Antigens for egg allergy

Abstract

The invention relates to antigens of egg allergy. The present invention provides a novel antigen for egg allergy, a diagnostic method and a diagnostic kit for egg allergy, a pharmaceutical composition containing the antigen, an egg from which the antigen has been removed or reduced, an egg processed product, or a bird produced or hatched from the egg, a method for producing the egg processed product from which the antigen has been removed or reduced, and a method for producing the egg processed product from which the antigen has been removed or reduced. And the detector is used for judging whether the egg antigen exists in the object or not.

Description

[0001] The present application is a divisional application of the invention patent application with application number 202210307092.2 and invention name “Antigens for Egg Allergy”, filed on June 2, 2017, with application number 201780034053.7 and invention name “Antigens for Egg Allergy”. Technical Field

[0002] The present invention relates to a novel antigen for egg allergy. The present invention also relates to a diagnostic kit, a diagnostic composition and a diagnostic method for egg allergy. The present invention also relates to a pharmaceutical composition containing the antigen, an egg or egg processed product with the antigen removed or reduced, or a bird produced or hatched from the egg. The present invention also relates to a method for producing an egg processed product with the antigen removed or reduced. The present invention further relates to a detector for determining whether an object has an egg antigen. Background Art

[0003] IgE antibodies specific to specific antigens (hereinafter, also referred to as allergens) are produced in the serum and tissues of allergy patients. The physiological consequences of the interaction between the IgE antibodies and the specific antigens cause allergic reactions. Antigens in a broad sense refer to foods / ingredients that cause allergic symptoms; in a narrow sense, they refer to proteins contained in foods / ingredients that are bound by specific IgE antibodies (hereinafter, also referred to as allergen components).

[0004] Most of the existing allergy detection reagents are prepared by simply grinding allergen candidate foods / ingredients to prepare antigen reagents (Patent Document 1). Therefore, among the allergen components contained in the existing antigen reagents, a positive reaction can only be detected in the allergy test when the binding with IgE antibodies exceeds the content that can determine a positive reaction threshold, and the diagnostic efficiency is not yet sufficiently high.

[0005] As egg allergens, the substances shown in the following table are currently known (Non-Patent Document 1).

[0006] [Table 1]

[0007]

[0008] Several allergen components have been suggested in food / ingredients that are candidates for allergens and have been commercialized as test kits. However, in order to improve the reliability of allergy detection, it is necessary to comprehensively limit the allergen components, but the patient detection rate by the determination of the above allergen components is still insufficient. Identification of new egg allergens is not only necessary to improve the accuracy of diagnostic reagents, but also very important as a target for hypoallergenic foods, hypoallergenic ingredients, and therapeutic drugs.

[0009] On the other hand, regarding the separation / purification of proteins, in recent years, as a method for separating and purifying a plurality of proteins from a small amount of sample, a two-dimensional electrophoresis method is used, in which isoelectric electrophoresis is performed in the first dimension and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is performed in the second dimension. The applicant has so far developed a two-dimensional electrophoresis method with high separation capability (Patent Documents 2 to 5).

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent Application Laid-Open No. 2002-286716

[0013] Patent Document 2: Japanese Patent Application Laid-Open No. 2011-33544

[0014] Patent Document 3: Japanese Patent Application Laid-Open No. 2011-33546

[0015] Patent Document 4: Japanese Patent Application Laid-Open No. 2011-33547

[0016] Patent Document 5: Japanese Patent Application Laid-Open No. 2011-33548

[0017] Non-patent literature

[0018] Non-patent document 1: Allergen Nomenclature, WHO / IUIS Allergen Nomenclature Sub-Committee, [May 31, 2018], インターネット<URL: http: / / www.allergen.org / search.php? allergenname=&allergensource=gallus+dome sticus&TaxSource=&TaxOrder=&foodallerg=all&bioname=> Summary of the invention

[0019] Problem that the invention aims to solve

[0020] The present invention provides a novel antigen for egg allergy. The present invention also provides a method for diagnosing egg allergy and a diagnostic kit. The present invention also relates to a pharmaceutical composition containing the antigen, an egg or egg processed product with the antigen removed or reduced, or a bird that produces the egg or is hatched from the egg. The present invention further provides a detector for determining whether an object has an egg antigen.

[0021] Solutions for solving problems

[0022] The present inventors have conducted intensive research on the identification of antigens that are the cause of egg allergy in order to solve the above problems. As a result, they have successfully identified a novel antigen to which IgE antibodies in the serum of patients with egg allergy specifically bind. Based on this finding, the present invention has been completed.

[0023] That is, in one embodiment, the present invention is as follows.

[0024] [1] A diagnostic kit for egg allergy, comprising at least one of the following proteins (1) to (3) as an antigen:

[0025] (1) (1A) A protein comprising the C-terminal portion of vitellogenin-3 or a mutant thereof, which is any one of the following (1A-a) to (1A-e) as an antigen for egg allergy:

[0026] (1A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 2;

[0027] (1A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 2;

[0028] (1A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 1;

[0029] (1A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence shown in SEQ ID NO: 1; or

[0030] (1A-e) a protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1; or

[0031] (1B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 11;

[0032] (2) (2A) A protein comprising the C-terminal portion of vitellogenin-3 or a mutant thereof, which is any one of the following (2A-a) to (2A-e) that is an antigen for egg allergy:

[0033] (2A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:13;

[0034] (2A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 13;

[0035] (2A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 12;

[0036] (2A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 12; or

[0037] (2A-e) a protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 12; or

[0038] (2B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 9 to 11, 13, 14 and 16;

[0039] (3) (3A) A protein comprising the central portion of vitellogenin-1 or the C-terminal portion of lipovitellin-1, or a mutant thereof, which is any one of the following proteins (3A-a) to (3A-e) that is an antigen for egg allergy:

[0040] (3A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:18;

[0041] (3A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 18;

[0042] (3A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 17;

[0043] (3A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 17; or

[0044] (3A-e) a protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence set forth in SEQ ID NO: 17; or

[0045] (3B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 18 to 45.

[0046] [2] A composition for diagnosing egg allergy, comprising at least one of the proteins specified in (1) to (3) in [1] above as an antigen.

[0047] [3] A method for providing an indicator for diagnosing egg allergy in a subject, comprising the following steps:

[0048] (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies;

[0049] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0050] (iii) when the subject's IgE antibody is detected to be bound to the antigen, providing an indication that the subject has an egg allergy;

[0051] Here, the antigen is at least one of the proteins specified in (1) to (3) in [1] above.

[0052] [4] A pharmaceutical composition comprising at least one of the proteins defined in (1) to (3) in [1] above.

[0053] [5] The pharmaceutical composition according to [4] above, which is used for treating egg allergy.

[0054] [6] An egg, an egg product or a bird, characterized in that it is an egg, an egg product or a bird that produces or is hatched from an antigen removed or reduced, and the antigen is at least one of the proteins specified in (1) to (3) in [1] above.

[0055] [7] A detector for determining the presence or absence of an egg antigen in an object, characterized in that it contains an antibody that binds to at least one of the proteins specified as (1) to (3) in [1] above.

[0056] [8] A detector for determining the presence or absence of an antigen causing an egg allergy in an object, characterized in that it comprises a primer having a base sequence complementary to at least one of the base sequences shown in SEQ ID NOs. 1, 12 or 17.

[0057] [9] A diagnostic kit for egg allergy, comprising at least one of the following proteins (4) to (10) as an antigen:

[0058] (4) (4A) A protein comprising vitellogenin-3 or a mutant thereof, which is any one of the following proteins (4A-a) to (4A-e) that is an antigen for egg allergy:

[0059] (4A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:47;

[0060] (4A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 47;

[0061] (4A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 46;

[0062] (4A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 46; or

[0063] (4A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO:46; or

[0064] (4B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 47 to 57;

[0065] (5) (5A) A protein comprising vitellogenin-2 precursor or a mutant thereof, which is any one of the following proteins (5A-a) to (5A-e) that is an antigen for egg allergy:

[0066] (5A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 59;

[0067] (5A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 59;

[0068] (5A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 58.

[0069] (5A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 58; or

[0070] (5A-e) a protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence set forth in SEQ ID NO: 58; or

[0071] (5B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 59 to 104;

[0072] (6) (6A) Vitellogenin-2 precursor or a mutant thereof, which is any one of the following proteins (6A-a) to (6A-e) that is an antigen for egg allergy:

[0073] (6A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:106;

[0074] (6A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 106;

[0075] (6A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 105;

[0076] (6A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 105; or

[0077] (6A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 105; or

[0078] (6B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 106 to 150;

[0079] (7) (7A) Apolipoprotein B precursor or a mutant thereof, which is any one of the following proteins (7A-a) to (7A-e) that is an antigen for egg allergy:

[0080] (7A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:152;

[0081] (7A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 152;

[0082] (7A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 151;

[0083] (7A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 151; or

[0084] (7A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 151; or

[0085] (7B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 152 to 235;

[0086] (8) (8A) Apolipoprotein B precursor or a mutant thereof, which is any one of the following proteins (8A-a) to (8A-e) that is an antigen for egg allergy:

[0087] (8A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 237;

[0088] (8A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 237;

[0089] (8A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 236;

[0090] (8A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 236; or

[0091] (8A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence set forth in SEQ ID NO: 236; or

[0092] (8B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 237 to 249;

[0093] (9) (9A) Apolipoprotein B precursor or a mutant thereof, which is any one of the following proteins (9A-a) to (9A-e) that is an antigen for egg allergy:

[0094] (9A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 251;

[0095] (9A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 251;

[0096] (9A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 250;

[0097] (9A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 250; or

[0098] (9A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 250; or

[0099] (9B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 251 to 260;

[0100] (10) (10A) Vitellogenin-2 precursor or a mutant thereof, which is any one of the following proteins (10A-a) to (10A-e) that is an antigen for egg allergy:

[0101] (10A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 262;

[0102] (10A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 262;

[0103] (10A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 261;

[0104] (10A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 261; or

[0105] (10A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 261; or

[0106] (10B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 262 to 271.

[0107]

[10] A composition for diagnosing egg allergy, comprising at least one of the proteins defined in (4) to (10) in [9] above as an antigen.

[0108]

[11] A method for providing an indicator for diagnosing egg allergy in a subject, comprising the following steps:

[0109] (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies;

[0110] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0111] (iii) when the subject's IgE antibody is detected to be bound to the antigen, providing an indication that the subject has an egg allergy;

[0112] Here, the antigen is at least one of the proteins defined as (4) to (10) in [9] above.

[0113]

[12] A pharmaceutical composition comprising at least one of the proteins defined in (4) to (10) in [9] above.

[0114]

[13] The pharmaceutical composition according to the above-mentioned

[12] , which is used for treating egg allergy.

[0115]

[14] An egg, an egg product or a bird, characterized in that it is an egg, an egg product or a bird that produces or is hatched from an antigen removed or reduced, and the antigen is at least one of the proteins defined as (4) to (10) in [9] above.

[0116]

[15] A detector for determining the presence or absence of an egg antigen in an object, characterized in that it contains an antibody that binds to at least one of the proteins defined as (4) to (10) in [9] above.

[0117]

[16] A detector for determining the presence or absence of an antigen causing an egg allergy in an object, characterized in that it comprises a primer having a base sequence complementary to at least one of the base sequences shown in sequence numbers 46, 58, 105, 151, 236, 250 or 261.

[0118]

[17] A method for producing an egg processed product from which antigens have been removed or reduced, comprising the steps of: confirming that antigens have been removed or reduced during the production process of the processed product;

[0119] Here, the antigen is at least one of the proteins specified as (1) to (3) in [1] above, or at least one of the proteins specified as (4) to (10) in [9] above.

[0120]

[18] An egg-derived antigen which is at least one of the proteins specified as (1) to (3) in [1] above, or at least one of the proteins specified as (4) to (10) in [9] above, and which causes an egg allergy.

[0121] Effects of the Invention

[0122] According to the present invention, a novel antigen of egg allergy can be provided. In the present invention, a novel antigen (allergen component) causing egg allergy is identified, and thus a highly sensitive diagnosis method and diagnosis kit for egg allergy, a pharmaceutical composition containing the antigen, an egg or egg processed product with the antigen removed or reduced, or a bird producing the egg or hatched from the egg, or an egg processed product with the antigen removed or reduced, and a detector for determining whether an egg antigen is present in an object can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 This is a gel photo showing the electrophoresis of proteins contained in the yolk of an egg (raw egg) using two-dimensional electrophoresis. The bands on the left side of the photo are bands of molecular weight markers, and the values ​​on the left side of the photo are the molecular weights (KDa) of each molecular weight marker. The values ​​on the top of the photo indicate the isoelectric point.

[0124] Figure 2 This is a photograph of a two-dimensional electrophoresis diagram of proteins contained in the yolk of an egg (raw egg), obtained by immunoblotting using the serum of the egg allergy patient 1.

[0125] Figure 3 This is a gel photograph showing a two-dimensional electrophoresis pattern of proteins contained in egg yolk (raw egg). Spots 1, 2, and 3 to which the serum of egg allergy patient 1 specifically reacted are shown enclosed by squares.

[0126] Figure 4 This is a photograph of a two-dimensional electrophoresis diagram of proteins contained in egg yolk (raw egg), obtained by immunoblotting using the serum of egg allergy patient 2. Spots 4 to 8, to which the serum of egg allergy patient 2 specifically reacted, are shown enclosed by boxes.

[0127] Figure 5 This is a photograph of a two-dimensional electrophoresis diagram of proteins contained in egg yolk (raw egg), obtained by immunoblotting using the serum of egg allergy patient 3. Spots 9 and 10, where the serum of egg allergy patient 3 specifically reacted, are respectively indicated by being enclosed by squares.

[0128] Figure 6This is a photograph of a two-dimensional electrophoresis diagram of proteins contained in egg yolk (raw egg), obtained by immunoblotting using the serum of egg allergy patient 4. The spot 11 to which the serum of egg allergy patient 4 specifically reacted is indicated by a box. DETAILED DESCRIPTION

[0129] The present invention will be described in detail below, but the present invention is not limited to these contents.

[0130] In this specification, unless otherwise defined, scientific terms and technical terms used in connection with the present invention have the meanings generally understood by those skilled in the art.

[0131] Allergy in this specification refers to: in an organism sensitive to a certain antigen, the state of allergic reaction that is produced when the antigen enters again and shows discomfort to the organism. Most of the allergic diseases in food produce IgE antibodies specific to antigens in blood and tissues. IgE antibodies are combined with mast cells or basophils. When the antigen specific to this IgE antibody enters the body of an allergic disease patient again, this antigen is combined with the IgE antibody combined with mast cells or basophils, and then this IgE antibody is cross-linked on the cell surface, producing the physiological effect of IgE antibody-antigen interaction. As their physiological effects, the release of histamine, 5-hydroxytryptamine, heparin, eosinophil chemotactic factor or various leukotrienes etc. can be listed. These released substances cause the allergic reaction produced by the combination of IgE antibodies and specific antigens. The allergic reaction produced by specific antigens is realized by the above-mentioned path.

[0132] In the present specification, the term "egg" refers to bird eggs, preferably chicken eggs.

[0133] In this specification, egg allergy refers to a state in which an allergic reaction occurs when a protein or the like contained in an egg is used as an antigen. Egg allergy refers to an allergic reaction that occurs when an antigen contained in an egg is contacted or ingested. Generally, an allergic reaction that occurs when a food is ingested is particularly referred to as a food allergy. Egg allergy may also be a food allergy.

[0134] In this specification, an antigen refers to a substance that causes an allergic reaction and is also called an allergen component. The antigen is preferably a protein.

[0135] In this specification, protein is a molecule having a structure in which natural amino acids are connected by peptide bonds. There is no particular limitation on the number of amino acids contained in a protein, and a protein in which about 2 to 50 amino acids are connected by peptide bonds is sometimes referred to as a peptide. In addition, with respect to amino acids, when optical isomers may exist, L-forms are indicated unless otherwise specified. The notation of the amino acid sequence of a protein or peptide used in this specification is as follows: Based on standard usage and the notation commonly used in the art, amino acids are indicated by a single character notation, with the left direction being the amino terminal direction and the right direction being the carboxyl terminal direction.

[0136] Antigen limitation

[0137] The above-mentioned method was used to define the antigens of egg allergy among the proteins contained in the egg. Specifically, raw eggs were separated into yolk and egg white, and the egg proteins contained therein were subjected to two-dimensional electrophoresis under the following conditions.

[0138] The first dimension electrophoresis uses a gel with a gel length of 5 to 10 cm and a pH range of 3 to 10 as a gel for isoelectric point electrophoresis, and a pH gradient of the gel relative to the electrophoresis direction, when the total length of the gel is set as 1, the gel length to pH 5 is set as a, the gel length of pH 5 to 7 is set as b, and the gel length above pH 7 is set as c, a is in the range of 0.15 to 0.3, b is in the range of 0.4 to 0.7, and c is in the range of 0.15 to 0.3. Specifically, IPG gel Immobiline Drystrip (pH 3-10 NL) manufactured by GE Healthcare Bioscience (hereinafter referred to as GE) is used for isoelectric point electrophoresis. The electrophoresis apparatus used is IPGphor manufactured by GE. The upper limit of the electrophoresis current value was set to 75 μA per gel, and the voltage program was set as follows: (1) the constant voltage was increased from 300 V to 750 Vhr to perform a constant voltage process (the current variation amplitude during 30 minutes of electrophoresis before the end of this process was 5 μA); (2) the voltage was gradually increased from 300 Vhr to 1000 V; (3) further, the voltage was gradually increased from 4500 Vhr to 5000 V; (4) thereafter, the first-dimensional isoelectric point electrophoresis was performed at a constant voltage of 5000 V until Vhr reached 12000.

[0139] The second dimension electrophoresis uses a polyacrylamide gel in which the gel concentration at the base end of the swimming direction is set to 3-6% and the gel concentration at the front end of the swimming direction is set to be higher than the gel concentration at the base end of the swimming direction. Specifically, NuPAGE 4-12% Bris-Tris Gels IPG well mini 1mm manufactured by Life Technologies was used for SDS-PAGE. The electrophoresis apparatus used was XCell SureLock Mini-Cell manufactured by Life Technologies. As the swimming buffer, 50mM MOPS, 50mM Tris base, 0.1% (w / v) SDS, 1mM EDTA were used, and the swimming was carried out for about 45 minutes at a constant voltage of 200V.

[0140] The results showed that when the egg protein was subjected to two-dimensional electrophoresis under the above conditions, the antigens in spots 1 to 3 specifically bound to the IgE antibodies of patients with egg allergy who were diagnosed with food-dependent exercise-induced allergy (Food-Dependent Exercise-Induced Anaphylaxis). In addition, it was found that in patients with other egg allergies, the antigens in spots 4 to 11 specifically bound to the IgE antibodies of the patients ( Figures 4 to 6 ).

[0141] antigen

[0142] (1) Spot 1 antigen

[0143] As a result of sequence identification of spot 1 by mass spectrometry, amino acid sequences of SEQ ID NOs: 3 to 11 were detected.

[0144] In addition, for spot 1, the mass spectrum data (sequence numbers 3 to 11) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and identified as the C-terminal part of vitellogenin-3 (amino acid sequence: sequence number 2, base sequence encoding it: sequence number 1).

[0145] Therefore, in the present application, the antigen of spot 1 is any one of the following (1A-a) to (1A-e) and (1B).

[0146] (1A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 2.

[0147] (1A-b) A protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 2.

[0148] (1A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 1.

[0149] (1A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 1.

[0150] (1A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1.

[0151] (1B) A protein containing at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs. 2 to 11, preferably a protein containing at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences. More preferably, a protein containing at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs. 2, 4 and SEQ ID NOs. 9 to 11, preferably a protein containing at least 2, 3, 4 or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs. 3 to 11 may have one or more amino acids deleted, substituted, inserted or added.

[0152] The proteins of (1A-a) to (1A-e) and (1B) mentioned above may also be proteins that appear as spots with a molecular weight of 20 kDa to 40 kDa, preferably a molecular weight of 30 kDa to 35 kDa and an isoelectric point of 4.0 to 10.0, preferably 4.0 to 9.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0153] (2) Spot 2 antigen

[0154] As a result of sequence identification of spot 2 by mass spectrometry, amino acid sequences of SEQ ID NOs: 9 to 11, 14, and 16 were detected.

[0155] In addition, for spot 2, the mass spectrum data obtained by the mass spectrometer (sequence numbers 9 to 11, 14, and 16) were compared and analyzed with the protein data of NCBI, and identified as the C-terminal part of vitellogenin-3 (amino acid sequence: sequence number 13, base sequence encoding it: sequence number 12).

[0156] Therefore, in the present application, the antigen of spot 2 is any one of the following (2A-a) to (2A-e) and (2B).

[0157] (2A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO:13.

[0158] (2A-b) A protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence shown in SEQ ID NO: 13.

[0159] (2A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 12.

[0160] (2A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 12.

[0161] (2A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 12.

[0162] (2B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 9 to 11, 13, 14, and 16, preferably a protein comprising at least 2, 3, 4, 5, or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 9 to 11, 13, and 16, preferably a protein comprising at least 2, 3, 4, or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs: 9 to 11, 13, 14, and 16 may have one or more amino acids deleted, substituted, inserted, or added.

[0163] The proteins of (2A-a) to (2A-e) and (2B) mentioned above may also be proteins that appear as spots with a molecular weight of 30 kDa to 60 kDa, preferably around 35 kDa to 40 kDa, and an isoelectric point of 5.0 to 10.0, preferably 6.0 to 9.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0164] (3) Spot 3 antigen

[0165] As a result of sequence identification of spot 3 by mass spectrometry, amino acid sequences of SEQ ID NOs: 19 to 45 were detected.

[0166] In addition, for spot 3, the mass spectrum data (sequence numbers 19 to 45) obtained by the mass spectrometer were compared and analyzed with the protein data of UniProt, and it was identified as the central part of vitellogenin-1 or the C-terminal part of yolk lipophosphoprotein-1 (Lipovitellin-1) that can be obtained by cutting on the N-terminal side of its protein (amino acid sequence: sequence number 18, base sequence encoding it: sequence number 17).

[0167] Therefore, in the present application, the antigen of spot 3 is any one of the following (3A-a) to (3A-e) and (3B).

[0168] (3A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO:18.

[0169] (3A-b) A protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence shown in SEQ ID NO: 18.

[0170] (3A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 17.

[0171] (3A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 17.

[0172] (3A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 17.

[0173] (3B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 18 to 45, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 18, 19, 22 to 31, 33 to 35, 37, 39, 40, 42, 43 and 45, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs: 18 to 45 may contain one or more amino acids deleted, substituted, inserted or added.

[0174] The proteins of (3A-a) to (3A-e) and (3B) mentioned above may also be proteins that appear as spots with a molecular weight of 35 kDa to 60 kDa, preferably around 40 kDa to 50 kDa, and an isoelectric point of 5.0 to 10.0, preferably 6.0 to 9.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0175] (4) Spot 4 antigen

[0176] As a result of sequence identification of spot 4 by mass spectrometry, amino acid sequences of SEQ ID NOs: 48 to 57 were detected.

[0177] In addition, for spot 4, the mass spectrum data (SEQ ID NOs. 48 to 57) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and it was identified as vitellogenin-3 (amino acid sequence: SEQ ID NO. 47, base sequence encoding it: SEQ ID NO. 46).

[0178] Therefore, in the present application, the antigen of spot 4 is any one of the following (4A-a) to (4A-e) and (4B).

[0179] (4A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 47.

[0180] (4A-b) A protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence shown in SEQ ID NO: 47.

[0181] (4A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 46.

[0182] (4A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 46.

[0183] (4A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 46.

[0184] (4B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 47 to 57, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 47, 50, 51, 53 and 54, preferably a protein comprising at least 2, 3, 4 or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs: 47 to 57 may have one or more amino acids deleted, substituted, inserted or added.

[0185] The proteins of (4A-a) to (4A-e) and (4B) mentioned above may also be proteins that appear as spots with a molecular weight of 20 kDa to 50 kDa, preferably a molecular weight of 25 kDa to 40 kDa, more preferably around a molecular weight of 30 kDa to 35 kDa, and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, and further preferably 4.0 to 6.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0186] (5) Antigens of spots 5 and 6

[0187] As a result of sequence identification of spots 5 and 6 by mass spectrometry, amino acid sequences of SEQ ID NOs: 60 to 104 were detected.

[0188] In addition, for spots 5 and 6, the mass spectrum data (sequence numbers 60 to 104) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and identified as vitellogenin-2 precursor (amino acid sequence: sequence number 59, base sequence encoding it: sequence number 58).

[0189] Therefore, in the present application, the antigens of spots 5 and 6 are any of the following (5A-a) to (5A-e) and (5B).

[0190] (5A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 59.

[0191] (5A-b) A protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence shown in SEQ ID NO: 59.

[0192] (5A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 58.

[0193] (5A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 58.

[0194] (5A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 58.

[0195] (5B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 59 to 104, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 59, 60, 63, 65, 68, 69, 71, 73, 75 to 79, 81, 83 to 88, 91 to 94, 96 to 99 and 101 to 103, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs: 59 to 104 may have one or more amino acids deleted, substituted, inserted or added.

[0196] The proteins of (5A-a) to (5A-e) and (5B) mentioned above may also be proteins that appear as spots with a molecular weight of 80 kDa to 260 kDa, preferably a molecular weight of 90 kDa to 200 kDa, and more preferably a molecular weight of about 110 kDa to 160 kDa, and an isoelectric point of 1.0 to 8.0, preferably 2.0 to 7.0, and even more preferably 3.0 to 6.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0197] (6) Spot 7 antigen

[0198] As a result of sequence identification of spot 7 by mass spectrometry, an amino acid sequence of SEQ ID NOs: 107 to 150 was detected.

[0199] In addition, for spot 7, the mass spectrum data (SEQ ID NOs. 107 to 150) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and it was identified as vitellogenin-2 precursor (amino acid sequence: SEQ ID NO. 106, base sequence encoding it: SEQ ID NO. 105).

[0200] Therefore, in the present application, the antigen of spot 7 is any one of the following (6A-a) to (6A-e) and (6B).

[0201] (6A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 106.

[0202] (6A-b) A protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence shown in SEQ ID NO: 106.

[0203] (6A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 105.

[0204] (6A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 105.

[0205] (6A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 105.

[0206] (6B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 106 to 150, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 44 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 106, 108, 110, 111, 113, 114, 116, 117, 120 to 124, 126 to 131, 133, 134, 136 to 138, 140 to 143, 145 to 147 and 149, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31 or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs: 106 to 150 may have one or more amino acids deleted, substituted, inserted or added.

[0207] The proteins of (6A-a) to (6A-e) and (6B) mentioned above may also be proteins that appear as spots with a molecular weight of 110 kDa to 300 kDa, preferably a molecular weight of 130 kDa to 280 kDa, and more preferably a molecular weight of about 160 kDa to 260 kDa, and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, and even more preferably 4.0 to 6.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0208] (7) Spot 8 antigen

[0209] As a result of sequence identification of spot 8 by mass spectrometry, an amino acid sequence of SEQ ID NOs: 153 to 235 was detected.

[0210] In addition, for spot 8, mass spectrum data (SEQ ID NOs. 153 to 235) obtained by a mass spectrometer were compared and analyzed with protein data from NCBI, and it was identified as apolipoprotein B precursor (amino acid sequence: SEQ ID NO. 152, base sequence encoding it: SEQ ID NO. 151).

[0211] Therefore, in the present application, the antigen of spot 8 is any one of the following (7A-a) to (7A-e) and (7B).

[0212] (7A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO:152.

[0213] (7A-b) A protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 152.

[0214] (7A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 151.

[0215] (7A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 151.

[0216] (7A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 151.

[0217] (7B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 152 to 235, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 83 sequences or all of the sequences. More preferably, the protein comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 152, 154, 157 to 160, 163, 164, 167 to 172, 174, 175, 177, 178, 180, 182 to 185, 187, 189, 190, 192, 194 to 196, 198 to 200, 203 to 207, 209, 211, 212, 214 to 216, 220 to 229, 231 and 232, and preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs: 152 to 235 may have one or more amino acids deleted, substituted, inserted or added.

[0218] The proteins of (7A-a) to (7A-e) and (7B) mentioned above may also be proteins that appear as spots with a molecular weight of 160 kDa to 550 kDa, preferably a molecular weight of 180 kDa to 400 kDa, and more preferably a molecular weight of around 200 kDa to 300 kDa, and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, and even more preferably 4.0 to 6.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0219] (8) Spot 9 antigen

[0220] As a result of sequence identification of spot 9 by mass spectrometry, amino acid sequences of SEQ ID NOs: 238 to 249 were detected.

[0221] In addition, for spot 9, mass spectrum data (SEQ ID NOs. 238 to 249) obtained by a mass spectrometer were compared and analyzed with protein data from NCBI, and it was identified as apolipoprotein B precursor (amino acid sequence: SEQ ID NO. 237, base sequence encoding it: SEQ ID NO. 236).

[0222] Therefore, in the present application, the antigen of spot 9 is any one of the following (8A-a) to (8A-e) and (8B).

[0223] (8A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 237.

[0224] (8A-b) A protein comprising an amino acid sequence that is 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequence shown in SEQ ID NO: 237.

[0225] (8A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 236.

[0226] (8A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 236.

[0227] (8A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 236.

[0228] (8B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 237 to 249, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 237 to 239, 241, 242, 244 and 246 to 249, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs: 237 to 249 may have one or more amino acids deleted, substituted, inserted or added.

[0229] The proteins of (8A-a) to (8A-e) and (8B) mentioned above may also be proteins that appear as spots with a molecular weight of 20 kDa to 50 kDa, preferably a molecular weight of 25 kDa to 45 kDa, more preferably around a molecular weight of 30 kDa to 40 kDa, and an isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, and even more preferably 9.0 to 10.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0230] (9) Spot 10 antigen

[0231] As a result of sequence identification of spot 10 by mass spectrometry, an amino acid sequence of SEQ ID NOs: 252 to 260 was detected.

[0232] In addition, the mass spectrum data (SEQ ID NOs. 252 to 260) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and spot 10 was identified as apolipoprotein B precursor (amino acid sequence: SEQ ID NO. 251, base sequence encoding it: SEQ ID NO. 250).

[0233] Therefore, in the present application, the antigen of spot 10 is any one of the following (9A-a) to (9A-e) and (9B).

[0234] (9A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 251.

[0235] (9A-b) A protein comprising an amino acid sequence that is 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequence shown in SEQ ID NO: 251.

[0236] (9A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 250.

[0237] (9A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 250.

[0238] (9A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 250.

[0239] (9B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 251 to 260, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 251 to 254, 256, 257 and 260, preferably a protein comprising at least 2, 3, 4, 5, 6 or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs: 251 to 260 may have one or more amino acids deleted, substituted, inserted or added.

[0240] The proteins of (9A-a) to (9A-e) and (9B) mentioned above may also be proteins that appear as spots with a molecular weight of 20 kDa to 50 kDa, preferably a molecular weight of 25 kDa to 45 kDa, more preferably around a molecular weight of 30 kDa to 40 kDa, and an isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, and even more preferably 9.0 to 10.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0241] (10) Antigen of spot 11

[0242] As a result of sequence identification of spot 11 by mass spectrometry, the amino acid sequence of SEQ ID NOs: 263 to 271 was detected.

[0243] In addition, for spot 11, the mass spectrum data (sequence numbers 263 to 271) obtained by the mass spectrometer were compared and analyzed with the protein data of NCBI, and it was identified as vitellogenin-2 precursor (amino acid sequence: sequence number 262, base sequence encoding it: sequence number 261).

[0244] Therefore, in the present application, the antigen of spot 11 is any one of the following (10A-a) to (10A-e) and (10B).

[0245] (10A-a) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 262.

[0246] (10A-b) A protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 262.

[0247] (10A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 261.

[0248] (10A-d) A protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the base sequence shown in SEQ ID NO: 261.

[0249] (10A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 261.

[0250] (10B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 262 to 271, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences. More preferably, a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 262 to 265, 268 and 269, preferably a protein comprising at least 2, 3, 4, 5 or all of the amino acid sequences. The amino acid sequence represented by any one of SEQ ID NOs: 262 to 271 may have one or more amino acids deleted, substituted, inserted or added.

[0251] The proteins (10A-a) to (10A-e) and (10B) mentioned above may also be proteins that appear as spots with a molecular weight of 15 kDa to 40 kDa, preferably a molecular weight of 17 kDa to 35 kDa, more preferably around a molecular weight of 20 kDa to 30 kDa, and an isoelectric point of 6.0 to 11.0, preferably 7.0 to 10.0, and even more preferably 8.0 to 10.0, in the gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned "Limitation of Antigens" item.

[0252] The proteins as antigens described in (1) to (3) and (4) to (10) above also include proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring opening, deamination, etc.

[0253] Preferably, the protein as an antigen in (1) to (3) and (4) to (10) above is an antigen for egg allergy.

[0254] In the present specification, the situation of "one or more amino acids are deleted, substituted, inserted or added" in an amino acid sequence means that in the amino acid sequence of interest, one or more amino acids are deleted (for example, 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1% of the amino acids relative to the total length of the amino acid sequence), or are substituted with other amino acids, or other amino acids are inserted, and / or other amino acids are added to form an amino acid sequence.

[0255] In the above, the substitution is preferably a conservative substitution. Conservative substitution refers to replacing a specific amino acid residue with a residue having similar physical and chemical characteristics; any substitution can be made as long as the characteristics related to the original sequence structure are not substantially changed. For example, the substitution of amino acids can be made as long as the helix present in the original sequence is not destroyed, or other types of secondary structures that are characteristics of the original sequence are not destroyed. In the following, conservative substitutions of amino acid residues are exemplified according to the classification of residues that can be substituted, but the amino acid residues that can be substituted are not limited to the substances described below.

[0256] Group A: leucine, isoleucine, valine, alanine, methionine

[0257] Group B: Aspartic acid, Glutamic acid

[0258] Group C: Asparagine, Glutamine

[0259] Group D: lysine, arginine

[0260] Group E: Serine, Threonine

[0261] Group F: Phenylalanine, Tyrosine

[0262] In the case of non-conservative substitutions, one of the above-mentioned types can also be replaced with a member of another type. For example, in order to exclude unexpected sugar chain modifications, the amino acids in the above-mentioned B, D, and E groups can also be replaced with amino acids in other groups. Alternatively, in order to prevent folding in the three-dimensional protein, cysteine ​​can be deleted or replaced with other amino acids. Alternatively, in order to maintain the hydrophilicity / hydrophobicity balance, or to facilitate synthesis, the hydrophilicity index (hydropathy index) of amino acids related to amino acids as a hydrophobicity / hydrophilicity index (J. Kyte and R. Doolittle, J. Mol. Biol., Vol. 157, p. 105-132, 1982) can also be replaced with amino acids in order to increase the hydrophilicity.

[0263] In this specification, the % identity of two amino acid sequences can be determined by visual inspection and mathematical calculation. In addition, computer programs can also be used to determine the % identity. As such computer programs, for example, BLAST and ClustalW can be listed. In particular, the various conditions (parameters) of the identity search using the BLAST program can be recorded according to Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997), from the National Center for Biotechnology Information (NCBI), DNA Data Bank of Japan (DDBJ) website, etc., publicly obtained (BLAST manual, Altschul et al. NCB / NLM / NIH Bethesda, MD 20894; Altschul et al.). In addition, programs such as genetic information processing software GENETYX Ver. 7 (GENETYX), DINASIS Pro (Hitachi Soft, Ltd.), Vector NTI (Infomax) can also be used to determine.

[0264] In this specification, the situation of "deletion, substitution, insertion or addition of one or more nucleotides" in the base sequence means that in the base sequence to be targeted, one or more nucleotides are deleted (for example, 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1% of the amino acids relative to the total length of the base sequence), or replaced with other nucleotides, or inserted with other nucleotides, and / or the base sequence formed by the addition of other nucleotides. It is preferred that no frame shift is generated in the sequence encoding the amino acid by the deletion, substitution, insertion or addition of the above nucleotides.

[0265] In this specification, the % identity of two base sequences can be determined by visual inspection and mathematical calculation. In addition, the % identity can also be determined using a computer program. As such a sequence comparison computer program, for example, the BLASTN program (Altschul et al. (1990) J. Mol. Biol. 215: 403-10) available from the website of the National Library of Medicine of the United States can be used, and the default parameters (Default Parameter) are used to determine.

[0266] In this specification, "stringent conditions" refers to hybridization under moderate or highly stringent conditions. Specifically, as moderately stringent conditions, for example, a person skilled in the art with general skills can easily determine based on the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Chapter 6-7, Cold Spring Harbor Laboratory Press, 2001. Preferably, moderately stringent conditions as hybridization conditions include: 1×SSC to 6×SSC, 42°C to 55°C; more preferably 1×SSC to 3×SSC, 45°C to 50°C; most preferably 2×SSC, 50°C. When the hybridization solution contains, for example, about 50% formamide, a temperature 5 to 15°C lower than the above temperature can be used. Washing conditions include: 0.5×SSC to 6×SSC, 40°C to 60°C. During hybridization and washing, 0.05% to 0.2%, preferably about 0.1% SDS is usually added. As highly stringent conditions, it can also be easily determined by a person skilled in the art based on the length of the DNA, for example. Generally, highly stringent conditions include hybridization and / or washing with a higher temperature and / or a lower salt concentration than moderately stringent conditions. For example, as hybridization conditions, there can be listed: 0.1×SSC~2×SSC, 55°C~65°C; more preferably 0.1×SSC~1×SSC, 60°C~65°C; most preferably 0.2×SSC, 63°C. As washing conditions, there can be listed: 0.2×SSC~2×SSC, 50°C~68°C, more preferably 0.2×SSC, 60~65°C.

[0267] Antigens can also be obtained by separation / purification from eggs (preferably chicken eggs) by combining protein purification methods known to those skilled in the art. Alternatively, antigens can also be obtained by expressing antigens as recombinant proteins using gene recombination techniques known to those skilled in the art, and then by separation / purification using protein purification methods known to those skilled in the art.

[0268] Examples of protein purification methods include methods utilizing solubility such as salting out and solvent precipitation; methods utilizing differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods utilizing charge such as ion exchange chromatography and hydroxyapatite chromatography; methods utilizing specific affinity such as affinity chromatography; methods utilizing differences in hydrophobicity such as reversed-phase high performance liquid chromatography; and methods utilizing differences in isoelectric points such as isoelectric point electrophoresis.

[0269] The preparation of proteins using genetic recombination technology can be carried out as follows: prepare an expression vector containing a nucleic acid encoding an antigen, introduce the expression vector into a suitable host cell by gene introduction or transformation, culture the host cell under conditions suitable for the expression of the recombinant protein, and then recover the recombinant protein expressed in the host cell.

[0270] A "vector" is a nucleic acid that can be used to introduce a nucleic acid connected to it into a host cell; an "expression vector" is a vector that can guide the expression of a protein encoded by a nucleic acid introduced by the vector. Vectors include plasmid vectors, viral vectors, etc. Those skilled in the art can select an expression vector suitable for recombinant protein expression according to the type of host cell used.

[0271] "Host cell" is a cell into which a gene has been introduced or transformed by a vector. The host cell can be appropriately selected by a person skilled in the art according to the vector used. The host cell can be derived from a prokaryotic organism such as Escherichia coli (E. coli). When a prokaryotic cell such as Escherichia coli is used as a host, in order to facilitate the expression of the recombinant protein in the prokaryotic cell, the antigen of the present invention preferably contains an N-terminal methionine residue. The N-terminal methionine can also be cut off from the recombinant protein after expression. Alternatively, it can be a unicellular eukaryotic organism such as yeast; a cell from a eukaryotic organism such as a plant cell, an animal cell (for example, a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell or an insect cell), or a silkworm.

[0272] The gene introduction or transformation of the expression vector to the host cell can be suitably carried out by methods well known to those skilled in the art. In addition, those skilled in the art will suitably select conditions suitable for expressing the recombinant protein to cultivate the host cell according to the type of the host cell, so that the recombinant protein can be expressed. Then, the host cell expressing the recombinant protein is homogenized, and the method of purifying the above-mentioned protein by the homogenous mixture obtained is suitably combined, so that the antigen expressed as the recombinant protein can be separated / purified.

[0273] Diagnostic kits / diagnostic methods

[0274] The present invention provides a method for diagnosing an indicator of egg allergy in a subject, comprising the following steps:

[0275] (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies;

[0276] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0277] (iii) when binding of the subject's IgE antibodies to the antigen is detected, providing an indication that the subject has an egg allergy;

[0278] Here, the antigen is at least one of the proteins specified as the antigens in (1) to (3) and (4) to (10) above.

[0279] The sample obtained from the object refers to a solution containing IgE antibodies collected from the object. Such a solution contains, for example, blood, saliva, sputum, nasal mucus, urine, sweat, and tears. Before contacting with the antigen, the sample obtained from the object may also be subjected to a pretreatment for increasing the concentration of IgE antibodies in the sample. As a pretreatment of the sample, it may also include, for example, obtaining serum or plasma from blood. In addition, the Fab portion as the antigen-binding portion may also be further purified. In a particularly preferred embodiment, the above-mentioned step (i) is performed by contacting the IgE antibodies in the serum obtained from the object with the antigen.

[0280] The IgE antibody may be the IgE antibody itself, or may be a mast cell or the like to which the IgE antibody binds.

[0281] The contact of the sample obtained from the object with the antigen and the detection of their combination can be carried out by known methods. As such a method, for example, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, protein immunoblotting, immunoprecipitation, and immunochromatography can be used. These are all methods of contacting and combining the antigen with the object IgE antibody, causing it to act on the secondary antibody formed by enzyme labeling of the IgE antibody specifically bound to the antigen, adding the substrate of the enzyme (usually a color developing or luminescent reagent) to detect the product of the enzyme reaction, thereby being able to detect the method of binding the antigen to the object IgE antibody. Or a method for detecting a fluorescently labeled secondary antibody. Alternatively, it is also possible to use surface plasmon resonance (SPR) and other assays that can evaluate the binding of antigens to IgE antibodies for detection. It is also possible to mix a variety of antigen-specific IgE antibodies.

[0282] The antigen may also be in a state where the separated antigen is fixed to a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance may be used in the above steps (i) and (ii); in addition, in the above step (i), the sample obtained from the object is contacted with the surface on which the antigen is fixed. The separated antigen can be obtained by separation / purification from eggs (preferably chicken eggs) in combination with a protein purification method known to those skilled in the art, or it can also be prepared by genetic recombination technology. In addition, the antibody may be fixed.

[0283] The antigen may also be in a state of being fixed to a carrier. In this case, in the above steps (i) and (ii), a flow cytometer or the like may be used, and the presence of the antigen bound to the antibody may be confirmed by laser. Examples thereof include a basophil activation test (BAT) and the like. In addition, a histamine free test (HRT) may also be used to confirm whether histamine is freed by further contacting the antigen with the blood cells in the sample.

[0284] In addition, the antigen can also be transferred from a state separated by two-dimensional electrophoresis and detected by protein immunoblotting. Two-dimensional electrophoresis is a method of separating protein samples by performing isoelectric point electrophoresis in the first dimension and SDS-PAGE (SDS-polyacrylamide gel electrophoresis) in the second dimension. In this case, the conditions of two-dimensional electrophoresis are not particularly limited as long as they are conditions that can separate the antigens of the present invention. For example, the conditions of two-dimensional electrophoresis described in the above-mentioned "Limitation of Antigens" item can be used. Alternatively, the electrophoresis conditions can be set with reference to the records of the above-mentioned patent documents 1 to 4, for example, two-dimensional electrophoresis is performed under at least one condition selected from the group consisting of the following:

[0285] (A) As a first-dimensional isoelectric point electrophoresis gel, when the gel length is in the range of 5 to 10 cm and the pH range of the gel is 3 to 10, the gel length of the pH gradient of the gel relative to the migration direction up to pH 5 is set as a, the gel length of pH 5 to 7 is set as b, and the gel length of pH 7 or above is set as c, the relationship of "a < b" and "b > c" is satisfied;

[0286] (B) In the case of (A), when the total length of the gel is 1, a is in the range of 0.15 to 0.3, b is in the range of 0.4 to 0.7, and c is in the range of 0.15 to 0.3;

[0287] (C) in the first-dimensional isoelectric point electrophoresis, a constant voltage of 100 V to 600 V is applied to each gel containing the sample to perform a constant voltage step, and after the electrophoresis amplitude is within the range of 5 μA for every 30 minutes of electrophoresis, a voltage increase step is started to increase the voltage from the constant voltage;

[0288] (D) In ​​the case of (C), the final voltage of the voltage increasing step is set within the range of 3000V to 6000V;

[0289] (E) the length of the first-dimension isoelectric point electrophoresis gel in the long-side direction is set to 5 to 10 cm, and the gel concentration at the base end of the second-dimension electrophoresis gel in the migration direction is set to 3 to 6%; and

[0290] (F) In the case of (E), the gel concentration at the front end side portion in the migration direction of the second-dimensional electrophoresis gel is set higher than the gel concentration at the base end portion in the migration direction.

[0291] The antigens of (1) to (3) and (4) to (10) above are antigens that specifically bind to IgE antibodies of patients with egg allergy. Therefore, if the binding of the subject's IgE antibodies to the antigen is detected, it provides an indicator that the subject is egg allergic.

[0292] The present invention also provides a diagnostic kit for egg allergy comprising at least one of the above-mentioned antigens (1) to (3) and (4) to (10). The diagnostic kit of the present invention can be used in the above-mentioned method for providing indicators for diagnosing egg allergy or the following diagnostic method. In addition to containing at least one of the above-mentioned antigens (1) to (3) and (4) to (10), the diagnostic kit of the present invention may also contain an enzyme-labeled anti-IgE antibody and a colorimetric substrate or a luminescent substrate that serves as a substrate for the enzyme. In addition, a fluorescently labeled anti-IgE antibody may also be used. In the diagnostic kit of the present invention, the antigen may also be provided in a state where the antigen is immobilized on a carrier. The diagnostic kit of the present invention may also be provided together with instructions regarding the steps used for diagnosis and a method package containing the instructions.

[0293] In another embodiment, the diagnostic kit includes a companion diagnostic drug for egg allergy. A companion diagnostic drug refers to a substance used for the following purposes: identifying patients who expect the effect of a drug or patients who are at risk of severe side effects of a drug, or studying the reactivity of the drug in order to optimize the treatment using the drug. Here, the optimization of treatment includes, for example, determining the dosage volume, determining the suspension of drug administration, and confirming whether immune tolerance is obtained by using which allergen component.

[0294] The present invention also provides a composition for diagnosing egg allergy, comprising at least one of the above-mentioned antigens (1) to (3) and (4) to (10). The composition for diagnosing the present invention can be used in the following diagnostic method. The composition for diagnosing the present invention can also contain pharmaceutically acceptable carriers and additives that are usually used together with the antigen of the present invention, as required.

[0295] In one embodiment, the present invention is a method for diagnosing egg allergy in a subject, comprising the following steps:

[0296] (i) contacting a sample obtained from a subject with an antigen;

[0297] (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen;

[0298] (iii) when the subject's IgE antibody is detected to be bound to the antigen, the subject is judged to have an egg allergy;

[0299] Here, the antigen is at least one of the proteins defined as the antigens in (1) to (3) and (4) to (10) above. The steps (i) and (ii) are performed according to the description of the steps of the method for providing an indicator for diagnosing egg allergy.

[0300] In another embodiment, the present invention provides a method for diagnosing an egg allergy in a subject, comprising administering to the subject at least one of the antigens of (1) to (3) and (4) to (10) above. The method may also be performed in the form of a skin test characterized in that the antigen is administered to the skin. The skin test includes the following forms: a prick test in which a diagnostic composition is applied to the skin, the skin is lightly scratched to a level that does not bleed, the skin is penetrated with the antigen, and then the skin reaction is observed; a scratch test in which the skin is lightly scratched after the diagnostic composition is applied and then the reaction is observed; a patch test in which a diagnostic composition in the form of a cream, ointment, etc. is applied to the skin and then the reaction is observed; an intradermal test in which the antigen is administered intradermally and then the reaction is observed; etc. In the case where a skin reaction such as swelling occurs on the part of the skin to which the antigen is applied, the subject is diagnosed as having an egg allergy. Here, the amount of antigen administered to the skin may be, for example, an amount of 100 μg or less per time.

[0301] In the diagnosis of allergy, a load test for the purpose of limiting antigens is often performed. At least one of the antigens (1) to (3) and (4) to (10) above can be used as an effective component of a load test for diagnosing egg allergy. Here, the antigen protein used in the load test may be a purified protein or a protein expressed in food / ingredients, such as pollen rice in which a gene for a Chinese fir pollen antigen is transformed into rice and the antigen protein is expressed in rice.

[0302] In another embodiment, the present invention provides at least one of the antigens (1) to (3) and (4) to (10) for use in diagnosing egg allergy. This also includes providing at least one of the antigens (1) to (3) and (4) to (10) mixed with a known antigen.

[0303] In yet another embodiment, the present invention provides use of at least one of the antigens of (1) to (3) and (4) to (10) above in the preparation of a diagnostic drug for egg allergy.

[0304] Pharmaceutical compositions / therapeutic methods

[0305] The present invention provides a pharmaceutical composition comprising at least one of the antigens described in (1) to (3) and (4) to (10).

[0306] In one embodiment, the above-mentioned pharmaceutical composition is used to treat egg allergy.

[0307] The present invention also provides a method for treating egg allergy, comprising administering at least one of the antigens (1) to (3) and (4) to (10) above to a patient in need of treatment for egg allergy.

[0308] In another embodiment, the present invention provides at least one of the antigens (1) to (3) and (4) to (10) for use in the treatment of egg allergy. In yet another embodiment, the present invention provides use of at least one of the antigens (1) to (3) and (4) to (10) for the preparation of a therapeutic drug for egg allergy.

[0309] In the treatment of allergies, desensitization therapy is often performed in which the purpose is to induce immune tolerance by administering antigens to patients. At least one of the antigens (1) to (3) and (4) to (10) can be used as an active ingredient in the desensitization therapy for treating egg allergies. Here, the antigen protein used in the desensitization therapy may be a purified protein or a protein expressed in food / ingredients, such as pollen rice in which a gene for a cedar pollen antigen is transformed into rice and the antigen protein is expressed in rice.

[0310] The pharmaceutical composition of the present invention can be administered via common administration routes, including, for example, oral, sublingual, transdermal, intradermal, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, and intrarectal administration.

[0311] The pharmaceutical composition of the present invention can be used as needed together with the antigen of the present invention by conventional methods to add commonly used pharmaceutically permitted adjuvants, excipients, or various additives (such as stabilizers, cosolvents, emulsifiers, buffers, storage agents, colorants, etc.) to the pharmaceutical composition. The dosage form of the pharmaceutical composition can be appropriately selected by a person skilled in the art according to the route of administration. It can also be in the form of, for example, tablets, capsules, syrups, sublingual tablets, injections, nasal sprays, ointments, solutions, creams, lotions, etc. The dosage, number of administrations and / or administration period of the pharmaceutical composition of the present invention can be appropriately selected by a physician according to the characteristics of the patient such as the route of administration, symptoms, age, and weight. For example, in the case of adults, it can also be administered in an amount of less than 100 μg per time. The dosing interval can be, for example, once a day, once a week, twice a month, or once every three months. The administration period can be, for example, several weeks to several years. During the administration period, it can also be an administration method that increases the dosage in a gradient.

[0312] Detector

[0313] The present invention provides a detector comprising an antibody against at least one of the antigens of (1) to (3) and (4) to (10) above.

[0314] The antibody can be prepared by conventional methods. For example, a mammal such as a rabbit can be immunized with the antigens of (1) to (3) and (4) to (10) above. The antibody can be an IgE antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab') 2 , Fab').

[0315] In addition, in the above-mentioned detector, the antibody can be provided in the form of being bound to a carrier. The carrier is not particularly limited as long as it can be used in the detection of antibody-antigen binding. Any carrier known to those skilled in the art can be used.

[0316] Examples of methods for examining whether an antigen is contained include the following methods.

[0317] A method in which a detector containing the prepared IgE antibody is brought into contact with a sample obtained from a food / ingredient, etc., and the binding of the IgE antibody to an antigen in the sample is detected using, for example, an ELISA method, and when the binding of the IgE antibody to the antigen is detected, it is determined that the antigen remains in the target food / ingredient, etc.

[0318] A method in which food / ingredients are wrapped with filter paper or the like and reacted with an antibody solution to detect antigens contained therein.

[0319] Another embodiment of the present invention includes a detector for determining the presence or absence of an antigen causing an egg allergy in an object, characterized in that it includes a primer having a base sequence complementary to at least a portion of the base sequence shown in SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250, or 261. In a non-limiting manner, the primer has, for example, a base sequence complementary to a portion of the 3' end or the central portion of at least one of the base sequences shown in SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250, or 261, preferably 12 bases, 15 bases, 20 bases, or 25 bases. In particular, when mRNA is used as the object, the primer has a complementary primer of a poly(A) tail. In a preferred embodiment, the detector comprising the above-mentioned primers further comprises a primer comprising a base sequence at the 5' end portion of at least one of the base sequences shown in sequence numbers 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261, preferably a base sequence consisting of 12 bases, 15 bases, 20 bases or 25 bases.

[0320] For example, cDNA is amplified by PCR (polymerase chain reaction) including RT-PCR using DNA or mRNA obtained from eggs or birds as a template and the complementary primers, and the presence or absence of the antigen is determined by comparing the sequence of the amplified cDNA with SEQ ID NOs: 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261. Examples of methods for amplification by PCR include RACE method and the like. At this time, when a point mutation encoding the same amino acid is present in the comparison between the amplified cDNA and sequence number 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261; or, even if there is a base insertion, deletion, substitution or addition in the base sequence of the amplified cDNA to the base sequence of sequence number 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261, the antigen is judged to be present when the amino acid sequence encoding the cDNA has an identity of 70% or more, preferably 80, 90, 95, 98, 99% or more, to the amino acid sequence of sequence number 2, 13, 18, 47, 59, 106, 152, 237, 251 or 262.

[0321] In one embodiment, the detector can be used to detect whether antigens are contained in food (eggs or birds) or in objects in food production lines. The detector can be used by manufacturers to check the quality of products on production lines and before shipment; it can also be used by tasters to check whether the target food / ingredients contain antigens.

[0322] Antigen-removed foods, etc.

[0323] The present invention provides an egg, an egg processed product, or a bird producing the egg or hatched from the egg, characterized in that at least one of the antigens (1) to (3) and (4) to (10) is removed or reduced.

[0324] The method for removing or reducing antigens of the present invention is not limited to eggs, processed egg products, or birds that produce or hatch eggs. The removal or reduction of antigens is not limited to the removal or reduction of antigens of the present invention, and any method may be used.

[0325] For example, eggs in which the expression of the antigen of the present invention is deleted or reduced can be prepared using gene knockout technology to produce eggs in which the expression of the antigen of the present invention is knocked out.

[0326] The gene knockout technology can use any of the methods known to those skilled in the art. For example, Oishi, et al. (Scientific Reports, Vol. 6, Article number: 23980, 2016, doi: 10.1038 / srep23980) describes the application of CRISPER / Cas9 to primordial germ cells of chickens as a genome editing technology to obtain individuals with ovomucoid gene deletions. The same method can also be used to obtain eggs from which antigens have been removed in the present invention. In addition, birds or eggs that do not contain antigens or have low antigen content can also be mated through artificial insemination to obtain birds or eggs from which antigens have been removed / reduced in the present invention. Artificial mating of birds or eggs can be performed by conventional methods.

[0327] The processed egg product with antigens removed or reduced in the present invention may also be a processed product using as a raw material an egg from which the antigens of the present invention have been removed or reduced. When ordinary eggs are used as raw materials, the treatment for removing or reducing antigens of the present invention is performed before or after the preparation of the processed egg product. In the processed egg product using ordinary eggs as raw materials, methods for removing or reducing antigens of the present invention include: methods for removing protein components in food / ingredients by high pressure treatment and elution with neutral salt solutions, high temperature steam, etc.; methods for hydrolysis, denaturation, and amino acid denaturation (chemical modification / detachment of side chains, etc.) by heat treatment and acid treatment.

[0328] The bird from which the antigen is removed or reduced in the present invention can also be a bird hatched and grown from an egg from which the antigen is removed or reduced in the present invention. Birds include birds at various growth stages, including chicks, young birds, young birds, adult birds, and old birds. In this specification, a bird refers to an animal belonging to the avian species, preferably a chicken.

[0329] Method for producing antigen-removed egg processed product

[0330] The present invention is a method for producing an egg processed product with antigens removed or reduced, comprising the steps of: confirming that the antigens have been removed or reduced during the production process of the processed product;

[0331] Here, the antigen is at least one of the antigens described in (1) to (3) and (4) to (10).

[0332] In the production process of the antigen-removed or reduced egg processed product, the step of confirming the removal or reduction of antigens can be performed by confirming whether antigens are contained by the method described in the above item "Detector".

[0333] In addition, the production of egg processed products from which antigens have been removed or reduced can be carried out by the method described in the above-mentioned section "Antigen-removed foods, etc."

[0334] Example

[0335] Hereinafter, examples of the present invention will be described, but the technical scope of the present invention is not limited to these examples.

[0336] Example 1: Confirmation of protein profile

[0337] The proteins contained in the eggs were detected using the two-dimensional electrophoresis method described below.

[0338] Protein extraction

[0339] Separate raw eggs into egg whites and yolks.

[0340] The extraction and purification of proteins contained in egg white and egg yolk are performed as follows. After adding a solubilizing agent to the egg white to extract the protein, water or urea buffer is added to obtain a protein extract. The composition of the urea buffer is as follows.

[0341] 30mM Tris

[0342] 2M Thiourea

[0343] 7M Urea

[0344] 4% (w / v) CHAPS:

[0345] 3-[3-(Cholamidopropyl)dimethylamino]propanesulfonic acid inner salt

[0346] Appropriate amount of dilute hydrochloric acid

[0347] Distilled water was added to adjust the total volume to 100 mL and the pH to 8.5.

[0348] Then, 25 μg of each was mixed based on the weight of protein to obtain an extract. A surfactant buffer (Mammalian Lysis Buffer (MCLI), SIGMA) was added to the egg yolk as a solubilizing agent to extract the protein.

[0349] After that, two precipitation operations were performed using the 2D-CleanUP kit (manufactured by GE). In the first precipitation operation, TCA (trichloroacetic acid) was added to the recovered protein extract to precipitate, and the precipitate (TCA precipitate) produced by the operation was recovered. In the second precipitation operation, acetone was added to the recovered TCA precipitate to precipitate, and the precipitate (test sample) obtained by the operation was recovered.

[0350] Preparation of sample solution

[0351] A portion of the obtained sample (50 μg in terms of protein weight) was dissolved in 150 μl of DeStreak Rehydration Solution (GE), a swelling buffer for gel for first-dimensional isoelectric electrophoresis, to prepare a sample solution for first-dimensional isoelectric electrophoresis (swelling sample solution). The composition of DeStreak Rehydration Solution is as follows.

[0352] 7M Thiourea

[0353] 2M Urea

[0354] 4%(w / v)CHAPS

[0355] 0.5% (v / v) IPG buffer; manufactured by GE

[0356] Appropriate amount of BPB (bromophenol blue)

[0357] Penetration of the specimen into the first-dimension isoelectric electrophoresis gel

[0358] A gel for first-dimensional isoelectric electrophoresis (IPG gel Immobiline Drystrip (pH 3-10 NL, manufactured by GE) was immersed in 140 μl of the above-mentioned sample solution for first-dimensional isoelectric electrophoresis (sample solution for expansion) and allowed to permeate overnight at room temperature.

[0359] In this example, IPGphor manufactured by GE was used as the electrophoresis apparatus.

[0360] The swimming lane was filled with silicone oil. Filter papers moistened with water were placed at both ends of the gel soaked with the specimen, and the gel was placed in the swimming lane so that the gel was covered with silicone oil. Electrodes were placed between the gel and the filter paper.

[0361] The upper limit of the current value of the isoelectric point electrophoresis instrument is set to 75 μA per gel, and the voltage program is set as follows: (1) the constant voltage is increased from 300 V to 750 Vhr to perform a constant voltage process (the current change amplitude during the 30 minutes of electrophoresis before the end of this process is 5 μA); (2) the voltage is gradually increased from 300 Vhr to 1000 V; (3) further, the voltage is gradually increased from 4500 Vhr to 5000 V; (4) thereafter, the first dimension isoelectric point electrophoresis is performed at a constant voltage of 5000 V until Vhr reaches 12000.

[0362] SDS equilibration of isoelectric point electrophoresis gel

[0363] After the first-dimensional isoelectric electrophoresis, the gel was removed from the isoelectric electrophoresis apparatus and immersed in an equilibration buffer containing a reducing agent and shaken at room temperature for 15 minutes. The composition of the equilibration buffer containing a reducing agent is as follows.

[0364] 100 mM Tris-HCl (pH 8.0)

[0365] 6M urea

[0366] 30% (v / v) glycerol

[0367] 2%(w / v)SDS

[0368] 1%(w / v)DTT

[0369] Next, the equilibration buffer containing the reducing agent was removed, and the gel was immersed in an equilibration buffer containing an alkylating agent and shaken at room temperature for 15 minutes to obtain an SDS-equilibrated gel. The composition of the equilibration buffer containing an alkylating agent is as follows.

[0370] 100 mM Tris-HCl (pH 8.0)

[0371] 6M urea

[0372] 30% (v / v) glycerol

[0373] 2%(w / v)SDS

[0374] 2.5% (w / v) iodoacetamide

[0375] SDS-PAGE in the second dimension

[0376] In this example, the electrophoresis apparatus used was XCell SureLock Mini-Cell manufactured by Life Technologies, Inc. The second-dimensional electrophoresis gel used was NuPAGE 4-12% Bis-Tris Gels manufactured by Life Technologies, Inc. In addition, a running buffer having the following composition was prepared and used.

[0377] 50mM MOPS

[0378] 50mM Tris base

[0379] 0.1%(w / v)SDS

[0380] 1mM EDTA

[0381] In this example, an agarose solution for bonding was used in which 0.5% (w / v) agarose S (manufactured by Nippongene Co., Ltd.) and an appropriate amount of BPB (bromophenol blue) were dissolved in a running buffer.

[0382] After the wells of SDS-PAGE were fully cleaned with the above-mentioned electrophoresis buffer, the buffer used in the washing was removed. Then, a fully dissolved agarose solution was added to the wells for bonding. Then, the gel that had been subjected to SDS equilibration was immersed in agarose, and the gel that had been subjected to SDS equilibration was closely fitted with the second dimension electrophoresis gel using tweezers. Under the closely fitted state of the two gels, it was confirmed that agarose was fully solidified, and electrophoresis was performed for about 45 minutes with a constant voltage of 200V.

[0383] Fluorescent staining of gels

[0384] The gel was fluorescently stained using SYPRO Ruby (manufactured by Life Technologies).

[0385] First, the sealed container used was thoroughly cleaned with 98% (v / v) ethanol in advance. The following treatment was performed twice: the second-dimensional electrophoresis gel was removed from the SDS-PAGE instrument after electrophoresis, placed in a clean sealed container, and immersed in an aqueous solution containing 50% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes. After that, the aqueous solution was replaced with water and immersed for 10 minutes. Next, the second-dimensional electrophoresis gel was immersed in 40 ml of SYPRO Ruby and shaken at room temperature overnight. Next, SYPRO Ruby was removed, the second-dimensional electrophoresis gel was washed with water, and then shaken with an aqueous solution containing 10% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes. Furthermore, the aqueous solution was replaced with water and shaken for more than 30 minutes.

[0386] Analysis

[0387] The gel for the second-dimensional electrophoresis subjected to the above series of treatments was subjected to scanning of a fluorescent image using Typhoon 9400 (manufactured by GE). The results of the two-dimensional electrophoresis of the proteins contained in the egg yolk are shown in FIG. Figure 1 The results for proteins are not shown. Molecular weight marker bands are visible on the left side of the gel photograph, and the positions of the bands indicate specific molecular weights (KDa).

[0388] Example 2: Confirmation of antigen by immunoblotting (1)

[0389] Antigen confirmation by immunoblotting was performed by performing the steps described in Example 1 up to "second-dimensional SDS-PAGE" and then performing the following operations of "transfer to membrane", "immunoblotting" and "analysis".

[0390] Transfer to membrane

[0391] Transfer to the membrane was performed using the following transfer apparatus and transfer buffer.

[0392] Blotting device: XCell SureLock Mini-Cell and XCell II blot module (Lifetechnologies)

[0393] Transfer buffer: NuPAGE transfer buffer (×20) (manufactured by Life Technologies) was diluted 20-fold with milliQ water and used.

[0394] Specifically, the proteins in the two-dimensional electrophoresis gel were transferred to a membrane (PVDF membrane) according to the following procedure.

[0395] (1) The PVDF membrane was immersed in 100% methanol, then immersed in milliQ water, and then transferred to a transfer buffer to perform a hydrophilization treatment on the PVDF membrane.

[0396] (2) The sponge, filter paper, second-dimensional SDS-PAGE gel, hydrophilized PVDF membrane, filter paper, and sponge were placed in the order described above and energized at a constant voltage of 30 V for 1 hour in a transfer device.

[0397] Immunoblotting

[0398] The serum of a patient with egg allergy (patient 1) or the serum of a non-egg allergy subject was used as the first antibody for immunoblotting of the membrane. The patient with egg allergy was diagnosed with food-dependent exercise-induced allergy (FDEIA) caused by eggs. It should be noted that for this patient, in the puncture test, raw eggs, boiled eggs, and hot spring eggs were all negative; in the specific IgE antibody test, the egg white, egg yolk, and ovomucoid were negative.

[0399] Immunoblotting of membranes was performed as follows.

[0400] (1) The transferred membrane was shaken in a 5% skim milk / PBST solution (PBS buffer containing 0.1% nonionic surfactant Tween 20) at room temperature for 1 hour.

[0401] (2) As the primary antibody, the cells were incubated in a 5% serum / 5% skim milk / PBST solution at room temperature for 1 hour.

[0402] (3) Wash with PBST solution (5 minutes x 3 times).

[0403] (4) As a secondary antibody, anti-human IgE-HRP (horseweed peroxidase) was diluted 5000 times with a 5% skim milk / PBST solution and allowed to stand at room temperature for 1 hour.

[0404] (5) Wash with PBST solution (5 minutes x 3 times).

[0405] (6) The sample was allowed to stand in Pierce Western Blotting Substrate Plus (manufactured by Thermo Fisher Scientific) for 5 minutes.

[0406] Analysis

[0407] The membrane subjected to the above series of treatments was subjected to scanning of a fluorescent image using Typhoon 9500 (manufactured by GE).

[0408] The immunoblot using the serum of the patient with egg allergy was compared with the immunoblot using the serum of the non-egg allergy subject as a control. The protein contained in the raw egg yolk was immunoblotted using the serum of the patient with egg allergy ( Figure 2 ), unlike the case where the serum of a non-egg allergic subject was used, three spots different from the known egg allergen proteins were detected.

[0409] The molecular weights and isoelectric points of the above three spots are as follows: Figure 3 ).

[0410] Spot 1: molecular weight 20-40 kDa, pI 4.0-10.0

[0411] Spot 2: molecular weight 30-60 kDa, pI 5.0-10.0

[0412] Spot 3: molecular weight 35-60 kDa, pI 5.0-10.0

[0413] Example 3: Mass spectrometry analysis and identification of antigens (1)

[0414] The amino acid sequence of the antigen producing three spots in Example 2 was identified by mass spectrometry.

[0415] Specifically, protein extraction and mass spectrometry analysis were performed according to the following steps.

[0416] (1) Protein extraction, two-dimensional electrophoresis and membrane transfer were performed on raw egg yolks according to the procedures of Examples 1 and 2, and the yolks were stained with 0.008% direct blue / 40% ethanol / 10% acetic acid by shaking.

[0417] (2) Thereafter, the sample was treated with 40% ethanol / 10% acetic acid for 5 minutes three times for decolorization, washed with water for 5 minutes, and then air-dried.

[0418] (3) Cut the target spot with a clean cutter and add it to a centrifuge tube. After hydrophilizing the membrane with 50 μL of methanol, wash it twice with 100 μL of water and remove it by centrifugation. Add 20 μL of 20 mM NH 4 HCO 3 50% acetonitrile.

[0419] (4) Add 1 μL of 1 pmol / μL lysine endopeptidase (WAKO), let stand at 37°C for 60 minutes, and then collect the solution into a new centrifuge tube. Add 20 μL of 20 mM NH 4 HCO 3 · 70% acetonitrile, soak at room temperature for 10 minutes, and further recover. Dissolve in 10 μL of 0.1% formic acid and 4% acetonitrile, and transfer to a test tube.

[0420] (5) The recovered solution was dried under reduced pressure, dissolved in 15 μl of solution A (0.1% formic acid, 4% acetonitrile solution), and subjected to mass spectrometry analysis (ESI-TOF6600, manufactured by AB Sciex).

[0421] (6) Protein identification based on mass spectrometry data obtained by a mass spectrometer was performed by searching NCBI or UniProt.

[0422] result

[0423] As a result of mass spectrometry analysis of each spot, the following amino acid sequences were detected.

[0424] Spot 1: Amino acid sequence shown in SEQ ID NOs: 3 to 11

[0425] Spot 2: amino acid sequence shown in SEQ ID NOs: 9 to 11, 14, and 16

[0426] Spot 3: Amino acid sequence shown in SEQ ID NOs: 19 to 45

[0427] Furthermore, for each spot, the mass spectrum data obtained by the mass spectrometer were analyzed using NCBI for spot 1 and spot 2, and the mass spectrum data obtained by the mass spectrometer were analyzed using UniProt for spot 3. As a result, each spot was identified as the following protein.

[0428] Spot 1: C-terminal portion of vitellogenin-3 (amino acid sequence: NCBI accession number XP_015146355, base sequence encoding it: GenBank accession number XM_015290869.1) (amino acid sequence: sequence number 2, base sequence encoding it: sequence number 1)

[0429] Spot 2: C-terminal portion of vitellogenin-3 (amino acid sequence: NCBI accession number XP_015146355, base sequence encoding it: GenBank accession number XM_015290869.1) (amino acid sequence: sequence number 13, base sequence encoding it: sequence number 12)

[0430] Spot 3: The central part of vitellogenin-1 (amino acid sequence: UniProt accession number P87498, base sequence encoding it: ENA (EMBL) accession number D89547.1), or the C-terminal part of lipovitellin-1 (amino acid sequence: sequence number 18, base sequence encoding it: sequence number 17)

[0431] Example 4: Confirmation of antigen by immunoblotting (2)

[0432] Similar to Example 2, antigen confirmation was performed by immunoblotting using sera from three other egg allergy patients (patient 2, patient 3, and patient 4). For proteins contained in raw egg yolk, immunoblotting using sera from egg allergy patients (patient 2 was Figure 4 For patient 3, Figure 5 For patient 4, Figure 6 ), unlike the case where the serum of a non-egg allergic subject was used, spots different from the known egg allergen protein were detected. Specifically, spots 4 to 8 were detected for patient 2, spots 9 and 10 were detected for patient 3, and spot 11 was detected for patient 4.

[0433] The molecular weights and isoelectric points of the above spots 4 to 11 are as follows: Figures 4 to 6 ).

[0434] Spot 4: molecular weight 20-50 kDa, pI 3.0-8.0

[0435] Spots 5 and 6: molecular weight 80-260 kDa, pI 1.0-8.0

[0436] Spot 7: molecular weight 110-300 kDa, pI 3.0-8.0

[0437] Spot 8: molecular weight 160-550 kDa, pI 3.0-8.0

[0438] Spot 9: molecular weight 20-50 kDa, pI 7.0-11.0

[0439] Spot 10: molecular weight 20-50 kDa, pI 7.0-11.0

[0440] Spot 11: molecular weight 15-40 kDa, pI 6.0-11.0

[0441] Example 5: Mass spectrometry analysis and identification of antigens (2)

[0442] In the same manner as in Example 3, the amino acid sequences of the antigens producing spots 4 to 11 in Example 4 were identified by mass spectrometry.

[0443] As a result of mass spectrometry analysis of each spot, the following amino acid sequences were detected.

[0444] Spot 4: amino acid sequence shown in SEQ ID NOs: 48 to 57

[0445] Spots 5 and 6: Amino acid sequences shown in SEQ ID NOs: 60 to 104

[0446] Spot 7: amino acid sequence shown in SEQ ID NOs: 107 to 150

[0447] Spot 8: Amino acid sequence shown in SEQ ID NOs: 153 to 235

[0448] Spot 9: amino acid sequence shown in SEQ ID NOs: 238 to 249

[0449] Spot 10: amino acid sequence shown in SEQ ID NOs: 252 to 260

[0450] Spot 11: Amino acid sequence shown in SEQ ID NOs: 263 to 271

[0451] Furthermore, as a result of analyzing the mass spectrum data obtained by the mass spectrometer for each spot using NCBI, each spot was identified as the following protein.

[0452] Spot 4: Vitellogenin-3 (amino acid sequence: NCBI accession number XP_015146355, base sequence encoding it: GenBank accession number XM_015290869.1) (amino acid sequence: sequence number 47, base sequence encoding it: sequence number 46)

[0453] Spots 5 and 6: Vitellogenin-2 precursor (amino acid sequence: NCBI accession number NP_001026447.1, base sequence encoding it: GenBank accession number NM_001031276.1) (amino acid sequence: sequence number 59, base sequence encoding it: sequence number 58)

[0454] Spot 7: Vitellogenin-2 precursor (amino acid sequence: NCBI accession number NP_001026447.1, base sequence encoding it: GenBank accession number NM_001031276.1) (amino acid sequence: sequence number 106, base sequence encoding it: sequence number 105)

[0455] Spot 8: Apolipoprotein B precursor (amino acid sequence: NCBI accession number NP_001038098.1, base sequence encoding it: GenBank accession number NM_001044633.1) (amino acid sequence: sequence number 152, base sequence encoding it: sequence number 151)

[0456] Spot 9: Apolipoprotein B precursor (amino acid sequence: NCBI accession number NP_001038098.1, base sequence encoding it: GenBank accession number NM_001044633.1) (amino acid sequence: sequence number 237, base sequence encoding it: sequence number 236)

[0457] Spot 10: Apolipoprotein B precursor (amino acid sequence: NCBI accession number NP_001038098.1, base sequence encoding it: GenBank accession number NM_001044633.1) (amino acid sequence: sequence number 251, base sequence encoding it: sequence number 250)

[0458] Spot 11: Vitellogenin-2 precursor (amino acid sequence: NCBI accession number NP_001026447.1, base sequence encoding it: GenBank accession number NM_001031276.1) (amino acid sequence: sequence number 262, base sequence encoding it: sequence number 261)

[0459] Industrial Applicability

[0460] The present invention provides a novel antigen for egg allergy, a method for diagnosing egg allergy and a diagnostic kit, a pharmaceutical composition containing the antigen, an egg or egg processed product with the antigen removed or reduced, or a bird producing the egg or hatched from the egg, and a method for producing an egg processed product with the antigen removed or reduced. The present invention can also provide a detector for determining whether an object has an egg antigen.

Claims

1. A diagnostic kit for egg allergy, comprising at least one of the following proteins (1) to (3) as an antigen: (1) (1A) A protein comprising the C-terminal portion of vitellogenin-3 or a mutant thereof, which is any one of the following (1A-a) to (1A-e) as an antigen for egg allergy: (1A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in SEQ ID NO: 2; (1A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence set forth in SEQ ID NO: 2; (1A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 1; (1A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence shown in SEQ ID NO: 1; or (1A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1; or (1B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 11; (2) (2A) A protein comprising the C-terminal portion of vitellogenin-3 or a mutant thereof, which is any one of the following proteins (2A-a) to (2A-e) that is an antigen for egg allergy: (2A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:13; (2A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 13; (2A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 12; (2A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 12; or (2A-e) a protein containing an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 12; or (2B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 9 to 11, 13, 14 and 16; (3) (3A) A protein comprising the central portion of vitellogenin-1 or the C-terminal portion of lipovitellin-1, or a mutant thereof, which is any one of the following (3A-a) to (3A-e) as an antigen for egg allergy: (3A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:18; (3A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 18; (3A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 17; (3A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 17; or (3A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 17; or (3B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 18 to 45.

2. A diagnostic kit for egg allergy, comprising at least one of the following proteins (4) to (10) as an antigen: (4) (4A) A protein comprising vitellogenin-3 or a mutant thereof, which is any one of the following proteins (4A-a) to (4A-e) that is an antigen for egg allergy: (4A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:47; (4A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 47; (4A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 46; (4A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 46; or (4A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO:46; or (4B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 47 to 57; (5) (5A) A protein comprising vitellogenin-2 precursor or a mutant thereof, which is any one of the following proteins (5A-a) to (5A-e) that is an antigen for egg allergy: (5A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 59; (5A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 59; (5A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 58; (5A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 58; or (5A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 58; or (5B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 59 to 104; (6) (6A) Vitellogenin-2 precursor or a mutant thereof, which is any one of the following proteins (6A-a) to (6A-e) that is an antigen for egg allergy: (6A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:106; (6A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 106; (6A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 105; (6A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 105; or (6A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 105; or (6B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 106 to 150; (7) (7A) Apolipoprotein B precursor or a mutant thereof, which is any one of the following proteins (7A-a) to (7A-e) that is an antigen for egg allergy: (7A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO:152; (7A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 152; (7A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 151; (7A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 151; or (7A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 151; or (7B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 152 to 235; (8) (8A) Apolipoprotein B precursor or a mutant thereof, which is any one of the following proteins (8A-a) to (8A-e) that is an antigen for egg allergy: (8A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 237; (8A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence of SEQ ID NO: 237; (8A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 236; (8A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 236; or (8A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence set forth in SEQ ID NO: 236; or (8B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 237 to 249; (9) (9A) Apolipoprotein B precursor or a mutant thereof, which is any one of the following proteins (9A-a) to (9A-e) that is an antigen for egg allergy: (9A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 251; (9A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 251; (9A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 250; (9A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 250; or (9A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 250; or (9B) a protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 251 to 260; (10) (10A) Vitellogenin-2 precursor or a mutant thereof, which is any one of the following proteins (10A-a) to (10A-e) that is an antigen for egg allergy: (10A-a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in SEQ ID NO: 262; (10A-b) a protein comprising an amino acid sequence having an identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 262; (10A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or more nucleotides are deleted, substituted, inserted, or added in SEQ ID NO: 261; (10A-d) a protein comprising an amino acid sequence encoded by a base sequence having an identity of 70% or more to the base sequence set forth in SEQ ID NO: 261; or (10A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 261; or (10B) A protein comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 262 to 271.

3. A composition for diagnosing egg allergy, comprising: as an antigen, at least one of the proteins defined as (1) to (3) in claim 1 or (4) to (10) in claim 2.

4. A method for providing an index for diagnosing egg allergy in a subject, comprising the following steps: (i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing IgE antibodies; (ii) detecting the binding of IgE antibodies in a sample obtained from the subject to the antigen; (iii) when the subject's IgE antibody is detected to be bound to the antigen, providing an indication that the subject has an egg allergy; Here, the antigen is at least one of the proteins defined as (1) to (3) in claim 1 or (4) to (10) in claim 2.

5. A pharmaceutical composition comprising: at least one of the proteins defined as (1) to (3) in claim 1 or (4) to (10) in claim 2.

6. The pharmaceutical composition according to claim 5, for use in the treatment of egg allergy.

7. An egg, an egg product or a bird, characterized in that: It is an egg, an egg processed product, or a bird producing or hatching the egg from which an antigen has been removed or reduced, and the antigen is at least one of the proteins defined as (1) to (3) in claim 1 or (4) to (10) in claim 2.

8. A detector for determining whether an object contains egg antigen, characterized in that: It comprises an antibody that binds to at least one of the proteins defined as (1) to (3) in claim 1 or (4) to (10) in claim 2.

9. A detector for determining whether an object contains an antigen causing an egg allergy, characterized in that: It contains a primer having a base sequence complementary to at least one of the base sequences shown in SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261.

10. A method for producing an egg processed product with antigens removed or reduced, comprising the steps of: confirming that the antigens have been removed or reduced during the production process of the processed product, Here, the antigen is at least one of the proteins specified in (1) to (3) of claim 1, or at least one of the proteins specified in (4) to (10) of claim 2.

11. An egg-derived antigen which is at least one of the proteins specified in (1) to (3) of claim 1 or at least one of the proteins specified in (4) to (10) of claim 2 and causes an egg allergy.

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