WO2015166689A1 - Molécule d'acide nucléique qui se lie à un allergène de crevette et son application - Google Patents

Molécule d'acide nucléique qui se lie à un allergène de crevette et son application Download PDF

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WO2015166689A1
WO2015166689A1 PCT/JP2015/054536 JP2015054536W WO2015166689A1 WO 2015166689 A1 WO2015166689 A1 WO 2015166689A1 JP 2015054536 W JP2015054536 W JP 2015054536W WO 2015166689 A1 WO2015166689 A1 WO 2015166689A1
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nucleic acid
acid molecule
allergen
shrimp
shrimp allergen
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PCT/JP2015/054536
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English (en)
Japanese (ja)
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朋子 横山
行大 白鳥
克紀 堀井
宏貴 皆川
穣 秋冨
金子　直人
嘉仁 吉田
巌 和賀
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Ｎｅｃソリューションイノベータ株式会社
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Priority to JP2016515877A priority Critical patent/JP6399611B2/ja
Publication of WO2015166689A1 publication Critical patent/WO2015166689A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/40—Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to nucleic acid molecules that bind to shrimp allergens and uses thereof.
  • Shrimp is a food that is frequently consumed on a daily basis, but in recent years, the number of shrimp allergies has increased and is regarded as a problem.
  • processed foods that use shrimp as raw materials and shrimp are foods for seafood, so processed foods made from seafood may contain shrimp components. For this reason, it is extremely important to analyze whether shrimp are mixed as a raw material in processed foods and production lines thereof.
  • Non-Patent Document 1 Non-Patent Document 2
  • an antibody is a protein and has a problem in stability, it is difficult to use the antibody for a simple test method at a low cost.
  • an object of the present invention is to provide a new nucleic acid molecule that can be used for the detection of shrimp allergen.
  • the shrimp allergen-binding nucleic acid molecule of the present invention is a nucleic acid molecule having a dissociation constant for shrimp allergen of 50 nM or less.
  • the sensor for analyzing shrimp allergen of the present invention is characterized by including the shrimp allergen-binding nucleic acid molecule of the present invention.
  • the method for analyzing shrimp allergens of the present invention comprises contacting a sample with the shrimp allergen-binding nucleic acid molecule of the present invention, and binding the shrimp allergen in the sample and the nucleic acid molecule to thereby remove the shrimp allergen in the sample.
  • the shrimp allergen-binding nucleic acid molecule of the present invention can bind to shrimp allergen with the dissociation constant as described above. Therefore, according to the shrimp allergen-binding nucleic acid molecule of the present invention, shrimp allergen can be detected with excellent accuracy, for example, by the presence or absence of binding with shrimp allergen in the sample. Therefore, the shrimp allergen-binding nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting shrimp allergens in the fields of food production, food management, food distribution, and the like.
  • FIG. 1 is a schematic diagram showing an example of a predicted secondary structure of a shrimp allergen-binding nucleic acid molecule of the present invention.
  • FIG. 2 is a graph showing aptamer binding ability to shrimp allergen in Example 1 of the present invention.
  • the shrimp allergen is tropomyosin or a subunit thereof.
  • the shrimp allergen is a native allergen or a heat-denatured allergen.
  • the nucleic acid molecule of the present invention includes, for example, at least one polynucleotide selected from the group consisting of the following (a) to (d).
  • C a polynucleotide that binds to the shrimp allergen, and (c) a base sequence that has an identity of 80% or more to any of the base sequences of (a).
  • a polynucleotide that binds to an allergen (d) a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of (a) above, comprising a complementary nucleotide sequence, and Binding polynucleotide
  • the polynucleotide is DNA.
  • the analysis sensor of the present invention further includes, for example, a nucleic acid molecule that forms a G quartet structure.
  • the nucleic acid molecule forming the G quartet structure is DNAzyme or RNAzyme.
  • the analysis sensor of the present invention further includes porferin, for example.
  • the sample is at least one selected from the group consisting of food, food raw materials, and food additives.
  • the shrimp allergen-binding nucleic acid molecule of the present invention is a nucleic acid molecule having a dissociation constant for shrimp allergen of 50 nM or less as described above.
  • the nucleic acid molecule of the present invention binds to, for example, tropomyosin, a subunit, or a domain thereof, which is a major shrimp allergen.
  • the type of tropomyosin is not particularly limited, and examples thereof include various shrimp-derived tropomyosin.
  • the shrimp for example, penaeidae (Penaeidae) can be mentioned, as specific examples, Penaeus monodon genus (Penaeus), Metapenaeus Ensis genus (Metapenaeus) and the like.
  • Specific examples of the genus Shrimp include brown shrimp ( Penaeus aztecus ) and shrimp ( Penaeus indicus ), and specific examples of the genus Shrimp include Metapenaeus enis . Specific examples of each shrimp tropomyosin include Pen a 1, Pen i 1, Met e 1, and the like.
  • the shrimp allergen may be, for example, an unmodified allergen that is not denatured by heating or a denatured allergen that is denatured by heating.
  • the nucleic acid molecule of the present invention can bind to any allergen.
  • the dissociation constant for the shrimp allergen is, for example, 50 nM or 40 nM or less.
  • the detection limit concentration of the shrimp allergen is, for example, 300 nM, 150 nM, or 75 nM.
  • the nucleic acid molecule of the present invention has a dissociation constant for the tropomyosin of, for example, 50 nM or 40 nM or less.
  • the detection limit concentration of the tropomyosin is, for example, 300 nM, 150 nM, or 75 nM.
  • the binding between the nucleic acid molecule of the present invention and the shrimp allergen can be determined, for example, by surface plasmon resonance molecular interaction (SPR) analysis.
  • SPR surface plasmon resonance molecular interaction
  • ProteON trade name, BioRad
  • BioRad BioRad
  • the nucleic acid molecule of the present invention is a nucleic acid molecule comprising at least one polynucleotide selected from the group consisting of (a) to (d) below, for example.
  • A a polynucleotide comprising at least one of the nucleotide sequences of SEQ ID NOs: 1, 2 and 3
  • B in the nucleotide sequence of any one of (a), one or several bases are deleted, substituted, inserted and
  • C a polynucleotide that binds to the shrimp allergen, and
  • c a base sequence that has an identity of 80% or more to any of the base sequences of (a).
  • a polynucleotide that binds to an allergen (d) a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of (a) above, comprising a complementary nucleotide sequence, and Binding polynucleotide
  • the structural unit of the polynucleotide is, for example, a nucleotide residue, and examples thereof include a deoxyribonucleotide residue and a ribonucleotide residue.
  • the polynucleotide is, for example, DNA composed of deoxyribonucleotide residues, DNA containing deoxyribonucleotide residues and ribonucleotide residues, and may further contain non-nucleotide residues.
  • the shrimp allergen-binding nucleic acid molecule of the present invention is hereinafter also referred to as a DNA aptamer, for example.
  • the nucleic acid molecule of the present invention may be, for example, a molecule composed of any of the polynucleotides (a) to (d) or a molecule containing the polynucleotide.
  • the nucleic acid molecule of the present invention may contain two or more of any of the polynucleotides (a) to (d) as described later.
  • the two or more polynucleotides may have the same sequence or different sequences.
  • the nucleic acid molecule of the present invention may further have, for example, a linker and / or an additional sequence.
  • the polynucleotide (a) is a polynucleotide comprising the base sequence of at least one of SEQ ID NOs: 1, 2, and 3.
  • SEQ ID NO: 2 and SEQ ID NO: 3 are miniaturized sequences of SEQ ID NO: 1, respectively.
  • Table 1 the underlined region of SEQ ID NO: 1 corresponds to the base sequence of SEQ ID NO: 2, and the lower case region of SEQ ID NO: 1 corresponds to the base sequence of SEQ ID NO: 3.
  • FIG. 1 shows a predicted secondary structure of a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 2 or 3, but the present invention is not limited to this.
  • “one or several” may be, for example, within a range in which the polynucleotide of (b) binds to a shrimp allergen.
  • the “one or several” is, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 or 2 in any one of the base sequences of (a).
  • the numerical range of numbers such as the number of bases and the number of sequences, for example, discloses all positive integers belonging to the range. That is, for example, the description “1 to 5 bases” means all disclosures of “1, 2, 3, 4, 5 bases” (the same applies hereinafter).
  • the “identity” may be, for example, a range in which the polynucleotide (c) binds to a shrimp allergen.
  • the identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).
  • the “hybridizable polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide in (a).
  • the hybridization can be detected by, for example, various hybridization assays.
  • the hybridization assay is not particularly limited. For example, “Molecular Cloning: A Laboratory Manual 2nd Ed.” (Cold Spring Harbor Laboratories) edited by Sambrook et al. (1989)] and the like can also be employed.
  • the “stringent condition” may be, for example, a low stringent condition, a medium stringent condition, or a highly stringent condition.
  • “Low stringent conditions” are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C.
  • “Medium stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C.
  • “High stringent conditions” are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C.
  • the degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like. “Stringent conditions” are, for example, the above-mentioned edition of Sambrook et al. “Molecular Cloning: A Laboratory Manual Second Edition (Molecular Cloning: A Laboratory Manual 2nd Ed.)” ((Cold Spring Harbor Lab (Cold Spring Harbor Lab)). 1989)]] and the like.
  • the nucleic acid molecule of the present invention may include, for example, one of the polynucleotide sequences (a) to (d) or a plurality of the polynucleotide sequences. In the latter case, it is preferable that a plurality of polynucleotide sequences are linked to form a single-stranded polynucleotide.
  • the sequences of the plurality of polynucleotides may be directly linked to each other or indirectly linked via a linker.
  • the polynucleotide sequences are preferably linked directly or indirectly at the respective ends.
  • the sequences of the plurality of polynucleotides may be the same or different, for example.
  • sequences of the plurality of polynucleotides are preferably the same, for example.
  • the number of the sequences is not particularly limited, and is, for example, 2 or more, 2 to 20, 2 to 10, 2 or 3.
  • the linker is not particularly limited.
  • the length of the linker is not particularly limited, and is, for example, 1 to 200 bases long, 1 to 20 bases long, 3 to 12 bases long, and 5 to 9 bases long.
  • the structural unit of the linker is, for example, a nucleotide residue, and examples thereof include a deoxyribonucleotide residue and a ribonucleotide residue.
  • the linker is not particularly limited, and examples thereof include polynucleotides such as DNA consisting of deoxyribonucleotide residues and DNA containing ribonucleotide residues.
  • linker examples include polydeoxythymine (poly dT), polydeoxyadenine (poly dA), poly dAdT which is a repeating sequence of A and T, and preferably poly dT and poly dAdT.
  • the polynucleotide is preferably a single-stranded polynucleotide.
  • the single-stranded polynucleotide is preferably capable of forming a stem structure and a loop structure by, for example, self-annealing.
  • the polynucleotide is preferably capable of forming a stem loop structure, an internal loop structure, and / or a bulge structure, for example.
  • the nucleic acid molecule of the present invention may be, for example, double stranded.
  • one single-stranded polynucleotide includes any of the polynucleotides (a) to (d), and the other single-stranded polynucleotide is not limited.
  • the other single-stranded polynucleotide include a polynucleotide comprising a base sequence complementary to any one of the polynucleotides (a) to (d).
  • the nucleic acid molecule of the present invention is double-stranded, it is preferably dissociated into a single-stranded polynucleotide by denaturation or the like prior to use.
  • the dissociated single-stranded polynucleotide of any one of (a) to (d) preferably has, for example, a stem structure and a loop structure as described above.
  • the stem structure and the loop structure can be formed means, for example, that the stem structure and the loop structure are actually formed, and even if the stem structure and the loop structure are not formed, the stem structure depending on the conditions. And the ability to form a loop structure.
  • a stem structure and a loop structure can be formed includes, for example, both experimental confirmation and prediction by a computer simulation.
  • the structural unit of the nucleic acid molecule of the present invention is, for example, a nucleotide residue.
  • the nucleotide residue include deoxyribonucleotide residue and ribonucleotide residue.
  • Examples of the nucleic acid molecule of the present invention include DNA composed only of deoxyribonucleotide residues, DNA containing one or several ribonucleotide residues, and the like. In the latter case, “one or several” is not particularly limited. For example, in the polynucleotide, for example, 1 to 91, 1 to 30, 1 to 15, 1 to 7, 1 to 3 One or two.
  • the polynucleotide may contain a modified base.
  • the modified base is not particularly limited, and examples thereof include a base modified with a natural base (non-artificial base), and preferably has the same function as the natural base.
  • the natural base is not particularly limited, and examples thereof include a purine base having a purine skeleton and a pyrimidine base having a pyrimidine skeleton.
  • the purine base is not particularly limited, and examples thereof include adenine (a) and guanine (g).
  • the pyrimidine base is not particularly limited, and examples thereof include cytosine (c), thymine (t), uracil (u) and the like.
  • the base modification site is not particularly limited.
  • examples of the purine base modification site include the 7th and 8th positions of the purine skeleton.
  • examples of the modification site of the pyrimidine base include the 5th and 6th positions of the pyrimidine skeleton.
  • modified uracil or modified thymine when “ ⁇ O” is bonded to carbon at position 4 and a group other than “—CH 3” or “—H” is bonded to carbon at position 5, it is called modified uracil or modified thymine. it can.
  • the modifying group of the modifying base is not particularly limited, and examples thereof include a methyl group, a fluoro group, an amino group, a thio group, a benzylaminocarbonyl group represented by the following formula (1), and a tryptaminocarbonyl represented by the following formula (2).
  • the modified base is not particularly limited.
  • modified adenine modified with adenine, modified thymine modified with thymine, modified guanine modified with guanine, modified cytosine modified with cytosine and modified modified with uracil examples include uracil and the like, and the modified thymine, the modified uracil and the modified cytosine are preferable.
  • modified adenine examples include 7'-deazaadenine and the like.
  • modified guanine examples include, for example, 7'-deazaguanine.
  • modified cytosine examples include 5'-methylcytosine (5-Me-dC).
  • modified thymine examples include 5'-benzylaminocarbonylthymine, 5'-tryptaminocarbonylthymine, 5'-isobutylaminocarbonylthymine and the like.
  • modified uracil examples include 5'-benzylaminocarbonyluracil (BndU), 5'-tryptaminocarbonyluracil (TrpdU), 5'-isobutylaminocarbonyluracil and the like.
  • the exemplified modified uracil can also be referred to as a modified base of thymine.
  • the polynucleotide may contain, for example, only one of the modified bases or two or more kinds of the modified bases.
  • the nucleic acid molecule of the present invention may contain, for example, a modified nucleotide.
  • the modified nucleotide may be a nucleotide having the modified base described above, a nucleotide having a modified sugar in which a sugar residue is modified, It may be a nucleotide having a modified base and the modified sugar.
  • the sugar residue is not particularly limited, and examples thereof include deoxyribose residue or ribose residue.
  • the modification site in the sugar residue is not particularly limited, and examples thereof include the 2'-position and the 4'-position of the sugar residue, and both of them may be modified.
  • Examples of the modifying group of the modified sugar include a methyl group, a fluoro group, an amino group, and a thio group.
  • the base when the base is a pyrimidine base, for example, the 2'-position and / or the 4'-position of the sugar residue is preferably modified.
  • Specific examples of the modified nucleotide residue include, for example, a 2′-methylated-uracil nucleotide residue and a 2′-methylated-cytosine nucleotide residue in which the deoxyribose residue or the 2 ′ position of the ribose residue is modified.
  • the number of the modified nucleotides is not particularly limited, and is, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70 in the polynucleotide.
  • the modified nucleotides in the full length of the nucleic acid molecule including the polynucleotide are not particularly limited, and are, for example, 1 to 91 or 1 to 78, preferably the same as the above-mentioned range.
  • the nucleic acid molecule of the present invention may contain, for example, one or several artificial nucleic acid monomer residues.
  • the “one or several” is not particularly limited, and is, for example, 1 to 100, 1 to 50, 1 to 30, or 1 to 10 in the polynucleotide.
  • Examples of the artificial nucleic acid monomer residue include PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylenebridged Nucleic Acids) and the like.
  • the nucleic acid in the monomer residue is the same as described above, for example.
  • the nucleic acid molecule of the present invention is preferably nuclease resistant, for example.
  • the nucleic acid molecule of the present invention preferably has, for example, the modified nucleotide residue and / or the artificial nucleic acid monomer residue for nuclease resistance. Since the nucleic acid molecule of the present invention is nuclease resistant, for example, tens of kDa PEG (polyethylene glycol) or deoxythymidine may be bound to the 5 'end or 3' end.
  • the nucleic acid molecule of the present invention may further have an additional sequence, for example.
  • the additional sequence is preferably bound to, for example, at least one of the 5 'end and the 3' end of the nucleic acid molecule, and more preferably the 3 'end.
  • the additional sequence is not particularly limited.
  • the length of the additional sequence is not particularly limited, and is, for example, 1 to 200 bases long, 1 to 50 bases long, 1 to 25 bases long, or 18 to 24 bases long.
  • the structural unit of the additional sequence is, for example, a nucleotide residue, and examples thereof include a deoxyribonucleotide residue and a ribonucleotide residue.
  • the additional sequence is not particularly limited, and examples thereof include polynucleotides such as DNA consisting of deoxyribonucleotide residues and DNA containing ribonucleotide residues. Specific examples of the additional sequence include poly dT and poly dA.
  • the nucleic acid molecule of the present invention can be used, for example, immobilized on a carrier.
  • a carrier for example, either the 5 'end or the 3' end is preferably immobilized, and more preferably the 3 'end.
  • the nucleic acid molecule may be immobilized directly or indirectly on the carrier. In the latter case, for example, it is preferable to immobilize via the additional sequence.
  • the method for producing the nucleic acid molecule of the present invention is not particularly limited, and can be synthesized by, for example, a genetic engineering technique such as a nucleic acid synthesis method using chemical synthesis or a known method.
  • the nucleic acid molecule of the present invention can also be obtained, for example, by the so-called SELEX method.
  • the target is preferably tropomyosin, which is a shrimp allergen.
  • the nucleic acid molecule of the present invention exhibits binding properties to the shrimp allergen.
  • the use of the nucleic acid molecule of the present invention is not particularly limited as long as it uses the binding property to the shrimp allergen.
  • the nucleic acid molecule of the present invention can be used in various methods, for example, instead of the antibody against the shrimp allergen.
  • the sensor for analyzing shrimp allergen of the present invention includes the shrimp allergen-binding nucleic acid molecule of the present invention as described above.
  • the sensor of the present invention only needs to contain the above-described shrimp allergen-binding nucleic acid molecule of the present invention, and other configurations are not limited at all.
  • the sensor of the present invention becomes active, for example, when the shrimp allergen binds to the shrimp allergen-binding nucleic acid molecule, and becomes inactive when the shrimp allergen binds to the shrimp allergen-binding nucleic acid molecule.
  • a nucleic acid molecule for detecting binding that detects the binding may be further included.
  • the binding of the shrimp allergen to the shrimp allergen-binding nucleic acid molecule depends on whether the nucleic acid molecule for binding detection is an active type or an inactive type. The presence or absence can be confirmed, thereby analyzing the presence or absence of the shrimp allergen.
  • binding detection nucleic acid molecule examples include a nucleic acid molecule that forms a G quartet structure.
  • the nucleic acid molecule that forms the G-culted structure is, for example, an active type when a G-culted structure is formed, and an inactive state when a G-culted structure is not formed.
  • nucleic acid molecule forming the G quartet structure examples include DNAzyme and RNAzyme, and preferably DNAzyme.
  • An active DNAzyme having a G-culted structure exhibits a peroxidase-like activity that catalyzes a redox reaction, for example. Therefore, when the sensor of the present invention has DNAzyme, the presence or amount of the shrimp allergen bound to the shrimp allergen-binding nucleic acid molecule can be analyzed by detecting the catalytic activity of the DNAzyme.
  • the sensor of the present invention coexists, for example, porphyrin.
  • the porphyrin is not particularly limited, and examples thereof include unsubstituted porphyrin and derivatives thereof.
  • the derivatives include substituted porphyrins and metal porphyrins complexed with metal elements.
  • Examples of the substituted porphyrin include N-methylmesoporphyrin.
  • Examples of the metal porphyrin include hemin, which is a trivalent iron complex.
  • the porphyrin is, for example, preferably the metal porphyrin, more preferably hemin.
  • active DNAzyme having a G-culted structure generates fluorescence by forming a complex with porphyrin, for example. Therefore, when the sensor of the present invention has DNAzyme, the porphyrin is allowed to coexist, and the fluorescence due to the complex formation of the DNAzyme and the porphyrin is detected, thereby binding the shrimp allergen to the shrimp allergen-binding nucleic acid molecule. The presence or absence or the amount of binding can be analyzed.
  • the porphyrin is not particularly limited, and for example, N-methylmesoporphyrin (NMM), Zn-DIGP, ZnPP9, and TMPyP are preferable.
  • the sensor of the present invention may further include a labeling substance, for example.
  • the labeling substance is preferably bound to, for example, at least one of the 5 'end and the 3' end of the nucleic acid molecule, and more preferably the 5 'end.
  • the labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes and enzymes. Examples of the fluorescent substance include pyrene, TAMRA, fluorescein, Cy3 dye, Cy5 dye, FAM dye, rhodamine dye, Texas red dye, JOE, MAX, HEX, TYE and the like, and the dye includes, for example, And Alexa dyes such as Alexa 488 and Alexa 647.
  • the labeling substance may be linked directly to the nucleic acid molecule or indirectly via a linker, for example.
  • the linker is not particularly limited, and for example, the above examples can be used.
  • the analysis method of the present invention is a method for analyzing shrimp allergen, wherein the sample is brought into contact with the shrimp allergen-binding nucleic acid molecule of the present invention, and the shrimp allergen in the sample and the aforementioned It comprises a step of detecting shrimp allergen in the sample by binding with a nucleic acid molecule.
  • the analysis method of the present invention is characterized by using the nucleic acid molecule of the present invention, and other steps and conditions are not particularly limited.
  • the sensor for analyzing shrimp allergen of the present invention may be used as the nucleic acid molecule of the present invention.
  • the nucleic acid molecule of the present invention specifically binds to a shrimp allergen, for example, by detecting the binding between the shrimp allergen and the nucleic acid molecule, the shrimp allergen in the sample is specifically detected. Can be detected. Specifically, for example, since the presence or absence of shrimp allergen in the sample or the amount of shrimp allergen can be analyzed, it can be said that qualitative or quantitative determination is also possible.
  • the sample is not particularly limited.
  • the sample include foods, food materials, food additives, and the like.
  • Examples of the sample include a deposit in a food processing shop or a cooking place, a cleaning liquid after cleaning, and the like.
  • the sample may be, for example, a liquid sample or a solid sample.
  • the sample is preferably a liquid sample because it is easy to contact with the nucleic acid molecule and is easy to handle.
  • a mixed solution, an extract, a dissolved solution, and the like may be prepared using a solvent and used.
  • the solvent is not particularly limited, and examples thereof include water, physiological saline, and buffer solution.
  • the detection step for example, a contact step in which the sample and the nucleic acid molecule are brought into contact with each other and a shrimp allergen in the sample is bound to the nucleic acid molecule, and a binding between the shrimp allergen and the nucleic acid molecule is detected.
  • the detection step further includes, for example, a step of analyzing the presence or amount of shrimp allergen in the sample based on the result of the binding detection step.
  • the method for contacting the sample and the nucleic acid molecule is not particularly limited.
  • the contact between the sample and the nucleic acid molecule is preferably performed in a liquid, for example.
  • the liquid is not particularly limited, and examples thereof include water, physiological saline, and buffer solution.
  • the contact condition between the sample and the nucleic acid molecule is not particularly limited.
  • the contact temperature is, for example, 4 to 37 ° C. or 18 to 25 ° C.
  • the contact time is, for example, 10 to 120 minutes or 30 to 60 minutes.
  • the nucleic acid molecule may be, for example, an immobilized nucleic acid molecule immobilized on a carrier or an unfixed free nucleic acid molecule.
  • the sample is contacted in a container.
  • the nucleic acid molecule is preferably, for example, the immobilized nucleic acid molecule because of its excellent handleability.
  • the carrier is not particularly limited, and examples thereof include a substrate, a bead, and a container. Examples of the container include a microplate and a tube.
  • the nucleic acid molecule is immobilized as described above, for example.
  • the binding detection step is a step of detecting the binding between the shrimp allergen in the sample and the nucleic acid molecule as described above.
  • the presence or absence of binding between the two for example, the presence or absence of shrimp allergen in the sample can be analyzed (qualitative), and by detecting the degree of binding (binding amount) between the two, for example, The amount of shrimp allergen in the sample can be analyzed (quantified).
  • the binding between the shrimp allergen and the nucleic acid molecule cannot be detected, it can be determined that there is no shrimp allergen in the sample. If the binding is detected, the shrimp allergen is present in the sample. It can be judged.
  • the method for analyzing the binding between the shrimp allergen and the nucleic acid molecule is not particularly limited.
  • a conventionally known method for detecting the binding between substances can be adopted, and specific examples include the above-mentioned SPR, fluorescence polarization method and the like.
  • the binding may be, for example, detection of a complex of the shrimp allergen and the nucleic acid molecule.
  • the detection of the binding between the shrimp allergen and the nucleic acid molecule by the fluorescence polarization method can be performed, for example, as follows.
  • the fluorescence polarization method is generally characterized in that when the labeling substance is irradiated with polarized excitation light, the fluorescence emitted from the labeling substance exhibits different degrees of polarization depending on the molecular weight of the molecule labeled with the labeling substance.
  • Is a measurement method based on In the present invention for example, by using the nucleic acid molecule labeled with the labeling substance (labeled nucleic acid molecule), the binding between the shrimp allergen and the nucleic acid molecule can be detected by the fluorescence polarization method. .
  • the former when the labeled nucleic acid molecule is compared with a state in which it is not bound to shrimp allergen and a state in which it is bound to shrimp allergen, the former has a relatively low molecular weight, and therefore has a relatively low degree of polarization.
  • the latter has a relatively high degree of polarization due to its relatively high molecular weight. Therefore, for example, by comparing the degree of polarization of the labeled nucleic acid molecule before contact with the sample and the degree of polarization of the labeled nucleic acid molecule after contact with the sample, the shrimp allergen and the label Binding to the activated nucleic acid molecule can be detected.
  • the polarization of the labeled nucleic acid molecule after contact with the sample is evaluated using the polarization degree of at least one of the labeled nucleic acid molecule unbound to shrimp allergen and the labeled nucleic acid molecule bound to shrimp allergen as an evaluation standard. By evaluating the degree, the binding between the shrimp allergen and the labeled nucleic acid molecule can be detected.
  • the nucleic acid molecule of the present invention can be easily used as a sensor only by labeling with the labeling substance.
  • the detection wavelength of the labeling substance varies depending on the type thereof, for example, the influence of the fluorescence derived from the sample can be reduced by selecting the labeling substance according to the type of the sample.
  • the labeled nucleic acid molecule is not particularly limited as long as the nucleic acid molecule of the present invention is labeled with the labeling substance, for example.
  • Examples of the labeled nucleic acid molecule include a form in which the labeling substance is linked to the nucleic acid molecule of the present invention.
  • the labeling substance may be directly linked to the nucleic acid molecule of the present invention, or the labeling substance is indirectly linked via a linker or the like as described above. You may connect to.
  • the length of the linker is not particularly limited and is, for example, 0 to 10 bases, 0 to 7 bases, or 0 to 5 bases.
  • the labeling substance may be linked to, for example, any part of the nucleic acid molecule of the present invention, and specific examples include 5 ′ end and 3 ′ end, and may be linked to both ends. It may be linked to any one of the ends, preferably the 5 ′ end.
  • labeled nucleic acid molecule examples include, for example, the nucleic acid molecule of the present invention and a complementary strand that is complementary to the nucleic acid molecule (hereinafter also referred to as “labeled complementary strand”). And a hybrid molecule in which the nucleic acid molecule and the labeled complementary strand are hybridized.
  • the complementary strand only needs to have a sequence complementary to a part of the nucleic acid molecule of the present invention, for example, may be composed of only the complementary sequence, or includes the complementary sequence. But you can.
  • the complementary strand may be complementary to any region of the nucleic acid molecule of the present invention, and is preferably complementary to the 5 'end region or 3' end region.
  • the nucleic acid molecule of the present invention preferably has a linker at the 5 'end or 3' end, and the complementary sequence is preferably complementary to the linker.
  • the length of the linker is not particularly limited, and is, for example, 10 to 30 bases long, 15 to 25 bases long, or 18 to 24 bases long.
  • the length of the complementary strand is not particularly limited, and is, for example, 10 to 30 bases long, 15 to 25 bases long, or 18 to 24 bases long.
  • the labeling substance may be linked to, for example, any part of the complementary strand, and specific examples include the 5 ′ end and the 3 ′ end. Alternatively, it may be linked to either one of the ends.
  • the labeling substance is preferably linked to the 5 ′ end of the complementary strand
  • the labeled complementary strand is When complementary to the 5 ′ end region of the nucleic acid molecule of the present invention, the labeling substance is preferably linked to the 3 ′ end of the complementary strand.
  • the labeling substance is not particularly limited, and the examples described above can be used, and among these, the fluorescent substance and the dye are preferable.
  • the analysis method of the present invention includes, for example, a contact step in which the sample and the labeled nucleic acid molecule are brought into contact with each other, and a shrimp allergen in the sample and the labeled nucleic acid molecule are bound together.
  • the labeling nucleic acid molecule is irradiated with polarized excitation light to measure the degree of polarization of the labeled nucleic acid molecule, and the measurement result in the measurement step and the evaluation standard are compared, and the shrimp allergen and the labeling are compared. It is preferable to include a detection step of detecting a step of detecting binding with the nucleic acid molecule.
  • the wavelength of the polarized excitation light and the detection wavelength of the polarization degree are not particularly limited, and can be appropriately set according to, for example, the type of the labeling substance.
  • the wavelength of the polarized excitation light is, for example, 620 to 680 nm
  • the detection wavelength of the degree of polarization is, for example, 660 to 800 nm.
  • the irradiation time of the polarized excitation light is not particularly limited, and examples thereof include 1 nanosecond to 5 nanoseconds.
  • the evaluation criterion may be determined in advance or may be determined for each measurement, for example.
  • a criterion for unshrimp allergen binding or a criterion for shrimp allergen binding can be set.
  • the former criterion is, for example, the degree of polarization of only the labeled nucleic acid molecule to which no shrimp allergen is bound
  • the latter criterion is, for example, the degree of polarization of the labeled nucleic acid molecule to which shrimp allergen is bound.
  • the former standard for example, if the measured value in the measurement step is higher than the standard, it can be determined that shrimp allergen is present, and if the measured value is relatively higher than the standard, relatively many It can be determined that shrimp allergen is present. On the other hand, if the measurement value in the measurement step is about the same as or lower than the reference, it can be determined that there is no shrimp allergen.
  • the former criterion may be, for example, the degree of polarization of the labeled nucleic acid molecule before the contacting step.
  • the latter standard when the latter standard is used, for example, if the measured value in the measurement step is lower than the standard, it can be determined that no shrimp allergen is present. On the other hand, if the measurement value in the measurement step is the same or higher than the reference, it can be determined that there is a shrimp allergen, and if it is relatively higher than the reference, it is determined that a relatively large amount of shrimp allergen is present. it can.
  • the reference may be a correlation between the amount of shrimp allergen and the degree of polarization.
  • the correlation is shown by contacting a plurality of known concentrations of shrimp allergen with a predetermined amount of the labeled nucleic acid molecule and measuring the degree of polarization of the labeled nucleic acid molecule bound to each concentration of shrimp allergen. A correlation equation is obtained. The amount of shrimp allergen in the sample can be determined from the correlation formula and the measured value in the measuring step.
  • the shrimp allergen when used as the nucleic acid molecule of the present invention, the shrimp allergen can be detected by, for example, detection of redox reaction or detection of fluorescence generation.
  • the DNAzyme is G by the binding of the shrimp allergen to the shrimp allergen binding nucleic acid molecule. It forms a culted structure and becomes an active form showing the catalytic activity of a peroxidase-like redox reaction. Therefore, by detecting the oxidation-reduction reaction, the binding of the shrimp allergen to the shrimp allergen-binding nucleic acid molecule can be detected. In this case, for example, it is preferable to use a substrate for the oxidation-reduction reaction in combination.
  • the substrate is not particularly limited, and for example, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 1,2-phenylenediamine (OPD), 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic Acid Ammonium Salt (ABTS), 3,3'-Diaminobenzodinine (DAB), 3,3'-Diaminobenzoidine Tetrahhydrochloride Hydrate (DAB4HCl), 3-Amino-9-ethylChalo-1 (EC) 4-N, 4-C 2,4,6-Tribromo-3-hydroxybenzoic Acid, 2, 4-Dichlorophenol, 4-Aminoantipyrine, 4-Aminoantipyrine Hydrochloride, luminol and the like.
  • TMB 5,5′-tetramethylbenzidine
  • OPD 1,2-phenylenediamine
  • 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic Acid Ammonium Salt
  • DAB 3,
  • the DNAzyme when the sensor of the present invention has a DNAzyme that forms a G-culted structure as the binding detection nucleic acid molecule, the DNAzyme has a G quartet structure due to the binding of the shrimp allergen to the shrimp allergen-binding nucleic acid molecule. Fluorescence is generated by forming a complex with porphyrin. For this reason, the shrimp allergen to the shrimp allergen-binding nucleic acid molecule can be detected by detecting the fluorescence.
  • the detection kit of the present invention comprises the above-described shrimp allergen-binding nucleic acid molecule of the present invention.
  • the detection kit of the present invention is not limited as long as it contains the nucleic acid molecule of the present invention. If the detection kit of the present invention is used, for example, the detection of the shrimp allergen can be performed as described above.
  • the detection kit of the present invention may include, for example, the sensor of the present invention as the nucleic acid molecule of the present invention.
  • the detection kit of the present invention may include other components in addition to the nucleic acid molecule of the present invention, for example. Examples of the component include the carrier, the porphyrin, a buffer solution, and instructions for use.
  • Example 1 For each aptamer, the binding ability and kinetic parameters to shrimp allergen were confirmed.
  • the aptamer was added with polydeoxyadenine (poly dA) having a length of 24 bases at the end, and used as a poly dA addition aptamer in SPR described later.
  • poly dA was added to the 5 'end.
  • a chip product name: ProteOn NLC Sensor Chip, BioRad
  • streptavidin was immobilized
  • 5 ⁇ mol / L of biotinylated poly dT was injected into the flow cell of the sensor chip using ultrapure water (DDW), and was bound until the signal intensity (RU: Resonance Unit) was about 900 RU.
  • the biotinylated poly dT was prepared by biotinylating the 5 'end or 3' end of deoxythymidine having a length of 24 bases.
  • an aptamer solidification measurement value (A) as a signal indicating the amount of solidification of the aptamer to the sensor chip.
  • the sample was injected with an SPR buffer at a flow rate of 50 ⁇ L / min for 120 seconds, and then washed by flowing the SPR buffer under the same conditions. In parallel with the injection of the sample and the washing with the SPR buffer, the signal intensity was measured.
  • a protein binding measurement value (B) As a signal indicating the binding amount of the aptamer and protein.
  • the concentration of the sample was 300 nmol / L, 150 nmol / L, 75 nmol / L, 37.5 nmol / L, 18.75 nmol / L.
  • the composition of the SPR buffer was 40 mmol / L HEPES, 125 mmol / L NaCl, 5 mmol / L KCl, 1 mmol / L MgCl 2 and 0.01% Tween (registered trademark) 20, and the pH was 7.5.
  • FIG. 2 is a graph showing the binding ability of aptamers to the tropomyosin.
  • the horizontal axis represents measurement time (seconds), and the vertical axis represents signal intensity (RU).
  • RU signal intensity
  • 0 to 120 seconds are the injection time of the sample, and 120 seconds and after are the time for washing with the SPR buffer (the same applies hereinafter).
  • the plots indicate that the concentrations of tropomyosin protein are 300 nmol / L, 150 nmol / L, 75 nmol / L, 37.5 nmol / L, and 18.75 nmol / L from the top.
  • the shrimp allergen-binding nucleic acid molecule of the present invention can bind to shrimp allergen with the dissociation constant as described above. Therefore, according to the shrimp allergen-binding nucleic acid molecule of the present invention, shrimp allergen can be detected with excellent accuracy, for example, by the presence or absence of binding with shrimp allergen in the sample. Therefore, the shrimp allergen-binding nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting shrimp allergens in the fields of food production, food management, food distribution, and the like.

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Abstract

La présente invention se rapporte à une nouvelle molécule d'acide nucléique qui peut être utilisée pour détecter un allergène de crevette. L'acide nucléique qui se lie à un allergène de crevette selon la présente invention est caractérisé en ce qu'il s'agit d'une molécule d'acide nucléique présentant une constante de dissociation pour l'allergène de crevette inférieure ou égale à 50 nM, et, par exemple, comporte, de préférence, un polynucléotide comprenant une séquence de bases d'au moins l'une parmi SED ID NO: 1, 2 et 3.
PCT/JP2015/054536 2014-04-30 2015-02-19 Molécule d'acide nucléique qui se lie à un allergène de crevette et son application WO2015166689A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN110684772A (zh) * 2019-09-23 2020-01-14 浙江工商大学 一种特异性结合甲壳类原肌球蛋白的核酸适配体及其应用

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