Patent Application
20230190924
The present invention relates to a novel antigen of an allergy to eggs. The present invention also relates to a kit, a composition, and a method for diagnosing allergy to eggs. The present invention also relates to a pharmaceutical composition comprising such an antigen, egg or processed products of egg in which such an antigen is eliminated or reduced, and bird that delivers such egg or is born from such egg. The present invention further relates to a method for producing a processed product of egg in which such an antigen is eliminated or reduced. The present invention further relates to a tester for determining the presence or absence of an egg antigen in an object of interest.
The present invention also relates to a polypeptide comprising an epitope of an antigen. The present invention also relates to a kit, a composition and a method for diagnosing an allergy, comprising such a polypeptide. The present invention also relates to a pharmaceutical composition comprising such a polypeptide, and a raw material or a processed product in which such a polypeptide is eliminated or reduced. The present invention further relates to a method for producing a processed product in which such a polypeptide is eliminated or reduced. The present invention further relates to a tester for determining the presence or absence of an antigen comprising such a polypeptide in an object of interest.
In sera and tissues of allergic patients, IgE antibodies specific to particular antigens (hereinafter also referred to as allergens) are produced. Physiological consequences caused by interaction between such IgE antibodies and such particular antigens elicit allergic reactions. The antigens refer to food or cooking ingredients, etc. that cause allergic symptoms in a broad sense, and refer to proteins (hereinafter also referred to as allergen components) contained in food or cooking ingredients, etc. to which specific IgE antibodies bind in a narrow sense.
In the process of production of conventional allergy testing agents, antigen reagents are commonly prepared simply by grinding a candidate allergenic food, cooking ingredients or the like (Patent Literature 1). For this reason, the only case where conventional allergy tests have permitted detection of a positive allergic reaction is when a conventional antigen reagent contains an allergen component in an amount exceeding a threshold that allows determination of a positive reaction for binding to an IgE antibody. Thus, diagnosis efficiency is not sufficiently high.
Egg allergens shown in the table below, etc. are currently known (Non Patent Literature 1).
Gallus domesticus
Some allergen components have been suggested for allergen candidate food or cooking ingredients, and have also been commercialized as testing kits. However, while it is necessary to exhaustively identify allergen components in order to enhance the reliability of allergy tests, the patient detection rate by the measurement of such allergenic components is far from sufficient. Identification of novel allergens in eggs is very important not only for increasing the precision of diagnosis, but also for determining targets of therapeutic agents, low allergenic foods, and low allergenic cooking ingredients.
Meanwhile, as for protein separation and purification, in recent years, a method for separating and purifying many different proteins from a small amount of sample has been used, which is more specifically a two-dimensional electrophoresis consisting of isoelectric focusing in the first dimension, followed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in the second dimension. The present applicant has conventionally developed some 2D electrophoresis methods with high separation ability (Patent Literature 2-5).
Allergen-specific IgE antibodies recognize and bind to epitopes that are particular amino acid sequences in allergen components. However, only a slight number of analyses have been made on epitopes as to the allergen components (Non Patent Literature 2), but such analyses are still totally quite rare. Furthermore, any kit for diagnosing an allergy using a polypeptide comprising an epitope has not yet emerged in the market.
The present invention provides novel antigens of an allergy to eggs. The present invention also provides methods and kits for diagnosing allergy to eggs. The present invention also provides a pharmaceutical composition comprising such an antigen, egg or processed products of egg in which such an antigen is eliminated or reduced, and bird that delivers such egg or is born from such egg. The present invention further provides testers for determining the presence or absence of an egg antigen in an object of interest.
The present invention also provides polypeptides comprising an epitope of an antigen. The present invention also provides kits, compositions and methods for diagnosing an allergy including such polypeptides. The present invention also provides pharmaceutical compositions comprising such a polypeptide, and raw materials or processed products in which an antigen comprising such a polypeptide is eliminated or reduced. The present invention further relates to methods for producing a processed product in which such an antigen is eliminated or reduced. The present invention further relates to testers for determining the presence or absence of an antigen comprising such a polypeptide in an object of interest.
In order to solve the aforementioned problems, the present inventors had made intensive studies to identify causative antigens of an allergy to eggs. As a result, the inventors succeeded in identifying novel antigens to which an IgE antibody in the serum of an egg allergic patient specifically binds. The present invention has been completed based on this finding.
Thus, in one embodiment, the present invention can be as defined below.
[1] A kit for diagnosing an allergy to an egg, the kit comprising, as an antigen, at least one of proteins as defined below in any one of (4) to (10):
(4) (4A) a protein comprising vitellogenin-3 or a variant thereof, which is an antigen of an allergy to an egg and is defined below in any of (4A-a) to (4A-e):
(4A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 47;
(4A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 47;
(4A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 46;
(4A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 46; or
(4A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 46; or
(4B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 47-57;
(5) (5A) a protein comprising vitellogenin-2 precursor (hereinafter also referred to as vitellogenin-2) or a variant thereof, which is an antigen of an allergy to an egg and is defined below in any of (5A-a) to (5A-e):
(5A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 59;
(5A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 59;
(5A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 58;
(5A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 58; or
(5A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 58; or
(5B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 59-104;
(6) (6A) vitellogenin-2 precursor (hereinafter also referred to as vitellogenin-2) or a variant thereof, which is an antigen of an allergy to an egg and is defined below in any of (6A-a) to (6A-e):
(6A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 106;
(6A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 106;
(6A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 105;
(6A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 105; or
(6A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 105; or
(6B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 106-150;
(7) (7A) apolipoprotein B precursor (hereinafter also referred to as apolipoprotein B) or a variant thereof, which is an antigen of an allergy to an egg and is defined below in any of (7A-a) to (7A-e):
(7A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 152;
(7A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 152;
(7A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 151;
(7A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 151; or
(7A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 151; or
(7B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 152-235;
(8) (8A) apolipoprotein B precursor (hereinafter also referred to as apolipoprotein B) or a variant thereof, which is an antigen of an allergy to an egg and is defined below in any of (8A-a) to (8A-e):
(8A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 237;
(8A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 237;
(8A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 236;
(8A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 236; or
(8A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 236; or
(8B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 237-249;
(9) (9A) apolipoprotein B precursor (hereinafter also referred to as apolipoprotein B) or a variant thereof, which is an antigen of an allergy to an egg and is defined below in any of (9A-a) to (9A-e):
(9A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 251;
(9A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 251;
(9A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 250;
(9A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 250; or
(9A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 250; or
(9B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 251-260;
(10) (10A) vitellogenin-2 precursor (hereinafter also referred to as vitellogenin-2) or a variant thereof, which is an antigen of an allergy to an egg and is defined below in any of (10A-a) to (10A-e):
(10A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 262;
(10A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 262;
(10A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 261;
(10A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 261; or
(10A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 261; or
(10B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 262-271;
[2] A composition for diagnosing an allergy to eggs, comprising, as an antigen, at least one of proteins as defined above in any of (4) to (10) of [1].
[3] A method for providing an indicator for diagnosing an allergy to eggs in a subject, the method comprising the steps of:
(i) contacting a sample obtained from the subject with an antigen, wherein the sample is a solution comprising an IgE antibody;
(ii) detecting binding between the IgE antibody present in the sample from the subject and the antigen; and
(iii) when the binding between the IgE antibody in the subject and the antigen is detected, an indicator of the fact that the subject is allergic to eggs is provided; wherein the antigen is at least one of proteins as defined above in any of (4) to (10) of [1].
[4] A pharmaceutical composition comprising at least one of proteins as defined above in any of (4) to (10) of [1].
[5] The pharmaceutical composition as set forth in [4], wherein the pharmaceutical composition is intended for the treatment of an allergy to eggs.
[6] Egg or processed products of egg in which an antigen is eliminated or reduced, or bird that delivers the egg or is born from the egg, wherein the antigen is at least one of proteins as defined above in any of (4) to (10) of [1].
[7] A tester for determining the presence or absence of an egg antigen in an object of interest, comprising an antibody that binds to at least one of proteins as defined above in any of (4) to (10) of [1].
[8] A tester for determining the presence or absence of an antigen of an allergy to eggs in an object of interest, comprising a primer having a nucleotide sequence complementary to at least one of the nucleotide sequences of SEQ ID NOs: 46, 58, 105, 151, 236, 250 and 261.
[9] A method for producing a processed product of egg in which an antigen is eliminated or reduced, the method comprising the step of confirming that the antigen is eliminated or reduced, in a production process of the processed product, wherein the antigen is at least one of proteins as defined above in any of (4) to (10) of [1].
[10] An egg-derived antigen which is at least one of proteins as defined above in any of (4) to (10) of [1] and is causative of an allergy to eggs.
The present inventors also succeeded in finding epitopes as to egg-derived antigens including the antigens described above.
Since the epitopes have a relatively short amino acid sequence, the IgE antibodies are capable of binding to different allergen components if the same amino acid sequence is present in the different allergen components. Since different allergen components have a common epitope so that IgE antibodies from allergic patients bind to both of them, the antigens have cross-reactivity. Thus, the epitopes defined in the present invention enable diagnosis or treatment of an allergy including cross-reactivity, and detection of a plurality of allergen components comprising the epitopes, etc.
The present invention has been completed based on this finding. Thus, in another embodiment, the present invention can be as defined below.
[11] A polypeptide specifically binding to an IgE antibody from an allergic patient, the polypeptide being any one of the following:
(1β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 272-295 and 319-525;
(2β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 296-303 and 526-552;
(3β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 304-311 and 553-625; and
(4β) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 312-318 and 626-682.
[12] The polypeptide according to the above [11], wherein the number of amino acid residues is 500 or less.
[13] A kit for diagnosing an allergy, comprising at least one of polypeptides according to the above [11] or [12].
[14] A composition for diagnosing an allergy, the composition comprising at least one of polypeptides according to the above [11] or [12] as an antigen.
[15] A method for providing an indicator for diagnosing an allergy in a subject, the method comprising the steps of:
(i) contacting a sample obtained from the subject with an antigen, wherein the sample is a solution comprising an IgE antibody;
(ii) detecting binding between the IgE antibody present in the sample obtained from the subject and the antigen; and
(iii) when the binding between the IgE antibody in the subject and the antigen is detected, an indicator of the fact that the subject is allergic is provided;
wherein the antigen is at least one of polypeptides according to any one of the above [11] or [12].
[16] A pharmaceutical composition comprising at least one of polypeptides according to the above [11] or [12].
[17] The pharmaceutical composition according to the above [16], wherein the pharmaceutical composition is intended for the treatment of an allergy.
[18] A tester for determining the presence or absence of an antigen in an object of interest, the tester comprising an antibody that binds to at least one of polypeptides according to the above [11] or [12].
[19] A tester for determining the presence or absence of an antigen in an object of interest, the tester comprising any of the following primers:
(a) a primer comprising a portion of the nucleotide sequence of a nucleic acid encoding a polypeptide according to the above [11] or [12], and/or a complementary strand thereof; and
(b) a primer which is a portion of at least one of the nucleotide sequences of SEQ ID NOs: 46, 58, 105, 151, 236, 250 and 261 and/or a primer which is a portion of a sequence complementary to at least one of the nucleotide sequences of SEQ ID NOs: 46, 58, 105, 151, 236, 250 and 261.
[20] A raw material or a processed product in which an antigen is eliminated or reduced, wherein the antigen is at least one of polypeptides according to the above [11] or [12].
[21] A method for producing a processed product in which an antigen is eliminated or reduced, the method comprising the step of confirming that the antigen is eliminated or reduced in a production process of the processed product, wherein the antigen is at least one of polypeptides according to the above [11] or [12].
The present invention can provide novel antigens of an allergy to eggs. Since the novel antigens (allergen components) that trigger an egg allergy were identified according to this invention, this invention can provide highly sensitive methods and kits for diagnosing an allergy to egg, pharmaceutical compositions comprising such an antigen, egg or processed products of egg in which such an antigen is eliminated or reduced, bird that delivers such egg or is born from such egg, a method for producing processed products of egg in which such an antigen is eliminated or reduced, and testers for determining the presence or absence of an egg antigen in an object of interest.
The present invention can provide novel polypeptides comprising an epitope of an antigen. Use of the polypeptide of the present invention enables provision of highly sensitive kits, compositions and methods for diagnosing an allergy, pharmaceutical compositions comprising such a polypeptide, testers for determining the presence or absence of an antigen comprising such a polypeptide in an object of interest, raw materials or processed products in which such a polypeptide is eliminated or reduced, and a method for producing the processed products.
The present invention will be described in detail below, but the present invention is not limited to them.
Unless otherwise defined herein, all scientific and technical terms used in relation to the present invention shall have meanings commonly understood by those skilled in the art.
As referred to herein, the “allergy” refers to the state in which, when a certain antigen enters the body of a living individual sensitized to said antigen, the living individual shows a hypersensitive reaction detrimental to him/her. An allergic reaction can be produced upon contact with an antigen or consumption of the antigen. Here, the contact refers to touch to an object and, particularly, as for the human body, refers to attachment to the skin, the mucosa (eyes, lips, etc.) or the like. The consumption refers to incorporation into the body and refers to incorporation by inhalation, through an oral route, or the like. In general, allergic reactions caused by consumption of foods are particularly referred to as food allergies. In a preferred embodiment, embodiment, the allergy may be a food allergy. In blood and tissues of individuals with many food-allergic diseases, IgE antibodies specific to antigens are produced. IgE antibodies bind to mast cells or basophils. When an antigen specific to such an IgE antibody enters again the body of a patient with an allergic disease, said antigen combines with the IgE antibody bound to mast cells or basophils, and the IgE antibody crosslinks said antigen on the cell surface, resulting in physiological effects of IgE antibody-antigen interaction. Examples of such physiological effects include release of histamine, serotonin, heparin, eosinophil chemotactic factors, leucotrienes, or the like. These released substances provoke an allergic reaction resulting from the combination of an IgE antibody with particular antigens. Specifically, IgE antibodies recognize and bind to epitopes that are particular amino acid sequences in particular antigens. Allergic reactions caused by such antigens occur through the aforementioned pathway.
In the present invention, the allergy of interest is not particularly limited as long as it is an allergy to an allergen comprising an epitope to be used. Such an allergy may include allergies to plants of the family Oleaceae, the family Compositae, the family Poaceae, the family Bromeliaceae, the family Juglandaceae, the family Cucurbitaceae, and the family Leguminosae, allergies to animals of the family Phasianidae and the family Bovidae, allergies to seafood of the family Carangidae, the family Sparidae, the family Salmonidae, and the family Penaeidae. Examples of the plant of the family Oleaceae include olive (scientific name: Olea europaea). Examples of the plant of the family Compositae include artichoke (scientific name: Cynara scolymus). Examples of the plant of the family Poaceae include bread wheat (scientific name: Triticum aestivum) and Tausch's goatgrass (scientific name: Aegilops tauschii). Examples of the plant of the family Bromeliaceae include pineapple (scientific name: Ananas comosus). Examples of the plant of the family Juglandaceae include walnut (scientific name: Juglans regia). Examples of the plant of the family Cucurbitaceae include squash (scientific name: Cucurbita moschata). Examples of the plant of the family Leguminosae include soybean (scientific name: Glycine max). Examples of the animal of the family Phasianidae include Japanese quail (scientific name: Coturnix japonica) and wild turkey (scientific name: Meleagris gallopavo). Examples of the animal of the family Bovidae include cattle (scientific name: Bos taurus). Examples of the seafood of the family Carangidae include greater amberjack (scientific name: Seriola dumerili). Examples of the seafood of the family Sparidae include red seabream (scientific name: Pagrus major). Examples of the seafood of the family Salmonidae include rainbow trout (scientific name: Oncorhynchus mykiss). Examples of the seafood of the family Penaeidae include whiteleg shrimp (scientific name: Litopenaeus vannamei).
As referred to herein, the egg means an egg of a bird, preferably a chicken egg, unless otherwise specified. As referred to herein, an egg of a fish is referred to as “fish egg” and is discriminated from the “egg”, which typically means an egg of a bird.
As referred to herein, the allergy to eggs refers to the state in which an individual has an allergic reaction caused by proteins, etc. present in eggs which act as an antigen. The allergy to eggs can produce an allergic reaction upon contact with an antigen present in eggs or consumption of the antigen. In general, allergic reactions caused by consumption of foods are particularly referred to as food allergies. The allergy to eggs may be a food allergy.
As referred to herein, the “antigen” refers to a substance that provokes an allergic reaction, and is also referred to as an “allergen component”. The antigen is preferably a protein.
As referred to herein, the “protein” refers to a molecule having a structure in which naturally occurring amino acids are joined together by peptide bond. The number of amino acids present in a protein is not particularly limited. As referred to herein, the term “polypeptide” also means a molecule having a structure in which naturally occurring amino acids are joined together by peptide bond. The number of amino acids present in a polypeptide is not particularly limited. The “polypeptide” conceptually includes the “protein”. Also, polypeptides having about 2 to 50 amino acids joined together by peptide bond are in some cases called “peptides”, especially. In the case where amino acids can form different enantiomers, the amino acids are understood to form an L-enantiomer, unless otherwise indicated. The amino acid sequences of proteins, polypeptides, or peptides as used herein are represented by one-letter symbols of amino acids in accordance with standard usage and the notational convention commonly used in the art. The leftward direction represents the amino-terminal direction, and the rightward direction represents the carboxy-terminal direction. In the one-letter symbols of amino acids, X can be any substance having an amino group and a carboxyl group that can bind to amino acids at both ends, and particularly represents that any of 20 types of naturally occurring amino acids are acceptable. The residue of X is an amino acid residue at a site where binding activity against IgE antibodies from allergic patients was maintained even after substitution by alanine in alanine scanning described in Example 6. It is well known to those skilled in the art that even when such a site is substituted by any other amino acids, it is highly probable that this binding activity against IgE antibodies is maintained.
Identification of Antigens
Proteins contained in eggs were analyzed by the aforementioned technique to identify antigens of an allergy to eggs. To be specific, raw chicken eggs were separated into yolk and albumen, and egg proteins contained in each of them were subjected to two-dimensional electrophoresis under the conditions described below.
The electrophoresis in the first dimension was isoelectric focusing, which was performed using isoelectric focusing gels with a gel-strip length of 5 to 10 cm and a gel pH range of 3 to 10. The pH gradient of the gels in the direction of electrophoresis was as follows: when the total gel-strip length is taken as 1, the gel-strip length up to pH 5 is taken as “a”, the gel-strip length from pH 5 to 7 is taken as “b”, and the gel-strip length above pH 7 is taken as “c”, “a” is in the range of 0.15 to 0.3, “b” is in the range of 0.4 to 0.7, and “c” is in the range of 0.15 to 0.3. More specifically, the isoelectric focusing was performed using the IPG gels, Immobiline Drystrip (pH3-10 NL), produced by GE Healthcare Bio-Sciences Corporation (hereinafter abbreviated as “GE”). The electrophoresis system used was IPGphor produced by GE. The maximum current of the electrophoresis system was limited to 75 μA per gel strip. The voltage program adopted to perform the first-dimensional isoelectric focusing was as follows: (1) a constant voltage step was performed at a constant voltage of 300 V until the volt-hours reached 750 Vhr (the current variation width during electrophoresis for 30 minutes before the end of this step was 5 μA); (2) the voltage was increased gradually to 1000 V for 300 Vhr; (3) the voltage was further increased gradually to 5000 V for 4500 Vhr; and then (4) the voltage was held at a constant voltage of 5000 V until the total Vhr reached 12000.
The electrophoresis in the second dimension was SDS-PAGE, which was performed using polyacrylamide gels whose gel concentration at the distal end in the direction of electrophoresis was set to 3 to 6% and whose gel concentration at the proximal end was set to a higher value than that at the distal end. More specifically, the SDS-PAGE was performed using NuPAGE 4-12% Bris-Tris Gels (IPG well, Mini, 1 mm) produced by Life Technologies. The electrophoresis system used was XCell SureLock Mini-Cell produced by Life Technologies. The electrophoresis was run at a constant voltage of 200 V for about 45 minutes using an electrophoresis buffer composed of 50 mM MOPS, 50 mM Tris base, 0.1% (w/v) SDS and 1 mM EDTA.
As a result, antigens in spots 1 to 3 in a two-dimensional electrophoresis gel run under the conditions described above for proteins in chicken egg have been revealed to specifically bind to IgE antibodies from egg-allergic patients diagnosed with food-dependent exercise-induced anaphylaxis (
Antigen
As the result of sequence identification of the antigen in spot 4 by mass spectroscopy, the amino acid sequences of SEQ ID NOs: 48-57 were detected.
Also, the mass spectroscopic data (SEQ ID NOs: 48-57) obtained for spot 4 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, vitellogenin-3 (amino acid sequence: SEQ ID NO: 47, encoding nucleotide sequence: SEQ ID NO: 46) was identified.
Accordingly, the antigen in spot 4 in the present invention can be any of below in (4A-a) to (4A-e), and (4B):
The proteins as defined above in (4A-a) to (4A-e) and (4B) can be proteins that are found in a protein spot with a molecular weight of 20 to 50 kDa, preferably around 25 to 40 kDa, more preferably 30 to 35 kDa, and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, more preferably 4.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
Epitopes of the aforementioned antigens (4A-a) to (4A-e) and (4B) reside in amino acids 14-28 (SEQ ID NO: 296), amino acids 164-178 (SEQ ID NO: 297), amino acids 204-218 (SEQ ID NO: 298), amino acids 214-228 (SEQ ID NO: 299), amino acids 224-238 (SEQ ID NO: 300), amino acids 244-258 (SEQ ID NO: 301), amino acids 254-268 (SEQ ID NO: 302), and amino acids 284-298 (SEQ ID NO: 303) of SEQ ID NO: 47. When the antigen in spot 4 contains a variation in the amino acid sequence of SEQ ID NO: 47, amino acids corresponding to at least one of these epitopes may retain the sequence in SEQ ID NO: 47.
From the viewpoint that binding activity against IgE antibodies such as sensitivity (a degree to which a patient can be diagnosed as being positive) or specificity (a degree to which a healthy subject is not diagnosed as being positive) remains even if the amino acid sequence is varied, the antigen in spot 4 may be the following variant.
In the epitope having the sequence of SEQ ID NO: 296, sites of amino acids other than X in SEQ ID NO: 526 or 527 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 4 contains a variation in the amino acid sequence of SEQ ID NO: 47, the antigen may have an amino acid sequence of SEQ ID NO: 47 in which amino acids 14-28 are SEQ ID NO: 526 or 527.
In the epitope having the sequence of SEQ ID NO: 297, sites of amino acids other than X in SEQ ID NO: 529, 530 or 532 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 4 contains a variation in the amino acid sequence of SEQ ID NO: 47, the antigen may have an amino acid sequence of SEQ ID NO: 47 in which amino acids 164-178 are SEQ ID NO: 529, 530 or 532.
In the epitope having the sequence of SEQ ID NO: 298, sites of amino acids other than X in SEQ ID NO: 536, 537, 545 or 546 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 4 contains a variation in the amino acid sequence of SEQ ID NO: 47, the antigen may have an amino acid sequence of SEQ ID NO: 47 in which amino acids 204-218 are SEQ ID NO: 536, 537, 545 or 546.
In the epitope having the sequence of SEQ ID NO: 299, sites of amino acids other than X in SEQ ID NO: 547, 548 or 549 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 4 contains a variation in the amino acid sequence of SEQ ID NO: 47, the antigen may have an amino acid sequence of SEQ ID NO: 47 in which amino acids 214-228 are SEQ ID NO: 547, 548 or 549.
In the epitope having the sequence of SEQ ID NO: 301, sites of amino acids other than X in SEQ ID NO: 552 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 4 contains a variation in the amino acid sequence of SEQ ID NO: 47, the antigen may have an amino acid sequence of SEQ ID NO: 47 in which amino acids 244-258 are SEQ ID NO: 552.
(5) Antigen in Spots 5 and 6
As the result of sequence identification of the antigen in spots 5 and 6 by mass spectroscopy, the amino acid sequences of SEQ ID NOs: 60-104 were detected.
Also, the mass spectroscopic data (SEQ ID NOs: 60-104) obtained for spots 5 and 6 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, Vitellogenin-2 precursor (amino acid sequence: SEQ ID NO: 59, encoding nucleotide sequence: SEQ ID NO: 58) was identified.
Accordingly, the antigens in spots 5 and 6 in the present invention can be any of below in (5A-a) to (5A-e) and (5B):
The proteins as defined above in (5A-a) to (5A-e) and (5B) can be proteins that are found in a protein spot with a molecular weight of 80 to 260 kDa, preferably around 90 to 200 kDa, more preferably 110 to 160 kDa, and an isoelectric point of 1.0 to 8.0, preferably 2.0 to 7.0, more preferably 3.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
Epitopes of the aforementioned antigens (5A-a) to (5A-e) and (5B) reside in amino acids 71-85 (SEQ ID NO: 272), amino acids 141-155 (SEQ ID NO: 273), amino acids 471-485 (SEQ ID NO: 274), amino acids 521-535 (SEQ ID NO: 275), amino acids 611-625 (SEQ ID NO: 276), amino acids 651-665 (SEQ ID NO: 277), amino acids 661-675 (SEQ ID NO: 278), amino acids 711-725 (SEQ ID NO: 279), amino acids 741-755 (SEQ ID NO: 280), amino acids 751-765 (SEQ ID NO: 281), amino acids 881-895 (SEQ ID NO: 282), amino acids 1011-1025 (SEQ ID NO: 283), amino acids 1071-1085 (SEQ ID NO: 284), and amino acids 1101-1115 (SEQ ID NO: 285) of SEQ ID NO: 59. When the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, amino acids corresponding to at least one of these epitopes may retain the sequence in SEQ ID NO: 59.
From the viewpoint that binding activity against IgE antibodies such as sensitivity or specificity remains even if the amino acid sequence is varied, the antigen in spots 5 and 6 may be the following variant.
In the epitope having the sequence of SEQ ID NO: 272, sites of amino acids other than X in SEQ ID NO: 320 or 321 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 71-85 are SEQ ID NO: 320 or 321.
In the epitope having the sequence of SEQ ID NO: 273, sites of amino acids other than X in SEQ ID NO: 328 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 141-155 are SEQ ID NO: 328.
In the epitope having the sequence of SEQ ID NO: 274, sites of amino acids other than X in SEQ ID NO: 332 or 337 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 471-485 are SEQ ID NO: 332 or 337.
In the epitope having the sequence of SEQ ID NO: 275, sites of amino acids other than X in SEQ ID NO: 341, 348 or 352 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 521-535 are SEQ ID NO: 341, 348 or 352.
In the epitope having the sequence of SEQ ID NO: 276, sites of amino acids other than X in SEQ ID NO: 358 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 611-625 are SEQ ID NO: 358.
In the epitope having the sequence of SEQ ID NO: 277, sites of amino acids other than X in SEQ ID NO: 361, 361, 370 or 371 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 651-665 are SEQ ID NO: 361, 361, 370 or 371.
In the epitope having the sequence of SEQ ID NO: 278, sites of amino acids other than X in SEQ ID NO: 376 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 661-675 are SEQ ID NO: 376.
In the epitope having the sequence of SEQ ID NO: 279, sites of amino acids other than X in SEQ ID NO: 382, 383, 394, 395 or 403 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 711-725 are SEQ ID NO: 382, 383, 394, 395 or 403.
In the epitope having the sequence of SEQ ID NO: 280, sites of amino acids other than X in SEQ ID NO: 408 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 741-755 are SEQ ID NO: 408.
In the epitope having the sequence of SEQ ID NO: 281, sites of amino acids other than X in SEQ ID NO: 415 or 416 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 751-765 are SEQ ID NO: 415 or 416.
In the epitope having the sequence of SEQ ID NO: 282, sites of amino acids other than X in SEQ ID NO: 420, 421, 427 or 428 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 881-895 are SEQ ID NO: 420, 421, 427 or 428.
In the epitope having the sequence of SEQ ID NO: 285, sites of amino acids other than X in SEQ ID NO: 431 or 432 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 5 or 6 contains a variation in the amino acid sequence of SEQ ID NO: 59, the antigen may have an amino acid sequence of SEQ ID NO: 59 in which amino acids 1101-1115 are SEQ ID NO: 431 or 432.
(6) Antigen in Spot 7
As the result of sequence identification of the antigen in spot 7 by mass spectroscopy, the amino acid sequences of SEQ ID NOs: 107-150 were detected.
Also, the mass spectroscopic data (SEQ ID NOs: 107-150) obtained for spot 7 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, Vitellogenin-2 precursor (amino acid sequence: SEQ ID NO: 106, encoding nucleotide sequence: SEQ ID NO: 105) was identified.
Accordingly, the antigen in spot 7 in the present invention can be any of below in (6A-a) to (6A-e), and (6B):
The proteins as defined above in (6A-a) to (6A-e) and (6B) can be proteins that are found in a protein spot with a molecular weight of 110 to 300 kDa, preferably around 130 to 280 kDa, more preferably 160 to 260 kDa, and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, more preferably 4.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
Epitopes of the aforementioned antigens (6A-a)-(6A-e) and (6B) reside in amino acids 71-85 (SEQ ID NO: 272), amino acids 141-155 (SEQ ID NO: 273), amino acids 471-485 (SEQ ID NO: 274), amino acids 521-535 (SEQ ID NO: 275), amino acids 611-625 (SEQ ID NO: 276), amino acids 651-665 (SEQ ID NO: 277), amino acids 661-675 (SEQ ID NO: 278), amino acids 711-725 (SEQ ID NO: 279), amino acids 741-755 (SEQ ID NO: 280), amino acids 751-765 (SEQ ID NO: 281), amino acids 881-895 (SEQ ID NO: 282), amino acids 1011-1025 (SEQ ID NO: 283), amino acids 1071-1085 (SEQ ID NO: 284), amino acids 1101-1115 (SEQ ID NO: 285), amino acids 1207-1221 (SEQ ID NO: 286), amino acids 1337-1351 (SEQ ID NO: 287), amino acids 1417-1431 (SEQ ID NO: 288), amino acids 1537-1551 (SEQ ID NO: 289), amino acids 1587-1601 (SEQ ID NO: 290), amino acids 1627-1641 (SEQ ID NO: 291), amino acids 1687-1701 (SEQ ID NO: 292), amino acids 1727-1741 (SEQ ID NO: 293), amino acids 1757-1771 (SEQ ID NO: 294), and amino acids 1797-1811 (SEQ ID NO: 295) of SEQ ID NO: 106. When the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, amino acids corresponding to at least one of these epitopes may retain the sequence in SEQ ID NO: 106.
From the viewpoint that binding activity against IgE antibodies such as sensitivity or specificity remains even if the amino acid sequence is varied, the antigen in spot 7 may be the following variant.
In the epitope having the sequence of SEQ ID NO: 272, sites of amino acids other than X in SEQ ID NO: 320 or 321 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 71-85 are SEQ ID NO: 320 or 321.
In the epitope having the sequence of SEQ ID NO: 273, sites of amino acids other than X in SEQ ID NO: 328 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 141-155 are SEQ ID NO: 328.
In the epitope having the sequence of SEQ ID NO: 274, sites of amino acids other than X in SEQ ID NO: 332 or 337 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 471-485 are SEQ ID NO: 332 or 337.
In the epitope having the sequence of SEQ ID NO: 275, sites of amino acids other than X in SEQ ID NO: 341, 348 or 352 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 521-535 are SEQ ID NO: 341, 348 or 352.
In the epitope having the sequence of SEQ ID NO: 276, sites of amino acids other than X in SEQ ID NO: 358 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 611-625 are SEQ ID NO: 358.
In the epitope having the sequence of SEQ ID NO: 277, sites of amino acids other than X in SEQ ID NO: 361, 361, 370 or 371 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 651-665 are SEQ ID NO: 361, 361, 370 or 371.
In the epitope having the sequence of SEQ ID NO: 278, sites of amino acids other than X in SEQ ID NO: 376 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 661-675 are SEQ ID NO: 376.
In the epitope having the sequence of SEQ ID NO: 279, sites of amino acids other than X in SEQ ID NO: 382, 383, 394, 395 or 403 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 711-725 are SEQ ID NO: 382, 383, 394, 395 or 403.
In the epitope having the sequence of SEQ ID NO: 280, sites of amino acids other than X in SEQ ID NO: 408 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 741-755 are SEQ ID NO: 408.
In the epitope having the sequence of SEQ ID NO: 281, sites of amino acids other than X in SEQ ID NO: 415 or 416 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 751-765 are SEQ ID NO: 415 or 416.
In the epitope having the sequence of SEQ ID NO: 282, sites of amino acids other than X in SEQ ID NO: 420, 421, 427 or 428 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 881-895 are SEQ ID NO: 420, 421, 427 or 428.
In the epitope having the sequence of SEQ ID NO: 285, sites of amino acids other than X in SEQ ID NO: 431 or 432 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 1101-1115 are SEQ ID NO: 431 or 432.
In the epitope having the sequence of SEQ ID NO: 286, sites of amino acids other than X in SEQ ID NO: 437, 441 or 442 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 1207-1221 are SEQ ID NO: 437, 441 or 442.
In the epitope having the sequence of SEQ ID NO: 287, sites of amino acids other than X in SEQ ID NO: 450 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 1337-1351 are SEQ ID NO: 450.
In the epitope having the sequence of SEQ ID NO: 288, sites of amino acids other than X in SEQ ID NO: 459 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 1417-1431 are SEQ ID NO: 459.
In the epitope having the sequence of SEQ ID NO: 290, sites of amino acids other than X in SEQ ID NO: 467 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 1587-1601 are SEQ ID NO: 467.
In the epitope having the sequence of SEQ ID NO: 291, sites of amino acids other than X in SEQ ID NO: 474, 475, 477, 478, 488 or 489 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 1627-1641 are SEQ ID NO: 474, 475, 477, 478, 488 or 489.
In the epitope having the sequence of SEQ ID NO: 292, sites of amino acids other than X in SEQ ID NO: 496, 501, 502 or 510 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 1687-1701 are SEQ ID NO: 496, 501, 502 or 510.
In the epitope having the sequence of SEQ ID NO: 293, sites of amino acids other than X in SEQ ID NO: 513 or 514 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 1727-1741 are SEQ ID NO: 513 or 514.
In the epitope having the sequence of SEQ ID NO: 294, sites of amino acids other than X in SEQ ID NO: 524 or 525 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 7 contains a variation in the amino acid sequence of SEQ ID NO: 106, the antigen may have an amino acid sequence of SEQ ID NO: 106 in which amino acids 1757-1771 are SEQ ID NO: 524 or 525.
(7) Antigen in Spot 8
As the result of sequence identification of the antigen in spot 8 by mass spectroscopy, the amino acid sequences of SEQ ID NOs: 153-235 were detected.
Also, the mass spectroscopic data (SEQ ID NOs: 153-235) obtained for spot 8 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, Apolipoprotein B precursor (amino acid sequence: SEQ ID NO: 152, encoding nucleotide sequence: SEQ ID NO: 151) was identified.
Accordingly, the antigen in spot 8 in the present invention can be any of the proteins as defined below in (7A-a) to (7A-e) and (7B).
The proteins as defined above in (7A-a) to (7A-e) and (7B) can be proteins that are found in a protein spot with a molecular weight of 160 to 550 kDa, preferably around 180 to 400 kDa, more preferably 200 to 300 kDa, and an isoelectric point of 3.0 to 8.0, preferably 3.5 to 7.0, more preferably 4.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
Epitopes of the aforementioned antigens (7A-a)-(7A-e) and (7B) reside in amino acids 2556-2570 (SEQ ID NO: 304), amino acids 131-145 (SEQ ID NO: 305), amino acids 631-645 (SEQ ID NO: 306), amino acids 681-695 (SEQ ID NO: 307), amino acids 841-855 (SEQ ID NO: 308), amino acids 1131-1145 (SEQ ID NO: 309), amino acids 1201-1215 (SEQ ID NO: 310), and amino acids 1571-1585 (SEQ ID NO: 311) of SEQ ID NO: 152. When the antigen in spot 8 contains a variation in the amino acid sequence of SEQ ID NO: 152, amino acids corresponding to at least one of these epitopes may retain the sequence in SEQ ID NO: 152.
From the viewpoint that binding activity against IgE antibodies such as sensitivity or specificity remains even if the amino acid sequence is varied, the antigen in spot 8 may be the following variant.
In the epitope having the sequence of SEQ ID NO: 304, sites of amino acids other than X in SEQ ID NO: 558 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 8 contains a variation in the amino acid sequence of SEQ ID NO: 152, the antigen may have an amino acid sequence of SEQ ID NO: 152 in which amino acids 2556-2570 are SEQ ID NO: 558.
In the epitope having the sequence of SEQ ID NO: 305, sites of amino acids other than X in SEQ ID NO: 563, 564, 574 or 575 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 8 contains a variation in the amino acid sequence of SEQ ID NO: 152, the antigen may have an amino acid sequence of SEQ ID NO: 152 in which amino acids 131-145 are SEQ ID NO: 563, 564, 574 or 575.
In the epitope having the sequence of SEQ ID NO: 306, sites of amino acids other than X in SEQ ID NO: 582 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 8 contains a variation in the amino acid sequence of SEQ ID NO: 152, the antigen may have an amino acid sequence of SEQ ID NO: 152 in which amino acids 631-645 are SEQ ID NO: 582.
In the epitope having the sequence of SEQ ID NO: 308, sites of amino acids other than X in SEQ ID NO: 590, 596 or 603 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 8 contains a variation in the amino acid sequence of SEQ ID NO: 152, the antigen may have an amino acid sequence of SEQ ID NO: 152 in which amino acids 841-855 are SEQ ID NO: 590, 596 or 603.
In the epitope having the sequence of SEQ ID NO: 309, sites of amino acids other than X in SEQ ID NO: 610, 611 or 612 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 8 contains a variation in the amino acid sequence of SEQ ID NO: 152, the antigen may have an amino acid sequence of SEQ ID NO: 152 in which amino acids 1131-1145 are SEQ ID NO: 610, 611 or 612.
In the epitope having the sequence of SEQ ID NO: 310, sites of amino acids other than X in SEQ ID NO: 620 or 621 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 8 contains a variation in the amino acid sequence of SEQ ID NO: 152, the antigen may have an amino acid sequence of SEQ ID NO: 152 in which amino acids 1201-1215 are SEQ ID NO: 620 or 621.
In the epitope having the sequence of SEQ ID NO: 311, sites of amino acids other than X in SEQ ID NO: 625 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 8 contains a variation in the amino acid sequence of SEQ ID NO: 152, the antigen may have an amino acid sequence of SEQ ID NO: 152 in which amino acids 1571-1585 are SEQ ID NO: 625.
(8) Antigen in Spot 9
As the result of sequence identification of the antigen in spot 9 by mass spectroscopy, the amino acid sequences of SEQ ID NOs: 238-249 were detected.
Also, the mass spectroscopic data (SEQ ID NOs: 238-249) obtained for spot 9 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, Apolipoprotein B precursor (amino acid sequence: SEQ ID NO: 237, encoding nucleotide sequence: SEQ ID NO: 236) was identified.
Accordingly, the antigen in spot 9 in the present invention can be any of the proteins as defined below in (8A-a) to (8A-e) and (8B).
The proteins as defined above in (8A-a) to (8A-e) and (8B) can be proteins that are found in a protein spot with a molecular weight of 20 to 50 kDa, preferably around 25 to 45 kDa, more preferably 30 to 40 kDa, and an isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, more preferably 9.0 to 10.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
Epitopes of the aforementioned antigens (8A-a) to (8A-e) and (8B) reside in amino acids 131 to 145 (SEQ ID NO: 305) of SEQ ID NO: 237. When the antigen in spot 9 contains a variation in the amino acid sequence of SEQ ID NO: 237, amino acids corresponding to at least one of these epitopes may retain the sequence in SEQ ID NO: 237.
From the viewpoint that binding activity against IgE antibodies such as sensitivity or specificity remains even if the amino acid sequence is varied, the antigen in spot 9 may be the following variant.
In the epitope having the sequence of SEQ ID NO: 305, sites of amino acids other than X in SEQ ID NO: 563, 564, 574 or 575 corresponding to the positions of the amino acids at positions 5-13 in SEQ ID NO: 198 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 9 contains a variation in the amino acid sequence of SEQ ID NO: 237, the antigen may have an amino acid sequence of SEQ ID NO: 237 in which amino acids 131-145 are SEQ ID NO: 563, 564, 574 or 575.
(9) Antigen in Spot 10
As the result of sequence identification of the antigen in spot 10 by mass spectroscopy, the amino acid sequences of SEQ ID NOs: 252-260 were detected.
Also, the mass spectroscopic data (SEQ ID NOs: 252-260) obtained for spot 10 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, apolipoprotein B precursor (amino acid sequence: SEQ ID NO: 251, encoding nucleotide sequence: SEQ ID NO: 250) was identified.
Accordingly, the antigen in spot 10 in the present invention can be any of below in (9A-a) to (9A-e), and (9B):
The proteins as defined above in (9A-a) to (9A-e) and (9B) can be proteins that are found in a protein spot with a molecular weight of 20 to 50 kDa, preferably around 25 to 45 kDa, more preferably 30 to 40 kDa, and an isoelectric point of 7.0 to 11.0, preferably 8.0 to 10.0, more preferably 9.0 to 10.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
(10) Antigen in Spot 11
As the result of sequence identification of the antigen in spot 11 by mass spectroscopy, the amino acid sequences of SEQ ID NOs: 263-271 were detected.
Also, the mass spectroscopic data (SEQ ID NOs: 263-271) obtained for spot 11 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, vitellogenin-2 precursor (amino acid sequence: SEQ ID NO: 262, encoding nucleotide sequence: SEQ ID NO: 261) was identified.
Accordingly, the antigen in spot 11 in the present invention can be any of below in (10A-a) to (10A-e), and (10B):
The proteins as defined above in (10A-a) to (10A-e) and (10B) can be proteins that are found in a protein spot with a molecular weight of 15 to 40 kDa, preferably around 17 to 35 kDa, more preferably 20 to 30 kDa, and an isoelectric point of 6.0 to 11.0, preferably 7.0 to 10.0, more preferably 8.0 to 10.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled “Identification of antigens”.
Epitopes of the aforementioned antigens (10A-a) to (10A-e) and (10B) reside in amino acids 41-55 (SEQ ID NO: 288), amino acids 161-175 (SEQ ID NO: 289), and amino acids 211-255 (SEQ ID NO: 290) of SEQ ID NO: 262. When the antigen in spot 11 contains a variation in the amino acid sequence of SEQ ID NO: 262, amino acids corresponding to at least one of these epitopes may retain the sequence in SEQ ID NO: 262.
From the viewpoint that binding activity against IgE antibodies such as sensitivity or specificity remains even if the amino acid sequence is varied, the antigen in spot 11 may be the following variant.
In the epitope having the sequence of SEQ ID NO: 288, sites of amino acids other than X in SEQ ID NO: 459 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 11 contains a variation in the amino acid sequence of SEQ ID NO: 262, the antigen may have an amino acid sequence of SEQ ID NO: 262 in which amino acids 41-55 are SEQ ID NO: 459.
In the epitope having the sequence of SEQ ID NO: 290, sites of amino acids other than X in SEQ ID NO: 467 are important for binding to IgE antibodies from allergic patients. Thus, in another preferred embodiment, embodiment, when the antigen in spot 11 contains a variation in the amino acid sequence of SEQ ID NO: 262, the antigen may have an amino acid sequence of SEQ ID NO: 262 in which amino acids 211-255 are SEQ ID NO: 467.
The proteins that are the aforementioned antigens (4) to (10) include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.
The proteins that are the aforementioned antigens (4) to (10) are preferably antigens of an allergy to egg.
By stating herein “deletion, substitution, insertion or addition of one or several amino acids” in relation to amino acid sequence, it is meant that in an amino acid sequence of interest, one or several amino acids (e.g., 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1%, of amino acids with respect to the total length of the amino acid sequence) are deleted, one or several amino acids are substituted by any other amino acids, any other amino acids are inserted, and/or any other amino acids are added.
Among the aforementioned modifications, substitution is preferably conservative substitution. The “conservative substitution” refers to the substitution of a certain amino acid residue by a different amino acid residue having similar physicochemical characteristics, and can be any type of substitution as long as it does not substantially change the characteristics of the structure of the original sequence for example, any type of substitution is acceptable as long as any substituted amino acids do not disrupt the helical structure of the original sequence or other secondary structures that characterize the original sequence. The following gives examples of separate groups of amino acid residues that are conservatively substitutable with each other, but substitutable amino acid residues are not limited to the examples given below.
In the case of non-conservative substitution, one member belonging to one of the aforementioned groups can be replaced with a member belong to any other group. For example, in order to eliminate the possibility of unwanted sugar-chain modification, amino acids of group B, D or E as listed above may be substituted by those of any other group. Also, cysteines may be deleted or substituted by any other amino acids to prevent them from being folded into a protein in its tertiary structure. Also, in order to maintain the balance between hydrophilicity and hydrophobicity or to increase hydrophilicity for the purpose of facilitating synthesis, any amino acids may be substituted in consideration of the hydropathy scales of amino acids, which are a measure of the hydrophilic and hydrophobic properties of amino acids (J. Kyte and R. Doolittle, J. Mol. Biol., Vol. 157, p. 105-132, 1982).
In another embodiment, embodiment, substitution of the original amino acid by an amino acid with less steric hindrance, for example, substitution of group F by group A, B, C, D or E; or substitution of an amino acid having an electric charge by an amino acid having no electric charge, for example, substitution of group B by group C, may be performed. This may improve binding activity against IgE antibodies.
As referred to herein, the percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity can be determined using a computer program. Examples of such computer programs include BLAST and ClustalW. In particular, various conditions (parameters) for identity searches with the BLAST program are described in Altschul, et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997), and are publicly available on the websites of the National Center for Biotechnology Information (NCBI) and DNA Data Bank of Japan (DDBJ) (Altschul, et al., BLAST Handbook, Altschul, et al., NCB/NLM/NIH Bethesda, Md. 20894). Also, the percent identity can be determined using a genetic information processing software program, such as GENETYX Ver. 7 (Genetyx Corporation), DNASIS Pro (Hitachi Software Engineering Co., Ltd.), or Vector NTI (Infomax Inc.).
By stating herein “deletion, substitution, insertion or addition of one or several nucleotides” in relation to nucleotide sequence, it is meant that in a nucleotide sequence of interest, one or several nucleotides (e.g., 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1%, of amino acid with respect to the total length of the nucleotide sequence, or e.g., 1, 5, 10, 15, 20, 25 or 30 nucleotides) are deleted, one or several nucleotides are substituted by any other nucleotides, any other nucleotides are inserted, and/or any other nucleotides are added. It is preferable that such a nucleotide deletion, substitution, insertion or addition should not give rise to a frame shift in an amino acid coding sequence.
As referred to herein, the percent identity between two nucleotide sequences can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity can be determined using a computer program. The BLASTN program (Altschul et al., (1990) J. Mol. Biol. 215: 403-10) available on the website: https://blast.ncbi.nlm.nih.gov/Blast.cgi of the National Library of Medicine can be used as such a sequence comparison computer program, and the percent identity can be determined using default parameters.
As referred to herein, “under stringent conditions” means that hybridization takes place under moderately or highly stringent conditions. To be specific, the moderately stringent conditions can be easily determined by those having ordinary skill in the art on the basis of, for example, the length of DNA. Basic conditions are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd ed., ch. 6-7, Cold Spring Harbor Laboratory Press, 2001. The moderately stringent conditions include hybridization under the conditions of preferably 1×SSC to 6×SSC at 42° C. to 55° C., more preferably 1×SSC to 3×SSC at 45° C. to 50° C., most preferably 2×SSC at 50° C. In the case of using a hybridization solution containing, for example, about 50% formamide, a temperature around 5 to 15° C. lower than the foregoing should be adopted. Washing is also carried out under the conditions of 0.5×SSC to 6×SSC at 40° C. to 60° C. In the process of hybridization and washing, generally 0.05% to 0.2% SDS, preferably about 0.1% SDS, may be added. Likewise, the highly stringent conditions can be easily determined by those having ordinary skill in the art on the basis of, for example, the length of DNA. Generally, the highly stringent (high stringent) conditions include hybridization and/or washing at a higher temperature and/or a lower salt concentration than those adopted under the moderately stringent conditions. For example, hybridization is carried out under the conditions of 0.1×SSC to 2×SSC at 55° C. to 65° C., more preferably 0.1×SSC to 1×SSC at 60° C. to 65° C., most preferably 0.2×SSC at 63° C. Washing is carried out under the conditions of 0.2×SSC to 2×SSC at 50° C. to 68° C., more preferably 0.2×SSC at 60 to 65° C.
Antigens may be obtained by separating and purifying them from eggs (preferably chicken eggs) using a combination of protein purification methods well known to those skilled in the art. Also, antigens may be obtained by expressing them as recombinant proteins using a genetic recombination technique well known to those skilled in the art and by separating and purifying them using protein purification methods well known to those skilled in the art.
Exemplary protein purification methods include: solubility-based purification methods such as salt precipitation and solvent precipitation; purification methods based on molecular weight difference, such as dialysis, ultrafiltration, gel filtration and SDS-PAGE; charge-based purification methods such as ion exchange chromatography and hydroxylapatite chromatography; specific affinity-based purification methods such as affinity chromatography; purification methods based on hydrophobicity difference, such as reverse-phase high-performance liquid chromatography; and purification methods based on isoelectric point difference, such as isoelectric focusing.
Preparation of a protein by a genetic recombination technique is carried out by preparing an expression vector comprising an antigen-encoding nucleic acid, introducing the expression vector into appropriate host cells by gene transfer or genetic transformation, culturing the host cells under suitable conditions for expression of a recombinant protein, and recovering the recombinant protein expressed in the host cells.
The “vector” refers to a nucleic acid that can be used to introduce a nucleic acid attached thereto into host cells. The “expression vector” is a vector that can induce the expression of a protein encoded by a nucleic acid introduced therethrough. Exemplary vectors include plasmid vectors and viral vectors. Those skilled in the art can select an appropriate expression vector for the expression of a recombinant protein depending on the type of host cells to be used.
The “host cells” refers to cells that undergo gene transfer or genetic transformation by a vector. The host cells can be appropriately selected by those skilled in the art depending on the type of the vector to be used. The host cells can be derived from prokaryotes such as E. coli. When prokaryotic cells like E. coli are used as host cells, the antigen of the present invention may be designed to contain an N-terminal methionine residue in order to facilitate the expression of a recombinant protein in the prokaryotic cells. The N-terminal methionine can be cleaved from the recombinant protein after expression. Also, the host cells may be cells derived from eukaryotes, such as single-cell eukaryotes like yeast, plant cells and animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, murine cells or insect cells) or silkworm.
Gene transfer or genetic transformation of an expression vector into host cells can be carried out as appropriate by following a technique known to those skilled in the art. Those skilled in the art can make possible the expression of a recombinant protein by selecting suitable conditions for the expression of the recombinant protein as appropriate depending on the type of host cells and culturing the host cells under the selected conditions. Then, those skilled in the art can homogenize the host cells having the expressed recombinant protein, and separate and purify an antigen expressed as the recombinant protein from the resulting homogenate by using an appropriate combination of such protein purification methods as mentioned above.
Diagnosis Kit and Method (1)
The present invention provides a method for providing an indicator for diagnosing an allergy to eggs in a subject, the method comprising the steps of:
The sample obtained from a subject is a solution containing an IgE antibody, as collected from the subject. Examples of such solutions include blood, saliva, sputum, snivel, urine, sweat, and tear. The sample obtained from the subject may be subjected to pretreatment for increasing the concentration of an IgE antibody in the sample before being contacted with an antigen. The pretreatment of a sample may involve, for example, collection of the serum or the plasma from the blood. Furthermore, a Fab moiety that is an antigen-binding moiety may be purified. In a particularly preferred embodiment, embodiment, the step (i) mentioned above is carried out by contacting an IgE antibody present in the serum obtained from a subject with an antigen.
The IgE antibody may be the IgE antibody itself or may be mast cells or the like bound to IgE antibodies.
Detection of contact and binding between the sample obtained from a subject and an antigen can be carried out by using a known method. Examples of such methods that can be used include detection by ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, or immunochromatography. These are all techniques for detecting the binding between an antigen and an IgE antibody from a subject by contacting and binding the IgE antibody from a subject with the antigen, allowing an enzymatically labelled secondary antibody to act on the IgE antibody specifically bound to the antigen, and adding an enzyme substrate (generally, chromogenic or luminescent reagent) to detect an enzymatic reaction product. Alternatively, they are methods of detecting a fluorescently labeled secondary antibody. Alternatively, detection by a measurement method capable of evaluating binding between an antigen and an IgE antibody, such as surface plasmon resonance (SPR), can also be used. A plurality of antigen-specific IgE antibodies may be mixed.
The antigen may be provided as an isolated antigen in a state immobilized on a carrier. In this case, the steps (i) and (ii) mentioned above can be carried out using ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, or the like. Also, the step (i) mentioned above can be carried out by contacting the sample obtained from a subject with an antigen-immobilized surface. The isolated antigen may be obtained by separating and purifying it from eggs (preferably chicken eggs) using a combination of protein purification methods well known to those skilled in the art, or by preparing it using a genetic recombination technique. Alternatively, the antibody may be used in a state immobilized on a carrier.
The antigen may be in a state unimmobilized on a carrier. In this case, flow cytometry or the like can be used in the aforementioned steps (i) and (ii), and the presence of the antigen bound to the antibody can be confirmed with laser beam. Examples of this method include a basophil activation test (BAT). Another example includes a histamine release test (HRT) which examines whether histamine is released by further contacting the antigen with blood cells in a sample.
The antigen may be detected by immunoblotting method by transferring from a state separated by two-dimensional electrophoresis. The two-dimensional electrophoresis is a technique for separating a protein sample by performing isoelectric focusing in the first dimension and performing SDS-PAGE (SDS-polyacrylamide gel electrophoresis) in the second dimension. The conditions for two-dimensional electrophoresis are not particularly limited as long as the conditions permit the separation of the antigen of the present invention. For example, the conditions for two-dimensional electrophoresis as described above in the subsection titled “Identification of antigens” can be adopted. Also, the electrophoresis conditions may be defined by reference to the descriptions in Patent Literatures 1 to 4 mentioned above. For example, two-dimensional electrophoresis can be carried out under the conditions that satisfy at least one selected from the group consisting of the following requirements:
The aforementioned antigens (4) to (10) are antigens that specifically bind to IgE antibodies from patients with allergy to eggs. Therefore, when binding between an IgE antibody from a subject and the antigen is detected, an indicator of the fact that the subject is allergic to eggs is provided.
The present invention further provides a kit for diagnosing an allergy to eggs, comprising at least one of the aforementioned antigens (4) to (10). The diagnosis kit of this invention may be used in the aforementioned method for providing an indicator for diagnosing an allergy to eggs or in a diagnosis method as described later. The diagnosis kit of this invention may comprise not only the at least one of the aforementioned antigens (4) to (10), but also an anti-IgE antibody labeled with an enzyme and a chromogenic or luminescent substrate serving as a substrate for the enzyme. Also, a fluorescent-labeled anti-IgE antibody may be used. In the diagnosis kit of this invention, the antigen may be provided in a state immobilized on a carrier. The diagnosis kit of this invention may also be provided together with instructions on the procedure for diagnosis or a package containing said instructions.
In another embodiment, embodiment, the aforementioned diagnosis kit comprises a companion diagnostic agent for an allergy to eggs. The companion diagnostic agent is used for identifying patients expected to respond to pharmaceutical products or identifying patients having the risk of severe adverse reactions to pharmaceutical products, or for studying the reactivity of pharmaceutical products in order to optimize treatment using the pharmaceutical products. Here, the optimization of treatment includes, for example, determination of dosage and administration, judgment regarding discontinuation of administration, and confirmation of an allergen component that is used to cause immunological tolerance.
The present invention further provides a composition for diagnosing an allergy to eggs, comprising at least one of the aforementioned antigens (4) to (10). The diagnosis composition of this invention can be used in a diagnosis method as described below. The diagnosis composition of this invention may further comprise a pharmaceutically acceptable carrier and/or additives commonly used with the antigen of this invention depending on the need.
In one embodiment, embodiment, the present invention provides a method for diagnosing an allergy to eggs in a subject, the method comprising:
In another embodiment, embodiment, the present invention provides a method for diagnosing an allergy to eggs in a subject, the method comprising administering to the subject at least one of the aforementioned antigens (4) to (10). This method may be performed in the form of a skin test characterized by applying the antigen onto the skin. Examples of the skin test include various forms of tests, such as: a prick test in which a diagnosis composition is applied onto the skin and then a tiny prick to such an extent as not to provoke bleeding is made in the skin to allow an antigen to penetrate the skin, thereby observing a skin reaction; a scratch test in which a diagnosis composition is applied onto the skin and then the skin is lightly scratched to observe a reaction; a patch test in which a diagnosis composition in the form of cream, ointment, etc. is applied onto the skin to observe a reaction; and an intracutaneous test in which an antigen is administered intracutaneously to observe a reaction. If a skin reaction such as swelling occurs in a skin portion to which the antigen has been applied, the subject of interest is diagnosed as having an allergy to eggs. The amount of the antigen to be applied to the skin in such tests can be, for example, not more than 100 μg per dose.
In the process of allergy diagnosis, a load test aiming to identify an antigen is often adopted. At least one of the aforementioned antigens (4) to (10) can be used as an active ingredient for a load test to diagnose an allergy to eggs. Here, the antigen protein to be used in the load test may be a protein that has been expressed and purified and may be a protein that has been expressed in food or cooking ingredients, such as rice-based vaccine expressing pollen allergens which are obtained by transforming rice with a gene of a cedar pollen antigen and expressing the antigen protein in the rice.
In yet another embodiment, embodiment, the present invention provides at least one of the aforementioned antigens (4) to (10), intended for use in the diagnosis of an allergy to eggs. This also includes the provision of at least one of the aforementioned antigens (4) to (10) mixed with a known antigen.
In still another embodiment, embodiment, the present invention provides use of at least one of the aforementioned antigens (4) to (10) for the production of a diagnostic agent for an allergy to eggs.
Pharmaceutical Composition and Treatment Method (1)
The present invention provides a pharmaceutical composition comprising at least one of the aforementioned antigens (4) to (10).
In one embodiment, embodiment, the aforementioned pharmaceutical composition is used for the treatment and diagnosis of an allergy to eggs. The treatment of an allergy increases the limit amount of an antigen in which the allergy does not develop even if the antigen is incorporated into the body, and finally aims for the state where the allergy does not develop by the common amount of the antigen to be consumed (remission).
The present invention also provides a method for treating an allergy to eggs, the method comprising administering at least one of the aforementioned antigens (4) to (10) to a patient in need of a treatment for an allergy to eggs.
In another embodiment, embodiment, the present invention provides at least one of the aforementioned antigens (4) to (10), intended for use in the treatment for an allergy to eggs. In yet another embodiment, embodiment, the present invention provides use of at least one of the aforementioned antigens (4) to (10) for the production of a therapeutic agent for an allergy to eggs.
In the process of allergy treatment, a hyposensitization therapy aiming to induce immunological tolerance by administering an antigen to a patient is often adopted. The at least one of the aforementioned antigens (4) to (10) can be used as an active ingredient for a hyposensitization therapy for an allergy to eggs. Here, the antigen protein to be used in the hyposensitization therapy may be a protein that has been expressed and purified and may be a protein that has been expressed in food or cooking ingredients, such as rice-based vaccine expressing pollen allergens which are obtained by transforming rice with a gene of a cedar pollen antigen and expressing the antigen protein in the rice.
The pharmaceutical composition of the present invention can be administered by common administration routes. Examples of common administration routes include oral, sublingual, percutaneous, intracutaneous, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, and intrarectal administrations.
The pharmaceutical composition of the present invention can be used as a pharmaceutical composition to which a commonly used pharmaceutically acceptable adjuvant or excipient or any other additives (e.g., stabilizer, solubilizer, emulsifier, buffer, preservative, colorant) are added by a conventional method together with the antigen of this invention depending on the need. The dosage form of the pharmaceutical composition can be selected by those skilled in the art as appropriate depending on the administration route. The pharmaceutical composition can be in the form of, for example, tablet, capsule, troche, sublingual tablet, parenteral injection, intranasal spray, poultice, solution, cream, lotion, or suppository. The administration dose, frequency and/or period of the pharmaceutical composition of this invention can be selected by a physician as appropriate depending on the administration route and the patient's condition and characteristics such as age and body weight. For example, the pharmaceutical composition may be administered to an adult patient at a dose of not more than 100 μg per dose. The administration interval can be, for example, once daily, once weekly, twice weekly, once per three months or so. The administration period can be, for example, several weeks to several years. The pharmaceutical composition may be administered in such a manner that the dose is increased in incremental steps over the administration period.
Tester (1)
The present invention provides a tester comprising an antibody for at least one of the aforementioned antigens (4) to (10).
The antibody can be prepared by a conventional method. For example, the antibody may be prepared by immunizing a mammal such as rabbit with one of the aforementioned antigens (4) to (10). The antibody may be an IgE antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab′)2, Fab′).
Further, in the aforementioned tester, the antibody may be provided in a form bound to a carrier. The type of the carrier is not particularly limited as long as it is usable for detection of binding between an antibody and an antigen. Any given carrier known to those skilled in the art can be used.
Examples of a method for determining the presence or absence of an antigen include the following methods:
Another embodiment of the present invention includes a tester for determining the presence or absence of an antigen of an allergy to eggs in an object of interest, the tester comprising a primer having a nucleotide sequence complementary to a portion of at least one of the nucleotide sequences of SEQ ID NOs: 46, 58, 105, 151, 236, 250 and 261. The primer has, for example, but not limited to, a nucleotide sequence complementary to, preferably, 12 residues, 15 bases, 20 bases, or 25 bases, in a 3′-terminal portion or central sequence in a sequence of at least one of the nucleotide sequences of SEQ ID NOs: 46, 58, 105, 151, 236, 250 and 261. Particularly, when mRNA is of interest, the tester has a complementary primer of a poly-A tail. In a preferred embodiment, embodiment, the tester comprising the primer mentioned above may further comprise a primer comprising a 5′-terminal nucleotide sequence, preferably a nucleotide sequence of 12 bases, 15 bases, 20 bases, or 25 bases, of at least one of the nucleotide sequences of SEQ ID NOs: 46, 58, 105, 151, 236, 250 and 261.
For example, DNA or cDNA is amplified by PCR (Polymerase Chain Reaction) including RT-PCR using templated DNA or mRNA obtained from an egg or a bird and the aforementioned complementary primer, and the sequence of the amplified cDNA is compared with the nucleotide sequence of SEQ ID NO: 46, 58, 105, 151, 236, 250 or 261 to determine the presence or absence of the antigen. Amplification methods by PCR can be exemplified by RACE. In this respect, even if there exists a point mutation encoding the same amino acid in the comparison of the amplified cDNA with the nucleotide sequence of SEQ ID NO: 46, 58, 105, 151, 236, 250 or 261, or even if the nucleotide sequence of the amplified cDNA has insertion, deletion, substitution or addition of bases in the nucleotide sequence of SEQ ID NO: 46, 58, 105, 151, 236, 250 or 261, it is determined that the antigen is present when the amino acid sequence encoded by the cDNA has at least 70%, preferably at least 80, 90, 95, 98, or 99% identity to the amino acid sequence of SEQ ID NO: 47, 59, 106, 152, 237, 251 or 262.
In one embodiment, embodiment, the aforementioned tester is used to determine the presence or absence of an antigen in cooking ingredients (an egg or a bird) or in products of interest in a food production line. The tester may also be used for quality inspection of production lines and pre-shipment products by manufacturers, or may be used for self-checking of the presence or absence of an antigen in a food or cooking ingredients of interest by consumers.
Antigen-Free Food and the Like (1)
The present invention provides an egg or a processed product of egg in which at least one of the aforementioned antigens (4) to (10) is eliminated or reduced, or a bird that delivers such egg or is born from such egg.
The method for eliminating or reducing the antigen of the present invention in an egg or processed products of egg, a bird that delivers such egg or is born from such egg is not limited. The elimination or reduction of the antigen may be conducted by any method, as long as the method permits the elimination or reduction of the antigen.
For example, the egg of the present invention whose antigen is eliminated or reduced may be obtained by preparing an egg in which the expression of the antigen of the present invention is knocked out, using a gene knock-out technique.
Any technique known to those skilled in the art can be used as the gene knock-out technique. For example, Oishi, et al. (Scientific Reports, Vol. 6, Article number: 23980, 2016, doi:10.1038/srep23980) states that individuals with ovomucoid gene deletion are obtained by applying a genome editing technique CRISPER/Cas9 to primordial germ cells of chickens. The egg of the present invention whose antigen is eliminated may be obtained through the use of a similar technique. The bird or the egg of the present invention whose antigen is eliminated or reduced may be obtained by mating through artificial insemination with birds or eggs containing no antigen or containing the antigen in a small amount. The artificial crossing of birds or eggs can be performed by a conventional method.
The processed products of egg of the present invention in which an antigen is eliminated or reduced may be a processed product using the egg of the present invention in which an antigen is eliminated or reduced as a source ingredient. In the case of using an ordinary egg as a source ingredient, a treatment for removing or reducing the antigen of this invention is performed before or after preparation of a processed product of egg. The methods for removing or reducing the antigen of the present invention in the processed products of egg which assumed an ordinary egg raw material include a method to remove protein component in food or a cooking ingredient such as a high pressure treatment and elution with the neutral salt solution or the high temperature steam, and a method to perform hydrolysis, denaturation, or amino acid alteration (chemical modification, elimination, or the like of a side chain) by heat treatment and acid treatment.
The bird of the present invention whose antigen is eliminated or reduced may be a bird grown from hatched eggs in which the aforementioned antigen of the present invention is eliminated or reduced. The bird includes each stage of growth of baby bird, child bird, young bird, adult bird and aged bird. As referred to herein, the bird means an animal belonging to avian species, preferably a chicken.
Method for Producing Antigen-Free Processed Product (1)
The present invention provides a method for producing a processed product of egg in which an antigen is eliminated or reduced, the method comprising the step of confirming that the antigen is eliminated or reduced, in a production process of the processed product, wherein the antigen is at least one of the aforementioned antigens (4) to (10).
The step of confirming that the antigen is eliminated or reduced, in a production process of the processed product of egg in which an antigen is eliminated or reduced may be performed by confirming the presence or absence of an antigen by the method described above in the subsection titled “Tester (1)”.
The production of the processed product of egg in which an antigen is eliminated or reduced may be performed by the method described above in the subsection titled “Antigen-free food and the like (1)”.
Epitope of Antigen
Epitopes and amino acids important for binding activity against IgE antibodies from allergic patients within the epitopes were identified as shown in Example 6 as to antigens identified as shown in Examples 2 to 5.
The present invention provides polypeptides of the following (1β) to (4β) as polypeptides comprising an amino acid sequence specifically binding to an IgE antibody from an allergic patient.
(1β) Epitope of Vitellogenin-2
In the present invention, the polypeptide (1β) can be any polypeptide selected from the group consisting of (1β-1) to (1β-21) as defined below:
(1β-1) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 272-295, 319, 323, 324, 327, 330, 331, 333, 335, 336, 339, 340, 342, 343, 344, 346, 347, 350, 351, 353, 355, 356, 357, 359, 360, 363, 366, 369, 374, 375, 377, 379, 380, 381, 384, 385, 388, 391, 392, 393, 397, 400, 401, 402, 404, 405, 406, 407, 409, 412, 414, 419, 422, 425, 426, 430, 434, 436, 440, 444, 446, 449, 451, 452, 455, 458, 461, 462, 464, 465, 466, 470, 471, 473, 476, 479, 480, 482, 483, 486, 487, 491, 492, 494, 495, 497, 498, 500, 503, 505, 507, 509, 510, 512, 515, 516, 517, 520, 522 and 523.
(1β-2) a polypeptide comprising the amino acid sequence of XXXXXQXEEYNGXWP (SEQ ID NO: 320), preferably a polypeptide comprising the amino acid sequence of XXXSPQVEEYNGVWP (SEQ ID NO: 321), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1-5, 7 and 13 of SEQ ID NO: 272 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 1-3 of SEQ ID NO: 272 are substituted by other amino acids, preferably alanine.
In another embodiment, embodiment, a polypeptide comprising the amino acid sequence of XEEYNG (SEQ ID NO: 322), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 1 of SEQ ID NO: 323 is substituted by another amino acid, preferably alanine.
(1β-3) a polypeptide comprising the amino acid sequence of XXXXXXXXNVYEXXX (SEQ ID NO: 328), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1-8 and 13-15 of SEQ ID NO: 273 are substituted by other amino acids, preferably alanine.
In another embodiment, embodiment, a polypeptide comprising the amino acid sequence of XXXXXXXXNV (SEQ ID NO: 325), preferably a polypeptide comprising the amino acid sequence of XXXIKXXXNV (SEQ ID NO: 326), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1-8 of SEQ ID NO: 327 are substituted by other amino acids, preferably alanine, more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1-3 and 6-8 of SEQ ID NO: 327 are substituted by other amino acids, preferably alanine.
(1β-4) a polypeptide comprising the amino acid sequence of RXRXXKXXXXXXXXX (SEQ ID NO: 332), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 2, 4, 5 and 7-15 of SEQ ID NO: 274 are substituted by other amino acids, preferably alanine.
In another embodiment, embodiment, a polypeptide comprising the amino acid sequence of RXXXXKXXXALXXXX (SEQ ID NO: 337), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 2-5, 7-9 and 12-15 of SEQ ID NO: 274 are substituted by other amino acids, preferably alanine.
In yet another embodiment, embodiment, a polypeptide comprising the amino acid sequence of RXRXXKXX (SEQ ID NO: 329), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2, 4, 5, 7 and 8 of SEQ ID NO: 330 are substituted by other amino acids, preferably alanine.
In yet another embodiment, embodiment, a polypeptide comprising the amino acid sequence of KXXXALXXXX (SEQ ID NO: 334), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 2-4 and 7-10 of SEQ ID NO: 335 are substituted by other amino acids, preferably alanine.
(1β-5) a polypeptide comprising the amino acid sequence of AXXKIAXXXPKTVXX (SEQ ID NO: 341), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 2, 3, 7-9, 14 and 15 of SEQ ID NO: 275 are substituted by other amino acids, preferably alanine.
In another embodiment, embodiment, a polypeptide comprising the amino acid sequence of AXXXXAXXXXXTVQX (SEQ ID NO: 348), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 2-5, 7-11 and 15 of SEQ ID NO: 275 are substituted by other amino acids, preferably alanine.
In yet another embodiment, embodiment, a polypeptide comprising the amino acid sequence of AXXXXAXKXXXXXQG (SEQ ID NO: 352), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 2-5, 7 and 9-13 of SEQ ID NO: 275 are substituted by other amino acids, preferably alanine.
In yet another embodiment, embodiment, a polypeptide comprising the amino acid sequence of AXXKIAXXXP (SEQ ID NO: 338), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2, 3 and 7-9 of SEQ ID NO: 339 are substituted by other amino acids, preferably alanine.
In yet another embodiment, embodiment, a polypeptide comprising the amino acid sequence of AWKDPKXXXG (SEQ ID NO: 345), preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 7-9 of SEQ ID NO: 346 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of AXKXXXXX (SEQ ID NO: 349), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2 and 4-8 of SEQ ID NO: 350 are substituted by other amino acids, preferably alanine.
(1β-6) a polypeptide comprising the amino acid sequence of XALXXLSPKLDSMSY (SEQ ID NO: 358), preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 1, 4 and 5 of SEQ ID NO: 276 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XLSPKLDS (SEQ ID NO: 354), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 1 of SEQ ID NO: 655 is substituted by another amino acid, preferably alanine.
(1β-7) a polypeptide comprising the amino acid sequence of VXXXRTMFPXAXISK (SEQ ID NO: 361), preferably a polypeptide comprising the amino acid sequence of VNXXRTMFPSAXISK (SEQ ID NO: 362), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2-4, 10 and 12 of SEQ ID NO: 277 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 3, 4 and 12 of SEQ ID NO: 277 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXSXXXMXPXAXXXK (SEQ ID NO: 370), preferably a polypeptide comprising the amino acid sequence of VNSPRTMXPXAXXXK (SEQ ID NO: 371), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1, 2, 4-6, 8, 10 and 12-14 of SEQ ID NO: 277 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 8, 10 and 12-14 of SEQ ID NO: 277 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of SXXXMXPXAX (SEQ ID NO: 364), preferably a polypeptide comprising the amino acid sequence of SPRTMXPXAX (SEQ ID NO: 365), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2-4, 6, 8 and 10 of SEQ ID NO: 366 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 3, 4 and 12 of SEQ ID NO: 366 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XMXPXAXXXK (SEQ ID NO: 367), preferably a polypeptide comprising the amino acid sequence of TMXPXAXXXK (SEQ ID NO: 368), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1, 3, 5 and 7-9 of SEQ ID NO: 369 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 3, 5 and 7-9 of SEQ ID NO: 369 are substituted by other amino acids, preferably alanine.
(1β-8) a polypeptide comprising the amino acid sequence of AIISKLXAXXAXSXA (SEQ ID NO: 376), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 7, 9, 10, 12 and 14 of SEQ ID NO: 278 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of IXKLXAXXA (SEQ ID NO: 372), preferably a polypeptide comprising the amino acid sequence of ISKLXAXXA (SEQ ID NO: 373), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 2, 5, 7 and 8 of SEQ ID NO: 374 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 5, 7 and 8 of SEQ ID NO: 374 are substituted by other amino acids, preferably alanine.
(1β-9) a polypeptide comprising the amino acid sequence of GKALQXXKELPTXTP (SEQ ID NO: 382), preferably a polypeptide comprising the amino acid sequence of GKALQXXKELPTETP (SEQ ID NO: 383), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 6, 7 and 13 of SEQ ID NO: 279 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 6 and 7 of SEQ ID NO: 279 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXAXXXXXEXXXXXP (SEQ ID NO: 394), preferably a polypeptide comprising the amino acid sequence of XXALXXXXEXXXXXP (SEQ ID NO: 395), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 1, 2, 4-8 and 10-14 of SEQ ID NO: 279 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1, 2, 5-8 and 10-14 of SEQ ID NO: 279 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of GKAXXXXKEXXXXXX (SEQ ID NO: 403), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 4-7 and 10-15 of SEQ ID NO: 279 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of KALQXXKELP (SEQ ID NO: 378), preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 5 and 6 of SEQ ID NO: 379 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XAXXXXXEX (SEQ ID NO: 386), preferably a polypeptide comprising the amino acid sequence of XALXXXXEX (SEQ ID NO: 387), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1, 3-7 and 9 of SEQ ID NO: 388 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1, 4-7 and 9 of SEQ ID NO: 388 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXEXXXXXP (SEQ ID NO: 389), preferably a polypeptide comprising the amino acid sequence of XXXEXXXEXP (SEQ ID NO: 390), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1-3 and 5-9 of SEQ ID NO: 392 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1-3, 5-7 and 9 of SEQ ID NO: 392 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of KAXXXXKEXX (SEQ ID NO: 396), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 3-6, 9 and 10 of SEQ ID NO: 397 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXKEXXXXXX (SEQ ID NO: 398), preferably a polypeptide comprising the amino acid sequence of GXKEXPXXTX (SEQ ID NO: 399), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1, 2 and 5-10 of SEQ ID NO: 400 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2, 5, 7, 8 and 10 of SEQ ID NO: 400 are substituted by other amino acids, preferably alanine.
(1β-10) a polypeptide comprising the amino acid sequence of XXXNKELXQQVMXXV (SEQ ID NO: 408), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1-3, 8, 13 and 14 of SEQ ID NO: 280 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of ELXQQ (SEQ ID NO: 406), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 3 of SEQ ID NO: 407 is substituted by another amino acid, preferably alanine.
(1β-11) a polypeptide comprising the amino acid sequence of XXXXVVXPADXNAAI (SEQ ID NO: 415), preferably a polypeptide comprising the amino acid sequence of XXXTVVEPADXNAAI (SEQ ID NO: 416), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1-4, 7 and 11 of SEQ ID NO: 281 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1-3 and 11 of SEQ ID NO: 281 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXVVXPA (SEQ ID NO: 410), preferably a polypeptide comprising the amino acid sequence of XXTVVEPA (SEQ ID NO: 411), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1-3 and 6 of SEQ ID NO: 412 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of ADXNA (SEQ ID NO: 413), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 3 of SEQ ID NO: 413 is substituted by another amino acid, preferably alanine.
(1β-12) a polypeptide comprising the amino acid sequence of KXXAKXXXXXKNXXX (SEQ ID NO: 420), preferably a polypeptide comprising the amino acid sequence of KXXAKXDVXXKNLXX (SEQ ID NO: 421), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 2, 3, 6-10 and 13-15 of SEQ ID NO: 282 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 2, 3, 6, 9, 10, 14 and 15 of SEQ ID NO: 282 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of KFXAKXDXKXKNXKX (SEQ ID NO: 427), preferably a polypeptide comprising the amino acid sequence of KFDAKXDVKXKNXKX (SEQ ID NO: 428), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 3, 6, 8, 10, 13 and 15 of SEQ ID NO: 282 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 6, 10, 13 and 15 of SEQ ID NO: 282 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXXKNLX (SEQ ID NO: 417), preferably a polypeptide comprising the amino acid sequence of DVXXKNLX (SEQ ID NO: 418), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1-4 and 8 of SEQ ID NO: 419 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 3, 4 and 8 of SEQ ID NO: 419 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of DXKXKNXKX (SEQ ID NO: 423), preferably a polypeptide comprising the amino acid sequence of DVKXKNXKX (SEQ ID NO: 424), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 2, 4, 7 and 9 of SEQ ID NO: 425 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 4, 7 and 9 of SEQ ID NO: 425 are substituted by other amino acids, preferably alanine.
(1β-13) a polypeptide comprising the amino acid sequence of NXXXKKXNXVXAXXX (SEQ ID NO: 431), preferably a polypeptide comprising the amino acid sequence of NRXXKKXNTVLAXXX (SEQ ID NO: 432), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 2-4, 7, 9, 11 and 13-15 of SEQ ID NO: 285 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 3, 4, 7 and 13-15 of SEQ ID NO: 285 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXKKXNTVLA (SEQ ID NO: 429), preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 1, 2 and 5 of SEQ ID NO: 430 are substituted by other amino acids, preferably alanine.
(1β-14) a polypeptide comprising the amino acid sequence of SSSSSSSXSNSKSSS (SEQ ID NO: 437), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 8 of SEQ ID NO: 286 is substituted by another amino acid, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXSXSSSXXXSKSSS (SEQ ID NO: 441), preferably a polypeptide comprising the amino acid sequence of XXSSSSSXXNSKSSS (SEQ ID NO: 442), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1, 2, 4 and 8-10 of SEQ ID NO: 286 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1, 2, 8 and 9 of SEQ ID NO: 286 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of SSSXSNSKS (SEQ ID NO: 433), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 4 of SEQ ID NO: 434 is substituted by another amino acid, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of SXSNSKSSSS (SEQ ID NO: 435), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 2 of SEQ ID NO: 436 is substituted by another amino acid, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXSKSS (SEQ ID NO: 438), preferably a polypeptide comprising the amino acid sequence of XXNSKSS (SEQ ID NO: 439), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 1-3 of SEQ ID NO: 440 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 1 and 2 of SEQ ID NO: 400 are substituted by other amino acids, preferably alanine.
(1β-15) a polypeptide comprising the amino acid sequence of XEXXXXKXPXXXAXX (SEQ ID NO: 450), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1, 3-6, 8, 10-12, 14 and 15 of SEQ ID NO: 287 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XEXXXXKXPX (SEQ ID NO: 443), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1, 3-6, 8 and 10 of SEQ ID NO: 444 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXKXPXXXA (SEQ ID NO: 445), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1-3, 5 and 7-9 of SEQ ID NO: 446 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XKXPXXXAXX (SEQ ID NO: 447), preferably a polypeptide comprising the amino acid sequence of XKXPXXXAXS (SEQ ID NO: 448), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1, 3, 5-7, 9 and 10 of SEQ ID NO: 449 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1, 3, 5-7 and 9 of SEQ ID NO: 449 are substituted by other amino acids, preferably alanine.
(1β-16) a polypeptide comprising the amino acid sequence of XXTXXXKXXLXADAX (SEQ ID NO: 459), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1, 2, 4-6, 8, 9, 11 and 15 of SEQ ID NO: 288 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of TXXXKXXLXA (SEQ ID NO: 453), preferably a polypeptide comprising the amino acid sequence of TXXXKXKLCA (SEQ ID NO: 454), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2-4, 6, 7 and 9 of SEQ ID NO: 455 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of KXXLXADAX (SEQ ID NO: 456), preferably a polypeptide comprising the amino acid sequence of KXKLCADAX (SEQ ID NO: 457), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 2, 3, 5 and 9 of SEQ ID NO: 458 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 2 and 9 of SEQ ID NO: 458 are substituted by other amino acids, preferably alanine.
(1β-17) a polypeptide comprising the amino acid sequence of KIKTXNEXXFXXSMP (SEQ ID NO: 467), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 5, 8, 9, 11 and 12 of SEQ ID NO: 290 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of KIKTX (SEQ ID NO: 460), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 5 of SEQ ID NO: 461 is substituted by another amino acid, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XNEXXFNYSM (SEQ ID NO: 463), preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 1, 4 and 5 of SEQ ID NO: 464 are substituted by other amino acids, preferably alanine.
(1β-18) a polypeptide comprising the amino acid sequence of XAXXXXAXXIXIGXH (SEQ ID NO: 474), preferably a polypeptide comprising the amino acid sequence of EAXXXXAXXIXIGXH (SEQ ID NO: 475), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1, 3-6, 8, 9, 11 and 14 of SEQ ID NO: 291 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 3-6, 8, 9, 11 and 14 of SEQ ID NO: 291 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XAXNXXAXXXKIGXH (SEQ ID NO: 477), preferably a polypeptide comprising the amino acid sequence of XAXNXKAXXXKIGXH (SEQ ID NO: 478), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1, 3, 5, 6, 8-10 and 14 of SEQ ID NO: 291 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1, 3, 5, 8-10 and 14 of SEQ ID NO: 291 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XAXXLXAXNIXXGXH (SEQ ID NO: 488), preferably a polypeptide comprising the amino acid sequence of EAXXLKAXNIXXGXH (SEQ ID NO: 489), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1, 3, 4, 6, 11, 12 and 14 of SEQ ID NO: 291 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 3, 4, 8, 11, 12 and 14 of SEQ ID NO: 291 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XAXNXXAXX (SEQ ID NO: 468), preferably a polypeptide comprising the amino acid sequence of EAXNLXAXX (SEQ ID NO: 469), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1, 3, 5, 6, 8 and 9 of SEQ ID NO: 470 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 3, 6, 8 and 9 of SEQ ID NO: 470 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXIXIGXH (SEQ ID NO: 472), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1, 2, 4 and 7 of SEQ ID NO: 473 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of AXXLKAXNI (SEQ ID NO: 481), preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 2, 3 and 7 of SEQ ID NO: 482 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XAXNIXXGX (SEQ ID NO: 484), preferably a polypeptide comprising the amino acid sequence of KAXNIXXGX (SEQ ID NO: 485), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1, 3, 6, 7 and 9 of SEQ ID NO: 486 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 3, 6, 7 and 9 of SEQ ID NO: 486 are substituted by other amino acids, preferably alanine.
(1β-19) a polypeptide comprising the amino acid sequence of AAPXXGXDKXXXXGK (SEQ ID NO: 496), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 4, 5, 7 and 10-13 of SEQ ID NO: 292 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of AAPXXGIDKLXFDGK (SEQ ID NO: 501), preferably a polypeptide comprising the amino acid sequence of AAPXXGIDKLYFDGK (SEQ ID NO: 502), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 4, 5 and 11 of SEQ ID NO: 292 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 4 and 5 of SEQ ID NO: 292 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of AAXXXXIDKXXXXXK (SEQ ID NO: 510), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 3-6 and 10-14 of SEQ ID NO: 292 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of AAPXXG (SEQ ID NO: 490), preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 4 and 5 of SEQ ID NO: 491 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of DKXXXX (SEQ ID NO: 493), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 3-6 of SEQ ID NO: 494 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of DKLXFDG (SEQ ID NO: 499), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 4 of SEQ ID NO: 500 is substituted by another amino acid, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of AAXXXXID (SEQ ID NO: 504), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 3-6 of SEQ ID NO: 505 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXXIDKXXX (SEQ ID NO: 506), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1-4 and 8-10 of SEQ ID NO: 507 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of DKXXXXXKT (SEQ ID NO: 508), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 3-7 of SEQ ID NO: 509 are substituted by other amino acids, preferably alanine.
(1β-20) a polypeptide comprising the amino acid sequence of XXEXXMXNGYXAKNA (SEQ ID NO: 513), preferably a polypeptide comprising the amino acid sequence of XQEXXMXNGYLAKNA (SEQ ID NO: 514), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1, 2, 4, 5, 7 and 11 of SEQ ID NO: 293 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1, 4, 5 and 7 of SEQ ID NO: 293 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of MXNGYLAKNA (SEQ ID NO: 511), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 2 of SEQ ID NO: 512 is substituted by another amino acid, preferably alanine.
(1β-21) a polypeptide comprising the amino acid sequence of XAXXLXXXFVKLXXX (SEQ ID NO: 524), preferably a polypeptide comprising the amino acid sequence of XAXXLXXSFVKLXXX (SEQ ID NO: 525), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1, 3, 4, 6-8 and 13-15 of SEQ ID NO: 294 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1, 3, 4, 6, 7 and 13-15 of SEQ ID NO: 294 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of AXXLXXXFVK (SEQ ID NO: 518), preferably a polypeptide comprising the amino acid sequence of AXXLXXSFVK (SEQ ID NO: 519), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2, 3 and 5-7 of SEQ ID NO: 520 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 2, 3, 5 and 6 of SEQ ID NO: 520 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XSFVKLX (SEQ ID NO: 521), preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 1 and 7 of SEQ ID NO: 522 are substituted by other amino acids, preferably alanine.
(2β) Epitope of Vitellogenin-3
In the present invention, the polypeptide (2β) can be any polypeptide selected from the group consisting of (2β-1) to (2β-6) as defined below:
(2β-1) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 296-303, 528, 531, 533, 534, 535, 539, 541, 542, 544, 550 and 551.
(2β-2) a polypeptide comprising the amino acid sequence of XPRTCDXXXKXXXXX (SEQ ID NO: 526), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1, 7-9 and 11-15 of SEQ ID NO: 296 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of SPRXXXXXLXLPXXX (SEQ ID NO: 527), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 4-8, 10 and 13-15 of SEQ ID NO: 296 are substituted by other amino acids, preferably alanine.
(2β-3) a polypeptide comprising the amino acid sequence of SXXXXXXEXXXXXXX (SEQ ID NO: 529), preferably a polypeptide comprising the amino acid sequence of SXXXXSXEXXXXXXX (SEQ ID NO: 530), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the amino acids at positions 2-7 and 9-15 of SEQ ID NO: 297 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 2-5, 7 and 9-15 of SEQ ID NO: 297 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXLXSXXXXXXXXX (SEQ ID NO: 532), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the amino acids at positions 1-3, 5 and 7-15 of SEQ ID NO: 297 are substituted by other amino acids, preferably alanine.
(2β-4) a polypeptide comprising the amino acid sequence of GKXXXXXXXXXXXKK (SEQ ID NO: 536), preferably a polypeptide comprising the amino acid sequence of GKXXXXXXXXXXEKK (SEQ ID NO: 537), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 3-13 of SEQ ID NO: 298 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 3-12 of SEQ ID NO: 298 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXTCXXXXXXXXEKK (SEQ ID NO: 545), preferably a polypeptide comprising the amino acid sequence of XKTCXIXXXXXXEKK (SEQ ID NO: 546), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1, 2 and 5-12 of SEQ ID NO: 298 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1, 5 and 7-12 of SEQ ID NO: 298 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of CXIXXXXXX (SEQ ID NO: 538), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 2 and 4-9 of SEQ ID NO: 539 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of IXXXXXXEKK (SEQ ID NO: 540), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2-7 of SEQ ID NO: 541 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXEKK (SEQ ID NO: 543), preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 1-3 of SEQ ID NO: 544 are substituted by other amino acids, preferably alanine.
(2β-5) a polypeptide comprising the amino acid sequence of XAXXXREYQMPSGYL (SEQ ID NO: 547), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1 and 3-5 of SEQ ID NO: 299 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XAXXXXXXQMPSXXL (SEQ ID NO: 548), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1, 3-8, 13 and 14 of SEQ ID NO: 299 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of DAXXXXXXQXXXXYL (SEQ ID NO: 549), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 3-8 and 10-13 of SEQ ID NO: 299 are substituted by other amino acids, preferably alanine.
(2β-6) a polypeptide comprising the amino acid sequence of XTXXXACKLXXXXXX (SEQ ID NO: 552), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1, 3-5 and 10-15 of SEQ ID NO: 301 are substituted by other amino acids, preferably alanine. In one embodiment, the polypeptide (2β) may not comprise a variant or a homolog having 100% or at least 90%, at least 80% or at least 70% identity to the C-terminal portion (SEQ ID NOs: 2 and 13) of vitellogenin-3 or the full-length amino acid sequence thereof.
(3β) Epitope of Apolipoprotein B
In the present invention, the polypeptide (3β) can be any polypeptide selected from the group consisting of (3β-1) to (3β-8) as defined below:
(3β-1) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 304-311, 553, 555, 556, 557, 561, 562, 565, 569, 571, 573, 576, 577, 579, 581, 583, 584, 585, 587, 589, 592, 594, 595, 598, 600, 602, 304, 605, 608, 609, 613, 616, 617, 619, 622, 623 and 624.
(3β-2) a polypeptide comprising the amino acid sequence of AEXXXXXXXXXXLNX (SEQ ID NO: 558), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 3-12 and 15 of SEQ ID NO: 304 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of AEXXXXXX (SEQ ID NO: 554), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 3-8 of SEQ ID NO: 555 are substituted by other amino acids, preferably alanine.
(3β-3) a polypeptide comprising the amino acid sequence of XXXXXXXXXXXXXNV (SEQ ID NO: 563), preferably a polypeptide comprising the amino acid sequence of XXXXXXXXXDEPLNV (SEQ ID NO: 564), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the amino acids at positions 1-13 of SEQ ID NO: 305 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1-9 of SEQ ID NO: 305 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXXXXXXKXEXXNX (SEQ ID NO: 574), preferably a polypeptide comprising the amino acid sequence of XXXKLYPXKXEXXNX (SEQ ID NO: 575), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 1-8, 10, 12, 13 and 15 of SEQ ID NO: 305 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1-3, 8, 10, 12, 13 and 15 of SEQ ID NO: 305 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XEPLN (SEQ ID NO: 559), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 1 of SEQ ID NO: 561 is substituted by another amino acid, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XEPLNV (SEQ ID NO: 560), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 1 of SEQ ID NO: 562 is substituted by another amino acid, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXXXXKXEX (SEQ ID NO: 567), preferably a polypeptide comprising the amino acid sequence of XKLYPXKXXX (SEQ ID NO: 568), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1-6, 8 and 10 of SEQ ID NO: 569 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1, 6 and 8-10 of SEQ ID NO: 569 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of KXEXXNX (SEQ ID NO: 570), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 2, 4, 5 and 7 of SEQ ID NO: 571 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of KXEX (SEQ ID NO: 572), preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 2 and 4 of SEQ ID NO: 573 are substituted by other amino acids, preferably alanine.
(3β-4) a polypeptide comprising the amino acid sequence of VSKRXXVXXXXXXXX (SEQ ID NO: 582), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 5, 6 and 8-15 of SEQ ID NO: 306 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of VSKRXX (SEQ ID NO: 578), preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 5 and 6 of SEQ ID NO: 579 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of SVPXXXX (SEQ ID NO: 580), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 4-7 of SEQ ID NO: 581 are substituted by other amino acids, preferably alanine.
(3β-5) a polypeptide comprising the amino acid sequence of XXXGXXXPXAXXAXX (SEQ ID NO: 590), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1-3, 5-7, 9, 11 and 12 of SEQ ID NO: 308 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXGXXXXGAKVAVX (SEQ ID NO: 596), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1-3, 5-8 and 15 of SEQ ID NO: 308 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXXIXXPXAXXAXK (SEQ ID NO: 603), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1-4, 6, 7, 9, 11, 12 and 14 of SEQ ID NO: 308 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of SGXX (SEQ ID NO: 586), preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 3 and 4 of SEQ ID NO: 587 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXPXAXXAX (SEQ ID NO: 588), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1-3, 5, 7, 8 and 10 of SEQ ID NO: 589 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XGXXXXGAK (SEQ ID NO: 591), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1 and 3-6 of SEQ ID NO: 592 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXXGAKVAV (SEQ ID NO: 593), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1-4 of SEQ ID NO: 594 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXIXXPXA (SEQ ID NO: 597), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1, 2, 4, 5 and 7 of SEQ ID NO: 598 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of IXXPXAXXAX (SEQ ID NO: 599), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2, 3, 5, 7, 8 and 10 of SEQ ID NO: 600 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XPXAXXAX (SEQ ID NO: 601), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1, 3, 5, 6 and 8 of SEQ ID NO: 602 are substituted by other amino acids, preferably alanine.
(3β-6) a polypeptide comprising the amino acid sequence of XXXXPXXXYXXMSSS (SEQ ID NO: 610), preferably a polypeptide comprising the amino acid sequence of XXXXPXXXYLXMSSS (SEQ ID NO: 611), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1-4, 6-8, 10 and 11 of SEQ ID NO: 309 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7 or 8 of the amino acids at positions 1-4, 6-8 and 11 of SEQ ID NO: 309 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXXXXKXYXQMSSX (SEQ ID NO: 612), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1-6, 8, 10 and 15 of SEQ ID NO: 309 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of PXXXYXXMSS (SEQ ID NO: 606), preferably a polypeptide comprising the amino acid sequence of PXXXYLXMSS (SEQ ID NO: 607), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2-4, 6 and 7 of SEQ ID NO: 608 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 2-4 and 7 of SEQ ID NO: 608 are substituted by other amino acids, preferably alanine.
(3β-7) a polypeptide comprising the amino acid sequence of XXLXDXXXXXXXXXX (SEQ ID NO: 620), preferably a polypeptide comprising the amino acid sequence of XXLXDXXXXLXXMXX (SEQ ID NO: 621), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the amino acids at positions 1, 2, 4 and 6-15 of SEQ ID NO: 310 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1, 2, 4, 6-9, 11, 12, 14 and 15 of SEQ ID NO: 310 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of LXDXXXXXX (SEQ ID NO: 614), preferably a polypeptide comprising the amino acid sequence of LXDXXXXLX (SEQ ID NO: 615), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 2 and 4-9 of SEQ ID NO: 616 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 2, 4-7 and 9 of SEQ ID NO: 616 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of DXXXXLXXMX (SEQ ID NO: 618), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 2-5, 7, 8 and 10 of SEQ ID NO: 619 are substituted by other amino acids, preferably alanine.
(3β-8) a polypeptide comprising the amino acid sequence of LHLXXXXXXXXXXXX (SEQ ID NO: 625), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 4-15 of SEQ ID NO: 311 are substituted by other amino acids, preferably alanine.
(4β) Epitope of Vitellogenin-1/Lipovitellin-1
In the present invention, the polypeptide (4β) can be any polypeptide selected from the group consisting of (4β-1) to (4β-7) as defined below:
(4β-1) a polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 312-318, 627, 630, 631, 632, 636, 637 (amino acid sequence: ELL), 641, 642, 644, 647, 649, 657, 658, 659, 660, 663, 664, 665, 668, 669, 672, 674, 676, 680 and 681.
(4β-2) a polypeptide comprising the amino acid sequence of XXXXXMFXQELLFGX (SEQ ID NO: 633), preferably a polypeptide comprising the amino acid sequence of XXXXXMFGQELLFGX (SEQ ID NO: 634), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1-5, 8 and 15 of SEQ ID NO: 312 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1-5 and 15 of SEQ ID NO: 312 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXXXMXXXXXLXXX (SEQ ID NO: 639), preferably a polypeptide comprising the amino acid sequence of SXXXXMFXXXXLXXX (SEQ ID NO: 638), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 1-5, 8-11 and 13-15 of SEQ ID NO: 312 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 2-5, 7-11 and 13-15 of SEQ ID NO: 312 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXYXXMFXXXXXFXX (SEQ ID NO: 643), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 1, 2, 4, 5, 8-12, 14 and 15 of SEQ ID NO: 312 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXXXMFG (SEQ ID NO: 626), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1-5 of SEQ ID NO: 627 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXMFXQELL (SEQ ID NO: 628), preferably a polypeptide comprising the amino acid sequence of XXXMFGQELL (SEQ ID NO: 629), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1-3 and 6 of SEQ ID NO: 630 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2 or 3 of the amino acids at positions 1-3 of SEQ ID NO: 630 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of SXXXXMFX (SEQ ID NO: 635), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 2-5 and 8 of SEQ ID NO: 636 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXYXXMF (SEQ ID NO: 640), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 1, 2, 4 and 5 of SEQ ID NO: 641 are substituted by other amino acids, preferably alanine.
(4β-3) a polypeptide comprising the amino acid sequence of XXXXXXXXDTLXXXX (SEQ ID NO: 650), preferably a polypeptide comprising the amino acid sequence of XXXXRXXXDTLXXXX (SEQ ID NO: 651), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the amino acids at positions 1-8 and 12-15 of SEQ ID NO: 313 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1-4, 6-8 and 12-15 of SEQ ID NO: 313 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXXDTLX (SEQ ID NO: 645), preferably a polypeptide comprising the amino acid sequence of RXXXDTLX (SEQ ID NO: 646), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1-4 and 8 of SEQ ID NO: 647 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 2-4 and 8 of SEQ ID NO: 647 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of DTLXXXXX (SEQ ID NO: 648), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 4-8 of SEQ ID NO: 649 are substituted by other amino acids, preferably alanine.
(4β-4) a polypeptide comprising the amino acid sequence of XXXSKTAXVXXXXXX (SEQ ID NO: 652), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1-3, 8 and 10-15 of SEQ ID NO: 315 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXSXXFAXXRXXXX (SEQ ID NO: 653), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1-3, 5, 6, 9, 10 and 12-15 of SEQ ID NO: 315 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXSXXFAXXXXIXD (SEQ ID NO: 654), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1-3, 5, 6, 9-12 and 14 of SEQ ID NO: 315 are substituted by other amino acids, preferably alanine.
(4β-5) a polypeptide comprising the amino acid sequence of XNIXXXAAXXXXPXX (SEQ ID NO: 655), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1, 4-6, 9-12, 14 and 15 of SEQ ID NO: 316 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXXXXAASXMXPXX (SEQ ID NO: 656), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 1-6, 10, 12, 14 and 15 of SEQ ID NO: 316 are substituted by other amino acids, preferably alanine.
(4β-6) a polypeptide comprising the amino acid sequence of PXXXXMXXDXXXASG (SEQ ID NO: 661), preferably a polypeptide comprising the amino acid sequence of PXIMXMXFDSXSASG (SEQ ID NO: 662), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 2-5, 7, 8 and 10-12 of SEQ ID NO: 317 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3 or 4 of the amino acids at positions 2, 5, 7 and 11 of SEQ ID NO: 317 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XXXXXXXXXXXSASG (SEQ ID NO: 666), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1-11 of SEQ ID NO: 317 are substituted by other amino acids, preferably alanine.
(4β-7) a polypeptide comprising the amino acid sequence of XXXXXXXDVPIEKIQ (SEQ ID NO: 670), preferably a polypeptide comprising the amino acid sequence of XXXXXYSDVPIEKIQ (SEQ ID NO: 671), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6 or 7 of the amino acids at positions 1-7 of SEQ ID NO: 318 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1-5 of SEQ ID NO: 318 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of XFXXVXSXXXXXXXQ (SEQ ID NO: 675), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acids at positions 1, 3, 4, 6 and 8-14 of SEQ ID NO: 318 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXXXXXSXVXIXKXQ (SEQ ID NO: 677), preferably a polypeptide comprising the amino acid sequence of XXXXXXSDVXIXKIQ (SEQ ID NO: 678), more preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the amino acids at positions 1-6, 8, 10 and 12 of SEQ ID NO: 318 are substituted by other amino acids, preferably alanine, further preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5 or 6 of the amino acids at positions 1-6, 10 and 12 of SEQ ID NO: 318 are substituted by other amino acids, preferably alanine.
In another embodiment, a polypeptide comprising the amino acid sequence of SFKPVXXXXXXXXXX (SEQ ID NO: 682), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids at positions 6-15 of SEQ ID NO: 318 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XXDVPIEK (SEQ ID NO: 667), preferably a polypeptide comprising an amino acid sequence in which any 1 or 2 of the amino acids at positions 1 and 2 of SEQ ID NO: 668 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of XSXVXXEX (SEQ ID NO: 673), preferably a polypeptide comprising an amino acid sequence in which any 1, 2, 3, 4 or 5 of the amino acids at positions 1, 3, 5, 6 and 8 of SEQ ID NO: 674 are substituted by other amino acids, preferably alanine.
In yet another embodiment, a polypeptide comprising the amino acid sequence of SFKPVX (SEQ ID NO: 679), preferably a polypeptide comprising an amino acid sequence in which the amino acid at position 6 of SEQ ID NO: 680 is substituted by another amino acid, preferably alanine.
In one embodiment, the polypeptide (4β) may not comprise a variant or a homolog having 100% or at least 90%, at least 80% or at least 70% identity to the central sequence of vitellogenin-1 or the C-terminal moiety (SEQ ID NO: 18) of lipovitellin-1 or the full-length amino acid sequence thereof.
The lengths of the aforementioned polypeptides ((1β) to (4β) are not particularly limited. In a preferred embodiment, the lengths of the aforementioned polypeptides ((1β) to (4β) can be 500 amino acids or less, 300 amino acids or less, 200 amino acids or less, 100 amino acids or less, 50 amino acids or less, 30 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less.
The aforementioned polypeptides (1β) to (4β) may be prepared by a technique of chemical synthesis such as solid-phase peptide synthesis. Alternatively, polypeptides comprising an epitope may be obtained by expressing them as recombinant proteins using a genetic recombination technique well known to those skilled in the art and by separating and producing them using protein producing methods well known to those skilled in the art.
Diagnosis Kit and Method (2)
The present invention provides a method for providing an indicator for diagnosing an allergy in a subject, the method comprising the steps of:
Hereinafter, the polypeptide that is at least one of the aforementioned polypeptides (1β) to (4β), or the polypeptide in which two or more of the aforementioned polypeptides (1β) to (4β) are joined together via or without a spacer is referred to as the “antigen including (1β) to (4β)” in the present specification. The type of the spacer is not particularly limited, and an ordinary spacer that is used by those skilled in the art for joining together two or more peptides can be used. The spacer may be, for example, a hydrocarbon chain such as Acp(6)—OH.
The sample obtained from a subject is as described above in the subsection titled “Diagnosis kit and method (1)”.
Detection of contact and binding between the sample obtained from a subject and the antigen can be carried out by using a known method described above in the subsection titled “Diagnosis kit and method (1)”, such as ELISA (Enzyme-Linked Immunosorbent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, or immunochromatography.
The antigen including (1β) to (4β) may be provided in a state immobilized on a carrier. In this case, the steps (i) and (ii) mentioned above can be carried out using ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, or the like. The step (i) mentioned above can be carried out by contacting the sample obtained from a subject with a surface on which the antigen including (1β) to (4β) is immobilized. The IgE antibody from the subject may be used in a state immobilized on a carrier, and binding to the antigen including (1β) to (4β) may be detected by the aforementioned technique.
The antigen including (1β) to (4β) may be in a state unimmobilized on a carrier. In this case, flow cytometry or the like can be used in the aforementioned steps (i) and (ii), and the presence of IgE antibody-bound antigen including (1β) to (4β) can be confirmed with laser beam. Examples of this method include a basophil activation test (BAT) which is a method in which a surface antigen CD203c that appears when basophils are activated by the contact of the antigen including (1β) to (4β) is detected. Another example includes a histamine release test (HRT) which examines whether histamine is released by further contacting the antigen including (1β) to (4β) with blood cells in a sample.
The antigen including (1β) to (4β) is an antigen specifically binding to IgE antibodies from allergic patients. Therefore, when binding between an IgE antibody from a subject and the antigen is detected, an indicator of the fact that the subject is allergic to egg is provided.
The present invention further provides a kit for diagnosing an allergy, comprising at least one antigen including (1β) to (4β). The diagnosis kit of this invention may be used in the aforementioned method for providing an indicator for diagnosing an allergy or in a diagnosis method as described later. The diagnosis kit of this invention may comprise not only the at least one antigen including (1β) to (4β), but also an anti-IgE antibody labeled with an enzyme and a chromogenic or luminescent substrate serving as a substrate for the enzyme. Also, a fluorescent-labeled anti-IgE antibody may be used. In the diagnosis kit of this invention, the antigen including (1β) to (4β) may be provided in a state immobilized on a carrier. The diagnosis kit of this invention may also be provided together with instructions on the procedure for diagnosis or a package containing said instructions.
In another embodiment, the aforementioned diagnosis kit comprises a companion diagnostic agent for an allergy. The companion diagnostic agent is used for identifying patients expected to respond to pharmaceutical products or identifying patients having the risk of severe adverse reactions to pharmaceutical products, or for studying the reactivity of pharmaceutical products in order to optimize treatment using the pharmaceutical products. Here, the optimization of treatment includes, for example, determination of dosage and administration, judgment regarding discontinuation of administration, and confirmation of an allergen component that is used to cause immunological tolerance.
The present invention further provides a composition for diagnosing an allergy, comprising at least one antigen including (1β) to (4β). The diagnosis composition of this invention can be used in a diagnosis method as described below. The diagnosis composition of this invention may further comprise a pharmaceutically acceptable carrier and/or additives commonly used with the antigen of this invention depending on the need.
In one embodiment, the present invention provides a method for diagnosing an allergy in a subject, the method comprising:
In another embodiment, the present invention provides a method for diagnosing an allergy in a subject, the method comprising administering to the subject at least one antigen including (1β) to (4β). This method may be performed in the form of a skin test characterized by applying the antigen including (1β) to (4β) onto the skin. Examples of the skin test include various forms of tests, such as: a prick test in which a diagnosis composition is applied onto the skin and then a tiny prick to such an extent as not to provoke bleeding is made in the skin to allow the antigen including (1β) to (4β) to penetrate the skin, thereby observing a skin reaction; a scratch test in which a diagnosis composition is applied onto the skin and then the skin is lightly scratched to observe a reaction; a patch test in which a diagnosis composition in the form of cream, ointment, etc. is applied onto the skin to observe a reaction; and an intracutaneous test in which the antigen including (1β) to (4β) is administered intracutaneously to observe a reaction. If a skin reaction such as swelling occurs in a skin portion to which the antigen including (1β) to (4β) has been applied, the subject of interest is diagnosed as having an allergy. The amount of the antigen including (1β) to (4β) to be applied to the skin in such tests can be, for example, not more than 100 μg per dose.
In the process of allergy diagnosis, a load test aiming to identify an antigen is often adopted. At least one antigen including (1β) to (4β) can be used as an active ingredient for a load test to diagnose an allergy. Here, the allergen component to be used in the load test may be a polypeptide that has been expressed and purified and may be a protein that has been expressed in food or cooking ingredients, such as rice-based vaccine expressing pollen allergens which are obtained by transforming rice with a gene of a cedar pollen antigen and expressing the antigen protein in the rice.
In still another embodiment, the present invention provides at least one antigen including (1β) to (4β), intended for use in the diagnosis of an allergy.
In still another embodiment, the present invention provides use of at least one antigen including (1β) to (4β) for the production of a diagnostic agent for an allergy.
In this subsection, the allergy to be diagnosed or detected can be allergies to the antigen including (1β) to (4β). Thus, detection of the allergy can be detection of not only an allergy to a single antigen including (1β) to (4β), but also allergies including cross-reactivity.
Pharmaceutical Composition and Treatment Method (2)
The present invention provides a pharmaceutical composition comprising at least one antigen including (1β) to (4β). In one embodiment, the aforementioned pharmaceutical composition is used for the treatment of an allergy. The treatment of an allergy increases the limit amount of an antigen in which the allergy does not develop even if the antigen is incorporated into the body, and finally aims for the state where the allergy does not develop by the common amount of the antigen to be consumed (remission).
The present invention also provides a method for treating an allergy, the method comprising administering at least one antigen including (1β) to (4β) to a patient in need of a treatment for an allergy.
In another embodiment, the present invention provides at least one antigen including (1β) to (4β), intended for use in the treatment for an allergy. In yet another embodiment, the present invention provides use of at least one antigen including (1β) to (4β) for the production of a therapeutic agent for an allergy.
In the process of allergy treatment, a hyposensitization therapy aiming to induce immunological tolerance by administering an antigen to a patient is often adopted. The at least one antigen including (1β) to (4β) can be used as an active ingredient for hyposensitization therapy for an allergy. Here, the allergen component to be used in the hyposensitization therapy may be a polypeptide that has been expressed and purified and may be a polypeptide that has been expressed in food or a cooking ingredient, such as rice-based vaccine expressing pollen allergens.
The administration route, administration dose, frequency and/or period of the pharmaceutical composition of this invention, and other ingredients to be contained in the pharmaceutical composition, and the dosage form can be as described above in the subsection titled “Pharmaceutical composition and treatment method (1)”. In the case of using the antigen including (1β) to (4β), for example, the dose to an adult patient may be a dose of not more than 100 μg per dose.
In this subsection, the allergy to be treated can be allergies to the antigen including (1β) to (4β). Thus, treatment of the allergy can be treatment of not only an allergy to a single antigen including (1β) to (4β), but also allergies including cross-reactivity.
Tester (2)
The present invention provides a tester comprising an antibody for at least one antigen including (1β) to (4β).
The antibody can be prepared by a conventional method. For example, the antibody may be prepared by immunizing a mammal such as rabbit with the aforementioned antigen including (1β) to (4β). The antibody may be an Ig antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab′)2, Fab′).
Further, in the aforementioned tester, the antibody may be provided in a form bound to a carrier. The type of the carrier is not particularly limited as long as it is usable for detection of binding between an antibody and the antigen including (1β) to (4β). Any given carrier known to those skilled in the art can be used. Also, the antibody for the antigen including (1β) to (4β) is preferably an antibody for the epitopes described above in the subsection titled “Epitope of antigen”. This can attain a tester that can also detect cross-reactivity.
Examples of a method for determining the presence or absence of the antigen including (1β) to (4β) include the following methods:
Another embodiment of the present invention includes a tester for determining the presence or absence of the antigen including (1β) to (4β) of an allergy in an object of interest, the tester comprising a primer appropriate for an epitope. The primer is not limited and may be designed so as to comprise, for example, a portion of the nucleotide sequence of a nucleic acid encoding any of the amino acid sequences defined above in (1β) to (4β), or a complementary strand thereof. Alternatively, the primer may be designed so as to be the nucleotide sequence of a region upstream of a portion encoding an epitope that is any of the amino acid sequences defined above in (1β) to (4β), in a nucleic acid encoding a protein comprising the epitope, or the nucleotide sequence of a complementary strand of a region downstream of the portion encoding the epitope. Examples of such a primer include a primer which is a portion of at least one of the nucleotide sequences of SEQ ID NOs: 46, 58, 105, 151, 236, 250 and 261 and/or a primer which is a portion of a sequence complementary to at least one of the nucleotide sequences of SEQ ID NOs: 46, 58, 105, 151, 236, 250 and 261. Here, the position of the epitope in the full-length sequence of an antigen is as defined in Table 2 of Example 6 given below. Particularly, when mRNA is of interest, the tester has a complementary primer of a poly-A tail.
For example, DNA is amplified by PCR (Polymerase Chain Reaction) including RT-PCR using templated DNA or mRNA obtained from a sample and the aforementioned primer, and the presence or absence of a nucleic acid encoding the amino acid sequences defined above in (1β) to (4β) in the sequence of the amplified DNA is determined to determine the presence or absence of the antigen including (1β) to (4β). Amplification methods by PCR for mRNA of interest can be exemplified by RACE.
In one embodiment, the aforementioned tester is used to determine the presence or absence of the antigen including (1β) to (4β) in raw materials or processed products of interest in a food production line. The raw material may be a cooking ingredient or may be a cosmetic raw material, a pharmaceutical raw material or the like. The processed product may be an edible processed product or may be a cosmetic, a pharmaceutical product or the like. The tester may also be used for search for organism species contained in raw materials, may be used for quality inspection of production lines and pre-shipment products by manufacturers, or may be used for self-checking of the presence or absence of an antigen in raw materials or processed products of interest by consumers or users.
Allergen-Free Raw Material and the Like (2)
The present invention provides a raw material or a processed product in which at least one antigen including (1β) to (4β) is eliminated or reduced.
The method for eliminating or reducing the antigen of the present invention in a raw material or a processed product is not limited. The elimination or reduction of the antigen may be conducted by any method, as long as the method permits the elimination or reduction of the antigen including (1β) to (4β). For example, the techniques described above in the subsection titled “Allergen-free food and the like (1)” may be used.
Elimination or reduction of at least one antigen including (1β) to (4β) may be achieved by eliminating or reducing the whole antigen or may be achieved by cleaving or removing the sequence moiety defined in (1β) to (4β) from the antigen protein. The “removal” includes deletion and modification of the whole or a portion of a sequence moiety defined above in (1) to (6).
For example, the raw material in which the antigen including (1β) to (4β) is eliminated or reduced may be obtained by preparing a raw material in which the expression of the antigen including (1β) to (4β) is knocked out, using a gene knock-out technique. Any technique known to those skilled in the art such as genetic modification can be used as the gene knock-out technique.
The processed product in which the antigen including (1β) to (4β) is eliminated or reduced may be a processed product of the raw material in which the antigen including (1β) to (4β) is eliminated or reduced, such as powdered milk obtained with purified peptide as a raw material. In the case of using an ordinary raw material, a treatment for eliminating or reducing the antigen including (1β) to (4β) is performed before or after preparation of a processed product. The techniques described in the subsection titled “Allergen-free food and the like (1)” may be used as methods for eliminating or reducing the antigen including (1β) to (4β) in a processed product obtained with an ordinary raw material. Examples of the method for cleaving the antigen including (1β) to (4β) include a method in which the antigen is treated by cleavage with a particular digestive enzyme.
Method for Producing Allergen-Free Processed Product (2)
The present invention provides a method for producing a processed product in which an antigen is eliminated or reduced, the method comprising the step of confirming that the antigen is eliminated or reduced, in a production process of the processed product, wherein the antigen is at least one of the aforementioned antigens including (1β) to (4β).
In the production method, elimination or reduction of an antigen means that at least one antigen including (1β) to (4β) is eliminated or reduced, or the sequence moiety defined in (1β) to (4β) are cleaved or removed from the antigen.
A technique of confirming that the antigen is eliminated or reduced in the production process of the processed product is not particularly limited, and any technique capable of detecting at least one of the aforementioned antigen including (1β) to (4β) may be used. For example, the presence or absence of the polypeptide or the antigen in the processed product may be confirmed from the binding activity of an antibody for at least one of the aforementioned antigen including (1β) to (4β) against a sample containing a material resulting from the production process of the processed product. Details of such a method are as described above in the subsection titled “Diagnosis kit and method (2)”. Thus, in the production method, the “IgE antibody from a subject” described above in the subsection titled “Diagnosis kit and method (2)” is replaced with the “antibody for at least one of the aforementioned antigen including (1β) to (4β)”, and the “antigen” described above in the subsection titled “Diagnosis kit and method (2)” is replaced with the “sample containing a material resulting from the production process of the processed product”. The techniques described above in the subsection titled “Diagnosis kit and method (2)” can be used to confirm that the antigen is eliminated or reduced in the production process of the processed product. The testers described above in the subsection titled “Tester (2)” can also be used.
The following describes examples of the present invention. The technical scope of this invention is not limited by these examples.
Proteins contained in eggs were investigated using a two-dimensional electrophoresis method described below.
Protein Extraction
Raw chicken eggs were separated into albumen and yolk.
Extraction and purification of proteins contained in albumen and yolk were carried out as follows. The proteins were extracted by adding a solubilizer to albumen. Then, water or a urea buffer was added thereto to obtain a liquid protein extract. The constituents of the urea buffer are as mentioned below.
30 mM Tris
2 M thiourea
7 M urea
4% (w/v) CHAPS:
3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate
Moderate Amount of Dilute Hydrochloric Acid
The total amount was adjusted to 100 mL by the addition of distilled water. The pH was 8.5.
Then, 25 μg (by weight) each of the proteins was mixed to obtain a liquid extract. The proteins were extracted by adding a surfactant buffer (Mammalian Lysis Buffer (MCLI), Sigma-Aldrich Co. LLC) as a solubilizer to yolk.
Thereafter, the precipitation procedure was repeated twice using a 2D-CleanUP Kit (produced by GE). In the first round of precipitation, the collected liquid protein extract was precipitated by adding TCA (trichloroacetic acid) thereto and the precipitated product produced by this procedure (TCA-precipitated product) was collected. In the second round of precipitation, the TCA-precipitated product collected above was further precipitated by adding acetone thereto and the precipitated product (sample) produced by this procedure was collected.
Preparation of a Sample Solution
Part of the collected sample (50 μg on a protein weight basis) was dissolved in 150 μL of a DeStreak Rehydration Solution (produced by GE), which is a swelling buffer for first-dimensional isoelectric focusing gels, thereby obtaining a sample solution for first-dimensional isoelectric focusing (sample solution for swelling). The constituents of the DeStreak Rehydration Solution are as mentioned below.
7 M thiourea
2 M urea
4% (w/v) of CHAPS
0.5% (v/v) IPG buffer; produced by GE
Moderate amount of BPB (bromophenol blue)
Penetration of the Sample into First-Dimensional Isoelectric Focusing Gels
First-dimensional isoelectric focusing gel strips (Immobiline Drystrip IPG gels (pH3-10 NL); produced by GE) were immersed in 140 μL of the foregoing sample solution for first-dimensional isoelectric focusing (sample solution for swelling) and impregnated with the solution at room temperature overnight.
In this example, an IPGphor electrophoresis system produced by GE was used.
An electrophoresis tray was filled with silicone oil. Filter paper moisten with water was positioned at both ends of the gel strips impregnated with the sample, and the gel strips were set in the electrophoresis tray such that the gel strips were covered with silicone oil. Electrodes were placed on the gel strips with the filter paper intervening therebetween.
The maximum current of the isoelectric focusing system was set to 75 μA per gel strip, and the first-dimensional isoelectric focusing was carried out according to the following voltage program: (1) a constant voltage step was performed at a constant voltage of 300 V until the volt-hours reached 750 Vhr (the current variation width during electrophoresis for 30 minutes before the end of this step was 5 μA); (2) the voltage was increased gradually to 1000 V for 300 Vhr; (3) the voltage was further increased gradually to 5000 V for 4500 Vhr; and then (4) the voltage was held at a constant voltage of 5000 V until the total Vhr reached 12000.
SDS Equilibration of Isoelectric Focusing Gels
After the aforementioned first-dimensional isoelectric focusing was done, the gel strips were taken out of the isoelectric focusing system, immersed in an equilibration buffer containing a reducing agent, and shaken at room temperature for 15 minutes. The constituents of the equilibration buffer containing the reducing agent are as mentioned below.
100 mM Tris-HCl (pH 8.0)
6 M urea
30% (v/v) glycerol
2% (w/v) SDS
1% (w/v) DTT
Next, the equilibration buffer containing the reducing agent was removed, and then the gel strips were immersed in an equilibration buffer containing an alkylating agent and shaken at room temperature for 15 minutes to obtain SDS-equilibrated gels. The constituents of the equilibration buffer containing the alkylating agent are as mentioned below.
100 mM Tris-HCl (pH 8.0)
6 M urea
30% (v/v) glycerol
2% (w/v) SDS
2.5% (w/v) iodoacetamide
Second-Dimensional SDS-PAGE
In this example, the XCell SureLock Mini-Cell electrophoresis system produced by Life Technologies was used. The second-dimensional electrophoresis gels used were NuPAGE 4-12% Bis-Tris Gels produced by Life Technologies. Also, an electrophoresis buffer composed of the following constituents was prepared and used.
50 mM MOPS
50 mM Tris base
0.1% (w/v) SDS
1 mM EDTA
Further, an agarose solution for gel adhesion was used in this example, which was prepared by dissolving 0.5% (w/v) Agarose S (produced by Nippon Gene Co., Ltd.) and a moderate amount of BPB (bromophenol blue) in the electrophoresis buffer.
SDS-PAGE wells were washed well with the electrophoresis buffer, and then the buffer used for the washing was removed. Next, the washed wells were charged with the fully dissolved agarose solution for gel adhesion. Next, the SDS-equilibrated gel strips were immersed in agarose and closely adhered to second-dimensional electrophoresis gels using tweezers. After it was confirmed that agarose was fully fixed with the gels being closely adhered to each other, electrophoresis was performed at a constant voltage of 200 V for about 45 minutes.
Fluorescent Staining of Gels
The gels were fluorescently stained with SYPRO Ruby (produced by Life Technologies).
First, an airtight container to be used was washed well in advance with 98% (v/v) ethanol. The electrophoresed second-dimensional electrophoresis gel strips were taken out of the SDS-PAGE system, placed onto the washed airtight container, and treated twice by immersion in 50% (v/v) methanol and 7% (v/v) aqueous solution containing acetic acid for 30 minutes. Then, a further immersion treatment was done for 10 minutes, with the solution being replaced by water. Next, the second-dimensional electrophoresis gel strips were immersed in 40 mL of SYPRO Ruby and shaken at room temperature overnight. Thereafter, the SYPRO Ruby was removed, and then the second-dimensional electrophoresis gel strips were washed with water and shaken in 10% (v/v) methanol and 7% (v/v) aqueous solution containing acetic acid for 30 minutes. Further shaking was done for at least 30 minutes, with the solution being replaced by water.
Analysis
The second-dimensional electrophoresis gels obtained through the foregoing series of treatments were subjected to fluorescent image scanning on Typhoon9400 (produced by GE). The results of the two-dimensional electrophoresis as to the proteins contained in the yolk are shown in
Identification of antigens by immunoblotting was carried out by taking all the steps up to the step of “Second-dimensional SDS-PAGE” as described above in Example 1, followed by the steps of “Transfer to membrane”, “Immunoblotting” and “Analysis” as described below.
Transfer to Membrane
Transfer to membrane was done using the following transfer system and transfer buffer.
To be specific, proteins in the two-dimensional electrophoresis gels were transferred to a membrane (PVDF membrane) according to the following procedure.
(1) The PVDF membrane was immersed in 100% methanol followed by milliQ water, and then moved into the transfer buffer to hydrophilize the PVDF membrane.
(2) After sponge, filter paper, gels treated by second-dimensional SDS-PAGE, the hydrophilized PVDF membrane, filter paper, and sponge were put in place in this order, the transfer system was energized at a constant voltage of 30 V for one hour.
Immunoblotting
Immunoblotting of the membrane was carried out using, as a primary antibody, a serum sample from patient (patient 1) with an egg allergy or a serum sample from a non-egg-allergic subject. This egg-allergic patient was a patient diagnosed with food-dependent exercise-induced anaphylaxis (FDEIA) caused by chicken eggs. This patient exhibited negativity to all of raw eggs, boiled eggs, and hot spring eggs in the prick test, and also exhibited negativity to albumen, yolk, and ovomucoid in the specific IgE antibody test.
Immunoblotting of the membrane was carried out according to the following procedure.
Analysis
The membrane obtained through the foregoing series of treatments was subjected to fluorescent image scanning on Typhoon9500 (produced by GE).
The immunoblots obtained with the serum from the egg-allergic patient were compared with those obtained with the control serum from the non-egg-allergic subject. By immunoblotting of the proteins contained in the raw yolk using the serum from the egg-allergic patient (
The molecular weights and isoelectric points of the 3 spots are as follows (
The amino acid sequences of the antigens that form the 3 protein spots of Example 2 were identified by mass spectroscopy.
To be specific, protein extraction and mass spectroscopy were done by the following procedure.
Results
The mass spectrometry of the spots resulted in the detection of the following amino acid sequences.
Furthermore, the spots were identified as the following proteins by the analysis of the mass data obtained from the mass spectrometer for the spots at NCBI for spots 1 and 2, and at UniProt for spot 3.
Identification of antigens by immunoblotting was carried out using the sera of three other egg-allergic patients (patient 2, patient 3 and patient 4) in the same manner as in Example 2. By immunoblotting of the proteins contained in the raw yolk using the serum from the egg-allergic patients (
The molecular weights and isoelectric points of the spots 4 to 11 are as follows (
The amino acid sequences of the antigens that form spots 4 to 11 of Example 4 were identified by mass spectroscopy in the same manner as in Example 3.
The mass spectrometry of the spots resulted in the detection of the following amino acid sequences.
Furthermore, the spots were identified as the following proteins by the analysis of the mass data obtained from the mass spectrometer for the spots at NCBI.
Epitopes of Chicken-Egg Allergen Components
Identification of epitopes was carried out by the following procedure as to the chicken-egg allergen components.
(A) Epitope Mapping (1)
Epitope mapping was carried out using a library of overlapping peptides (length: 15 amino acids) corresponding to the amino acid sequences identified as allergy components of chicken egg. Specifically, the library of overlapping peptides was prepared on the basis of the amino acid sequences of SEQ ID NOs: 59, 106 and 262 for vitellogenin-2 precursor, SEQ ID NOs: 2, 13 and 47 for vitellogenin-3, SEQ ID NOs: 152, 237 and 251 for apolipoprotein B precursor, and SEQ ID NO: 18 for vitellogenin-1 or lipovitellin-1.
The peptides to be synthesized were shifted by 10 amino acids. Thus, each peptide had an overlap of 5 amino acids with each of the peptides previous and subsequent thereto.
For preparation of peptide arrays, the Intavis CelluSpots™ technique was used. Specifically, the peptide arrays were prepared by the following procedure: (1) synthesizing peptides of interest on amino-modified cellulose disks using an automated chemical synthesis apparatus (Intavis MultiPep RS), (2) dissolving the amino-modified cellulose disks to obtain a cellulose-bound peptide solution, and (3) spotting the cellulose-bound peptides onto coated glass slides. The details of each procedure are as described below.
(1) Synthesis of Peptide
Peptide synthesis was carried out in incremental steps through 9-fluorenylmethoxycarbonyl (Fmoc) chemical reaction on amino-modified cellulose disks in 384-well synthesis plates. Specifically, amino acids in which a Fmoc group is bonded to an amino group were activated in a solution of N,N′-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF) and added dropwise to the cellulose disks so that the Fmoc group-bound amino acids were bound to the amino groups on the cellulose disks (coupling). Unreacted amino groups were capped with acetic anhydride and washed with DMF. Furthermore, the Fmoc groups were eliminated from the amino groups of the amino acids bound to the amino groups on the cellulose disks by treatment with piperidine and washing with DMF. The amino acids bound to the amino groups on the cellulose disks were repetitively subjected to the coupling, the capping, and the Fmoc group elimination described above to elongate the amino terminus for peptide synthesis.
(2) Dissolution of Amino-Modified Cellulose Disk
The peptides-bound cellulose disks of interest obtained above in the subsection titled “(1) Synthesis of peptide” were transferred to 96-well plates and treated with a side chain deprotection mixed solution of trifluoroacetic acid (TFA), dichloromethane, triisopropylsilane (TIPS), and water for deprotection of amino acid side chains. Then, the deprotected cellulose-bound peptides were dissolved in a mixed solution of TFA, perfluoromethanesulfonic acid (TFMSA), TIPS, and water and precipitated in tetrabutyl methyl ether (TBME), and the precipitate was resuspended in dimethyl sulfoxide (DMSO) and mixed with a mixed solution of NaCl, sodium citrate, and water to obtain a peptide solution for slide spotting.
(3) Spotting of Cellulose-Bound Peptide Solution
The peptide solution for slide spotting obtained above in the subsection titled “(2) Dissolution of amino-modified cellulose disk” was spotted onto Intavis CelluSpots™ slides using Intavis Slide Spotting Robot, and the slides were dried to prepare peptide arrays.
The presence or absence of binding, to each peptide fragment, of an IgE antibody present in the serum of a WDEIA patient was measured through antigen-antibody reaction using the peptide arrays. The measurement was carried out according to the following procedure.
Chemiluminescence intensity in images obtained by the measurement described above in (7) was converted into a numeric value using ImageQuant TL (GE Healthcare). The maximum value of numeric values determined from images obtained from results about the serum of 5 non-egg-allergic subjects was defined as a Nmax value as a value from the non-egg-allergic subjects. Numeric values determined from images obtained from results about the serum of 6 patients were divided by the Nmax value of each peptide. It was determined that a peptide having the resulting value larger than 0 and having a value of at least 30,000 in terms of the numeric values determined from images obtained from results about the serum of the patients was a peptide bound to an IgE antibody in a patient-specific manner.
(B) Epitope Mapping (2): Overlapping
On the basis of the sequences (SEQ ID NOs: 272-295 for vitellogenin-2 precursor, SEQ ID NOs: 296-303 for vitellogenin-3, SEQ ID NOs: 304-311 for apolipoprotein B precursor, and SEQ ID NOs: 312-318 for vitellogenin-1 or lipovitellin-1) of the peptides bound to an IgE antibody in serum in a patient-specific manner as described above in (A), a library of overlapping peptide fragments (length: 10 amino acids) was prepared using the sequences of the peptides and sequences in which sequences were added so as to flank each peptide in the amino acid sequences of allergen components (amino acid sequences of SEQ ID NO: 59, 106 or 262 for vitellogenin-2 precursor, SEQ ID NO: 2, 13 or 47 for vitellogenin-3, SEQ ID NO: 152, 237 or 251 for apolipoprotein B, and SEQ ID NO: 18 for vitellogenin-1 or lipovitellin-1) comprising the sequences of the peptide to carry out epitope mapping.
The peptides to be synthesized were shifted by one amino acid. Thus, each peptide had an overlap of 9 amino acids with each of the peptides previous and subsequent thereto.
The library was prepared by the same procedure as described above in (A), and the presence or absence of binding of an IgE antibody present in the serum of a patient and a non-egg-allergic subject to each peptide fragment was measured by the same technique as described above. Amino acids at positions where the binding activity against IgE antibodies from patients were lost or decreased in the peptides shifted by one amino acid while binding activity against IgE antibodies from non-egg-allergic subjects arose were confirmed as amino acids adversely affecting the specificity of binding to IgE antibodies.
Chemiluminescence intensity in images obtained by measurement was converted into a numeric value in the same manner as in (A) using ImageQuant TL (GE Healthcare). Numeric values determined from images obtained as a result of using only anti-human IgE antibody-HRP antibody without carrying out the procedures (2) and (3) described above in the subsection titled (3) Spotting of cellulose-bound peptide solution were divided by numeric values determined from images obtained from results about the serum of 6 patients. It was determined that a peptide having the resulting value larger than 0 had no binding activity against IgE antibodies when having less than 20% of values obtained using the sequences (amino acid sequences of SEQ ID NO: 59, 106 or 262 for vitellogenin-2, SEQ ID NO: 2, 13 or 47 for vitellogenin-3, SEQ ID NO: 152, 237 or 251 for apolipoprotein B, and SEQ ID NO: 18 for vitellogenin-1 or lipovitellin-1) serving as the basis of overlapping, had binding activity, albeit poor, against IgE antibodies when having 20% or more and less than 50% thereof, had binding activity, albeit somewhat poor, against IgE antibodies when having 50% or more and less than 70% thereof, had almost equivalent binding activity against IgE antibodies when having 70% or more and less than 90% thereof, and had equivalent or good binding activity against IgE antibodies and thus had remaining binding activity against IgE antibodies when having 90% or more thereof.
(C) Epitope Mapping (3): Alanine Scanning
From the amino acid sequences identified above in (A), a library of peptide fragments in which N-terminal amino acids were substituted one by one by alanine according to a technique called alanine scanning (Non Patent Literature 1) was prepared by the same technique as described above. The presence or absence of binding of an IgE antibody present in the serum of a patient and a non-egg-allergic subject to each peptide fragment was measured by the same technique as described above. Amino acids at positions where the binding activity against IgE antibodies from patients were lost or decreased by the substitution by alanine while binding activity against IgE antibodies from non-egg-allergic subjects arose were confirmed as amino acids important for exertion of original antigenicity, or amino acids influencing exertion of original antigenicity. Also, amino acids at positions where the binding activity against IgE antibodies from patients was maintained even after the substitution by alanine while binding activity against IgE antibodies from non-egg-allergic subjects did not arise were confirmed as substitutable positions that were not important for exertion of original antigenicity,
Chemiluminescence intensity in images obtained by measurement was converted into a numeric value in the same manner as in (A) using ImageQuant TL (GE Healthcare). Numeric values determined from images obtained as a result of using only anti-human IgE antibody-HRP antibody without carrying out the procedures (2) and (3) described above in the subsection titled (3) Spotting of cellulose-bound peptide solution were divided by numeric values determined from images obtained from results about the serum of 6 patients. It was determined that a peptide having the resulting value larger than 0 had no binding activity against IgE antibodies when having less than 20% of values obtained using the sequences (amino acid sequences of SEQ ID NO: 59, 106 or 262 for vitellogenin-2, SEQ ID NO: 2, 13 or 47 for vitellogenin-3, SEQ ID NO: 152, 237 or 251 for apolipoprotein B, and SEQ ID NO: 18 for vitellogenin-1 or lipovitellin-1) serving as the basis of overlapping, had binding activity, albeit poor, against IgE antibodies when having 20% or more and less than 50% thereof, had binding activity, albeit somewhat poor, against IgE antibodies when having 50% or more and less than 70% thereof, had almost equivalent binding activity against IgE antibodies when having 70% or more and less than 90% thereof, and had equivalent or good binding activity against IgE antibodies and thus had remaining binding activity against IgE antibodies when having 90% or more thereof.
By this analysis, common sequences important for exertion of original antigenicity were found.
(D) Results
As a result of the epitope mapping described above in (A), an IgE antibody was confirmed to bind in a patient-specific manner to each of epitope Nos. 1-24 (peptides having the amino acid sequences of SEQ ID NOs: 272-295, respectively) for vitellogenin-2 precursor, epitope Nos. 25-32 (peptides having the amino acid sequences of SEQ ID NOs: 296-303, respectively) for vitellogenin-3, epitope Nos. 33-40 (peptides having the amino acid sequences of SEQ ID NOs: 304-311, respectively) for apolipoprotein B precursor, and epitope Nos. 41-47 (peptides having the amino acid sequences of SEQ ID NOs: 312-318, respectively) for vitellogenin-1 or lipovitellin-1. Portions to which the peptide sequences of these epitopes correspond in the sequences of the allergen components identified in Examples 2 to 5 are shown in Table 2.
Amino acids important for binding of the epitope sequences to IgE antibodies from allergic patients were identified by the procedures of (B) and (C) mentioned above as to each of the epitopes.
(1) Epitope 1
In epitope 1 (SEQ ID NO: 272), SEQ ID NO: 323 was identified as a region important for binding to IgE antibodies from patients. Further, SEQ ID NO: 319 was identified as a sequence comprising a region amino-terminally adjacent to epitope 1 and binding to IgE antibodies from patients.
In SEQ ID NO: 272, the amino acids at positions 6, 8-12, 14 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 4, 5, 7 and 13 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-3 of SEQ ID NO: 272 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 323, the amino acids at positions 2-6 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 1 was identified as an amino acid influencing binding to IgE antibodies from allergic patients.
(2) Epitope 2
In epitope 2 (SEQ ID NO: 273), SEQ ID NO: 327 was identified as a region important for binding to IgE antibodies from patients. Further, SEQ ID NO: 324 was identified as a sequence comprising a region amino-terminally adjacent to epitope 2 and binding to IgE antibodies from patients.
In SEQ ID NO: 273, the amino acids at positions 9-12 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-8 and 13-15 of SEQ ID NO: 273 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 327, the amino acids at positions 9 and 10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 4 and 5 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-3 and 6-8 of SEQ ID NO: 327 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(3) Epitope 3
In epitope 3 (SEQ ID NO: 274), SEQ ID NOs: 330, 331, 333 and 335 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 336 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 3 and binding to IgE antibodies from patients.
In SEQ ID NO: 274, the amino acids at positions 1, 3 and 6 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2, 4, 5 and 7-15 of SEQ ID NO: 274 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 274, the amino acids at positions 1, 6, 10 and 11 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 2-5, 7-9 and 12-15 of SEQ ID NO: 274 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 330, the amino acids at positions 1, 3 and 6 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2, 4, 5, 7 and 8 of SEQ ID NO: 330 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 335, the amino acids at positions 1, 5 and 6 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2-4 and 7-10 of SEQ ID NO: 335 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(4) Epitope 4
In epitope 4 (SEQ ID NO: 275), SEQ ID NOs: 339, 343, 344, 346, 347 and 350 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 342 was identified as a sequence comprising a region amino-terminally adjacent to epitope 4 and binding to IgE antibodies from patients, and SEQ ID NO: 351 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 4 and binding to IgE antibodies from patients.
In SEQ ID NO: 275, the amino acids at positions 1, 4-6 and 10-13 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2, 3, 7-9, 14 and 15 of SEQ ID NO: 275 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 275, the amino acids at positions 1, 6, 12-14 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 2-5, 7-11 and 15 of SEQ ID NO: 275 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 275, the amino acids at positions 1, 6, 8, 14 and 15 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 2-5, 7, 9-13 of SEQ ID NO: 275 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 339, the amino acids at positions 1, 4-6 and 10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2, 3 and 7-9 of SEQ ID NO: 339 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 346, the amino acids at positions 1-6 and 10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 7-9 of SEQ ID NO: 346 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 350, the amino acids at positions 1 and 3 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2 and 4-8 of SEQ ID NO: 350 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(5) Epitope 5
In epitope 5 (SEQ ID NO: 276), SEQ ID NOs: 355 and 356 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 353 was identified as a sequence comprising a region amino-terminally adjacent to epitope 5 and binding to IgE antibodies from patients, and SEQ ID NO: 357 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 5 and binding to IgE antibodies from patients.
In SEQ ID NO: 276, the amino acids at positions 2, 3 and 6-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 4 and 5 of SEQ ID NO: 276 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 355, the amino acids at positions 2-8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 1 of SEQ ID NO: 355 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(6) Epitope 6
In epitope 6 (SEQ ID NO: 277), SEQ ID NOs: 366 and 369 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NOs: 359 and 363 were identified as sequences comprising a region amino-terminally adjacent to epitope 6 and binding to IgE antibodies from patients, and SEQ ID NO: 360 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 6 and binding to IgE antibodies from patients.
In SEQ ID NO: 277, the amino acids at positions 1, 5-9, 11 and 13-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 2 and 10 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 3, 4 and 12 of SEQ ID NO: 277 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 277, the amino acids at positions 3, 7, 9, 11 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 1, 2 and 4-6 were identified as possible amino acids influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 8, 10 and 12-14 of SEQ ID NO: 277 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 366, the amino acids at positions 1, 5, 7 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 2-4 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 6, 8 and 10 of SEQ ID NO: 366 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 369, the amino acids at positions 2, 4, 6 and 10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 1 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 3, 5 and 7-9 of SEQ ID NO: 369 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(7) Epitope 7
In epitope 7 (SEQ ID NO: 278), SEQ ID NO: 374 was identified as a region important for binding to IgE antibodies from patients. Further, SEQ ID NO: 375 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 7 and binding to IgE antibodies from patients.
In SEQ ID NO: 278, the amino acids at positions 1-6, 8, 11, 13 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 7, 9, 10, 12 and 14 of SEQ ID NO: 278 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 374, the amino acids at positions 1, 3, 4, 6 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 2 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 5, 7 and 8 of SEQ ID NO: 374 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(8) Epitope 8
In epitope 8 (SEQ ID NO: 279), SEQ ID NOs: 379, 388, 391, 392, 397 and 400 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NOs: 377, 384 and 385 were identified as sequences comprising a region amino-terminally adjacent to epitope 8 and binding to IgE antibodies from patients, and SEQ ID NOs: 380, 381, 393, 401 and 402 were identified as sequences comprising a region carboxy-terminally adjacent to epitope 8 and binding to IgE antibodies from patients.
In SEQ ID NO: 279, the amino acids at positions 1-5, 8-12, 14 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 13 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 6 and 7 of SEQ ID NO: 279 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 279, the amino acids at positions 3, 9 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 4 was identified as a possible amino acid influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1, 2, 5-8 and 10-14 of SEQ ID NO: 279 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 279, the amino acids at positions 1-3, 8 and 9 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 4-7 and 10-15 of SEQ ID NO: 279 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 379, the amino acids at positions 1-4 and 7-10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 5 and 6 of SEQ ID NO: 379 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 388, the amino acids at positions 2 and 8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 3 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1, 4-7 and 9 of SEQ ID NO: 388 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 392, the amino acids at positions 4 and 10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 8 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-3, 5-7 and 9 of SEQ ID NO: 392 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 397, the amino acids at positions 1, 2, 7 and 8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 3-6, 9 and 10 of SEQ ID NO: 397 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 400, the amino acids at positions 3 and 4 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 1, 6 and 9 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2, 5, 7, 8 and 10 of SEQ ID NO: 400 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(9) Epitope 9
In epitope 9 (SEQ ID NO: 280), SEQ ID NO: 407 was identified as a region important for binding to IgE antibodies from patients. Further, SEQ ID NO: 404 was identified as a sequence comprising a region amino-terminally adjacent to epitope 9 and binding to IgE antibodies from patients.
In SEQ ID NO: 280, the amino acids at positions 4-7, 9-12 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-3, 8, 13 and 14 of SEQ ID NO: 280 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 407, the amino acids at positions 1, 2, 4 and 5 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 3 of SEQ ID NO: 407 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(10) Epitope 10
In epitope 10 (SEQ ID NO: 281), SEQ ID NOs: 412 and 414 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 409 was identified as a sequence comprising a region amino-terminally adjacent to epitope 10 and binding to IgE antibodies from patients.
In SEQ ID NO: 281, the amino acids at positions 5, 6, 8-10 and 12-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 4 and 7 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-3 and 11 of SEQ ID NO: 281 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 412, the amino acids at positions 4-8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 3 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1 and 2 of SEQ ID NO: 412 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 414, the amino acids at positions 1, 2, 4 and 5 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 3 of SEQ ID NO: 414 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(11) Epitope 11
In epitope 11 (SEQ ID NO: 282), SEQ ID NOs: 419 and 425 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 422 was identified as a sequence comprising a region amino-terminally adjacent to epitope 11 and binding to IgE antibodies from patients, and SEQ ID NO: 426 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 11 and binding to IgE antibodies from patients.
In SEQ ID NO: 282, the amino acids at positions 1, 4, 5, 11 and 12 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 7, 8 and 13 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2, 3, 6, 9, 10, 14 and 15 of SEQ ID NO: 282 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 282, the amino acids at positions 1, 2, 4, 5, 7, 9, 11, 12 and 14 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 3 and 8 were identified as possible amino acids influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 6, 10, 13 and 15 of SEQ ID NO: 282 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 419, the amino acids at positions 5-7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 1 and 2 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 3, 4 and 8 of SEQ ID NO: 419 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 425, the amino acids at positions 1, 3, 5, 6 and 8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 2 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 4, 7 and 9 of SEQ ID NO: 425 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(14) Epitope 14
In epitope 14 (SEQ ID NO: 285), SEQ ID NO: 430 was identified as a region important for binding to IgE antibodies from patients.
In SEQ ID NO: 285, the amino acids at positions 1, 5, 6, 8, 10 and 12 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 2, 9 and 11 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 3, 4, 7 and 13-15 of SEQ ID NO: 285 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 430, the amino acids at positions 3, 4 and 6-9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 2 and 5 of SEQ ID NO: 430 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(15) Epitope 15
In epitope 15 (SEQ ID NO: 286), SEQ ID NOs: 434, 436 and 440 were identified as regions important for binding to IgE antibodies from patients.
In SEQ ID NO: 286, the amino acids at positions 1-7 and 9-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 8 of SEQ ID NO: 286 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 286, the amino acids at positions 3, 5-7 and 11-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 4 and 10 were identified as possible amino acids influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1, 2, 8 and 9 of SEQ ID NO: 286 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 434, the amino acids at positions 1-3 and 5-9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 4 of SEQ ID NO: 434 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 436, the amino acids at positions 1 and 3-10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 2 of SEQ ID NO: 436 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 440, the amino acids at positions 4-7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 3 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1 and 2 of SEQ ID NO: 440 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(16) Epitope 16
In epitope 16 (SEQ ID NO: 287), SEQ ID NOs: 444, 446 and 449 were identified as regions important for binding to IgE antibodies from patients.
In SEQ ID NO: 287, the amino acids at positions 2, 7, 9 and 13 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 3-6, 8, 10-12, 14 and 15 of SEQ ID NO: 287 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 444, the amino acids at positions 2, 7 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 3-6, 8 and 10 of SEQ ID NO: 444 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 446, the amino acids at positions 4, 6 and 10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-3, 5 and 7-9 of SEQ ID NO: 446 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 449, the amino acids at positions 2, 4 and 8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 10 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1, 3, 5-7 and 9 of SEQ ID NO: 449 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(17) Epitope 17
In epitope 17 (SEQ ID NO: 288), SEQ ID NOs: 455 and 458 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NOs: 451 and 452 were identified as sequences comprising a region amino-terminally adjacent to epitope 17 and binding to IgE antibodies from patients.
In SEQ ID NO: 288, the amino acids at positions 3, 7, 10 and 12-14 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 2, 4-6, 8, 9, 11 and 15 of SEQ ID NO: 288 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 455, the amino acids at positions 1, 5, 8 and 10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 7 and 9 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2-4 and 6 of SEQ ID NO: 455 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 458, the amino acids at positions 1, 4 and 6-8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 3 and 5 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2 and 9 of SEQ ID NO: 458 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(19) Epitope 19
In epitope 19 (SEQ ID NO: 290), SEQ ID NOs: 461 and 464 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 462 was identified as a sequence comprising a region amino-terminally adjacent to epitope 19 and binding to IgE antibodies from patients, and SEQ ID NOs: 465 and 466 were identified as sequences comprising a region carboxy-terminally adjacent to epitope 19 and binding to IgE antibodies from patients.
In SEQ ID NO: 290, the amino acids at positions 1-4, 6, 7, 10 and 13-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 5, 8, 9, 11 and 12 of SEQ ID NO: 290 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 461, the amino acids at positions 1-4 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 5 of SEQ ID NO: 461 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 464, the amino acids at positions 2, 2 and 6-10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 4 and 5 of SEQ ID NO: 464 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(20) Epitope 20
In epitope 20 (SEQ ID NO: 291), SEQ ID NOs: 470, 473, 482, 483 and 486 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NOs: 471, 479 and 480 were identified as sequences comprising a region amino-terminally adjacent to epitope 20 and binding to IgE antibodies from patients, and SEQ ID NOs: 476 and 487 were identified as sequences comprising a region carboxy-terminally adjacent to epitope 20 and binding to IgE antibodies from patients.
In SEQ ID NO: 291, the amino acids at positions 2, 7, 10, 12, 13 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 1 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 3-6, 8, 9, 11 and 14 of SEQ ID NO: 291 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 291, the amino acids at positions 2, 4, 7, 11-13 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 6 was identified as a possible amino acid influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1, 3, 5, 8-10 and 14 of SEQ ID NO: 291 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 291, the amino acids at positions 2, 5, 7, 9, 10, 13 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 1 and 6 were identified as possible amino acids influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 3, 4, 8, 11, 12 and 14 of SEQ ID NO: 291 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 470, the amino acids at positions 2, 4 and 7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 1 and 5 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 3, 6, 8 and 9 of SEQ ID NO: 470 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 473, the amino acids at positions 3, 5, 6 and 8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 2, 4 and 7 of SEQ ID NO: 473 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 482, the amino acids at positions 1, 4-6, 8 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2, 3 and 7 of SEQ ID NO: 483 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 486, the amino acids at positions 2, 4, 5 and 8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 1 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 3, 6, 7 and 9 of SEQ ID NO: 486 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(21) Epitope 21
In epitope 21 (SEQ ID NO: 292), SEQ ID NOs: 491, 494, 500, 505, 507 and 509 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NOs: 492, 497, 498 and 503 were identified as sequences comprising a region amino-terminally adjacent to epitope 21 and binding to IgE antibodies from patients, and SEQ ID NO: 495 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 21 and binding to IgE antibodies from patients.
In SEQ ID NO: 292, the amino acids at positions 1-3, 6, 8, 9, 14 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 4, 5, 7 and 10-13 of SEQ ID NO: 292 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 292, the amino acids at positions 1-3, 6-10 and 12-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 11 was identified as a possible amino acid influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 4 and 5 of SEQ ID NO: 292 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 292, the amino acids at positions 1, 2, 7-9 and 15 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 3-6 and 10-14 of SEQ ID NO: 292 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 491, the amino acids at positions 1-3 and 6 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 4 and 5 of SEQ ID NO: 491 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 494, the amino acids at positions 1 and 2 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 3-6 of SEQ ID NO: 494 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 500, the amino acids at positions 1-3 and 5-7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 4 of SEQ ID NO: 500 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 505, the amino acids at positions 1, 2, 7 and 8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 3-6 of SEQ ID NO: 505 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 507, the amino acids at positions 5-7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-4 and 8-10 of SEQ ID NO: 507 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 509, the amino acids at positions 1, 2, 8 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 3-7 of SEQ ID NO: 509 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(22) Epitope 22
In epitope 22 (SEQ ID NO: 293), SEQ ID NO: 512 was identified as a region important for binding to IgE antibodies from patients. Further, SEQ ID NO: 515 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 22 and binding to IgE antibodies from patients.
In SEQ ID NO: 293, the amino acids at positions 3, 6, 8-10 and 12-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 2 and 11 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1, 4, 5 and 7 of SEQ ID NO: 293 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 512, the amino acids at positions 1 and 3-10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 2 of SEQ ID NO: 512 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(23) Epitope 23
In epitope 23 (SEQ ID NO: 294), SEQ ID NOs: 520 and 522 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NOs: 516 and 517 were identified as sequences comprising a region amino-terminally adjacent to epitope 23 and binding to IgE antibodies from patients, and SEQ ID NO: 523 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 23 and binding to IgE antibodies from patients.
In SEQ ID NO: 294, the amino acids at positions 2, 5 and 9-12 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 8 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1, 3, 4, 6, 7 and 13-15 of SEQ ID NO: 294 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 520, the amino acids at positions 1, 4 and 8-10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 7 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2, 3, 5 and 6 of SEQ ID NO: 520 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 522, the amino acids at positions 2-6 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1 and 7 of SEQ ID NO: 522 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(25) Epitope 25
In SEQ ID NO: 296 (epitope 25), the amino acids at positions 2-6 and 10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 7-9 and 11-15 of SEQ ID NO: 296 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 296, the amino acids at positions 1-3, 9, 11 and 12 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 4-8, 10 and 13-15 of SEQ ID NO: 296 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(26) Epitope 26
In epitope 26 (SEQ ID NO: 297), SEQ ID NOs: 528 and 531 were identified as regions important for binding to IgE antibodies from patients.
In SEQ ID NO: 297, the amino acids at positions 1 and 8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 6 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2-5, 7 and 9-15 of SEQ ID NO: 297 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 297, the amino acids at positions 4 and 6 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1-3, 5 and 7-15 of SEQ ID NO: 297 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(27) Epitope 27
In epitope 27 (SEQ ID NO: 298), SEQ ID NOs: 533, 534, 539, 541 and 544 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NOs: 535 and 542 were identified as sequences comprising a region carboxy-terminally adjacent to epitope 27 and binding to IgE antibodies from patients.
In SEQ ID NO: 298, the amino acids at positions 1, 2, 14 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 13 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 3-12 of SEQ ID NO: 298 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 298, the amino acids at positions 3, 4, and 13-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 2 and 6 were identified as possible amino acids influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1, 5 and 7-12 of SEQ ID NO: 298 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 539, the amino acids at positions 1 and 3 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2 and 4-9 of SEQ ID NO: 539 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 541, the amino acids at positions 1 and 8-10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2-7 of SEQ ID NO: 541 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 544, the amino acids at positions 4-6 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-3 of SEQ ID NO: 544 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(28) Epitope 28
In SEQ ID NO: 299 (epitope 28), the amino acids at positions 2 and 6-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1 and 3-5 of SEQ ID NO: 299 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 299, the amino acids at positions 2, 9-12 and 15 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1, 3-8, 13 and 14 of SEQ ID NO: 299 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 299, the amino acids at positions 1, 2, 9, 14 and 15 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 3-8 and 10-13 of SEQ ID NO: 299 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(30) Epitope 30
SEQ ID NO: 550 was identified as a sequence comprising a region amino-terminally adjacent to epitope 30 (SEQ ID NO: 301) and binding to IgE antibodies from patients, and SEQ ID NO: 551 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 30 and binding to IgE antibodies from patients.
In SEQ ID NO: 301, the amino acids at positions 2 and 6-9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 3-5 and 10-15 of SEQ ID NO: 301 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(33) Epitope 33
In epitope 33 (SEQ ID NO: 304), SEQ ID NO: 555 was identified as a region important for binding to IgE antibodies from patients. Further, SEQ ID NO: 553 was identified as a sequence comprising a region amino-terminally adjacent to epitope 33 and binding to IgE antibodies from patients, and SEQ ID NOs: 556 and 557 were identified as sequences comprising a region carboxy-terminally adjacent to epitope 33 and binding to IgE antibodies from patients.
In SEQ ID NO: 304, the amino acids at positions 1, 2, 13 and 14 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 3-12 and 15 of SEQ ID NO: 304 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 555, the amino acids at positions 1 and 2 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 3-8 of SEQ ID NO: 555 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(34) Epitope 34
In epitope 34 (SEQ ID NO: 305), SEQ ID NOs: 561, 562, 569, 571 and 573 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 565 was identified as a sequence comprising a region amino-terminally adjacent to epitope 34 and binding to IgE antibodies from patients.
In SEQ ID NO: 305, the amino acids at positions 14 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 10-13 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-9 of SEQ ID NO: 305 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 305, the amino acids at positions 9, 11 and 14 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 4-7 were identified as possible amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-3, 8, 10, 12, 13 and 15 of SEQ ID NO: 305 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 561, the amino acids at positions 2-5 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 1 of SEQ ID NO: 561 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 562, the amino acids at positions 2-6 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acid at position 1 of SEQ ID NO: 562 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 569, the amino acids at positions 7 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 2-5 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1, 6, 8 and 10 of SEQ ID NO: 569 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 571, the amino acids at positions 1, 3 and 6 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2, 4, 5 and 7 of SEQ ID NO: 571 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 573, the amino acids at positions 1 and 3 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2 and 4 of SEQ ID NO: 573 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(35) Epitope 35
In epitope 35 (SEQ ID NO: 306), SEQ ID NOs: 579 and 581 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 576 was identified as a sequence comprising a region amino-terminally adjacent to epitope 35 and binding to IgE antibodies from patients, and SEQ ID NO: 577 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 35 and binding to IgE antibodies from patients.
In SEQ ID NO: 306, the amino acids at positions 1-4 and 7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 5, 6 and 8-15 of SEQ ID NO: 306 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 579, the amino acids at positions 1-4 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 5 and 6 of SEQ ID NO: 579 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 581, the amino acids at positions 1-3 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 4-7 of SEQ ID NO: 581 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(36) Epitope 36
In epitope 36 (SEQ ID NO: 307), SEQ ID NO: 585 was identified as a region important for binding to IgE antibodies from patients. Further, SEQ ID NO: 583 was identified as a sequence comprising a region amino-terminally adjacent to epitope 36 and binding to IgE antibodies from patients, and further, SEQ ID NO: 584 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 36 and binding to IgE antibodies from patients.
(37) Epitope 37
In epitope 37 (SEQ ID NO: 308), SEQ ID NOs: 587, 589, 592, 594, 598, 600 and 602 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 595 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 37 and binding to IgE antibodies from patients.
In SEQ ID NO: 308, the amino acids at positions 4, 8, 10 and 13 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-3, 5-7, 9, 11, 12, 14 and 15 of SEQ ID NO: 308 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 308, the amino acids at positions 4 and 9-14 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1-3, 5-8 and 15 of SEQ ID NO: 308 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 308, the amino acids at positions 5, 8, 10, 13 and 15 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1-4, 6, 7, 9, 11, 12 and 14 of SEQ ID NO: 308 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 587, the amino acids at positions 1 and 2 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 3 and 4 of SEQ ID NO: 587 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 589, the amino acids at positions 4, 6 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-3, 5, 7, 8 and 10 of SEQ ID NO: 589 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 592, the amino acids at positions 2 and 7-9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1 and 3-6 of SEQ ID NO: 592 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 594, the amino acids at positions 5-10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-4 of SEQ ID NO: 594 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 598, the amino acids at positions 3, 6 and 8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 2, 4, 5 and 7 of SEQ ID NO: 598 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 600, the amino acids at positions 1, 4, 6 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2, 3, 5, 7, 8 and 10 of SEQ ID NO: 600 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 602, the amino acids at positions 2, 4 and 7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 3, 5, 6 and 8 of SEQ ID NO: 602 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(38) Epitope 38
In epitope 38 (SEQ ID NO: 309), SEQ ID NO: 608 was identified as a region important for binding to IgE antibodies from patients. Further, SEQ ID NOs: 604 and 605 were identified as sequences comprising a region amino-terminally adjacent to epitope 38 and binding to IgE antibodies from patients, and further, SEQ ID NO: 609 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 38 and binding to IgE antibodies from patients.
In SEQ ID NO: 309, the amino acids at positions 5, 9 and 12-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 10 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-4, 6-8 and 11 of SEQ ID NO: 309 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 309, the amino acids at positions 7, 9 and 11-14 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1-6, 8, 10 and 15 of SEQ ID NO: 309 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 608, the amino acids at positions 1, 5 and 8-10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 6 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2-4 and 7 of SEQ ID NO: 608 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(39) Epitope 39
In epitope 39 (SEQ ID NO: 310), SEQ ID NOs: 616, 617 and 619 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 613 was identified as a sequence comprising a region amino-terminally adjacent to epitope 39 and binding to IgE antibodies from patients.
In SEQ ID NO: 310, the amino acids at positions 3 and 5 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 10 and 13 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1, 2, 4, 6-9, 11, 12, 14 and 15 of SEQ ID NO: 310 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 616, the amino acids at positions 1 and 3 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 8 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2, 4-7 and 9 of SEQ ID NO: 616 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 619, the amino acids at positions 1, 6 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2-5, 7, 8 and 10 of SEQ ID NO: 619 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(40) Epitope 40
SEQ ID NOs: 622 and 623 were identified as sequences comprising a region amino-terminally adjacent to epitope 40 (SEQ ID NO: 311) and binding to IgE antibodies from patients, and SEQ ID NO: 624 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 40 and binding to IgE antibodies from patients.
In SEQ ID NO: 311, the amino acids at positions 1-3 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 4-15 of SEQ ID NO: 311 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(41) Epitope 41
In epitope 41 (SEQ ID NO: 312), SEQ ID NOs: 627, 630, 636, 637 (amino acid sequence: ELL), 641 and 642 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NOs: 631 and 632 were identified as sequences comprising a region carboxy-terminally adjacent to epitope 41 and binding to IgE antibodies from patients.
In SEQ ID NO: 312, the amino acids at positions 6, 7 and 9-14 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 8 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-5 and 15 of SEQ ID NO: 312 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 312, the amino acids at positions 6 and 12 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 1 and 7 were identified as possible amino acids influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 2-5, 8-11 and 13-15 of SEQ ID NO: 312 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 312, the amino acids at positions 3, 6, 7 and 13 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1, 2, 4, 5, 8-12, 14 and 15 of SEQ ID NO: 312 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 627, the amino acids at positions 6-8 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-5 of SEQ ID NO: 627 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 630, the amino acids at positions 4, 5 and 7-10 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 6 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-3 of SEQ ID NO: 630 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 636, the amino acids at positions 1, 6 and 7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 2-5 and 8 of SEQ ID NO: 636 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 641, the amino acids at positions 3, 6 and 7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 2, 4 and 5 of SEQ ID NO: 641 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(42) Epitope 42
In epitope 42 (SEQ ID NO: 313), SEQ ID NOs: 647 and 649 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 644 was identified as a sequence comprising a region amino-terminally adjacent to epitope 42 and binding to IgE antibodies from patients.
In SEQ ID NO: 313, the amino acids at positions 9-11 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 4 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-4, 6-8 and 12-15 of SEQ ID NO: 313 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 647, the amino acids at positions 5-7 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acid at position 1 was identified as an amino acid influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2-4 and 8 of SEQ ID NO: 647 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 649, the amino acids at positions 1-3 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 4-8 of SEQ ID NO: 649 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(44) Epitope 44
In SEQ ID NO: 315 (epitope 44), the amino acids at positions 4-7 and 9 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1-3, 8 and 10-15 of SEQ ID NO: 315 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 315, the amino acids at positions 4, 7, 8 and 11 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1-3, 5, 6, 9, 10 and 12-15 of SEQ ID NO: 315 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 315, the amino acids at positions 4, 7, 8, 13 and 15 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1-3, 5, 6, 9-12 and 14 of SEQ ID NO: 315 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(45) Epitope 45
In SEQ ID NO: 316 (epitope 45), the amino acids at positions 2, 3, 7, 8 and 13 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients. The amino acids at positions 1, 4-6, 9-12, 14 and 15 of SEQ ID NO: 316 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 316, the amino acids at positions 7-9, 11 and 13 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1-6, 10, 12, 14 and 15 of SEQ ID NO: 316 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(46) Epitope 46
In epitope 46 (SEQ ID NO: 317), SEQ ID NOs: 658, 659, 663 and 664 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 657 was identified as a sequence comprising a region amino-terminally adjacent to epitope 46 and binding to IgE antibodies from patients, and further, SEQ ID NOs: 660 and 665 were identified as sequences comprising a region carboxy-terminally adjacent to epitope 46 and binding to IgE antibodies from patients.
In SEQ ID NO: 317, the amino acids at positions 1, 6, 9 and 13-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 3, 4, 8, 10 and 12 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 2, 5, 7 and 11 of SEQ ID NO: 317 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 317, the amino acids at positions 12-15 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1-11 of SEQ ID NO: 317 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
(47) Epitope 47
In epitope 47 (SEQ ID NO: 318), SEQ ID NOs: 669, 674, 680 and 681 were identified as regions important for binding to IgE antibodies from patients. Further, SEQ ID NO: 672 was identified as a sequence comprising a region amino-terminally adjacent to epitope 47 and binding to IgE antibodies from patients, and further, SEQ ID NO: 676 was identified as a sequence comprising a region carboxy-terminally adjacent to epitope 47 and binding to IgE antibodies from patients.
In SEQ ID NO: 318, the amino acids at positions 8-15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 6 and 7 were identified as amino acids influencing binding to IgE antibodies from allergic patients. The amino acids at positions 1-5 of SEQ ID NO: 318 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 318, the amino acids at positions 2, 5, 7 and 15 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1, 3, 4, 6 and 8-14 of SEQ ID NO: 318 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 318, the amino acids at positions 7, 9, 11, 13 and 15 were identified as amino acids particularly important for binding to IgE antibodies from allergic patients, and the amino acids at positions 8 and 14 were identified as possible amino acids influencing binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 1-6, 10 and 12 of SEQ ID NO: 318 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
In SEQ ID NO: 318, the amino acids at positions 1-5 were identified as possible amino acids particularly important for binding to IgE antibodies from allergic patients. In this case, the amino acids at positions 6-15 of SEQ ID NO: 318 had no influence on the binding activity against IgE antibodies from allergic patients when substituted by alanine.
Cross-reactivity based on amino acid sequences was studied by PHI-BLAST analysis with initial set parameters of PHI-BLAST using the query sequences and the PHI pattern sequences shown below in Table 3.
As a result, proteins derived from the species shown in Table 3 were identified. This indicates that SEQ ID NO: 1 can be used for detecting antigen cross-reactivity irrespective of species, genera, or plant kingdom.
Phasianidae etc.
Phasianidae etc., Olive of family Oleaceae, Artichoke of
Bromeliaceae, Shinano walnut of family Juglandaceae,
Salmonidae, Squash of family Cucurbitaceae etc.
Phasianidae etc., Whiteleg shrimp of family Penaeidae,
Leguminosae etc.
Phasianidae, Soybean of family Leguminosae, Shinano
The peptides of the aforementioned SEQ ID NOs were prepared by the Fmoc method and studied for their binding activity against IgE antibodies in serum by ELISA. On the basis of RXRXXKXX (SEQ ID NO: 329), the peptides of chicken egg RGREDKMK and soybean RFREDKEE were prepared, and their binding activity against IgE antibodies were confirmed by ELISA using the serum of cases having food allergies to chicken egg and soybean in combination. As a result, patient-specific binding activity was able to be confirmed.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/020654 | Jun 2017 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/043877 | 12/6/2017 | WO |
