WO2018092374A1 - Molécule d'acide nucléique, et son utilisation - Google Patents

Molécule d'acide nucléique, et son utilisation Download PDF

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WO2018092374A1
WO2018092374A1 PCT/JP2017/030180 JP2017030180W WO2018092374A1 WO 2018092374 A1 WO2018092374 A1 WO 2018092374A1 JP 2017030180 W JP2017030180 W JP 2017030180W WO 2018092374 A1 WO2018092374 A1 WO 2018092374A1
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nucleic acid
acid molecule
polynucleotide
modified
lysozyme
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PCT/JP2017/030180
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Japanese (ja)
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行大 白鳥
あすみ 稲熊
晃尚 清水
金子　直人
嘉仁 吉田
穣 秋冨
藤田　智子
克紀 堀井
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 JP2018551037A priority Critical patent/JP6687264B2/ja
Publication of WO2018092374A1 publication Critical patent/WO2018092374A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to nucleic acid molecules that bind to egg-derived lysozyme and uses 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.
  • lysozyme which is an egg white protein is known as an allergen.
  • ELISA method As an analysis method for lysozyme, a method using an ELISA method 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. Further, electrophoresis, blotting on a nitrocellulose membrane, and the like 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.
  • an object of the present invention is to provide a new nucleic acid molecule that binds to egg-derived lysozyme.
  • the nucleic acid molecule of the present invention is a nucleic acid molecule that binds to lysozyme, characterized in that it comprises any of the following polynucleotides (a) or (b).
  • (b) comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence of (a)
  • the egg-derived lysozyme detection reagent of the present invention contains the nucleic acid molecule of the present invention.
  • the method for detecting an egg-derived lysozyme of the present invention comprises contacting the nucleic acid molecule of the present invention or the detection reagent of the present invention with a sample, the egg-derived lysozyme in the sample, the nucleic acid molecule or the detection reagent, and Forming a complex of: A step of detecting the complex.
  • the nucleic acid molecule of the present invention can bind to egg-derived lysozyme. Therefore, according to the nucleic acid molecule of the present invention, egg-derived lysozyme can be detected based on the presence or absence of binding to the allergen in the sample. Therefore, the nucleic acid molecule of the present invention can be said to be a very useful tool for detecting, for example, allergens derived from eggs in fields such as food production, food management, and food distribution.
  • FIG. 1 shows a presumed secondary structure of aptamer 1 in Example 1 of the present invention.
  • FIG. 2 is a graph showing aptamer 1 binding to egg-derived lysozyme in Example 1 of the present invention.
  • FIG. 3 is a graph showing the binding property of aptamer 1 to an egg sample in Example 1 of the present invention.
  • FIG. 4 is a graph showing the binding property of aptamer 1 to lysozyme in Example 1 of the present invention.
  • FIG. 5 shows the predicted secondary structures of aptamers 2 to 7 in Example 2 of the present invention.
  • FIG. 6 is a graph showing the binding property of each aptamer to egg-derived lysozyme in Example 2 of the present invention.
  • FIG. 7 is a graph showing the measurement results of the light emission amount in Example 3 of the present invention.
  • FIG. 8 is a graph showing the measurement result of the light emission amount in Example 3 of the present invention.
  • nucleic acid molecule of this invention is a nucleic acid molecule couple
  • (b) comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence of (a)
  • the target is egg-derived lysozyme.
  • the eggs are, for example, chicken eggs and quail eggs.
  • the egg-derived lysozyme is, for example, chicken egg-derived lysozyme, and specifically, for example, egg white-derived lysozyme of chicken egg.
  • commercially available lysozyme can be used as lysozyme for confirming the binding ability, and specific examples include lysozyme derived from egg white of chicken eggs (120-02674, manufactured by Wako Pure Chemical Industries, Ltd.).
  • the lysozyme may be, for example, an unmodified allergen that has not been denatured by heating or the like, or a denatured allergen that has been denatured by heating or the like.
  • the egg-derived lysozyme is hereinafter simply referred to as lysozyme.
  • the nucleic acid molecule of the present invention can bind to lysozyme as described above.
  • binding to lysozyme means, for example, having a binding property to lysozyme or having a binding activity to lysozyme.
  • the binding between the nucleic acid molecule of the present invention and lysozyme can be determined by, for example, surface plasmon resonance molecular interaction (SPR) analysis.
  • SPR surface plasmon resonance molecular interaction
  • BIACORE 3000 (trade name, GE Healthcare UK Ltd.) can be used. Since the nucleic acid molecule of the present invention binds to lysozyme, it can be used, for example, for detection of lysozyme.
  • the nucleic acid molecule of the present invention has a dissociation constant indicating a binding force to lysozyme, for example, 1.04 nM or less.
  • the nucleic acid molecule of the present invention is also called a DNA molecule or a DNA aptamer.
  • the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (a) or (b) or a molecule containing the polynucleotide.
  • the polynucleotide (a) may be, for example, a polynucleotide containing the base sequence of SEQ ID NO: 1, a polynucleotide comprising the base sequence of SEQ ID NO: 1, or a nucleotide sequence of SEQ ID NO: 1. It may be a polynucleotide containing a partial sequence or a polynucleotide comprising the partial sequence.
  • the polynucleotide of SEQ ID NO: 1 is shown below.
  • Lys391TR8m4 (SEQ ID NO: 1) GGTTAATCCCGACAAGCCCGTTAAGGGTTAACACGACATTTCGCTGTTGTAACAGGTCATAGTCACCACGGCTCATTTG
  • the partial sequence is not particularly limited, and examples thereof include the nucleotide sequences of SEQ ID NOs: 2 to 7.
  • Lys391TR8m4_s64 (SEQ ID NO: 2) GGTTAATCCCGACAAGCCCGTTAAGGGTTAACACGACATTTCGCTGTTGTAACAGGTCATAGTC Lys391TR8m4_s54 (SEQ ID NO: 3) GACAAGCCCGTTAAGGGTTAACACGACATTTCGCTGTTGTAACAGGTCATAGTC Lys391TR8m4_s52 (SEQ ID NO: 4) CAAGCCCGTTAAGGGTTAACACGACATTTCGCTGTTGTAACAGGTCATAGTC Lys391TR8m4_s69 (SEQ ID NO: 5) GACAAGCCCGTTAAGGGTTAACACGACATTTCGCTGTTGTAACAGGTCATAGTCACCACGGCTCATTTG Lys391TR8m4_s67 (SEQ ID NO: 6) CAAGCCCGTTAAGGGTTAACACGACATTTCGCTGTTGTAACAGGTCATAGTCACCACGGCTCATTTG Lys391TR8m4_s67 (S
  • the “identity” is not particularly limited as long as the polynucleotide of (b) is bound to lysozyme.
  • the identity is, for example, 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, 96% or more, 97% or more, particularly preferably 98% or more, and most preferably 99%. % Or more.
  • the identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).
  • the polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (c) below.
  • the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (c) or a molecule containing the polynucleotide.
  • C A polynucleotide comprising a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to the polynucleotide comprising the base sequence of (a) and binding to egg-derived lysozyme
  • the “hybridizing polynucleotide” is not particularly limited, and is, for example, a polynucleotide that is completely or partially complementary to the base sequence of (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, medium stringent conditions, and high 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.
  • the polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (d) below.
  • the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (d) or a molecule containing the polynucleotide.
  • D a polynucleotide comprising a base sequence in which one or several bases are deleted, substituted, inserted and / or added in the base sequence of (a), and binding to egg-derived lysozyme
  • “one or several” may be, for example, within a range in which the polynucleotide of (d) binds to egg-derived lysozyme.
  • the “one or several” in the base sequence of (a) is, for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, further preferably 1 to 3, particularly preferably. Is one or two.
  • the polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (e) below.
  • the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (e) or a molecule containing the polynucleotide.
  • E a polynucleotide that binds to egg-derived lysozyme, comprising a base sequence having at least 80% identity to the base sequence of (a), comprising any one of SEQ ID NOs: 2 to 7
  • identity is not particularly limited, and is the same as (b), for example.
  • the polynucleotide in the nucleic acid molecule of the present invention may be, for example, the polynucleotide (f) below.
  • the nucleic acid molecule of the present invention may be, for example, a molecule composed of the polynucleotide (f) or a molecule containing the polynucleotide.
  • F a base sequence having 80% or more identity to at least one base sequence selected from the group consisting of SEQ ID NOs: 1 to 7, represented by the following formulas (I) to (VII): That bind to egg-derived lysozyme capable of forming a secondary structure
  • “identity” is not particularly limited, and is the same as (b), for example.
  • “can form a secondary structure” means, for example, that the polynucleotide of (f) can form a stem structure and a loop structure in the above formula. The stem structure and loop structure will be described later.
  • the nucleic acid molecule of the present invention may include, for example, one of the polynucleotide sequences (a) to (f) 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, but are preferably the same.
  • the number of the sequences is not particularly limited, and is, for example, 2 or more, preferably 2 to 12, more preferably 2 to 6, and further preferably 2. is there.
  • the linker is, for example, a polynucleotide, and the structural unit is, for example, a nucleotide residue.
  • the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue.
  • the length of the linker is not particularly limited, and is, for example, 1 to 24 bases long, preferably 12 to 24 bases long, more preferably 16 to 24 bases long, and further preferably 20 to 24 bases long. It is long.
  • 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 is any one of the polynucleotides (a) to (f), and the other single-stranded polynucleotide is not limited.
  • the other single-stranded polynucleotide include a polynucleotide having a base sequence complementary to any one of the polynucleotides (a) to (f).
  • 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 (f) preferably has a stem structure and a loop structure as described above, for example.
  • 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 length of the nucleic acid molecule is not particularly limited, and the lower limit thereof is, for example, 15 base length, preferably 75 base length or 80 base length, and the upper limit thereof is, for example, 1000 base length, preferably Is 200 bases, 100 bases or 90 bases long.
  • nucleotide residue examples include deoxyribonucleotide residue and ribonucleotide residue.
  • nucleic acid molecule of the present invention examples include DNA composed only of deoxyribonucleotide residues, DNA containing one or several ribonucleotide residues, and the like. In the latter case, “1 or several” is not particularly limited, and is, for example, 1 to 3 in the polynucleotide.
  • 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 3 bases” means all disclosures of “1, 2, 3 bases” (hereinafter the same).
  • the polynucleotide includes, for example, at least one 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.
  • 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 polynucleotide is not particularly limited, and may include, for example, only one type of the modified base, or may include two or more types of the modified base.
  • the number of the modified base is not particularly limited.
  • the number of the modified base is, for example, one or more.
  • the modified base is, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly preferably 1 to 30, and most preferably. 1 to 20 and all the bases may be the modified bases.
  • 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 modified base in the entire length of the nucleic acid molecule containing the polynucleotide is not particularly limited, and is, for example, 1 to 80, 1 to 50, or 1 to 20, preferably in the same range as described above. It is.
  • the ratio of the modified base is not particularly limited.
  • the ratio of the modified base is, for example, 1/100 or more, preferably 1/40 or more, more preferably 1/20 or more, still more preferably 1/10 or more, particularly preferably, of the total number of bases of the polynucleotide. 1/4 or more, most preferably 1/3 or more.
  • the ratio of the modified base in the entire length of the nucleic acid molecule containing the polynucleotide is not particularly limited, and is the same as the above range.
  • the total number of bases is, for example, the total number of natural bases and modified bases in 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 number of the modified thymine is not particularly limited.
  • natural thymine can be substituted for the modified thymine.
  • the number of the modified thymine is, for example, one or more.
  • the modified thymine is, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly preferably 1 to 30, and most preferably. 1 to 21 and all the thymines may be the modified thymines.
  • the ratio of the modified thymine is not particularly limited.
  • the ratio of the modified thymine is, for example, 1/100 or more, preferably 1/40 or more, more preferably 1/20 or more, further preferably 1 out of the total number of the natural thymine and the modified thymine. / 10 or more, particularly preferably 1/4 or more, and most preferably 1/3 or more.
  • the number of the modified uracil is not particularly limited.
  • natural thymine can be substituted for the modified uracil.
  • the number of the modified uracil is, for example, one or more.
  • the modified uracil is, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly preferably 1 to 30, and most preferably. 1 to 21 and all the uracils may be the modified uracils.
  • the ratio of the modified uracil is not particularly limited.
  • the ratio of the modified uracil is, for example, 1/100 or more, preferably 1/40 or more, more preferably 1/20 or more, and further preferably 1 out of the total number of the natural thymines and the number of the modified uracils. / 10 or more, particularly preferably 1/4 or more, and most preferably 1/3 or more.
  • Examples of the number of the modified thymine and the modified uracil may be, for example, the total number of both.
  • the number of the modified cytosines is not particularly limited.
  • natural cytosine can be substituted for the modified cytosine.
  • the number of the modified cytosines is, for example, one or more.
  • the modified cytosine is, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly preferably 1 to 30, and most preferably. 1 to 21 and all cytosines may be the modified cytosine.
  • the ratio of the modified cytosine is not particularly limited.
  • the ratio of the modified cytosine is, for example, 1/100 or more, preferably 1/40 or more, more preferably 1/20 or more, further preferably 1 out of the total number of the natural cytosine and the modified cytosine. / 10 or more, particularly preferably 1/4 or more, and most preferably 1/3 or more.
  • the modified base is the modified adenine or the modified guanine
  • cytosine and modified cytosine are referred to as “adenine” and “modified adenine” or “guanine” and It can be read as “modified guanine”.
  • natural adenine can be substituted with the modified adenine
  • natural guanine can be substituted with the modified guanine.
  • the nucleic acid molecule of the present invention may contain 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, or a nucleotide having the 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.
  • the polynucleotide for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40, particularly The number is preferably 1 to 30, and most preferably 1 to 21.
  • the modified nucleotides in the entire length of the nucleic acid molecule including the polynucleotide are not particularly limited, and are, for example, 1 to 80, 1 to 50, and 1 to 20, preferably in the same range as described above. It is.
  • 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 for example, in the polynucleotide, for example, 1 to 80, preferably 1 to 70, more preferably 1 to 50, still more preferably 1 to 40 Particularly preferred is 1 to 30, most preferably 1 to 21.
  • 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 artificial nucleic acid monomer residue in the entire length of the nucleic acid molecule containing the polynucleotide is not particularly limited, and is, for example, 1 to 80, 1 to 50, or 1 to 20, preferably the above-mentioned Similar to range.
  • the nucleic acid molecule of the present invention is preferably nuclease resistant, for example.
  • the nucleic acid molecule of the present invention preferably has, for example, the modified nucleotide residue and / or the artificial nucleic acid monomer residue for nuclease resistance. Since the nucleic acid molecule of the present invention is nuclease resistant, for example, tens of kDa PEG (polyethylene glycol) or deoxythymidine may be bound to the 5 'end or 3' end.
  • the nucleic acid molecule of the present invention may further have an additional sequence, for example.
  • the additional sequence is preferably bound to, for example, at least one of the 5 'end and the 3' end of the nucleic acid molecule, and more preferably the 3 'end.
  • the additional sequence is not particularly limited.
  • the length of the additional sequence is not particularly limited, and is, for example, 1 to 200 bases long, preferably 1 to 50 bases long, more preferably 1 to 25 bases long, and further preferably 18 to 24 bases long. It is.
  • 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 In the nucleic acid molecule of the present invention, for example, either the 5 'end or the 3' end can be immobilized.
  • the nucleic acid molecule may be immobilized directly or indirectly on the carrier. In the latter case, the nucleic acid molecule of the present invention is immobilized on the carrier, for example, via the additional sequence.
  • the carrier include beads, plates, filters, columns, substrates, containers and the like.
  • the nucleic acid molecule of the present invention may further have a labeling substance, and specifically, the labeling substance may be bound to the nucleic acid molecule.
  • the nucleic acid molecule to which the labeling substance is bound can also be referred to as a nucleic acid sensor of the present invention, for example.
  • the labeling substance may be bound to, for example, at least one of the 5 'end and the 3' end of the nucleic acid molecule.
  • the labeling with the labeling substance may be, for example, binding or chemical modification.
  • the labeling substance is not particularly limited, and examples thereof include enzymes, fluorescent substances, dyes, isotopes, drugs, toxins, and antibiotics.
  • Examples of the enzyme include luciferase and SA-Lucia luciferase.
  • 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 is, for example, a polynucleotide linker.
  • the method for producing the nucleic acid molecule of the present invention is not particularly limited, and can be synthesized by, for example, a known method such as a nucleic acid synthesis method using chemical synthesis or a genetic engineering technique.
  • the nucleic acid molecule of the present invention exhibits binding to lysozyme as described above. For this reason, the use of the nucleic acid molecule of the present invention is not particularly limited as long as it uses binding to lysozyme.
  • the nucleic acid molecule of the present invention can be used in various methods in place of, for example, an antibody against lysozyme.
  • lysozyme can be detected.
  • the method for detecting lysozyme is not particularly limited, and can be performed by detecting the binding between lysozyme and the nucleic acid molecule.
  • the detection reagent of the present invention is a detection reagent for egg-derived lysozyme, and includes the nucleic acid molecule of the present invention.
  • the detection reagent of this invention should just contain the nucleic acid molecule of the said this invention, and another structure is not restrict
  • egg-derived lysozyme can be detected as described above.
  • the detection reagent of the present invention can be said to be a binder to egg-derived lysozyme, for example.
  • the detection reagent of the present invention may further have a labeling substance, for example, and the labeling substance may be bound to the nucleic acid molecule.
  • the labeling substance for example, the description in the nucleic acid molecule of the present invention can be used.
  • the detection reagent of the present invention may have, for example, a carrier, and the nucleic acid molecule may be immobilized on the carrier.
  • the carrier for example, the description in the nucleic acid molecule of the present invention can be incorporated.
  • the detection kit of the present invention includes the nucleic acid molecule of the present invention or the detection reagent of the present invention.
  • the detection kit of the present invention may further include other components, for example.
  • the constituent element include a buffer solution for preparing the sample, an instruction manual, and the like.
  • the detection kit of the present invention is, for example, A kit containing a nucleic acid sensor and an allergen-labeled carrier (lysozyme-labeled carrier) can be obtained.
  • the description of the nucleic acid molecule of the present invention can be used, and the nucleic acid molecule of the present invention and the detection method of the present invention described later can also be used for the method of use. .
  • the method for detecting an egg-derived lysozyme of the present invention comprises contacting the nucleic acid molecule of the present invention or the detection reagent of the present invention with a sample, and then egg-derived lysozyme in the sample. And a step of forming a complex with the nucleic acid molecule or the detection reagent, and a step of detecting the complex.
  • the detection method of the present invention is characterized by using the nucleic acid molecule of the present invention or the detection reagent, and the other steps and conditions are not particularly limited.
  • the use of the nucleic acid molecule of the present invention will be described as an example, but the nucleic acid molecule of the present invention can be read as the detection reagent of the present invention.
  • the nucleic acid molecule of the present invention specifically binds to lysozyme, for example, by detecting the binding of lysozyme and the nucleic acid molecule or the detection reagent, lysozyme in a sample is detected. It can be detected specifically. Specifically, for example, since the amount of lysozyme in a sample can be analyzed, it can be said that qualitative analysis or quantitative analysis is also possible.
  • the sample is not particularly limited.
  • the sample include foods, food materials, food additives, and the like.
  • Examples of the sample include a deposit in a food processing shop or a cooking place, a cleaning liquid after cleaning, and the like.
  • the sample may be, for example, a liquid sample or a solid sample.
  • the sample is preferably a liquid sample because it is easy to contact with the nucleic acid molecule and is easy to handle.
  • a mixed solution, an extract, a dissolved solution, and the like may be prepared using a solvent and used.
  • the solvent is not particularly limited, and examples thereof include water, physiological saline, and buffer solution.
  • the detection method of the present invention will be described with reference to an example of a method for detecting lysozyme using the nucleic acid sensor of the present invention labeled with a labeling substance as the nucleic acid molecule of the present invention.
  • this invention is not restrict
  • the detection step further includes, for example, a step of analyzing the presence or amount of lysozyme in the sample based on the detection result of the complex.
  • 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., preferably 18 to 25 ° C.
  • the contact time is, for example, 10 to 120 minutes, preferably 30 to 60 minutes.
  • the nucleic acid molecule may be, for example, an immobilized nucleic acid molecule (solid phase carrier) immobilized on a carrier or an unfixed free nucleic acid molecule.
  • the sample is contacted in a container.
  • the carrier is not particularly limited, and examples thereof include a plate, a filter, a column, 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 detection step is a step of detecting the binding between the lysozyme 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 lysozyme in the sample can be analyzed (qualitative), and by detecting the degree of binding (binding amount) between the two, for example, the sample
  • the amount of lysozyme can be analyzed (quantified).
  • the method for detecting the binding between lysozyme and the nucleic acid molecule is not particularly limited.
  • a conventionally known method for detecting binding between substances can be adopted, and specific examples thereof include the SPR described above.
  • a method using a nucleic acid sensor in which a luciferase that is a labeling substance is bound to the nucleic acid molecule and an egg-derived lysozyme labeling carrier will be described below.
  • the nucleic acid sensor and the sample are mixed.
  • the nucleic acid molecule in the nucleic acid sensor binds to the target egg-derived lysozyme.
  • the nucleic acid molecule in the nucleic acid sensor is in an unbound state with the target.
  • the lysozyme labeled carrier is removed.
  • the carrier include beads.
  • the nucleic acid molecule in the nucleic acid sensor cannot bind to the egg-derived lysozyme in the lysozyme-labeled carrier. Therefore, when a luciferase substrate is added to the fraction from which the lysozyme-labeled carrier has been removed to perform a luminescence reaction, luminescence is generated by the luciferase catalytic reaction in the nucleic acid sensor.
  • the nucleic acid molecule in the nucleic acid sensor binds to egg-derived lysozyme in the lysozyme-labeled carrier. For this reason, by removing the lysozyme labeled carrier, the nucleic acid sensor is also removed while bound to the lysozyme labeled carrier. For this reason, when the luciferase substrate is added to the fraction from which the lysozyme-labeled carrier has been removed and the luminescence reaction is performed, luminescence due to the luciferase catalytic reaction occurs because the nucleic acid sensor does not exist. Absent.
  • the presence or absence of egg-derived lysozyme in the sample can be analyzed (qualitative analysis) based on the presence or absence of luminescence.
  • the amount of egg-derived lysozyme in the sample and the amount of the nucleic acid sensor remaining in the fraction after removing the lysozyme-labeled carrier have a correlation, depending on the intensity of luminescence,
  • the amount of egg-derived lysozyme can also be analyzed (quantitative analysis).
  • egg-derived lysozyme that is an allergen can be detected.
  • Example 1 About the aptamer of this invention, the binding property with respect to the egg white origin lysozyme of a chicken egg was confirmed by SPR analysis.
  • Aptamer Aptamer 1 of the following polynucleotide was synthesized as an aptamer of Examples.
  • T is all deoxyribonucleotide residues having 5′-tryptaminocarbonyluracil (TrpdU) in which 5-position of thymine is substituted in place of natural thymine (T)
  • C Were all deoxyribonucleotide residues having 5′-methylcytosine substituted in position 5 of cytosine in place of natural cytosine (C).
  • Aptamer 1 Lys391TR8m4 (SEQ ID NO: 1) GGTTAATCCCGACAAGCCCGTTAAGGGTTAACACGACATTTCGCTGTTGTAACAGGTCATAGTCACCACGGCTCATTTG
  • the estimated secondary structure of aptamer 1 is shown in FIG. However, it is not limited to this.
  • the aptamer was added with polydeoxyadenine (poly dA) having a length of 20 bases at the 3 'end and used as a poly dA added aptamer in SPR described later.
  • poly dA polydeoxyadenine
  • the poly dA-added aptamer used was heat-denatured at 95 ° C. for 5 minutes.
  • the composition of the SB1T 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.
  • each sample was prepared from the materials shown below for confirmation of the cross-reaction of the aptamer.
  • the preparation of the gliadin sample, the gluten sample, and the ⁇ -casein sample was performed in the same manner as the preparation of the lysozyme sample.
  • a milk sample, a raw peanut sample, and a roasted peanut sample were performed in the same manner as the preparation of the egg sample.
  • Gliadin (101778, manufactured by MP Biomedicals) Wheat-derived gluten (073-00575, manufactured by Wako Pure Chemical Industries, Ltd.) Milk-derived ⁇ -casein (C6780-19, manufactured by SIGMA) Milk (made by Ashigara Dairy Co., Ltd.) Raw peanuts (made by Indian curry shop Earl Tee) Roasted peanut (KFV Fruit)
  • a ProteON dedicated sensor chip a chip (trade name: ProteOn NLC Sensor Chip, BioRad) on which streptavidin was immobilized was set in the ProteON XPR36.
  • 1 ⁇ mol / L of biotinylated poly dT was injected into the flow cell of the sensor chip using ultrapure water (DDW) and allowed to bind until the signal intensity (RU: Resonance Unit) was about 900 RU.
  • the biotinylated poly dT was prepared by biotinylating the 5 ′ end of 20 base deoxythymidine.
  • FIG. 2 is a graph showing the binding property of aptamer 1 to egg white-derived lysozyme, in which the horizontal axis represents each sample and the vertical axis represents signal intensity (RU).
  • lysozyme sample, egg sample, gliadin sample, gluten sample, ⁇ -casein sample, milk sample, raw peanut sample, and roasted peanut sample are shown in order from the left.
  • the concentration (ppm) in each sample indicates the concentration of each protein for lysozyme sample, gliadin sample, gluten sample, and ⁇ -casein sample, and for egg sample, milk sample, raw peanut sample, and roast peanut sample, The concentration of total protein contained in the sample is shown.
  • FIG. 1 shows the concentration of total protein contained in the sample.
  • aptamer 1 showed binding properties to lysozyme samples and egg samples. Since aptamer 1 selectively binds to egg white-derived lysozyme, it can be said that the ability of aptamer 1 to bind to an egg sample showed binding to egg white-derived lysozyme contained in the egg sample. On the other hand, aptamer 1 has a signal intensity of 0.02 or less and exhibits no binding to gliadin samples, gluten samples, ⁇ -casein samples, milk samples, raw peanut samples, and roasted peanut samples. It was.
  • binding analysis was performed in the same manner except that the egg sample was used and the protein concentration in the sample was changed to 0.37, 1.1, 3.3, 10 and 30 ppm.
  • FIG. 3 is a graph showing the binding property of aptamer 1 to an egg sample, the horizontal axis indicates the protein concentration (ppm) in the egg sample, and the vertical axis indicates the signal intensity (RU).
  • the signal intensity of the aptamer 1 increased as the protein concentration in the egg sample increased. From this result, it was found that the lysozyme concentration in the egg sample can be quantitatively analyzed by measuring the signal intensity using the aptamer of the present invention.
  • the binding analysis was performed in the same manner except that the concentration of lysozyme in the sample was 3.125, 6.25, 12.5, and 25 nmol / L, The signal intensity at a predetermined time after the start of sample injection was determined.
  • FIG. 4 is a graph showing the binding property of aptamer 1 to lysozyme, the horizontal axis indicates the elapsed time (seconds) after the start of injection of the sample, and the vertical axis indicates the signal intensity (RU). As shown in FIG. 4, the signal intensity of the aptamer 1 increased as the concentration of lysozyme increased.
  • aptamer 1 has a dissociation constant (KD) for lysozyme of 1.04 ⁇ 10 ⁇ 9 mol / L, and has excellent binding properties.
  • KD dissociation constant
  • Example 2 About the miniaturized aptamer of this invention, the binding property with respect to an egg white origin lysozyme was confirmed by SPR analysis.
  • the following polynucleotide aptamers 2 to 7 were synthesized as miniaturized aptamers of the examples.
  • the aptamers 2 to 7 are aptamers obtained by downsizing the aptamer 1 (SEQ ID NO: 1) of Example 1.
  • SEQ ID NO: 1 SEQ ID NO: 1
  • T is all deoxyribonucleotide residues having 5′-benzylaminocarbonyluracil (BndU) substituted at the 5-position of thymine in place of natural thymine (T)
  • BndU deoxyribonucleotide residues having 5′-methylcytosine substituted in position 5 of cytosine in place of natural cytosine (C).
  • the estimated secondary structure of aptamers 2 to 7 is shown in FIG. However, it is not limited to this.
  • the aptamer 1 of Example 1 and the miniaturized aptamers 2 to 7 were used.
  • the lysozyme sample prepared in Example 1 25 nmol / L or 100 nmol / L
  • ⁇ casein sample 100 nmol / L or 400 nmol / L
  • FIG. 6 is a graph showing aptamer binding to lysozyme (25 nmol / L or 100 nmol / L), gliadin (4 ⁇ mol / L), and ⁇ -casein (100 nmol / L or 400 nmol / L).
  • the results of the miniaturized aptamer 2, the miniaturized aptamer 3, the miniaturized aptamer 4, the miniaturized aptamer 5, the miniaturized aptamer 6, and the aptamer 1 are shown.
  • (B) shows the results of the miniaturized aptamer 4 and the miniaturized aptamer 7. Show.
  • the horizontal axis represents each sample, and the vertical axis represents signal intensity (RU).
  • Each graph shows a miniaturized aptamer 2, a miniaturized aptamer 3, a miniaturized aptamer 4, a miniaturized aptamer 5, a miniaturized aptamer 6, and an aptamer 1 in order from the left in (A), and in FIG.
  • the miniaturized aptamer 4 and the miniaturized aptamer 7 are shown in order.
  • miniaturized aptamers 2 to 7 in which the aptamer 1 was miniaturized exhibited high binding ability to lysozyme derived from egg white (25 nmol / L or 100 nmol / L).
  • the miniaturized aptamers 2 to 7 each had a signal intensity of 0.00 or less and showed no binding to gliadin and ⁇ -casein. From these results, it was found that the miniaturized aptamers 2 to 7 bind with high specificity to lysozyme.
  • Example 3 A nucleic acid sensor in which a labeling substance luciferase was bound to the aptamer of the present invention was prepared, and the binding property of the nucleic acid sensor to egg white-derived lysozyme was confirmed. The confirmation of the binding was performed using target solid-phased beads in which the target egg white-derived lysozyme was solid-phased and the nucleic acid sensor.
  • the nucleic acid molecule in the nucleic acid sensor cannot bind to lysozyme immobilized on the target immobilized beads. For this reason, when a luminescence reaction is performed on the fraction from which the target solid-phased beads have been removed, luminescence is generated by the catalytic reaction of luciferase in the nucleic acid sensor.
  • the reaction solution when the nucleic acid sensor is not bound to the target, the nucleic acid molecule in the nucleic acid sensor is bound to lysozyme immobilized on the target immobilized beads.
  • the nucleic acid sensor is also removed while bound to the target-immobilized beads.
  • the luminescence due to the luciferase catalytic reaction is not caused by the absence of the nucleic acid sensor. Does not occur. Therefore, egg white-derived lysozyme can be detected by detecting luminescence by luciferase using the nucleic acid sensor and the target solid-phased beads.
  • the nucleic acid sensor uses the fluorescent substance SA-Lucia (trademark) luciferase (Invitrogen, cat # rep-strlc), and according to the instructions for use, the 5 ′ end of the aptamer 1 (Lys391TR8m4) of Example 1 described above. Was prepared by labeling.
  • Example 1 As the sample, the lysozyme sample of Example 1 was used.
  • the target solid-phased beads were prepared using NHS-activated Sepharose 4 Fast Flow Lab Packs (manufactured by GE Healthcare) using the lysozyme sample as a target.
  • the binding property of the nucleic acid sensor to egg white lysozyme was confirmed as follows. First, a filter plate (manufactured by Millipore, cat # MSGVN2250) was set on a 96-well U-bottom plate, and the target solid-phased beads were added to each well of the U-bottom plate to 20 ⁇ L / well. .
  • Infinite M1000 Pro (TECAN) was used according to the instruction manual.
  • QuantiLuc (trade name, manufactured by Invitrogen, cat # rep-qlc1) was used as a substrate.
  • FIG. 7 shows the measurement results of light emission.
  • FIG. 7 is a graph showing the measurement results of the luminescence amount for the lysozyme sample.
  • the horizontal axis indicates the concentration of the lysozyme sample
  • the vertical axis indicates the light emission amount (RLU).
  • RLU light emission amount
  • the detection limit (LOD) of the nucleic acid sensor for egg white-derived lysozyme was 0.039 ppm. From this, it was found that the nucleic acid sensor can detect a small amount of lysozyme derived from egg white.
  • Example 1 the egg sample prepared in Example 1 and the milk sample, raw peanut sample, and gluten sample prepared in Example 1 (final concentration 0) were confirmed to confirm the cross reaction. 1.56, 3.14, 6.25, 12.5, 25, 50, 100 ppm), and the same measurement was performed.
  • FIG. 8 is a graph showing the measurement results of the amount of luminescence when eggs, milk, raw peanuts, and gluten are used as samples.
  • the horizontal axis indicates the concentration of each sample, and the vertical axis indicates the light emission amount (RLU).
  • RLU light emission amount
  • the nucleic acid sensor can specifically detect the egg sample, specifically, egg white-derived lysozyme in the egg sample.
  • the detection limit (LOD) of the nucleic acid sensor in the egg sample was 3.125 ppm. From this, it was found that the nucleic acid sensor can sufficiently detect the egg white-derived lysozyme in the egg sample.
  • Example 4 About the aptamer of this invention, cross-reactivity with respect to various foodstuffs was confirmed.
  • binding properties were analyzed in the same manner as in Example 3 except that the following measurement conditions were used.
  • the aptamer 1 did not show binding properties to any of the above samples.
  • the aptamer of the present invention specifically binds to egg-derived lysozyme, and it can be detected by measurement, and according to the aptamer of the present invention, the intensity of luminescence of the egg-derived lysozyme in the sample It was found that the amount could be analyzed.
  • the nucleic acid molecule of the present invention can bind to egg-derived lysozyme. Therefore, according to the nucleic acid molecule of the present invention, egg-derived lysozyme can be detected based on the presence or absence of binding to the allergen in the sample. For this reason, the nucleic acid molecule of the present invention can be said to be an extremely useful tool for detecting, for example, allergens derived from eggs in fields such as food production, food management, and food distribution.

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Abstract

L'invention concerne une nouvelle molécule d'acide nucléique qui se lie au lysozyme dérivé d'œuf. Cette molécule d'acide nucléique, qui se lie au lysozyme dérivé d'œuf, comprend n'importe quel polynucléotide présenté en (a) ou (b) ci-dessous : (a) un polynucléotide comprenant la séquence de bases de SEQ ID NO : 1 ou une séquence partielle de la séquence de bases de SEQ ID NO : 1 ; ou (b) un polynucléotide qui comprend une séquence de bases ayant au moins 90 % d'identité avec la séquence de bases en (a), et qui se lie au lysozyme dérivé d'œuf.
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WO2016117701A1 (fr) * 2015-01-22 2016-07-28 Ｎｅｃソリューションイノベータ株式会社 Appareil d'analyse de cible et procédé d'analyse de cible

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Title
BAYRAMOGLU, GULAY ET AL.: "Lysozyme specific aptamer immobilized MCM-41 silicate for single-step purification and quartz crystal microbalance (QCM) -based determination of lysozyme from chicken egg white", MICROPOROUS AND MESOPOROUS MATERIALS, vol. 207, 2015, pages 95 - 104, XP029142016, ISSN: 1387-1811, DOI: doi:10.1016/j.micromeso.2015.01.009 *
POTTY, AJISH S.R. ET AL.: "Biophysical characterization of DNA and RNA aptamer interactions with hen egg lysozyme", INT. J. BIOL. MACROMOL., vol. 48, 2011, pages 392 - 397, XP028186149, ISSN: 0141-8130, DOI: doi:10.1016/j.ijbiomac.2010.12.007 *
TRAN, DINH T. ET AL.: "Selection and characterization of DNA aptamers for egg white lysozyme", MOLECULES, vol. 15, 2010, pages 1127 - 1140, XP055229152, ISSN: 1420-3049, DOI: doi:10.3390/molecules15031127 *

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