WO2015151350A1 - Egg-allergen-binding nucleic acid molecule and use therefor - Google Patents

Egg-allergen-binding nucleic acid molecule and use therefor Download PDF

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Publication number
WO2015151350A1
WO2015151350A1 PCT/JP2014/083352 JP2014083352W WO2015151350A1 WO 2015151350 A1 WO2015151350 A1 WO 2015151350A1 JP 2014083352 W JP2014083352 W JP 2014083352W WO 2015151350 A1 WO2015151350 A1 WO 2015151350A1
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nucleic acid
acid molecule
egg allergen
allergen
egg
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PCT/JP2014/083352
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French (fr)
Japanese (ja)
Inventor
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 JP2016511324A priority Critical patent/JP6347498B2/en
Publication of WO2015151350A1 publication Critical patent/WO2015151350A1/en

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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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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 nucleic acid molecule that binds to an egg allergen and use thereof.
  • Eggs are a food that is frequently consumed on a daily basis, but in recent years, there has been an increase in the number of patients with egg allergies, which is regarded as a problem. Since many processed foods use eggs, it is extremely important to analyze whether or not eggs are mixed as raw materials in processed foods and their production lines.
  • Allergic allergens are generally proteins and their degradation products (peptides), and analysis methods using antibodies using these as antigens are the mainstream.
  • eggs for example, ovotransferrin, which is an egg white protein, is known as an allergen, and a method for detecting the egg allergen using an antibody has been reported (Non-patent Document 1).
  • 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. Moreover, electrophoresis and blotting on a nitrocellulose membrane are necessary, and the operation is complicated. For this reason, in recent years, attention has been focused on nucleic acid molecules that specifically bind to antigens instead of antibodies. However, no nucleic acid molecule for egg allergen has been reported so far.
  • an object of the present invention is to provide a new nucleic acid molecule that can be used for detection of egg allergen.
  • the egg allergen-binding nucleic acid molecule of the present invention is a nucleic acid molecule having a dissociation constant for egg allergen of 20 nM or less.
  • the egg allergen analysis sensor of the present invention comprises the egg allergen-binding nucleic acid molecule of the present invention.
  • the method for analyzing an egg allergen according to the present invention comprises contacting a sample with the egg allergen-binding nucleic acid molecule of the present invention, and binding the egg allergen in the sample with the egg allergen in the sample. A step of detecting.
  • the egg allergen-binding nucleic acid molecule of the present invention can bind to an egg allergen with the dissociation constant as described above. Therefore, according to the egg allergen-binding nucleic acid molecule of the present invention, for example, the egg allergen can be detected with excellent accuracy depending on the presence or absence of binding to the egg allergen in the sample. Therefore, the egg allergen-binding nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting egg allergens in fields such as food production, food management, and food distribution.
  • FIG. 1 is a schematic diagram showing an example of a predicted secondary structure of an egg allergen-binding nucleic acid molecule of the present invention.
  • FIG. 2 is a graph showing the binding ability between aptamer and egg allergen in Example 1 of the present invention.
  • the egg allergen is Ovotransferrin or a subunit thereof.
  • the egg 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).
  • A a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1-4
  • B one or several bases deleted, substituted, inserted and / or added in any one of the nucleotide sequences of (a)
  • C a polynucleotide that binds to the egg allergen
  • D a polynucleotide comprising a complementary base sequence and binding to an egg allergen to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the base sequences of (a) above nucleotide
  • the polynucleotide is DNA.
  • the polynucleotide includes a modified base.
  • 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 porphyrin, for example.
  • the sample is at least one selected from the group consisting of food, food raw materials, and food additives.
  • Egg allergen-binding nucleic acid molecule The egg allergen-binding nucleic acid molecule of the present invention is a nucleic acid molecule having a dissociation constant for egg allergen of 20 nM or less as described above.
  • the nucleic acid molecule of the present invention binds to, for example, Ovotransferrin, a subunit thereof, or a domain thereof, which is a major allergen of eggs.
  • Ovotransferrin is an egg white protein, has a molecular weight of 77.8 kDa, and is considered to be composed of two domains (TF1, TF2).
  • the amount of ovotransferrin per egg total protein is generally about 12%.
  • the egg allergen may be, for example, a non-denatured 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 nucleic acid molecule of the present invention has a dissociation constant for the egg allergen of, for example, 20 nM or less, 10 nM or less, or 3 nM or less.
  • the detection limit concentration of the egg allergen is, for example, 3.125 nM, 12.5 nM.
  • the nucleic acid molecule of the present invention has a dissociation constant for ovotransferrin of, for example, 20 nM or less, 10 nM or less, or 3 nM or less.
  • the detection limit concentration of the ovotransferrin is, for example, 3.125 nM, 12.5 nM.
  • the binding between the nucleic acid molecule of the present invention and the egg allergen can be determined by, for example, 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 any one of the nucleotide sequences of SEQ ID NOS: 1-4
  • B one or several bases deleted, substituted, inserted and / or added in any one of the nucleotide sequences of (a)
  • C a polynucleotide that binds to the egg allergen
  • C a polynucleotide that binds to the egg allergen
  • D a polynucleotide comprising a complementary base sequence and binding to an egg allergen to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the base sequences of (a) above nucleotide
  • 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 egg 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 any one of the nucleotide sequences of SEQ ID NOs: 1-4 shown in Table 1 below.
  • the polynucleotide OVTF188R8m1s41_1 of SEQ ID NO: 2 is a miniaturized sequence of the polynucleotide OVTF188R8m1 of SEQ ID NO: 1.
  • Table 1 the underlined portion of the base sequence of SEQ ID NO: 1 indicates the region of SEQ ID NO: 2.
  • the presumed secondary structure of said OVTF188R8m1 and said OVTF188R8m1s41_1 is shown in FIG. 1, it is not limited to this.
  • “one or several” may be, for example, within a range in which the polynucleotide of (b) binds to an egg 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, within a range in which the polynucleotide (c) binds to an egg 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, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.
  • the “stringent conditions” may be, for example, any of low stringent conditions, moderate stringent conditions, and highly stringent conditions.
  • 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, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.
  • 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 include 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. Can do.
  • 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 number of the modified base is not particularly limited.
  • the number of the modified base is not particularly limited.
  • the modified base is, for example, 1 to 96, 1 to 76, 1 to 70, 1 to 60, 1 to 45, 41, and all the bases are It may be a modified base.
  • the number of the modified bases may be, for example, the number of any one of the modified bases or the total number of the two or more modified bases.
  • the ratio of the modified base is not particularly limited.
  • the ratio of the modified base is, for example, 1/100 or more, 1/40 or more, 1/20 or more, 1/10 or more, 1/4 or more, or 1/3 or more of the total number of bases of the polynucleotide. .
  • the ratio of the modified base is expressed as a fraction, and the total number of bases and the number of modified bases that satisfy this are positive integers.
  • the polynucleotide comprising the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 preferably contains the modified base, and in particular, thymine is the modified thymine (or the modified uracil), and cytosine is the modified cytosine. Is preferred.
  • the number of the modified thymine (or modified uracil) is not particularly limited, and the lower limit is, for example, 1, 3, 5, and the upper limit is, for example, 17, 15, and 13 and all the thymines may be the modified thymines.
  • the ratio of the modified thymine (or modified uracil) is not particularly limited.
  • the ratio of the modified thymine (or modified uracil) is, for example, 1/100 or more, 1/40 or more, 1/20 or more of the total number of the natural thymine and the modified thymine (or modified uracil). 1/10 or more, 1/4 or more, or 1/3 or more.
  • a modified polynucleotide (SEQ ID NO: 5 or 6) in which a part of thymine is the modified uracil and a part of cytosine is the modified cytosine in the polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 4, Illustrate.
  • X is the modified uracil, for example, the BndUTP
  • Z is the modified cytosine, for example, the 5-Me-dC.
  • 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 5 '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, more preferably the 5 '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 ovotransferrin, which is an egg allergen.
  • the nucleic acid molecule of the present invention exhibits binding property to the egg allergen. Therefore, 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 egg allergen.
  • the nucleic acid molecule of the present invention can be used in various methods, for example, instead of the antibody against the egg allergen.
  • the sensor for egg allergen analysis of the present invention includes the egg allergen-binding nucleic acid molecule of the present invention as described above.
  • the sensor of the present invention only needs to contain the egg 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 when the egg allergen-binding nucleic acid molecule is bound to the egg allergen, and becomes inactive when the egg allergen-bound nucleic acid molecule is not bound to the egg allergen.
  • a nucleic acid molecule for detecting binding that detects the binding may be further included.
  • the binding detection nucleic acid molecule the binding of the egg allergen to the egg allergen binding nucleic acid molecule depends on whether the binding detection nucleic acid molecule is active or inactive. The presence or absence can be confirmed, thereby analyzing the presence or absence of the egg 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. For this reason, when the sensor of the present invention has DNAzyme, the presence or amount of binding of the egg allergen to the egg 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, binding of the egg allergen to the egg allergen binding nucleic acid molecule by detecting the fluorescence due to the complex formation of the DNAzyme and the porphyrin in the presence of the porphyrin 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 an egg allergen analysis method, wherein a sample is contacted with the egg allergen-binding nucleic acid molecule of the present invention, and the egg allergen in the sample and the It includes a step of detecting an egg allergen in the sample by binding to 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 egg allergen analysis sensor 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 the egg allergen
  • the egg allergen in the sample is specifically detected by detecting the binding between the egg allergen and the nucleic acid molecule, for example. Can be detected. Specifically, for example, since it is possible to analyze the presence or absence of egg allergen or the amount of egg allergen in a sample, it can be said that qualitative or quantitative determination is 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 detects, for example, a contact step of bringing the sample and the nucleic acid molecule into contact with each other to bind the egg allergen and the nucleic acid molecule in the sample, and binding between the egg allergen and the nucleic acid molecule.
  • the detection step further includes, for example, a step of analyzing the presence or amount of egg 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 egg 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 egg allergen in the sample can be analyzed (qualitative), and by detecting the degree of binding between the two (binding amount), for example, The amount of egg allergen in the sample can be analyzed (quantified).
  • the binding between the egg allergen and the nucleic acid molecule cannot be detected, it can be determined that the egg allergen is not present in the sample. If the binding is detected, the egg allergen is present in the sample. It can be judged.
  • the method for analyzing the binding between the egg 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 egg allergen and the nucleic acid molecule.
  • the detection of the binding between the egg 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 egg 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 an unbound state with an egg allergen and a bound state with an egg allergen, the former has a relatively low molecular weight and thus 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 contacting with the sample and the degree of polarization of the labeled nucleic acid molecule after contacting with the sample, the egg allergen and the label are compared. 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 based on the polarization degree of at least one of the labeled nucleic acid molecule not bound to the egg allergen and the labeled nucleic acid molecule bound to the egg allergen. By evaluating the degree, the binding between the egg allergen and the labeled nucleic acid molecule can also 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 of bringing the sample and the labeled nucleic acid molecule into contact with each other, and binding the egg allergen and the labeled nucleic acid molecule in the sample. , Irradiating the labeled nucleic acid molecule with polarized excitation light to measure the degree of polarization of the labeled nucleic acid molecule, comparing the measurement result in the measurement step and the evaluation criteria, and comparing the egg allergen and the labeling 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.
  • the evaluation standard for example, a standard for egg allergen unbinding and a standard for egg allergen binding can be set.
  • the former criterion is, for example, the degree of polarization of the labeled nucleic acid molecule to which egg allergen is not bound
  • the latter criterion is, for example, the degree of polarization of the labeled nucleic acid molecule to which egg 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 egg allergen is present. It can be determined that egg allergen is present. On the other hand, if the measurement value in the measurement step is the same or lower than the reference, it can be determined that no egg allergen is present.
  • the former criterion may be, for example, the degree of polarization of the labeled nucleic acid molecule before the contacting step.
  • the measurement value in the measurement step is lower than the standard, it can be determined that the egg allergen is not present.
  • the measurement value in the measurement step is the same or higher than the reference, it can be determined that egg allergen is present, and if it is relatively higher than the reference, it is determined that relatively many egg allergens are present. it can.
  • the reference may be a correlation between the amount of egg allergen and the degree of polarization.
  • the correlation is shown by contacting a plurality of egg allergens at known concentrations with a predetermined amount of the labeled nucleic acid molecule and measuring the degree of polarization of the labeled nucleic acid molecules bound to each concentration of egg allergen. A correlation equation is obtained. The amount of egg allergen in the sample can be determined from this correlation equation and the measured value in the measurement step.
  • the egg allergen analysis sensor of the present invention When the egg allergen analysis sensor of the present invention is used as the nucleic acid molecule of the present invention, the egg allergen can be detected by, for example, detecting a redox reaction or detecting fluorescence.
  • the DNAzyme is G by the binding of the egg allergen to the egg 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 redox reaction, the binding of the egg allergen to the egg 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 egg allergen to the egg allergen-binding nucleic acid molecule. Fluorescence is generated by forming a complex with porphyrin. For this reason, the egg allergen to the egg allergen binding nucleic acid molecule can be detected by detecting the fluorescence.
  • the detection kit of the present invention comprises the egg 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 egg allergen can be detected 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 for egg allergen were confirmed.
  • the aptamer was added with polydeoxyadenine (poly dA) having a length of 24 bases at the 5 'end and used as a poly dA addition aptamer for SPR described later.
  • poly dA polydeoxyadenine
  • 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 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 100 nmol / L, 50 nmol / L, 25 nmol / L, 12.5 nmol / L, 6.25 nmol / L, 3.125 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 heated ovotransferrin, where the horizontal axis indicates measurement time (seconds) and the vertical axis indicates signal intensity (RU). On the horizontal axis, 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). Moreover, in each graph of FIG. 2 (A) and (B), the plot is from the top, ovotransferrin concentration is 100 nmol / L, 50 nmol / L, 25 nmol / L, 12.5 nmol / L, FIG.
  • the plots from the top are ovotransferrin concentrations of 50 nmol / L, 25 nmol / L, 12.5 nmol / L, 6.25 nmol / L, 3.125 nmol / L. is there.
  • the egg allergen-binding nucleic acid molecule of the present invention can bind to an egg allergen with the dissociation constant as described above. Therefore, according to the egg allergen-binding nucleic acid molecule of the present invention, for example, the egg allergen can be detected with excellent accuracy depending on the presence or absence of binding to the egg allergen in the sample. Therefore, the egg allergen-binding nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting egg allergens in fields such as food production, food management, and food distribution.

Abstract

Provided is a novel nucleic acid molecule that can be used to detect an egg allergen. An egg-allergen-binding nucleic acid according to the present invention is characterized by being a nucleic acid molecule that has a dissociation constant with regard to an egg allergen of 20 nM or less. For example, the nucleic acid ideally includes a polynucleotide that comprises a base sequence that is any of SEQ ID NO: 1-4.

Description

卵アレルゲンに結合する核酸分子およびその用途Nucleic acid molecules that bind to egg allergens and uses thereof
　本発明は、卵アレルゲンに結合する核酸分子およびその用途に関する。 The present invention relates to a nucleic acid molecule that binds to an egg allergen and use thereof.
　卵は、日常的に頻繁に摂取される食品であるが、近年、卵アレルギーの患者が増加しており、問題視されている。加工食品等は、卵を使用するものが多く存在するため、加工食品やその製造ライン等においては、原料として卵が混入しているか否かを分析することは、極めて重要である。 Eggs are a food that is frequently consumed on a daily basis, but in recent years, there has been an increase in the number of patients with egg allergies, which is regarded as a problem. Since many processed foods use eggs, it is extremely important to analyze whether or not eggs are mixed as raw materials in processed foods and their production lines.
　アレルギーのアレルゲンは、一般的に、タンパク質やその分解物（ペプチド）であり、これらを抗原とする抗体を使用した分析方法が、主流である。そして、卵に関しても、例えば、卵白タンパク質であるオボトランスフェリンがアレルゲンとして知られており、抗体を使用して前記卵アレルゲンを検出する方法が報告されている（非特許文献１）。 Allergic allergens are generally proteins and their degradation products (peptides), and analysis methods using antibodies using these as antigens are the mainstream. Regarding eggs, for example, ovotransferrin, which is an egg white protein, is known as an allergen, and a method for detecting the egg allergen using an antibody has been reported (Non-patent Document 1).
　しかし、抗体は、タンパク質であり、安定性に問題があるため、低コストで簡易な検査法に抗体を用いることが難しい。また、電気泳動とニトロセルロース膜へのブロッティング等が必要であり、操作が煩雑である。このため、近年、抗体に代えて、抗原と特異的に結合する核酸分子が注目されている。しかしながら、これまでに卵アレルゲンに対する核酸分子は報告されていない。 However, since 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. Moreover, electrophoresis and blotting on a nitrocellulose membrane are necessary, and the operation is complicated. For this reason, in recent years, attention has been focused on nucleic acid molecules that specifically bind to antigens instead of antibodies. However, no nucleic acid molecule for egg allergen has been reported so far.
　そこで、本発明の目的は、卵アレルゲンの検出に利用可能な新たな核酸分子を提供することにある。 Therefore, an object of the present invention is to provide a new nucleic acid molecule that can be used for detection of egg allergen.
　本発明の卵アレルゲン結合核酸分子は、卵アレルゲンに対する解離定数が、２０ｎＭ以下の核酸分子であることを特徴とする。 The egg allergen-binding nucleic acid molecule of the present invention is a nucleic acid molecule having a dissociation constant for egg allergen of 20 nM or less.
　本発明の卵アレルゲン分析用センサは、前記本発明の卵アレルゲン結合核酸分子を含むことを特徴とする。 The egg allergen analysis sensor of the present invention comprises the egg allergen-binding nucleic acid molecule of the present invention.
　本発明の卵アレルゲンの分析方法は、試料と前記本発明の卵アレルゲン結合核酸分子とを接触させ、前記試料中の卵アレルゲンと前記核酸分子とを結合させることにより、前記試料中の卵アレルゲンを検出する工程を含むことを特徴とする。 The method for analyzing an egg allergen according to the present invention comprises contacting a sample with the egg allergen-binding nucleic acid molecule of the present invention, and binding the egg allergen in the sample with the egg allergen in the sample. A step of detecting.
　本発明の卵アレルゲン結合核酸分子は、卵アレルゲンに対して前述のような解離定数で結合することができる。このため、本発明の卵アレルゲン結合核酸分子によれば、例えば、試料中の卵アレルゲンとの結合の有無によって、優れた精度で、卵アレルゲンを検出できる。したがって、本発明の卵アレルゲン結合核酸分子は、例えば、食品製造、食品管理、食品の流通等の分野における卵アレルゲンの検出に、極めて有用なツールといえる。 The egg allergen-binding nucleic acid molecule of the present invention can bind to an egg allergen with the dissociation constant as described above. Therefore, according to the egg allergen-binding nucleic acid molecule of the present invention, for example, the egg allergen can be detected with excellent accuracy depending on the presence or absence of binding to the egg allergen in the sample. Therefore, the egg allergen-binding nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting egg allergens in fields such as food production, food management, and food distribution.
図１は、本発明の卵アレルゲン結合核酸分子の推定二次構造の一例を示す概略図である。FIG. 1 is a schematic diagram showing an example of a predicted secondary structure of an egg allergen-binding nucleic acid molecule of the present invention. 図２は、本発明の実施例１において、アプタマーと卵アレルゲンとの結合能を示すグラフである。FIG. 2 is a graph showing the binding ability between aptamer and egg allergen in Example 1 of the present invention.
　本発明の核酸分子は、例えば、前記卵アレルゲンが、オボトランスフェリン（Ovotransferrin）またはそのサブユニットである。 In the nucleic acid molecule of the present invention, for example, the egg allergen is Ovotransferrin or a subunit thereof.
　本発明の核酸分子は、例えば、前記卵アレルゲンが、未変性アレルゲンまたは加熱変性アレルゲンである。 In the nucleic acid molecule of the present invention, for example, the egg allergen is a native allergen or a heat-denatured allergen.
　本発明の核酸分子は、例えば、下記（ａ）～（ｄ）からなる群から選択された少なくとも一つのポリヌクレオチドを含む。
（ａ）配列番号１－４のいずれかの塩基配列からなるポリヌクレオチド
（ｂ）前記（ａ）のいずれかの塩基配列において、１もしくは数個の塩基が欠失、置換、挿入および／または付加された塩基配列からなり、前記卵アレルゲンに結合するポリヌクレオチド
（ｃ）前記（ａ）のいずれかの塩基配列に対して、８０％以上の同一性を有する塩基配列からなり、前記卵アレルゲンに結合するポリヌクレオチド
（ｄ）前記（ａ）のいずれかの塩基配列からなるポリヌクレオチドに対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドに、相補的な塩基配列からなり、卵アレルゲンに結合するポリヌクレオチド 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).
(A) a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1-4 (b) one or several bases deleted, substituted, inserted and / or added in any one of the nucleotide sequences of (a) (C) a polynucleotide that binds to the egg allergen, and (c) a polynucleotide that has 80% or more identity to the nucleotide sequence of (a) and binds to the egg allergen (D) a polynucleotide comprising a complementary base sequence and binding to an egg allergen to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the base sequences of (a) above nucleotide
　本発明の核酸分子は、例えば、前記ポリヌクレオチドが、ＤＮＡである。 In the nucleic acid molecule of the present invention, for example, the polynucleotide is DNA.
　本発明の核酸分子は、例えば、前記ポリヌクレオチドが、修飾塩基を含む。 In the nucleic acid molecule of the present invention, for example, the polynucleotide includes a modified base.
　本発明の分析用センサは、例えば、さらに、Ｇカルテット構造を形成する核酸分子を含む。 The analysis sensor of the present invention further includes, for example, a nucleic acid molecule that forms a G quartet structure.
　本発明の分析用センサは、例えば、前記Ｇカルテット構造を形成する核酸分子が、ＤＮＡｚｙｍｅまたはＲＮＡｚｙｍｅである。 In the analytical sensor of the present invention, for example, the nucleic acid molecule forming the G quartet structure is DNAzyme or RNAzyme.
　本発明の分析用センサは、例えば、さらに、ポルフィリンを含む。 The analysis sensor of the present invention further includes porphyrin, for example.
　本発明の分析方法は、例えば、前記試料が、食品、食品原料および食品添加物からなる群から選択された少なくとも一つである。 In the analysis method of the present invention, for example, the sample is at least one selected from the group consisting of food, food raw materials, and food additives.
（１）卵アレルゲン結合核酸分子
　本発明の卵アレルゲン結合核酸分子は、前述のように、卵アレルゲンに対する解離定数が、２０ｎＭ以下の核酸分子であることを特徴とする。 (1) Egg allergen-binding nucleic acid molecule The egg allergen-binding nucleic acid molecule of the present invention is a nucleic acid molecule having a dissociation constant for egg allergen of 20 nM or less as described above.
　本発明の核酸分子は、例えば、卵の主要アレルゲンである、オボトランスフェリン（Ovotransferrin）、そのサブユニット、またはそのドメインに結合する。オボトランスフェリンは、卵白タンパク質であり、分子量が７７．８ｋＤａであり、２つのドメイン（ＴＦ１、ＴＦ２）から構成されると考えられている。卵の総タンパク質量あたりのオボトランスフェリン量は、一般に、約１２％程度である。 The nucleic acid molecule of the present invention binds to, for example, Ovotransferrin, a subunit thereof, or a domain thereof, which is a major allergen of eggs. Ovotransferrin is an egg white protein, has a molecular weight of 77.8 kDa, and is considered to be composed of two domains (TF1, TF2). The amount of ovotransferrin per egg total protein is generally about 12%.
　前記卵アレルゲンは、例えば、加熱による変性が生じていない未変性アレルゲンでもよいし、加熱による変性が生じた変性アレルゲンでもよい。本発明の核酸分子は、例えば、いずれのアレルゲンに対しても結合可能である。 The egg allergen may be, for example, a non-denatured allergen that is not denatured by heating or a denatured allergen that is denatured by heating. For example, the nucleic acid molecule of the present invention can bind to any allergen.
　本発明の核酸分子は、前記卵アレルゲンに対する解離定数が、例えば、２０ｎＭ以下、１０ｎＭ以下、３ｎＭ以下である。本発明の核酸分子は、前記卵アレルゲンの検出限界濃度が、例えば、３．１２５ｎＭ、１２．５ｎＭである。 The nucleic acid molecule of the present invention has a dissociation constant for the egg allergen of, for example, 20 nM or less, 10 nM or less, or 3 nM or less. In the nucleic acid molecule of the present invention, the detection limit concentration of the egg allergen is, for example, 3.125 nM, 12.5 nM.
　本発明の核酸分子は、前記オボトランスフェリンに対する解離定数が、例えば、２０ｎＭ以下、１０ｎＭ以下、３ｎＭ以下である。本発明の核酸分子は、前記オボトランスフェリンの検出限界濃度が、例えば、３．１２５ｎＭ、１２．５ｎＭである。 The nucleic acid molecule of the present invention has a dissociation constant for ovotransferrin of, for example, 20 nM or less, 10 nM or less, or 3 nM or less. In the nucleic acid molecule of the present invention, the detection limit concentration of the ovotransferrin is, for example, 3.125 nM, 12.5 nM.
　本発明の核酸分子と前記卵アレルゲンとの結合は、例えば、表面プラズモン共鳴分子相互作用（ＳＰＲ；Surface　Plasmon　resonance）解析等により決定できる。前記解析は、例えば、ＰｒｏｔｅＯＮ（商品名　BioRad社）が使用できる。 The binding between the nucleic acid molecule of the present invention and the egg allergen can be determined by, for example, surface plasmon resonance molecular interaction (SPR) analysis. For example, ProteON (trade name, BioRad) can be used for the analysis.
　本発明の卵アレルゲン結合核酸分子について、具体例を以下に示す。本発明の核酸分子は、例えば、下記（ａ）～（ｄ）からなる群から選択された少なくとも一つのポリヌクレオチドを含む核酸分子である。
（ａ）配列番号１－４のいずれかの塩基配列からなるポリヌクレオチド
（ｂ）前記（ａ）のいずれかの塩基配列において、１もしくは数個の塩基が欠失、置換、挿入および／または付加された塩基配列からなり、前記卵アレルゲンに結合するポリヌクレオチド
（ｃ）前記（ａ）のいずれかの塩基配列に対して、８０％以上の同一性を有する塩基配列からなり、前記卵アレルゲンに結合するポリヌクレオチド
（ｄ）前記（ａ）のいずれかの塩基配列からなるポリヌクレオチドに対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドに、相補的な塩基配列からなり、卵アレルゲンに結合するポリヌクレオチド Specific examples of the egg allergen-binding nucleic acid molecule of the present invention are shown below. 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 any one of the nucleotide sequences of SEQ ID NOS: 1-4 (b) one or several bases deleted, substituted, inserted and / or added in any one of the nucleotide sequences of (a) (C) a polynucleotide that binds to the egg allergen, and (c) a polynucleotide that has 80% or more identity to the nucleotide sequence of (a) and binds to the egg allergen (D) a polynucleotide comprising a complementary base sequence and binding to an egg allergen to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the base sequences of (a) above nucleotide
　本発明の核酸分子において、前記ポリヌクレオチドの構成単位は、例えば、ヌクレオチド残基であり、デオキシリボヌクレオチド残基およびリボヌクレオチド残基があげられる。前記ポリヌクレオチドは、後述するように、例えば、デオキシリボヌクレオチド残基からなるＤＮＡ、デオキシリボヌクレオチド残基およびリボヌクレオチド残基を含むＤＮＡであり、さらに、非ヌクレオチド残基を含んでもよい。本発明の卵アレルゲン結合核酸分子は、例えば、以下、ＤＮＡアプタマーともいう。 In the nucleic acid molecule of the present invention, the structural unit of the polynucleotide is, for example, a nucleotide residue, and examples thereof include a deoxyribonucleotide residue and a ribonucleotide residue. As will be described later, 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 egg allergen-binding nucleic acid molecule of the present invention is hereinafter also referred to as a DNA aptamer, for example.
　本発明の核酸分子は、例えば、前記（ａ）～（ｄ）のいずれかのポリヌクレオチドからなる分子でもよいし、前記ポリヌクレオチドを含む分子でもよい。後者の場合、本発明の核酸分子は、例えば、後述するように、前記（ａ）～（ｄ）のいずれかのポリヌクレオチドを２つ以上含んでもよい。前記２つ以上のポリヌクレオチドは、同じ配列でもよいし、異なる配列でもよい。また、後者の場合、本発明の核酸分子は、例えば、さらに、リンカーおよび／または付加配列等を有してもよい。 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. In the latter case, 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. In the latter case, the nucleic acid molecule of the present invention may further have, for example, a linker and / or an additional sequence.
　前記（ａ）のポリヌクレオチドは、下記表１に示す配列番号１－４のいずれかの塩基配列からなるポリヌクレオチドである。 The polynucleotide (a) is a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOs: 1-4 shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　配列番号２のポリヌクレオチドOVTF188R8m1s41_1は、配列番号１のポリヌクレオチドOVTF188R8m1の小型化配列であり、前記表１において、配列番号１の塩基配列の下線部は、配列番号２の領域を示す。また、前記OVTF188R8m1および前記OVTF188R8m1s41_1の推定二次構造を図１に示すが、これには限定されない。 The polynucleotide OVTF188R8m1s41_1 of SEQ ID NO: 2 is a miniaturized sequence of the polynucleotide OVTF188R8m1 of SEQ ID NO: 1. In Table 1, the underlined portion of the base sequence of SEQ ID NO: 1 indicates the region of SEQ ID NO: 2. Moreover, although the presumed secondary structure of said OVTF188R8m1 and said OVTF188R8m1s41_1 is shown in FIG. 1, it is not limited to this.
　前記（ｂ）において、「１もしくは数個」は、例えば、前記（ｂ）のポリヌクレオチドが、卵アレルゲンに結合する範囲であればよい。前記「１もしくは数個」は、前記（ａ）のいずれかの塩基配列において、例えば、１～１０個、１～７個、１～５個、１～３個、１または２個である。本発明において、塩基数および配列数等の個数の数値範囲は、例えば、その範囲に属する正の整数を全て開示するものである。つまり、例えば、「１～５塩基」との記載は、「１、２、３、４、５塩基」の全ての開示を意味する（以下、同様）。 In the above (b), “one or several” may be, for example, within a range in which the polynucleotide of (b) binds to an egg 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). In the present invention, 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).
　前記（ｃ）において、「同一性」は、例えば、前記（ｃ）のポリヌクレオチドが、卵アレルゲンに結合する範囲であればよい。前記同一性は、例えば、８０％以上、８５％以上、９０％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上である。前記同一性は、例えば、ＢＬＡＳＴ、ＦＡＳＴＡ等の解析ソフトウェアを用いて、デフォルトのパラメータにより算出できる（以下、同様）。 In (c), the “identity” may be, for example, within a range in which the polynucleotide (c) binds to an egg 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).
　前記（ｄ）において、「ハイブリダイズ可能なポリヌクレオチド」は、例えば、前記（ａ）のポリヌクレオチドに対して、完全または部分的に相補的なポリヌクレオチドである。前記ハイブリダイズは、例えば、各種ハイブリダイゼーションアッセイにより検出できる。前記ハイブリダイゼーションアッセイは、特に制限されず、例えば、ザンブルーク（Ｓａｍｂｒｏｏｋ）ら編「モレキュラー・クローニング：ア・ラボラトリーマニュアル第２版（Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　２ｎｄ　Ｅｄ．）」〔（Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ　（１９８９）〕等に記載されている方法を採用することもできる。 In (d), 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, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.
　前記（ｄ）において、「ストリンジェントな条件」は、例えば、低ストリンジェントな条件、中ストリンジェントな条件、高ストリンジェントな条件のいずれでもよい。「低ストリンジェントな条件」は、例えば、５×ＳＳＣ、５×デンハルト溶液、０．５％ＳＤＳ、５０％ホルムアミド、３２℃の条件である。「中ストリンジェントな条件」は、例えば、５×ＳＳＣ、５×デンハルト溶液、０．５％ＳＤＳ、５０％ホルムアミド、４２℃の条件である。「高ストリンジェントな条件」は、例えば、５×ＳＳＣ、５×デンハルト溶液、０．５％ＳＤＳ、５０％ホルムアミド、５０℃の条件である。ストリンジェンシーの程度は、当業者であれば、例えば、温度、塩濃度、プローブの濃度および長さ、イオン強度、時間等の条件を適宜選択することで、設定可能である。「ストリンジェントな条件」は、例えば、前述したザンブルーク（Ｓａｍｂｒｏｏｋ）ら編「モレキュラー・クローニング：ア・ラボラトリーマニュアル第２版（Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　２ｎｄ　Ｅｄ．）」〔（Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ　（１９８９）〕等に記載の条件を採用することもできる。 In (d) above, the “stringent conditions” may be, for example, any of low stringent conditions, moderate stringent conditions, and highly stringent conditions. “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, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.
　本発明の核酸分子は、例えば、前記（ａ）～（ｄ）のいずれかのポリヌクレオチドの配列を１つ含んでもよいし、前記ポリヌクレオチドの配列を複数含んでもよい。後者の場合、複数のポリヌクレオチドの配列が連結して、一本鎖のポリヌクレオチドを形成していることが好ましい。前記複数のポリヌクレオチドの配列は、例えば、それぞれが直接的に連結してもよいし、リンカーを介して、それぞれが間接的に連結してもよい。前記ポリヌクレオチドの配列は、それぞれの末端において、直接的または間接的に連結していることが好ましい。前記複数のポリヌクレオチドの配列は、例えば、同じでもよいし、異なってもよい。前記複数のポリヌクレオチドの配列は、例えば、同じであることが好ましい。前記ポリヌクレオチドの配列を複数含む場合、前記配列の数は、特に制限されず、例えば、２以上、２～２０、２～１０、２または３である。 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. For example, 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. The sequences of the plurality of polynucleotides are preferably the same, for example. When a plurality of the polynucleotide sequences are included, 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.
　前記リンカーは、特に制限されない。前記リンカーの長さは、特に制限されず、例えば、１～２００塩基長、１～２０塩基長、３～１２塩基長、５～９塩基長である。前記リンカーの構成単位は、例えば、ヌクレオチド残基であり、デオキシリボヌクレオチド残基およびリボヌクレオチド残基等があげられる。前記リンカーは、特に制限されず、例えば、デオキシリボヌクレオチド残基からなるＤＮＡ、リボヌクレオチド残基を含むＤＮＡ等のポリヌクレオチドがあげられる。前記リンカーの具体例として、例えば、ポリデオキシチミン（ポリｄＴ）、ポリデオキシアデニン（ポリｄＡ）、ＡとＴの繰り返し配列であるポリｄＡｄＴ等があげられ、好ましくはポリｄＴ、ポリｄＡｄＴである。 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. Specific examples of the linker 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.
　本発明の核酸分子において、前記ポリヌクレオチドは、一本鎖ポリヌクレオチドであることが好ましい。前記一本鎖ポリヌクレオチドは、例えば、自己アニーリングによりステム構造およびループ構造を形成可能であることが好ましい。前記ポリヌクレオチドは、例えば、ステムループ構造、インターナルループ構造および／またはバルジ構造等を形成可能であることが好ましい。 In the nucleic acid molecule of the present invention, 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.
　本発明の核酸分子は、例えば、二本鎖でもよい。二本鎖の場合、例えば、一方の一本鎖ポリヌクレオチドは、前記（ａ）～（ｄ）のいずれかのポリヌクレオチドを含み、他方の一本鎖ポリヌクレオチドは、制限されない。前記他方の一本鎖ポリヌクレオチドは、例えば、前記（ａ）～（ｄ）のいずれかのポリヌクレオチドに相補的な塩基配列を含むポリヌクレオチドがあげられる。本発明の核酸分子が二本鎖の場合、例えば、使用に先立って、変性等により、一本鎖ポリヌクレオチドに解離させることが好ましい。また、解離した前記（ａ）～（ｄ）のいずれかの一本鎖ポリヌクレオチドは、例えば、前述のように、ステム構造およびループ構造を形成していることが好ましい。 The nucleic acid molecule of the present invention may be, for example, double stranded. In the case of a double strand, for example, one single-stranded polynucleotide includes any of the polynucleotides (a) to (d), and the other single-stranded polynucleotide is not limited. Examples of the other single-stranded polynucleotide include a polynucleotide comprising a base sequence complementary to any one of the polynucleotides (a) to (d). When 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. Further, 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.
　本発明において、「ステム構造およびループ構造を形成可能」とは、例えば、実際にステム構造およびループ構造を形成すること、ならびに、ステム構造およびループ構造が形成されていなくても、条件によってステム構造およびループ構造を形成可能なことも含む。「ステム構造およびループ構造を形成可能」とは、例えば、実験的に確認した場合、および、コンピュータ等のシミュレーションで予測した場合の双方を含む。 In the present invention, “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.
　本発明の核酸分子の構成単位は、例えば、ヌクレオチド残基である。前記ヌクレオチド残基は、例えば、デオキシリボヌクレオチド残基およびリボヌクレオチド残基があげられる。本発明の核酸分子は、例えば、デオキシリボヌクレオチド残基のみから構成されるＤＮＡ、１もしくは数個のリボヌクレオチド残基を含むＤＮＡ等があげられる。後者の場合、「１もしくは数個」は、特に制限されず、例えば、前記ポリヌクレオチドにおいて、例えば、１～９１個、１～３０個、１～１５個、１～７個、１～３個、１または２個である。 The structural unit of the nucleic acid molecule of the present invention is, for example, a nucleotide residue. Examples of 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.
　前記ポリヌクレオチドは、修飾塩基を含んでもよい。前記修飾塩基は、特に制限されず、例えば、天然塩基（非人工塩基）が修飾された塩基があげられ、前記天然塩基と同様の機能を有することが好ましい。前記天然塩基は、特に制限されず、例えば、プリン骨格を有するプリン塩基、ピリミジン骨格を有するピリミジン塩基等があげられる。前記プリン塩基は、特に制限されず、例えば、アデニン（ａ）、グアニン（ｇ）があげられる。前記ピリミジン塩基は、特に制限されず、例えば、シトシン（ｃ）、チミン（ｔ）、ウラシル（ｕ）等があげられる。前記塩基の修飾部位は、特に制限されない。前記塩基がプリン塩基の場合、前記プリン塩基の修飾部位は、例えば、前記プリン骨格の７位および８位があげられる。前記塩基がピリミジン塩基の場合、前記ピリミジン塩基の修飾部位は、例えば、前記ピリミジン骨格の５位および６位があげられる。前記ピリミジン骨格において、４位の炭素に「＝Ｏ」が結合し、５位の炭素に「－ＣＨ３」または「－Ｈ」以外の基が結合している場合、修飾ウラシルまたは修飾チミンということができる。 The polynucleotide may include 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. When the base is a purine base, examples of the purine base modification site include the 7th and 8th positions of the purine skeleton. When the base is a pyrimidine base, examples of the modification site of the pyrimidine base include the 5th and 6th positions of the pyrimidine skeleton. In the pyrimidine skeleton, 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. Can do.
　前記修飾塩基の修飾基は、特に制限されず、例えば、メチル基、フルオロ基、アミノ基、チオ基、下記式（１）のベンジルアミノカルボニル基（benzylaminocarbonyl）、下記式（２）のトリプタミノカルボニル基（tryptaminocarbonyl）およびイソブチルアミノカルボニル基（isobutylaminocarbonyl）等があげられる。 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). Group (tryptaminocarbonyl), isobutylaminocarbonyl group (isobutylaminocarbonyl) and the like.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
　前記修飾塩基は、特に制限されず、例えば、アデニンが修飾された修飾アデニン、チミンが修飾された修飾チミン、グアニンが修飾された修飾グアニン、シトシンが修飾された修飾シトシンおよびウラシルが修飾された修飾ウラシル等があげられ、前記修飾チミン、前記修飾ウラシルおよび前記修飾シトシンが好ましい。 The modified base is not particularly limited. For example, 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.
　前記修飾アデニンの具体例としては、例えば、７’－デアザアデニン等があげられる。 Specific examples of the modified adenine include 7'-deazaadenine and the like.
　前記修飾グアニンの具体例としては、例えば、７’－デアザグアニン等があげられる。 Specific examples of the modified guanine include, for example, 7'-deazaguanine.
　前記修飾シトシンの具体例としては、例えば、５’－メチルシトシン（５－Ｍｅ－ｄＣ）等があげられる。 Specific examples of the modified cytosine include 5'-methylcytosine (5-Me-dC).
　前記修飾チミンの具体例としては、例えば、５’－ベンジルアミノカルボニルチミン、５’－トリプタミノカルボニルチミン、５’－イソブチルアミノカルボニルチミン等があげられる。 Specific examples of the modified thymine include 5'-benzylaminocarbonylthymine, 5'-tryptaminocarbonylthymine, 5'-isobutylaminocarbonylthymine and the like.
　前記修飾ウラシルの具体例としては、例えば、５’－ベンジルアミノカルボニルウラシル（ＢｎｄＵ）、５’－トリプタミノカルボニルウラシル（ＴｒｐｄＵ）および５’－イソブチルアミノカルボニルウラシル等があげられる。例示した前記修飾ウラシルは、チミンの修飾塩基ということもできる。 Specific examples of the modified uracil 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.
　前記ポリヌクレオチドは、例えば、いずれか１種類の前記修飾塩基のみを含んでもよいし、２種類以上の前記修飾塩基を含んでもよい。 The polynucleotide may contain, for example, only one of the modified bases or two or more kinds of the modified bases.
　前記修飾塩基の個数は、特に制限されない。前記ポリヌクレオチドにおいて、前記修飾塩基の個数は、特に制限されない。前記修飾塩基は、前記ポリヌクレオチドにおいて、例えば、１～９６個、１～７６個、１～７０個、１～６０個、１～４５個、４１個であり、また、全ての塩基が、前記修飾塩基でもよい。前記修飾塩基の個数は、例えば、いずれか１種類の前記修飾塩基の個数であってもよいし、２種類以上の前記修飾塩基の個数の合計であってもよい。 The number of the modified base is not particularly limited. In the polynucleotide, the number of the modified base is not particularly limited. In the polynucleotide, the modified base is, for example, 1 to 96, 1 to 76, 1 to 70, 1 to 60, 1 to 45, 41, and all the bases are It may be a modified base. The number of the modified bases may be, for example, the number of any one of the modified bases or the total number of the two or more modified bases.
　前記ポリヌクレオチドが前記修飾塩基を含む場合、前記修飾塩基の割合は、特に制限されない。前記修飾塩基の割合は、前記ポリヌクレオチドの全塩基数のうち、例えば、１／１００以上、１／４０以上、１／２０以上、１／１０以上、１／４以上、１／３以上である。前記修飾塩基の割合を分数で示すが、これを満たす全塩基数と修飾塩基数とは、それぞれ正の整数である。 When the polynucleotide contains the modified base, the ratio of the modified base is not particularly limited. The ratio of the modified base is, for example, 1/100 or more, 1/40 or more, 1/20 or more, 1/10 or more, 1/4 or more, or 1/3 or more of the total number of bases of the polynucleotide. . The ratio of the modified base is expressed as a fraction, and the total number of bases and the number of modified bases that satisfy this are positive integers.
　前記配列番号３、配列番号４の塩基配列からなるポリヌクレオチドは、前記修飾塩基を含むことが好ましく、中でも、チミンが前記修飾チミン（または前記修飾ウラシル）であり、シトシンが前記修飾シトシンであることが好ましい。前記配列番号３、配列番号４のポリヌクレオチドにおいて、前記修飾チミン（または修飾ウラシル）の個数は、特に制限されず、下限は、例えば、１個、３個、５個であり、上限は、例えば、１７個、１５個、１３個であり、また、全てのチミンが、前記修飾チミンでもよい。 The polynucleotide comprising the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 preferably contains the modified base, and in particular, thymine is the modified thymine (or the modified uracil), and cytosine is the modified cytosine. Is preferred. In the polynucleotides of SEQ ID NO: 3 and SEQ ID NO: 4, the number of the modified thymine (or modified uracil) is not particularly limited, and the lower limit is, for example, 1, 3, 5, and the upper limit is, for example, 17, 15, and 13 and all the thymines may be the modified thymines.
　前記配列番号３のポリヌクレオチドまたは配列番号４のポリヌクレオチドにおいて、前記修飾チミン（または修飾ウラシル）の割合は、特に制限されない。前記修飾チミン（または修飾ウラシル）の割合は、前記天然チミンの個数と前記修飾チミン（または修飾ウラシル）の個数との合計のうち、例えば、１／１００以上、１／４０以上、１／２０以上、１／１０以上、１／４以上、１／３以上である。 In the polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 4, the ratio of the modified thymine (or modified uracil) is not particularly limited. The ratio of the modified thymine (or modified uracil) is, for example, 1/100 or more, 1/40 or more, 1/20 or more of the total number of the natural thymine and the modified thymine (or modified uracil). 1/10 or more, 1/4 or more, or 1/3 or more.
　配列番号３のポリヌクレオチドまたは配列番号４のポリヌクレオチドにおいて、一部のチミンが前記修飾ウラシルであり、一部のシトシンが前記修飾シトシンである修飾ポリヌクレオチド（配列番号５または６）を、以下に例示する。下記配列において、Ｘは、前記修飾ウラシルであり、例えば、前記ＢｎｄＵＴＰであり、Ｚは、前記修飾シトシンであり、例えば、前記５－Ｍｅ－ｄＣである。 A modified polynucleotide (SEQ ID NO: 5 or 6) in which a part of thymine is the modified uracil and a part of cytosine is the modified cytosine in the polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 4, Illustrate. In the following sequence, X is the modified uracil, for example, the BndUTP, and Z is the modified cytosine, for example, the 5-Me-dC.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　本発明の核酸分子は、例えば、修飾ヌクレオチドを含んでもよい、前記修飾ヌクレオチドは、前述の前記修飾塩基を有するヌクレオチドでもよいし、糖残基が修飾された修飾糖を有するヌクレオチドでもよいし、前記修飾塩基および前記修飾糖を有するヌクレオチドでもよい。 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.
　前記糖残基は、特に制限されず、例えば、デオキシリボース残基またはリボース残基があげられる。前記糖残基における修飾部位は、特に制限されず、例えば、前記糖残基の２’位または４’位があげられ、いずれか一方でも両方が修飾されてもよい。前記修飾糖の修飾基は、例えば、メチル基、フルオロ基、アミノ基、チオ基等があげられる。 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.
　前記修飾ヌクレオチド残基において、塩基がピリミジン塩基の場合、例えば、前記糖残基の２’位および／または４’位が修飾されていることが好ましい。前記修飾ヌクレオチド残基の具体例は、例えば、デオキシリボース残基またはリボース残基の２’位が修飾された、２’－メチル化－ウラシルヌクレオチド残基、２’－メチル化－シトシンヌクレオチド残基、２’－フルオロ化－ウラシルヌクレオチド残基、２’－フルオロ化－シトシンヌクレオチド残基、２’－アミノ化－ウラシルヌクレオチド残基、２’－アミノ化－シトシンヌクレオチド残基、２’－チオ化－ウラシルヌクレオチド残基、２’－チオ化－シトシンヌクレオチド残基等があげられる。 In the modified nucleotide residue, 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. 2'-fluorinated-uracil nucleotide residues, 2'-fluorinated-cytosine nucleotide residues, 2'-aminated-uracil nucleotide residues, 2'-aminated-cytosine nucleotide residues, 2'-thiolated -Uracil nucleotide residue, 2'-thiolated-cytosine nucleotide residue and the like.
　前記修飾ヌクレオチドの個数は、特に制限されず、例えば、前記ポリヌクレオチドにおいて、例えば、１～１００個、１～９０個、１～８０個、１～７０個である。また、前記ポリヌクレオチドを含む前記核酸分子の全長における前記修飾ヌクレオチドも、特に制限されず、例えば、１～９１個または１～７８個であり、好ましくは、前述の範囲と同様である。 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. In addition, 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.
　本発明の核酸分子は、例えば、１もしくは数個の人工核酸モノマー残基を含んでもよい。前記「１もしくは数個」は、特に制限されず、例えば、前記ポリヌクレオチドにおいて、例えば、１～１００個、１～５０個、１～３０個、１～１０個である。前記人工核酸モノマー残基は、例えば、ＰＮＡ（ペプチド核酸）、ＬＮＡ（Ｌｏｃｋｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ）、ＥＮＡ（２’－Ｏ，４’－Ｃ－Ｅｔｈｙｌｅｎｅｂｒｉｄｇｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ）等があげられる。前記モノマー残基における核酸は、例えば、前述と同様である。 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.
　本発明の核酸分子は、例えば、ヌクレアーゼ耐性であることが好ましい。本発明の核酸分子は、ヌクレアーゼ耐性のため、例えば、前記修飾化ヌクレオチド残基および／または前記人工核酸モノマー残基を有することが好ましい。本発明の核酸分子は、ヌクレアーゼ耐性のため、例えば、５’末端または３’末端に、数１０ｋＤａのＰＥＧ（ポリエチレングリコール）またはデオキシチミジン等が結合してもよい。 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.
　本発明の核酸分子は、例えば、さらに付加配列を有してもよい。前記付加配列は、例えば、前記核酸分子の５’末端および３’末端の少なくとも一方に結合していることが好ましく、より好ましくは５’末端である。前記付加配列は、特に制限されない。前記付加配列の長さは、特に制限されず、例えば、１～２００塩基長、１～５０塩基長、１～２５塩基長、１８～２４塩基長である。前記付加配列の構成単位は、例えば、ヌクレオチド残基であり、デオキシリボヌクレオチド残基およびリボヌクレオチド残基等があげられる。前記付加配列は、特に制限されず、例えば、デオキシリボヌクレオチド残基からなるＤＮＡ、リボヌクレオチド残基を含むＤＮＡ等のポリヌクレオチドがあげられる。前記付加配列の具体例として、例えば、ポリｄＴ、ポリｄＡ等があげられる。 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 5 '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.
　本発明の核酸分子は、例えば、担体に固定化して使用できる。前記本発明の核酸分子は、例えば、５’末端および３’末端のいずれかを固定化することが好ましく、より好ましくは５’末端である。本発明の核酸分子を固定化する場合、例えば、前記核酸分子は、前記担体に、直接的に固定化してもよいし、間接的に固定化してもよい。後者の場合、例えば、前記付加配列を介して固定化することが好ましい。 The nucleic acid molecule of the present invention can be used, for example, immobilized on a carrier. In the nucleic acid molecule of the present invention, for example, either the 5 'end or the 3' end is preferably immobilized, more preferably the 5 'end. When immobilizing the nucleic acid molecule of the present invention, for example, 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.
　本発明の核酸分子の製造方法は、特に制限されず、例えば、化学合成を利用した核酸合成方法等、遺伝子工学的手法、公知の方法により合成できる。本発明の核酸分子は、例えば、いわゆるＳＥＬＥＸ法によっても得ることができる。この場合、ターゲットは、卵アレルゲンであるオボトランスフェリンが好ましい。 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. In this case, the target is preferably ovotransferrin, which is an egg allergen.
　本発明の核酸分子は、前述のように、前記卵アレルゲンに結合性を示す。このため、本発明の核酸分子の用途は、前記卵アレルゲンへの結合性を利用する用途であれば、特に制限されない。本発明の核酸分子は、例えば、前記卵アレルゲンに対する抗体に代えて、種々の方法に使用できる。 As described above, the nucleic acid molecule of the present invention exhibits binding property to the egg allergen. Therefore, 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 egg allergen. The nucleic acid molecule of the present invention can be used in various methods, for example, instead of the antibody against the egg allergen.
（２）卵アレルゲン分析用センサ
　本発明の卵アレルゲン分析用センサは、前述のように、本発明の卵アレルゲン結合核酸分子を含むことを特徴とする。本発明のセンサは、前記本発明の卵アレルゲン結合核酸分子を含んでいればよく、その他の構成は、何ら制限されない。 (2) Sensor for egg allergen analysis The sensor for egg allergen analysis of the present invention includes the egg allergen-binding nucleic acid molecule of the present invention as described above. The sensor of the present invention only needs to contain the egg allergen-binding nucleic acid molecule of the present invention, and other configurations are not limited at all.
　本発明のセンサは、例えば、前記卵アレルゲン結合核酸分子に前記卵アレルゲンが結合した状態で、活性型となり、前記卵アレルゲン結合核酸分子に前記卵アレルゲンが未結合の状態で、不活性型となる、前記結合を検出する結合検出用核酸分子を、さらに含んでもよい。本発明のセンサは、前記結合検出用核酸分子を含む場合、前記結合検出用核酸分子が活性型であるか不活性型であるかによって、前記卵アレルゲン結合核酸分子への前記卵アレルゲンの結合の有無を確認でき、それによって、前記卵アレルゲンの有無を分析することができる。 The sensor of the present invention, for example, becomes active when the egg allergen-binding nucleic acid molecule is bound to the egg allergen, and becomes inactive when the egg allergen-bound nucleic acid molecule is not bound to the egg allergen. Further, a nucleic acid molecule for detecting binding that detects the binding may be further included. When the sensor of the present invention includes the binding detection nucleic acid molecule, the binding of the egg allergen to the egg allergen binding nucleic acid molecule depends on whether the binding detection nucleic acid molecule is active or inactive. The presence or absence can be confirmed, thereby analyzing the presence or absence of the egg allergen.
　前記結合検出用核酸分子としては、例えば、Ｇカルテット構造を形成する核酸分子があげられる。前記Ｇカルテッド構造を形成する核酸分子は、例えば、Ｇカルテッド構造を形成した状態が、活性型であり、Ｇカルテッド構造を非形成の状態が、不活性型である。 Examples of the binding detection nucleic acid molecule 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.
　前記Ｇカルテット構造を形成する核酸分子は、例えば、ＤＮＡｚｙｍｅまたはＲＮＡｚｙｍｅ等があげられ、好ましくはＤＮＡｚｙｍｅである。 Examples of the nucleic acid molecule forming the G quartet structure include DNAzyme and RNAzyme, and preferably DNAzyme.
　Ｇカルテッド構造を形成した活性型ＤＮＡｚｙｍｅは、例えば、酸化還元反応を触媒するペルオキシダーゼ様の活性を示す。このため、本発明のセンサがＤＮＡｚｙｍｅを有する場合、前記ＤＮＡｚｙｍｅの触媒活性を検出することによって、前記卵アレルゲン結合核酸分子への前記卵アレルゲンの結合の有無または結合量を分析することができる。 An active DNAzyme having a G-culted structure exhibits a peroxidase-like activity that catalyzes a redox reaction, for example. For this reason, when the sensor of the present invention has DNAzyme, the presence or amount of binding of the egg allergen to the egg allergen-binding nucleic acid molecule can be analyzed by detecting the catalytic activity of the DNAzyme.
　この場合、本発明のセンサは、例えば、ポルフィリンを共存させることが好ましい。前記ポルフィリンは、特に制限されず、例えば、無置換体のポルフィリン、その誘導体があげられる。前記誘導体は、例えば、置換体のポルフィリンおよび金属元素と錯体を形成した金属ポルフィリン等があげられる。前記置換体のポルフィリンは、例えば、Ｎ－メチルメソポルフィリン等があげられる。前記金属ポルフィリンは、例えば、三価鉄錯体であるヘミン等があげられる。前記ポルフィリンは、例えば、前記金属ポルフィリンが好ましく、より好ましくはヘミンである。 In this case, it is preferable that 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. Examples of 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.
　また、Ｇカルテッド構造を形成した活性型のＤＮＡｚｙｍｅは、例えば、ポルフィリンと複合体を形成することで、蛍光を生じる。このため、本発明のセンサがＤＮＡｚｙｍｅを有する場合、前記ポルフィリンを共存させ、前記ＤＮＡｚｙｍｅと前記ポルフィリンとの複合体形成による蛍光を検出することによって、前記卵アレルゲン結合核酸分子への前記卵アレルゲンの結合の有無または結合量を分析することができる。 In addition, 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, binding of the egg allergen to the egg allergen binding nucleic acid molecule by detecting the fluorescence due to the complex formation of the DNAzyme and the porphyrin in the presence of the porphyrin The presence or absence or the amount of binding can be analyzed.
　前記ポルフィリンは、特に制限されず、例えば、Ｎ－メチルメソポルフィリン（ＮＭＭ）、Ｚｎ－ＤＩＧＰ、ＺｎＰＰ９およびＴＭＰｙＰ等が好ましい。 The porphyrin is not particularly limited, and for example, N-methylmesoporphyrin (NMM), Zn-DIGP, ZnPP9, and TMPyP are preferable.
　本発明のセンサは、例えば、さらに標識物質を有してもよい。前記標識物質は、例えば、前記核酸分子の５’末端および３’末端の少なくとも一方に結合していることが好ましく、より好ましくは５’末端である。前記標識物質は、特に制限されず、例えば、蛍光物質、色素、同位体、酵素等があげられる。前記蛍光物質は、例えば、ピレン、ＴＡＭＲＡ、フルオレセイン、Ｃｙ３色素、Ｃｙ５色素、ＦＡＭ色素、ローダミン色素、テキサスレッド色素、ＪＯＥ、ＭＡＸ、ＨＥＸ、ＴＹＥ等の蛍光団があげられ、前記色素は、例えば、Ａｌｅｘａ４８８、Ａｌｅｘａ６４７等のＡｌｅｘａ色素等があげられる。 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.
　前記標識物質は、例えば、前記核酸分子に直接的に連結してもよいし、リンカーを介して、間接的に連結してもよい。前記リンカーは、特に制限されず、例えば、前述の例示を援用できる。 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.
（３）分析方法
　本発明の分析方法は、前述のように、卵アレルゲンの分析方法であって、試料と前記本発明の卵アレルゲン結合核酸分子とを接触させ、前記試料中の卵アレルゲンと前記核酸分子とを結合させることにより、前記試料中の卵アレルゲンを検出する工程を含むことを特徴とする。本発明の分析方法は、前記本発明の核酸分子を使用することが特徴であって、その他の工程および条件等は、特に制限されない。また、本発明の分析方法は、前記本発明の核酸分子として、前記本発明の卵アレルゲン分析用センサを使用してもよい。 (3) Analysis method As described above, the analysis method of the present invention is an egg allergen analysis method, wherein a sample is contacted with the egg allergen-binding nucleic acid molecule of the present invention, and the egg allergen in the sample and the It includes a step of detecting an egg allergen in the sample by binding to 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. In the analysis method of the present invention, the egg allergen analysis sensor of the present invention may be used as the nucleic acid molecule of the present invention.
　本発明によれば、前記本発明の核酸分子が、卵アレルゲンに特異的に結合することから、例えば、卵アレルゲンと前記核酸分子との結合を検出することによって、試料中の卵アレルゲンを特異的に検出可能である。具体的には、例えば、試料中の卵アレルゲンの有無または卵アレルゲンの量を分析可能であることから、定性または定量も可能といえる。 According to the present invention, since the nucleic acid molecule of the present invention specifically binds to the egg allergen, the egg allergen in the sample is specifically detected by detecting the binding between the egg allergen and the nucleic acid molecule, for example. Can be detected. Specifically, for example, since it is possible to analyze the presence or absence of egg allergen or the amount of egg allergen in a sample, it can be said that qualitative or quantitative determination is possible.
　本発明において、前記試料は、特に制限されない。前記試料は、例えば、食品、食品原料、食品添加物等があげられる。また、前記試料は、例えば、食品加工場または調理場等における付着物、洗浄後の洗浄液等があげられる。 In the present invention, the sample is not particularly limited. Examples of 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.
　前記試料は、例えば、液体試料でもよいし、固体試料でもよい。前記試料は、例えば、前記核酸分子と接触させ易く、取扱いが簡便であることから、液体試料が好ましい。前記固体試料の場合、例えば、溶媒を用いて、混合液、抽出液、溶解液等を調製し、これを使用してもよい。前記溶媒は、特に制限されず、例えば、水、生理食塩水、緩衝液等があげられる。 The sample may be, for example, a liquid sample or a solid sample. For example, the sample is preferably a liquid sample because it is easy to contact with the nucleic acid molecule and is easy to handle. In the case of the solid sample, for example, 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.
　前記検出工程は、例えば、前記試料と前記核酸分子とを接触させて、前記試料中の卵アレルゲンと前記核酸分子とを結合させる接触工程と、前記卵アレルゲンと前記核酸分子との結合を検出する結合検出工程とを含む。また、前記検出工程は、例えば、さらに、前記結合検出工程の結果に基づいて、前記試料中の卵アレルゲンの有無または量を分析する工程を含む。 The detection step detects, for example, a contact step of bringing the sample and the nucleic acid molecule into contact with each other to bind the egg allergen and the nucleic acid molecule in the sample, and binding between the egg allergen and the nucleic acid molecule. A binding detection step. The detection step further includes, for example, a step of analyzing the presence or amount of egg allergen in the sample based on the result of the binding detection step.
　前記接触工程において、前記試料と前記核酸分子との接触方法は、特に制限されない。前記試料と前記核酸分子との接触は、例えば、液体中で行われることが好ましい。前記液体は、特に制限されず、例えば、水、生理食塩水、緩衝液等があげられる。 In the contacting 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.
　前記接触工程において、前記試料と前記核酸分子との接触条件は、特に制限されない。接触温度は、例えば、４～３７℃、１８～２５℃であり、接触時間は、例えば、１０～１２０分、３０～６０分である。 In the contacting step, 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., and the contact time is, for example, 10 to 120 minutes or 30 to 60 minutes.
　前記接触工程において、前記核酸分子は、例えば、担体に固定化された固定化核酸分子でもよいし、未固定の遊離した核酸分子でもよい。後者の場合、例えば、容器内で、前記試料と接触させる。前記核酸分子は、例えば、取扱性に優れることから、前記固定化核酸分子が好ましい。前記担体は、特に制限されず、例えば、基板、ビーズ、容器等があげられ、前記容器は、例えば、マイクロプレート、チューブ等があげられる。前記核酸分子の固定化は、例えば、前述の通りである。 In the contacting step, the nucleic acid molecule may be, for example, an immobilized nucleic acid molecule immobilized on a carrier or an unfixed free nucleic acid molecule. In the latter case, for example, 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.
　前記結合検出工程は、前述のように、前記試料中の卵アレルゲンと前記核酸分子との結合を検出する工程である。前記両者の結合の有無を検出することによって、例えば、前記試料中の卵アレルゲンの有無を分析（定性）でき、また、前記両者の結合の程度（結合量）を検出することによって、例えば、前記試料中の卵アレルゲンの量を分析（定量）できる。 The binding detection step is a step of detecting the binding between the egg allergen in the sample and the nucleic acid molecule as described above. By detecting the presence or absence of binding between the two, for example, the presence or absence of egg allergen in the sample can be analyzed (qualitative), and by detecting the degree of binding between the two (binding amount), for example, The amount of egg allergen in the sample can be analyzed (quantified).
　そして、前記卵アレルゲンと前記核酸分子との結合が検出できなかった場合は、前記試料中に卵アレルゲンは存在しないと判断でき、前記結合が検出された場合は、前記試料中に卵アレルゲンが存在すると判断できる。 If the binding between the egg allergen and the nucleic acid molecule cannot be detected, it can be determined that the egg allergen is not present in the sample. If the binding is detected, the egg allergen is present in the sample. It can be judged.
　前記卵アレルゲンと前記核酸分子との結合の分析方法は、特に制限されない。前記方法は、例えば、物質間の結合を検出する従来公知の方法が採用でき、具体例として、前述のＳＰＲ、蛍光偏光法等があげられる。また、前記結合は、例えば、前記卵アレルゲンと前記核酸分子との複合体の検出でもよい。 The method for analyzing the binding between the egg allergen and the nucleic acid molecule is not particularly limited. As the method, for example, 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 egg allergen and the nucleic acid molecule.
　前記蛍光偏光法による前記卵アレルゲンと前記核酸分子との結合の検出は、例えば、以下のようにして行うことができる。 The detection of the binding between the egg allergen and the nucleic acid molecule by the fluorescence polarization method can be performed, for example, as follows.
　前記蛍光偏光法は、一般に、偏光励起光を前記標識物質に照射した際、前記標識物質から発せられる蛍光が、前記標識物質で標識された分子の分子量に応じて異なった偏光度を示すという特性に基づく測定方法である。本発明においては、例えば、前記標識物質で標識化した前記核酸分子（標識化核酸分子）を使用することで、前記蛍光偏光法により前記卵アレルゲンと前記核酸分子との結合を検出することができる。具体的には、前記標識化核酸分子は、卵アレルゲンと未結合の状態と、卵アレルゲンと結合した状態とを比較した場合、前者は、相対的に分子量が小さいため、相対的に偏光度が低く、一方、後者は、相対的に分子量が大きいため、相対的に偏光度が高い。このため、例えば、試料と接触させる前の前記標識化核酸分子の偏光度と、前記試料と接触させた後の前記標識化核酸分子の前記偏光度とを比較することで、卵アレルゲンと前記標識化核酸分子との結合を検出できる。また、卵アレルゲンと未結合の前記標識化核酸分子および卵アレルゲンと結合した前記標識化核酸分子の少なくとも一方の偏光度を評価基準として、前記試料と接触させた後の前記標識化核酸分子の偏光度を評価することでも、卵アレルゲンと前記標識化核酸分子との結合を検出できる。 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 egg allergen and the nucleic acid molecule can be detected by the fluorescence polarization method. . Specifically, when the labeled nucleic acid molecule is compared with an unbound state with an egg allergen and a bound state with an egg allergen, the former has a relatively low molecular weight and thus has a relatively low degree of polarization. On the other hand, 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 contacting with the sample and the degree of polarization of the labeled nucleic acid molecule after contacting with the sample, the egg allergen and the label are compared. Binding to the activated nucleic acid molecule can be detected. In addition, the polarization of the labeled nucleic acid molecule after contact with the sample is evaluated based on the polarization degree of at least one of the labeled nucleic acid molecule not bound to the egg allergen and the labeled nucleic acid molecule bound to the egg allergen. By evaluating the degree, the binding between the egg allergen and the labeled nucleic acid molecule can also be detected.
　前記蛍光偏光法によれば、例えば、前記本発明の核酸分子を、前記標識物質で標識化するのみで、センサとして容易に使用できる。また、前記標識物質は、その種類によって検出波長が異なるため、例えば、試料の種類に応じて前記標識物質を選択することで、前記試料由来の蛍光の影響を低減することもできる。 According to the fluorescence polarization method, for example, the nucleic acid molecule of the present invention can be easily used as a sensor only by labeling with the labeling substance. In addition, since 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.
　前記標識化核酸分子は、例えば、前記本発明の核酸分子が前記標識物質で標識化されていればよく、その標識方法は、特に制限されない。 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.
　前記標識化核酸分子としては、例えば、前記本発明の核酸分子に前記標識物質が連結した形態があげられる。この形態は、例えば、前述の記載が援用でき、前記本発明の核酸分子に、前記標識物質が直接的に連結してもよいし、前述のようにリンカー等を介して前記標識物質が間接的に連結してもよい。前記リンカーの長さは、特に制限されず、例えば、０～１０塩基長、０～７塩基長、、０～５塩基長である。前記標識物質は、例えば、前記本発明の核酸分子のいずれの部位に連結されてもよく、具体例としては、５’末端および３’末端があげられ、両末端に連結してもよいし、いずれか一方の末端に連結してもよく、好ましくは、５’末端である。 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. For example, the above description can be used for this form, and 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.
　前記標識化核酸分子としては、この他に、例えば、前記本発明の核酸分子と、これに相補的であって且つ標識物質が連結した相補鎖（以下、「標識化相補鎖」ともいう）とを含み、前記核酸分子と前記標識化相補鎖とがハイブリダイズしたハイブリッド分子があげられる。 Other examples of the labeled nucleic acid molecule 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.
　前記相補鎖は、例えば、前記本発明の核酸分子の一部に相補的な配列を有していればよく、前記相補的な配列のみから構成されてもよいし、前記相補的な配列を含んでもよい。前記相補鎖は、前記本発明の核酸分子のどの領域に対して相補的でもよく、好ましくは、５’末端領域または３’末端領域に相補的である。また、例えば、前記本発明の核酸分子が、その５’末端または３’末端にリンカーを有し、前記相補的な配列は、前記リンカーに相補的であることが好ましい。前記リンカーの長さは、特に制限されず、例えば、１０～３０塩基長、１５～２５塩基長、１８～２４塩基長である。前記相補鎖の長さは、特に制限されず、例えば、１０～３０塩基長、１５～２５塩基長、１８～２４塩基長である。 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. In addition, for example, 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.
　前記標識化相補鎖において、前記標識物質は、例えば、前記相補鎖のいずれの部位に連結されてもよく、具体例としては、５’末端および３’末端があげられ、両末端に連結してもよいし、いずれか一方の末端に連結してもよい。前記標識化相補鎖が、前記本発明の核酸分子の３’末端領域に相補的な場合、前記標識物質は、前記相補鎖の５’末端に連結することが好ましく、前記標識化相補鎖が、前記本発明の核酸分子の５’末端領域に相補的な場合、前記標識物質は、前記相補鎖の３’末端に連結することが好ましい。 In the labeled complementary strand, 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. When the labeled complementary strand is complementary to the 3 ′ end region of the nucleic acid molecule of the present invention, the labeling substance is preferably linked to the 5 ′ end of the complementary strand, and 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.
　前記標識物質は、特に制限されず、前述した例示を援用でき、中でも前記蛍光物質および前記色素が好ましい。 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.
　前記蛍光偏光法を採用する場合、本発明の分析方法は、例えば、前記試料と前記標識化核酸分子とを接触させ、前記試料中の卵アレルゲンと前記標識化核酸分子とを結合させる接触工程と、前記標識化核酸分子に偏光励起光を照射して、前記標識化核酸分子の偏光度を測定する測定工程と、前記測定工程における測定結果と評価基準とを比較し、卵アレルゲンと前記標識化核酸分子との結合を検出する工程を検出する検出工程を含むことが好ましい。 When employing the fluorescence polarization method, the analysis method of the present invention includes, for example, a contact step of bringing the sample and the labeled nucleic acid molecule into contact with each other, and binding the egg allergen and the labeled nucleic acid molecule in the sample. , Irradiating the labeled nucleic acid molecule with polarized excitation light to measure the degree of polarization of the labeled nucleic acid molecule, comparing the measurement result in the measurement step and the evaluation criteria, and comparing the egg allergen and the labeling It is preferable to include a detection step of detecting a step of detecting binding with the nucleic acid molecule.
　前記測定工程において、前記偏光励起光の波長および前記偏光度の検出波長は、特に制限されず、例えば、前記標識物質の種類に応じて適宜設定できる。具体例として、前記標識物質がＡｌｅｘａ６４７の場合、前記偏光励起光の波長は、例えば、６２０～６８０ｎｍであり、偏光度の検出波長は、例えば、６６０～８００ｎｍである。前記偏光励起光の照射時間は、特に制限されず、例えば、１ナノ～５ナノ秒があげられる。 In the measurement step, 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. As a specific example, when the labeling substance is Alexa647, the wavelength of the polarized excitation light is, for example, 620 to 680 nm, and 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.
　前記検出工程において、前記評価基準は、例えば、予め決定してもよいし、測定ごとに決定してもよい。前記評価基準としては、例えば、卵アレルゲン未結合の基準、卵アレルゲン結合の基準が設定できる。前者の基準は、例えば、卵アレルゲンが結合していない前記標識化核酸分子のみの偏光度であり、後者の基準は、例えば、卵アレルゲンが結合した前記標識化核酸分子の偏光度である。 In the detection step, the evaluation criterion may be determined in advance or may be determined for each measurement, for example. As the evaluation standard, for example, a standard for egg allergen unbinding and a standard for egg allergen binding can be set. The former criterion is, for example, the degree of polarization of the labeled nucleic acid molecule to which egg allergen is not bound, and the latter criterion is, for example, the degree of polarization of the labeled nucleic acid molecule to which egg allergen is bound.
　前者の基準を用いる場合は、例えば、前記測定工程における測定値が、前記基準よりも高ければ、卵アレルゲンが存在すると判断でき、また、前記基準よりも相対的に高ければ、相対的に多くの卵アレルゲンが存在すると判断できる。他方、前記測定工程における測定値が、前記基準と同程度または低ければ、卵アレルゲンが存在しないと判断できる。前者の基準は、例えば、前記接触工程前の前記標識化核酸分子の偏光度でもよい。 In the case of using the former standard, for example, if the measured value in the measurement step is higher than the standard, it can be determined that egg allergen is present. It can be determined that egg allergen is present. On the other hand, if the measurement value in the measurement step is the same or lower than the reference, it can be determined that no egg allergen is present. The former criterion may be, for example, the degree of polarization of the labeled nucleic acid molecule before the contacting step.
　また、後者の基準を用いる場合は、例えば、前記測定工程における測定値が、前記基準よりも低ければ、卵アレルゲンが存在しないと判断できる。他方、前記測定工程における測定値が、前記基準と同程度または高ければ、卵アレルゲンが存在すると判断でき、また、前記基準よりも相対的に高ければ、相対的に多くの卵アレルゲンが存在すると判断できる。 Further, when the latter standard is used, for example, if the measurement value in the measurement step is lower than the standard, it can be determined that the egg allergen is not 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 egg allergen is present, and if it is relatively higher than the reference, it is determined that relatively many egg allergens are present. it can.
　また、前記基準は、卵アレルゲンの量と偏光度との相関関係であってもよい。例えば、複数の既知濃度の卵アレルゲンと所定量の前記標識化核酸分子とを接触させ、各濃度の卵アレルゲンに結合した前記標識化核酸分子の偏光度を測定することにより、前記相関関係を示す相関式が得られる。そして、この相関式と、前記測定工程における測定値とから、前記試料における卵アレルゲンの量を判断することができる。 Further, the reference may be a correlation between the amount of egg allergen and the degree of polarization. For example, the correlation is shown by contacting a plurality of egg allergens at known concentrations with a predetermined amount of the labeled nucleic acid molecule and measuring the degree of polarization of the labeled nucleic acid molecules bound to each concentration of egg allergen. A correlation equation is obtained. The amount of egg allergen in the sample can be determined from this correlation equation and the measured value in the measurement step.
　また、本発明の核酸分子として、前記本発明の卵アレルゲン分析用センサを使用する場合、例えば、酸化還元反応の検出または蛍光発生の検出によって、前記卵アレルゲンの検出を行うことができる。 When the egg allergen analysis sensor of the present invention is used as the nucleic acid molecule of the present invention, the egg allergen can be detected by, for example, detecting a redox reaction or detecting fluorescence.
　前述のように、前記本発明のセンサが、前記結合検出用核酸分子としてＧカルテッド構造を形成するＤＮＡｚｙｍｅを有する場合、前記卵アレルゲン結合核酸分子への前記卵アレルゲンの結合によって、前記ＤＮＡｚｙｍｅは、Ｇカルテッド構造を形成し、ペルオキシダーゼ様の酸化還元反応の触媒活性を示す活性型となる。このため、前記酸化還元反応を検出することによって、前記卵アレルゲン結合核酸分子への前記卵アレルゲンの結合を検出できる。この場合、例えば、前記酸化還元反応の基質を併用することが好ましい。 As described above, when the sensor of the present invention has a DNAzyme that forms a G-carted structure as the nucleic acid molecule for detection of binding, the DNAzyme is G by the binding of the egg allergen to the egg 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 redox reaction, the binding of the egg allergen to the egg 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.
　前記基質は、特に制限されず、例えば、３，３’，５，５’－Ｔｅｔｒａｍｅｔｈｙｌｂｅｎｚｉｄｉｎｅ（ＴＭＢ）、１，２－Ｐｈｅｎｙｌｅｎｅｄｉａｍｉｎｅ（ＯＰＤ）、２，２’－Ａｚｉｎｏｂｉｓ（３－ｅｔｈｙｌｂｅｎｚｏｔｈｉａｚｏｌｉｎｅ－６－ｓｕｌｆｏｎｉｃ　Ａｃｉｄ　Ａｍｍｏｎｉｕｍ　Ｓａｌｔ（ＡＢＴＳ）、３，３’－Ｄｉａｍｉｎｏｂｅｎｚｉｄｉｎｅ（ＤＡＢ）、３，３’－Ｄｉａｍｉｎｏｂｅｎｚｉｄｉｎｅ　Ｔｅｔｒａｈｙｄｒｏｃｈｌｏｒｉｄｅ　Ｈｙｄｒａｔｅ（ＤＡＢ４ＨＣｌ）、３－Ａｍｉｎｏ－９－ｅｔｈｙｌｃａｒｂａｚｏｌｅ（ＡＥＣ）、４－Ｃｈｌｏｒｏ－１－ｎａｐｈｔｈｏｌ（４Ｃ１Ｎ）、２，４，６－Ｔｒｉｂｒｏｍｏ－３－ｈｙｄｒｏｘｙｂｅｎｚｏｉｃ　Ａｃｉｄ、２，４－Ｄｉｃｈｌｏｒｏｐｈｅｎｏｌ、４－Ａｍｉｎｏａｎｔｉｐｙｒｉｎｅ、４－Ａｍｉｎｏａｎｔｉｐｙｒｉｎｅ　Ｈｙｄｒｏｃｈｌｏｒｉｄｅ、ルミノール等があげられる。 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.
　また、前記本発明のセンサが、前記結合検出用核酸分子としてＧカルテッド構造を形成するＤＮＡｚｙｍｅを有する場合、前記卵アレルゲン結合核酸分子への前記卵アレルゲンの結合によって、前記ＤＮＡｚｙｍｅは、Ｇカルテット構造を形成し、ポルフィリンとの複合体を形成することで、蛍光を発生する。このため、前記蛍光を検出することによって、前記卵アレルゲン結合核酸分子への卵アレルゲンを検出できる。 Further, 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 egg allergen to the egg allergen-binding nucleic acid molecule. Fluorescence is generated by forming a complex with porphyrin. For this reason, the egg allergen to the egg allergen binding nucleic acid molecule can be detected by detecting the fluorescence.
（４）検出キット
　本発明の検出キットは、前記本発明の卵アレルゲン結合核酸分子を含むことを特徴とする。本発明の検出キットは、前記本発明の核酸分子を含んでいればよく、その他の構成は何ら制限されない。本発明の検出キットを使用すれば、前述のように、例えば、前記卵アレルゲンの検出等を行うことができる。 (4) Detection kit The detection kit of the present invention comprises the egg 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 egg allergen can be detected as described above.
　本発明の検出キットは、例えば、前記本発明の核酸分子として、前記本発明のセンサを含んでもよい。また、前記本発明の検出キットは、例えば、前記本発明の核酸分子の他に、その他の構成要素を含んでもよい。前記構成要素は、例えば、前記担体、前記ポルフィリン、緩衝液、使用説明書等があげられる。 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. In addition, 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.
　つぎに、本発明の実施例について説明する。ただし、本発明は、下記実施例により制限されない。市販の試薬は、特に示さない限り、それらのプロトコールに基づいて使用した。 Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents were used based on their protocol unless otherwise indicated.
［実施例１］
　各アプタマーについて、卵アレルゲンに対する結合能および動態パラメータを確認した。 [Example 1]
For each aptamer, the binding ability and kinetic parameters for egg allergen were confirmed.
（１）アプタマー
　下記ポリヌクレオチドを合成し、実施例のアプタマーとした。なお、配列番号５および６において、Ｘは、天然チミンに代えて、前記修飾ウラシルの前記ＢｎｄＵＴＰであり、Ｚは、天然シトシンに代えて、前記修飾シトシンの５－Ｍｅ－ｄＣである。 (1) Aptamer The following polynucleotides were synthesized and used as aptamers in the examples. In SEQ ID NOS: 5 and 6, X is the BndUTP of the modified uracil instead of natural thymine, and Z is the 5-Me-dC of the modified cytosine instead of natural cytosine.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　前記アプタマーは、その５’末端に、２４塩基長のポリデオキシアデニン（ポリｄＡ）を付加し、ポリｄＡ付加アプタマーとして、後述するＳＰＲに使用した。 The aptamer was added with polydeoxyadenine (poly dA) having a length of 24 bases at the 5 'end and used as a poly dA addition aptamer for SPR described later.
（２）試料
　市販の加熱オボトランスフェリン（商品名C0755-100MG、Conalbumin　from　chicken　egg　white/heat　denatured、別名　オボトランスフェリン、SIGMA-ALDRICH社製)を、試料として、以下の試験に使用した。 (2) Sample Commercially available heated ovotransferrin (trade name C0755-100MG, Conalbumin from chicken egg white / heat denatured, also known as ovotransferrin, manufactured by SIGMA-ALDRICH) was used as a sample in the following tests.
（３）ＳＰＲによる結合能の解析
　結合能の解析には、ProteON　XPR36（BioRad社）を、その使用説明書にしたがって使用した。 (3) Analysis of binding ability by SPR For analysis of binding ability, ProteON XPR36 (BioRad) was used according to the instruction manual.
　まず、前記ProteON専用のセンサーチップとして、ストレプトアビジンが固定化されたチップ（商品名　ProteOn　NLC　Sensor　Chip、BioRad社）を、前記ProteON　XPR36にセットした。前記センサーチップのフローセルに、超純水（ＤＤＷ）を用いて、５μｍｏｌ／Ｌのビオチン化ポリｄＴをインジェクションし、シグナル強度（ＲＵ：Ｒｅｓｏｎａｎｃｅ　Ｕｎｉｔ）が約９００ＲＵになるまで結合させた。前記ビオチン化ポリｄＴは、２４塩基長のデオキシチミジンの３’末端をビオチン化して調製した。そして、前記チップの前記フローセルに、ＳＰＲバッファーを用いて、４００ｎｍｏｌ／Ｌの前記ポリｄＡ付加アプタマーを、流速２５μＬ／ｍｉｎで８０秒間インジェクションし、シグナル強度が約８００ＲＵになるまで結合させた。この結果を、アプタマーのセンサーチップへの固層化量を示すシグナルとして、アプタマー固層化測定値（Ａ）という。続いて、前記試料を、ＳＰＲバッファーを用いて、流速５０μＬ／ｍｉｎで１２０秒間インジェクションし、引き続き、同じ条件で、ＳＰＲバッファーを流して洗浄を行った。前記試料のインジェクションおよび前記ＳＰＲバッファーによる洗浄に並行して、シグナル強度の測定を行った。この結果を、前記アプタマーとタンパクの結合量を示すシグナルとして、タンパク質結合測定値（Ｂ）という。前記試料の濃度は、１００ｎｍｏｌ／Ｌ、５０ｎｍｏｌ／Ｌ、２５ｎｍｏｌ／Ｌ、１２．５ｎｍｏｌ／Ｌ、６．２５ｎｍｏｌ／Ｌ、３．１２５ｎｍｏｌ／Ｌとした。 First, as a ProteON dedicated sensor chip, a chip (product name: ProteOn NLC Sensor Chip, BioRad) on which streptavidin was immobilized was set in the ProteON XPR36. 5 μmol / 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 3 'end of deoxythymidine having a length of 24 bases. Then, 400 nmol / L of the poly dA-added aptamer was injected into the flow cell of the chip at a flow rate of 25 μL / min for 80 seconds using an SPR buffer, and was bound until the signal intensity reached about 800 RU. This result is referred to as an aptamer solidification measurement value (A) as a signal indicating the amount of solidification of the aptamer to the sensor chip. Subsequently, the sample was injected with an SPR buffer at a flow rate of 50 μL / 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. This result is referred to as a protein binding measurement value (B) as a signal indicating the binding amount of the aptamer and protein. The concentration of the sample was 100 nmol / L, 50 nmol / L, 25 nmol / L, 12.5 nmol / L, 6.25 nmol / L, 3.125 nmol / L.
　前記ＳＰＲバッファーの組成は、４０ｍｍｏｌ／Ｌ　ＨＥＰＥＳ、１２５ｍｍｏｌ／Ｌ　ＮａＣｌ、５ｍｍｏｌ／Ｌ　ＫＣｌ、１ｍｍｏｌ／Ｌ　ＭｇＣｌ２および０．０１％　Ｔｗｅｅｎ（登録商標）２０とし、ｐＨは、７．５とした。 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.
　これらの結果を図２に示す。図２は、前記加熱オボトランスフェリンに対するアプタマーの結合能を示すグラフであり、横軸は、測定時間（秒）を示し、縦軸は、シグナル強度（ＲＵ）を示す。横軸において、０～１２０秒が、前記試料のインジェクション時間であり、１２０秒以降が、前記ＳＰＲバッファーによる洗浄の時間である（以下、同様）。また、図２の（Ａ）および（Ｂ）の各グラフにおいて、プロットは、上から、オボトランスフェリン濃度が、１００ｎｍｏｌ／Ｌ、５０ｎｍｏｌ／Ｌ、２５ｎｍｏｌ／Ｌ、１２．５ｎｍｏｌ／Ｌであり、図２の（Ｃ）および（Ｄ）の各グラフにおいて、プロットは、上から、オボトランスフェリン濃度が、５０ｎｍｏｌ／Ｌ、２５ｎｍｏｌ／Ｌ、１２．５ｎｍｏｌ／Ｌ、６．２５ｎｍｏｌ／Ｌ、３．１２５ｎｍｏｌ／Ｌである。 These results are shown in FIG. FIG. 2 is a graph showing the binding ability of aptamers to the heated ovotransferrin, where the horizontal axis indicates measurement time (seconds) and the vertical axis indicates signal intensity (RU). On the horizontal axis, 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). Moreover, in each graph of FIG. 2 (A) and (B), the plot is from the top, ovotransferrin concentration is 100 nmol / L, 50 nmol / L, 25 nmol / L, 12.5 nmol / L, FIG. In the graphs (C) and (D), the plots from the top are ovotransferrin concentrations of 50 nmol / L, 25 nmol / L, 12.5 nmol / L, 6.25 nmol / L, 3.125 nmol / L. is there.
　図２に示すように、いずれのアプタマーを使用した場合も、前記加熱変性オボトランスフェリンに対して、結合性を示した。 As shown in FIG. 2, when any aptamer was used, it showed binding properties to the heat-denatured ovotransferrin.
　また、前記図２のＳＰＲ解析の結果から、動態パラメータを算出した。これらの結果を下記表４に示す。下記表４に示すように、いずれのアプタマーを使用した場合も、オボトランスフェリンに対する解離定数（ＫＤ）は、２０ｎＭ以下であり、非常に優れた結合性であることがわかった。
In addition, kinetic parameters were calculated from the results of the SPR analysis in FIG. These results are shown in Table 4 below. As shown in Table 4 below, when any aptamer was used, the dissociation constant (KD) for ovotransferrin was 20 nM or less, and it was found that the binding property was very excellent.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　以上、実施形態および実施例を参照して本願発明を説明したが、本願発明は、上記実施形態および実施例に限定されるものではない。本願発明の構成や詳細には、本願発明のスコープ内で当業者が理解しうる様々な変更をできる。 As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.
　この出願は、２０１４年３月３１日に出願された日本出願特願２０１４－０７３７３９を基礎とする優先権を主張し、その開示のすべてをここに取り込む。 This application claims priority based on Japanese Patent Application No. 2014-073739 filed on Mar. 31, 2014, the entire disclosure of which is incorporated herein.
　本発明の卵アレルゲン結合核酸分子は、卵アレルゲンに対して前述のような解離定数で結合することができる。このため、本発明の卵アレルゲン結合核酸分子によれば、例えば、試料中の卵アレルゲンとの結合の有無によって、優れた精度で、卵アレルゲンを検出できる。したがって、本発明の卵アレルゲン結合核酸分子は、例えば、食品製造、食品管理、食品の流通等の分野における卵アレルゲンの検出に、極めて有用なツールといえる。 The egg allergen-binding nucleic acid molecule of the present invention can bind to an egg allergen with the dissociation constant as described above. Therefore, according to the egg allergen-binding nucleic acid molecule of the present invention, for example, the egg allergen can be detected with excellent accuracy depending on the presence or absence of binding to the egg allergen in the sample. Therefore, the egg allergen-binding nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting egg allergens in fields such as food production, food management, and food distribution.

Claims (12)

  1. 卵アレルゲンに対する解離定数が、２０ｎＭ以下の核酸分子であることを特徴とする卵アレルゲン結合核酸分子。 An egg allergen-binding nucleic acid molecule, wherein the nucleic acid molecule has a dissociation constant for egg allergen of 20 nM or less.
  2. 前記卵アレルゲンが、オボトランスフェリン（Ovotransferrin）またはそのサブユニットである、請求項１記載の卵アレルゲン結合核酸分子。 The egg allergen-binding nucleic acid molecule according to claim 1, wherein the egg allergen is Ovotransferrin or a subunit thereof.
  3. 前記卵アレルゲンが、未変性アレルゲンまたは加熱変性アレルゲンである、請求項１または２記載の卵アレルゲン結合核酸分子。 The egg allergen-binding nucleic acid molecule according to claim 1 or 2, wherein the egg allergen is a native allergen or a heat-denatured allergen.
  4. 下記（ａ）～（ｄ）からなる群から選択された少なくとも一つのポリヌクレオチドを含む、請求項１から３のいずれか一項に記載の卵アレルゲン結合核酸分子。
    （ａ）配列番号１－４のいずれかの塩基配列からなるポリヌクレオチド
    （ｂ）前記（ａ）のいずれかの塩基配列において、１もしくは数個の塩基が欠失、置換、挿入および／または付加された塩基配列からなり、前記卵アレルゲンに結合するポリヌクレオチド
    （ｃ）前記（ａ）のいずれかの塩基配列に対して、８０％以上の同一性を有する塩基配列からなり、前記卵アレルゲンに結合するポリヌクレオチド
    （ｄ）前記（ａ）のいずれかの塩基配列からなるポリヌクレオチドに対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドに、相補的な塩基配列からなり、卵アレルゲンに結合するポリヌクレオチド     The egg allergen-binding nucleic acid molecule according to any one of claims 1 to 3, comprising at least one polynucleotide selected from the group consisting of the following (a) to (d):
    (A) a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1-4 (b) one or several bases deleted, substituted, inserted and / or added in any one of the nucleotide sequences of (a) (C) a polynucleotide that binds to the egg allergen, and (c) a polynucleotide that has 80% or more identity to the nucleotide sequence of (a) and binds to the egg allergen (D) a polynucleotide comprising a complementary base sequence and binding to an egg allergen to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the base sequences of (a) above nucleotide
  5. 前記ポリヌクレオチドが、ＤＮＡである、請求項４記載の卵アレルゲン結合核酸分子。 The egg allergen-binding nucleic acid molecule according to claim 4, wherein the polynucleotide is DNA.
  6. 前記ポリヌクレオチドが、修飾塩基を含む、請求項４または５記載の卵アレルゲン結合核酸分子。 The egg allergen-binding nucleic acid molecule according to claim 4 or 5, wherein the polynucleotide comprises a modified base.
  7. 請求項１から６のいずれか一項に記載の卵アレルゲン結合核酸分子を含むことを特徴とする、卵アレルゲン分析用センサ。 A sensor for egg allergen analysis, comprising the egg allergen-binding nucleic acid molecule according to any one of claims 1 to 6.
  8. さらに、Ｇカルテット構造を形成する核酸分子を含む、請求項７記載の卵アレルゲン分析用センサ。 Furthermore, the egg allergen analysis sensor of Claim 7 containing the nucleic acid molecule which forms G quartet structure.
  9. 前記Ｇカルテット構造を形成する核酸分子が、ＤＮＡｚｙｍｅまたはＲＮＡｚｙｍｅである、請求項８記載の卵アレルゲン分析用センサ。 The sensor for egg allergen analysis according to claim 8, wherein the nucleic acid molecule forming the G quartet structure is DNAzyme or RNAzyme.
  10. さらに、ポルフィリンを含む、請求項７から９のいずれか一項に記載の卵アレルゲン分析用センサ。 Furthermore, the sensor for egg allergen analysis as described in any one of Claim 7 to 9 containing a porphyrin.
  11. 試料と請求項１から６のいずれか一項に記載の卵アレルゲン結合核酸分子とを接触させ、前記試料中の卵アレルゲンと前記核酸分子とを結合させることにより、前記試料中の卵アレルゲンを検出する工程を含むことを特徴とする、卵アレルゲンの分析方法。 An egg allergen in the sample is detected by bringing the sample into contact with the egg allergen-binding nucleic acid molecule according to any one of claims 1 to 6 and binding the egg allergen in the sample and the nucleic acid molecule. A method for analyzing an egg allergen, comprising the step of:
  12. 前記試料が、食品、食品原料および食品添加物からなる群から選択された少なくとも一つである、請求項１１記載の卵アレルゲンの分析方法。 12. The method for analyzing an egg allergen according to claim 11, wherein the sample is at least one selected from the group consisting of foods, food ingredients, and food additives.
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