。进行非线性拟合得到的结果如图10所示，实验结果如下表。
表6 所筛十条适配体的亲合力常数K S
Figure PCTCN2020090634-appb-000009
7、NK核酸适配体特异性测定
分别将不同浓度的牛血清蛋白和胰蛋白酶流经芯片，相同条件下得到的响应值分别如图11所示。
结果表明随着蛋白浓度的提高，SPR响应值没有相应变大。可推断此适配体与牛血清蛋白和胰蛋白酶没有结合或结合力很弱。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种纳豆激酶核酸适配体的筛选方法，包括以下步骤：
    设计构建两端带有固定序列的随机寡聚核苷酸文库；
    所设计核苷酸文库与纳豆激酶混合孵育，并进行毛细管电泳分离，收集结合的复合物；
    以收集的复合物作为模板进行不对称PCR扩增，并制备次级文库；
    对次级文库进行多轮筛选，以最后一轮筛选得到的复合物进行PCR扩增，得到与纳豆激酶结合的核苷酸文库；
    对核苷酸文库纯化后测序，获得候选适配体；
    对候选适配体进行结构、与纳豆激酶亲和力以及特异性检测，获得高亲和力、高特异性纳豆激酶核酸适配体。
  2. 根据权利要求1所述的一种纳豆激酶核酸适配体的筛选方法，其特征在于：所述毛细管电泳分离的条件为，毛细管温度25℃，0.5psi，20s进样，分离电压25KV，毛细管电泳的运行缓冲液为pH7.8、50mmol/L硼酸-硼砂缓冲液，检测波长为280nm。
  3. 根据权利要求2所述的一种纳豆激酶核酸适配体的筛选方法，其特征在于：所述毛细管电泳分离过程中，纳豆激酶在3.3min出峰，ssDNA在9.9min出峰，NK-ssDNA复合物的峰出现15.2min，收集时间约为18.76min～24.56min。
  4. 根据权利要求1所述的一种纳豆激酶核酸适配体的筛选方法，其特征在于：使用69nt随机序列文库与纳豆激酶结合，所述69nt随机序列为5′-TTGAGCCTACGAGCGATACC-29N-GATGTCAGGTGTCTCGTCGT-3′，其中29N表示有29个T,G,A,C随机的序列。
  5. 根据权利要求1所述的一种纳豆激酶核酸适配体的筛选方法，其特征在于：不对称PCR涉及的引物序列分别见SEQ ID NO：18和SEQ ID NO：19所示，其中下游引物的浓度为0.5μmol/L，上、下游引物比例为30:1。
  6. 根据权利要求1所述的一种纳豆激酶核酸适配体的筛选方法，其特征在于：所述以最后一轮筛选得到的复合物进行PCR扩增涉及的引物序列见SEQ ID NO：23和SEQ ID NO：24所示。
  7. 根据权利要求1所述的一种纳豆激酶核酸适配体的筛选方法，其特征在于：所述核酸纯化的方法包括磁珠法纯化。
  8. 纳豆激酶核酸适配体，所述适配体的核苷酸序列为SEQ ID NO：1～SEQ ID NO：17中的任一条。
  9. 根据权利要求8所述纳豆激酶核酸适配体，其特征在于，所述纳豆激酶核酸适配体利用权 利要求1-7任一所述的筛选方法获得。
  10. 一种纳豆激酶蛋白质分子识别试剂盒，包括权利要求8所述的纳豆激酶核酸适配体。
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