WO2016072653A1 - Dna aptamer specifically binding to surface of live cells of vibrio fischeri, and use thereof - Google Patents

Dna aptamer specifically binding to surface of live cells of vibrio fischeri, and use thereof 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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French (fr)
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.

Abstract

The present invention relates to a DNA aptamer specifically binding to the surface of live cells of Vibrio fischeri, and a use thereof. The DNA aptamer of the present invention can bind, with high specificity, to the surface of live cells of Vibrio fischeri. Whether live cells of Vibrio fischeri are present in a specimen sample can be detected and checked when using the DNA aptamer of the present invention. When using a DNA aptamer-antibiotic complex in which an antibiotic is conjugated to the DNA aptamer of the present invention, growth of Vibrio fischeri can inhibited and a biofilm formed by this bacterium can also be effectively inhibited.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
비브리오 피셔리 생균의 표면에 특이적으로 결합하는 DNA 앱타머 및 이의 용도  DNA aptamers that specifically bind to the surface of vibrio fishery bacteria and their use
【기술 분야】 [Technical field]
본 발명은 비브리오 피셔리 생균의 표면에 특이적으로 결합하는 DNA 앱타머 및 이의 용도에 관한 것이다. 【배경 기술】  The present invention relates to a DNA aptamer specifically binding to the surface of vibrio fishery bacteria and its use. [Background technology]
비브리오 피셔리 (Vibr io f ischer i )는 비브리오 (Vibrio) 속 중에서 심해 오징어의 발광기관에 붙어 생장하는 균주로 통성 혐기성 그람 음성 간균이며 운동성이 있고 하나 이상의 편모가 있는 특징을 갖는 균이다. 비브리오 피셔리는 해양 생태계에서 서식하는 다양한 균주 중 하나이며 생존력을 증가하기 위한 전략으로 바이오필름을 형성하는데 이는 여러 스트레스에 대한 저항성이 비형성군에 비해 현저히 증가되어 생존전략에 있어서 필수적인 단계라고 인식되고 있다. 하지만 바이오필름이 형성되면 유영상태일 때에 비해 비브리오 피셔리에 대한 치료제 및 항생제의 내성이 약 500 배가 증가하게 되는 문제가 발생한다. 따라서 바이오필름 형성은 생물체의 생존전략의 수단으로 자연스런 현상일지라도 경제 산업적으로 많은 악영향을 낳는다. 바이오필름의 형성은 대표적으로 임플란트 시술에서 발생하는 부작용 원인의 80%를 차지하는 것으로 밝혀져 있다. 종래의 바이오필름의 생성을 제어하는 방법은 항생제나 방오제에 의한 세균사멸에 기초하여 시도되었으나, 내성이 증가된 균주에 대한 항생제의 무분별한 사용은 항생제 내성 균주로의 변이를 유발할 수 있어 그 사용이 매우 제한적이다. 또한, 방오제는 타 생물체에 독성을 나타내는 비선택적 생물독소이므로 방오제의 사용은 환경 생태계에 큰 피해를 안길 뿐만 아니라 이를 회복하기 위한 경제적인 피해도 심각하다. 방오제의 위해성은 1970 년대 이후 선박이나 양식어업에 가장 많이 사용된 화학 방오물질인 TBT (tr ibutyl t in)가 각종 해양생물의 암컷에 수컷 생식기가 발생하는 것과 같은 성변이 현상을 유발하는 것을 확인하면서 해양환경을 심각하게 위협하는 환경 호르몬인 것으로 지목되었다. TBT는 1 ppt 이하의 농도에서도 성변이를 유발할 수 있으며， 남해안에서도 TBT 오염에 의한 성변이 현상이 지속적으로 발견되고 있다. 이에 따라 선진국에서는 1982년부터 대형선박에 방오제로서 사용되는 TBT농도를 규제하였고 연안을 운항하는 소형선박에는 사용을 금지시켜 왔다. 국내에서는 2005 년부터 TBT 규제가 시작되었으며， 대체 물질로는 황산구리 등의 물질이 사용되고 있지만 이 또한 오염물질로 지목되어 TBT와 같은 방오제의 대체물질 개발이 필요한 상황이다. 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. . However, when 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. Therefore, 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. In addition, since 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. The risk of antifouling agents has been confirmed that TBT (tr ibutyl t in), the most commonly used chemical antifouling substance in ships and aquaculture since the 1970s, causes sexual alterations such as male genitalia in females of various marine organisms. 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. As a result, 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. In Korea, 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.
앱타머는 짧은 길이의 을리고머로 구성되어 자체적인 다양한 삼차원 구조를 형성하게 되는데 이 올리고머 구조체 라이브러리들은 다양한 표적물질들과 구조적으로 결합할 수 있는 능력이 있으며 이 중 표적물질에만 특이적인 앱타머를 선별하는 과정을 거쳐 확보하게 된다. 선별된 앱타머는 서열분석을 통해 서열을 획득하고 화학적 합성 기법을 이용하여 단시간， 저비용으로 대량 생산이 가능하며 한번 제작， 생산 후 동일한 능력을 가지는 앱타머를 지속적으로 생산 가능한 탁월한 장점을 갖고 있다. 뿐만 아니라， DNA 로 구성되어 있고 부가적인 화학적 치환 과정을 거쳐 보다 안정성을 부여할 수 있어 주변의 pH 와 은도에 매우 안정하며 바이오틴과 같은 화합물을 부착시켜 표적 물질의 탐지 및 질환 진단 센서 개발 등 환경 의료 등 다양한 분야에서 활용될 수 있어 그 가능성이 높이 평가되고 있다. 본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.  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. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
【발명의 상세한 설명】 [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
본 발명자들은 생균 상태의 비브리오 피셔리 (Vibr io f i scheri )균에 특이적으로 결합함으로써 이 균의 검출과 성장 억제 등 다양한 용도로 사용될 수 있는 DNA 앱타머 (aptamer)를 개발하기 위해 연구 노력하였다. 그 결과， 비브리오 피셔리의 생균 표면에 높은 특이도로 결합할 수 있는The 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.
DNA 앱타머를 합성 및 선별하는데 성공하였으며， 이 DNA 앱타머를 이용하여 시료 중에서 비브리오 피셔리를 쉽게 검출할 수 있는 래피드 키트 플랫품을 개발하였고， 비브리오 피셔리의 생장을 억제하여 비브리오 피셔리가 생성하는 바이오필름을 효율적으로 차단할 수 있는 가능성올 실험적으로 확인함으로써 본 발명을 완성하였다. We succeeded in synthesizing and screening DNA aptamers, and developed a kit for rapid kits that can easily detect Vibrio fisheries in samples using DNA aptamers. The present invention was completed by experimentally confirming the possibility of effectively blocking the film.
따라서， 본 발명의 목적은 비브리오 피셔리 (Vibrio fischeri) 생균 표면에 특이적으로 결합하는 DNA 앱타머를 제공하는데 있다.  Accordingly, it is an object of the present invention to provide a DNA aptamer that specifically binds to Vibrio fischeri viable surface.
본 발명의 다른 목적은 상기 DNA 앱타머를 유효성분으로 포함하는 비브리오 피셔리 (Vibrio fischeri) 생균의 검출용 조성물 및 키트를 제공하는데 있다.  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.
본 발명의 또 다른 목적은 비브리오 피셔리 (Vibrio fischeri) 생균의 검출 방법을 제공하는데 있다. 본 발명의 목적 및 장점은 하기의 발명의 상세한 설명， 청구의 범위 및 도면에 의해 보다 명확하게 된다.  Still another object of the present invention is to provide a method for detecting Vibrio fischeri bacteria. The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.
【기술적 해결방법】 Technical Solution
본 발명의 일 양태에 따르면， 본 발명은 비브리오 피셔리 fischeri) 생균 표면에 특이적으로 결합하는 DNA 앱타머를 제공한다.  According to one aspect of the present invention, the present invention provides a DNA aptamer specifically binding to the vibrio fishery fischeri) microbial surface.
본 발명의 일 구현예에 따르면， 본.발명의 DNA 앱타머는 서열번호 1 내지 13의 서열 중 어느 하나에 개시된 염기서열과 90% 이상의 동일성을 갖는 염기서열을 갖는 올리고뉴클레오타이드이다. According to one embodiment of the invention, the present . 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.
본 발명의 다른 일 구현예에 따르면, 본 발명의 DNA 앱타머는 서열번호 1 내지 13 서열 중 어느 하나에 개시된 염기서열을 갖는 을리고뉴클레오타이드이다.  According to another embodiment of the present invention, the DNA aptamer of the present invention is an oligonucleotide having a nucleotide sequence disclosed in any one of SEQ ID NOs: 1-13.
본 발명의 다른 일 구현예에 따르면ᅵ 본 발명의 DNA 앱타머는 이의 5' 말단 또는 3' 말단이 화학적으로 변형， 개질 또는 표지물질로 결합되어 있으며， 바람직하게는 바이오틴 (biotin), 아민기 티올기 Cy3, Cy5, 플루오레세인 (fulorescein), 또는 방사성 물질이 결합된 올리고뉴클레오타이드이다. 본 발명의 다른 일 양태에 따르면， 본 발명은 상기 DNA 앱타머를 유효성분으로 포함하는 비브리오 피셔리 ( 7Zv/o fischeri) 생균의 검출용 조성물을 제공한다. According to another embodiment of the present invention, 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. According to another aspect of the present invention, the present invention provides a composition for detecting vibrio fishery (7Zv / o fischeri) live bacteria comprising the DNA aptamer as an active ingredient.
본 발명의 다른 일 양태에 따르면, 본 발명은 상기 DNA 앱타머를 유효성분으로 포함하는 비브리오 피셔리 ( /b/ o fischeri) 생균의 검출용 키트를 제공한다.  According to another aspect of the present invention, the present invention provides a kit for detecting vibrio fishery (/ b / o fischeri) bacteria comprising the DNA aptamer as an active ingredient.
본 발명의 일 구현예에 따르면， 본 발명의 키트는 DNA 앱타머가 칩상에 고정화된 칩 형태이다.  According to one embodiment of the invention, the kit of the present invention is in the form of a chip in which the DNA aptamer is immobilized on the chip.
본 발명의 다른 일 구현예에 따르면 , 본 발명의 키트는 DNA 앱타머가 칩상에 고정화된 마이크로 어레이 형태이다.  According to another embodiment of the present invention, the kit of the present invention is in the form of a micro array in which DNA aptamer is immobilized on a chip.
본 발명의 다른 일 양태에 따르면, 본 발명은 다음의 단계를 포함하는 비브리오 피셔리 ( /br/o fischeri) 생균의 검출 방법을 제공한다: (a) 비브리오 피셔리 ( / / ο fischeri) 생균을 함유하는 것으로 예상되는 시료와 상기 DNA 앱타머를 접촉시키는 단계; 및 (b) 상기 DNA 앱타머와 결합된 비브리오 피셔리 ( / r/o fischeri) 생균을 확인하는 단계 .  According to another aspect of the present invention, the present invention provides a method for detecting vibrio fishery (/ br / o fischeri) microorganisms comprising the following steps: (a) Vibrio fishery (/ / ο 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.
본 발명의 다른 일 양태에 따르면, 본 발명은 (i) 상기 DNA 앱타머; 및 (ii) 상기 DNA 앱타머에 링커 (linker)를 통해 접합된 항균물질을 포함하는 ¾타머-항균물질 복합체를 제공한다.  According to another aspect of the invention, the invention (i) the DNA aptamer; And (ii) provides a ¾ tammer-antimicrobial complex comprising an antimicrobial conjugated to the DNA aptamer through a linker (linker).
본 발명의 다른 일 양태에 따르면， 본 발명은 상기 앱타머-항균물질 복합체를 비브리오 피셔리 ( 76 /o fischeri) 생균을 함유하는 것으로 예상되는 시료와 접촉시키는 단계를 포함하는 비브리오 피셔리 ( / /o fischeri)^ 생장을 억제하는 방법을 제공한다. 이하에서 본 발명을 보다 상세하게 설명한다. 비브리오 피셔리 fischeri) 생균 표면에 결합하는 DNA 앱타머 본 발명은 비브리오 피셔리 Ζν/σ fischeri) 생균의 표면에 높은 특이도로 결합하는 DNA 앱타머에 관한 것이다.  According to another aspect of the present invention, 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. Hereinafter, the present invention will be described in more detail. DNA aptamers that bind to vibrio fishery fischeri) viable surfaces The present invention relates to DNA aptamers that bind to vibrio fischerium vischeri) viable bacteria with high specificity.
본 명세서에서 용어 "DNA 앱타머 (aptamer)" 는 특정 타겟 분자에 고친화성 및 특이성을 갖고 결합할 수 있는 DNA 핵산 분자를 의미한다. 상기 용어 "DNA 앱타머" 는 "DNA 올리고뉴클레오타이드" 와 실질적으로 동등한 의미로서 흔용하여 사용될 수 있다 . As used herein, the term “DNA aptamer” refers to a DNA nucleic acid molecule capable of binding with high affinity and specificity to a particular target molecule. The term "DNA aptamer" is substantially equivalent to "DNA oligonucleotide" Commonly used as an equivalent meaning.
본 명세서에서 용어 "올리고뉴클레오타이드" 는 일반적으로 길이가 약 200개 미만을 갖는 뉴클레오타이드 중합체를 지칭하며, 이에는 DNA 및 RNA가 포함될 수 있으며， 바람직하게는 DNA 핵산 분자이다. 뉴클레오타이드는 데옥시리보뉴클레오타이드， 리보뉴클레오타이드， 변형된 뉴클레오타이드 또는 염기 및 /또는 이들의 유사체 또는 DNA 또는 RNA 폴리머라아제에 의해 또는 합성 반웅에 의해 증합체 내로 도입될 수 있는 모든 기질일 수 있다. 뉴클레오타이드 구조에 대한 변형이 존재하는 경우， 이러한 변형은 을리고뉴클레오타이드 중합체의 합성 전에 또는 후에 추가될 수 있다. 뉴클레오타이드 서열은 비-뉴클레오타이드 성분에 의해 중단될 수 있다. 올리고뉴클레오타이드는 합성 후에 형광물질과 같은 표지 (label) 물질이 결합될 수 있으며, 안정성 향상을 위해 화학적으로 변형 또는 개질될 수 있다.  As used herein, the term “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.
본 발명의 DNA 앱타머에는 예를 들어 바이오틴 (biotin), 아민기， 티을기， 방사성 물질， Cy3, Cy5, 플루오레세인과 같은 형광물질이 5' 말단 또는 3' 말단에 결합 또는 도입될 수 있다. 또한, DNA 앱타머의 안정성과 효율성 향상을 위해 뉴클레오타이드의 당 (sugar) 2번 탄소에 위치한 수소를 불소원자 (-F), 아미노기 (-N¾), 또는 메특시기 (-0CH3)로 치환할 수 있다. 본 발명의 DNA 앱타머는 전형적으로는 타겟 분자의 결합에 대한 시험관내 선택법에 의해 얻을 수 있다. 타겟 분자에 특이적으로 결합하는 앱타머를 선택하는 방법은 당분야에서 공지되어 있다. 예를 들어， 유기 분자， 뉴클레오타이드, 아미노산, 폴리펩타이드, 세포 표면상의 마커분자, 이온， 금속， 염， 다당류가 각 리간드에 특이적으로 결합할 수 있는 앱타머를 분리하는 적절한 표적 분자가 될 수 있다. In the DNA aptamer of the present invention, for example, 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. . In addition, to improve the stability and efficiency of DNA aptamers, hydrogen located at the sugar carbon number 2 of the nucleotides can be substituted with a fluorine atom (-F), an amino group (-N¾), or a mesogroup (-0CH 3 ). have. The DNA aptamers of the invention can typically be obtained by in vitro selection methods for binding of target molecules. Methods of selecting aptamers that specifically bind to a target molecule are known in the art. For example, 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. .
앱타머의 선별은 SELEX (Systematic Evolution of Ligands by Aptamers are screened by SELEX (Systematic Evolution of Ligands)
Exponential Enrichment) 방법으로 공지된 생체내 또는 시험관내 선택 기술을 이용할 수 있다 (Ellington et al. , Nature 346, 818-22, 1990； 및 Tuerk et al . Science 249, 505-10, 1990) . 본 명세서에서 용어 "SELEX 방법" 이란 임의적으로 합성된 DNA의 집합에서 특정 분자에 대해 높은 결합력을 지니는 DNA를 선별하여 증폭시킴으로써 해당 분자의 DNA 결합 서열을 알아내는 방법을 의미한다 (Louis et al. 1992. Nature 355, 564- 566). DNA 앱타머의 선별 및 제조에 대한 구체적인 방법은 미국특허 5,582,981, W0 00/20040， 미국특허 5,270,163， Lorsch and Szostak, Biochemistry, 33 :973(1994) , Mannironi et al ..Biochemistry 36:9726(1997)， Blind, Proc. Natl. Acad. Sci. USA 96:3606-3610(1999), Huizenga and Szostak, Biochemistry, 34:656-665(1995), W0 99/54506, W0 99/27133, W0 97/42317 및 미국특허 5，756，291에 기재되어 있으며， 상기 문헌들은 본 명세서에 참조로써 삽입된다. 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). As used herein, the term “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. 5,270,163, Lorsch and Szostak, Biochemistry, 33: 973 (1994), Mannironi et al. Biochemistry 36: 9726 (1997). , Blind, Proc. Natl. Acad. Sci. USA 96: 3606-3610 (1999), Huizenga and Szostak, Biochemistry, 34: 656-665 (1995), WO 99/54506, WO 99/27133, WO 97/42317 and US Pat. No. 5,756,291. And the documents are incorporated herein by reference.
본 발명의 DNA 앱타머는 바람직하게는 서열번호 1 내지 13 중 어느 하나에 개시된 염기서열을 갖는을리고뉴클레오타이드이다.  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.
한편， 본 발명의 서열번호 1 내지 13 중 어느 하나에 개시된 염기서열을 갖는 DNA 앱타머는 특정 2차 구조를 형성할 것으로 추정된다. 본 발명의 DNA 앱타머는 비브리오 피셔리 생균 표면에 특이적으로 결합하는 특성을 유지하면서， 상기 서열번호 1 내지 13에 개시된 염기서열 증 어느 하나의 염기서열과 실질적인 동일성을 나타내는 염기서열을 갖는 올리고뉴클레오타이드도 포함하는 것으로 해석된다.  On the other hand, 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.
상기의 실질적인 동일성은 상기한 본 발명의 뉴클레오타이드 서열과 임의의 다른 서열을 최대한 대웅되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘 (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. 8:155-65(1992) and Pearson et al . , Meth. Mol. Biol. 24:307- 31(1994))을 이용하쪄 얼라인된 서열을 분석한 경우에, 최소 90%의 동일성 보다 바람직하게는 최소 95%의 동일성, 가장 바람직하게는 최소 98%의 동일성을 나타내는 뉴클레오타이드 서열을 의미한다.  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. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994)), when analyzing the aligned sequences, at least 90% identity, more preferably A nucleotide sequence that exhibits 95% identity, most preferably at least 98% identity.
Live Cell SELEX 기법을 통한 비브리오 피셔리 생균 표면 결합 DNA 앱타머의 선별 Selection of Vibrio Fisher's Live Bacterial Surface Binding DNA Aptamers by Live Cell SELEX Technique
본 발명의 비브리오 피셔리 생균 표면에 특이적으로 결합하는 DNA 앱타머는 Cell-SELEX방법에 의해 선별된다. 하기 본 발명의 구체적인 일 실시예에 따르면， 본 발명의 DNA 앱타머는 다음의 단계를 통해 선별된다: ( i ) PCR(Polymerase Chain Reaction) 기법을 통해 DNA 앱타머를 증폭하고， ssDNA 앱타머를 제작하는 단계; (ii) 비브리오 피셔리 (Vibrio fischeri)를 배양하고 세척하는 단계; (iii) 비브리오 피셔리 생균을 이용한 Live Cell SELEX 기법을 통해 비브리오 피셔리 생균 표면에 특이적으로 결합하는 DNA 앱타머를 선별하는 단계 , DNA aptamers specifically binding to the vibrio fishery viable surface of the present invention are selected by the Cell-SELEX method. According to one specific embodiment of the present invention, 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;
( i ) PCR 방법을 이용하여 DNA 앱타머를 증폭하고, ssDNA 앱타머를 제작하는 단계 :  (i) amplifying the DNA aptamer using a PCR method and preparing the ssDNA aptamer:
먼저， PCR을 이용하여 랜덤 dsDNA 라이브러리를 증폭한다. 증폭된 랜던 dsDNA 라이브러리에서 ssDNA 만을 증폭하기 위해 비대칭 PCR을 수행한다. 비대칭 PCR은 정방향 프라이머와 역방향 프라이머를 10 ： 1의 비율, 예를 들면， 동일한 농도 (25 μΜ)에서 정방향 프라이머를 10 μϊ, 역방향 프라이머 1 峰 사용하여 PCR을 수행함으로써 ssDNA를 수득하는 방법이다. ssDNA 앱타머를 선별하는 방법은 예를 들면, PCR 수행시 역방향 프라이머에 바이오틴 (biotin)을 부착시켜 dsDNA를 증폭하고, 증폭 산물에 스트템트아비딘을 처리하여 바이오틴-스트렙트아비딘 복합체를 형성하여 복합체를 선택적으로 제거함으로써 ssDNA 앱타머만을 선별할 수 있게 된다. 제조한 ssDNA 앱타머는 SELEX 방법에 사용하기 위해 ssDNA를 가열하여 변성시킨 후 상온에서 천천히 식혀 3차원 구조를 형성시킨다.  First, amplify a random dsDNA library using PCR. 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 µϊ of the forward primer and 1 reverse of the reverse primer at the same concentration (25 µM). 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. By selective removal, only ssDNA aptamers can be screened. 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.
(ii) 비브리오 피셔리를 배양하고 세척하는 단계:  (ii) incubating and washing the Vibrio fishery:
비브리오 피셔리의 생균 표면에 결합하는 DNA 앱타머를 선별하기 위해， 비브리오 피셔리를 배양한다. 생균 상태를 유지하기 위해 비브리오 피셔리는 예컨대 LBS 배지 [트립톤 1% (w/v), 효모 추출물 0.5% (w/v), NaCl 2% (w/v) , 한천 1.5% (w/v) 및 20 mM Tris-HCl ( H 7.5)]에서 배양할 수 있다.  Vibrio Fisher is incubated to select DNA aptamers that bind to viable surface of Vibrio Fisher. In order to maintain viable conditions, 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).
(iii) 상기 배양한 비브리오 피셔리 생균을 이용하여 Live Cell SELEX 기법을 통해 비브리오 피셔리 생균 표면에 특이적으로 결합하는 DNA 앱타머를 선별하는 단계: .  (iii) screening the DNA aptamer specifically binding to the vibrio fishery viable surface using the Live Cell SELEX technique using the cultured vibrio fishery viable:.
상기 준비한 비브리오 피셔리 생균과 ssDNA 앱타머를 서로 접촉하여 반웅시킨 후, 결합하지 않는 ssDNA 앱타머는 세척하여 제거하고 특이적으로 결합하는 ssDNA만을 용출시킨다. 이 단계에서 다른 세균， 예를 들어 그람 음성 균인 대장균과 그람 양성균인 바실러스 균에 결합하는 ssDNA를 제거하는 네가티브 (Negat i ve) SELEX 단계를 포함할 수 있다. After the vibrio fishery probiotics prepared above and the ssDNA aptamer were contacted with each other, the non-binding ssDNA aptamer was washed and removed and specifically Only eluting ssDNA is eluted. 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.
본 발명의 방법에서는 비브리오 피셔리 생균 표면에 특이적으로 결합하는 DNA 앱타머가 최대로 용출되는 최적의 SELEX 라운드를 선별하기 위해， 용출된 ssDNA* 나노—드랍을 이용한 ssDNA 농도를 정량하는 방법을 사용할 수 있다.  In the method of the present invention, 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.
하기 본 발명의 구체적인 일 실시예에 따르면， 본 발명의 방법에서 최적 SELEX 라운드 증 비브리오 피셔리와 최적 결합력을 보이는 DNA 앱타머를 선별하기 위하여 최적 SELEX 라운드 선별과 마찬가지로 최적 라운드에서 확보한 ¾타머 서열 각각을 모두 확보하여 동일한 농도의 ssDNA로 제작 후 SELEX를 진행한 뒤 나노드랍 (Nano-drop)을 이용하여 용리된 ¾타머 농도를 나노드랍 방법을 통해 측정함으로서 최적 비브리오 피셔리 표면 결합 DNA 앱타머를 선별하였다. 비브리오 피셔리 생균의 표면에 결합하는 앱타머를 이용한 비브리오 피셔리 생균의 검출 방법 및 생장 억제 방법  According to one specific embodiment of the present invention, in order to select DNA aptamers exhibiting optimal binding force with the optimal SELEX round-enhanced Vibrio Fisheries in the method of the present invention, each of the ¾ 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 ¾ 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
본 발명은 상기 선별된 DNA 앱타머를 유효성분으로 포함하는 비브리오 피셔리 ( /Zv/o fischeri) 생균의 검출용 조성물에 관한 것이다. 하기 본 발명의 구체적인 일 실시예에서 입증되는 바와 같이, 본 발명의 DNA 앱타머는 비브리오 피셔리 생균 표면에 특이적으로 결합하므로， 시료 내에 살아있는 비브리오 피셔리균의 존재 여부를 검출하는데 유용하다. 본 발명은 상기 선별된 DNA 앱타머를 유효성분으로 포함하는 비브리오 피셔리 ( / v o fischeri) 생균의 검출용 키트에 관한 것이다.  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. As demonstrated in one specific example of the present invention, since 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.
상기 키트는 DNA 앱타머가 칩상에 고정화된 칩 형태일 수 있으며, 또는 DNA 앱타머가 기판상에 고정화된 마이크로어레이 형태일 수 있다. DNA 앱타머를 칩이나 기판상에 고정화시키는 것은 당업계에 공지된 방법을 사용할 수 있다.  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.
본 발명의 구체적인 일 실시예에 따르면， 칩 또는 기판을 스트렙트아비딘 ( st rept avi din)을 도입하여 개질하고, DNA 앱타머의 5' 말단은 바이오티닐화 (bi ot inyl at i on)시킨 후， DNA 앱타머의 바이오틴과 칩 또는 기판상에 도입된 스트랩트아비딘과의 결합을 이용하여 고정화한다. 본 명세서에서 용어 "마이크로 어레이 " 는 기판의 특정의 영역에According to a specific embodiment of the present invention, 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. As used herein, the term "micro array" refers to a particular area of the substrate.
DNA 핵산 물질이 고밀도로 부착된 어레이 (배열)를 의미한다. 본 명세서에서 용어 "마이크로어레이의 기판" 은 적합한 견고성 또는 반- 견고성을 갖는 지지체를 의미하며， 예컨대 유리， 막 (membrane) , 슬라이드， 필터, 칩， 웨이퍼， 파이버， 자기성 비드 또는 비자기성 비드， 겔, 류빙， 플레이트， 고분자, 미소입자 및 모세관을 포함하나 이에 한정되지 않는다. 본 발명의 DNA 앱타머는 상기 기판상에 배열되고 고정화된다. 이와 같은 고정화는 화학적 결합 방법 또는 UV와 같은 공유 결합적 방법에 의해 실시된다. 예를 들어， DNA 을리고뉴클레오타이드는 에폭시 화합물 또는 알데히드기를 포함하도록 변형된 유리 표면에 결합될 수 있고， 또한 폴리라이신 코팅 표면에서 UV에 의해 결합될 수 있다. 또한， 상기 DNA 올리고뉴클레오타이드는 링커 (예: 에틸렌 글리콜 올리고머 및 디아민)를 통해 기판에 결합될 수 있다. 본 발명의 DNA 앱타마는 예를 들어， 바이오티닐화 (biot inyl ated)될 수 있고， 이는 스트랩트아비딘이 코팅된 기판상에 성공적으로 결합될 수 있다. 기판 상에 고정화된 본 발명의 DNA 앱타머는 비브리오 피셔리 생균과 결합하여 이를 포획할 수 있으며， 이와 같이 포획된 비브리오 피셔리 생균은 다시 비브리오 피셔리 생균과 특이적으로 결합하는 DNA 앱타머를 이용하여 포획 유무를 시각화 할 수 있다. An array (array) to which DNA nucleic acid material is attached at a high density. As used herein, the term "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. For example, 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. In addition, 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.
본 발명의 키트는 시료 중의 비브리오 피셔리 생균 검출에 사용하기 위한 설명서 또는 표지 ( l abel ) 물질을 추가로 포함할 수 있다.  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.
본 발명은 비브리오 피셔리 ( /br/o fischeri) 생균을 함유하는 것으로 예상되는 시료와 상기 설명된 DNA 앱타머를 접촉시키고， 상기 DNA 앱타머와 결합된 비브리오 피셔리 ( /^/o fischeri) 생균을 확인하는 단계를 포함하는 비브리오 피셔리 ( / v o fischeri) 생균의 검출 방법에 관한 것이다.  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.
본 발명의 DNA 앱타머는 바브리오 피셔리 생균의 표면에 특이적으로 결합하므로 DNA 앱타머를 비브리오 피셔리 생균을 함유할 것으로 예상되는 시료를 접촉시켜 상기 비브리오 피셔리 생균을 검출하는 데 유용하게 사용될 수 있다. 상기 DNA 앱타머에 결합된 비브리오 피셔리의 검출은 DNA 앱타머와 비브리오 피셔리 결합 복합체를 검출하는 방법에 기초하여 행할 수 있다. 상기 복합체의 검출을 용이하게 하기 위해 DNA 앱타머가 형광물질， 예를 들어 플루오레세인 (fluorescein) Cy3 또는 Cy5; 방사성물질 또는 화학물질， 예를 들어 바이오틴 (biotin)으로 표지되거나 1차 아민 (primary amine)으로 변형된 뉴클레오타이드를 포함할 수 있다. 본 발명은 (i) 상기 설명된 DNA 앱타머; 및 (ii) 상기 DNA 앱타머에 링커 (linker)를 통해 접합된 항균물질을 포함하는 DNA 앱타머-항균물질 복합체에 관한 것이다. Since 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.
또한, 본 발명은 상기 DNA 앱타머-항균물질 복합체를 비브리오 피셔리 (ί^Τ /ο fischeri) 생균을 함유하는 것으로 예상되는 시료와 접촉시켜 비브리오 피셔리 ( /?r/o fischeri)^\ 생장을 억제하는 방법에 관한 것이다.  In addition, the present invention is to contact the DNA aptamer-antimicrobial complex with a sample that is expected to contain a vibrio fisher (ί ^ Τ / ο fischeri) viable bacteria growth (/? R / o fischeri) ^ \ It is about how to suppress.
'본 발명의 DNA 앱타머에 항균물질을 접합시켜 DNA 앱타머-항균물질 복합체를 제조하고, 상기 복합체를 비브리오 피셔리 ( / r/(? fischeri) 생균을 함유하는 것으로 예상되는 시료와 접촉시키면, 상기 복합체와 비브리오 피셔리 균간의 접촉 회수가 증가하여 비브리오 피셔리의 생장을 효과적으로 억제할 수 있다.  '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.
본 발명에서 DNA 앱타머와 항균물질간의 접합 (conjugate)은 당업계에 공지된 적합한 방법을 통해 행할 수 있으며, 바람직하게는 링커 (linker)를 통해 접합을 행할 수 있다. 상기 링커는 예를 들어 SPDP ((N-succinimidyl 3-(2-pyridyldi thio) propionate)) , S-Hynic (succinimidyl-6-hydrazino- nicotinamide) , Sᅳ 4FB (N-succinimidyl-4-formylbenzamide)¾- 단독 또는 2이상을 흔합하여 사용할 수 있으며， 결합하고자 하는 항균물질이 단백질인 경우 단백질의 N-말단과 DNA 앱타머 3' -말단 또는 5' -말단의 아민기， 바이오틴기 등의 다양한 변형을 이용하여 항균물질과 DNA 앱타머를 접합할 수 있으나, 이에 한정되지 않는다. In the present invention, 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 ᅳ 4FB (N-succinimidyl-4-formylbenzamide) ¾ - 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.
본 발명에서 사용될 수 있는 항균제는 비브리오 피셔리 균에 대해 항균활성을 나타내는 물질을 사용할 수 있으며, 예를 들어， 무기화합물 유래 무기계 항균제， 유기화합물 유래 유기계 항균제, 나노실버 (nano- silver), 나노촉매 (nano_catalyst )， 바이오세라믹스 (bioceramics) , 이온 방출 (ion emitters) 효과를 갖는 금속염， 천연 항균물질， 프로폴리스， 락토페린, 라이소자임, 키토산 등의 동물유래 항균물질 및 나이신， 폴리라이신 등의 미생물 유래 항균물질을 포함하나 이에 한정되지 않는다. The antimicrobial agent that can be used in the present invention may use a material that exhibits antimicrobial activity against Vibrio Fishery bacteria. For example, 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. (nano_catalyst), 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.
【유리한 효과】 Advantageous Effects
본 발명의 이점 및 효과를 요약하면 다음과 같다: The advantages and effects of the present invention are summarized as follows:
) 본 발명은 DNA 앱타머는 비브리오 피셔리 (Vibr io f i scher i ) 생균의 표면에 높은 특이도로 결합할 수 있다.  In the present invention, the DNA aptamer may bind with high specificity to the surface of Vibrio io scher i viable bacteria.
( ii ) 본 발명의 DNA 앱타머를 사용하면 표본 시료내에서 비브리오 피셔리 생균의 존재 여부를 검출 및 확인할 수 있다.  (ii) By using the DNA aptamer of the present invention, it is possible to detect and confirm the presence of vibrio fishery microorganisms in a specimen sample.
( iii ) 본 발명의 DNA 앱타머에 항균물질이 접합된 DNA 앱타머- 항균물질 복합체를 사용하면 비브리오 피셔리 균의 생장을 억제할 수 있으며, 이 균에 의해 형성되는 바이오필름도 효과적으로 저해할 수 있다.  (iii) The use of a 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.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1 은 랜덤 DNA 앱타머를 PCR 기법을 이용하여 증폭한 후， 스트렙트아비딘 아가로스 레진을 이용하여 ssDNA 만을 선택적으로 회수한 결과와 PCR 로만 증폭한 결과를 나타낸다. 레인 1 : lOObp DNA s i ze 마커; 레인 2 : DNA 앱타머를 PCR 기법을 이용하여 증폭한 결과; 레인 3 : PCR 기법으로 증폭한 후， 스트렙트아비딘 아가로스 레진을 이용하여 ssDNA 만을 회수한 결과이다.  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.
도 2 는 비브리오 피셔리 표면 결합 DNA 앱타머의 제작을 위한 SELEX 과정 전체 10 개 라운드 중 각 라운드에서 회수된 비브리오 피셔리 표면 '결합 DNA 앱타머군의 용리 액의 농도를 나노-드랍을 이용하여 정량적으로 측정한 결과이다. 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.
도 3 은 비브리오 피셔리 표면 결합 DNA 앱타머 제작을 위한 SELEX 과정 후 선별된 라운드에서 확보한 비브리오 피셔리 표면 결합 DNA 앱타머 후보군들 각각에 대한 1 차 (패널 (a) ) , 2 차 (패널 (b) ) 용리액을 나노- 드랍을 이용하여 정량적으로 측정한 결과이다.  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.
도 4 는 m-fold 프로그램을 이용하여 비브리오 피셔리 표면 결합 DNA 앱타머 WCA-03의 예상 2차 구조를 나타낸 것이다. 1 Figure 4 shows the expected secondary structure of the Vibrio Fisher surface binding DNA aptamer WCA-03 using the m-fold program. One
도 5 는 비브리오 피셔리 표면 결합 DNA 앱타머를 스트렙트아비딘이 코팅된 센서 칩 (Sensor chip) SA 의 표면에 고정시키고 비브리오 피셔리 생균을 결합시키는 과정올 나타낸 도면이다. . 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. .
도 6 은 비브리오 피셔리 표면 결합 DNA 앱타머를 스트렙트아비딘이 코팅된 센서 칩 (Sensor chip) SA (GE healthcare , USA)의 표면에 고정시키고 비브리오 피셔리 이외의 균인 쉬겔라 소네이， 리스테리아 모노사이토제네스, 대장균， 비브리오 파라해모라이티쿠스를 홀려서 비브리오 피셔리 표면 결합 DNA 앱타머의 특이성을 확인한 결과를 보여주는 그래프이다.  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.
도 7 은 비브리오 피셔리 표면 결합 DNA 앱타머를 이용하여 시료 상에 비브리오의 존재 '여부를 시각적으로 빠른 시간 내에 확인하기 위하여 만든 래피드 키트 (Rapid ki t )의 모식도이다. 골드 나노 입자 (Gold Nano Part i c le)에 DNA 앱타머를 부착시킨 후에 샘플 패드에 시료를 홀려주게 되면 시료 속의 비브리오 피셔리와 DNA 앱타머가 결합하게 되고 테스트라인의 다른 앱타머와 비브리오 피셔리가 고정되면서 라인이 나타나게 된다. 이를 통해 시료 상의 비브리오 피셔리의 존재 여부를 확인할 수 있다. 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. After attaching the DNA aptamer to the Gold Nano Particles, 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.
도 8 은 비브리오 피셔리 표면 결합 DNA 앱타머를 항균물질인 락토페린과 결합시킨 후， 균 배양액에 락토페린과 DNA 앱타머가 결합된 락토페린—앱타머 결합체를 첨가하였을 때 표적 균주와 락토페린 간의 접촉 기회가 늘어날 수 있다는 것을 보여주는 모식도이다.  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.
【발명의 실시를 위한 형태】 [Form for implementation of invention]
이하， 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로， 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. 실시예 실시예 1 : DNA 앱타머 풀의 준비 1-1. 프라이머 (primer) 및 DNA 앱타머 풀의 합성 Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . EXAMPLES Example 1 Preparation of DNA Aptamer Pools 1-1. Synthesis of Primers and DNA Aptamer Pools
비브리오 피셔리 (Vibrio fisheri) 균에 특이적으로 결합하는 비브리오 피셔리 표면결합 DNA 앱타머를 제작하기 위하여 양 말단에 앱타머 풀을 쉽게 증폭하기 위한 부위로서 -ATACCAGCTTATTCAATT의 부위와 AGATAGTMGTGCMTCT-3 를 구성하고 중심부에는 40개의 연속 랜덤 뉴클레오타이드를 dA ： dG ： dC ： dT = 1.5 ： 1.15 ： 1.25 ： 1의 비율로 가지는 서열 [5' -ATACCAGCTTATTCAATT-N40-AGATAGTAAGTGCAATCT-3' (서열번호 14)]과 이 DNA 라이브러리를 증폭할 수 있는 정방향 프라이머와 단일 가닥 DNA를 회수하기 위해 바이오티닐화된 역방향 프라이머를 바이오니아 (Bioneer, Korea)에 주문하여 제작하였다 [정방향 프라이머 ： 5' -ATACCAGCTTATTCAATT-3 ' (서열번호 15), 바이오티닐화된 (biot inylated) 역방향 프라이머 ： 5' -바이오틴 -AGATTGCACTTACTATCT-3' (서열번호 16)]. In order to easily amplify the aptamer pool at both ends, the site of -ATACCAGCTTATTCAATT and AGATAGTMGTGCMTCT-3 were constructed to fabricate the Vibrio fishery surface binding DNA aptamer specifically binding to Vibrio fisheri. the heart, 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)].
1-2. PCR 기법을 활용한 DNA 앱타머 풀의 증폭 1-2. Amplification of DNA Aptamer Pools Using PCR Technique
합성한 프라이머 및 40개의 연속 랜덤 서열을 가지는 임의의 DNA 라이브러리를 PCR (Bioneer, Korea)을 이용하여 증폭하였다. 76 bp의 DNA 라이브러리의 증폭을 위한 PCR 반웅 조성으로는 10X PCR 완충용액 5 ^， 각 2.5 mM dNTP 흔합물 (mixture) 4/ , 10 M 정방향 프라이머 2 βί, 바이오티닐화된 역방향 프라이머 2 ^， 주형 DNA 라이브러리 1 - 2 ≠, Ex Taq 중합효소 (TaKaRa, Japan) 0.S≠ (1 unit/^)와 증류수 34.7 - 35.7 ^로 구성하였다. PCR 반웅 조건은 먼저 94°C에서 5분간 변성시킨 후에， 94°C에서 30초， 52°C에서 30초, 그리고 72°C에서 30초간 반웅을 20주기를 반복한 후, 72°C에서 5분간 추가로 신장시키는 반웅을 이용하였다. PCR 반웅 후에 3 ^를 취하여， 2% 아가로스 젤을 이용하여 정확한 크기인 76 bp에서 밴드가 나타나는지 확인하였다. 확인된 DNA는 PCR'정제 키트 (Qiagen, USA)를 이용하여 DNA 앱타머 풀을 회수하였다. Any DNA library having synthesized primers and 40 consecutive random sequences was amplified using PCR (Bioneer, Korea). 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 βί, 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).
1-3. DNA 앱타머 풀의 ssDNA 제작 및 정제 1-3. SsDNA Construction and Purification of DNA Aptamer Pools
상기한 바와 같이 5' 말단에 바이오티닐화된 dsDNA 앱타머 풀을 ssDNA 앱타머 풀로 제작하기 위하여 5' 말단에 바이오티닐화된 dsDNA 앱타머 풀 200 를 85°C에서 5분간 끓인 후에 얼음에 신속히 냉각시켰다. 이후에 스트렙트아비딘 아가로스 레진 (Streptavidin agarose resin)을 dsDNA 앱타머 풀의 1/2배인 100 ^를 넣고 상온에서 스트렙트아비딘과 1시간 이상 반웅을 유도하였다. 이후에 원심분리 (4°C, 13,000rpm)를 10분간 수행하여 스트렙트아비딘 아가로스 레진을 침전시킴으로써 스트렙트아비딘과 바이오틴 결합을 통해 바이오티닐화된 dsDNA 앱타머 풀 증 한쪽 서열을 제거하여 ssDNA 앱타머 풀을 제작하였다. 이후 순수한 ssDNA 앱타머 풀을 확보하기 위하여 상층 200 ^를 새 튜브에 옮겼다. 통상적으로 본 업계에서 사용하는 PCI 처리 및 에탄을 침전법을 활용하였다. 본 방법은 옮긴 ssDNA 앱타머 풀에 동량의 PCI 200 ^를 처리하고 교반하여 ssDNA에 잔류하는 지질과 단백질을 제거한 후에 원심분리 (4°C, 13,000rpm)를 15분간 수행한 후에 상층 200 를 새 류브에 옮겨 페놀을 제거하였다. 이 후에 에탄올을 DNA양의 3배인 600 f 를 넣고 3M 소듐 아세테이트 (Sodium Acetate)를 1/10 배인 20 ≠ 그리고 1 - 의 tRNA를 첨가한 후에 -70°C의 초저온 넁동고 (Deep freezer)에 1시간 이상 반웅시켰다. 그 후에 원심분리 (4°C, 13,000rpm)를 20분간 수행하여 DNA를 침전시키고 상층을 제거한 후에 85°C 히팅블록 (Heat block)에서 건조시킨 후 증류수 를 첨가하여 ssDNA 앱타머 풀을 확보하였다. 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. Subsequently, centrifugation (4 ° C, 13,000 rpm) was performed for 10 minutes to precipitate streptavidin agarose resin, thereby removing one sequence of the biotinylated dsDNA aptamer pool through biotin binding with streptavidin to remove the ssDNA app. The tamer paste was produced. The upper 200 ^ was then transferred to a new tube to ensure a pure ssDNA aptamer pool. In general, the PCI treatment and ethane precipitation used in the art were utilized. In this method, the same amount of PCI 200 ^ was treated and stirred in the transferred ssDNA aptamer pool to remove lipids and proteins remaining in ssDNA, followed by centrifugation (4 ° C, 13,000rpm) for 15 minutes, and then the upper layer 200 was regenerated. Transferred to remove phenol. After this, ethanol was added 600 f, which is three times the amount of DNA, and 3M sodium acetate was added 1/10 times 20 ≠ and 1-tRNA, followed by 1 to -70 ° C deep freezer. Reaction over time. Thereafter, 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.
1-4. ssDNA 앱타머 풀 확인을 위한 PAGE 1-4. PAGE to identify the ssDNA aptamer pool
스트렙트아비딘과 바이오틴 결합을 통해 회수된 ssDNA 앱타머 풀을 SsDNA aptamer pool recovered through streptavidin and biotin binding
0.5X TBE 아크릴아마이드 젤 (Acrylamide gel) 상에서 전기영동하여 확인하였다. 0.5X TBE (Tris-borate-EDTA) 아크릴아마이드 젤의 조성으로는 40% 아크릴아마이드 3.75 ml, 10X TBE 완층액 0.75 ml을 넣고 증류수로 15 ml을 채운 후에 10% APS (Ammonium per sulfate) 용액 135 μί, TEMED (Tetramethylethylenediamine) 10.5 ^로 구성하였다. 아크릴아마이드 젤에 ssDNA 앱타머 풀 와 DNA 로딩 다이 (loading dye) 2 μί를 섞어 넣었고 래더 마커 (Ladder marker)로는 100bp DNA 마커를 사용하였다. 제작한 PAGE (Poly-acrylamide Gel Electrophoresis)용 ssDNA 앱타머 풀 확인용 아크릴아마이드 젤을 파워서플라이 (Major science, USA) 150 V(volt)에 45분간 전기영동하였다. 전기영동한 아크릴아마이드 젤은 ETBR 용액에 5분간 염색한 후에 Gel -doc (Bio-rad, USA)으로 자외선을 조사하여 관찰하였다 (도 1 참고). It was confirmed by electrophoresis on 0.5X TBE acrylamide gel. 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 μί of 10% APS (Ammonium per sulfate) solution. , TEMED (Tetramethylethylenediamine) 10.5 ^. The acrylamide gel was mixed with ssDNA aptamer pool and 2 μί 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).
1- 5. DNA 앱타머의 3차원 구조 형성 1- 5. Formation of 3-D Structure of DNA Aptamer
상기 실시예 1-4에서 PAGE로 확인한 ssDNA 앱타머의 풀은 자체 3차원의 구조를 형성하기 위하여 기 제작 확보한 ssDNA 앱타머 풀 100 ^와 동량의 2X LBS 배지 를 첨가한 후 85°C로 예열된 히팅 블록 (Heat block)에서 5분간 가열하여 ssDNA 앱타머 풀의 변성을 유도하였다. 변성된 ssDNA는 상온에서 2시간 이상 서서히 식혀 자체 3차원 구조 형성을 유도하였다. 실시예 2: 비브리오 피셔리( ^%"/0 fischeri) 생균 결합 DNA 앱타머 제작을 위한 생균 (Live-cell)의 준비 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
2- 1, 비브리오 피셔리의 준비 및 배양  2- 1, Preparation and Incubation of Vibrio Fisheries
비브리오 피셔리 생균 결합 DNA 앱타머를 제작하기 위하여 , 비브리오 피셔리는 배양을 통해 확보하였다. 배양 배지로는 LBS (10 mM Tris- HC1내의 1% 트립톤， 0.5% 효모 추출물 및 2% NaCl)를 사용하였으며， 371 교반배양기에서 12시간 동안 배양하였다. 또한， 생균 자체에만 결합하도록 배양시 형성되는 타 물질의 제거를 위하여 Tris base 10 mmol/L NaCl 0.85%, pH 8.0 조성의 세척용액을 사용하여 배양된 비브리오 피셔리를 세척하였다. 세척방법은 먼저 배양된 비브리오 피셔리를 원심분리 (4°C, 13,000rpm)를 10분간 수행하여 비브리오 피셔리를 침전시키고 상층액을 제거하였다. 그 후에 세척 용액 200 를 첨가하여 파이펫팅을 통해 비브리오 피셔리의 표면을 세척하고 원심분리 (4°C, 13,000rpm)를 10분간 수행하여 비브리오 피셔리를 침전시키고 상층을 제거하였다. 상기의 과정을 3회 반복하여 비브리오 피셔리 표면을 세척하였다. In order to produce vibrio fishery viable binding DNA aptamers, vibrio fishery was obtained through culture. LBS (1% tryptone, 0.5% yeast extract and 2% NaCl in 10 mM Tris-HC1) was used as the culture medium and incubated for 12 hours in a 371 agitator. In addition, in order to remove other substances formed during the cultivation to bind only to the viable bacteria itself, the cultured Vibrio fishery was washed using a washing solution of Tris base 10 mmol / L NaCl 0.85%, pH 8.0. In the washing method, the cultured Vibrio fishery was centrifuged (4 ° C, 13,000 rpm) for 10 minutes to precipitate the Vibrio fishery and the supernatant was removed. Thereafter, 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.
2-2. 네가티브 선별 (Negative selection) 진행 및 결합력 비교를 위한 타세균 배양 및 확보 2-2. Cultivate and secure other bacteria for negative selection and comparison of binding capacity
비브리오 피셔리에 특이적으로 결합하는 비브리오 피셔리 생균 특이 결합 DNA 앱타머 후보군 중에 다른 균주에도 결합하는 비특이적인 앱타머 후보군을 제거하기 위하여 대장균 및 바실러스 서브틸리스를 이용한 네가티브 선별 (negative selection)을 수행하였다. 또한, SPR측정에서 비브리오 피셔리에 대한 DNA 앱타머의 결합력을 비교하기 위해 배양된 타 균주들을 사용한 결합력 측정을 행하였다. Non-specific aptamers that bind to other strains among the Vibrio Fisher's live bacteria specific binding DNA aptamer candidates that specifically bind to Vibrio Fisheries Negative selection was performed using Escherichia coli and Bacillus subtilis to remove candidate groups. In addition, in order to compare the binding affinity of the DNA aptamer to the Vibrio fishery in the SPR measurement, binding affinity was measured using other cultured strains.
2-2-1. ^^균 {Escherichia co//)의 배양 및 확보 2-2-1. Cultivation and acquisition of ^ (Escherichia co //)
네가티브 선별 (Negative selection) 대상 균주인 대장균의 배양배지로는 LB broth를 사용하였다. LB broth 5 ml에 균을 접종하여 37°C에서 12시간동안 배양하였다. 그 후에 배양된 대장균을 원심분리 (4°C, 13,000rpm)를 10분간 수행하여 침전시키고 상층을 제거하였다. 그 후에 세척 용액 200 ^를 첨가하여 파이펫팅을 통해 대장균의 표면을 세척하고 원심분리 (4°C, 13,000rpm)를 10분간 수행하여 침전시키고 상층을 제거하였다. 상기 과정을 3회 반복하여 대장균 표면을 세척하였다. 2-2-2. 바실러스 서브틸리스 0¾c/ /"s subtUis)^ 배양 및 확보 네가티브 선별 (Negative selection) 대상 균주인 바실러스 서브틸리스의 배양배지로는 LB broth를 사용하였다. LB broth 5 ml에 바실러스 서브틸리스를 접종하여 37°C에서 12시간 동안 배양하였다. 그 후에 배양된 바실러스 서브틸리스를 원심분리 (4°C, 13,000rpi)를 10분간 수행하여 침전시키고 상층을 제거하였다. 그 후에 세척 용액 200 峰 첨가하여 파이펫팅을 통해 바실러스 서브틸리스의 표면을 세척하고 원심분리 (4°C, 13,000rpm)를 10분간 수행하여 침전시키고 상층을 제거하였다. 상기의 과정을 3회 반복하여 바실러스 서브틸리스 표면을 세척하였다. 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. Bacillus subtilis 0¾c / / "s subtUis) ^ Culture and Securing Negative Selection The culture medium of Bacillus subtilis, the target strain, was used as LB broth. Bacillus subtilis was inoculated into 5 ml of LB broth. the cells were cultured for 12 hours at 37 ° C. after the cultured Bacillus subtilis centrifugation (4 ° C, 13,000rpi) was carried out for 10 minutes to remove the precipitate and supernatant. after the addition of wash solution 200峰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.
2-2-3. 리스테리아 모노사이토제네스 ( /sfe /a monocytogene^^ 배양 및 확보 2-2-3. Listeria monocytogenes (/ sfe / a monocytogene ^^
SPR 측정시 앱타머의 비특이성을 측정하기 위한 균주인 리스테리아 모노사이토제네스의 배양배지로는 NA broth를 사용하였다. NA broth 5 ml에 리스테리아 모노사이토제네스를 접종하여 37°C에서 12시간동안 배양하였다. 그 후에 배양된 리스테리아 모노사이토제네스를 원심분리 (4V, 13,000rpm)를 10분간 수행하여 침전시키고 상층을 제거하였다. 그 후에 세척 용액 200 를 첨가하여 파이펫팅을 통해 리스테리아 모노사이토제네스의 표면을 세척하고 원심분리 (4°C, 13,000rpm)를 10분간 수행하여 침전시키고 상층을 제거하였다. 상기의 과정을 3회 반복하여 리스테리아 모노사이토제네스 표면을 세척하였다. NA broth was used as a culture medium of Listeria monocytogenes, a strain for measuring the specificity of aptamer when measuring SPR. Inoculated with Listeria monocytogenes in 5 ml of NA broth and incubated at 37 ° C for 12 hours. Subsequently centrifugation of the cultured Listeria monocytogenes (4V, 13,000 rpm) was performed for 10 minutes to precipitate and the upper layer was removed. Thereafter, 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.
2- 2-4. 비브리오 파라해모라이티쿠스 7b/ o parahaemolyticus)^ 배양 및 확보 2- 2-4. Vibrio parahaemolyticus 7b / o parahaemolyticus)
SP 측정시 앱타머의 비특이성을 확인하기 위한 균주인 비브리오 파라해모라이티쿠스의 배양배지로는 3% NaCl을 포함한 NA broth를 사용하였다. 3% NaCl을 포함한 NA 브로쓰 (broth) 5 ml에 비브리오 파라해모라이티쿠스를 접종하여 37°C에서 14시간동안 배양하였다. 그 후에 배양된 비브리오 파라해모라이티쿠스를 원심분리 (4°C, 13,000 rpm)를 10분간 수행하여 침전시키고 상층을 제거하였다. 그 후에 세척 용액 200 ^를 첨가하여 파이펫팅을 통해 비브리오 파라해모라이티쿠스의 표면을 세척하고 원심분리 (4°C, 13,000 rpm)를 10분간 수행하여 침전시키고 상층올 제거하였다. 상기의 과정을 3회 반복하여 비브리오 파라해모라이티쿠스 표면을 세척하였다. 실시예 3: 비브리오 피셔리와 특이적으로 결합하는 비브리오 피셔리 표면 결합 앱타머의 제작 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. Thereafter, 200 ^ of the washing solution was added to wash the surface of Vibrio parahaemoriaticus via pipetting, and centrifuged (4 ° C, 13,000 rpm) for 10 minutes to precipitate and remove the upper layer. The above procedure was repeated three times to wash the surface of Vibrio parahaemoriaticus. Example 3: Construction of a Vibrio Fisher surface binding aptamer specifically binding to a Vibrio Fisher
3- 1. 비브리오 피셔리 생균에 특이적으로 결합하는 DNA 앱타머 후보군의 선별 및 확보  3- 1. Selection and acquisition of DNA aptamer candidates that specifically bind to Vibrio Fisher's live bacteria
상기 실시예 1과 2에서 설명된 방법을 통해 확보한 비브리오 피셔리 생균을 세척한 후에， 자체 3차원 구조를 형성한 ssDNA 앱타머 풀 200 ^에 넣고 Thermo mixer (Eppendorf , USA) 4°C, 500rpm으로 1시간 동안 반응시켰다. 이 후 튜브를 꺼내 원심분리 (4°C, 13,000rpm)를 10분간 수행함으로서 ssDNA 앱타머 풀이 결합되어 있는 비브리오 피셔리를 침전시키고 상층액을 제거하였다. 비브리오 피셔리 생균 결합 DNA 앱타머는 비브리오 피셔리의 무게에 의해 침전되고， 결합되지 않은 ssDNA 앱타머들은 상층에 남아 제거하였다. 침전된 비브리오 피셔리와 비브리오 피셔리 생균 결합 DNA 앱타머 후보군 중 비브리오 피셔리와 결합하지 않은 ssDNA 앱타머 풀을 제거하기 위하여 세척용액 200 를 첨가하여 파이펫팅한 후에 원심분리 (4°C , 13 , 000rpm)를 10분간 수행하여 비브리오 피셔리를 침전시키고 상층액을 제거하였다. 상기의 과정을 3회 반복하여 비브리오 피셔리에 비특이적으로 결합하는 ssDNA 앱타머 풀을 제거하였다. 이 후에 침전물에 10 mM Tr i s (pH 7.5) , 1 mM EDTA로 구성된 DNA 앱타머 용출 용액 100 를 첨가하였다. 그 후에 851；로 예열된 히팅 블록 (Heat bl ock)에 5분간 반웅시킨 후 원심분리 (4°C , 13 , 000rpm)를 10분간 수행하여 비브리오 피셔리를 침전시키고 비브리오 피셔리 생균 특이 결합 DNA 앱타머가 용리된 상층액은 새로운 튜브에 옮겨 비브리오 피셔리에 특이적으로 결합하는 DNA 앱타머 후보군을 회수하였다. 상기의 과정을 2회 반복하여 DNA 앱타머 풀 각 100 ^를 회수하였다. 3-2. 비브리오 피셔리 생균 특이 결합 DNA 앱타머 후보군의 네가티브 선별 After washing the vibrio fishery probiotic secured by the method described in Examples 1 and 2, and placed in the ssDNA aptamer pool 200 ^ formed its own three-dimensional structure, Thermo mixer (Eppendorf, USA) 4 ° C, 500 rpm Reacted for 1 hour. Thereafter, the tube was taken out and centrifuged (4 ° C., 13,000 rpm) for 10 minutes to precipitate the Vibrio fishery to which the ssDNA aptamer pool was bound, and the supernatant was removed. Vibrio Fisher's live bacterial binding DNA aptamers were precipitated by the weight of Vibrio Fisher's, and unbound ssDNA aptamers remained on top and removed. To remove precipitated vibrio fisher and vibrio fisher probiotic-binding DNA aptamer candidate ssDNA aptamer pools that did not bind to vibrio fisher, pipette with 200 rinse solution (4 ° C, 13, 000 rpm) was performed for 10 minutes to precipitate the Vibrio fishery and remove the supernatant. The above procedure was repeated three times to remove the ssDNA aptamer pool that nonspecifically binds to Vibrio fishery. After this, 100 mM DNA aptamer elution solution consisting of 10 mM Tr is (pH 7.5) and 1 mM EDTA was added to the precipitate. After reaction for 5 minutes in a heating block preheated to 851; for 5 minutes, centrifugation (4 ° C, 13,000 rpm) was performed for 10 minutes to precipitate the Vibrio fishery and the Vibrio fishery specific bacteria-binding DNA apta. The supernatant from which the mer eluted was transferred to a new tube to recover the DNA aptamer candidate group that specifically binds to Vibrio fisheries. The above procedure was repeated twice to recover each 100 ^ DNA aptamer pool. 3-2. Negative Screening of Vibrio Fisher's Probiotic Specific Binding DNA Aptamer Candidates
제 6 라운드의 선별과정 후에 비특이적으로 결합하는 DNA 앱타머를 제거하기 위하여 네가티브 선별을 수행하였다. 상기 실시예 1과 2에서 설명된 방법을 통해 대장균 및 바실러스 서브틸리스 생균을 확보하고 먼저 대장균에 자체 3차원 구조를 형성한 ssDNA 앱타머 풀 200 ^를 넣고 Thermo mixer (Eppendor f , USA) 4°C , 500rpm으로 1시간 동안 반웅시켰다. 이 후 류브를 꺼내 원심분리 (4°C , 13 , 000rpm)를 10분간 수행함으로서 비특이적인 ssDNA 앱타머 풀이 결합되어 있는 대장균을 침전시키고 상층액올 획득하였다. 획득한 상층액은 준비된 바실러스 서브틸리스에 넣고 Thermo mixer (Eppendor f , USA) 4°C， 500rpm으로 1시간 동안 반웅시켰다. 표적 균주가 아닌 대장균과 바실러스 서브틸리스에 결합된 ¾타머는 상층액을 회수하는 과정에서 제거되었다. 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.
3-3. 비브리오 피서리 생균 특이 결합 DNA 앱타머 후보군의 정제 및 용리 농도 측정 3-3. Purification and Elution Concentration of Vibrio Fiery-Fly-Bacterial Specific Binding DNA Aptamer Candidates
비브리오 피셔리 생균 특이 결합 DNA 앱타머 풀에 잔류하는 단백질과 지질을 제거하고 DNA만을 회수하기 위하여 업계에서 통상적으로 사용되는 PCI 처리법 및 에탄올 침전법을 사용하였다. 회수한 각 DNA 앱타머 풀 10 에 PCI 용액 100 를 각각 처리하여 섞어주었다. 이 후 원심분리 (4°C , 13 , 000rpm)를 15분간 수행한 뒤에 상층액 100 ^를 새로운 류브에 옮겼다. 이후에 에탄올 ( 100% Ethanol ) 300 ^를 넣고 3M 소듐아세테이트 (sodium acetate) 10 μ 그리고 1 ^의 tRNA를 첨가한 후에 -70°C의 초저온 냉동고 (Deep freezer )에 1시간 이상 반응하였다. 그 후에 원심분리 (4°C , 13 , 000rpm)를 20분간 수행하여 DNA를 침전시키고 상층을 제거한 후에 85°C 히팅 블록 (Heat bl ock)에서 건조시킨 후 증류수 50 ^를 각각 첨가하여 용출된 DNA 앱타머 풀을 확보하였다. 확보한 DNA 앱타머 풀 1 ^를 나노- 드랍 (Nano-drop)에서 측정하여 DNA농도를 확인하였다. 실시예 4 : 비브리오 피셔리와 최적 결합력을 보이는 DNA 앱타머 후보군의 선별 Proteins remaining in vibrio fishery probiotic-specific binding DNA aptamer pools In order to remove lipids and recover only DNA, PCI treatment and ethanol precipitation, which are commonly used in the industry, were used. Each of the recovered DNA aptamer pools 10 was treated with PCI solution 100 and mixed. Thereafter, centrifugation (4 ° C, 13,000 rpm) was performed for 15 minutes, and then the supernatant 100 ^ was transferred to a new stream. After adding 300 ^ ethanol (100% Ethanol) 10 μl of 3M sodium acetate and 1 ^ tRNA were added, and then reacted in a deep freezer of -70 ° C. for more than 1 hour. Thereafter, centrifugation (4 ° C, 13, 000rpm) was performed for 20 minutes to precipitate the DNA, the upper layer was removed, dried in an 85 ° C heating block (Heat bl ock), and then eluted by adding 50 ^ of distilled water, respectively. Aptamer pool is secured. Obtained DNA aptamer pool 1 ^ was measured in the nano-drop (Nano-drop) to confirm the DNA concentration. Example 4 Screening of DNA Aptamer Candidates with Optimal Binding to Vibrio Fisheries
4-1. 각 라운드 (round) DNA 앱타머 풀의 선별 및 확보  4-1. Selection and acquisition of each round DNA aptamer pool
상기 기술한 방법을 이용하여 각 라운드 (round)의 용리 DNA 앱타머 풀을 이용하여 ssDNA를 제작한 후에 확보한 각 라운드 (round)의 ssDNA 앱타머 풀 50 ^를 상기 기술한 방법과 동일한 방식으로 제작한 비브리오 피셔리의 침전물에 넣고 Thermo mixer (Eppendorf , USA) 4°C , 500 rpm으로 1시간 동안 반응시켰다. 이 후 튜브를 꺼내 원심분리 (4°C , 13 , 000 rpm)를 10분간 수행하여 구조 형성 ssDNA 앱타머 풀이 결합되어 있는 비브리오 피셔리를 침전시키고 상층을 제거한 후에 세척용액 200 를 넣어 파이펫팅한 후에 원심분리 (4°C , 13 , 000 rpm)를 10분간 수행하여 상층을 제거하여 세척하는 과정을 3회 수행하고 용출용액 100 ^를 넣어 85°C에서 5분간 가열한 후에 원심분리 (4°C , 13 , 000 rpm)를 10분간 수행하여 각 라운드 (round)의 용출된 DNA 앱타머를 확보하였다. 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.
4-2. 나노 -드랍 (Nano-drop)을 이용한 각 라운드 (round) 용리 DNA 앱타머 풀의 분석 4-2. Analysis of each round eluting DNA aptamer pool using nano-drop
각 라운드 (round)의 DNA 앱타머 풀에 잔류하는 단백질과 지질을 제거하고 DNA만을 회수하기 위하여 당업계에서 통상적으로 사용하는 PCI 처리법 및 에탄올 침전법을 사용하여 정제하였다. 확보한 각 라운드의 DNA 앱타머 풀 를 나노 -드랍 (Nano-drop)으로 측정하여 DNA 농도를 확인하여 네가티브 선별 (Negative selection)이 끝난 후， 가장 농도가 높은 라운드인 8 라운드의 앱타머 후보군을 선별하였다 (도 2 참고). 실시예 5: 선별된 라운드 (round)에서 비브리오 피셔리에 특이적으로 결합하는 비브리오 피셔리 표면결합 DNA 앱타머 후보군 분석 PCI commonly used in the art to remove proteins and lipids remaining in each round of DNA aptamer pool and recover only DNA Purification was performed using treatment and ethanol precipitation. Each round of DNA aptamer pools obtained was measured by nano-drop to check the DNA concentration, and after the negative selection, the eight rounds of aptamer candidates were selected. (See FIG. 2). Example 5 Analysis of Vibrio Fishery Surface Binding DNA Aptamer Candidates Specificly Binding to Vibrio Fisheries in Selected Rounds
5-1. DNA 앱타머 후보군 확보를 위한 T-백터 클로닝 (T-vector cloning) 수행 및 선별  5-1. T-vector cloning and screening for DNA aptamer candidates
선별된 8 라운드에 대한 DNA 앱타머 후보군 확보를 위하여 8 라운드 Round 8 to secure DNA Aptamer candidates for the selected 8 rounds
DNA 앱타머 풀의 증폭을 위한 반웅 조성으로는 10X PCR 완층용액 5 각 2.5 mM dNTP 흔합물 (mixture) 4 μί, 10 Μ 정방향 프라이머 2 ≠, 역방향 프라이머 Κ 주형 DNA 라이브러리 1-2 μΐ, Ex Taq 중합효소 (TaKaRa, Japan) 0.3/^ (lunit/ ^)와 증류수 34.7—35.7 ^로 구성하였다. PCR 반웅 조건은 먼저 94°C에서 5분간 변성시킨 후에， 94°C에서 30초， 52°C에서 30초， 그리고 72°C에서 30초간 반웅을 20주기 반복한 후， 72°C에서 5분간 추가로 신장시키는 반웅올 이용하였다. 증폭시킨 DNA 앱타머를 T-백터 클로닝 키트 (Solgent, Korea)를 이용하여 T-블런트 백터 1 6X 클로닝 완층액 1 ≠, PC 산물 4 ^의 조성으로 25°C에서 10분간 리게이션 (ligation)을 실시한 후에 컴피턴트 세포 (Competent cell; E. col/ DH5a) 100 ^에 6 βί를 넣고 20분간 얼음 안에서 반웅시켰다. 그 후에 열층격법을 이용하여 42°C에서 30초간 반응시켜 형질전환하였고 암피실린 (ampicillin, 50 The reaction composition for the amplification of the DNA aptamer pool was 10X PCR complete solution 5 each 2.5 mM dNTP mixture 4 μί, 10 Μ forward primer 2 ≠, reverse primer Κ template DNA library 1-2 μΐ, 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. Using the T-vector cloning kit (Solgent, Korea), 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 βί 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.
카나마이신 (kanamycin, 50 /ig/n ), X— gal (50 ug/mi), IPTG(5 /g/m£)이 포함되어 있는 LB 배양 플레이트 (LB plate)에 스프레딩하여 배양하였다. 배지에 배양되어 자란 콜로니 중 흰색 콜로니 31개를 선별하여 각각 앰피실린과 카나마이신이 첨가된 LB broth 5 ml에 접종한 후 플라스미드 DNA prep kit (Intron, USA)을 이용하여 플라스미드를 추출하였다. 확보한 각 콜로니에 대한 플라스미드 DNA는 솔젠트 (Solgent, Korea)사에 서열분석을 의뢰하였다. 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).
5-2. Clustal-X를 통한 비브리오 피셔리 생균 특이 결합 DNA 앱타머 후보군 서열 간의 그룹핑 5-2. Vibrio Fisher's Probiotic Specific Binding DNA Aptamers Via Clustal-X Grouping Between Candidate Sequences
각 콜로니들에 대한 서열 증에서 클로닝올 통해 삽입된 DNA 앱타머 서열을 확인하고 나열하여 Clustal-X 프로그램을 이용해 분석하였다. 각 서열에 대한 그룹을 형성하여 각 서열간의 서열 유사도를 분석하고 동일 서열 7개를 포함한 6개로 그룹핑된 13개의 서열을 확보하였다 (아래 표 1 참고) .  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).
【표 1】  Table 1
Figure imgf000022_0001
실시예 6 : 비브리오 피셔리 생균 특이 결합 DNA 앱타머 후보군의 평가
Figure imgf000022_0001
Example 6 Evaluation of Vibrio Fisher Probiotic Specific Binding DNA Aptamer Candidates
6-1. 비브리오 피셔리 생균 특이 결합 DNA 앱타머 후보군의 정제 기 확보한 비브리오 피셔리 특이 결합 DNA 앱타머 후보군의 정제를 위하여 상기의 PCI 처리법 및 에탄을 침전법을 이용하여 정제된 순수 비브리오 피셔리 특이 결합 DNA 앱타머 후보군을 확보하였다. 6-1. Purification of Vibrio Fisher's Probiotic Specific Binding DNA Aptamer Candidates In order to secure the pure Vibrio Fishery specific binding DNA aptamer candidate group purified by the above PCI treatment and ethane precipitation method.
6- 2. Nano-drop을 활용한 비브리오 피셔리 특이 결합 DNA 앱타머 후보군의 친화성 평가 6- 2. Affinity Evaluation of Vibrio Fishery Specific Binding DNA Aptamer Candidates Using Nano-drop
기 확보한 정제된 비브리오 피셔리 특이 결합 DNA 앱타머 후보군들에 대한 친화성을 평가하기 위하여 확보한 각 비브리오 피셔리 특이 결합 DNA 앱타머 후보군 각 를 Nano-drop으로 측정하여 DNA 농도를 확인해 1차 용리시와 2차 용리시 가장 농도가 높게 측정된 DNA 앱타머 후보군인 2번과 3번 후보군을 선별하였다 (도 3 참고) . - 실시예 7 : 선별된 DNA 앱타머의 예상 구조 모식도 확보  In order to evaluate the affinity for the purified Vibrio Fisher-specific binding DNA aptamer candidates obtained, 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
7- 1 . 각 DNA 앱타머 후보군의 예상 2차 구조 분석  7- 1. Expected secondary structure analysis of each DNA aptamer candidate group
선별한 비브리오 피셔리 특이 결합 DNA 앱타머 중에서 특이성이 가장 높았던 WCA-03의 예상 구조를 확인하기 위하여 hUp : //mfo ld . rna . albany . edu/의 M-fo ld 프로그램을 이용하여 예상 구조를 분석하였다. 구조 분석 조건으로는 DNA 앱타머의 서열을 선형가닥으로 지정하였으며 구조 제작 온도는 상온인 25°C로 지정하였다. 구조에 영향을 줄 수 있는 나트륨의 농도는 배양 배지인 LBS의 농도인 0.3M로 지정하여 예상 구조를 제작하였다 (도 4 참고) . 실시예 8 : SPR측정을 위한 DNA 앱타머와 비브리오 피셔리의 준비To identify the expected structure of WCA-03, which had the highest specificity among the selected Vibrio Fishery specific binding DNA aptamers, hUp: // mfo ld. rna. albany. The expected structure was analyzed using M-fo ld program of edu /. As a structural analysis condition, the sequence of DNA aptamer was designated as a linear strand, and the structure fabrication temperature was designated as 25 ° C. at room temperature. The concentration of sodium, which may affect the structure, was set to 0.3 M, which is the concentration of LBS, which is a culture medium, to prepare an expected structure (see FIG. 4). Example 8 Preparation of DNA Aptamers and Vibrio Fisheries for SPR Measurement
8- 1 . SPR (Sur face Pl asmon Resonance ) 측정을 위한 DNA 앱타머의 준비 8- 1. Preparation of DNA Aptamer for Sur face Pl asmon Resonance (SPR) Measurement
실시예 1에 제시된 방법을 이용하되 PCR 시료 중 10 pM 바이오티닐화된 역방향 프라이머와 10 pM 정방향 프라이머 대신 10 pM 바이오티닐화된 정방향 프라이머와 10 pM 역방향 프라이머를 첨가한 시료를 이용하여 PCR을 진행하였다. 이후 과정은 실시예 1에 따라 진행하여 PCR 정제를 수행하였다. 기 확보한 PCR 시료는 ssDNA를 제작하기 위하여 비대칭 PCR을 사용하였다. 비대칭 PCR의 반웅 조성으로는 10X PCR 완층용액 10 ^， 각 2.5 mM dNTP 흔합물 8 μΐ , 10 M 바이오티닐화된 정방향 프라이머 10 10 M 역방향 프라이머 1 ， 주형 DNA 라이브러리 10 μί , Ex Taq 중합효소 (TaKaRa , Japan) 0.5 μΐ ( 1 uni t/ ^)와 증류수 60.5 ^로 구성하였다. PCR 반웅 조건은 먼저 94°C에서 5분간 변성시킨 후에， 94°C에서 30초， 52°C에서 30초， 그리고 72°C에서 30초간 반웅을 15주기 반복한 후， 72°C에서 5분간 추가로 신장시키는 반웅을 이용하였다. 이 후에 통상적으로 당업계에서 사용하는 PCI 처리법 및 에탄올 침전법을 이용하여 순수 DNA 앱타머를 정제 및 확보하였다. 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 μΐ, each 10 mM biotinylated 2.5 mM dNTP mixture Forward primer 10 10 M reverse primer 1, template DNA library 10 μί, Ex Taq polymerase (TaKaRa, Japan) 0.5 μΐ (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.
8- 2. ssDNA 앱타머의 구조 제작 8- 2. Construction of ssDNA Aptamer Structure
확보한 ssDNA는 나노 -드랍 (Nano— drop)에 1 ^를 넣고 ssDNA 농도를 측정한 후에 나머지 99 ^의 ssDNA 앱타머에 99 ^의 2X LBS를 첨가하여 85°C에서 5분간 가열하고 상온에서 1시간 이상 천천히 넁각하였다. 그 후에 DNA의 농도가 25 μ Μ이 되도톡 IX LBS에 희석하여 $비하였다. 8-3. SPR (Surface Pl asmon Resonance) 측정을 위한 비브리오 피셔리의 준비 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 μM and diluted in $ 1 to IX LBS. 8-3. Preparation of Vibrio Fisheries for Surface Pl asmon Resonance (SPR) Measurements
SPR 측정을 하기 위하여 실시예 2에 제시된 방법을 근거로 하여 비브리오 피셔리를 배양하였다. 이후 실시예 2에 따라 세척용액을 이용하여 생균 표면을 세척하였다. 실시예 9 : SPR측정을 통한 최적 DNA 앱타머의 선별  Vibrio Fisheries were incubated based on the method set forth in Example 2 to make SPR measurements. Thereafter, the living cell surface was washed using the washing solution according to Example 2. Example 9 Screening of Optimal DNA Aptamers by SPR Measurement
9- 1. 센서 칩 (Sensor chip) SA에 DNA 앱타머 고정  9-1.Fixing DNA Aptamers to Sensor Chip SAs
GE heal thcare에서 제공한 프로토콜 (protocol )에 제시된 방법에 의하여 금 (Gold) 판막 위에 텍스트란 (Dextran)으로 고정된 스트렙트아비딘을 활성화하였다. 활성화시킨 센서 칩 (Sensor chip) SA의 표면에 DNA 앱타머를 고정시키기 위해 시료의 유속을 10 /min으로 지정하였다. 센서 칩 (Sensor chip) SA의 4개 채널 중에서 1번은 검사용으로 앱타머를 붙이지 않았고, 2 , 3 , 4번의 채널에만 각각의 선별된 앱타머를 결합시켰다. DNA 앱타머의 결합 조건으로는 유속 10 z^/min으로 1분간 접종하는 일련의 과정을 각각 3회 반복 수행하였다. 그 후에 10분간 유속을 10 /min으로 HBS-EP 완층액 (buf fer )을 흘려주어 결합된 ssDNA 앱타머가 안정화되도록 하였다. 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. Of the four channels of 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.
9-2. 비브리오 피셔리의 결합 9-2. Vibrio Fisher's Bond
준비된 비브리오 피셔리를 배양한 배양액 1 을 튜브에 옮긴 후 센서 칩 (Sensor chip) SA 위에 유속 5 /min으로 20분간 홀려주는 일련의 과정을 2회 반복 수행하였다. 이 과정동안 1번 채널과 2， 3 , 4 채널의 Sensogram 비교， 분석을 통하여 결합력을 확인하였다. 이 후에 50 mM NaOH를 유속 10 /min으로 5분간 흘려주는 일련의 과정을 2회 반복 수행하여 결합된 비브리오 피셔리와 ssDNA 앱타머를 제거한 후에 상기의 과정과 동일하게 ssDNA 앱타머를 3회 결합시켰다 (도 5 참고) . 이 과정에서 최종 VPCA-03를 최적의 비브리오 피셔리 생균 결합 DNA 앱타머로 선별하였다 (표 2 참고) .  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).
【표 2】  Table 2
Figure imgf000025_0001
실시예 10 : 최적 비브리오 피셔리 생균 결합 DNA 앱타머의 특이성 평가
Figure imgf000025_0001
Example 10 Evaluation of Specificity of Optimal Vibrio Fisher Probiotic Binding DNA Aptamers
10-1 . 비브리오 파라해모라이티쿠스의 친화성 측정  10-1. Affinity Determination of Vibrio Parahaemoriticus
비브리오 피셔리와의 결합력 비교를 위하여 비브리오 파라해모라이티쿠스를 배양하였다. 비브리오 파라해모라이티쿠스의 배양은 상기 실시예 2에서 설명된 방법에 따라 행하였다. 그 후에 배양액 1 m을 튜브에 옮긴 후 센서 칩 (Sensor chip) SA 위에 유속 5 /min으로 20분간 흘려주는 일련의 과정을 2회 반복 수행하였다. 이 후에 50 mM NaOH를 유속 10 /min으로 5분간 홀려주는 일련의 과정을 2회 반복 수행하여 결합된 비브리오 파라해모라이티쿠스와 ssDNA 앱타머를 제거한 후에 상기의 과정과 동일하게 ssDNA 앱타머를 3회 결합시켰다.  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.
10-2. 리스테리아 모노사이토제네스의 친화성 측정 비브리오 피셔리와의 결합력 비교를 위하여 리스테리아 모노사이토제네스를 배양하였다. 리스테리아 모노사이토제네스의 배양은 상기 실시예 2에 설명된 방법에 따라 행하였다. 그 후에 배양액 1 m을 튜브에 옮긴 후 센서 칩 (Sensor chip) SA 위에 유속 5 /min으로 20분간 홀려주는 일련의 과정을 2회 반복 수행하였다. 이 후에 50 mM NaOH를 유속 10 / /min으로 5분간 흘려주는 일련의 과정을 2회 반복 수행하여 결합된 리스테리아 모노사이토제네스와 ssDNA 앱타머를 제거한 후에 상기의 과정과 동일하게 ssDNA 앱타머를 3회 결합시켰다. 10-3. 쉬겔라 소네이의 친화성 측정 10-2. Affinity Determination of Listeria monocytogenes Listeria monocytogenes was cultured for binding to Vibrio Fishery. Incubation of Listeria monocytogenes 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 procedures of 20 min of 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 performed twice to remove the combined Listeria monocytogenes and ssDNA aptamers, followed by three ssDNA aptamers in the same manner as above. Combined. 10-3. Affinity Measurement of Shigella Sonei
비브리오 피셔리와의 결합력 비교를 위하여 쉬겔라 소네이를 배양하였다. 쉬겔라 소네이의 배양방법은 상기 실시예 2에 설명된 방법을 따라 행하였다. 그 후에 배양액 1 을 류브에 옮긴 후 센서 칩 (Sensor chip) SA 위에 유속 5 /min으로 20 분간 흘려주는 일련의 과정을 2회 반복 수행하였다. 이 후에 50 mM NaOH를 유속 10 ^/min으로 5분간 흘려주는 일련의 과정을 2회 반복 수행하여 결합된 쉬겔라 소네이와 ssDNA 앱타머를 제거한 후에 상기의 과정과 동일하게 ssDNA 앱타머를 3회 결합시켰다. 10-4. 대장균의 친화성 측정  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
비브리오 피셔리와의 결합력 비교를 위하여 대장균을 배양하였다. 대장균의 배양은 상기 실시예 2에서 설명된 방법에 따라 행하였다. 그 후에 배양액 1 ^을 튜브에 옮긴 후 센서 칩 (Sensor chi p) SA 위에 유속 5 / /min으로 20분간 홀려주는 일련의 과정을 2회 반복 수행하였다. 이 후에 50 mM NaOH를 유속 10 £/min으로 5분간 홀려주는 일련의 과정을 2회 반복 수행하여 결합된 대장균과 ssDNA 앱타머를 제거한 후에 상기의 과정과 동일하게 ssDNA 앱타머를 3회 결합시켰다 (표 3 참고) .  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).
【표 3]  [Table 3]
표적 미생물 Ko 값  Target Microbe Ko Value
비브리오 피셔리 (Ύ. fischeri) 1.28e"8士 2.50 대장균 (E. coli) 4. 73e"8土 0. 15 리스테리아 모노사이토제네스 (L monocytogenes) 1.37e"5土 1.76 살모넬라 소네이 . soneii) 2. 17e"6土 0. 59 비브리오 파라해모라이티쿠스 ( V. parahaemolyticus) 4.75e"8土 3.33 실시예 11: 비브리오 피셔리의 신속한 검출을 위한 래피드 키트 (Rapid kit) 플랫폼 개발 Fibrio fisheries (Ύ.fischeri) 1.28e "8士 2.50 E. coli 4. 73e "8土 0. 15 Listeria monocytogenes 1.37e " 5土 1.76 Salmonella sonei. soneii) 2. 17e "6土 0. 59 Vibrio parahaemolyticus 4.75e " 8土 3.33 Example 11: Rapid kit platform for rapid detection of Vibrio fisheries
상기의 실시예를 통해 제작, 확보한 비브리오 피셔리 생균 특이 결합 DNA 앱타머의 산업적 활용을 위하여 비브리오 피셔리 생균 특이 결합 DNA 앱타머를 이용한 검출 방법 및 웅용 가능성을 확인하기 위하여 다양한 시료에서의 비브리오 피셔리의 검출 능력을 확인하기 위한 래피드 키트의 플랫품을 개발하였다. 래피드 키트는 스트렙트아비딘-바이오틴 결합을 이용할 수 있으며， 래피드 키트의 구성에 대한 간략한 모식도를 도 7에 나타내었다. 비브리오 피셔리의 래피드 키트를 이용하여 비브리오 피셔리를 검출하기 위하여 먼저 니트로셀를로오스 멤브레인에 비브리오 피셔리 생균 특이 결합 DNA 앱타머와 스트랩트아비딘을 고정하도록 설계하였다 (도 7(a) 참고) . 이후 비브리오 피셔리 생균이 포함된 시료를 처리하여 실제로 검출 가능한지 확인하기 위하여, 비브리오 피셔리 생균 특이 결합 DNA 앱타머의 5' -말단에 아민기를 치환하고 10 nm 카르복실화된 골드 나노 입자 (Carboxylated gold nanopart icles)에 아민결합 비브리오 피셔리 생균 특이 결합 DNA 앱타머를 고정하기 위해 NHS/EDC (Amine coupl ing ki t ) 완층움액을 넣어 1시간 동안 25°C에 처리한 후， 준비한 아민 결합 비브리오 피셔리 생균 특이 결합 DNA 앱타머와의 반응을 유도하여 확인할 수 있다. 본 과정으로 제작된 나노 입자에 고정된 비브리오 피셔리 생균 특이 결합 DNA 앱타머에 비브리오 피셔리가 포함되어있는 시료를 함께 흔합하여 1 시간 동안 251에서 처리한 후， 10X 로딩 완층액 ( loading buf fer , 2.5% Tri ton X-100 , 500 mM Tr i s-HCl , 10% Tween 20， 1.5M NaCl )를 IX가 되도록 흔합한 후， 샘플 패드에 로딩하여 처리하였다. 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. In order to detect the Vibrio fishery using the Vibrio Fisher's Rapid Kit, it was first designed to fix Vibrio Fisher's probiotic specific binding DNA aptamer and strapavidin to the nitrocell membrane (see FIG. 7 (a)). Subsequently, in order to check whether the sample containing Vibrio Fisher's live bacteria is actually detectable, 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 In order to fix amine-bound Vibrio Fisher's probiotic-specific binding DNA aptamer to nanopart icles, NHS / EDC (Amine coupling kit) complete supernatant was treated at 25 ° C for 1 hour, and then prepared amine-bound Vibrio Fisher It can be confirmed by inducing a reaction with probiotic specific binding DNA aptamers. 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.
시료 내에 비브리오 피셔리가 존재하는 경우 두 개의 선으로 나타나게 되며 (도 7(b) 참고) , 존재하지 않을 경우 하나의 선만 나타나도록 하였으며, 제작한 비브리오 피셔리 생균 검출 키트의 능력을 확인하기 위하여 시료를 넣지 않은 경우 비브리오 피셔리가 존재하지 않는 시료， 비브리오 피셔리가 존재하는 시료를 준비하여 컨트를 라인과 테스트 라인의 발색 여부를 확인할 수 있다. 상기의 실험을 통하여 제작한 비브리오 피셔리 검출 키트가 정상적으로 만들어진 것을 확인할 수 있게 설계하였다. 실시예 12 : 최적 비브리오 피셔리 생균 결합 DNA 앱타머에 항균물질인 락토페린올 접합시켜 비브리오 피셔리 제어 및 바이오필름 형성 억제능 확인 If 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
12-1. 비브리오 피셔리 생균 결합 DNA 앱타머와 락토페린 Oactoferr in)와 접합  12-1. Conjugation with Vibrio Fisher's Probiotic Binding DNA Aptamer and Lactoferrin Oactoferr in
비브리오 피셔리와 특이적으로 결합하는 DNA 앱타머와 락토페린을 접합시키기 위하여 아민기가 도입된 DNA 앱타머를 합성하였다. 합성된 DNA 앱타머는 바이오링커 (Biol inker)인 SPDP 시약 -3110( ^ 1^ 0^1 3-(2- pyr idyldithio) propionate, Thermo scient i f ic사)를 이용하여 양 말단을 활성화한 후 접합시켰다. 제조사 (Thermo scient i f i c)에서 제공한 프로토콜에 제시된 방법에 따라 SPDP 접합을 수행하였다. 락토페린의 농도는 10 rag/ ^로 고정하여 사용하였으며 표적 균주에 따른 농도 조절이 가능하다.  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.
12-2. 비브리오 피셔리 생균 배양액에 수정된 앱타머 처리 12-2. Modified Aptamer Treatment in Vibrio Fisher's Probiotic Cultures
비브리오 피셔리 생균을 실시예 2에 설명된 방법에 따라 배양하였다. 배양된 생균을 다시 3개의 100 ml의 LBS 배지 (medium)에 재접종하였으며 아무것도 첨가하지 않은 네가티브 대조군 (Negat ive control ) , 락토페린 및， 앱타머와 접합시킨 락토페린을 각각 첨가한 실험군을 비교하여 생장 억제를 확인하였다. 배양액들은 30°C 교반 배양기에서 24시간 동안 계속적으로 배양하고， 이 배양액들은 업계에서 통상적으로 사용하는 브래드포드 분석법을 이용하여 1시간 마다 O.D. (Opt ical Densi ty)를 측정하여 생장곡선으로 생장 억제 정도를 확인할 수 있다. 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.
12-3. 비브리오 피셔리 생장 억제 및 바이오필름 형성 억제 가능성 확인 12-3. Vibrio Fishery growth inhibition and biofilm formation potential Confirm
상기 실시예 12-2에서 수행하는 실험을 통해, 시간별로 측정된 O . D .를 이용하여 생장곡선을 도출하여 비브리오 피셔리의 생장이 억제되는 것을 확인할 수 있다. 본 실험에서는 앱타머와 접합시킨 락토페린을 첨가한 경우에서 가장 효과적으로 비브리오 피셔리의 생장이 억제되었고， 그 다음은 락토페린， 네가티브 대조군 (negat ive cont rol )의 순으로 측정되었다. 바이오필름의 형성량을 확인하기 위한 방법으로 크리스탈 바이올렛 (cryst al vi ol et )을 이용한 염색법을 수행하여 확인할 수 있다. 비브리오 피셔리를 5 ml LBS 배지 (medi um)에 접종하여 30 °C에서 밤새 배양한 후 0D600 값이 0. 1이 되도록 회석하여 96 웰 -플레이트에 200 ^씩 분주하였다. 24 시간 이상 층분히 배양한 후， 상층액을 제거하고 lx PBS를 이용하여 웰을 2-3회 세척하였다. 그 후, 1.0% 크리스탈 바이을렛 200 ^를 이용하여 20분간 염색하면 바이오필름 및 세포 추출물에 염색되었다. 염색된 웰을 lx PBS를 이용하여 2-3회 층분히 세척한 후 건조시켜 웰 표면에 부착된 바이오필름을 제외한 세포 추출물을 제거하였다. 100% 에탄을 200 ^를 이용하여 바이오필름에 염색된 크리스탈 바이을렛을 탈색하여 그 시료를 0D550 에서 측정하여 염색된 바이오매스를 정량화할 수 있었다. 본 실험에서는 생장억제와 유사하게 앱타머와 접합된 락토페린에서 가장 우수한 바이오필름 억제능을 가지는 것을 확인하였다. Through the experiment performed in 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. In this experiment, 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). As a method for confirming the formation amount of the biofilm, it may be confirmed by performing staining using crystal violet (cryst al vi ol et). 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. 100% ethane was used to decolorize the crystal vial stained on the biofilm using 200 ^ and the sample was measured at 0D 550 to quantify the dyed biomass. In this experiment, it was confirmed that lactoferrin conjugated with aptamer had the best biofilm inhibitory activity similar to growth inhibition.
.  .
실시예 13 : 최종 선별된 비브리오 피셔리 표면결합 DNA 앱타머의 바이오필름 형성 억제능의 산업적 활용을 위한 연구  Example 13 Study for Industrial Utilization of Biofilm Formation Inhibitory Activity of the Final Selected Vibrio Fisher Surface-bound DNA Aptamer
상기의 실시예들을 통해 제작 및 확보한 비브리오 피셔리 표면 결합 DNA 앱타머의 바이오필름 형성 억제능의 산업적 활용을 위하여 비브리오 피셔리가 감염된 환경에서의 비브리오 피셔리가 바이오필름 형성을 하는 초기 단계에 비브리오 피셔리 표면 결합 DNA 앱타머가 비브리오 피셔리의 생장을 억제하여 바이오필름을 제어하는 실험을 통하여 바이오필름 형성 억제에 대한 다양한 웅용 가능성을 확인하였다. 이를 통해 다양한 시료에서의 활용력을 이용하여 다양한 환경에서의 바이오필름의 제한이 가능할 것으로 기대된다. 이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바， 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며， 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다. Vibrio Fisher's surface at the initial stage of Vibrio Fisher's biofilm formation in Vibrio Fisher's infected environment for industrial utilization of biofilm formation inhibitory ability of Vibrio Fisher's surface binding DNA aptamer prepared and obtained through the above examples. Through experiments in which the binding DNA aptamer inhibits the growth of the Vibrio fishery to control the biofilm, various potentials for inhibition of biofilm formation were confirmed. Through this, it is expected that the limitation of biofilm in various environments can be made by utilizing the power of various samples. Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that this specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

【청구의 범위】 [Range of request]
【청구항 1】  [Claim 1]
비브리오 피셔리 ( /Z /o fischeri) 생균 표면에 특이적으로 결합하는 DNA 앱타머 .  Vibrio Fisher (/ Z / o fischeri) DNA aptamers that specifically bind to the surface of live cells.
【청구항 2】 [Claim 2]
제 1 항에 있어서， 상기 DNA 앱타머는 서열번호 1 내지 13 의 서열 중 어느 하나에 개시된 염기서열과 90% 이상의 동일성을 갖는 염기서열을 갖는 올리고뉴클레오타이드인 것을 특징으로 하는 DNA 앱타머.  The DNA aptamer according to claim 1, wherein the DNA aptamer is an oligonucleotide having a nucleotide sequence having 90% or more identity with a nucleotide sequence disclosed in any one of SEQ ID NOs: 1 to 13.
【청구항 3] [Claim 3]
제 1 항에 있어서， 상기 DNA 앱타머는 서열번호 1 내지 13 서열 중 어느 하나 개시된 염기서열을 갖는 을리고뉴클레오타이드인 것을 특징으로 하는 DNA 맵타머 .  According to claim 1, wherein the DNA aptamer is a DNA maptamer, characterized in that the ligonucleotide having a nucleotide sequence of any one of SEQ ID NO: 1 to 13.
【청구항 4] [Claim 4]
제 1 항에 있어서， 상기 DNA 앱타머는 5' 말단 또는 3' 말단에 바이오틴 (biotin), 아민기， 티을기， Cy3, Cy5, 플루오레세인 (fulorescein)， 또는 방사성 물질이 결합된 것을 특징으로 하는 DNA 앱타머 .  According to claim 1, wherein the DNA aptamer biotin (biotin), amine group, thiol group, Cy3, Cy5, fluorescein (fulorescein), or radioactive material is characterized in that the 5 'end or 3' end DNA aptamers.
【청구항 5] [Claim 5]
상기 제 1 항 내지 제 4 항 중 어느 한 항의 DNA 앱타머를 유효성분으로 포함하는 비브리오 피셔리 ( /b /o fischeri) 생균의 검출용 조성물.  A composition for detecting vibrio fishery (/ b / o fischeri) live bacteria comprising the DNA aptamer of any one of claims 1 to 4 as an active ingredient.
【청구항 6】 [Claim 6]
상기 제 1 항 내지 제 4 항 중 어느 한 항의 DNA 앱타머를 유효성분으로 포함하는 비브리오 피셔리 ( /v/o fischeri) 생균의 검출용 키트.  Kit for the detection of vibrio fishery (/ v / o fischeri) live bacteria comprising the DNA aptamer of claim 1 as an active ingredient.
【청구항 7】 제 6 항에 있어서, 상기 키트는 DNA 앱타머가 칩상에 고정화된 칩 형태인 것을 특징으로 하는 키트. [Claim 7] The kit of claim 6, wherein the kit is in the form of a chip in which DNA aptamers are immobilized on a chip.
【청구항 8】 [Claim 8]
게 6 항에 있어서， 상기 키트는 DNA 앱타머가 칩상에 고정화된 마이크로 어레이 형태인 것을 특징으로 하는 키트.  The kit of claim 6, wherein the kit is in the form of a micro array in which DNA aptamers are immobilized on a chip.
【청구항 9】 [Claim 9]
다음의 단계를 포함하는 비브리오 피셔리 ( /Ζν 'σ fischeri) 생균의 검출 방법 : Detection method of vibrio fishery (/ Ζν ' σ fischeri) live bacteria comprising the following steps:
(a) 비브리오 피셔리 ( /Zv o fischeri) 생균을 함유하는 것으로 예상되는 시료와 상기 게 1 항 내지 제 4 항 중 어느 한 항의 DNA 앱타머를 접촉시키는 단계; 및  (a) contacting the DNA aptamer of any one of claims 1 to 4 with a sample expected to contain vibrio fisher (/ Zv o fischeri) live bacteria; And
(b) 상기 DNA 앱타머와 결합된 비브리오 피셔리 ( /Zv/o fischeri) 생균을 확인하는 단계 .  (b) identifying the Vibrio fisher (/ Zv / o fischeri) viable bacteria bound to the DNA aptamer.
【청구항 10】 [Claim 10]
(i) 상기 제 1 항 내지 제 4 항 중 어느 한 항의 DNA 앱타머; 및 (i) the DNA aptamer of any one of claims 1 to 4; And
( ii) 상기 DNA 앱타머에 링커 (linker)를 통해 접합된 항균물질을 포함하는 DNA 앱타머 -항균물질 복합체. (ii) a DNA aptamer-antimicrobial complex comprising an antimicrobial conjugated to the DNA aptamer via a linker.
【청구항 111 [Claim 111]
상기 제 10 항 기재의 앱타머-항균물질 복합체를 비브리오 피셔리 ( /v/o fischeri) 생균을 함유하는 것으로 예상되는 시료와 접촉시키는 단계를 포함하는 비브리오 피셔리 ( /v/o fischeri)^ 생장을 억제하는 방법 .  Vibrio fishery (/ v / o fischeri) growth comprising contacting the aptamer-antimicrobial complex of claim 10 with a sample expected to contain vibrio fishery (/ v / o fischeri) bacteria How to suppress.
PCT/KR2015/011328 2014-11-06 2015-10-26 Dna aptamer specifically binding to surface of live cells of vibrio fischeri, and use thereof WO2016072653A1 (en)

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