WO2022088480A1 - Nucleic acid aptamer h10 with targeted inhibition of vibrio anguillarum and use thereof - Google Patents

Nucleic acid aptamer h10 with targeted inhibition of vibrio anguillarum and use thereof Download PDF

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WO2022088480A1
WO2022088480A1 PCT/CN2020/141236 CN2020141236W WO2022088480A1 WO 2022088480 A1 WO2022088480 A1 WO 2022088480A1 CN 2020141236 W CN2020141236 W CN 2020141236W WO 2022088480 A1 WO2022088480 A1 WO 2022088480A1
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nucleic acid
vibrio
aptamer
eel
aptamers
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郑江
林筱钧
江兴龙
鄢庆枇
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集美大学
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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

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  • the present invention relates to a nucleic acid aptamer, in particular to a nucleic acid aptamer H10 with targeted inhibition of Vibrio eel and application thereof.
  • Vibrio eel is one of the most common opportunistic pathogens in aquaculture.
  • the diseases of aquaculture animals it causes are prevalent worldwide and can infect more than 50 species of sea and freshwater organisms, including rainbow trout, eel, sweetfish, sea bass, cod, Turbot, flounder, yellow croaker, etc.
  • the number of farmed animals infected with vibrosis is also increasing, which brings huge economic losses to the aquaculture industry.
  • Vibrio eel Due to the serious harm caused by Vibrio eel to aquaculture, a large number of antibiotics are mainly used to control Vibrio, which not only leads to the emergence of bacterial resistance, but also brings huge risks and hidden dangers to the quality and safety of aquatic products.
  • Nucleic acid aptamer is a single-stranded oligonucleotide sequence including ssDNA and RNA obtained from in vitro synthetic screening by exponential enrichment ligand system evolution technology (Systematic Evolution of Ligands by Exponential Enrichment, SELEX). It has the characteristics of high affinity, specificity, easy modification, high stability, no immunogenicity, and no toxic and side effects. It has received extensive attention in many fields such as life science research, target identification, biomedicine, and environmental monitoring. With the improvement of the level of science and technology, the research on nucleic acid aptamers as therapeutic reagents has become more and more extensive. In 1992, Bock LC et al.
  • a coagulation-grade protease thrombin single-stranded DNA aptamer which can inhibit thrombin-catalyzed fibrin-thrombus formation in vitro.
  • Ng EW et al. conducted clinical experiments on the aptamer Pegaptanib, which proved that it is effective in the treatment of age-related macular degeneration and choroidal neovascularization. This is the first aptamer therapy approved for use in humans.
  • Shuhao Zhu et al. found that the aptamer BT200 has a good inhibitory effect on human von Willebrand factor in vitro and can prevent arterial occlusion in non-human primates. Therefore, the investigation of aptamers as inhibitors is a promising development with potentially broad therapeutic benefits.
  • the technical problem to be solved by the present invention is to provide a nucleic acid aptamer H10 with targeted inhibition of Vibrio eel and its application.
  • the present invention is realized in this way:
  • the present invention first provides a nucleic acid aptamer H10 targeting and inhibiting Vibrio eel, the nucleic acid sequence of which is shown in SEQ.NO.1.
  • the present invention also provides the application of the nucleic acid aptamer H10 in the preparation of an inhibitor of Vibrio eel.
  • the concentration of the nucleic acid aptamer H10 in the inhibitor is 0.5-1.5 ⁇ M.
  • the concentration of the nucleic acid aptamer H10 in the inhibitor is 1 ⁇ M.
  • Vibrio eel was diluted to OD 0.1 using LB liquid medium.
  • nucleic acid aptamer H10 was prepared and diluted with 1 ⁇ TE buffer when used.
  • the culture was cultured in a lighted incubator at 28° C. for 72 hours.
  • the present invention has the following advantages: the present invention takes Vibrio eel as the target, and selects the aptamer (H10) screened by the Cell-SELEX technology to analyze its antibacterial function. It has the functions of short screening period, simple chemical synthesis, and can replace antibiotics, and has good application prospects. It provides a theoretical basis for further research on inhibitory aptamers.
  • Figure 1 is a graph showing the relationship between the bacterial film of Vibrio eel and time.
  • Figure 2 shows the bacteriostatic rates of different aptamers.
  • Figure 3 shows the bacteriostatic rates of other types of aptamers.
  • Figure 4 shows the bacteriostatic rates of different concentrations of aptamers.
  • Bacterial treatment take the cultured Vibrio eel in a centrifuge tube, centrifuge at 6000 r/min for 5 min, discard the supernatant, wash the bacteria 3 times and add a sterile medium to mix evenly, measure the OD value of the bacterial solution, and then Take the Vibrio eel bacteria solution containing about 4 ⁇ 108 bacteria, centrifuge at 6000 r/min for 5 min, discard the supernatant, wash the bacterial precipitation three times with 0.9% saline, and wash the bacterial precipitate once with 1 ⁇ binding buffer. Add 100 ⁇ L of 2x binding buffer to resuspend.
  • Binding Take the ssDNA random library, dilute it with 2 ⁇ binding buffer to 3 ⁇ mol/L 100 ⁇ L, denature it in a constant temperature metal bath at 95°C for 5min, then ice bath for 10min, then add it to the previous bacterial suspension, mix well Combine in a shaker at 30°C, 100r/min for 2h.
  • Amplification Asymmetric PCR amplification is used to obtain the next-level ssDNA library.
  • Electrophoresis detection place the prepared agarose gel in an electrophoresis tank, and add 1 ⁇ TAE electrophoresis buffer solution to completely cover the agarose gel. Take 2 ⁇ L of Marker reference, take 5 ⁇ L of asymmetric PCR product and mix it with 2 ⁇ L of loading buffer, add it to the well, and conduct electrophoresis under the condition of voltage of 90V and 45min. Finally, observe and take pictures.
  • nucleic acid aptamers The sequences of nucleic acid aptamers are shown in Table 1 (underlined at both ends is the fixed sequence that binds to the primers).
  • the above-mentioned aptamers were synthesized by Sangon Bioengineering Co., Ltd.
  • the synthesized lyophilized powder product was prepared into a stock solution with a concentration of 10 ⁇ mol/L with 1 ⁇ TE buffer, and stored in a -20°C refrigerator for future use.
  • Vibrioanguillarum was identified and provided by the Disease Laboratory of Jimei University.
  • LB solid medium take 5 g of tryptone, 2.5 g of yeast extract, 5 g of NaCl, and 7.5 g of agar powder. Add ultrapure water to dissolve, adjust the pH to 7.0 with HCl or NaOH, the total volume to 500mL, and sterilize at 121°C for 45min before use.
  • 20 ⁇ binding buffer take NaCl 5.844g, KCl3.725g, Tris-HCl 6.06g, MgCl2 ⁇ 6H2O 2.033g. Add ultrapure water to dissolve, adjust pH to 7.0 with HCl or NaOH, and dilute to 100mL. Diluted into 2 ⁇ and 1 ⁇ binding buffer, sterilized at 121°C for later use.
  • Staining solution (0.1% crystal violet): Dissolve 0.1 g of crystal violet in 20 mL of 95% ethanol to prepare solution A; weigh 0.8 g of ammonium oxalate and dissolve it in 80 mL of ddH2O to prepare solution B. Mix liquid A and liquid B evenly, filter the liquid with analytical filter paper, and store at room temperature for later use.
  • Fixing agent (4% acetaldehyde) paraformaldehyde 40g, NaH 2 P0 4 2.965g, Na 2 HPO 4 29.00g, mixed in 500mL ddH 2 O, placed on a constant temperature magnetic stirrer, 60 °C, 2h to dissolve the paraformaldehyde Thoroughly, dilute the volume to 1000 mL with ddH 2 O, and store in a brown reagent bottle at room temperature away from light.
  • 33% acetic acid solution Measure 33 mL of acetic acid solution, add ddH 2 O to dilute to 100 mL, and store at 4°C for later use.
  • Phosphate buffer (pH7.4): NaCl 0.8g, KC1 0.02g, Na2HP04.12H20, 0.363g, KH2PO4 0.024g ddH 2 O, sterilized in volume of 100ml, and stored at 4°C for use.
  • Fixing the bacterial membrane Add 200 ⁇ L of fixative to each well for 15 minutes to fix the bacterial bacterial membrane structure on the surface of the culture well plate, absorb the fixative, and air dry at room temperature.
  • Staining Add 200 ⁇ L/well of staining solution, incubate for 5 min, remove the staining solution, wash the plate 3 times with phosphate buffer, and dry at room temperature.
  • Detection Add 200 ⁇ L/well of 33% acetic acid solution, incubate at room temperature for 15 minutes to elute and dissolve the bacterial membrane structure on the surface of the culture plate wall, and measure the absorbance value at 595 nm.
  • the aptamers were metal-denatured at 95 °C for 5 min, and then placed on ice for 10 min; in the experimental group, 100 ⁇ L/well of aptamers with a concentration of 1 ⁇ M was added to a 96-well plate, and fresh LB liquid medium was taken to dilute the bacterial solution to OD 0.1 , 200 ⁇ L/well was added to the 96-well plate for mixing and culture; for blank control, 100 ⁇ L/well 2 ⁇ buffer was added to 200 ⁇ L/well diluted bacterial solution for mixing culture; 1uM 100 ⁇ L/well was added for random library, and 200 ⁇ L/well diluted The bacterial liquid was mixed and cultivated in a light incubator at 28°C for 72h. The processing method after taking out the sample is the same as 2.2.1.
  • inhibition rate (%) (A C -A)/A C (A is the absorbance of the experimental group, and A C is the absorbance of the blank control group).
  • the aptamers were denatured in a water bath at 95°C for 5 min, and then placed on ice for 10 min; 100 ⁇ L/well of different concentrations of aptamers (0.5 ⁇ M, 1 ⁇ M, 1.5 ⁇ M) were mixed with 200 ⁇ L/well of bacterial liquid with an OD of 0.1, and 100 ⁇ L was taken for the blank control. Add 200 ⁇ L/well of 2 ⁇ buffer/well to mix and culture, and incubate at 28°C for 72h in a lighted incubator. Measure the absorbance value at 595nm and calculate the bacteriostatic rate, the methods are the same as 2.2.1 and 2.2.2.
  • Vibrio eel biofilm As shown in Figure 1, the formation of Vibrio eel biofilm was closely related to the incubation time. In the early stage of cultivation, the OD595 value of the biofilm formed by Vibrio eel increased with the extension of the cultivation time, and reached a peak value at about 48h. When the cultivation continued, the OD595 value decreased with the extension of the cultivation time; 60-72h tended to be relatively stable. , to reach a new balance.
  • the three selected aptamers can all reduce the formation of Vibrio eel's bacterial film, but the bacteriostatic effects are different. Among them, the antibacterial effect of H10 is the best, reaching 37.46%, which can replace the use of antibiotics. H7 had the lowest bacteriostatic rate of 16.59%. The inhibitory effect of the random library on the biofilm of Vibrio eel was not obvious, only 5.21%, which was lower than the inhibitory rate of the three aptamers.
  • bacterial motility flagella are closely related, and aptamer can specifically bind to the site on the bacterial flagella, restricting its movement and reducing the formation of biofilm, so as to achieve bacteriostatic effect.
  • the antibacterial effects of different aptamers are different, which may be due to the different binding abilities of different aptamers to bacterial targets.
  • the other three aptamers with good affinity for Vibrio eel were selected for bacteriostatic test, among which H1 and H5 were the aptamers with the highest affinity in the preliminary screening of Vibrio eel (CN110578010A), but they had poor antibacterial effect or even No effect, as shown in Figure 3. It can be seen that the strength of the affinity has nothing to do with the bacteriostasis, and the bacteriostasis is not the case that the affinity is strong.
  • the inhibitory rate of aptamers on Vibrio anguillarum membrane increased with the increase of concentration, reached a peak at 1 ⁇ M, and then gradually decreased with the increase of concentration.
  • aptamer H7 can promote the growth of biofilm instead.
  • the promoting effect at low concentration may be due to the fact that the lower concentration of aptamer does not achieve bacteriostatic effect but provides nutrients to the bacteria; the bacteriostatic effect at high concentration may be due to the fact that the number of bacteria in the experimental system is saturated with aptamer binding , the excess aptamers could not combine with bacteria, but provided nutrients to bacteria. It can be seen that the aptamer needs to control a certain concentration to produce a bacteriostatic effect, on the contrary, it produces a growth-promoting effect.

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Abstract

Provided are a nucleic acid aptamer H10 with the targeted inhibition of Vibrio anguillarum and the use thereof in the preparation of a Vibrio anguillarum inhibitor. The nucleic acid sequence of the nucleic acid aptamer H10 is as shown in SEQ.NO.1.

Description

一种具有靶向抑制鳗弧菌的核酸适配体H10及其应用A kind of nucleic acid aptamer H10 with targeted inhibition of Vibrio eel and its application 技术领域technical field
本发明涉及一种核酸适配体，尤其涉及一种具有靶向抑制鳗弧菌的核酸适配体H10及其应用。The present invention relates to a nucleic acid aptamer, in particular to a nucleic acid aptamer H10 with targeted inhibition of Vibrio eel and application thereof.
背景技术Background technique
前言foreword
鳗弧菌是水产养殖中最为常见的条件致病菌之一，其引起的水产养殖动物疾病在全球范围内流行，可感染50多种海淡水生物包括虹鳟、鳗鲡、香鱼、鲈鱼、鳕鱼、大菱鲆、牙鲆、黄鱼等。目前随着水产养殖向高密度、集约化发展的同时，养殖动物感染弧菌病数量也日益增加，给水产养殖业带来巨大的经济损失。由于鳗弧菌对水产养殖造成的严重危害，主要使用大量抗生素来防治弧菌，这不仅导致细菌耐药性产生，还给水产品质量安全带来巨大风险和隐患。Vibrio eel is one of the most common opportunistic pathogens in aquaculture. The diseases of aquaculture animals it causes are prevalent worldwide and can infect more than 50 species of sea and freshwater organisms, including rainbow trout, eel, sweetfish, sea bass, cod, Turbot, flounder, yellow croaker, etc. At present, with the high-density and intensive development of aquaculture, the number of farmed animals infected with vibrosis is also increasing, which brings huge economic losses to the aquaculture industry. Due to the serious harm caused by Vibrio eel to aquaculture, a large number of antibiotics are mainly used to control Vibrio, which not only leads to the emergence of bacterial resistance, but also brings huge risks and hidden dangers to the quality and safety of aquatic products.
核酸适配体是通过指数富集配体系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment,SELEX)从体外人工合成筛选得到的一种单链寡核苷酸序列包括ssDNA和RNA。它具有亲和特异性高、易于修饰，稳定性高、无免疫原性及毒副作用等特点，已在生命科学研究、靶点鉴定、生物医学、环境监测等多个领域受到广泛关注。随着科学技术水平的提高，核酸适配体作为治疗试剂方面的研究也越来越广泛。1992年Bock LC等人分离了凝血级的蛋白酶凝血酶单链DNA适配体，适配体能在体外抑制凝血酶催化的纤维蛋白-血栓形成。2006年Ng EW等人对适配体Pegaptanib进行临床实验，证明了其在治疗与年龄相关的黄斑变性脉络膜新生血管方面是有效的。这是第一个被批准用于人类的适配体治疗药物。2020年Shuhao Zhu等人发现适配体BT200在体外对人的血管性血友病因子有良好的抑制作用，并能预防非人灵长类动物的动脉阻塞。因此，研究适配体作为抑制剂是 一个很有前途的发展，具有潜在的广泛的治疗益处。Nucleic acid aptamer is a single-stranded oligonucleotide sequence including ssDNA and RNA obtained from in vitro synthetic screening by exponential enrichment ligand system evolution technology (Systematic Evolution of Ligands by Exponential Enrichment, SELEX). It has the characteristics of high affinity, specificity, easy modification, high stability, no immunogenicity, and no toxic and side effects. It has received extensive attention in many fields such as life science research, target identification, biomedicine, and environmental monitoring. With the improvement of the level of science and technology, the research on nucleic acid aptamers as therapeutic reagents has become more and more extensive. In 1992, Bock LC et al. isolated a coagulation-grade protease thrombin single-stranded DNA aptamer, which can inhibit thrombin-catalyzed fibrin-thrombus formation in vitro. In 2006, Ng EW et al. conducted clinical experiments on the aptamer Pegaptanib, which proved that it is effective in the treatment of age-related macular degeneration and choroidal neovascularization. This is the first aptamer therapy approved for use in humans. In 2020, Shuhao Zhu et al. found that the aptamer BT200 has a good inhibitory effect on human von Willebrand factor in vitro and can prevent arterial occlusion in non-human primates. Therefore, the investigation of aptamers as inhibitors is a promising development with potentially broad therapeutic benefits.
发明内容SUMMARY OF THE INVENTION
本发明要解决的技术问题，在于提供一种具有靶向抑制鳗弧菌的核酸适配体H10及其应用。The technical problem to be solved by the present invention is to provide a nucleic acid aptamer H10 with targeted inhibition of Vibrio eel and its application.
本发明是这样实现的：The present invention is realized in this way:
本发明首先提供了一种靶向抑制鳗弧菌的核酸适配体H10，其核酸序列如SEQ.NO.1所示。The present invention first provides a nucleic acid aptamer H10 targeting and inhibiting Vibrio eel, the nucleic acid sequence of which is shown in SEQ.NO.1.
本发明还提供了所述核酸适配体H10在制备鳗弧菌抑制剂中的应用。The present invention also provides the application of the nucleic acid aptamer H10 in the preparation of an inhibitor of Vibrio eel.
进一步地，所述核酸适配体H10在抑制剂中的浓度为0.5-1.5μM。Further, the concentration of the nucleic acid aptamer H10 in the inhibitor is 0.5-1.5 μM.
优选地，所述核酸适配体H10在抑制剂中的浓度为1μM。Preferably, the concentration of the nucleic acid aptamer H10 in the inhibitor is 1 μM.
具体应用时，将核酸适配体H10在95℃水浴变性5min,然后转于冰上放置10min；取0.5-1.5μM核酸适配体H10 100μL/孔与200μL/孔OD 0.1的鳗弧菌菌液混合培养。For specific applications, denature the aptamer H10 in a 95°C water bath for 5 min, then place it on ice for 10 min; take 100 μL/well of 0.5-1.5 μM aptamer H10 and 200 μL/well OD 0.1 of Vibrio eel bacteria solution mixed culture.
进一步地，采用LB液体培养基将鳗弧菌菌液稀释至OD 0.1。Further, the Vibrio eel was diluted to OD 0.1 using LB liquid medium.
进一步地，所述核酸适配体H10使用时用1×TE缓冲液配制稀释。Further, the nucleic acid aptamer H10 was prepared and diluted with 1×TE buffer when used.
进一步地，所述培养，在光照培养箱中28℃培养72h。Further, the culture was cultured in a lighted incubator at 28° C. for 72 hours.
本发明具有如下优点：本发明以鳗弧菌为靶标，选取Cell-SELEX技术筛选出的适配体(H10)对其进行抗菌功能进行分析。具有筛选周期短、化学合成简便、能替代抗生素等作用，具有较好的应用前景。为进一步开展抑制性适配体的研究提供理论基础。The present invention has the following advantages: the present invention takes Vibrio eel as the target, and selects the aptamer (H10) screened by the Cell-SELEX technology to analyze its antibacterial function. It has the functions of short screening period, simple chemical synthesis, and can replace antibiotics, and has good application prospects. It provides a theoretical basis for further research on inhibitory aptamers.
附图说明Description of drawings
下面参照附图结合实施例对本发明作进一步的说明。The present invention will be further described below with reference to the accompanying drawings and embodiments.
图1为鳗弧菌菌膜随时间变化关系图。Figure 1 is a graph showing the relationship between the bacterial film of Vibrio eel and time.
图2为不同适配体的抑菌率。Figure 2 shows the bacteriostatic rates of different aptamers.
图3为其它种类适配体的抑菌率。Figure 3 shows the bacteriostatic rates of other types of aptamers.
图4为不同浓度适配体的抑菌率。Figure 4 shows the bacteriostatic rates of different concentrations of aptamers.
具体实施方式Detailed ways
1核酸适配体的SELEX筛选1 SELEX screening of nucleic acid aptamers
(1)菌的处理：取培养好的鳗弧菌于离心管中，6000r/min离心5min，弃上清，洗涤菌3次后再加无菌培养液混合均匀，测菌液OD值，然后取含菌量约为4×108个的鳗弧菌菌液，6000r/min下离心5min，弃上清，0.9％生理盐水洗涤菌沉淀3次，1×结合缓冲液洗涤菌沉淀1次，最后加入100μL的2×结合缓冲液重悬。(1) Bacterial treatment: take the cultured Vibrio eel in a centrifuge tube, centrifuge at 6000 r/min for 5 min, discard the supernatant, wash the bacteria 3 times and add a sterile medium to mix evenly, measure the OD value of the bacterial solution, and then Take the Vibrio eel bacteria solution containing about 4 × 108 bacteria, centrifuge at 6000 r/min for 5 min, discard the supernatant, wash the bacterial precipitation three times with 0.9% saline, and wash the bacterial precipitate once with 1 × binding buffer. Add 100 μL of 2x binding buffer to resuspend.
(2)结合：取ssDNA随机文库，用2×结合缓冲液稀释到3μmol/L 100μL，在95℃恒温金属浴中变性5min后冰浴10min，然后加入到前面的菌悬液中，混匀后在30℃，100r/min的摇床中结合2h。(2) Binding: Take the ssDNA random library, dilute it with 2× binding buffer to 3μmol/L 100μL, denature it in a constant temperature metal bath at 95°C for 5min, then ice bath for 10min, then add it to the previous bacterial suspension, mix well Combine in a shaker at 30°C, 100r/min for 2h.
(3)洗涤：结合完成后，6000r/min离心5min，弃上清，含有菌及其结合ssDNA的菌沉淀，用200μL1×结合缓冲液洗涤1次。(3) Washing: After the binding is completed, centrifuge at 6000 r/min for 5 min, discard the supernatant, and wash the bacterial pellet containing bacteria and its binding ssDNA with 200 μL of 1× binding buffer once.
(4)分离：用100μL1×结合缓冲液溶液重悬菌沉淀，95℃加热5min，使ssDNA变性，从而与菌分离，冷却后在15000r/min离心10min，取上清液，则可分离到与目标菌结合的ssDNA，该ssDNA即为获得的筛选产物，将该筛选产物用PCR管分装成每管20μL，放于-20℃保存备用。(4) Separation: Resuspend the bacterial pellet with 100 μL of 1× binding buffer solution, heat at 95°C for 5 min to denature the ssDNA, thereby separating from the bacteria, centrifuge at 15000 r/min for 10 min after cooling, and take the supernatant to separate the The ssDNA bound by the target bacteria, the ssDNA is the obtained screening product, and the screening product is divided into 20 μL tubes in PCR tubes, and stored at -20° C. for future use.
(5)扩增：采用不对称PCR扩增来获取下一级ssDNA文库。(5) Amplification: Asymmetric PCR amplification is used to obtain the next-level ssDNA library.
(6)电泳检测：将制好的琼脂糖凝胶放置于电泳槽中，加入1×TAE电泳缓冲溶液使琼脂糖凝胶完全被覆盖。取2μL的Marker参考，取5μL的不对称PCR产物与2μL的上样缓冲液混合均匀后加入孔中，在电压为90V、45min的条件下进行电泳。最后进行观察和拍照。(6) Electrophoresis detection: place the prepared agarose gel in an electrophoresis tank, and add 1×TAE electrophoresis buffer solution to completely cover the agarose gel. Take 2 μL of Marker reference, take 5 μL of asymmetric PCR product and mix it with 2 μL of loading buffer, add it to the well, and conduct electrophoresis under the condition of voltage of 90V and 45min. Finally, observe and take pictures.
(7)重复筛选：如果电泳检测后显示PCR产物中有条带，表明PCR效果可以，则可将该轮PCR产物用作下一轮SELEX筛选的文库，重复上述(1)到(6)的筛选过程。筛选得到的适配体进行抑菌性能的验证。(7) Repeat screening: If the PCR product has a band after electrophoresis detection, indicating that the PCR effect is good, then this round of PCR product can be used as the library for the next round of SELEX screening, repeat the above (1) to (6) screening process. The aptamers obtained by screening were verified for bacteriostatic properties.
2抑菌性能的验证2 Verification of antibacterial properties
2.1材料2.1 Materials
2.1.1核酸适配体2.1.1 Nucleic acid aptamers
核酸适配体序列如表1(两端带下划线的是和引物结合的固定序列)。The sequences of nucleic acid aptamers are shown in Table 1 (underlined at both ends is the fixed sequence that binds to the primers).
上述适配体由生工生物工程股份有限公司合成。合成的冻干粉产物，用1×TE缓冲液配制成浓度为10μmol/L的贮存液，于-20℃冰箱保存备用。The above-mentioned aptamers were synthesized by Sangon Bioengineering Co., Ltd. The synthesized lyophilized powder product was prepared into a stock solution with a concentration of 10 μmol/L with 1×TE buffer, and stored in a -20°C refrigerator for future use.
表1鳗弧菌适配体的序列Table 1 Sequences of Vibrio eel aptamers
Figure PCTCN2020141236-appb-000001
Figure PCTCN2020141236-appb-000001
2.1.2实验用菌2.1.2 Experimental bacteria
鳗弧菌(Vibrioanguillarum)由集美大学病害实验室鉴定并提供。Vibrioanguillarum was identified and provided by the Disease Laboratory of Jimei University.
2.1.3培养基和试剂2.1.3 Culture medium and reagents
LB固体培养基：取胰蛋白胨5g，酵母提取物2.5g，NaCl5g，琼脂粉7.5g。加超纯水溶解，用HCl或NaOH调pH至7.0，总体积至500mL，经121℃灭菌45min后备用。LB solid medium: take 5 g of tryptone, 2.5 g of yeast extract, 5 g of NaCl, and 7.5 g of agar powder. Add ultrapure water to dissolve, adjust the pH to 7.0 with HCl or NaOH, the total volume to 500mL, and sterilize at 121°C for 45min before use.
20×结合缓冲液：取NaCl5.844g、KCl3.725g、Tris-HCl6.06g、MgCl2·6H2O 2.033g。加超纯水溶解，用HCl或NaOH调pH至7.0定容至100mL。稀释为2×和1×结合缓冲液，经121℃灭菌后备用。20× binding buffer: take NaCl 5.844g, KCl3.725g, Tris-HCl 6.06g, MgCl2·6H2O 2.033g. Add ultrapure water to dissolve, adjust pH to 7.0 with HCl or NaOH, and dilute to 100mL. Diluted into 2× and 1× binding buffer, sterilized at 121°C for later use.
染色液(0.1％crystal violet):称0.1g结晶紫溶于20mL95％的乙醇，配制成A液；称0.8g草酸铵溶于80mLddH2O中，配制成B液。将A液与B液混合均匀，使用分析滤纸对液体进行过滤，常温储存待用。Staining solution (0.1% crystal violet): Dissolve 0.1 g of crystal violet in 20 mL of 95% ethanol to prepare solution A; weigh 0.8 g of ammonium oxalate and dissolve it in 80 mL of ddH2O to prepare solution B. Mix liquid A and liquid B evenly, filter the liquid with analytical filter paper, and store at room temperature for later use.
固定剂(4％acetaldehyde)多聚甲醛40g,NaH 2P0 42.965g,Na 2HPO 429.00g， 混匀500mLddH 2O中，放置于恒温磁力搅拌器上，60℃、2h使多聚甲醛溶解彻底，ddH 2O定容至1000mL，放于棕色试剂瓶中常温避光储存。 Fixing agent (4% acetaldehyde) paraformaldehyde 40g, NaH 2 P0 4 2.965g, Na 2 HPO 4 29.00g, mixed in 500mL ddH 2 O, placed on a constant temperature magnetic stirrer, 60 ℃, 2h to dissolve the paraformaldehyde Thoroughly, dilute the volume to 1000 mL with ddH 2 O, and store in a brown reagent bottle at room temperature away from light.
33％醋酸溶液:量取33mL的醋酸溶液，加入ddH 2O定容至100mL,4℃储存待用。 33% acetic acid solution: Measure 33 mL of acetic acid solution, add ddH 2 O to dilute to 100 mL, and store at 4°C for later use.
磷酸缓冲液(pH7.4):NaCl 0.8g,KC1 0.02g,Na2HP04.12H20，0.363g,KH2PO4 0.024g ddH 2O定容100ml灭菌,4℃贮存待用。 Phosphate buffer (pH7.4): NaCl 0.8g, KC1 0.02g, Na2HP04.12H20, 0.363g, KH2PO4 0.024g ddH 2 O, sterilized in volume of 100ml, and stored at 4°C for use.
2.2实验方法2.2 Experimental method
2.2.1时间对于抑菌效果的影响2.2.1 The effect of time on the antibacterial effect
取新鲜的LB液体培养基将菌液稀释至OD 0.1后，200μL/孔加入96孔板中培养。培养不同时间(6、12、24、36、48、60、72h)时取出样品，除去孔板中的菌液,加入磷酸缓冲液清洗孔板3次,移液器尽量吸干孔板。Take fresh LB liquid medium to dilute the bacterial liquid to OD 0.1, and add 200 μL/well to the 96-well plate for culture. After culturing for different time (6, 12, 24, 36, 48, 60, 72h), take out the sample, remove the bacterial liquid in the well plate, add phosphate buffer to wash the well plate 3 times, and dry the well plate as much as possible with a pipette.
固定菌膜：每孔加入固定剂200μL处理15min,固定培养孔板壁表面的细菌菌膜结构，吸去固定剂，室温风干。Fixing the bacterial membrane: Add 200 μL of fixative to each well for 15 minutes to fix the bacterial bacterial membrane structure on the surface of the culture well plate, absorb the fixative, and air dry at room temperature.
染色：加入200μL/孔的染色液，孵育5min后去除染液，磷酸缓冲液清洗孔板3次，室温干燥。Staining: Add 200 μL/well of staining solution, incubate for 5 min, remove the staining solution, wash the plate 3 times with phosphate buffer, and dry at room temperature.
检测：加入200μL/孔33％的醋酸溶液,室温孵育15min将培养孔板壁表面的细菌菌膜结构洗脱溶解，测量595nm处吸光度值。Detection: Add 200 μL/well of 33% acetic acid solution, incubate at room temperature for 15 minutes to elute and dissolve the bacterial membrane structure on the surface of the culture plate wall, and measure the absorbance value at 595 nm.
2.2.2不同适配体的抑菌效果2.2.2 The antibacterial effect of different aptamers
适配体在95℃金属变性5min,转于冰上放置10min；实验组取浓度为1μM的适配体100μL/孔加入96孔板，再取新鲜的LB液体培养基将菌液稀释至OD 0.1，200μL/孔加入96孔板中混匀培养；空白对照取100μL/孔2×缓冲液加入200μL/孔稀释的菌液混匀培养；随机文库取1uM 100μL/孔，之后加入200μL/孔稀释的菌液混匀培养，在光照培养箱中28℃培养72h。取出样品后处理方法同2.2.1。The aptamers were metal-denatured at 95 °C for 5 min, and then placed on ice for 10 min; in the experimental group, 100 μL/well of aptamers with a concentration of 1 μM was added to a 96-well plate, and fresh LB liquid medium was taken to dilute the bacterial solution to OD 0.1 , 200μL/well was added to the 96-well plate for mixing and culture; for blank control, 100μL/well 2× buffer was added to 200μL/well diluted bacterial solution for mixing culture; 1uM 100μL/well was added for random library, and 200μL/well diluted The bacterial liquid was mixed and cultivated in a light incubator at 28°C for 72h. The processing method after taking out the sample is the same as 2.2.1.
计算菌膜抑制率:抑制率(％)＝(A C-A)/A C(A为实验组吸光度，A C为空白对照组所得吸光度)。 Calculate the inhibition rate of biofilm: inhibition rate (%)=(A C -A)/A C (A is the absorbance of the experimental group, and A C is the absorbance of the blank control group).
2.2.3不同浓度适配体的抑菌效果2.2.3 The antibacterial effect of different concentrations of aptamers
适配体95℃水浴变性5min,转于冰上放置10min；取不同浓度适配体 (0.5μM、1μM、1.5μM)100μL/孔与200μL/孔OD 0.1的菌液混合培养，空白对照取100μL/孔2×缓冲液加入200μL/孔的菌液混匀培养，在光照培养箱中28℃培养72h。测量595nm处吸光度值和计算抑菌率，方法同2.2.1和2.2.2。The aptamers were denatured in a water bath at 95°C for 5 min, and then placed on ice for 10 min; 100 μL/well of different concentrations of aptamers (0.5 μM, 1 μM, 1.5 μM) were mixed with 200 μL/well of bacterial liquid with an OD of 0.1, and 100 μL was taken for the blank control. Add 200 μL/well of 2× buffer/well to mix and culture, and incubate at 28°C for 72h in a lighted incubator. Measure the absorbance value at 595nm and calculate the bacteriostatic rate, the methods are the same as 2.2.1 and 2.2.2.
3结果与分析：3 Results and analysis:
3.1鳗弧菌菌膜随时间变化关系3.1 The relationship between Vibrio eel's bacterial film and time
如图1所示，鳗弧菌生物膜的形成与培养时间密切相关。在培养初期，鳗弧菌所形成的生物膜OD595值随着培养时间的延长而增加，约48h达到峰值，继续培养，OD595值随培养时间的延长而有所下降；60-72h趋于相对稳定，达到新的平衡。As shown in Figure 1, the formation of Vibrio eel biofilm was closely related to the incubation time. In the early stage of cultivation, the OD595 value of the biofilm formed by Vibrio eel increased with the extension of the cultivation time, and reached a peak value at about 48h. When the cultivation continued, the OD595 value decreased with the extension of the cultivation time; 60-72h tended to be relatively stable. , to reach a new balance.
根据此结果显示，由于菌膜形成前期的不稳定性，接下来的实验选择培养72h后测量鳗弧菌菌膜形成效果最佳。According to this result, due to the instability in the early stage of biofilm formation, the next experiment chose to measure the biofilm formation of Vibrio eel after 72h of culture.
3.2不同种类适配体的抑菌率3.2 Bacteriostatic rate of different aptamers
如图2所示，选取的三条适配体均能减少鳗弧菌菌膜的形成，但是抑菌效果各不相同。其中H10的抑菌效果最佳，达到了37.46％，，能替代抗生素使用。H7的抑菌率最低，为16.59％。随机文库对鳗弧菌的菌膜的抑制效果不明显，仅为5.21％，低于三条适配体的抑菌率。As shown in Figure 2, the three selected aptamers can all reduce the formation of Vibrio eel's bacterial film, but the bacteriostatic effects are different. Among them, the antibacterial effect of H10 is the best, reaching 37.46%, which can replace the use of antibiotics. H7 had the lowest bacteriostatic rate of 16.59%. The inhibitory effect of the random library on the biofilm of Vibrio eel was not obvious, only 5.21%, which was lower than the inhibitory rate of the three aptamers.
抑菌的原理：细菌运动鞭毛息息相关，而适配体可以与细菌鞭毛上的位点特异性结合，限制其运动并减少生物膜的形成，从而达到抑菌效果。The principle of bacteriostasis: bacterial motility flagella are closely related, and aptamer can specifically bind to the site on the bacterial flagella, restricting its movement and reducing the formation of biofilm, so as to achieve bacteriostatic effect.
不同适配体的抑菌效果有差异，可能是由于不同适配体结合细菌靶标能力不同，抑菌率高的适配体结合靶标能力较强，反之较弱。The antibacterial effects of different aptamers are different, which may be due to the different binding abilities of different aptamers to bacterial targets.
3.3其余种类适配体的抑菌率3.3 Bacteriostatic rate of other aptamers
选取另外三条对鳗弧菌有较好亲和力的适配体进行抑菌试验，其中H1、H5是鳗弧菌前期筛选里亲和力最高的适配体(CN110578010A)，但在抑菌方面效果较差甚至没有效果，如图3所示。可见，亲和力的强度与抑菌性没有关系，不是亲和力强抑菌性就强。The other three aptamers with good affinity for Vibrio eel were selected for bacteriostatic test, among which H1 and H5 were the aptamers with the highest affinity in the preliminary screening of Vibrio eel (CN110578010A), but they had poor antibacterial effect or even No effect, as shown in Figure 3. It can be seen that the strength of the affinity has nothing to do with the bacteriostasis, and the bacteriostasis is not the case that the affinity is strong.
3.4不同浓度适配体的抑菌率3.4 Bacteriostatic rate of different concentrations of aptamer
如图4所示，适配体对鳗弧菌菌膜的抑制率随着浓度的增加而升高，到 了1μM达到峰值，之后随着浓度的增加反而逐渐下降。适配体H7在0.5μM时反而出现了促进菌膜生长的情况。As shown in Figure 4, the inhibitory rate of aptamers on Vibrio anguillarum membrane increased with the increase of concentration, reached a peak at 1 μM, and then gradually decreased with the increase of concentration. At 0.5μM, aptamer H7 can promote the growth of biofilm instead.
低浓度出现促进效果可能是由于较低浓度的适配体达不到抑菌作用反而给细菌提供营养作用；高浓度出现抑菌效果可能是由于实验体系中细菌数量与适配体结合达到饱和状态,多余的适配体无法与细菌发生结合,反而给细菌提供了营养作用。可见，适配体需要控制一定的浓度才能产生抑菌作用，相反则产生促进生长的作用。The promoting effect at low concentration may be due to the fact that the lower concentration of aptamer does not achieve bacteriostatic effect but provides nutrients to the bacteria; the bacteriostatic effect at high concentration may be due to the fact that the number of bacteria in the experimental system is saturated with aptamer binding , the excess aptamers could not combine with bacteria, but provided nutrients to bacteria. It can be seen that the aptamer needs to control a certain concentration to produce a bacteriostatic effect, on the contrary, it produces a growth-promoting effect.
虽然以上描述了本发明的具体实施方式，但是熟悉本技术领域的技术人员应当理解，我们所描述的具体的实施例只是说明性的，而不是用于对本发明的范围的限定，熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化，都应当涵盖在本发明的权利要求所保护的范围内。Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments we describe are only illustrative, rather than used to limit the scope of the present invention. Equivalent modifications and changes made by a skilled person in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.

Claims (7)

  1. 一种靶向抑制鳗弧菌的核酸适配体H10，其其特征在于：核酸序列如SEQ.NO.1所示。A nucleic acid aptamer H10 targeting and inhibiting Vibrio eel is characterized in that: the nucleic acid sequence is shown in SEQ.NO.1.
  2. 如权利要求1所述的核酸适配体H10在制备鳗弧菌抑制剂中的应用。The application of the nucleic acid aptamer H10 according to claim 1 in the preparation of an inhibitor of Vibrio eel.
  3. 根据权利要求2所述的应用，其特征在于：所述核酸适配体H10在抑制剂中的浓度为0.5-1.5μM。The application according to claim 2, wherein the concentration of the nucleic acid aptamer H10 in the inhibitor is 0.5-1.5 μM.
  4. 根据权利要求2所述的应用，其特征在于：将核酸适配体H10在95℃水浴中变性5min,然后转于冰上放置10min；取0.5-1.5μM核酸适配体H10 100μL/孔与200μL/孔OD 0.1的鳗弧菌菌液混合培养。The application according to claim 2, characterized in that: the aptamer H10 is denatured in a water bath at 95°C for 5 min, and then placed on ice for 10 min; 100 μL/well of 0.5-1.5 μM aptamer H10 and 200 μL The mixed culture of Vibrio anguillarum with an OD of 0.1 per well.
  5. 根据权利要求4所述的应用，其特征在于：所述培养，在光照培养箱中28℃培养72h。The application according to claim 4, wherein the culture is cultured in a lighted incubator at 28°C for 72 hours.
  6. 根据权利要求4所述的应用，其特征在于：采用LB液体培养基将鳗弧菌菌液稀释至OD 0.1。Application according to claim 4, is characterized in that: adopt LB liquid medium to dilute Vibrio eel bacterium liquid to OD 0.1.
  7. 根据权利要求4所述的应用，其特征在于：所述核酸适配体H10使用时用1×TE缓冲液配制稀释。The application according to claim 4, wherein the nucleic acid aptamer H10 is prepared and diluted with 1×TE buffer when used.
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