WO2016072653A1 - Aptamère d'adn se liant spécifiquement à la surface de cellules vivantes de vibrio fischeri et son utilisation - Google Patents

Aptamère d'adn se liant spécifiquement à la surface de cellules vivantes de vibrio fischeri et son utilisation Download PDF

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WO2016072653A1
WO2016072653A1 PCT/KR2015/011328 KR2015011328W WO2016072653A1 WO 2016072653 A1 WO2016072653 A1 WO 2016072653A1 KR 2015011328 W KR2015011328 W KR 2015011328W WO 2016072653 A1 WO2016072653 A1 WO 2016072653A1
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vibrio
dna
dna aptamer
aptamer
fischeri
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Korean (ko)
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김양훈
이문종
이상희
조성진
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충북대학교 산학협력단
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • DNA aptamers that specifically bind to the surface of vibrio fishery bacteria and their use
  • the present invention relates to a DNA aptamer specifically binding to the surface of vibrio fishery bacteria and its use. [Background technology]
  • Vibrio io f ischer i is a strain that grows in the genus Vibrio and attaches to the light-emitting organs of deep-sea squids. It is a fungative anaerobic Gram-negative bacillus and has a motility and one or more flagellar characteristics.
  • Vibrio Fishery is one of the various strains in marine ecosystems and forms biofilms as a strategy to increase viability, which is recognized as an essential step in survival strategy because resistance to various stresses is significantly increased compared to non-formed groups. .
  • the biofilm is formed, there is a problem in that the resistance of the therapeutic agent and the antibiotic to the Vibrio fishery is increased by about 500 times compared to when swimming.
  • biofilm formation has many adverse effects on the economy and industry, even if it is a natural phenomenon as a means of living survival strategy.
  • the formation of biofilms has been shown to account for 80% of the side effects of the implant procedure.
  • Conventional methods for controlling the production of biofilms have been attempted based on bacterial killing by antibiotics or antifouling agents, but the indiscriminate use of antibiotics against increased strains can lead to mutations in antibiotic resistant strains. Very limited.
  • the antifouling agent is a non-selective biotoxin that is toxic to other organisms, the use of the antifouling agent not only causes great damage to the environmental ecosystem, but also economic damage to recover it.
  • TBT tr ibutyl t in
  • TBT tr ibutyl t in
  • the marine environment It has been identified as being a serious threat to environmental hormones.
  • TBT can cause sexual variation even at concentrations of less than 1 ppt, and sexual variation due to TBT contamination has been found in the south coast.
  • developed countries have regulated the concentration of TBT used as antifouling agent for large ships since 1982, and has banned the use of small ships operating along the coast.
  • TBT regulation began in 2005, and alternative materials such as copper sulfate are used, but this is also considered as a pollutant, so it is necessary to develop alternative substances for antifouling agents such as TBT.
  • the aptamers consist of short length ligomers to form their own various three-dimensional structures. These oligomer structure libraries have the ability to structurally bind to various target materials, and among them, aptamers that are specific to the target material are selected. It is secured through the process. Selected aptamers can be sequenced to obtain sequences through sequencing and can be mass produced in a short time and at low cost using chemical synthesis techniques. In addition, since it is composed of DNA and can be given more stability through additional chemical substitution process, it is very stable to the surrounding pH and silver, and it attaches compounds such as biotin to environmental medicine such as detection of target substance and development of disease diagnosis sensor. It can be used in various fields such as, and the possibility is highly appreciated.
  • the present inventors have tried to develop DNA aptamers that can be used for various purposes such as the detection and growth inhibition of the bacteria by specifically binding to Vibr io fi scheri bacteria in a live state. As a result, they can bind with high specificity to the viable surface of Vibrio Fishery.
  • Another object of the present invention is to provide a composition and kit for detecting Vibrio fischeri microorganisms comprising the DNA aptamer as an active ingredient.
  • Still another object of the present invention is to provide a method for detecting Vibrio fischeri bacteria.
  • the present invention provides a DNA aptamer specifically binding to the vibrio fishery fischeri) microbial surface.
  • the DNA aptamer of the invention is an oligonucleotide having a nucleotide sequence having at least 90% identity with a nucleotide sequence disclosed in any one of SEQ ID NOs: 1 to 13.
  • the DNA aptamer of the present invention is an oligonucleotide having a nucleotide sequence disclosed in any one of SEQ ID NOs: 1-13.
  • the DNA aptamer of the present invention is chemically modified, modified, or labeled at its 5 'end or 3' end, and is preferably a biotin or amine thiol group. Cy3, Cy5, fluorescein, or oligonucleotides bound to radioactive material.
  • the present invention provides a composition for detecting vibrio fishery (7Zv / o fischeri) live bacteria comprising the DNA aptamer as an active ingredient.
  • the present invention provides a kit for detecting vibrio fishery (/ b / o fischeri) bacteria comprising the DNA aptamer as an active ingredient.
  • the kit of the present invention is in the form of a chip in which the DNA aptamer is immobilized on the chip.
  • the kit of the present invention is in the form of a micro array in which DNA aptamer is immobilized on a chip.
  • the present invention provides a method for detecting vibrio fishery (/ br / o fischeri) microorganisms comprising the following steps: (a) Vibrio fishery (/ / ⁇ fischeri) Contacting said DNA aptamer with a sample expected to contain; And (b) identifying vibrio fisher (/ r / o fischeri) viable bacteria associated with the DNA aptamer.
  • the invention (i) the DNA aptamer; And (ii) provides a 3 ⁇ 4 tammer-antimicrobial complex comprising an antimicrobial conjugated to the DNA aptamer through a linker (linker).
  • the present invention comprises contacting the aptamer-antimicrobial complex with a sample expected to contain vibrio fisher (76 / o fischeri) microorganisms (/ / o fischeri) ⁇ Provides a way to inhibit growth.
  • a sample expected to contain vibrio fisher (76 / o fischeri) microorganisms (/ / o fischeri) ⁇ Provides a way to inhibit growth.
  • the present invention will be described in more detail.
  • the present invention relates to DNA aptamers that bind to vibrio fischerium vischeri) viable bacteria with high specificity.
  • DNA aptamer refers to a DNA nucleic acid molecule capable of binding with high affinity and specificity to a particular target molecule.
  • DNA aptamer is substantially equivalent to "DNA oligonucleotide” Commonly used as an equivalent meaning.
  • oligonucleotide generally refers to a nucleotide polymer having less than about 200 lengths, which may include DNA and RNA, and is preferably a DNA nucleic acid molecule.
  • Nucleotides can be any substrate that can be introduced into the polymer by deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs or DNA or RNA polymerases thereof or by synthetic reactions. If modifications to the nucleotide structure are present, such modifications may be added before or after the synthesis of the ligonucleotide polymer.
  • Nucleotide sequences can be interrupted by non-nucleotide components.
  • the oligonucleotide may be labeled with a labeling substance such as a fluorescent substance after synthesis, and may be chemically modified or modified to improve stability.
  • a biotin, an amine group, a thi group, a radioactive substance, a fluorescent substance such as Cy3, Cy5, and fluorescein may be bound or introduced at the 5 ' end or 3 ' end.
  • hydrogen located at the sugar carbon number 2 of the nucleotides can be substituted with a fluorine atom (-F), an amino group (-N3 ⁇ 4), or a mesogroup (-0CH 3 ).
  • the DNA aptamers of the invention can typically be obtained by in vitro selection methods for binding of target molecules.
  • aptamers that specifically bind to a target molecule are known in the art.
  • organic molecules, nucleotides, amino acids, polypeptides, marker molecules on the cell surface, ions, metals, salts, and polysaccharides can be suitable target molecules that separate aptamers that can specifically bind to each ligand. .
  • Exponential Enrichment methods In vivo or in vitro selection techniques known as Exponential Enrichment methods can be used (Ellington et al., Nature 346, 818-22, 1990; and Tuerk et al. Science 249, 505-10, 1990).
  • SELEX method refers to a method of determining the DNA binding sequence of a molecule by selecting and amplifying a DNA having a high binding capacity to a specific molecule in a collection of randomly synthesized DNA (Louis et al. 1992). Nature 355, 564- 566). Specific methods for the selection and preparation of DNA aptamers are described in US Pat. Nos. 5,582,981, W0 00/20040, US Pat. No.
  • the DNA aptamer of the present invention is preferably an oligonucleotide having a nucleotide sequence disclosed in any one of SEQ ID NOs: 1 to 13.
  • the DNA aptamer having a nucleotide sequence disclosed in any one of SEQ ID NOs: 1 to 13 of the present invention is assumed to form a specific secondary structure.
  • the DNA aptamer of the present invention is an oligonucleotide having a nucleotide sequence showing substantial identity with any one of the nucleotide sequences disclosed in SEQ ID NOS: 1 to 13, while maintaining the property of specifically binding to Vibrio Fisher's live bacteria surface It is to be interpreted as including.
  • Such substantial identity aligns the nucleotide sequence of the present invention with any other sequence as described above to the fullest extent, and includes algorithms commonly used in the art (Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Li man, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988) Higgins and Sharp, CAB I OS 5: 151-3 (1989); Corpet et al., Nuc.Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Ap l. BioSci.
  • DNA aptamers specifically binding to the vibrio fishery viable surface of the present invention are selected by the Cell-SELEX method.
  • the DNA aptamer of the present invention is selected through the following steps: (i) amplifying the DNA aptamer through the PCR (Polymerase Chain Reaction) technique, and producing the ssDNA aptamer step; (ii) incubating and washing Vibrio fischeri; (iii) screening DNA aptamers that specifically bind to Vibrio Fisher's live bacteria surface via Live Cell SELEX using Vibrio Fisher's live bacteria;
  • Asymmetric PCR is performed to amplify only ssDNA in the amplified Landon dsDNA library.
  • Asymmetric PCR is a method of obtaining ssDNA by performing a PCR using a forward primer and a reverse primer in a ratio of 10: 1, for example, 10 ⁇ of the forward primer and 1 reverse of the reverse primer at the same concentration (25 ⁇ M).
  • the method for screening ssDNA aptamer for example, amplifies dsDNA by attaching biotin to a reverse primer during PCR, and forms a biotin-streptavidin complex by treating the amplified product with streptavidin.
  • the prepared ssDNA aptamer is denatured by heating the ssDNA for use in the SELEX method and then cooled slowly at room temperature to form a three-dimensional structure.
  • Vibrio Fisher is incubated to select DNA aptamers that bind to viable surface of Vibrio Fisher.
  • Vibrio Fishery has for example LBS medium [Tryptone 1% (w / v), Yeast extract 0.5% (w / v), NaCl 2% (w / v), Agar 1.5% (w / v ) And 20 mM Tris-HCl (H 7.5).
  • This step may include a negative SEVE step of removing ssDNA that binds to other bacteria, for example, E. coli, which is Gram-negative, and Bacillus, which is Gram-positive.
  • a method of quantifying ssDNA concentration using eluted ssDNA * nano-drops can be used to select an optimal SELEX round where the DNA aptamer specifically binding to the vibrio fishery viable surface is maximally eluted. have.
  • each of the 3 ⁇ 4 timer sequences obtained in the optimal round as in the optimal SELEX round selection After obtaining all of the ssDNA of the same concentration and proceed with SELEX and then the nano-drop eluted 3 ⁇ 4 tammer concentration was measured by the nanodrop method to select the optimal Vibrio Fisher surface-bound DNA aptamer It was. Detection method and growth inhibition method of vibrio fishery viable bacteria using aptamer binding to vibrio fishery viable surface
  • the present invention relates to a composition for detecting vibrio fishery (/ Zv / o fischeri) live bacteria comprising the selected DNA aptamer as an active ingredient.
  • a composition for detecting vibrio fishery (/ Zv / o fischeri) live bacteria comprising the selected DNA aptamer as an active ingredient.
  • the DNA aptamer of the present invention specifically binds to the vibrio fishery viable surface, it is useful for detecting the presence of live vibrio fishery bacteria in a sample.
  • the present invention relates to a kit for detecting vibrio fishery (/ v o fischeri) live bacteria comprising the selected DNA aptamer as an active ingredient.
  • the kit may be in the form of a chip in which the DNA aptamer is immobilized on a chip, or in the form of a microarray in which the DNA aptamer is immobilized on a substrate.
  • the immobilization of DNA aptamers on chips or substrates can employ methods known in the art.
  • the chip or substrate is modified by introducing a streptavidin (st rept avi din), and the 5 ' end of the DNA aptamer is biotinylated (biot inyl at i on) , With DNA aptamer biotin Immobilization is performed using binding with strapavidin introduced on a chip or substrate.
  • streptavidin streptavidin
  • biotinylated biot inyl at i on
  • microarray substrate means a support having suitable rigidity or semi-rigidity, such as glass, membrane, slide, filter, chip, wafer, fiber, magnetic beads or nonmagnetic beads, Gels, flows, plates, polymers, microparticles and capillaries.
  • the DNA aptamer of the invention is arranged and immobilized on the substrate. This immobilization is carried out by chemical bonding methods or by covalent binding methods such as UV.
  • DNA oligonucleotides can be bound to glass surfaces modified to include epoxy compounds or aldehyde groups, and can also be bound by UV at the polylysine coating surface.
  • the DNA oligonucleotide may be bound to the substrate via a linker (eg, ethylene glycol oligomer and diamine).
  • the DNA aptamas of the invention can be biotinyled, for example, which can be successfully bound onto a strapavidin coated substrate.
  • the DNA aptamer of the present invention immobilized on a substrate can bind to and capture Vibrio Fisher's live bacteria, and the captured Vibrio Fisher's live bacteria is again using a DNA aptamer that specifically binds to Vibrio Fisher's live bacteria. Visualize the presence or absence of capture.
  • Kits of the present invention may further comprise instructions or label (l abel) materials for use in detecting Vibrio Fisher's live bacteria in a sample.
  • the present invention comprises contacting a sample that is expected to contain vibrio fisher (/ br / o fischeri) bacteria with the DNA aptamer described above, and vibrio fisher (/ ⁇ / o fischeri) bacteria combined with the DNA aptamer. It relates to a method for detecting vibrio fishery (/ vo fischeri) live bacteria comprising the step of confirming.
  • the DNA aptamer of the present invention specifically binds to the surface of the Babrio Fisher's live bacteria, the DNA aptamer may be usefully used for detecting the Vibrio Fisher's live bacteria by contacting a sample that is expected to contain the Vibrio Fisher's live bacteria. have. Detection of the Vibrio fishery bound to the DNA aptamer is DNA It can be performed based on a method for detecting an aptamer and a Vibrio Fishery binding complex. In order to facilitate detection of the complex, DNA aptamers may be selected from fluorescent materials, such as fluorescein Cy3 or Cy5; Radioactive substances or chemicals, for example nucleotides labeled with biotin or modified with primary amines.
  • the present invention provides a kit comprising (i) the DNA aptamer described above; And (ii) relates to a DNA aptamer-antimicrobial complex comprising an antimicrobial conjugated to the DNA aptamer via a linker.
  • the present invention is to contact the DNA aptamer-antimicrobial complex with a sample that is expected to contain a vibrio fisher ( ⁇ ⁇ ⁇ / ⁇ fischeri) viable bacteria growth (/? R / o fischeri) ⁇ ⁇ It is about how to suppress.
  • the DNA aptamer-antimicrobial complex was prepared by conjugating an antimicrobial substance to the DNA aptamer of the present invention and contacting the complex with a sample expected to contain vibrio fishery (/ r / (? Fischeri) probiotics, By increasing the number of contacts between the complex and the Vibrio Fisher bacteria, it is possible to effectively suppress the growth of the Vibrio Fisher.
  • the conjugate between the DNA aptamer and the antimicrobial agent may be performed through a suitable method known in the art, and preferably, a linker may be used.
  • the linker is for example SPDP ((N-succinimidyl 3- (2-pyridyldithio) propionate)), S-Hynic (succinimidyl-6-hydrazino- nicotinamide), S ⁇ 4FB (N-succinimidyl-4-formylbenzamide) 3 ⁇ 4 - various modifications such as an amine group, a biotin group of the end-alone or be combined to use the least common and, when the antibacterial substance to be N- terminal binding protein DNA and protein aptamers of 3'-end or 5 ' It can be used to conjugate the antimicrobial material and DNA aptamer, but is not limited thereto.
  • the antimicrobial agent that can be used in the present invention may use a material that exhibits antimicrobial activity against Vibrio Fishery bacteria.
  • an inorganic antimicrobial agent derived from an inorganic compound an organic antimicrobial agent derived from an organic compound, nano silver, and a nanocatalyst may be used.
  • nanocatalyst an inorganic antimicrobial agent derived from an inorganic compound
  • organic antimicrobial agent derived from an organic compound nano silver
  • a nanocatalyst nanocatalyst
  • bioceramics bioceramics
  • metal salts with ion emitters effect natural antibacterial substances
  • propolis Animal-derived antimicrobial substances such as lactoferrin, lysozyme, chitosan, and microorganisms derived from microorganisms such as niacin and polylysine.
  • the DNA aptamer may bind with high specificity to the surface of Vibrio io scher i viable bacteria.
  • DNA aptamer-antimicrobial complex conjugated with an antimicrobial substance to the DNA aptamer of the present invention can inhibit the growth of Vibrio fishery bacteria, and can also effectively inhibit the biofilm formed by the bacteria. have.
  • Figure 1 shows a result of amplifying random DNA aptamer using a PCR technique, selectively recovering only ssDNA using streptavidin agarose resin and amplification by PCR only.
  • Lane 1 lOObp DNA s i ze marker
  • Lane 2 amplification of the DNA aptamer using the PCR technique
  • Lane 3 After amplification by PCR, ssDNA was recovered using streptavidin agarose resin.
  • Figure 2 quantitatively using the nano-drop the concentration of the eluate of the Vibrio Fisher surface ' binding DNA aptamer group recovered in each round of the SELEX process for the production of the Vibrio Fisher surface-bound DNA aptamer It is a result of a measurement.
  • Figure 3 shows the primary (panel (a)), secondary (panel (a) for each of the Vibrio Fisher surface-binding DNA aptamer candidates obtained in the selected round after the SELEX procedure for the production of the Vibrio Fisher surface-binding DNA aptamer b)) Eluent was measured quantitatively using nano-drop.
  • Figure 4 shows the expected secondary structure of the Vibrio Fisher surface binding DNA aptamer WCA-03 using the m-fold program.
  • FIG. 5 shows a process of immobilizing Vibrio Fisher surface binding DNA aptamer on the surface of a Streptavidin-coated Sensor chip SA and binding Vibrio Fisher's live bacteria. .
  • Figure 6 is fixed to the surface of the Streptavidin-coated sensor chip SA (GE healthcare, USA) Vibrio Fisher surface binding DNA aptamer and Shigella sonei, Listeria monocyto, a bacterium other than Vibrio fishery Genes, Escherichia coli, Vibrio parahaemoriticus is a graph showing the specificity of the Vibrio Fishery surface binding DNA aptamer.
  • FIG. 7 is a schematic view of a rapid kit (Rapid ki t) made in order to confirm in the Vibrio Fisher Lee surface combined with a DNA aptamer presence of Vibrio in the sample, whether or not the visually quickly.
  • the sample pad is transferred to the sample pad, and the vibrio fisher and the DNA aptamer in the sample are combined and the other aptamer and vibrio fisher are fixed in the test line. The line will appear. This can confirm the presence of Vibrio Fisher on the sample.
  • FIG. 8 shows that after the Vibrio Fisher surface-binding DNA aptamer is combined with the antimicrobial lactoferrin, the addition of lactoferrin and aptamer conjugated to lactoferrin and aptamer in the bacterial culture may increase the chance of contact between the target strain and lactoferrin. It is a schematic that shows that there is.
  • the site of -ATACCAGCTTATTCAATT and AGATAGTMGTGCMTCT-3 were constructed to fabricate the Vibrio fishery surface binding DNA aptamer specifically binding to Vibrio fisheri.
  • the dA 40 contiguous random oligonucleotide: dG: dC: dT 1.5 : 1.15: 1.25: sequence having a rate of 1 [5 '-ATACCAGCTTATTCAATT-N40- AGATAGTAAGTGCAATCT-3' ( SEQ ID NO: 14)] and the DNA library the Bioneer bio T a reverse primer annealing to recover the forward primer and the single-stranded DNA that can be amplified (Bioneer, Korea) was produced in order to [forward primer to: 5 '-ATACCAGCTTATTCAATT-3' (SEQ ID NO: 15), the biotinylated (biot inylated) reverse primer: 5'-biotin -AGATTGCACTTACTATCT-3 '(SEQ ID NO: 16)].
  • PCR reactions for the amplification of the 76 bp DNA library include 10X PCR buffer 5 ⁇ , 2.5 mM dNTP mixtures 4 /, 10 M forward primer 2 ⁇ , biotinylated reverse primer 2 ⁇ , template DNA library 1-2 ⁇ , Ex Taq polymerase (TaKaRa, Japan) 0.S ⁇ (1 unit / ⁇ ) and distilled water 34.7-35.7 ⁇ .
  • PCR banung condition First after at 94 ° C 5 bungan modified, and then repeat 20 cycles for 30 seconds banung in 30 sec at 94 ° C, 30 sec at 52 ° C, and 72 ° C, at 72 ° C 5 A reaction was used to stretch further for a minute. After PCR reaction, 3 ⁇ was taken to confirm that the band appeared at the correct size of 76 bp using 2% agarose gel. Confirmed DNA was recovered from the DNA aptamer pool using the PCR ' purification kit (Qiagen, USA).
  • the one described as 5 biotinylated The dsDNA aptamer pool 200 to boil after rapidly cooling on ice for 5 minutes at 85 ° C at the terminal, the bio-T at the terminal biotinylated dsDNA aptamer pool to ssDNA aptamers 5 to manufacture pool " I was. Thereafter, streptavidin agarose resin (Streptavidin agarose resin) was added to 100 ⁇ , which is 1/2 times the dsDNA aptamer pool, to induce reaction with streptavidin at room temperature for 1 hour or more.
  • centrifugation (4 ° C, 13,000rpm) was performed for 20 minutes to precipitate the DNA, and after removing the upper layer, dried in an 85 ° C heating block (heat block) and distilled water was added to secure the ssDNA aptamer pool.
  • 0.5X TBE Tris-borate-EDTA
  • acrylamide gel is composed of 3.75 ml of 40% acrylamide, 0.75 ml of 10X TBE complete solution, and 15 ml of distilled water, followed by 135 ⁇ of 10% APS (Ammonium per sulfate) solution.
  • TEMED Tetramethylethylenediamine 10.5 ⁇ .
  • the acrylamide gel was mixed with ssDNA aptamer pool and 2 ⁇ of DNA loading dye, and a 100 bp DNA marker was used as a ladder marker.
  • the prepared acrylamide gel for ssDNA aptamer pool identification for PAGE (Poly-acrylamide Gel Electrophoresis) was electrophoresed for 45 minutes at 150 V (volt) of a power supply (Major science, USA). Electrophoretic acrylamide gel was dyed in ETBR solution for 5 minutes and then irradiated with UV light using Gel-doc (Bio-rad, USA). Observation was made (see FIG. 1).
  • the pool of ssDNA aptamer identified by PAGE in Example 1-4 is preheated to 85 ° C after adding 100 s of the ssDNA aptamer pool prepared in advance and the same amount of 2X LBS medium to form its own three-dimensional structure
  • the heating block was heated for 5 minutes to induce denaturation of the ssDNA aptamer pool.
  • the denatured ssDNA cooled slowly over 2 hours at room temperature to induce its own three-dimensional structure formation.
  • Example 2 Preparation of Live-Cells for Fabrication of Vibrio Fisheri ( ⁇ % " / 0 fischeri) Probiotic Binding DNA Aptamers
  • vibrio fishery was obtained through culture.
  • LBS 1% tryptone, 0.5% yeast extract and 2% NaCl in 10 mM Tris-HC1
  • the cultured Vibrio fishery was washed using a washing solution of Tris base 10 mmol / L NaCl 0.85%, pH 8.0.
  • the cultured Vibrio fishery was centrifuged (4 ° C, 13,000 rpm) for 10 minutes to precipitate the Vibrio fishery and the supernatant was removed.
  • washing solution 200 was added to wash the surface of the Vibrio Fischer through pipetting and centrifugation (4 ° C, 13,000 rpm) was performed for 10 minutes to precipitate the Vibrio Fischer and remove the upper layer. The above procedure was repeated three times to wash the Vibrio fishery surface.
  • LB broth was used as a culture medium for Escherichia coli, a strain of negative selection. Inoculated with 5 ml LB broth and incubated for 12 hours at 37 ° C. Thereafter, the cultured Escherichia coli was precipitated by centrifugation (4 ° C., 13,000 rpm) for 10 minutes, and the upper layer was removed. Thereafter, 200 ⁇ of the washing solution was added to wash the surface of E. coli through pipetting, and centrifuged (4 ° C, 13,000 rpm) for 10 minutes to settle and remove the upper layer. The process was repeated three times to wash the E. coli surface. 2-2-2.
  • wash solution 200 ⁇ The surface of the Bacillus subtilis was washed by pipetting, precipitated by centrifugation (4 ° C., 13,000 rpm) for 10 minutes, and the upper layer was removed. The above procedure was repeated three times to wash the Bacillus subtilis surface. It was.
  • NA broth was used as a culture medium of Listeria monocytogenes, a strain for measuring the specificity of aptamer when measuring SPR.
  • Subsequently centrifugation of the cultured Listeria monocytogenes (4V, 13,000 rpm) was performed for 10 minutes to precipitate and the upper layer was removed.
  • washing solution 200 was added to wash the surface of Listeria monocytogenes through pipetting, and centrifugation (4 ° C, 13,000 rpm) was performed for 10 minutes to precipitate and the upper layer was removed. The above procedure was repeated three times to wash the Listeria monocytogenes surface.
  • NA broth containing 3% NaCl was used as a culture medium for Vibrio parahaemolyticus, a strain for confirming nonspecificity of aptamers when measuring SP.
  • 5 ml of NA broth containing 3% NaCl was inoculated with Vibrio parahaemolyticus and incubated at 37 ° C for 14 hours. Thereafter, the cultured Vibrio parahaemoriticus was precipitated by centrifugation (4 ° C, 13,000 rpm) for 10 minutes, and the upper layer was removed.
  • Example 3 Construction of a Vibrio Fisher surface binding aptamer specifically binding to a Vibrio Fisher
  • Negative selection was performed to remove non-specifically binding DNA aptamers after the sixth round of selection.
  • E. coli and Bacillus subtilis live bacteria were secured by the method described in Examples 1 and 2, and ssDNA aptamer pool 200 ⁇ , which had its own three-dimensional structure formed in E. coli, was added to the thermo mixer (Eppendor f, USA) 4 ° The reaction was carried out at 500 rpm for 1 hour. Thereafter, the leucine was taken out, and centrifugation (4 ° C., 13, 000 rpm) was performed for 10 minutes to precipitate E. coli bound with a nonspecific ssDNA aptamer pool, and supernatant was obtained.
  • the obtained supernatant was placed in the prepared Bacillus subtilis and reacted for 1 hour at a Thermo mixer (Eppendor f, USA) 4 ° C, 500 rpm. E. coli and Bacillus subtilis bound to the non-target strain were removed during the supernatant recovery.
  • SsDNA was prepared using the eluted DNA aptamer pool of each round using the method described above, and then the ssDNA aptamer pool 50 ⁇ of each round was obtained in the same manner as described above.
  • Into the precipitate of a Vibrio Fishery was reacted for 1 hour at 4 ° C, 500 rpm Thermo mixer (Eppendorf, USA). After that, remove the tube and centrifuge (4 ° C, 13, 000 rpm) for 10 minutes to precipitate the Vibrio fishery with the structure-forming ssDNA aptamer pool, remove the upper layer, and pipette with 200 wash solution.
  • Centrifugation (4 ° C, 13, 000 rpm) was performed for 10 minutes to remove the upper layer and washed three times, and the eluent was added to 100 ⁇ and heated at 85 ° C for 5 minutes, then centrifuged (4 ° C) , 13, 000 rpm) was performed for 10 minutes to obtain each round of eluted DNA aptamer.
  • the reaction composition for the amplification of the DNA aptamer pool was 10X PCR complete solution 5 each 2.5 mM dNTP mixture 4 ⁇ , 10 ⁇ forward primer 2 ⁇ , reverse primer ⁇ template DNA library 1-2 ⁇ , Ex Taq polymerization Enzyme (TaKaRa, Japan) 0.3 / ⁇ (lunit / ⁇ ) and distilled water 34.7-35.7 ⁇ .
  • PCR banung terms first, after denaturing at 94 ° C 5 bungan, 94 ° C 30 seconds, 52 ° 30 sec at C, and then repeated cycles of 20 for 30 seconds banung at 72 ° C, 5 minutes at 72 ° C Further elongation was used.
  • the amplified DNA aptamer was subjected to ligation for 10 minutes at 25 ° C with a composition of T-blnt vector 1 6X cloning solution 1 ⁇ , PC product 4 ⁇ . After the test, 6 ⁇ was added to 100 ⁇ of competent cells (E. col / DH5a) and reacted in ice for 20 minutes. Subsequently, the cells were transformed by thermal stratification at 42 ° C. for 30 seconds, and ampicillin (50) was used.
  • T-vector cloning kit Solgent, Korea
  • the culture was carried out by spreading on an LB culture plate (LB plate) containing kanamycin (kanamycin, 50 / ig / n), X-gal (50 ug / mi), IPTG (5 / g / m £). Thirty-one colonies of the colonies grown in the medium were selected and inoculated into 5 ml of LB broth to which ampicillin and kanamycin were added, and plasmids were extracted using a plasmid DNA prep kit (Intron, USA). Plasmid DNA for each colony obtained was commissioned by Solgent (Solgent, Korea).
  • the DNA aptamer sequences inserted through cloningol were identified and sequenced in the sequencing for each colony and analyzed using the Clustal-X program. A group for each sequence was formed to analyze sequence similarity between each sequence and to obtain 13 sequences grouped into 6 including 7 identical sequences (see Table 1 below).
  • each of the obtained Vibrio Fisher-specific binding DNA aptamer candidate groups was measured by Nano-drop to confirm the DNA concentration.
  • Candidates 2 and 3 which were the highest concentrations of DNA aptamer candidates at the time of the second and second elution, were selected (see FIG. 3).
  • Example 7 Securing the expected structural schematic of selected DNA aptamers
  • PCR was performed using the method described in Example 1, but using a sample in which 10 pM biotinylated forward primer and 10 pM reverse primer were added instead of 10 pM biotinylated reverse primer and 10 pM forward primer. . After the process was carried out according to Example 1 to perform PCR purification. The secured PCR sample was used for asymmetric PCR to prepare the ssDNA.
  • the reaction composition of the asymmetric PCR was 10 ⁇ PCR complete solution 10 ⁇ , 8 ⁇ , each 10 mM biotinylated 2.5 mM dNTP mixture Forward primer 10 10 M reverse primer 1, template DNA library 10 ⁇ , Ex Taq polymerase (TaKaRa, Japan) 0.5 ⁇ (1 uni t / ⁇ ) and distilled water 60.5 ⁇ .
  • PCR banung terms first, after denaturing at 94 ° C 5 minutes, 94 ° C 30 seconds, 52 ° 30 sec at C, and then repeat 15 for 30 seconds banung at 72 ° C cycle, for 5 minutes at 72 ° C Further elongation was used. Thereafter, the pure DNA aptamer was purified and secured using the PCI treatment and the ethanol precipitation method commonly used in the art.
  • the obtained ssDNA was added with 1 ⁇ in the nano-drop and the concentration of ssDNA was measured, followed by heating at 85 ° C for 5 minutes by adding 99 ⁇ 2X LBS to the remaining 99 ⁇ ssDNA aptamer. It was slowly observed over time. Thereafter, the concentration of DNA was 25 ⁇ M and diluted in $ 1 to IX LBS. 8-3.
  • Example 9 Screening of Optimal DNA Aptamers by SPR Measurement
  • Streptavidin immobilized with Textran on gold valves was activated by the method presented in the protocol provided by GE heal thcare.
  • the flow rate of the sample was set to 10 / min to fix the DNA aptamer on the surface of the activated sensor chip SA.
  • the Sensor chip SA no one was attached to the aptamer for testing, and each of the selected aptamers was coupled to only two, three and four channels.
  • As a binding condition of the DNA aptamer a series of inoculation steps of 1 minute at a flow rate of 10 z ⁇ / min was repeated three times. After that, the ssDNA bound by flowing HBS-EP buf fer at a flow rate of 10 / min for 10 minutes.
  • the aptamers were allowed to stabilize.
  • Transferring the culture medium 1 incubated with the prepared Vibrio Fishery was carried out twice a series of procedures to be carried out for 20 minutes at a flow rate of 5 / min on the sensor chip (Sensor chip) SA. During this process, the binding force was confirmed by comparing and analyzing the sensograms of channels 1, 2, 3, and 4. Thereafter, a series of processes of flowing 50 mM NaOH at a flow rate of 10 / min for 5 minutes was repeated twice to remove bound Vibrio fisheries and ssDNA aptamers, and then ssDNA aptamers were bound three times in the same manner as above. (See Figure 5). In the process, the final VPCA-03 was selected as the optimal Vibrio Fisher's live bacterial binding DNA aptamer (see Table 2).
  • Vibrio parahaemoriaticus was cultured for binding to Vibrio Fishery. Incubation of Vibrio parahaemoriticus was performed according to the method described in Example 2 above. Thereafter, 1 m of the culture solution was transferred to a tube, and a series of processes of flowing 20 minutes at a flow rate of 5 / min on a sensor chip SA was repeated twice. Thereafter, a series of two-time procedures of blowing 50 mM NaOH at a flow rate of 10 / min for 5 minutes was repeated twice to remove the combined Vibrio parahaemoriticus and ssDNA aptamer, followed by ssDNA aptamer 3 in the same manner as described above. Combined twice.
  • Shigella soney were incubated for binding to Vibrio Fishery.
  • the culture method of Shigella sonei was performed according to the method described in Example 2 above. Thereafter, the culture solution 1 was transferred to the lyosphere, and then a series of procedures of flowing 20 minutes at a flow rate of 5 / min on a sensor chip SA was repeated twice. Thereafter, a series of two-time flows of 50 mM NaOH at a flow rate of 10 ⁇ / min was repeated twice to remove the combined Shigella sonei and ssDNA aptamers, followed by three ssDNA aptamers in the same manner as above. Combined. 10-4. Affinity Measurement of Escherichia Coli
  • Escherichia coli was cultured for comparison of the binding force with Vibrio Fishery. Incubation of E. coli was performed according to the method described in Example 2 above. Thereafter, the culture solution 1 ⁇ was transferred to a tube, and then a series of procedures were repeated twice for 20 minutes at a flow rate of 5 / / min on a sensor chip (Sensor chi p) SA. Thereafter, a series of two-time procedures of blowing 50 mM NaOH at a flow rate of 10 £ / min for 5 minutes was repeated twice to remove the bound E. coli and ssDNA aptamer, and then ssDNA aptamer was bound three times in the same manner as above. See Table 3).
  • Vibrio Fisher in various samples to confirm the detection method and the possibility of using Vibrio Fisher's live bacteria specific binding DNA aptamer for industrial use of Vibrio Fisher's live bacteria specific binding DNA aptamer prepared and obtained through the above examples.
  • a flat kit of rapid kit was developed to confirm the detection capability of li. Rapid kits can utilize streptavidin-biotin binding, and a brief schematic diagram of the construction of the rapid kit is shown in FIG. 7.
  • the amine group is substituted at the 5'-end of the Vibrio Fisher's live bacteria specific binding DNA aptamer, and 10 nm carboxylated gold nanoparticles (Carboxylated gold
  • NHS / EDC Amin coupling kit
  • Vibrio Fisher's probiotic specific binding DNA aptamer immobilized on the nanoparticles prepared in this process was mixed with a sample containing Vibrio Fischer and treated at 251 for 1 hour, followed by 10X loading buf fer (2.5 % Tri ton X-100, 500 mM Tr i s-HCl, 10% Tween 20, 1.5 M NaCl) was mixed to IX and then loaded into the sample pad for treatment.
  • 10X loading buf fer 2.5 % Tri ton X-100, 500 mM Tr i s-HCl, 10% Tween 20, 1.5 M NaCl
  • Vibrio Fisher is present in the sample, it will appear as two lines (see Fig. 7 (b)). If the sample is not added to confirm the capability of the produced Vibrio Fisher's live bacteria detection kit, a sample without Vibrio Fischer and a sample with Vibrio Fischer may be prepared to check the color of the control line and the test line. have. It was designed to confirm that the Vibrio Fishery detection kit produced through the above experiment was made normally.
  • Example 12 Confirmation of Vibrio Fishery Control and Inhibition of Biofilm Formation by Conjugation of Antimicrobial Lactoferrinol to Optimal Vibrio Fisher Probiotic Binding DNA Aptamer
  • a DNA aptamer introduced with an amine group was synthesized to conjugate lactoferrin with a DNA aptamer specifically binding to Vibrio fisheries.
  • the synthesized DNA aptamer was conjugated by activating both ends using a biolinker SPDP reagent -3110 ( ⁇ 1 ⁇ 0 ⁇ 1 3- (2-pyr idyldithio) propionate, Thermo scient if ic). .
  • SPDP conjugation was performed according to the method presented in the protocol provided by the manufacturer (Thermo scient i f i c). The concentration of lactoferrin was fixed at 10 rag / ⁇ and the concentration could be adjusted according to the target strain.
  • Vibrio Fisher's live cells were incubated according to the method described in Example 2.
  • the cultured bacteria were re-inoculated into three 100 ml LBS medium and growth inhibition was compared by adding the negative control (Negat ive control), lactoferrin and lactoferrin conjugated with aptamer, respectively. It was confirmed.
  • the cultures were continuously incubated for 24 hours in a 30 ° C stirred incubator, and the cultures were inhibited by growth curve by measuring OD (Optical Densi ty) every hour using the Bradford assay commonly used in the industry. You can check.
  • Example 12-2 O measured by time. It can be seen that the growth curve of Vibrio fishery is suppressed by deriving a growth curve using D.
  • the growth of vibrio fisheries was most effectively inhibited with the addition of lactoferrin conjugated with aptamer, followed by lactoferrin and negative control (negat ive control).
  • lactoferrin conjugated with aptamer followed by lactoferrin and negative control (negat ive control).
  • Vibrio Fisheries were inoculated in 5 ml LBS medium (medi um) and incubated overnight at 30 ° C., then distilled to a 0D 600 value of 0.1 and dispensed 200 ⁇ into 96 well-plates. After intensive incubation for at least 24 hours, the supernatant was removed and the wells were washed 2-3 times using lx PBS. Thereafter, staining was performed on biofilm and cell extracts by staining for 20 minutes using 1.0% crystal vial 200 ⁇ . The stained wells were washed layered 2-3 times with lx PBS and dried to remove cell extracts except for the biofilm attached to the well surface.
  • the binding DNA aptamer inhibits the growth of the Vibrio fishery to control the biofilm, various potentials for inhibition of biofilm formation were confirmed.
  • the limitation of biofilm in various environments can be made by utilizing the power of various samples.

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Abstract

La présente invention concerne un aptamère d'ADN qui se lie spécifiquement à la surface de cellules vivantes de Vibrio fischeri et une utilisation de celui-ci. L'aptamère d'ADN de la présente invention peut se lier, avec une spécificité élevée, à la surface de cellules vivantes de Vibrio fischeri. La présence de cellules vivantes de Vibrio fischeri dans un échantillon de spécimen peut être détectée et vérifiée au moyen de l'aptamère d'ADN de la présente invention. L'utilisation d'un complexe antibiotique-aptamère ADN dans lequel un antibiotique est conjugué à l'aptamère d'ADN de la présente invention peut inhiber la croissance de Vibrio fischeri ; un biofilm formé par cette bactérie peut également être efficacement inhibé.
PCT/KR2015/011328 2014-11-06 2015-10-26 Aptamère d'adn se liant spécifiquement à la surface de cellules vivantes de vibrio fischeri et son utilisation WO2016072653A1 (fr)

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