WO2017209287A1 - 卵アレルギーの抗原 - Google Patents
卵アレルギーの抗原 Download PDFInfo
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- WO2017209287A1 WO2017209287A1 PCT/JP2017/020654 JP2017020654W WO2017209287A1 WO 2017209287 A1 WO2017209287 A1 WO 2017209287A1 JP 2017020654 W JP2017020654 W JP 2017020654W WO 2017209287 A1 WO2017209287 A1 WO 2017209287A1
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Definitions
- the present invention relates to a novel antigen for allergy to eggs.
- the present invention also relates to an allergy diagnostic kit, diagnostic composition, and diagnostic method for eggs.
- the present invention also relates to a pharmaceutical composition comprising the antigen, and an egg, processed egg product, or bird born from or born from the egg from which the antigen has been removed or reduced.
- the present invention further relates to a method for producing a processed egg product from which the antigen is removed or reduced.
- the present invention further relates to a tester for determining the presence or absence of an egg antigen in an object.
- IgE antibodies specific for specific antigens are produced in the serum and tissues of allergic patients. Allergic reactions are triggered by the physiological results produced by the interaction of this IgE antibody with a specific antigen.
- antigen refers to foods / foods that cause allergic symptoms in a broad sense, and refers to proteins (hereinafter also referred to as allergen components) contained in foods / foods to which specific IgE antibodies bind in a narrow sense.
- allergen reagents are often prepared simply by grinding foods / food ingredients, etc., of allergen candidates (Patent Document 1). For this reason, a positive reaction in an allergy test is detected only when the content of the allergen component contained in the conventional antigen reagent exceeds the threshold at which a positive reaction can be determined for binding to an IgE antibody. However, the diagnostic efficiency was not sufficiently high.
- Non-Patent Document 1 Japanese Patent Document 1
- allergen components are suggested in foods and ingredients of candidate allergens and are commercialized as test kits, but it is necessary to comprehensively identify allergen components in order to increase the reliability of allergy tests
- the patient detection rate by the measurement of the allergen component is still insufficient. Identification of novel allergens in eggs not only enhances the accuracy of diagnostic agents, but is also very important as a target for low allergen foods, low allergen foodstuffs and therapeutic agents.
- JP 2002-286716 A JP2011-33544 JP2011-33546A JP2011-33547 JP2011-33548
- the present invention provides a novel antigen for allergy to eggs.
- the present invention also provides a diagnostic method and diagnostic kit for allergy to eggs.
- the present invention also provides a pharmaceutical composition comprising the antigen, and an egg from which the antigen has been removed or reduced, an egg product, or a bird that lays or is born from the egg.
- the present invention further provides a tester for determining the presence or absence of an egg antigen in an object.
- the present inventors have conducted intensive research on identification of the allergen causing the allergy to eggs. As a result, the inventors succeeded in identifying a novel antigen that specifically binds to an IgE antibody in the serum of a patient having egg allergy. Based on this finding, the present invention has been completed.
- the present invention may be as follows.
- An egg allergy diagnostic kit comprising at least one of the following proteins (1) to (3): (1) (1A) A protein containing a C-terminal part of vitellogenin-3 (Vitellogenin-3) or a variant thereof, which is an allergic antigen to eggs, the following (1A-a) to (1A-e) Any of the proteins: (1A-a) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 2; (1A-b) a protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 2; (1A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or several 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 70% or more identity with the base sequence represented by SEQ ID NO: 1; or (1A-e) complementary to the base sequence represented
- composition for diagnosis of egg allergy which comprises at least one of the proteins specified as (1) to (3) in [1] as an antigen.
- 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 binding between the IgE antibody and the antigen in a sample obtained from the subject; (Iii) if binding of the subject IgE antibody to the antigen is detected, an indication is provided that the subject is egg allergic; Wherein the antigen is at least one of the proteins identified as (1) to (3) in [1] above.
- a pharmaceutical composition comprising at least one of the proteins specified as (1) to (3) in [1] above.
- a tester for determining the presence or absence of an egg antigen in an object comprising an antibody that binds to at least one of the proteins specified as (1) to (3) in [1] above.
- a method for determining the presence or absence of an allergic antigen to an egg in an object comprising a primer having a base sequence complementary to at least one of the base sequences represented by SEQ ID NOs: 1, 12, or 17 tester.
- a diagnostic kit for egg allergy wherein at least one of the following proteins (4) to (10): (4) (4A) Any one of the following (4A-a) to (4A-e), which is a protein containing Vitellogenin-3 or a variant thereof and is an antigen of allergy to eggs: protein: (4A-a) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 47; (4A-b) a protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 47; (4A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or several 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 70% or more identity with the base sequence represented by SEQ ID NO: 46; or (4A-e) complementary to the base sequence represented by SEQ ID NO: 46 A
- composition for diagnosis of egg allergy comprising at least one of the proteins specified as (4) to (10) in [9] as an antigen.
- 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 binding between the IgE antibody and the antigen in a sample obtained from the subject; (Iii) if binding of the subject IgE antibody to the antigen is detected, an indication is provided that the subject is egg allergic; Wherein the antigen is at least one of the proteins identified as (4) to (10) in [9] above.
- a pharmaceutical composition comprising at least one of the proteins specified as (4) to (10) in [9] above.
- a tester for determining the presence or absence of an egg antigen in an object comprising an antibody that binds to at least one of the proteins specified as (4) to (10) in [9] above.
- Allergy to eggs in a subject comprising a primer having a base sequence complementary to at least one of the base sequences represented by SEQ ID NOs: 46, 58, 105, 151, 236, 250 or 261 A tester for determining the presence or absence of an antigen.
- a method for producing a processed egg product from which an antigen has been removed or reduced comprising the step of confirming that the antigen has been removed or reduced in the process of producing the processed product, wherein the antigen is the above-mentioned
- the production method which is at least one of the proteins specified as (1) to (3) in [1], or at least one of the proteins specified as (4) to (10) in [9] above.
- An antigen derived from an egg which is specified as at least one of the proteins specified as (1) to (3) in [1] above or as (4) to (10) in [9] above Said antigen which is at least one of the proteins and causes allergies to eggs.
- a novel antigen allergic to eggs can be provided. Since a novel antigen (allergen component) that causes egg allergy has been identified in the present invention, a highly sensitive diagnosis method and kit for allergy to eggs, a pharmaceutical composition containing the antigen, and the antigen has been removed or reduced.
- FIG. 1 is a photograph of a gel showing a protein migration pattern by two-dimensional electrophoresis for proteins contained in egg yolk of chicken eggs (raw eggs).
- the band on the left side of the photograph is the molecular weight marker band, and the value on the left side of the photograph is the molecular weight (KDa) of each molecular weight marker.
- the numerical value at the top of the photo represents the isoelectric point.
- FIG. 2 is a photograph of an immunoblot using the serum of egg allergy patient 1 against the two-dimensional electrophoresis pattern of the protein contained in the egg yolk of chicken eggs (live eggs).
- FIG. 3 is a gel photograph of a two-dimensional electrophoresis pattern of a protein contained in the egg yolk of a chicken egg (raw egg).
- FIG. 4 is a photograph of an immunoblot using the serum of egg allergy patient 2 against the two-dimensional electrophoresis pattern of the protein contained in the egg yolk of chicken eggs (live eggs). The spots 4 to 8 where the serum of the egg allergy patient 2 specifically reacted are shown by being surrounded by a square frame.
- FIG. 5 is a photograph of an immunoblot using the serum of egg allergic patient 3 with respect to the two-dimensional electrophoresis pattern of the protein contained in the egg yolk of a chicken egg (live egg). Spots 9 and 10 to which the serum of egg allergy patient 3 specifically reacted are shown by being surrounded by a square frame.
- FIG. 4 is a photograph of an immunoblot using the serum of egg allergy patient 2 against the two-dimensional electrophoresis pattern of the protein contained in the egg yolk of chicken eggs (live eggs). The spots 4 to 8 where the serum of the egg allergy patient 2 specifically reacted are shown by being surrounded by a square frame.
- FIG. 5 is a photograph of an immunoblot using the serum
- FIG. 6 is a photograph of an immunoblot using the serum of an egg allergic patient 4 with respect to a two-dimensional electrophoresis pattern of a protein contained in the egg yolk of a chicken egg (raw egg).
- a spot 11 to which the serum of the egg allergy patient 4 specifically reacted is shown surrounded by a square frame.
- allergy refers to a state in which a hypersensitivity reaction unfavorable to the living body, which occurs when the antigen enters the living body sensitized to a certain antigen again.
- IgE antibodies specific for antigens are produced in blood and tissues. IgE antibodies bind to mast cells or basophils. When the antigen specific to the IgE antibody enters the body of the allergic disease patient again, the antigen is combined with the IgE antibody bound to mast cells or basophils, and the IgE antibody crosslinks on the cell surface. -Produces the physiological effects of antigen interactions.
- an egg means an avian egg, preferably a chicken egg.
- allergy to an egg refers to a state having an allergic reaction that occurs using proteins contained in the egg as antigens. Allergy to eggs can cause an allergic reaction when in contact with an antigen contained in the egg or when the antigen is ingested. In general, an allergic reaction that occurs when food is ingested is called food allergy.
- the allergy to eggs may be food allergy.
- an antigen is a substance that induces an allergic reaction and is also referred to as an allergen component.
- the antigen is preferably a protein.
- a protein is a molecule having a structure in which natural amino acids are linked by peptide bonds.
- the number of amino acids contained in the protein is not particularly limited, but a protein in which about 2 to 50 amino acids are linked by peptide bonds may be called a peptide.
- L form is shown unless otherwise specified.
- the protein or peptide amino acid sequence notation used herein is based on standard usage and conventions commonly used in the art and is represented by a single letter of amino acid, the left direction is the amino terminal direction, and The right direction is the carboxy terminal direction.
- the antigen of the allergy to the egg was specified using the above-described method. Specifically, raw chicken eggs were divided into egg yolk and egg white, and the egg proteins contained in each were subjected to two-dimensional electrophoresis under the following conditions.
- the first dimension electrophoresis is an isoelectric focusing gel
- the gel length is in the range of 5-10 cm
- the gel pH range is 3-10
- the pH gradient of the gel with respect to the migration direction is
- a gel length up to pH 5 is a
- a gel length of pH 5-7 is b
- a gel length of pH 7 or higher is c
- a is in the range of 0.15-0.3
- b Is a gel having a range of 0.4 to 0.7
- c is in a range of 0.15 to 0.3.
- Isoelectric focusing was carried out using an IPG gel Immobiline® Drystrip® (pH 3-10NL).
- IPGphor manufactured by GE was used as the electrophoresis apparatus.
- the upper limit of the current value of the electrophoresis apparatus is set to 75 ⁇ A per gel, and the voltage program is (1) a constant voltage process is performed at a constant voltage of 300 V up to 750 Vhr (the current change width for 30 minutes before the end of the process is (2) Gradually increase the voltage to 1000V over 300Vhr, (3) Gradually increase the voltage to 5000V over 4500Vhr, and (4) then the total Vhr to 12000 at 5000V constant voltage Until then, isoelectric focusing of the first dimension was performed.
- the gel concentration at the base end of the migration direction is set to 3 to 6%, and the gel concentration at the tip end in the migration direction is set higher than the gel concentration at the base end of the migration direction.
- SDS-PAGE was performed using NuPAGE 4-12% Bris-Tris Gels IPG well mini 1 mm manufactured by Life Technologies.
- As an electrophoresis instrument XCell SureLock Mini-Cell manufactured by life Technologies was used.
- electrophoresis was performed at 200 V constant voltage for about 45 minutes.
- spots 1 to 3 were those of allergic patients to eggs and diagnosed as food-dependent exercise-induced anaphylaxis. It was revealed that it specifically binds to an IgE antibody (FIG. 3). In addition, in other allergic patients to eggs, it was revealed that the antigens of spots 4-11 specifically bind to the IgE antibodies of the patients (FIGS. 4 to 6).
- the antigen of spot 1 may be any of (1A-a) to (1A-e) and (1B) below.
- (1A-a) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 2.
- (1A-b) 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 with the amino acid sequence represented by SEQ ID NO: 2 , A protein comprising 99% or more amino acid sequence.
- (1A-d) 70% or more identity with the base sequence represented by SEQ ID NO: 1, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence.
- (1A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1.
- a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences
- amino acid sequence represented by any of SEQ ID NOs: 3 to 11 one or several amino acids may be deleted, substituted, inserted or added.
- the proteins (1A-a) to (1A-e) and (1B) have a molecular weight of 20 kDa to 40 kDa in a gel subjected to two-dimensional electrophoresis under the conditions described in the above-mentioned “Identification of antigen”, Preferably, the protein may appear as a spot having a molecular weight of 30 kDa to 35 kDa and an isoelectric point of 4.0 to 10.0, preferably an isoelectric point of 4.0 to 9.0.
- the mass data obtained from the mass spectrometer (SEQ ID NOs: 9 to 11, 14, and 16) was analyzed by collating with NCBI protein data.
- the C-terminal of vitellogenin-3 (Vitellogenin-3) was analyzed. It was identified to be a portion (amino acid sequence: SEQ ID NO: 13, base sequence encoding it: SEQ ID NO: 12).
- the antigen of spot 2 may be any of (2A-a) to (2A-e) and (2B) below.
- (2A-a) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 13.
- (2A-b) 70% or more identity with the amino acid sequence represented by SEQ ID NO: 13, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising 99% or more amino acid sequence.
- (2A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 12.
- (2A-d) 70% or more of identity with the base sequence represented by SEQ ID NO: 12, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence.
- (2A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 12.
- a protein containing a sequence More preferably, it comprises a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9-11, 13, and 16, preferably comprising at least 2, 3, 4 or all of the amino acid sequences. protein.
- the amino acid sequence represented by any of SEQ ID NOs: 9 to 11, 13, 14, and 16 one or several amino acids may be deleted, substituted, inserted, or added.
- the proteins (2A-a) to (2A-e) and (2B) are those having a molecular weight of 30 kDa to 60 kDa in gels subjected to two-dimensional electrophoresis under the conditions described in the above-mentioned “Identification of antigen”, Preferably, the protein may appear as a spot having a molecular weight of about 35 kDa to 40 kDa and an isoelectric point of 5.0 to 10.0, preferably an isoelectric point of 6.0 to 9.0.
- the mass data obtained from the mass spectrometer (SEQ ID NOs: 19 to 45) were analyzed by collating with UniProt protein data.
- the central portion of Vitellogenin-1 or the protein of the protein was analyzed. It was identified to be the C-terminal part (amino acid sequence: SEQ ID NO: 18, the base sequence encoding it: SEQ ID NO: 17) of lipoviterin-1 produced by cleavage at the N-terminal side.
- the antigen of spot 3 may be any one of (3A-a) to (3A-e) and (3B) below.
- (3A-a) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 18.
- (3A-b) 70% or more identity with the amino acid sequence represented by SEQ ID NO: 18, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising 99% or more amino acid sequence.
- (3A-d) 70% or more identity with the base sequence represented by SEQ ID NO: 17, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence.
- (3A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 17.
- a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 45, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, A protein comprising 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or all sequences. More preferably, a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 22-31, 33-35, 37, 39, 40, 42, 43 and 45, preferably the amino acid 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 sequences of the sequence.
- amino acid sequence represented by any of SEQ ID NOs: 18 to 45 one or several amino acids may be deleted, substituted, inserted or added.
- the proteins (3A-a) to (3A-e) and (3B) have molecular weights of 35 kDa to 60 kDa in gels subjected to two-dimensional electrophoresis under the conditions described in the above-mentioned “Identification of antigen”, It may be a protein that appears as a spot having a molecular weight of about 40 kDa to 50 kDa and an isoelectric point of 5.0 to 10.0, preferably an isoelectric point of 6.0 to 9.0.
- the mass data (SEQ ID NOs: 48 to 57) obtained from the mass spectrometer was analyzed against the NCBI protein data, and vitellogenin-3 (amino acid sequence: SEQ ID NO: 47) was analyzed. And the nucleotide sequence encoding it: SEQ ID NO: 46).
- the antigen of spot 4 may be any of the following (4A-a) to (4A-e) and (4B).
- (4A-d) 70% or more identity with the base sequence represented by SEQ ID NO: 46, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence.
- (4A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 46 under stringent conditions.
- a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 47 to 57, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10 of the amino acid sequence Or a protein containing all sequences. More preferably, a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 50, 51, 53, and 54, preferably at least 2, 3, 4 or all of the amino acid sequences. Containing protein.
- the amino acid sequence represented by any of SEQ ID NOs: 47 to 57 one or several amino acids may be deleted, substituted, inserted or added.
- the proteins (4A-a) to (4A-e) and (4B) have a molecular weight of 20 to 50 kDa in a gel subjected to two-dimensional electrophoresis under the conditions described in the above-mentioned section “Identification of antigen”. , Preferably 25 to 40 kDa, more preferably around 30 to 35 kDa, and spot having an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, more preferably 4.0 to 6.0
- the antigens of spots 5 and 6 may be any of the following (5A-a) to (5A-e) and (5B).
- (5A-c) a protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 58.
- (5A-d) 70% or more identity with the base sequence represented by SEQ ID NO: 58, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence.
- (5A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 58.
- a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 59 to 104, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
- a protein comprising 15, 20, 25, 30, 35, 40, 45 or all sequences. More preferably, it is 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.
- a protein comprising at least one of the amino acid sequences, preferably comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or all of the amino acid sequences. protein.
- amino acid sequence represented by any of SEQ ID NOs: 59 to 104 one or several amino acids may be deleted, substituted, inserted or added.
- the proteins (5A-a) to (5A-e) and (5B) have a molecular weight of 80 to 260 kDa in a gel subjected to two-dimensional electrophoresis under the conditions described in the item “Identification of antigen”. , Preferably 90 to 200 kDa, more preferably around 110 to 160 kDa, and spot having an isoelectric point of 1.0 to 8.0, preferably 2.0 to 7.0, more preferably 3.0 to 6.0
- the antigen of spot 7 may be any of (6A-a) to (6A-e) and (6B) below.
- (6A-a) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 106.
- (6A-b) 70% or more identity with the amino acid sequence represented by SEQ ID NO: 106, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising 99% or more amino acid sequence.
- (6A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 105.
- (6A-d) 70% or more identity with the base sequence represented by SEQ ID NO: 105, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence.
- (6A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 105.
- amino acid sequence represented by any of SEQ ID NOs: 106 to 150 one or several amino acids may be deleted, substituted, inserted or added.
- the proteins (6A-a) to (6A-e) and (6B) have a molecular weight of 110 to 300 kDa in a gel subjected to two-dimensional electrophoresis under the conditions described in the above-mentioned section “Identification of antigen”. , Preferably 130 to 280 kDa, more preferably around 160 to 260 kDa, and spot having an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, more preferably 4.0 to 6.0
- apolipoproteinsB precursor amino acid sequence: SEQ ID NO: 152, the base sequence encoding it: SEQ ID NO: 151).
- the antigen of spot 8 may be any of (7A-a) to (7A-e) and (7B) below.
- (7A-a) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 152.
- (7A-b) 70% or more identity with the amino acid sequence represented by SEQ ID NO: 152, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising 99% or more amino acid sequence.
- (7A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 151.
- (7A-d) 70% or more identity with the base sequence represented by SEQ ID NO: 151, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence.
- (7A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 151.
- a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 152 to 235, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
- a protein comprising 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 83 species or all sequences.
- amino acid sequence represented by any of SEQ ID NOs: 152 to 235 one or several amino acids may be deleted, substituted, inserted or added.
- the proteins (7A-a) to (7A-e) and (7B) have a molecular weight of 160 to 550 kDa in a gel subjected to two-dimensional electrophoresis under the conditions described in the item “Identification of antigen”. , Preferably 180 to 400 kDa, more preferably around 200 to 300 kDa, and spot having an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, more preferably 4.0 to 6.0
- the spot 9 was analyzed by comparing the mass data obtained from the mass spectrometer (SEQ ID NO: 238 to 249) with the protein data of NCBI.
- the apolipoprotein ⁇ ⁇ B precursor amino acid sequence: SEQ ID NO: 237, the base sequence encoding it: SEQ ID NO: 236).
- the antigen of spot 9 may be any of (8A-a) to (8A-e) and (8B) below.
- (8A-a) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 237.
- (8A-b) 70% or more of identity with the amino acid sequence represented by SEQ ID NO: 237, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising 99% or more amino acid sequence.
- (8A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 236.
- (8A-d) 70% or more of identity with the base sequence represented by SEQ ID NO: 236, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence.
- (8A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 236.
- a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 237 to 249, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, A protein comprising 11, 12 or all sequences. More preferably, a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 237 to 239, 241, 242, 244, and 246 to 249, preferably at least 2, 3, 4, A protein comprising 5, 6, 7, 8, 9 or all sequences.
- amino acid sequence represented by any of SEQ ID NOs: 237 to 249 one or several amino acids may be deleted, substituted, inserted or added.
- the proteins (8A-a) to (8A-e) and (8B) have a molecular weight of 20 to 50 kDa in a gel when two-dimensional electrophoresis is performed under the conditions described in the above-mentioned section “Identification of antigen”. , Preferably 25 to 45 kDa, more preferably around 30 to 40 kDa, and isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, more preferably 9.0 to 10.0.
- the spot 10 was analyzed by comparing the mass data obtained from the mass spectrometer (SEQ ID NO: 252 to 260) with the protein data of NCBI, and as a result, apolipoprotein B precursor (amino acid sequence: SEQ ID NO: 251 and the base sequence encoding it: SEQ ID NO: 250).
- the antigen of the spot 10 may be any of the following (9A-a) to (9A-e) and (9B).
- (9A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 250. (9A-d) 70% or more identity with the base sequence represented by SEQ ID NO: 250, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence. (9A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 250.
- a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 251 to 260, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences
- one or several amino acids may be deleted, substituted, inserted or added.
- the proteins (9A-a) to (9A-e) and (9B) have a molecular weight of 20 to 50 kDa in a gel subjected to two-dimensional electrophoresis under the conditions described in the above-mentioned section “Identification of antigen”. , Preferably 25 to 45 kDa, more preferably around 30 to 40 kDa, and isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, more preferably 9.0 to 10.0.
- the spot 11 was analyzed by comparing the mass data (SEQ ID NO: 263 to 271) obtained from the mass spectrometer with the protein data of NCBI.
- vitellogenin-2 precursor amino acid sequence: sequence
- No. 262 the base sequence encoding it: SEQ ID NO: 261.
- the antigen of spot 11 may be any of (10A-a) to (10A-e) and (10B) below.
- (10A-a) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NO: 262.
- (10A-b) 70% or more identity with the amino acid sequence represented by SEQ ID NO: 262, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising 99% or more amino acid sequence.
- (10A-c) A protein comprising an amino acid sequence encoded by a base sequence in which one or several nucleotides are deleted, substituted, inserted or added in SEQ ID NO: 261.
- (10A-d) 70% or more of identity with the base sequence represented by SEQ ID NO: 261, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more , A protein comprising an amino acid sequence encoded by 99% or more of the base sequence.
- (10A-e) A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 261.
- a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 262 to 271; preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or all of the amino acid sequences
- the amino acid sequence represented by any of SEQ ID NOs: 262 to 271 one or several amino acids may be deleted, substituted, inserted or added.
- the proteins of the above (10A-a) to (10A-e) and (10B) have a molecular weight of 15 to 40 kDa in a gel when subjected to two-dimensional electrophoresis under the conditions described in the above-mentioned section “Identification of antigen”. , Preferably 17 to 35 kDa, more preferably around 20 to 30 kDa, and spot having an isoelectric point of 6.0 to 11.0, preferably 7.0 to 10.0, more preferably 8.0 to 10.0
- Proteins that are antigens (1) to (3) and (4) to (10) above are modified by phosphorylation, sugar chain modification, aminoacylation, ring opening, deamination, etc. Proteins that have been subjected to are also included.
- the protein as the antigen (1) to (3) or (4) to (10) is an allergic antigen to eggs.
- amino acids when “one or several amino acids are deleted, substituted, inserted or added” in the amino acid sequence, one or several amino acids (for example, amino acid sequence) in the target amino acid sequence 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1% amino acids) of the total length of Or an amino acid sequence in which other amino acids are inserted and / or other amino acids are added.
- substitution is preferably a conservative substitution.
- a conservative substitution is the replacement of a particular amino acid residue with a residue having similar physicochemical characteristics, but any substitution that does not substantially change the structural characteristics of the original sequence. For example, any substitution may be made so long as the substituted amino acid does not destroy the helix present in the original sequence or other types of secondary structures characterizing the original sequence.
- conservative substitution of amino acid residues is exemplified for each substitutable residue, but the substitutable amino acid residues are not limited to those described below.
- Group A leucine, isoleucine, valine, alanine, methionine
- B group aspartic acid
- glutamic acid C group asparagine
- glutamine D group lysine
- Group E serine
- Group F phenylalanine, tyrosine
- one member of the above types can be exchanged for another type of member.
- the amino acids of the above groups B, D, and E may be substituted with amino acids of other groups.
- cysteines may be deleted or substituted with other amino acids to prevent folding in the protein with tertiary structure.
- the hydropathic index of amino acids (J.P.), which is a measure of hydrophobicity / hydrophilicity for amino acids, so that the hydrophilic / hydrophobic balance is maintained or the hydrophilicity is increased to facilitate synthesis.
- J.P. hydropathic index of amino acids
- the percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation.
- the percent identity can also be determined using a computer program. Examples of such computer programs include BLAST and ClustalW. In particular, various conditions (parameters) for identity search by the BLAST program are described in Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997). The National Center for Biotechnology Information (NCBI) ) And DNA Data Bank of Japan (DDBJ) website (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, MD20894; Altschul et al.). It can also be determined by using programs such as genetic information processing software GENETYX Ver.7 (Genetics), DNASIS Pro (Hitachi Software), Vector NTI (Infomax).
- nucleotides when “one or several nucleotides are deleted, substituted, inserted or added” in the base sequence, one or several nucleotides (for example, base sequence) in the target base sequence 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1% amino acids) are deleted or substituted with other nucleotides Or a nucleotide sequence in which other nucleotides are inserted and / or other nucleotides are added. It is preferable that no frame shift occurs in the sequence encoding the amino acid due to the deletion, substitution, insertion or addition of the nucleotide.
- the percent identity of two base sequences can be determined by visual inspection and mathematical calculation.
- the percent identity can also be determined using a computer program.
- sequence comparison computer program examples include the BLASTN program (Altschul et al. (1990) available from the website of the National Library of Medicine: https://blast.ncbi.nlm.nih.gov/Blast.cgi. ) J. Mol. Biol. 215: 403-10 can be used and can be determined using default parameters.
- under stringent conditions means to hybridize under moderately or highly stringent conditions.
- moderately stringent conditions can be easily determined by those skilled in the art having general techniques based on, for example, the length of DNA. The basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001.
- moderately stringent conditions are as hybridization conditions: 1 ⁇ SSC to 6 ⁇ SSC, 42 ° C. to 55 ° C., more preferably 1 ⁇ SSC to 3 ⁇ SSC, 45 ° C. to 50 ° C.
- the most preferable conditions are 2 ⁇ SSC and 50 ° C.
- the hybridization solution contains, for example, about 50% formamide
- a temperature 5 to 15 ° C. lower than the above temperature is adopted.
- Cleaning conditions include 0.5 ⁇ SSC to 6 ⁇ SSC, 40 ° C. to 60 ° C.
- 0.05% to 0.2%, preferably about 0.1% SDS may generally be added.
- Highly stringent conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA.
- highly stringent conditions include hybridization and / or washing at higher temperatures and / or lower salt concentrations than moderately stringent conditions.
- the hybridization conditions are 0.1 ⁇ SSC to 2 ⁇ SSC, 55 ° C.
- Washing conditions include 0.2 ⁇ SSC to 2 ⁇ SSC, 50 ° C. to 68 ° C., more preferably 0.2 ⁇ SSC, 60 to 65 ° C.
- the antigen may be obtained from an egg (preferably a chicken egg) by separating and purifying it by combining protein purification methods well known to those skilled in the art.
- the antigen may be obtained by expressing the antigen as a recombinant protein by a gene recombination technique well known to those skilled in the art, and separating and purifying it by a protein purification method well known to those skilled in the art.
- Protein purification methods include, for example, methods using solubility such as salting out, solvent precipitation, methods using molecular weight differences such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE, ion exchange chromatography and hydroxylation. Methods that use charge such as apatite chromatography, methods that use specific affinity such as affinity chromatography, methods that use the difference in hydrophobicity such as reversed-phase high-performance liquid chromatography, isoelectric focusing, etc. For example, a method using the difference in electric points can be used.
- an expression vector containing a nucleic acid encoding an antigen is prepared, the expression vector is introduced into an appropriate host cell by gene transfer or transformation, and the host cell is transformed into a recombinant protein. Culturing under conditions suitable for expression and recovering the recombinant protein expressed in the host cell.
- a “vector” is a nucleic acid that can be used to introduce a nucleic acid linked thereto into a host cell, and an “expression vector” can direct the expression of a protein encoded by the nucleic acid introduced by the vector. It is a vector. Vectors include plasmid vectors, viral vectors and the like. One skilled in the art can select an appropriate expression vector for expression of the recombinant protein depending on the type of host cell used.
- a “host cell” is a cell that is subjected to gene transfer or transformation with a vector.
- the host cell can be appropriately selected by those skilled in the art depending on the vector to be used.
- the host cell can be derived from a prokaryote such as, for example, E. coli.
- the antigen of the present invention may contain an N-terminal methionine residue to facilitate expression of the recombinant protein in the prokaryotic cell. This N-terminal methionine can also be cleaved from the recombinant protein after expression.
- cells or silkworms derived from eukaryotes such as unicellular eukaryotes such as yeast, plant cells, animal cells (eg, human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells).
- eukaryotes such as yeast, plant cells, animal cells (eg, human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells).
- Gene transfer or transformation of an expression vector into a host cell can be appropriately performed by a method known to those skilled in the art.
- those skilled in the art can express the recombinant protein by appropriately selecting conditions suitable for the expression of the recombinant protein according to the type of the host cell and culturing the host cell.
- the host cell which expressed the recombinant protein is homogenized,
- the antigen expressed as a recombinant protein can be isolate
- the present invention is a method for providing an index for diagnosing egg allergy in a subject, and comprises 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 binding between the IgE antibody and the antigen in a sample obtained from the subject; (Iii) if binding of the subject IgE antibody to the antigen is detected, an indication is provided that the subject is egg allergic; Wherein the antigen is at least one of the proteins specified as the antigens (1) to (3) and (4) to (10) above.
- the sample obtained from the subject is a solution containing IgE antibody collected from the subject.
- solutions include, for example, blood, saliva, sputum, runny nose, urine, sweat, tears.
- the sample obtained from the subject may be subjected to a pretreatment for increasing the IgE antibody concentration in the sample before contacting with the antigen.
- Sample pretreatment may include, for example, obtaining serum or plasma from blood.
- the Fab portion that is a binding portion with an antigen may be purified.
- the step (i) is performed by contacting an antigen with IgE antibody in serum obtained from the subject.
- the IgE antibody may be an IgE antibody itself or a mast cell to which the IgE antibody is bound.
- the contact between the sample obtained from the subject and the antigen and the detection of the binding can be performed by a known method.
- a known method for example, detection by ELISA (Enzyme-Linked Immunosorvent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, or immunochromatography can be used.
- the target IgE antibody is brought into contact with and bound to the antigen, and an enzyme-labeled secondary antibody is allowed to act on the IgE antibody specifically bound to the antigen, whereby the enzyme substrate (usually color development or luminescence).
- This is a technique for detecting the binding between an antigen and a target IgE antibody by adding a reagent) and detecting the product of the enzyme reaction.
- a fluorescently labeled secondary antibody it is a method for detecting a fluorescently labeled secondary antibody.
- detection by a measuring method capable of evaluating the binding between an antigen and an IgE antibody, such as surface plasmon resonance (SPR), can also be used.
- SPR surface plasmon resonance
- Plural types of antigen-specific IgE antibodies may be mixed.
- the antigen may be in a state where the isolated antigen is immobilized on a carrier.
- ELISA sandwich immunoassay
- immunochromatography immunochromatography
- surface plasmon resonance etc.
- an antigen is immobilized on the sample obtained from the subject. This is done by contacting the surface.
- An isolated antigen may be obtained by separating and purifying from an egg (preferably a chicken egg) by a combination of protein purification methods well known to those skilled in the art, or by preparing by genetic recombination techniques. Alternatively, the antibody may be fixed.
- the antigen may not be fixed to the carrier.
- flow cytometry or the like can be used in the above steps (i) and (ii), and the presence of the antigen bound by the antibody can be confirmed by laser light.
- BAT basophil activation test
- HRT histamine release test
- the antigen may be transferred from a state separated by two-dimensional electrophoresis and detected by immunoblotting.
- Two-dimensional electrophoresis is a technique for separating protein samples by performing isoelectric focusing in the first dimension and SDS-PAGE (SDS-polyacrylamide gel electrophoresis) in the second dimension.
- the conditions for two-dimensional electrophoresis are not particularly limited as long as the antigens of the present invention can be separated.
- the two-dimensional electrophoresis conditions described in the item “Identification of antigen” can be used.
- electrophoresis conditions can be determined with reference to the descriptions in Patent Documents 1 to 4 described above, for example:
- (A) As a first-dimension isoelectric focusing gel, the gel length is in the range of 5 to 10 cm, the pH range of the gel is 3 to 10, and the gel pH gradient with respect to the migration direction is up to pH 5. Satisfying the relationship of “a ⁇ b” and “b> c”, where a is the gel length of b, the gel length of pH 5-7 is b, and the gel length of pH 7 or higher is c;
- (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 0.
- a constant voltage step is performed by applying a constant voltage having a value in the range of 100 V to 600 V for each gel containing a specimen, and the change width of electrophoresis per 30 minutes of electrophoresis is Start the voltage increasing process to increase the voltage from the constant voltage after being in the range of 5 ⁇ A;
- the final voltage in the voltage raising step is within the range of 3000V to 6000V;
- the gel length in the longitudinal direction of the first-dimension isoelectric focusing gel is 5 to 10 cm, and the gel concentration at the proximal end in the migration direction of the second-dimensional electrophoresis gel is 3 to 6%;
- the gel concentration at the distal end portion in the migration direction of the second-dimensional electrophoresis gel is set higher than the gel concentration at the proximal end portion in the migration direction;
- Two-dimensional electrophoresis can be performed
- the antigens (1) to (3) and (4) to (10) above are antigens that specifically bind to IgE antibodies of patients who are allergic to eggs. Therefore, if binding of the subject IgE antibody to the antigen is detected, an indication that the subject is allergic to eggs is provided.
- the present invention also provides an allergy diagnostic kit for eggs containing at least one of the antigens (1) to (3) and (4) to (10).
- the diagnostic kit of the present invention may be used in a method for providing an index for diagnosing the above-mentioned egg allergy or the following diagnostic method.
- the diagnostic kit of the present invention contains at least one of the antigens (1) to (3) and (4) to (10) above, as well as an enzyme-labeled anti-IgE antibody and a chromogenic substrate serving as a substrate for the enzyme, A luminescent substrate may be included. Alternatively, a fluorescently labeled anti-IgE antibody may be used.
- the antigen may be provided in a state immobilized on a carrier.
- the diagnostic kit of the present invention may also be provided together with instructions for a procedure for diagnosis and a package containing the instructions.
- the diagnostic kit includes a companion diagnostic agent for allergy to eggs.
- Companion diagnostic agents are used to identify patients who are expected to benefit from the drug, to identify patients who are at risk of serious side effects of the drug, or to examine the reactivity of the drug to optimize treatment with the drug. It is used for this purpose.
- the optimization of treatment includes, for example, determination of dosage volume, determination of discontinuation of administration, confirmation of which allergen component is used for immune tolerance, and the like.
- the present invention also provides a composition for diagnosing allergy to an egg comprising at least one of the antigens (1) to (3) and (4) to (10).
- the diagnostic composition of the present invention can be used in the following diagnostic methods.
- the diagnostic composition of the present invention may contain a pharmaceutically acceptable carrier or additive generally used with the antigen of the present invention, if necessary.
- the present invention is a method for diagnosing allergy to an egg of a subject, comprising the following steps: (I) contacting a sample obtained from a subject with an antigen; (Ii) detecting binding between the IgE antibody and the antigen in a sample obtained from the subject; (Iii) If binding between the subject IgE antibody and the antigen is detected, it is determined that the subject is allergic to eggs; Wherein the antigen is at least one of the proteins specified as the antigens (1) to (3) and (4) to (10) above.
- steps (i) and (ii) are performed as described for each step of the method for providing an index for diagnosing allergy to eggs.
- the present invention relates to a method for diagnosing allergy to an egg of a subject, comprising administering at least one of the antigens (1) to (3) and (4) to (10) to the subject.
- the method may be performed in the form of a skin test characterized by applying an antigen to the skin.
- the prick test and diagnostic composition were applied to observe the skin reaction by infiltrating the skin with the antigen by making a minute wound so as not to bleed. Scratch test to observe reaction by scratching skin a little, patch test to observe reaction by applying diagnostic composition such as cream or ointment to skin, and observe reaction by administering antigen intradermally Includes forms such as an intradermal test.
- the amount of the antigen applied to the skin may be, for example, a dose of 100 ⁇ g or less per one time.
- the antigen protein used for the load test may be a protein that has been expressed and purified, such as pollen rice in which rice is transformed with a cedar pollen antigen gene and the antigen protein is expressed in rice. It may be expressed in foods / food ingredients.
- the present invention provides at least one of the antigens (1) to (3) and (4) to (10) described above for use in diagnosis of allergy to eggs.
- it also includes providing at least one of the antigens (1) to (3) and (4) to (10) mixed with a known antigen.
- the present invention provides the use of at least one of the antigens (1) to (3) and (4) to (10) above for the manufacture of a diagnostic agent for allergy to eggs.
- composition / Treatment Method The present invention provides a pharmaceutical composition comprising at least one of the antigens (1) to (3) and (4) to (10).
- the pharmaceutical composition is used to treat an allergy to eggs.
- the present invention also relates to an egg comprising administering at least one of the antigens (1) to (3) and (4) to (10) to a patient in need of treatment of an allergy to the egg.
- the present invention provides at least one of the above antigens (1) to (3) and (4) to (10) for use in the treatment of allergy to eggs. In still another aspect, the present invention provides use of at least one of the antigens (1) to (3) and (4) to (10) above for the manufacture of a therapeutic agent for allergy to eggs.
- desensitization therapy is often performed with the goal of inducing immune tolerance by administering an antigen to a patient.
- At least one of the above antigens (1) to (3) and (4) to (10) can be used as an active ingredient for desensitization therapy against allergy to eggs.
- the antigen protein used for the desensitization therapy may be an expression-purified protein.
- a pollen rice obtained by transforming a rice cedar pollen antigen gene into rice and expressing the antigen protein in rice. In this way, it may be expressed in foods / foodstuffs.
- the pharmaceutical composition of the present invention can be administered by a usual administration route.
- Common routes of administration include, for example, oral, sublingual, transdermal, intradermal, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, and rectal administration.
- the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable adjuvant, excipient, or various additives (for example, a stabilizer, a solubilizing agent, milk, etc.) that are generally used together with the antigen of the present invention as necessary.
- a suspending agent, a buffering agent, a preservative, a coloring agent, etc. can be used as a pharmaceutical composition added by a conventional method.
- the dosage form of the pharmaceutical composition can be appropriately selected by those skilled in the art depending on the administration route. For example, it may be in the form of tablets, capsules, troches, sublingual tablets, injections, intranasal sprays, poultices, solutions, creams, lotions, suppositories, and the like.
- the dosage, frequency and / or administration period of the pharmaceutical composition of the present invention can be appropriately selected by a doctor according to the administration route, symptoms, patient characteristics such as age and weight, and the like. For example, in the case of an adult, it may be administered at a dose of 100 ⁇ g or less per dose.
- the dosing interval may be, for example, about once a week, once a week, twice a month, or about once every three months.
- the administration period can be, for example, several weeks to several years. It may be an administration method in which the dosage is increased stepwise within the administration period.
- Tester The present invention provides a tester comprising an antibody against at least one of the antigens (1) to (3) and (4) to (10).
- the antibody can be prepared by a conventional method. For example, it may be prepared by immunizing a mammal such as a rabbit with the antigens (1) to (3) and (4) to (10).
- the antibody may be an IgE antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (eg, Fab, F (ab ′) 2 , Fab ′).
- the antibody may be provided in a form bound to a carrier.
- the carrier is not particularly limited as long as it is a carrier that can be used for detecting the binding between the antibody and the antigen. Any carrier known to those skilled in the art can be utilized.
- Examples of the method for examining the presence or absence of an antigen include the following methods. -A tester containing the prepared IgE antibody is brought into contact with a sample obtained from food, food, etc., and the binding between the IgE antibody and the antigen in the sample is detected using, for example, an ELISA method, and the IgE antibody and the antigen are detected. A method for determining that the antigen remains in the target food / food material, etc. when binding to is detected. -A method of allowing an antibody solution to react so that food or food material is soaked in filter paper and the antigen contained therein is detected.
- a primer having a base sequence complementary to at least a part of the base sequence represented by SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261 A tester for determining the presence or absence of an allergic antigen to the egg in the object.
- the primer may be, for example, the 3 ′ end or the middle of a part of at least one sequence of the base sequence represented by SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261
- the partial sequence preferably has a base sequence complementary to 12 residues, 15 bases, 20 bases and 25 bases. In particular, when targeting mRNA, it has a complementary primer of poly A tail.
- the tester comprising the above primer further comprises a 5 ′ terminal portion of at least one sequence of the base sequence represented by SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261.
- a primer comprising a base sequence consisting of a base sequence of preferably 12 bases, 15 bases, 20 bases and 25 bases may be included.
- the complementary primer For example, using DNA or mRNA obtained from an egg or bird as a template, using the complementary primer, amplifying cDNA by PCR (Polymerase Chain Reaction) including RT-PCR, and the amplified cDNA sequence is represented by SEQ ID NO: By comparing with 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261, the presence or absence of the antigen is determined. Examples of the amplification method using PCR include the RACE method. At this time, when the amplified cDNA is compared with SEQ ID NO: 1, 12, 17, 46, 58, 105, 151, 236, 250 or 261, a point mutation encoding the same amino acid is present or amplified.
- the cDNA has an identity of 70% or more with respect to the amino acid sequence of SEQ ID NO: 2, 13, 18, 47, 59, 106, 152, 237, 251 or 262, preferably 80, 90, 95, 98, 99. If it is% or more, it is determined that the antigen is present.
- the above tester is used to examine the presence or absence of an antigen in a food material (egg or bird) or a target such as in a food production line.
- the tester may be used for quality inspection of a production line and a product before shipment by a manufacturer, or may be used for checking the presence or absence of antigens in the target food / food by the eater.
- the present invention provides an egg, an egg processed product or the egg, wherein at least one of the antigens (1) to (3) and (4) to (10) is removed or reduced. Provide birds that lay or are born from the eggs.
- the method of removing or reducing the antigen of the present invention is not limited in eggs, egg processed products, or birds that lay eggs or were born from such eggs.
- the removal or reduction of the antigen may be performed by any method as long as the antigen of the present invention is removed or reduced.
- an egg from which the antigen of the present invention has been removed or reduced may be prepared using a gene knockout technique to prepare an egg in which the expression of the antigen of the present invention has been knocked out.
- any method known to those skilled in the art can be used as the gene knockout technique.
- Oishi, et al. (Scientific Reports, Vol.6, Article number: 23980, 2016, doi: 10.1038 / srep23980) applied CRISPER / Cas9, a genome editing technology, to chick primordial germ cells, and developed an ovomucoid. It describes obtaining genetically deleted individuals.
- a similar technique may be used to obtain an egg from which the antigen of the present invention has been removed.
- Artificial mating of birds or eggs can be performed by conventional methods.
- the processed egg product from which the antigen of the present invention has been removed or reduced may be a processed product made from an egg from which the antigen of the present invention has been removed or reduced.
- a treatment for removing or reducing the antigen of the present invention is performed before or after preparation of processed egg products.
- a method for removing or reducing the antigen of the present invention in processed egg products using ordinary eggs as raw materials a method for removing protein components in foods and ingredients such as high-pressure treatment and elution with a neutral salt solution, high-temperature steam, etc. And a method of hydrolysis / denaturation / amino acid change (chemical modification / elimination of side chains, etc.) by heat treatment and acid treatment.
- the bird from which the antigen of the present invention has been removed or reduced may be a bird that has hatched and grown eggs from which the antigen of the present invention has been removed or reduced.
- Birds include those from the growth stages of chicks, young birds, young chickens, adult birds and old birds.
- a bird means an animal belonging to birds, preferably a chicken.
- the present invention antigens removing processed egg products, steps to ensure that a method for producing a processed egg products antigen is removed or reduced, an antigen in the production process of the workpiece is removed or reduced Wherein the antigen is at least one of the antigens (1) to (3) and (4) to (10).
- the step of confirming that the antigen has been removed or reduced during the manufacturing process of the processed egg product from which the antigen has been removed or reduced is to confirm the presence or absence of the antigen by the method described in the above “Tester” section. May be performed.
- processed egg product from which the antigen has been removed or reduced may be produced by the method described in the above item “antigen-removed foods”.
- Example 1 Confirmation of protein pattern Proteins contained in eggs were examined using the following two-dimensional electrophoresis method.
- Extraction and purification of proteins contained in egg white and egg yolk were performed as follows. Proteins were extracted from egg white by adding a solubilizing agent, and then water or urea buffer was added to obtain a protein extract.
- the composition of the urea buffer is as follows. 30 mM Tris 2M thiourea 7M urea 4% (w / v) CHAPS: 3-[(3-Colamidopropyl) dimethylammonio] propanesulfonate An appropriate amount of dilute hydrochloric acid distilled water was added to adjust the whole to 100 mL. The pH was 8.5. Thereafter, 25 ⁇ g each of protein weight was mixed to obtain an extract.
- Surfactant buffer (Mammalian Lysis Buffer (MCLI), SIGMA) was added to egg yolk as a solubilizer to extract proteins.
- precipitation was performed twice using a 2D-CleanUP kit (manufactured by GE).
- TCA trichloroacetic acid
- TCA precipitation was recovered.
- acetone was added to the recovered TCA precipitate to perform precipitation, and the precipitate (specimen) obtained by the operation was recovered.
- DeStreak Rehydration Solution a swelling buffer for gel for first-dimensional isoelectric focusing.
- a specimen solution for second-order isoelectric focusing was used.
- the composition of DeStreak Rehydration Solution is as follows. 7M thiourea 2M urea 4% (w / v) CHAPS 0.5% (v / v) IPG buffer; appropriate amount of BPB (bromophenol blue) manufactured by GE
- First-order isoelectric focusing of the first-dimension isoelectric focusing gel The first-dimension isoelectric focusing gel (GE: IPG gel Immobiline Drystrip (pH 3-10NL)) described above
- the sample solution for electrophoresis was immersed in 140 ⁇ l and allowed to permeate overnight at room temperature.
- IPGphor manufactured by GE was used as an electrophoresis apparatus.
- the electrophoresis tray was filled with silicon oil.
- a filter paper moistened with water was provided at both ends of the gel infiltrated with the specimen, and the gel was set on an electrophoresis tray so as to be covered with silicone oil, and an electrode was set with the filter paper sandwiched between the gel and the gel.
- the upper limit of the current value of the isoelectric focusing device is set to 75 ⁇ A per gel, and the voltage program is set to (1) a constant voltage step to 750 Vhr at a constant voltage of 300 V (current for 30 minutes before the end of the step) (2) The voltage was gradually increased to 1000V over 300Vhr), (3) the voltage was gradually increased to 5000V over 4500Vhr, and (4) the total Vhr at 5000V constant voltage thereafter. The first-dimension isoelectric focusing was performed until the value reached 12000.
- the composition of the equilibration buffer containing the alkylating agent is as follows. 100 mM Tris-HCl (pH 8.0) 6M urea 30% (v / v) glycerol 2% (w / v) SDS 2.5% (w / v) iodoacetamide
- Second dimension SDS-PAGE In this example, XCell SureLock Mini-Cell manufactured by life technologies was used as an electrophoresis apparatus. As the gel for the second dimension electrophoresis, NuPAGE 4-12% Bis-Tris Gels manufactured by life technologies was used. In addition, an electrophoresis buffer having the following composition was prepared and used. 50 mM MOPS 50 mM Tris base 0.1% (w / v) SDS 1 mM EDTA
- an agarose solution for adhesion in which 0.5% (w / v) agarose S (manufactured by Nippon Gene) and an appropriate amount of BPB (bromophenol blue) were dissolved in the electrophoresis buffer was used.
- the sealed container to be used was thoroughly washed with 98% (v / v) ethanol in advance.
- Remove the gel for the second dimensional electrophoresis after electrophoresis from the SDS-PAGE instrument, place it in a washed sealed container, and immerse it in an aqueous solution containing 50% (v / v) methanol and 7% (v / v) acetic acid for 30 minutes was performed twice. Thereafter, the aqueous solution was replaced with water and immersed for 10 minutes.
- the 2D gel was immersed in 40 ml of SYPRO Ruby and shaken overnight at room temperature.
- the two-dimensional gel for electrophoresis subjected to the above-described series of treatments was subjected to a fluorescence image scan using Typhoon 9400 (manufactured by GE).
- the results of two-dimensional electrophoresis for proteins contained in egg yolk are shown in FIG. 1 (results for egg white are not shown).
- a molecular weight marker band is seen, and the position of the band indicates a specific molecular weight (KDa).
- Example 2 Antigen confirmation by immunoblot (1) Confirming the antigen by immunoblotting should be carried out by performing the procedures described in Example 1 up to “second-dimensional SDS-PAGE” and then performing the following “transfer to membrane”, “immunoblot” and “analysis”. It went by.
- Transfer to the membrane Transfer to the membrane was performed using the following transfer device and transfer buffer.
- Transcriptor XCell SureLock Mini-Cell and XCell II Blot Module (life technologies)
- Transfer buffer NuPAGE transfer buffer ( ⁇ 20) (manufactured by life technologies) was diluted 20 times with milliQ water and used.
- the protein in the two-dimensional electrophoresis gel was transferred to a membrane (PVDF membrane) according to the following procedure.
- the PVDF membrane was dipped in 100% methanol, then dipped in milliQ water, then transferred to a transfer buffer solution, and the PVDF membrane was hydrophilized.
- Sponge, filter paper, 2nd-dimensional SDS-PAGE finished gel, hydrophilized PVDF membrane, filter paper, sponge were set in this order, and energized for 1 hour at a constant voltage of 30 V in the transfer device.
- Immunoblotting of the immunoblot membrane was performed using the serum of a patient who has an allergy to eggs (patient 1) or the serum of a non-egg allergic subject as the primary antibody.
- Patients with this egg allergy are those diagnosed with food-dependent exercise-induced anaphylaxis (FDEIA) from chicken eggs.
- FDEIA food-dependent exercise-induced anaphylaxis
- the immunoblotting of the membrane was performed according to the following procedure.
- (1) The transferred membrane was shaken in a 5% skim milk / PBST solution (PBS buffer containing 0.1% of the nonionic surfactant Tween 20) at room temperature for 1 hour.
- (2) The primary antibody was allowed to stand at room temperature for 1 hour in a 5% serum / 5% skim milk / PBST solution.
- Anti-human IgE-HRP horsedish peroxidase
- (6) The mixture was allowed to stand for 5 minutes with Pierce Western Blotting Substrate Plus (manufactured by Thermo).
- Immunoblots using serum from egg allergy patients were compared with immunoblots using serum from non-egg allergic subjects as controls.
- the immunoblot using the serum of egg allergic patients for the protein contained in raw egg yolk Fig. 2
- three spots different from the case of using the serum of non-egg allergic subjects and different from the known chicken egg allergen protein was detected.
- the molecular weights and isoelectric points of the three spots are as follows (FIG. 3).
- Spot 1 molecular weight 20-40 kDa, pI 4.0-10.0
- Spot 2 molecular weight 30-60 kDa, pI 5.0-10.0
- Spot 3 molecular weight 35-60 kDa, pI 5.0-10.0
- Example 3 Mass spectrometry and antigen identification (1) The amino acid sequence of the antigen producing the three spots of Example 2 was identified by mass spectrometry.
- Example 4 Antigen confirmation by immunoblot (2) As in Example 2, the antigen was confirmed by immunoblotting using the sera of three other egg allergy patients (patient 2, patient 3, patient 4). In the immunoblot using the serum of egg allergic patients for the proteins contained in raw egg yolk (FIG. 4 for patient 2, FIG. 5 for patient 3 and FIG. 6 for patient 4) A spot different from the known chicken egg allergen protein was detected. Specifically, spots 4 to 8 were detected for patient 2, spots 9 and 10 for patient 3, and spot 11 for patient 4.
- the molecular weights and isoelectric points of the spots 4 to 11 are as follows (FIGS. 4 to 6).
- Spot 4 molecular weight 20-50 kDa, pI 3.0-8.0
- Spots 5 and 6 molecular weight 80-260 kDa, pI 1.0-8.0
- Spot 7 molecular weight 110-300 kDa, pI 3.0-8.0
- Spot 8 molecular weight 160-550 kDa, pI 3.0-8.0
- Spot 9 molecular weight 20-50 kDa, pI 7.0-11.0
- Spot 10 molecular weight 20-50 kDa, pI 7.0-11.0
- Spot 11 molecular weight 15-40 kDa, pI 6.0-11.0
- Example 5 Mass spectrometry and antigen identification (2) The amino acid sequence of the antigen that produced spots 4 to 11 of Example 4 was identified by mass spectrometry in the same manner as in Example 3.
- each spot was identified as the following protein.
- 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: SEQ ID NO: 47, base sequence encoding it) : SEQ ID NO: 46)
- 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: SEQ ID NO: 59, Base sequence encoding it: SEQ ID NO: 58)
- 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: 59, Base sequence encoding it: SEQ ID NO: 58)
- a novel antigen for an allergy to an egg a diagnostic method and a diagnostic kit for an allergy to an egg, a pharmaceutical composition containing the antigen, an egg from which the antigen has been removed or reduced, a processed egg product, or the egg or the egg And a method for producing egg processed products from which the antigen is removed or reduced can be provided.
- the present invention can further provide a tester for determining the presence or absence of an egg antigen in an object.
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Abstract
Description
(1)(1A)ビテロゲニン-3(Vitellogenin-3)のC末端部分を含むタンパク質又はその変異体であって、卵に対するアレルギーの抗原である、以下の(1A-a)~(1A-e)のいずれかのタンパク質:
(1A-a)配列番号2において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(1A-b)配列番号2で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(1A-c)配列番号1において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(1A-d)配列番号1で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(1A-e)配列番号1で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(1B)配列番号2~11からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(2)(2A)ビテロゲニン-3(Vitellogenin-3)のC末端部分を含むタンパク質又はその変異体であって、卵に対するアレルギーの抗原である、以下の(2A-a)~(2A-e)のいずれかのタンパク質:
(2A-a)配列番号13において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(2A-b)配列番号13で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(2A-c)配列番号12において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(2A-d)配列番号12で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(2A-e)配列番号12で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(2B)配列番号9~11、13、14、及び16からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(3)(3A)ビテロゲニン-1(Vitellogenin-1)の中央部分若しくはリポビテリン-1(lipovitelin-1)のC末端部分を含むタンパク質、又はそれらの変異体であって、卵に対するアレルギーの抗原である、以下の(3A-a)~(3A-e)のいずれかのタンパク質:
(3A-a)配列番号18において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(3A-b)配列番号18で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(3A-c)配列番号17において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(3A-d)配列番号17で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(3A-e)配列番号17で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(3B)配列番号18~45からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
を抗原として含む、前記診断キット。
(i)対象から得られた試料を抗原に接触させる、ここで当該試料はIgE抗体が含まれる溶液である;
(ii)対象から得られた試料中のIgE抗体と当該抗原との結合を検出する;
(iii)対象のIgE抗体と当該抗原との結合が検出された場合、対象が卵アレルギーであることの指標が提供される;
を含み、ここで当該抗原は、上記[1]において(1)~(3)として特定されるタンパク質の少なくとも一つである、前記方法。
(4)(4A)ビテロゲニン-3(Vitellogenin-3)を含むタンパク質又はその変異体であって、卵に対するアレルギーの抗原である、以下の(4A-a)~(4A-e)のいずれかのタンパク質:
(4A-a)配列番号47において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(4A-b)配列番号47で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(4A-c)配列番号46において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(4A-d)配列番号46で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(4A-e)配列番号46で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(4B)配列番号47~57からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(5)(5A)ビテロゲニン-2プレカーサー(Vitellogenin-2 precursor)を含むタンパク質又はその変異体であって、卵に対するアレルギーの抗原である、以下の(5A-a)~(5A-e)のいずれかのタンパク質:
(5A-a)配列番号59において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(5A-b)配列番号59で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(5A-c)配列番号58において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(5A-d)配列番号58で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(5A-e)配列番号58で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(5B)配列番号59~104からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(6)(6A)ビテロゲニン-2プレカーサー(Vitellogenin-2 precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(6A-a)~(6A-e)のいずれかのタンパク質:
(6A-a)配列番号106において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(6A-b)配列番号106で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(6A-c)配列番号105において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(6A-d)配列番号105で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(6A-e)配列番号105で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(6B)配列番号106~150からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(7)(7A)アポリポプロテインBプレカーサー(Apolipoprotein B precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(7A-a)~(7A-e)のいずれかのタンパク質:
(7A-a)配列番号152において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(7A-b)配列番号152で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(7A-c)配列番号151において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(7A-d)配列番号151で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(7A-e)配列番号151で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(7B)配列番号152~235からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(8)(8A)アポリポプロテインBプレカーサー(Apolipoprotein B precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(8A-a)~(8A-e)のいずれかのタンパク質:
(8A-a)配列番号237において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(8A-b)配列番号237で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(8A-c)配列番号236において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(8A-d)配列番号236で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(8A-e)配列番号236で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(8B)配列番号237~249からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(9)(9A)アポリポプロテインBプレカーサー(Apolipoprotein B precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(9A-a)~(9A-e)のいずれかのタンパク質:
(9A-a)配列番号251において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(9A-b)配列番号251で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(9A-c)配列番号250において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(9A-d)配列番号250で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(9A-e)配列番号250で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(9B)配列番号251~260からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(10)(10A)ビテロゲニン-2プレカーサー(Vitellogenin-2 precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(10A-a)~(10A-e)のいずれかのタンパク質:
(10A-a)配列番号262において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(10A-b)配列番号262で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(10A-c)配列番号261において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(10A-d)配列番号261で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(10A-e)配列番号261で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(10B)配列番号262~271からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
を抗原として含む、前記診断キット。
(i)対象から得られた試料を抗原に接触させる、ここで当該試料はIgE抗体が含まれる溶液である;
(ii)対象から得られた試料中のIgE抗体と当該抗原との結合を検出する;
(iii)対象のIgE抗体と当該抗原との結合が検出された場合、対象が卵アレルギーであることの指標が提供される;
を含み、ここで当該抗原は、上記[9]において(4)~(10)として特定されるタンパク質の少なくとも一つである、前記方法。
卵に含まれるタンパク質について、上記の手法を利用して卵に対するアレルギーの抗原の特定を行った。具体的には、生の鶏卵を卵黄と卵白とに分け、それぞれに含まれる卵タンパク質を下記の条件で2次元電気泳動に供した。
(1)スポット1の抗原
スポット1について質量分析による配列同定を行った結果、配列番号3~11のアミノ酸配列が検出された。
(1A-a)配列番号2において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(1A-b)配列番号2で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(1A-c)配列番号1において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(1A-d)配列番号1で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(1A-e)配列番号1で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(1B)配列番号2~11からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号2、4、及び9~11からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4種またはすべての配列を含むタンパク質。ここにおいて、配列番号3~11のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
スポット2について質量分析による配列同定を行った結果、配列番号9~11、14、16のアミノ酸配列が検出された。
(2A-a)配列番号13において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(2A-b)配列番号13で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(2A-c)配列番号12において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(2A-d)配列番号12で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(2A-e)配列番号12で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(2B)配列番号9~11、13、14、および16からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号9~11、13、および16からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4種またはすべての配列を含むタンパク質。ここにおいて、配列番号9~11、13、14、16のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
スポット3について質量分析による配列同定を行った結果、配列番号19~45のアミノ酸配列が検出された。
(3A-a)配列番号18において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(3A-b)配列番号18で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(3A-c)配列番号17において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(3A-d)配列番号17で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(3A-e)配列番号17で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(3B)配列番号18~45からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも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種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号18、19、22~31、33~35、37、39、40、42、43および45からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20種またはすべての配列を含むタンパク質。ここにおいて、配列番号18~45のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
スポット4について質量分析による配列同定を行った結果、配列番号48~57のアミノ酸配列が検出された。
(4A-a)配列番号47において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(4A-b)配列番号47で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(4A-c)配列番号46において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(4A-d)配列番号46で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(4A-e)配列番号46で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(4B)配列番号47~57からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9、10種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号47、50、51、53、および54からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4種またはすべての配列を含むタンパク質。ここにおいて、配列番号47~57のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
スポット5および6について質量分析による配列同定を行った結果、配列番号60~104のアミノ酸配列が検出された。
(5A-a)配列番号59において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(5A-b)配列番号59で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(5A-c)配列番号58において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(5A-d)配列番号58で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(5A-e)配列番号58で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(5B)配列番号59~104からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号59、60、63、65、68、69、71、73、75~79、81、83~88、91~94、96~99、および101~103からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9、10、15、20、25、30種またはすべての配列を含むタンパク質。ここにおいて、配列番号59~104のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
スポット7について質量分析による配列同定を行った結果、配列番号107~150のアミノ酸配列が検出された。
(6A-a)配列番号106において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(6A-b)配列番号106で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(6A-c)配列番号105において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(6A-d)配列番号105で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(6A-e)配列番号105で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(6B)配列番号106~150からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、44種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号106、108、110、111、113、114、116、117、120~124、126~131、133、134、136~138、140~143、145~147および149からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9、10、15、20、25、30、31種またはすべての配列を含むタンパク質。ここにおいて、配列番号106~150のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
スポット8について質量分析による配列同定を行った結果、配列番号153~235のアミノ酸配列が検出された。
(7A-a)配列番号152において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(7A-b)配列番号152で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(7A-c)配列番号151において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(7A-d)配列番号151で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(7A-e)配列番号151で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(7B)配列番号152~235からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、83種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号152、154、157~160、163、164、167~172、174、175、177、178、180、182~185、187、189、190、192、194~196、198~200、203~207、209、211、212、214~216、220~229、231および232からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55種またはすべての配列を含むタンパク質。ここにおいて、配列番号152~235のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
スポット9について質量分析による配列同定を行った結果、配列番号238~249のアミノ酸配列が検出された。
(8A-a)配列番号237において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(8A-b)配列番号237で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(8A-c)配列番号236において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(8A-d)配列番号236で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(8A-e)配列番号236で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(8B)配列番号237~249からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9、10、11、12種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号237~239、241、242、244、および246~249からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9種またはすべての配列を含むタンパク質。ここにおいて、配列番号237~249のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
スポット10について質量分析による配列同定を行った結果、配列番号252~260のアミノ酸配列が検出された。
(9A-a)配列番号251において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(9A-b)配列番号251で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(9A-c)配列番号250において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(9A-d)配列番号250で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(9A-e)配列番号250で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(9B)配列番号251~260からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号251~254、256、257および260からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6種またはすべての配列を含むタンパク質。ここにおいて、配列番号251~260のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
スポット11について質量分析による配列同定を行った結果、配列番号263~271のアミノ酸配列が検出された。
(10A-a)配列番号262において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質。
(10A-b)配列番号262で示されるアミノ酸配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上のアミノ酸配列を含むタンパク質。
(10A-c)配列番号261において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(10A-d)配列番号261で示される塩基配列と同一性が70%以上、好ましくは75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上、99%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質。
(10A-e)配列番号261で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質。
(10B)配列番号262~271からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5、6、7、8、9種またはすべての配列を含むタンパク質。さらに好ましくは、配列番号262~265、268および269からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質、好ましくは当該アミノ酸配列の少なくとも2、3、4、5種またはすべての配列を含むタンパク質。ここにおいて、配列番号262~271のいずれかで示されるアミノ酸配列は、1もしくは数個のアミノ酸が欠失、置換、挿入もしくは付加されていてもよい。
A群:ロイシン、イソロイシン、バリン、アラニン、メチオニン
B群:アスパラギン酸、グルタミン酸
C群:アスパラギン、グルタミン
D群:リジン、アルギニン、
E群:セリン、スレオニン
F群:フェニルアラニン、チロシン
非保存的置換の場合は、上記種類のうち、ある1つのメンバーと他の種類のメンバーとを交換することができる。例えば、不用意な糖鎖修飾を排除するために上記のB、D、E群のアミノ酸をそれ以外の群のアミノ酸に置換してもよい。または、3次構造でタンパク質中に折りたたまれることを防ぐためにシステインを欠失させるか、他のアミノ酸に置換してもよい。あるいは、親水性/疎水性のバランスが保たれるように、または合成を容易にするために親水度を上げるように、アミノ酸に関する疎水性/親水性の指標であるアミノ酸のハイドロパシー指数(J. Kyte 及びR. Doolittle, J. Mol. Biol., Vol.157, p.105-132, 1982)を考慮して、アミノ酸を置換してもよい。
本発明は、対象の卵アレルギーを診断するための指標を提供する方法であって、以下の工程:
(i)対象から得られた試料を抗原に接触させる、ここで当該試料はIgE抗体が含まれる溶液である;
(ii)対象から得られた試料中のIgE抗体と当該抗原との結合を検出する;
(iii)対象のIgE抗体と当該抗原との結合が検出された場合、対象が卵アレルギーであることの指標が提供される;
を含み、ここで当該抗原は上記(1)~(3)、(4)~(10)の抗原として特定されるタンパク質の少なくとも一つである、前記方法を提供する。
(A)1次元目の等電点電気泳動ゲルとして、ゲル長が5~10cmの範囲内であって、ゲルのpH範囲が3~10であり、泳動方向に対するゲルのpH勾配が、pH5までのゲル長をa、pH5~7のゲル長をb、pH7以上のゲル長をcとした場合において、「a<b]及び「b>c」の関係を満たす;
(B)(A)の場合であって、ゲルの全長を1とした場合、aが0.15~0.3の範囲内、bが0.4~0.7の範囲内、cが0.15~0.3の範囲内である;
(C)1次元目の等電点電気泳動において、検体を含むゲル1本につき100V~600Vの範囲内の値の定電圧の印加による定電圧工程を行い、泳動30分あたりの泳動変化幅が5μAの範囲内となった後に前記定電圧から電圧を上昇させる電圧上昇行程を始める;
(D)(C)の場合に、電圧上昇工程の最終電圧を3000V~6000Vの範囲内とする;
(E)1次元目の等電点電気泳動ゲルの長手方向のゲル長が5~10cmであって、2次元目電気泳動ゲルの泳動方向基端部のゲル濃度を3~6%とする;および
(F)(E)の場合に、2次元目電気泳動ゲルの泳動方向先端側の部分のゲル濃度が、泳動方向基端部のゲル濃度よりも高く設定する;
からなる群より選択される少なくとも一つを満たす条件で2次元電気泳動を行うことができる。
(i)対象から得られた試料を抗原に接触させる;
(ii)対象から得られた試料中のIgE抗体と当該抗原との結合を検出する;
(iii)対象のIgE抗体と当該抗原との結合が検出された場合、対象が卵に対するアレルギーであると判断する;
ことを含み、ここで当該抗原は上記(1)~(3)、(4)~(10)の抗原として特定されるタンパク質の少なくとも一つである、前記方法を提供する。ここにおいて(i)および(ii)の各工程は、卵に対するアレルギーを診断するための指標を提供する方法の各工程について説明したとおりに行われる。
本発明は、上記(1)~(3)、(4)~(10)の抗原の少なくとも一つを含む医薬組成物を提供する。
本発明は、上記(1)~(3)、(4)~(10)の抗原の少なくとも一つに対する抗体を含むテスターを提供する。
・作製したIgE抗体を含むテスターを食品・食材等から得られた試料に接触させて、例えばELISA法等を用いて当該IgE抗体と試料中の抗原との結合を検出し、当該IgE抗体と抗原との結合が検出された場合に、対象食品・食材等に当該抗原が残留していると判断する方法。
・ろ紙などに食品・食材をしみこませ、そこに含まれる抗原を検出するように、抗体溶液を反応させる方法。
本発明は、上記(1)~(3)、(4)~(10)の抗原の少なくとも一つが除去又は低減されていることを特徴とする卵、卵加工品または当該卵を産む若しくは当該卵から生まれた鳥を提供する。
本発明は、抗原が除去または低減されている卵加工品の製造方法であって、当該加工品の製造過程で抗原が除去又は低減されていることを確認するステップを有する、ここで当該抗原は上記(1)~(3)、(4)~(10)の抗原の少なくとも一つである、前記製造方法を提供する。
下記の二次元電気泳動方法を使用して、卵に含まれるタンパク質を調べた。
生の鶏卵を、卵白と卵黄に分けた。
30mM Tris
2M チオ尿素
7M 尿素
4%(w/v) CHAPS:
3-[(3-コラミドプロピル)ジメチルアンモニオ]プロパンスルホナート
適量の希塩酸
蒸留水を加えて全体を100mLに調整した。pHは8.5であった。
その後、タンパク質重量として25μgずつを混合して抽出液を得た。卵黄に可溶化剤として界面活性剤バッファー(Mammalian Lysis Buffer (MCLI), SIGMA社)を添加して、タンパク質を抽出した。
得られた検体の一部(タンパク質重量として50μg)を、1次元目等電点電気泳動用ゲルの膨潤用緩衝液であるDeStreak Rehydration Solution(GE社製)150μlに溶解し、1次元目等電点電気泳動用の検体溶液(膨潤用検体溶液)とした。DeStreak Rehydration Solutionの組成は以下の通りである。
7M チオ尿素
2M 尿素
4%(w/v) CHAPS
0.5%(v/v) IPGバッファー;GE社製
適量のBPB(ブロモフェノールブルー)
1次元目等電点電気泳動用ゲル(GE社製:IPGゲルImmobiline Drystrip (pH3-10NL))を前記した1次元目等電点電気泳動用の検体溶液(膨張用検体溶液)140μlに浸漬し、一晩室温にて浸透させた。
上記の1次元目の等電点電気泳動を行った後、等電点電気泳動機器からゲルを取り外し、還元剤を含む平衡化緩衝液に当該ゲルを浸漬して、15分・室温にて振とうした。上記還元剤を含む平衡化緩衝液の組成は以下の通りである。
100mM Tris-HCl(pH8.0)
6M 尿素
30%(v/v) グリセロール
2%(w/v) SDS
1%(w/v) DTT
100mM Tris-HCl(pH8.0)
6M 尿素
30%(v/v) グリセロール
2%(w/v) SDS
2.5%(w/v) ヨードアセトアミド
本実施例においては、電気泳動機器としてlife technologies社製のXCell SureLock Mini-Cellを使用した。2次元目泳動用ゲルはlife technologies社製NuPAGE 4-12% Bis-Tris Gelsを使用した。また、以下の組成の泳動用緩衝液を調製し、使用した。
50mM MOPS
50mM Tris塩基
0.1%(w/v) SDS
1mM EDTA
SYPRO Ruby(life technologies社製)を用いてゲルの蛍光染色を行った。
上記一連の処理を施した2次元目泳動用ゲルをTyphoon9400(GE社製)を使用した蛍光イメージのスキャンに供した。卵黄に含まれるタンパク質について、2次元電気泳動の結果を図1に示す(卵白についての結果は示さない)。ゲルの写真の左側には、分子量マーカーのバンドが見られ、バンドの位置が特定の分子量(KDa)を示す。
イムノブロットでの抗原確認は、実施例1に記載した手順を「2次元目のSDS-PAGE」まで行った後、以下の「メンブレンへの転写」「イムノブロット」「解析」の操作を行うことにより行った。
メンブレンへの転写は、以下の転写装置及び転写用緩衝液を用いて行った。
転写装置:XCell SureLock Mini-Cell およびXCell IIブロットモジュール(life technologies社製)
転写用緩衝液: NuPAGEトランスファーバッファー(×20)(life technologies社製)をmilliQ水で20倍希釈して使用した。
(2)スポンジ、ろ紙、2次元目のSDS-PAGEが終わったゲル、親水化処理済みPVDF膜、ろ紙、スポンジの順でセットし、転写装置中、30V定電圧で1時間通電した。
メンブレンのイムノブロットを、1次抗体として卵に対するアレルギーを有する患者(患者1)の血清または非卵アレルギー被験者の血清を用いて行った。この卵アレルギーの患者は、鶏卵による食物依存性運動誘発アナフィラキシー(FDEIA)と診断された患者である。なお、この患者は、プリックテストでは、生卵、ゆで卵、および温泉卵ともに陰性を示し、特異的IgE抗体検査でも、卵白、卵黄、オボムコイドについて陰性を示した。
(1)転写したメンブレンを5%スキムミルク/PBST溶液(非イオン界面活性剤Tween 20を0.1%含むPBSバッファー)中、室温で1時間振とうした。
(2)1次抗体として、5%血清/5%スキムミルク/PBST溶液中、室温で1時間静置した。
(3)PBST溶液で洗浄した(5分×3回)。
(4)2次抗体として抗ヒトIgE-HRP(セイヨウワサビペルオキシダーゼ)を5%スキムミルク/PBST溶液で5000倍に希釈した溶液中、室温で1時間静置した。
(5)PBST溶液で洗浄した(5分×3回)。
(6)Pierce Western Blotting Substrate Plus(Thermo社製)で、5分間静置した。
上記一連の処理を施したメンブレンをTyphoon9500(GE社製)を使用した蛍光イメージのスキャンに供した。
スポット1:分子量20~40kDa、pI4.0~10.0
スポット2:分子量30~60kDa、pI5.0~10.0
スポット3:分子量35~60kDa、pI5.0~10.0
実施例2の3つのスポットを生じる抗原について、質量分析によるアミノ酸配列の同定を行った。
(1)生の鶏卵の卵黄について、実施例1及び2の手順に従ってタンパク質抽出、2次元電気泳動及びメンブレン転写を行い、0.008%Direct blue/40%エタノール・10%酢酸で振とうにより染色した。
(2)その後、40%エタノール・10%酢酸で5分間の処理を3回行って脱色し、水で5分間洗浄後、風乾した。
(3)目的のスポットをきれいなカッター刃で切り取り、遠心チューブに入れた。50μLのメタノールでメンブレン親水処理後、100μLの水で2回洗浄後遠心除去し、20μLの20mM NH4HCO3・50%アセトニトリルを加えた。
(4)1pmol/μLリシルエンドペプチダーゼ(WAKO)1μLを加え、37℃で60分間静置した後、溶液を新しい遠心チューブに回収した。20μLの20mM NH4HCO3・70%アセトニトリルをメンブレンに加え、室温にて10分間浸し、さらに回収した。0.1%ギ酸、4%アセトニトリル10μLで溶解し、チューブに移した。
(5)回収した溶液を減圧乾燥した後、A液(0.1%ギ酸、4%アセトニトリル溶液)15μlで溶解し、質量分析(ESI-TOF6600, AB Sciex社製)を行った。
(6)質量分析計から得られた質量データに基づくタンパク質の同定は、NCBIまたはUniProtをサーチすることで行った。
各スポットについて、質量分析を行ったところ、以下のアミノ酸配列が検出された。
スポット1:配列番号3~11で示されるアミノ酸配列
スポット2:配列番号9~11、14、16で示されるアミノ酸配列
スポット3:配列番号19~45で示されるアミノ酸配列
スポット1:ビテロゲニン-3(Vitellogenin-3)(アミノ酸配列:NCBIアクセッション番号XP_015146355、それをコードする塩基配列:GenBankアクセッション番号XM_015290869.1)のC末端部分(アミノ酸配列:配列番号2、それをコードする塩基配列:配列番号1)
スポット2:ビテロゲニン-3(Vitellogenin-3)(アミノ酸配列:NCBIアクセッション番号XP_015146355、それをコードする塩基配列:GenBankアクセッション番号XM_015290869.1)のC末端部分(アミノ酸配列:配列番号13、それをコードする塩基配列:配列番号12)
スポット3:ビテロゲニン-1(Vitellogenin-1)(アミノ酸配列:UniProtアクセッション番号P87498、それをコードする塩基配列:ENA(EMBL)アクセッション番号 D89547.1)の中央部分または、リポビテリン-1(Lipovitelin-1)のC末端部分(アミノ酸配列:配列番号18、それをコードする塩基配列:配列番号17)
実施例2と同様に、別の卵アレルギー患者3名(患者2、患者3、患者4)の血清を用いてイムノブロットでの抗原確認を行った。生の卵黄に含まれるタンパク質について卵アレルギー患者の血清を用いたイムノブロット(患者2について図4、患者3について図5、患者4について図6)において、非卵アレルギー被験者の血清を用いた場合とは異なり、かつ公知の鶏卵アレルゲンタンパク質とは異なるスポットが検出された。具体的には、患者2についてスポット4~8が、患者3についてスポット9および10が、患者4についてスポット11が検出された。
スポット4:分子量20~50kDa、pI3.0~8.0
スポット5、6:分子量80~260kDa、pI1.0~8.0
スポット7:分子量110~300kDa、pI3.0~8.0
スポット8:分子量160~550kDa、pI3.0~8.0
スポット9:分子量20~50kDa、pI7.0~11.0
スポット10:分子量20~50kDa、pI7.0~11.0
スポット11:分子量15~40kDa、pI6.0~11.0
実施例4のスポット4~11を生じる抗原について、質量分析によるアミノ酸配列の同定を、実施例3と同様に行った。
スポット4:配列番号48~57で示されるアミノ酸配列
スポット5、6:配列番号60~104で示されるアミノ酸配列
スポット7:配列番号107~150で示されるアミノ酸配列
スポット8:配列番号153~235で示されるアミノ酸配列
スポット9:配列番号238~249で示されるアミノ酸配列
スポット10:配列番号252~260で示されるアミノ酸配列
スポット11:配列番号263~271で示されるアミノ酸配列
スポット4:ビテロゲニン-3(Vitellogenin-3)(アミノ酸配列:NCBIアクセッション番号XP_015146355、それをコードする塩基配列:GenBankアクセッション番号XM_015290869.1)(アミノ酸配列:配列番号47、それをコードする塩基配列:配列番号46)
スポット5、6:ビテロゲニン-2 プレカーサー(Vitellogenin-2 precursor)(アミノ酸配列:NCBIアクセッション番号NP_001026447.1、それをコードする塩基配列:GenBankアクセッション番号NM_001031276.1)(アミノ酸配列:配列番号59、それをコードする塩基配列:配列番号58)
スポット7:ビテロゲニン-2 プレカーサー(Vitellogenin-2 precursor)(アミノ酸配列:NCBIアクセッション番号NP_001026447.1、それをコードする塩基配列:GenBankアクセッション番号NM_001031276.1)(アミノ酸配列:配列番号106、それをコードする塩基配列:配列番号105)
スポット8:アポリポプロテイン B プレカーサー(Apolipoprotein B precursor)(アミノ酸配列:NCBIアクセッション番号NP_001038098.1、それをコードする塩基配列:GenBankアクセッション番号NM_001044633.1)(アミノ酸配列:配列番号152、それをコードする塩基配列:配列番号151)
スポット9:アポリポプロテイン B プレカーサー(Apolipoprotein B precursor)(アミノ酸配列:NCBIアクセッション番号NP_001038098.1、それをコードする塩基配列:GenBankアクセッション番号NM_001044633.1)(アミノ酸配列:配列番号237、それをコードする塩基配列:配列番号236)
スポット10:アポリポプロテイン B プレカーサー(Apolipoprotein B precursor)(アミノ酸配列:NCBIアクセッション番号NP_001038098.1、それをコードする塩基配列:GenBankアクセッション番号NM_001044633.1)(アミノ酸配列:配列番号251、それをコードする塩基配列:配列番号250)
スポット11:ビテロゲニン-2 プレカーサー(Vitellogenin-2 precursor)(アミノ酸配列:NCBIアクセッション番号NP_001026447.1、それをコードする塩基配列:GenBankアクセッション番号NM_001031276.1)(アミノ酸配列:配列番号262、それをコードする塩基配列:配列番号261)
Claims (11)
- 卵アレルギーの診断キットであって、以下(1)~(3)のタンパク質の少なくとも一つ:
(1)(1A)ビテロゲニン-3(Vitellogenin-3)のC末端部分を含むタンパク質又はその変異体であって、卵に対するアレルギーの抗原である、以下の(1A-a)~(1A-e)のいずれかのタンパク質:
(1A-a)配列番号2において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(1A-b)配列番号2で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(1A-c)配列番号1において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(1A-d)配列番号1で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(1A-e)配列番号1で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(1B)配列番号2~11からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(2)(2A)ビテロゲニン-3(Vitellogenin-3)のC末端部分を含むタンパク質又はその変異体であって、卵に対するアレルギーの抗原である、以下の(2A-a)~(2A-e)のいずれかのタンパク質:
(2A-a)配列番号13において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(2A-b)配列番号13で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(2A-c)配列番号12において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(2A-d)配列番号12で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(2A-e)配列番号12で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(2B)配列番号9~11、13、14、及び16からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(3)(3A)ビテロゲニン-1(Vitellogenin-1)の中央部分若しくはリポビテリン-1(Lipovitelin-1)のC末端部分を含むタンパク質、又はそれらの変異体であって、卵に対するアレルギーの抗原である、以下の(3A-a)~(3A-e)のいずれかのタンパク質:
(3A-a)配列番号18において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(3A-b)配列番号18で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(3A-c)配列番号17において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(3A-d)配列番号17で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(3A-e)配列番号17で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(3B)配列番号18~45からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
を抗原として含む、前記診断キット。 - 卵アレルギーの診断キットであって、以下(4)~(10)のタンパク質の少なくとも一つ:
(4)(4A)ビテロゲニン-3(Vitellogenin-3)を含むタンパク質又はその変異体であって、卵に対するアレルギーの抗原である、以下の(4A-a)~(4A-e)のいずれかのタンパク質:
(4A-a)配列番号47において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(4A-b)配列番号47で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(4A-c)配列番号46において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(4A-d)配列番号46で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(4A-e)配列番号46で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(4B)配列番号47~57からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(5)(5A)ビテロゲニン-2 プレカーサー(Vitellogenin-2 precursor)を含むタンパク質又はその変異体であって、卵に対するアレルギーの抗原である、以下の(5A-a)~(5A-e)のいずれかのタンパク質:
(5A-a)配列番号59において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(5A-b)配列番号59で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(5A-c)配列番号58において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(5A-d)配列番号58で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(5A-e)配列番号58で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(5B)配列番号59~104からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(6)(6A)ビテロゲニン-2 プレカーサー(Vitellogenin-2 precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(6A-a)~(6A-e)のいずれかのタンパク質:
(6A-a)配列番号106において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(6A-b)配列番号106で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(6A-c)配列番号105において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(6A-d)配列番号105で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(6A-e)配列番号105で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(6B)配列番号106~150からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(7)(7A)アポリポプロテイン B プレカーサー(Apolipoprotein B precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(7A-a)~(7A-e)のいずれかのタンパク質:
(7A-a)配列番号152において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(7A-b)配列番号152で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(7A-c)配列番号151において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(7A-d)配列番号151で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(7A-e)配列番号151で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(7B)配列番号152~235からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(8)(8A)アポリポプロテイン B プレカーサー(Apolipoprotein B precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(8A-a)~(8A-e)のいずれかのタンパク質:
(8A-a)配列番号237において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(8A-b)配列番号237で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(8A-c)配列番号236において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(8A-d)配列番号236で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(8A-e)配列番号236で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(8B)配列番号237~249からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(9)(9A)アポリポプロテイン B プレカーサー(Apolipoprotein B precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(9A-a)~(9A-e)のいずれかのタンパク質:
(9A-a)配列番号251において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(9A-b)配列番号251で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(9A-c)配列番号250において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(9A-d)配列番号250で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(9A-e)配列番号250で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(9B)配列番号251~260からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
(10)(10A)ビテロゲニン-2 プレカーサー(Vitellogenin-2 precursor)又はその変異体であって、卵に対するアレルギーの抗原である、以下の(10A-a)~(10A-e)のいずれかのタンパク質:
(10A-a)配列番号262において1又は数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列を含むタンパク質;
(10A-b)配列番号262で示されるアミノ酸配列と同一性が70%以上のアミノ酸配列を含むタンパク質;
(10A-c)配列番号261において1又は数個のヌクレオチドが欠失、置換、挿入又は付加された塩基配列によりコードされるアミノ酸配列を含むタンパク質;
(10A-d)配列番号261で示される塩基配列と同一性が70%以上の塩基配列によりコードされるアミノ酸配列を含むタンパク質;もしくは
(10A-e)配列番号261で示される塩基配列に対し相補的な塩基配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸によりコードされるアミノ酸配列を含むタンパク質;又は
(10B)配列番号262~271からなる群より選択されるアミノ酸配列の少なくとも一つを含むタンパク質;
を抗原として含む、前記診断キット。 - 卵アレルギーの診断用組成物であって、請求項1において(1)~(3)または請求項2において(4)~(10)として特定されるタンパク質の少なくとも一つを抗原として含む、前記診断用組成物。
- 対象の卵アレルギーを診断するための指標を提供する方法であって、以下の工程:
(i)対象から得られた試料を抗原に接触させる、ここで当該試料はIgE抗体が含まれる溶液である;
(ii)対象から得られた試料中のIgE抗体と当該抗原との結合を検出する;
(iii)対象のIgE抗体と当該抗原との結合が検出された場合、対象が卵アレルギーであることの指標が提供される;
を含み、ここで当該抗原は、請求項1において(1)~(3)または請求項2において(4)~(10)として特定されるタンパク質の少なくとも一つである、前記方法。 - 請求項1において(1)~(3)または請求項2において(4)~(10)として特定されるタンパク質の少なくとも一つを含む医薬組成物。
- 卵アレルギーを治療するための、請求項5に記載の医薬組成物。
- 抗原が除去又は低減されていることを特徴とする卵、卵加工品または当該卵を産む若しくは当該卵から生まれた鳥であって、当該抗原は請求項1において(1)~(3)または請求項2において(4)~(10)として特定されるタンパク質の少なくとも一つである、前記卵、卵加工品または鳥。
- 請求項1において(1)~(3)または請求項2において(4)~(10)として特定されるタンパク質の少なくとも一つと結合する抗体を含むことを特徴とする、対象物における卵抗原の有無を判定するためのテスター。
- 配列番号1、12、17、46、58、105、151、236、250または261で示される塩基配列の少なくとも1つと相補的な塩基配列を有するプライマーを含むことを特徴とする、対象物における卵に対するアレルギーの抗原の有無を判定するためのテスター。
- 抗原が除去または低減されている卵加工品の製造方法であって、当該加工品の製造過程で抗原が除去又は低減されていることを確認するステップを有する、ここで当該抗原は請求項1において(1)~(3)として特定されるタンパク質の少なくとも一つ、または請求項2において(4)~(10)として特定されるタンパク質の少なくとも一つである、前記製造方法。
- 卵由来の抗原であって、請求項1において(1)~(3)として特定されるタンパク質の少なくとも一つ、または請求項2において(4)~(10)として特定されるタンパク質の少なくとも一つであり、そして卵に対するアレルギーの原因となる、前記抗原。
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EP3467499A4 (en) | 2020-02-19 |
KR20220104296A (ko) | 2022-07-26 |
US20230190924A1 (en) | 2023-06-22 |
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JP2019002934A (ja) | 2019-01-10 |
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US20240000924A1 (en) | 2024-01-04 |
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