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WO2015046737A1 - Aptamère d'adn se liant spécifiquement à la protéine prfa du facteur régulateur de virulence de listeria, et son utilisation - Google Patents

Aptamère d'adn se liant spécifiquement à la protéine prfa du facteur régulateur de virulence de listeria, et son utilisation Download PDF

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WO2015046737A1
WO2015046737A1 PCT/KR2014/007044 KR2014007044W WO2015046737A1 WO 2015046737 A1 WO2015046737 A1 WO 2015046737A1 KR 2014007044 W KR2014007044 W KR 2014007044W WO 2015046737 A1 WO2015046737 A1 WO 2015046737A1
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listeria monocytogenes
dna aptamer
prfa
prfa protein
aptamer
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PCT/KR2014/007044
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Korean (ko)
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김양훈
이상희
엄현주
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충북대학교 산학협력단
한국보건산업진흥원
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Publication of WO2015046737A1 publication Critical patent/WO2015046737A1/fr

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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the present invention relates to a DNA aptamer specifically binding to PrfA (virulence regulator PrfA), which is a pathogenic regulatory protein of Listeria monocytogenes that causes Listeriosis.
  • PrfA viral regulatory protein of Listeria monocytogenes that causes Listeriosis.
  • Listeriosis a common acquired disease, occurs when a person eats food contaminated with Listeria monocytogenes .
  • Prostate symptoms including fever, anxiety, and typical listeriosis, such as meningitis, sepsis or encephalitis, is a high-risk disease that threatens human life.
  • patients with immune diseases newborns and pregnant women are more than 20 times more likely to be infected than normal people.
  • infected women premature delivery, spontaneous abortion, stillbirth, and fetal infections occur, and infected mothers and fetuses have meningitis or endocarditis. It is known to cause serious complications and lead to death.
  • Listeria caused by Listeria monocytogenes is a fatal disease with the lowest morbidity but high mortality compared to other food poisoning bacteria. It is the PrfA protein that takes contaminated food and controls pathogenic outbreaks in the body. When activated, it causes pathogenicity in the body.
  • Listeria monocytogenes which causes fatal diseases, is the number of occurrences of health and environmental diseases caused by various pathogenic microorganisms, human infections and public due to the increase of imported livestock, processed processed foods and the increase of domestic average temperature due to global warming and extreme weather. The rate of transmission to the environment is increasing rapidly. Accordingly, there is a demand for developing a technology capable of effectively detecting Listeria monocytogenes contained in various foods such as processed foods, imported livestock products, dairy products, and drinking water.
  • PCR polymerase chain reaction
  • immunological diagnostic methods using antigen antibodies have been developed and used for the rapid and sensitive diagnosis of Listeriosis.
  • These methods target specific pathogenic factors of Shigella Sonei, which can detect specific causative organisms more sensitively and faster than conventional sample culture methods.
  • the main pathogenic factors mainly used for the diagnosis of listeriosis are proteins expressed by genes such as prfA, inlA, inlB, and actA.
  • PrfA encoded by the prfA gene is known. Proteins regulate the onset through quorum recognition of Listeria monocytogenes.
  • PCR and immunological diagnostic methods based on pathogenic genes and proteins encoded therein have the advantages of being more sensitive and faster than conventional culture-based detection methods.
  • Aptamers are short-length oligomers that form specific tertiary structures with high affinity to specific targets.
  • chemical synthesis techniques it is possible to mass-produce in a short time and at low cost, and has an excellent advantage of continuously producing aptamers having the same ability once produced and produced.
  • it is highly stable to the surrounding pH and temperature, and recently, the possibility of use in various fields, such as the detection of target substances and the development of disease diagnosis sensors, is widely appreciated.
  • the present inventors have succeeded in developing a DNA aptamer capable of early diagnosis of Listeriosis, and have succeeded in synthesizing and selecting a DNA Aptamer specifically binding to the PrfA protein that controls the pathogenicity of Listeriosis.
  • the present invention was completed by making a kit for early diagnosis of Listeriosis using the same.
  • Another object of the present invention is a Listeria monocytogenes detection or listeria monocytogenes ( Listeria monocytogenes) comprising a DNA aptamer that specifically binds to the PrfA protein as an active ingredient, Listeria monocytogenes detection or diagnostic composition, diagnostic kit and diagnostic bio To provide a chip.
  • It is still another object of the present invention to provide a method for preparing a biologically active product comprising (a) contacting a biological sample isolated from a living body with a DNA aptamer that specifically binds to the PrfA protein of Listeria monocytogenes ; And (b) confirming the presence of Listeria monocytogenes through a PrfA protein specific binding reaction between the biological sample and the DNA aptamer.
  • Listeria monocytogenes detection method comprising: It is to provide the information necessary for the diagnosis of Listeriosis.
  • the present invention provides a PrfA protein specific DNA aptamer that specifically binds to the PrfA protein of Listeria monocytogenes.
  • the DNA aptamer is an oligonucleotide having a nucleotide sequence having at least 90% identity with any one nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 7 to SEQ ID NO: 19 Can be.
  • the DNA aptamer may be an oligonucleotide having any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 7 to SEQ ID NO: 19.
  • the DNA ampamer may further comprise a labeling material.
  • the DNA aptamer may be one of the labeling material selected from the group consisting of fluorescent material, amine group, biotin and thiol group is labeled at the 5 'end or 3' end of the DNA aptamer. have.
  • the DNA aptamer is any one of the group consisting of a hydrogen atom, a fluorine atom, -OR, -OCOR and a hydroxyl group of the ribose 2 'position of one or more nucleotides constituting the DNA aptamer It may be substituted with one.
  • the present invention also provides a composition for detecting Listeria monocytogenes or diagnosing Listeria, comprising as an active ingredient a DNA aptamer that specifically binds to PrfA protein of Listeria monocytogenes .
  • the present invention provides a kit for detecting Listeria monocytogenes detection or Listeria syndrome comprising DNA aptamer specifically binding to PrfA protein of Listeria monocytogenes as an active ingredient.
  • the present invention is for detecting Listeria monocytogenes detecting or diagnosing Listeriosis in which a DNA aptamer that specifically binds to the PrfA protein of Listeria monocytogenes is integrated on a substrate and specifically reacts to a sample containing the PrfA protein.
  • a DNA aptamer that specifically binds to the PrfA protein of Listeria monocytogenes is integrated on a substrate and specifically reacts to a sample containing the PrfA protein.
  • the substrate may be selected from the group consisting of paper, plastic, glass, metal and silicon.
  • the present invention comprises the steps of (a) contacting a biological sample isolated from the living body to a DNA aptamer that specifically binds to the PrfA protein of Listeria monocytogenes ; And (b) confirming the presence of Listeria monocytogenes through a PrfA protein specific binding reaction between the biological sample and the DNA aptamer.
  • Listeria monocytogenes detection method comprising a to provide.
  • the present invention comprises the steps of (a) contacting a biological sample isolated from the living body to a DNA aptamer that specifically binds to the PrfA protein of Listeria monocytogenes ( Listeria monocytogenes ); And (b) confirming the presence or absence of Listeria monocytogenes through a PrfA protein-specific binding reaction between the biological sample and the DNA aptamer.
  • the information necessary for diagnosing Listeriosis includes Provide a way to provide.
  • the DNA aptamer according to the present invention has an activity of specifically binding to Listeria monocytogenes PrfA protein, thereby early detection of Listeria monocytogenes, a harmful bacterium existing in the natural environment, food and human body, and premature listeriosis. This can be useful for diagnosis.
  • the DNA aptamer of the present invention has a high specificity for the PrfA protein of Listeria monocytogenes can increase the sensitivity of the diagnosis, and can be effectively used economically in the mass production of the same quality aptamer at a low cost could be.
  • Lane 1 is a result of transducing the expression vector containing the Listeria monocytogenes prfA gene into E. coli and expressing the GST fusion PrfA protein produced therefrom, and confirmed by SDS-PAGE (10%).
  • Lane M protein size marker (Elpis, Korea) (lane 1: water soluble protein (1 mM IPTG induction, 18 ° C. culture), lane 2: pellet protein (1 mM IPTG induction, 18 ° C. culture), lane 3: water soluble protein (18 ° C.) culture), lane 4: pellet protein (18 ° C culture))
  • Figure 2 shows the results of amplifying the random DNA aptamer using the PCR technique and the result of the ssDNA production using the heat-cooling (Heating-cooling) technique by comparing the result by 10% acrylamide gel electrophoresis.
  • Lane M 100 bp DNA marker (Elpis, Korea)
  • Lane 1 DNA aptamer was amplified by PCR technique.
  • Lane 2 DNA aptamer pool obtained by PCR amplification was heated-cooled to ssDNA. As a result)
  • Figure 3 shows the SELEX process for the production of DNA aptamers specifically binding to Listeria monocytogenes PrfA protein.
  • Figure 4 is a quantitative measurement of the concentration of Listeria monocytogenes PrfA protein specific binding DNA aptamer candidates eluted in each SELEX round after the SELEX process to produce Listeria monocytogenes PrfA protein specific binding DNA aptamer using a Nanodrop spectrophotometer One result.
  • Figure 5 illustrates a method for evaluating the binding capacity of Listeria monocytogenes PrfA protein and Listeria monocytogenes PrfA protein specific binding DNA aptamer.
  • each process of activating the surface of the carboxyl sensor chip, CM5, through EDC / NHS buffer solution and fixing Listeria monocytogenes PrfA protein to bind to Listeria monocytogenes PrfA protein specific binding DNA aptamer Is a diagram showing.
  • LMPA5 Listeria monocytogenes PrfA protein specific binding DNA aptamer
  • LMPA7 SPR measurement sensorgram (Sensorgram) showing the binding capacity of Listeria monocytogenes PrfA protein by concentration.
  • LMPA9 Listeria monocytogenes PrfA protein specific binding DNA aptamer
  • D Listeria monocytogenes PrfA protein specific binding DNA aptamer
  • Sensorgram SPR measurement sensorgram showing the binding capacity of Listeria monocytogenes PrfA protein by concentration.
  • FIG. 7 is a diagram showing the secondary structure of a total of four Listeria monocytogenes PrfA protein specific binding DNA aptamers selected as having a Listeria monocytogenes PrfA protein specific binding ability.
  • FIG. 8 is a schematic diagram of a diagnostic kit for Listeriosis using an aptamer specifically binding to Listeria monocytogenes PrfA protein.
  • Panel (a) is a diagram showing the configuration of a simple diagnostic kit capable of early detection of Listeria monocytogenes PrfA protein, and panel (b) shows Listeria monocytogenes PrfA for early detection of Listeria monocytogenes PrfA protein.
  • This is a schematic of the principles of a simple kit for diagnosing Listeria and detecting Listeria monocytogenes using protein-specific binding DNA aptamers.
  • the present invention relates to DNA aptamers that specifically bind to PrfA proteins that regulate listeriosis.
  • DNA aptamer refers to a DNA nucleic acid molecule capable of binding to a specific molecule with high affinity and specificity.
  • DNA aptamer is used interchangeably with “DNA oligonucleotide”.
  • oligonucleotide generally refers to a nucleotide polymer having less than about 200 lengths, which may include DNA and RNA, and is preferably a DNA molecule.
  • a nucleotide may be any substrate that can be introduced into a polymer by deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or by DNA or RNA polymerase or by synthetic reactions. If modifications to the nucleotide structure are present, such modifications may be added before or after the synthesis of the oligonucleotide polymer.
  • Nucleotide sequences can be interrupted by non-nucleotide components. Oligonucleotides can be further modified after synthesis, for example by binding to a label.
  • 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.
  • organic molecules, nucleotides, amino acids, polypeptides, marker molecules on the cell surface, ions, metals, salts, polysaccharides can be suitable target molecules that separate aptamers that can specifically bind to each ligand.
  • Screening of aptamers can utilize in vivo or in vitro selection techniques known as the Systematic Evolution of Ligands of Exponential enrichment (SELEX) method (Ellington et al., Nature 346, 818-22, 1990; and Tuerk et al.
  • SELEX Systematic Evolution of Ligands of Exponential enrichment
  • SELEX method refers to a method of determining a DNA binding sequence of a molecule by selecting and amplifying a DNA having a high binding strength to a specific molecule in a randomly synthesized DNA set (Louis et al., 1992. Nature 355,564-566). Specific methods for the selection and preparation of aptamers are described in US Pat. No. 5,582,981, WO 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.
  • the DNA aptamer of the present invention is selected through the following steps; (i) extracting and expressing the prfA gene in Listeria monocytogenes; (ii) amplifying DNA aptamers using PCR techniques and preparing ssDNA aptamers through heat treatment and streptavidin binding; (iii) screening DNA aptamers that specifically bind to PrfA protein after immobilizing PrfA, a listeriosis regulatory protein, on an immobilization column (see FIG. 3); (iv) analyzing specificity of selected DNA aptamers.
  • PrfA which is a listeriosis pathogenic regulatory protein
  • genomic DNA is extracted from listeria monocytogenes and amplified genes encoding PrfA protein.
  • Primers are prepared using the sequence information of prfA gene, which is known from the National Center for Biotechnology Information (NCBI) database, and amplified using PCR technique.
  • the amplified prfA gene is treated with the same restriction enzyme as the protein expression vector pGEX-4T-1, ligation and transformed into E. coli BL21.
  • Recombinant transformants are cultured in LB broth and treated with IPTG to induce overexpression of PrfA protein encoding pathogenic regulatory proteins.
  • the overexpressed PrfA protein is purified using Glutathione-S-Transferase (GST) protein, a fusion protein present in pGEX 4T-1, to obtain pure PrfA protein.
  • GST Glutathione-S-Transferase
  • the method for screening ssDNA aptamer for example, amplifies dsDNA by attaching biotin to a reverse primer during PCR, induces the amplification product to fall to a single strand, and then treats streptavidin. Only the ssDNA aptamer can be selected by forming a biotin-streptavidin complex to selectively remove the complex. The obtained ssDNA aptamer can be confirmed by electrophoresis on acrylamide gel (see Fig. 1). The prepared ssDNA aptamer is heated for denaturation for use in the SELEX method and then slowly reacts at room temperature to induce the formation of a three-dimensional structure.
  • the ssDNA aptamer and PrfA protein prepared above react with each other by inducing contact with each other, and the ssDNA aptamer which does not bind is washed and removed, and only ssDNA that specifically binds to PrfA protein is eluted.
  • This step may include a Negative SELEX step to remove ssDNA that binds to conditions other than PrfA, such as protein immobilization resin and buffer components.
  • the eluted ssDNA was selected from the nano-Drop spectrophotometer (Thermo Scientific.) To select an optimal SELEX round in which DNA aptamers specifically binding to the listeriosis control protein PrfA were eluted. Can be quantified using.
  • each of the aptamer sequences is secured for the selection of the PrfA protein-specific binding DNA aptamer during the optimal SELEX round to confirm the similarity between the obtained sequences.
  • the acquired sequences can be analyzed for homology between the sequences using the Cluster X program.
  • the DNA aptamer of the present invention may include any DNA aptamer that binds to the PrfA protein, but preferably may be an oligonucleotide having a nucleotide sequence disclosed in any one of SEQ ID NOS: 7-19.
  • DNA aptamer of the present invention is to include an oligonucleotide having a nucleotide sequence showing a substantial identity to the base sequence of any one of SEQ ID NO: 7 to 19, while maintaining the property of binding specifically to PrfA, a listeriosis regulator protein Interpreted
  • the substantial identity above aligns the nucleotide sequence of the present invention with any other sequence to the maximum correspondence, and the algorithm 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 Lipman, Methods in Mol. Biol.
  • the "% sequence homology" for a polynucleotide is determined by comparing the comparison regions with two optimally arranged sequences, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
  • surface plasmon resonance may be used to quantitatively provide the binding force of the listeriosis regulatory protein PrfA and the DNA aptamer specifically binding thereto.
  • the Biacore 3000 a device capable of measuring SPR-based binding forces, is used to process EDC / NHS on a commonly used CM5 chip and react with Ethanolamine to encode an unactivated gold surface. After fixing the PrfA protein having an amine group, it is possible to measure the binding force with the PrfA protein while inducing the reaction by flowing the each aptamer secured.
  • the DNA aptamer belonging to SEQ ID NO: 7 sequence 19 of the present invention that binds with a high affinity to the PrfA protein through the above experiment can form a secondary structure shown in FIG.
  • the invention also provides compositions and methods for diagnosing Listeria monocytogenes PrfA specific binding DNA aptamer based Listeriasis and detecting Listeria monocytogenes.
  • the detection of the PrfA protein is based on the method for detecting the binding complex of the DNA aptamer and the PrfA protein proposed in the present invention.
  • the DNA aptamer may be a coloring material, for example, a fluorescent material such as fluorescein, Cy3, or Cy5, and may include nanoparticles; Nucleotides modified with radioactive substances or chemicals such as biotin or modified to have primary amines, thiol groups can be included in the DNA aptamers.
  • the DNA aptamers of the invention can be biotinylated, for example, which can be successfully immobilized on a streptavidin coated substrate.
  • the DNA aptamer of the present invention immobilized on a substrate can bind to and capture the PrfA protein, and the captured PrfA protein can be visualized by using a DNA aptamer that specifically binds to the PrfA protein. have.
  • the present invention also relates to a method for detecting diseased PrfA protein of Listeria monocytogenes comprising the steps of: (a) reacting the DNA aptamer with a sample expected to contain PrfA protein; (b) identifying the DNA aptamer bound to the protein PrfA protein. Since the DNA aptamer of the present invention has a property of specifically binding to PrfA protein in Listeria monocytogenes, preferably Listeria monocytogenes, early detection of Listeria monocytogenes is possible.
  • the present invention provides a sensor for diagnosing Listeriosis comprising the DNA aptamer as an active ingredient.
  • the DNA aptamer may be immobilized on a chip or a substrate, and specifically, the DNA aptamer may be in the form of a microarray immobilized on a substrate.
  • microarray refers to a microarray in which oligonucleotide groups are immobilized at a high density on a substrate, and the oligonucleotide groups are immobilized in a predetermined region, respectively.
  • microarrays are well known in the art. For example, microarrays are disclosed in US Pat. Nos.
  • microarray refers to specific regions of the substrate. Refers to an array (array) to which DNA nucleic acid material is attached at a high density.
  • substrate of a microarray is a support having suitable rigidity or semi-rigidity, where “substrate” refers to any substrate to which a DNA aptamer can be attached under conditions where the background level of binding is kept low.
  • 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 that have been 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 aptamers of the present invention can be biotinylated, for example, which can be successfully bound onto a substrate that has been encoded with strapavidin.
  • the sensor of the present invention is a sensor for use in detecting Listeria monocytogenes PrfA protein in a sample can be used for the diagnosis of Listeriosis, and the use of a sensor chip for the diagnosis and the instructions or labels for using the sensor further May contain
  • the present invention provides a diagnostic kit for detecting the presence of PrfA protein of Listeria monocytogenes in patients and biological samples suspected of Listeria infection to provide information necessary for diagnosing Listeria (see FIG. 8).
  • biological sample may include blood, saliva, tear fluid, urea, vigor, mucus, cells, tissues, and other tissues and body fluids, as well as cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems. It also includes, but is not limited to, water samples and food in the environment.
  • the present invention provides a diagnostic method for a kit for early diagnosis of Listeriosis using DNA aptamer as an active ingredient: (a) containing a target substance in a first aptamer fixed to a solid phase and specifically binding to the target substance Reacting by adding a second aptamer specifically bound to the sample and the target material and to which a labeling substance is attached; And (b) analyzing the label to detect the target.
  • the "solid phase” is a solid support in which the aptamer is fixed, and the shape or material is not limited as long as the aptamer can be fixed.
  • nanoparticles that can be identified with the naked eye, but the color factor is not limited to nanoparticles having ordinary skill in the art. Will be self-evident.
  • multi-well type microplates can be generally used for the convenience of performing analytical methods, but other shapes such as sensor chips, plastics, polypropylene, or columns filled with beads such as Sepharose or Agarose can also be used.
  • a fluorescent material is used as a label, emission or color change occurs in the presence of the target material, and thus the target material can be detected by measuring the same.
  • an image scanner capable of detecting a fluorescent dye may be scanned to determine whether a target substance is detected by scanning a well that caused a reaction, and the amount of detection may be measured by measuring the degree of thickening of the image through software.
  • the method according to the invention may be provided in the form of a kit, in order to increase portability. That is, in another aspect, the present invention includes a detection reagent containing a solid phase in which a first aptamer specifically binding to a target material is immobilized on a solid phase, and a second aptamer specifically binding to the target material.
  • the present invention relates to a kit for detecting a target substance.
  • the detection reagent containing the second aptamer may be provided in a separate container or in a reaction part to perform sandwich bonding.
  • the detection kit may include a detection buffer solution and the like, and may further include a tool for mixing the detection reagent and a sample solution to be detected.
  • the optimal Listeria monocytogenes PrfA protein binding DNA aptamer according to the present invention can be utilized for early detection of Listeria in the medical field and the environment and food industry for the diagnosis of Listeriosis.
  • a recombinant expression vector including a Listeria monocytogenes prfA gene was constructed as a first step for the production of recombinant strains.
  • a pair of primers containing a gene sequence and a restriction enzyme site were prepared by Bioneer, Korea. [Forward primer: 5'-ATTGTCGACAGATGAACGCTCAAGCAGAA-3 ', (SEQ ID NO: 1), reverse primer: 5'-ATAGCGGCCGCATTTAATTTTCCCCAAGTA-3', (SEQ ID NO: 2)].
  • the Listeria monocytogenes prfA gene was amplified and the reaction composition was 1 to 2 ⁇ l of template DNA, 5 ⁇ l of 10X PCR buffer, 4 ⁇ l of each 2.5 mM dNTP mixture, 2 ⁇ l of 25 uM forward primer and 25 ⁇ M reverse primer. 2 ⁇ l, Ex Taq polymerase (TaKaRa, Japan) 0.3 ⁇ l (1 unit / ⁇ l) and 35.7 to 34.7 ⁇ l of water. PCR reaction conditions were first denatured at 94 ° C. for 5 minutes, repeated 30 cycles at 94 ° C. for 30 seconds, 65 ° C. for 30 seconds, and 72 ° C.
  • the amplified Listeria monocytogenes prfA gene and pGEX 4T-1 vector were cut with the same restriction enzyme (SalI / NotI), and then ligated to prepare a recombinant expression vector.
  • Recombinant expression vector induces transformation in E. coli BL21 hosts by electroporation, and selects colonies that are resistant to ampicillin and chloramphenicol antibiotics to select recombinant Listeria monocytogenes prfA gene. An expression vector having was obtained.
  • PrfA protein was induced by using the Lac operon system in a recombinant expression vector containing a prfA gene.
  • Culture conditions at the time of passage were added 10 ml LB broth, 1 mM IPTG (isopropylthio- ⁇ -D-galactoside).
  • IPTG isopropylthio- ⁇ -D-galactoside
  • 100 ⁇ l of transformed E. coli BL21 was inoculated into 10 ml of LB broth, and incubated at 18 ° C. for 10 hours.
  • IPTG was 0 mM, 0.1 mM, and 1 mM, respectively. IPTG induction to be incubated for 20 hours at 18 °C.
  • the cell culture was centrifuged at 13,000 rpm for 10 minutes at 4 ° C to separate only cells.
  • the cells obtained through centrifugation were resuspended in 10 mM Tris-HCl (pH 8.0) and centrifuged again to wash the cells. Thereafter, the cells were disrupted using an ultrasonic grinder, and then centrifuged at 13,000 rpm for 10 minutes at 4 ° C to separate soluble proteins and insoluble proteins. Each result was confirmed using a 10% SDS-PAGE gel (see Fig. 1).
  • the site of 5 ⁇ -ATACCAGCTTATTCAATT and 3 ⁇ -AGATTGCACTTACTATCT are composed of 40 sites at the center to easily amplify the aptamer pool at both ends.
  • a biotinylated reverse primer to be used for the purpose of recovering amplified forward primer and single-stranded DNA was ordered to Bioneer, Korea.
  • PCR reaction composition for amplification of 76 bp DNA library includes 5 ⁇ l of 10X PCR buffer, 4 ⁇ l of each 2.5 mM dNTP mixture, 2 ⁇ l of 10 pM forward primer (SEQ ID NO: 4), and a biotinylated reverse primer (SEQ ID NO: 6). ) 2 ⁇ l, template DNA library (SEQ ID NO: 3) 1-2 ⁇ l, Ex Taq polymerase (TaKaRa, Japan) 0.3 ⁇ l (1 unit / ⁇ l) and distilled water 34.7-35.7 ⁇ l.
  • PCR reaction conditions were first denatured at 94 ° C. for 5 minutes, followed by 20 cycles of reaction for 30 seconds at 94 ° C., 30 seconds at 52 ° C., and 30 seconds at 72 ° C., followed by further extension at 72 ° C. for 5 minutes. Was used.
  • 3 ⁇ l 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 a PCR purification kit (Qiagen, USA). The recovered aptamer pool was confirmed by using a 10% acrylamide gel to show the band of the correct size (see Figure 2, lane 1).
  • dsDNA was denatured to ssDNA using a heating-cooling technique. Specifically, the dsDNA obtained in Example was reacted at 85 ° C. for 5 minutes to denature the dsDNA to ssDNA, and immediately after the reaction was completed, the reaction solution was cooled to 4 ° C. to prepare ssDNA.
  • the strept was added to 100 ul of the reaction solution obtained in the present example.
  • 50 ⁇ l of avidin agarose resin (Streptavidin agarose resin, Thermo Scientific, USA) was added and reacted at room temperature for 1 hour.
  • the supernatant was recovered by centrifugation at 13,000 rpm for 15 minutes at. 1/100 volume of tRNA (sigma aldrich, USA), 1/10 volume of 3 M sodium acetate (pH 4.5) and 3 volumes of 100% ethanol were added to the supernatant for at least 1 hour at -70 ° C. I was. After the reaction was centrifuged for 20 minutes at 13,000 rpm at 4 °C to recover only ssDNA. The recovered ssDNA was dried at 65 ° C. and then dissolved in 50 ⁇ l of distilled water. 10 ⁇ l of the recovered ssDNA was taken, and 10% acrylamide gel was used to confirm that the band of the correct size appeared compared to dsDNA (see FIG. 2 and lane 2).
  • Each solution used in SELEX was used a GST bulk kit (GE healthcare, UK), the composition was as follows. Namely, 1 X aptamer selection solution: 10 mM PBS (pH 7.4), 2 X aptamer selection solution: 20 mM PBS (pH 7.4), washing solution: 1 X aptamer selection solution, DNA aptamer elution solution: 50 mM Tris-HCl (pH 8.0), 10 mM glutathione.
  • Example 3.2 10 ⁇ l of a GST fusion IpaH protein obtained through the method described in Example 1 was prepared in Example 3.2. After mixing with the ssDNA aptamer pool obtained through the method described in the section, the total reaction volume was adjusted to 100 ⁇ l with 1 X aptamer selection solution, and then reacted at 4 ° C. for at least 12 hours.
  • 200 ml of glutathione sepharose 4B was added with 1 ml of 1 X PBS, stirred at 4 ° C. for 20 minutes, and reacted, followed by centrifugation at 4 ° C. at 13,000 rpm for 10 minutes. The supernatant was removed.
  • reaction solution of the GST fusion PrfA protein and the ssDNA aptamer pool was reacted with activated glutathione Sepharose 4B at 4 ° C. for 1 hour with stirring. Thereafter, the supernatant was removed by centrifugation at 13,000 rpm for 10 minutes at 4 ° C., followed by three times of washing with 1 ml of 1 ⁇ PBS to remove ssDNA aptamer that did not bind with GST fusion PrfA protein.
  • DNA aptamer elution solution 50 mM Tris-HCl (pH 8.0), 10 mM glutathione
  • 100 ⁇ l of DNA aptamer elution solution 50 mM Tris-HCl (pH 8.0), 10 mM glutathione
  • the reaction was stirred for 30 minutes at, followed by centrifugation at 13,000 rpm for 10 minutes at 4 ° C. to obtain an upper elution solution. This procedure was repeated twice to obtain an eluting solution of the upper layer.
  • SELEX was carried out up to 10 times in the same manner as in 3.3.
  • the binding conditions of SELEX decreased the reaction time of ssDNA aptamer pool and GST fusion PrfA protein as the number of times increased. To obtain an aptamer that binds more specifically to the target material PrfA protein.
  • the concentration of ssDNA bound to PrfA protein, a pathogenic regulator of Listeria monocytogenes recovered in each round was measured. Measurement was performed using a Nano-drop spectrophotometer (Thermo Scientific). As a result of measuring the concentration of ssDNA aptamer eluted in each round, the highest concentration of 87 rounds was 877.0 ng / ⁇ l, and the concentration of 10 rounds was 467.8 ng / ⁇ l. Through this, it was confirmed that the optimal aptamer pool that specifically binds to the pathogenic regulatory protein PrfA of Listeria monocytogenes is a 9 round pool (see FIG. 4).
  • Cloning was performed by mixing 1 ⁇ l of T-vector (10 ng / ⁇ l), 4 ⁇ l of PCR product (20 ng / ⁇ l), and 1 ⁇ l of 6 ⁇ T-blunt buffer and reacting at 25 ° C. for 5 minutes. 10 ⁇ l of the ligate TA cloned was mixed with 100 ⁇ l of DH5 ⁇ and subjected to a heat shock at 42 ° C. for 1 minute and 30 seconds.
  • Nhydroxysuccinimide (NHS) and 0.2 M Nethyl-N '-(dimethylaminopropyl) carbodiimide (EDC) mixed solution were flowed into the sensor chip CM5 for 10 minutes at a rate of 10 ⁇ l / min to make the carboxyl group on the surface of the sensor chip more reactive.
  • Activated with hydroxysuccinimide ester N-Hydroxy-succinimide ester; NHS-ester).
  • PrfA protein-binding DNA aptamer candidates obtained for screening aptamers having the highest affinity with PrfA protein were dissolved in HBS-EP buffer (GE Healthcare, UK) at concentrations of 300 nM, 500 nM, 1000 nM and 2000 nM, respectively. Ready.
  • the prepared PrfA binding DNA aptamer is bound to PrfA of various concentrations (300 nM, 500 nM, 1000 nM, 2000 nM) to a sensor chip (channel 1) to which nothing is bound, and a sensor chip (channel 2) to which PrfA protein is fixed.
  • Affinity between the PrfA protein and the DNA aptamer candidate group specifically binding thereto was quantified by injecting the DNA aptamer candidate group.
  • Rensselear polytechnic institute provides the structure of LMPA-5, LMPA-7, LMPA-9, and LMPA-12, among the PrfA-specific binding DNA aptamer candidates of Listeria monocytogenes, with high affinity to the PrfA protein. Imaging was possible using the DNA mfold program (see FIG. 7).
  • the present invention is reported in the form of Rapid test kit. Diagnostic method was used.
  • a membrane was produced to show the results.
  • the membrane is composed of a sample pad to react the sample and an absorbing pad to draw a solution of the sample pad so that the sample can react with the membrane to form a line on the membrane. It consists of capture line and control line.
  • the control line was prepared to fix the streptavidin to confirm that the reaction occurred even if the PrfA protein was not present in the sample to form a control line by combining the sample with the biotinylated aptamer and nanoparticle complexes. .
  • PrfA protein-specific binding aptamer LMPA-9 was fixed in front of the control line of the membrane. If PrfA protein is present in the sample, the band is designed to appear in the capture line. The relevant figure is shown in detail in FIG. 8.
  • the Listeria simple detection or diagnostic kit using PrfA protein specific binding aptamer is used for the detection of Listeria monocytogenes using streptavidin-biotin binding to LMPA-9 having high affinity with PrfA protein in Example 5. Biotin was labeled at the 3 'end, and an amine group was labeled at the 5' end. Then, LMPA-9 modified at both ends is fixed to the membrane coated with streptavidin obtained through the above process using biotin labeled at the 3 ′ end. After mixing LMPA-12 and Listeria monocytogenes with a sample that can be colored and nanoparticles that can be colored, drop them onto the sample pad of the LMPA-9 immobilized membrane.

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Abstract

La présente invention concerne l'aptamère d'ADN se liant spécifiquement à la protéine PrfA, qui est un facteur régulateur de virulence de Listeria, et son utilisation, et plus précisément un procédé pour la détection de Listeria monocytogenes par utilisation d'un aptamère d'ADN se liant spécifiquement à la protéine PrfA de Listeria monocytogenes, et sur une composition pour le diagnostic de la listériose. L'aptamère d'ADN selon la présente invention a une activité de liaison spécifique à la protéine PrfA de Listeria monocytogenes et peut donc être utilisé pour la détection précoce de Listeria monocytogenes, qui est la bactérie nocive présente dans l'environnement naturel classique, les aliments et l'organisme humain, et permet le diagnostic précoce d'une listériose. En outre, l'aptamère d'ADN de la présente invention a une grande spécificité pour la protéine PrfA de Listeria monocytogenes, ce qui améliore la sensibilité du diagnostic et la production en grande série d'une même qualité de l'aptamère pour un faible coût, de sorte qu'il peut être utilisé d'une manière efficace d'un point de vue économique.
PCT/KR2014/007044 2013-09-26 2014-07-31 Aptamère d'adn se liant spécifiquement à la protéine prfa du facteur régulateur de virulence de listeria, et son utilisation WO2015046737A1 (fr)

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KR102108432B1 (ko) * 2018-12-12 2020-05-08 주식회사 세니젠 리스테리아 모노사이토제니스와 리스테리아 이노쿠아의 동시 검출을 위한 프라이머세트 및 이를 포함하는 중합효소연쇄반응 키트

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KR20100129709A (ko) * 2009-06-01 2010-12-09 성균관대학교산학협력단 췌장암 세포 또는 조직에 특이적으로 결합할 수 있는 핵산 압타머 및 그 용도
KR20120092938A (ko) * 2011-02-14 2012-08-22 충북대학교 산학협력단 알파태아단백질에 특이적으로 결합하는 ｄｎａ 앱타머 및 이의 용도
KR20120133408A (ko) * 2011-05-31 2012-12-11 충북대학교 산학협력단 리스테리아 모노사이토제네스 생균의 표면에 특이적으로 결합하는 ｄｎａ 앱타머 및 이의 용도

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KR20100101248A (ko) * 2009-03-09 2010-09-17 이기성 리스테리아 오염 탐지용 특이단백질 부위 및 그에 대한 항체제조법과 활용
KR20100129709A (ko) * 2009-06-01 2010-12-09 성균관대학교산학협력단 췌장암 세포 또는 조직에 특이적으로 결합할 수 있는 핵산 압타머 및 그 용도
KR20120092938A (ko) * 2011-02-14 2012-08-22 충북대학교 산학협력단 알파태아단백질에 특이적으로 결합하는 ｄｎａ 앱타머 및 이의 용도
KR20120133408A (ko) * 2011-05-31 2012-12-11 충북대학교 산학협력단 리스테리아 모노사이토제네스 생균의 표면에 특이적으로 결합하는 ｄｎａ 앱타머 및 이의 용도

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