Antigens and epitopes of allergy
阅读说明：本技术 ***反应的抗原及其表位 (Antigens and epitopes of allergy ) 是由 松永佳世子 矢上晶子 中村政志 下條尚志 佐藤奈由 青木祐治 酒井智美 于 2018-05-01 设计创作，主要内容包括：本发明提供虾变态反应的新型抗原、虾变态反应的诊断方法和诊断试剂盒、包含该抗原的药物组合物、去除了该抗原的虾或虾加工品、以及用于判断对象物中有无虾抗原的检测组合物。本发明还涉及包含抗原表位的多肽、包含该多肽的变态反应的诊断试剂盒、诊断用组合物和诊断方法、包含该多肽的药物组合物、以及包含该多肽的抗原被去除或降低了的原料或者加工品。本发明进而还涉及用于判断对象物中有无抗原的检测组合物。(The present invention provides a novel antigen for shrimp allergy, a method and a kit for diagnosing shrimp allergy, a pharmaceutical composition containing the antigen, a shrimp or a processed shrimp product from which the antigen has been removed, and a detection composition for determining the presence or absence of a shrimp antigen in a subject. The present invention also relates to a polypeptide comprising an epitope, a diagnostic kit for allergy comprising the polypeptide, a diagnostic composition and a diagnostic method, a pharmaceutical composition comprising the polypeptide, and a raw material or processed product from which an antigen comprising the polypeptide is removed or reduced. The present invention further relates to a detection composition for determining the presence or absence of an antigen in a subject.)
1. A diagnostic kit for allergy comprising at least one of the following polypeptides:
(E1) (ii) (i) a polypeptide comprising the amino acid sequence of sequence No. 558-565;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 4, 5, 7, 8, 10, 11, 12, 13, 14 and 15 of SEQ ID NO. 558 and 565 have been substituted with an arbitrary amino acid residue;
(E2) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 566-581, 949;
(ii) a polypeptide comprising an amino acid sequence wherein 1 or more amino acid residues corresponding to the amino acid residues at positions 1, 3, 4, 5, 6, 7, 8, 9, 11 and 12 of SEQ ID NO. 566 have been substituted with an arbitrary amino acid residue in SEQ ID NO. 566-581, 949;
(E3) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 582-;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 9 th, 10 th, 11 th, 12 th and 13 th amino acid residues of SEQ ID NO. 582 are substituted with arbitrary amino acid residues in SEQ ID NO. 582-;
(E4) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 586-;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 2, 4, 5, 6, 7, 10, 11 and 12 of SEQ ID NO. 586 are substituted with arbitrary amino acid residues in SEQ ID NO. 586 and 593 and 951;
(E5) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 594-598, 952;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 9 th, 11 th and 12 th amino acid residues of SEQ ID NO. 594 are substituted with arbitrary amino acid residues in SEQ ID NO. 594, 598 and 952;
(E6) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 599-;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 of SEQ ID NO. 599-613, 953 and 954 of SEQ ID NO. 599 are substituted with an arbitrary amino acid residue;
(E7) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 614-623, 955;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14 and 15 of SEQ ID NO. 614-623 and 955 are substituted with arbitrary amino acid residues;
(E8) (i) a polypeptide comprising the amino acid sequence of SEQ ID No. 624-633;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 3, 5, 6, 7, 8, 10, 11, 12 and 14 of SEQ ID NO. 624 are substituted with arbitrary amino acid residues in the sequence of SEQ ID NO. 624-;
(E9) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 634-639;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 4, 6, 8, 10 and 11 of SEQ ID NO. 634 and 639 are substituted with arbitrary amino acid residues;
(E10) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 640-653, 956, 957;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 4, 6, 8, 9, 11, 14 and 15 of SEQ ID NO. 640 are substituted with arbitrary amino acid residues in SEQ ID NO. 640, 653, 956 and 957;
(E11) (i) a polypeptide comprising the amino acid sequence of seq id No. 654-670;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 2, 4, 5, 6, 7, 8, 9, 10, 12, 13 and 15 of SEQ ID NO. 654 and 670 are substituted with arbitrary amino acid residues;
(E12) a polypeptide comprising the amino acid sequence of seq id No. 671;
(E13) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 672-677, 958;
(ii) in SEQ ID NOS 672-677 and 958, 1 or more amino acid residues corresponding to the amino acid residues at positions 6, 7, 8, 9, 10 and 14 of SEQ ID NO 672 are optionally substituted with an arbitrary amino acid residue;
(E14) a polypeptide comprising the amino acid sequence of SEQ ID NO. 678-680;
(E15) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 681-685;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 10 th amino acid residue of SEQ ID NO. 681 are substituted with arbitrary amino acid residues in SEQ ID NO. 681 and 685;
(E16) (i) a polypeptide comprising the amino acid sequence of seq id no 686 690, 959, 960;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 4, 5, 6, 7, 9, 10 and 12 of SEQ ID NO. 686 are substituted with arbitrary amino acid residues in SEQ ID NO. 686-690, 959 and 960;
(E17) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 691-696, 961;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 7, 10 and 12 of SEQ ID NO. 691, 10 and 12 are substituted with arbitrary amino acid residues in SEQ ID NO. 691-;
(E18) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 697-703;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 1, 3 and 5 of SEQ ID NO. 697 are substituted with arbitrary amino acid residues in SEQ ID NO. 697-703;
(E19) a polypeptide comprising the amino acid sequence of seq id No. 704;
(E20) a polypeptide comprising the amino acid sequence of SEQ ID NO. 705-707;
(E21) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 708-716, 962, 963;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 1, 2, 4, 5, 7, 8, 9, 10, 12, 13 and 15 of SEQ ID NO. 708-716, 962 and 963 are substituted with arbitrary amino acid residues;
(E22) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 717-724, 964;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 2, 4, 5, 6, 8, 9, 10 and 12 of SEQ ID NO. 717 are substituted with arbitrary amino acid residues in SEQ ID NO. 717, 724 and 964;
(E23) a polypeptide comprising the amino acid sequence of SEQ ID NO. 725-728;
(E24) (i) a polypeptide comprising the amino acid sequence of SEQ ID Nos. 729-740, 965, 966;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 4, 5, 6, 7, 8, 9, 11 and 12 of SEQ ID NO. 729-740, 965 and 966 are substituted with an arbitrary amino acid residue;
(E25) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 741-749, 967, 968;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 3, 5, 6, 8, 9, 10, 11 and 13 of SEQ ID NO 741-749, 967 and 968 are substituted with arbitrary amino acid residues;
(E26) a polypeptide comprising the amino acid sequence of SEQ ID NO. 750-751;
(E27) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 752-764, 969, 970;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 3, 4, 5, 7, 8, 9, 10 and 11 of SEQ ID NO. 752 and 764, 969 and 970 have been substituted with an arbitrary amino acid residue;
(E28) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 765-769;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 3, 5 and 6 of SEQ ID NO. 765 are substituted with arbitrary amino acid residues in SEQ ID NO. 765-;
(E29) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 770-777, 971, 972;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 1, 2, 5, 6, 7, 8, 9, 10, 11 and 12 of SEQ ID NO. 770 are substituted with arbitrary amino acid residues in SEQ ID NO. 770-777, 971 and 972;
(E30) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 778-787, 973;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 2, 3, 4, 6, 7, 8, 9, 10 and 14 of SEQ ID NO. 778-787 and 973 are substituted with an arbitrary amino acid residue;
(E31) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 788-792;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 4, 7, 8, 9, 10, 11 and 13 of SEQ ID NO. 788-792 are substituted with arbitrary amino acid residues;
(E32) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 793-809, 974, 975;
(ii) a polypeptide comprising an amino acid sequence obtained by substituting 1 or more amino acid residues corresponding to the amino acid residues at positions 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 of SEQ ID NO. 793 and residues 809, 974 and 975 of SEQ ID NO. 793 with arbitrary amino acid residues;
(E33) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 810-816;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 2, 4, 5, 6, 7, 8, 9, 10, 12 and 14 of SEQ ID NO. 810 are substituted with arbitrary amino acid residues in SEQ ID NO. 810 and 816;
(E34) (i) a polypeptide comprising the amino acid sequence of seq id No. 817-825;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 2, 4, 5, 7, 8, 9, 11, 12, 13 and 14 of SEQ ID NO. 817 are substituted with arbitrary amino acid residues in SEQ ID NO. 817-825;
(E35) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 826-832;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 2, 3, 5, 8, 9, 10, 11, 12 and 14 of SEQ ID NO. 826 are substituted with arbitrary amino acid residues in SEQ ID NO. 826-;
(E36) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 833-;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 of SEQ ID NO. 833-846 and 976 are substituted with an arbitrary amino acid residue;
(E37) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 847-857, 977;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 5, 6, 7, 9, 10, 11, 12, 13 and 14 of SEQ ID NO. 847-857 and 977 are substituted with arbitrary amino acid residues;
(E38) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 858-864;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 6, 7, 8, 9, 13 and 14 of SEQ ID NO. 858 are substituted with arbitrary amino acid residues in SEQ ID NO. 858 and 864;
(E39) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 865-878, 984, 978;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 1, 2, 4, 5, 6, 8, 9, 10, 11 and 12 of SEQ ID NO 865 are substituted with arbitrary amino acid residues in SEQ ID NO 865-878, 984 and 978;
(E40) a polypeptide comprising the amino acid sequence of SEQ ID NO 879-880;
(E41) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 881-891, 979;
(ii) a polypeptide having an amino acid sequence wherein 1 or more amino acid residues corresponding to the amino acid residues at positions 2, 3, 4, 6, 9, 10, 11, 12, 13 and 15 of SEQ ID NO. 881 and/or SEQ ID NO. 881 are substituted with an arbitrary amino acid residue in sequence Nos. 881-891 and 979;
(E42) (i) a polypeptide comprising the amino acid sequence of seq id nos 892-907, 980;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14 and 15 of SEQ ID NO. 892-907 and 980 are substituted with arbitrary amino acid residues;
(E43) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 908-911, 981;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 7 th amino acid residue of SEQ ID NO 908 are substituted with arbitrary amino acid residues in SEQ ID NO 908, 911, 981;
(E44) a polypeptide comprising the amino acid sequence of SEQ ID NO 912-914;
(E45) a polypeptide comprising the amino acid sequence of SEQ ID NO. 915-916;
(E46) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 917-;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 4, 7, 8, 9, 10 and 15 of SEQ ID NO. 917 are substituted with an arbitrary amino acid residue in SEQ ID NO. 917, 927, 982 and 983;
(E47) (i) a polypeptide comprising the amino acid sequence of seq id No. 928-934;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 6, 7, 8, 10, 12 and 14 of SEQ ID NO. 928 have been substituted with an arbitrary amino acid residue in SEQ ID NO. 928-934;
(E48) (i) a polypeptide comprising the amino acid sequence of SEQ ID No. 935-938;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 5 th and 6 th amino acid residues of SEQ ID NO. 935-938 are substituted with arbitrary amino acid residues in SEQ ID NO. 935-938;
(E49) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 939-942;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 3, 6, 7, 10 and 11 of SEQ ID NO. 939 are substituted with arbitrary amino acid residues in SEQ ID NO. 939-942;
(E50) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 943-948;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at positions 1, 2, 3, 4, 5, 8, 10 and 11 of SEQ ID NO. 943-948 are substituted with an arbitrary amino acid residue.
2. A diagnostic composition for allergy comprising at least one of the polypeptides of claim 1.
3. A polypeptide which is any one of the polypeptides of claim 1 which specifically binds to IgE antibodies of allergy patients.
4. A method of providing an indicator for diagnosing allergy in a subject, comprising the steps of:
(i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing an IgE antibody;
(ii) detecting binding of an IgE antibody in a sample obtained from the subject to the antigen;
(iii) providing an indication that the subject is allergic upon detection of binding of IgE antibodies to the antigen in the subject;
here, the antigen is at least one of the polypeptides according to claim 3.
5. A pharmaceutical composition comprising at least one of the polypeptides of claim 3.
6. The pharmaceutical composition according to claim 5, for use in the treatment of allergy.
7. A detection composition for determining the presence or absence of an antigen in a subject, comprising an antibody that binds to at least one of the polypeptides according to claim 3.
8. A detection composition for determining the presence or absence of an antigen in a subject, comprising any of the following primers:
(a) a primer comprising a part of a base sequence of a nucleic acid encoding the polypeptide of claim 3 and/or a part of a complementary strand thereof; or
(b) A primer that is a part of at least one sequence of the base sequences represented by SEQ ID Nos. 1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540, or 548; and/or
A primer that is a part of a sequence complementary to at least one sequence of the base sequences represented by SEQ ID Nos. 1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540, or 548.
9. A method for determining the presence or absence of the polypeptide of claim 3 in a raw material/processed product, comprising: detecting the polypeptide of claim 3 in the feedstock/processed product.
10. A raw material or processed product from which an antigen comprising at least one of the polypeptides according to claim 3 has been removed or reduced.
11. A method for producing a processed product from which an antigen has been removed or reduced, the method comprising a step of confirming that the antigen has been removed or reduced in a process for producing the processed product, wherein the antigen is at least one of the polypeptides according to claim 3.
Technical Field
The present invention relates to novel antigens of shrimp allergy. The invention also relates to a diagnostic kit, a diagnostic composition and a diagnostic method for shrimp allergy. The invention also relates to a pharmaceutical composition comprising the antigen, and a shrimp or shrimp-processed product from which the antigen is removed or reduced. The present invention further relates to a detection composition for determining the presence or absence of a shrimp antigen in a subject.
The invention also relates to polypeptides comprising the antigenic epitopes. The invention also relates to a diagnostic kit, a diagnostic composition and a diagnostic method for allergy containing the polypeptide. The invention also relates to a pharmaceutical composition containing the polypeptide, and a raw material or a processed product containing the polypeptide which is removed or reduced. The present invention also relates to a method for producing a processed product in which a polypeptide is removed or reduced. The present invention also relates to a detection composition for determining the presence or absence of an antigen containing the polypeptide in a subject.
Background
IgE antibodies specific to a specific antigen (hereinafter, also referred to as allergen) are produced in the serum and tissues of allergy patients. Allergic reactions are caused by the physiological consequences of the interaction of this IgE antibody with specific antigens. The antigen broadly refers to food/food materials and the like that cause allergy symptoms; in a narrow sense, the term refers to a protein (hereinafter, also referred to as an allergen component) contained in food or food materials or the like to which a specific IgE antibody binds.
In many conventional allergy-detecting reagents, allergen candidate foods, foods and the like are simply ground to prepare an antigen reagent (patent document 1). Therefore, among a plurality of allergen components contained in the conventional antigen reagent, a positive reaction can be detected in an allergy test only when the binding of the allergen component to an IgE antibody exceeds a threshold content at which a positive reaction can be judged, and the diagnosis efficiency is not said to be sufficiently high.
Among foods and food materials that are candidates for allergens, several allergen components are suggested and commercialized as a detection kit. However, in order to improve the reliability of the allergy test, it is necessary to comprehensively identify the allergen components, and as a result, the patient detection rate by the allergen component measurement is not sufficient. Identification of a novel allergen in shrimp is not only required to improve the accuracy of a diagnostic reagent, but also important as a target for hypoallergenic foods, and therapeutic drugs.
On the other hand, in recent years, as a method for separating and purifying a plurality of proteins from a small amount of sample, a two-dimensional electrophoresis method of performing isoelectric point electrophoresis in a first dimension and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in a second dimension has been used. The applicant has developed a two-dimensional electrophoresis method having high separation ability (
Allergen-specific IgE antibodies recognize and bind to epitopes that are specific amino acid sequences in allergen components. However, there are only a few examples of the epitope-resolved allergen components (non-patent document 1), and the resolution is extremely limited. In addition, there is no diagnostic kit for allergy using epitope-containing polypeptides on the market at present.
Disclosure of Invention
Problems to be solved by the invention
The present invention provides novel antigens of shrimp allergy. The invention also provides a diagnostic method and a diagnostic kit for shrimp allergy. The invention also provides a pharmaceutical composition comprising the antigen, and a shrimp or shrimp-processed product with the antigen removed or reduced. The present invention further provides a detection composition for determining the presence or absence of a shrimp antigen in a subject.
The invention also provides polypeptides comprising the antigenic epitopes. The present invention also provides a diagnostic kit for allergy, a diagnostic composition and a diagnostic method comprising the polypeptide. The invention also provides a pharmaceutical composition containing the polypeptide, and a raw material or a processed product with the antigen of the polypeptide removed or reduced. The present invention also relates to a method for producing a processed product in which the antigen is removed or reduced. The present invention further provides a detection composition for determining the presence or absence of an antigen comprising the polypeptide in a subject.
Means for solving the problems
The present inventors have intensively studied the identification of antigens responsible for shrimp allergy in order to solve the above problems. The results successfully identified a novel antigen specifically bound by IgE antibodies in the serum of patients with shrimp allergy. The present invention has been completed based on this finding.
That is, in one embodiment, the present invention is as follows.
[1] A diagnostic kit for shrimp allergy, comprising at least one of the following proteins (1) to (11) as an antigen:
(1) (1A) a protein comprising the C-terminal portion of myosin heavy chain type 1(myosin heavy chain type 1) or the C-terminal portion of myosin heavy chain type a (myosin heavy chain type a) or a mutant thereof, which is any one of the following proteins (1A-a) to (1A-e) as an antigen for shrimp allergy:
(1A-a) a protein comprising an amino acid sequence wherein 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 2, 45 or 87;
(1A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 2, 45 or 87;
(1A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO.1, 44 or 86 in which 1 or more nucleotides are deleted, substituted, inserted or added;
(1A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO.1, 44 or 86; or
(1A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO.1, 44, or 86; or
(1B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 2 to 43, 45 to 85, and 87 to 115;
(2) (2A) a protein comprising the N-terminal portion of myosin
(2A-a) a protein comprising an amino acid sequence wherein 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 117, 141 or 146;
(2A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 117, 141, or 146;
(2A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 116, 140 or 145 in which 1 or more nucleotides are deleted, substituted, inserted or added;
(2A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 116, 140 or 145; or
(2A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 116, 140, or 145; or
(2B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 117 to 139, 141 to 144, and 146 to 159;
(3) (3A) a protein comprising the C-terminal portion of myosin heavy chain type 2(myosin heavy chain type 2) or the C-terminal portion of myosin heavy chain type b (myosin heavy chain type b), which is any one of the following proteins (3A-a) to (3A-e) as an antigen for shrimp allergy, or a mutant thereof:
(3A-a) a protein comprising an amino acid sequence in which 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 161, 179 or 230;
(3A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 161, 179 or 230;
(3A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 160, 178 or 229 in which 1 or more nucleotides are deleted, substituted, inserted or added;
(3A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 160, 178 or 229; or
(3A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 160, 178 or 229; or
(3B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 161 to 177, SEQ ID Nos. 179 to 228, and SEQ ID Nos. 230 to 274;
(4) (4A) a protein comprising the N-terminal portion of myosin
(4A-a) a protein comprising an amino acid sequence in which 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 276, 300 or 306;
(4A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 276, 300 or 306;
(4A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 275, 299 or 305 in which 1 or more nucleotides are deleted, substituted, inserted or added;
(4A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 275, 299 or 305; or
(4A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 275, 299, or 305; or
(4B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 276 to 298, 300 to 304, or 306 to 320;
(5) (5A) a protein comprising glycogen phosphorylase (glycogen phosphorylase) or a mutant thereof, which is an antigen for shrimp allergy, comprising any one of the following proteins (5A-a) to (5A-e):
(5A-a) a protein comprising the amino acid sequence of SEQ ID NO. 362 in which 1 or more amino acids are deleted, substituted, inserted, or added.
(5A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 362;
(5A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 361 in which 1 or more nucleotides are deleted, substituted, inserted, or added;
(5A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 361; or
(5A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 361; or
(5B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 321 to 336, 337 to 360, and 362 to 379;
(6) (6A) a protein comprising a part of hemocyanin subunit L1(hemocyanin subbunit L1), hemocyanin (hemocyanin) or hemocyanin subunit L (hemocyanin subbunit L), or a mutant thereof, which is any one of the following (6A-a) to (6A-e) proteins as an antigen of shrimp allergy:
(6A-a) a protein comprising an amino acid sequence in which 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 381, 399 or 414;
(6A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 381, 399 or 414;
(6A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 380, 398 or 413 by deletion, substitution, insertion or addition of 1 or more nucleotides;
(6A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 380, 398 or 413; or
(6A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 380, 398 or 413; or
(6B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 381 to 397, 399 to 412, and 414 to 419;
(7) (7A) a protein comprising pyruvate kinase 3(pyruvate kinase 3) or a mutant thereof, which is any one of the following proteins (7A-a) to (7A-e) as an antigen for shrimp allergy:
(7A-a) a protein comprising the amino acid sequence of SEQ ID NO. 421 in which 1 or more amino acids are deleted, substituted, inserted, or added;
(7A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 421;
(7A-c) a protein having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 420 in which 1 or more nucleotides are deleted, substituted, inserted, or added;
(7A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 420; or
(7A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 420; or
(7B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 421 to 435 or SEQ ID Nos. 436 to 441;
(8) (8A) a phosphopyruvate hydratase (phosphopyruvate hydratase) -containing protein which is any one of the following proteins (8A-a) to (8A-e) as an antigen of shrimp allergy, or a mutant thereof:
(8A-a) a protein comprising an amino acid sequence in which 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 455 or 481;
(8A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 455 or 481;
(8A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 454 or 480 by deletion, substitution, insertion or addition of 1 or more nucleotides;
(8A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 454 or 480; or
(8A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 454 or 480; or
(8B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 442 to 453, 455 to 479, and 481 to 484;
(9) (9A) mitochondrial ATP synthase subunit α precursor (mitochondal ATP synthase subunit precorsor) or a mutant thereof, which is any one of the following proteins (9A-a) to (9A-e) as an antigen of shrimp allergy:
(9A-a) a protein comprising an amino acid sequence wherein 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 486;
(9A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 486;
(9A-c) a protein having an amino acid sequence encoded by the base sequence of SEQ ID NO. 485 in which 1 or more nucleotides are deleted, substituted, inserted, or added;
(9A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 485; or
(9A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 485; or
(9B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 486 to 490, 491 to 497, and 498 to 518;
(10) (10A) a protein comprising troponin I (tropin I) or a mutant thereof, which is any one of the following proteins (10A-a) to (10A-e) as an antigen for shrimp allergy:
(10A-a) a protein comprising the amino acid sequence of SEQ ID NO. 520 in which 1 or more amino acids have been deleted, substituted, inserted, or added;
(10A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 520;
(10A-c) a protein having an amino acid sequence encoded by the base sequence of SEQ ID NO. 519 wherein 1 or more nucleotides are deleted, substituted, inserted, or added;
(10A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 519; or
(10A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 519; or
(10B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 520 to 527, 528 to 532, and 533 to 539;
(11) (11A) a protein comprising cyclophilin A (cyclophilin A) or a mutant thereof, which is any one of the following proteins (11A-a) to (11A-e) as an antigen for shrimp allergy:
(11A-a) a protein comprising the amino acid sequence of SEQ ID NO. 541 or 549 in which 1 or more amino acids are deleted, substituted, inserted, or added;
(11A-b) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO. 541 or 549;
(11A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 540 or 548 wherein 1 or more nucleotides are deleted, substituted, inserted, or added;
(11A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more identity to the base sequence represented by SEQ ID NO. 540 or 548; or
(11A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 540 or 548; or
(11B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 541 to 547, 549 to 553, and 554 to 557.
[2] A composition for diagnosing allergy in shrimp, which comprises at least one of the proteins defined in (1) to (11) in the above [1] as an antigen.
[3] A method for providing an index for diagnosing shrimp allergy in a subject, comprising the steps of:
(i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing an IgE antibody;
(ii) detecting binding of an IgE antibody in a sample obtained from the subject to the antigen;
(iii) providing an indication that the subject is a shrimp allergy upon detection of binding of IgE antibodies of the subject to the antigen;
here, the antigen is at least one of the proteins defined in [1] to [ 11 ].
[4] A pharmaceutical composition comprising at least one of the proteins defined in [1] above as (1) to (11).
[5] The pharmaceutical composition according to the above [4], which is used for treating shrimp allergy.
[6] A shrimp or a processed shrimp product, wherein an antigen is removed or reduced, and the antigen is at least one of the proteins defined in (1) to (11) in the above [1 ].
[7] A detection composition for determining the presence or absence of a shrimp antigen in a subject, comprising an antibody that binds to at least one of the proteins defined as (1) to (11) in the above [1 ].
[8] A detection composition for determining the presence or absence of an antigen that causes shrimp allergy in a subject, comprising a primer having a nucleotide sequence complementary to a part of at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540, and 548.
The present inventors have also succeeded in finding epitopes of antigens derived from shrimp, which comprise the above antigens.
Since the epitope has a relatively short amino acid sequence, the IgE antibody can bind to a plurality of allergen components if the same amino acid sequence is present in different allergen components. Common epitopes are present in different allergen components and as a result the antigens are cross-reactive as both bind IgE antibodies of allergy sufferers. Therefore, the epitope defined in the present application enables diagnosis and treatment of allergy including crossability, detection of a plurality of allergen components including the epitope, and the like.
The present invention has been completed based on this finding. That is, in another embodiment, the present invention is as follows.
[ means 1]
A diagnostic kit for allergy comprising at least one of the following polypeptides:
(E1) (ii) (i) a polypeptide comprising the amino acid sequence of sequence No. 558-565;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E2) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 566-581, 949;
(ii) a polypeptide comprising an amino acid sequence wherein 1 or more amino acid residues corresponding to the amino acid residues at
(E3) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 582-;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 9 th, 10 th, 11 th, 12 th and 13 th amino acid residues of SEQ ID NO. 582 are substituted with arbitrary amino acid residues in SEQ ID NO. 582-;
(E4) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 586-;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E5) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 594-598, 952;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 9 th, 11 th and 12 th amino acid residues of SEQ ID NO. 594 are substituted with arbitrary amino acid residues in SEQ ID NO. 594, 598 and 952;
(E6) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 599-;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E7) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 614-623, 955;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E8) (i) a polypeptide comprising the amino acid sequence of SEQ ID No. 624-633;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E9) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 634-639;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E10) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 640-653, 956, 957;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E11) (i) a polypeptide comprising the amino acid sequence of seq id No. 654-670;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E12) a polypeptide comprising the amino acid sequence of seq id No. 671;
(E13) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 672-677, 958;
(ii) in SEQ ID NOS 672-677 and 958, 1 or more amino acid residues corresponding to the amino acid residues at
(E14) A polypeptide comprising the amino acid sequence of SEQ ID NO. 678-680;
(E15) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 681-685;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 10 th amino acid residue of SEQ ID NO. 681 are substituted with arbitrary amino acid residues in SEQ ID NO. 681 and 685;
(E16) (i) a polypeptide comprising the amino acid sequence of seq id no 686 690, 959, 960;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E17) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 691-696, 961;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E18) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 697-703;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E19) a polypeptide comprising the amino acid sequence of seq id No. 704;
(E20) a polypeptide comprising the amino acid sequence of SEQ ID NO. 705-707;
(E21) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 708-716, 962, 963;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E22) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 717-724, 964;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E23) a polypeptide comprising the amino acid sequence of SEQ ID NO. 725-728;
(E24) (i) a polypeptide comprising the amino acid sequence of SEQ ID Nos. 729-740, 965, 966;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E25) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 741-749, 967, 968;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E26) a polypeptide comprising the amino acid sequence of SEQ ID NO. 750-751;
(E27) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 752-764, 969, 970;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E28) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 765-769;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E29) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 770-777, 971, 972;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E30) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 778-787, 973;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E31) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 788-792;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E32) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 793-809, 974, 975;
(ii) a polypeptide comprising an amino acid sequence obtained by substituting 1 or more amino acid residues corresponding to the amino acid residues at
(E33) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 810-816;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E34) (i) a polypeptide comprising the amino acid sequence of seq id No. 817-825;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E35) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 826-832;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E36) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 833-;
(ii) a polypeptide comprising an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E37) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 847-857, 977;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E38) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 858-864;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E39) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 865-878, 984, 978;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E40) a polypeptide comprising the amino acid sequence of SEQ ID NO 879-880;
(E41) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 881-891, 979;
(ii) a polypeptide having an amino acid sequence wherein 1 or more amino acid residues corresponding to the amino acid residues at
(E42) (i) a polypeptide comprising the amino acid sequence of seq id nos 892-907, 980;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E43) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO 908-911, 981;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 7 th amino acid residue of SEQ ID NO 908 are substituted with arbitrary amino acid residues in SEQ ID NO 908, 911, 981;
(E44) a polypeptide comprising the amino acid sequence of SEQ ID NO 912-914;
(E45) a polypeptide comprising the amino acid sequence of SEQ ID NO. 915-916;
(E46) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 917-;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E47) (i) a polypeptide comprising the amino acid sequence of seq id No. 928-934;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E48) (i) a polypeptide comprising the amino acid sequence of SEQ ID No. 935-938;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the 5 th and 6 th amino acid residues of SEQ ID NO. 935-938 are substituted with arbitrary amino acid residues in SEQ ID NO. 935-938;
(E49) (ii) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 939-942;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
(E50) (i) a polypeptide comprising the amino acid sequence of SEQ ID NO. 943-948;
(ii) a polypeptide having an amino acid sequence in which 1 or more amino acid residues corresponding to the amino acid residues at
[ means 2]
A diagnostic composition for allergy comprising at least one of the polypeptides according to
[ means 3]
A polypeptide any one of the polypeptides of
[ means 4]
A method of providing an indicator for diagnosing allergy in a subject, comprising the steps of:
(i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing an IgE antibody;
(ii) detecting binding of an IgE antibody in a sample obtained from the subject to the antigen;
(iii) providing an indication that the subject is allergic upon detection of binding of IgE antibodies to the antigen in the subject;
here, the antigen is at least one of the polypeptides described in
[ means 5]
A pharmaceutical composition comprising at least one of the polypeptides of
[ means 6]
The pharmaceutical composition according to mode 5 for use in the treatment of allergy.
[ means 7]
A detection composition for determining the presence or absence of an antigen in a subject, comprising an antibody that binds to at least one of the polypeptides according to
[ means 8]
A detection composition for determining the presence or absence of an antigen in a subject, comprising any of the following primers:
(a) a primer comprising a part of the base sequence of a nucleic acid encoding the polypeptide described in
(b) A primer that is a part of at least one sequence of the base sequences represented by SEQ ID Nos. 1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540, or 548; and/or
A primer that is a part of a sequence complementary to at least one sequence of the base sequences represented by SEQ ID Nos. 1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540, or 548.
[ means 9]
A method for determining the presence or absence of a polypeptide according to
[ means 10]
A raw material or a processed product from which an antigen, which is at least one of the polypeptides described in the above-mentioned
[ means 11]
A method for producing a processed product from which an antigen has been removed or reduced, the method comprising a step of confirming that the antigen has been removed or reduced in a process for producing the processed product, wherein the antigen is at least one of the polypeptides described in
ADVANTAGEOUS EFFECTS OF INVENTION
The present invention can provide a novel antigen for shrimp allergy. In the present invention, since a novel antigen (allergen component) that causes shrimp allergy is identified, a highly sensitive diagnostic method and diagnostic kit for shrimp allergy, a pharmaceutical composition containing the antigen, a shrimp or a processed shrimp product from which the antigen is removed or reduced, and a detection composition for determining the presence or absence of a shrimp antigen in a subject can be provided.
In addition, by the present invention, a novel polypeptide comprising an epitope can be provided. By using the polypeptide of the present invention, a highly sensitive diagnostic kit, a diagnostic composition and a diagnostic method for allergy, a pharmaceutical composition containing the polypeptide, a detection composition for determining the presence or absence of an antigen containing the polypeptide in a subject, a raw material or processed product from which the polypeptide has been removed or reduced, and a method for producing the processed product can be provided.
Drawings
FIG. 1 is a gel photograph showing an electrophoretic pattern of a protein obtained by two-dimensional electrophoresis of a protein contained in Penaeus vannamei (white leg shrimp). The band on the left of the photograph is the band of the molecular weight markers, and the numerical value on the left of the photograph is the molecular weight (KDa) of each molecular weight marker. The numerical value at the top of the photograph represents the isoelectric point.
FIG. 2 is a photograph of a two-dimensional electrophoretogram of proteins contained in Penaeus vannamei, which was immunoblotted with the serum of a shrimp allergy patient.
FIG. 3 is a gel photograph showing an electrophoretic pattern of a protein obtained by two-dimensional electrophoresis of a protein contained in Penaeus monodon (giant tiger prawn). The band on the left of the photograph is the band of the molecular weight markers, and the numerical value on the left of the photograph is the molecular weight (KDa) of each molecular weight marker. The numerical value at the top of the photograph represents the isoelectric point.
FIG. 4 is a photograph of a two-dimensional electrophoresis image of proteins contained in Penaeus monodon, which was immunoblotted with the serum of a shrimp allergy patient.
FIG. 5 is a gel photograph showing an electrophoretic image of a protein obtained by two-dimensional electrophoresis of a protein contained in a Japanese Pacific shrimp (kuruma shrimp). The band on the left of the photograph is the band of the molecular weight markers, and the numerical value on the left of the photograph is the molecular weight (KDa) of each molecular weight marker. The numerical value at the top of the photograph represents the isoelectric point.
FIG. 6 is a photograph of a two-dimensional electrophoretogram of proteins contained in Penaeus japonicus by immunoblotting with the serum of a shrimp allergy patient.
FIG. 7 shows the results of investigation of cross-reactivity by ELISA using sera of patients who developed shrimp and wheat or crab allergy to peptides having the amino acid sequences of the respective epitopes.
FIG. 8 shows the results of investigation of cross-reactivity by ELISA using sera of patients who developed shrimp and wheat or crab allergy to peptides having the amino acid sequences of the respective epitopes.
FIG. 9 shows the results of investigation of cross-reactivity by ELISA using sera of patients who developed shrimp and wheat or crab allergy to peptides having the amino acid sequences of the respective epitopes.
FIG. 10 shows the results of investigation of cross-reactivity by ELISA using sera of patients who developed shrimp and wheat or crab allergy to peptides having the amino acid sequences of the respective epitopes.
FIG. 11 shows the results of investigation of cross-reactivity by ELISA using sera of patients who developed shrimp and wheat or crab allergy to peptides having the amino acid sequences of the respective epitopes.
FIG. 12 shows the results of investigation of cross-reactivity by ELISA for peptides having amino acid sequences of the respective epitopes using sera of patients who developed shrimp and wheat or crab allergy.
Detailed Description
The present invention will be specifically described below, but the present invention is not limited to these.
In the present specification, unless otherwise defined, scientific terms and technical terms used in connection with the present invention have meanings commonly understood by those skilled in the art.
The allergy in the present specification means: in an organism sensitive to an antigen, the antigen is caused to reenter and an uncomfortable allergic reaction is caused to the organism. Allergic reactions can occur when contacted with or ingested by an antigen. Here, the contact means that an object is sensed, and particularly, in the case of a human body, it means that the object is attached to skin, mucous membrane (eye, lip, etc.), and the like. The term "ingested" means absorbed into the body by means such as inhalation or oral administration. The allergy occurring when food is ingested is often referred to as food allergy in particular. In a preferred manner, the allergy may also be a food allergy. Most allergic diseases in food produce IgE antibodies specific to antigens in blood and tissues. IgE antibodies bind to mast cells or basophils. When an antigen specific for the IgE antibody reenters the body of a patient with an allergic disorder, the antigen combines with the IgE antibody bound to mast cells or basophils to produce the physiological effects of an IgE antibody-antigen interaction. Examples of physiological effects include the release of histamine, 5-hydroxytryptamine, heparin, eotaxin, and various leukotrienes. These released substances cause an allergic reaction generated by the combination of IgE antibodies with specific antigens. Specifically, IgE antibodies recognize and bind to epitopes that are specific amino acid sequences in specific antigens, and allergy caused by the specific antigens is achieved through the above pathway.
The allergy to be treated in the present invention is not particularly limited as long as it is an allergy of an allergen including the epitope to be used. In one embodiment, the allergen includes seafood, fruit, vegetables, nuts (seeds and fruits), food herbs, grains, livestock meat, milk, dairy products, and the like ingested by an organism (particularly a human), or parasites parasitic on an organism (particularly a human).
The seafood may include, but is not limited to, shrimps and crabs belonging to the order of the ten-legged order (order of shrimps). Most of the crustaceans generally recognized as "crustaceans" are included in the order of decapod (shrimp). The order of the Octopus (order of the Macropoda) includes the order of the Octopus Brachyura (alias: Suborder Brachyura) and the order of the Octopus anomala (suborder Anomura). All of the members of the order of the ten-podales (order of the shrimps), except the sub-order of the short-tailed order (alias: crab Brachyura) and the sub-order of the heterotrophe (suborder Anomura), are collectively called "shrimps". For shrimp, the following is described. The order brachypoda (alternative name: Eriocheir) includes, without limitation, the family Cariranaceae (Cheiraconidae) (e.g.Elimace eriocheir sinensis (Erimacrus isebeckii)), the family Eriocheir sinensis (Oregonidae) (e.g.Chionoecetes opilio), the family Bisaceae (Carcinidae) (Portunus trituberculatus). Without limitation, the order decapod Anomura (suborder Anomura) comprises the family lithospermaceae (Lithodidae) (e.g., alaska delbrueckea (Paralithodes camtschaticus)).
The seafood may be squid or octopus belonging to the order of Teuthida or Octopoda. The seafood also includes fish belonging to Scombridae and Gadidae. The seafood may further include shellfish belonging to Veneridae (Veneridae). Without limitation, squid, such as squid, comprises pacific plectropod (Todarodespacificus). Pacific plectropods are also known as Japanese squid (Japanese squid). Octopus Octopus includes common Octopus (Octopus vulgaris). The fish of Scombridae family comprises Oriental blue Fin tuna (Thunnus orientalis), Scomber japonicus (Scomber japonica). The fish of the family Gadidae comprises Gadus macrocephalus. Shellfish of the family Veneruleaceae include Mactra Manila (Manila clam), Mactra orientalis (common origin clam), Mactra basket (Basjet clam), etc. In one embodiment, the extract comprises Ruditapes philippinarum.
The fruit includes, but is not limited to, fruits belonging to the families Actinidiaceae, Anacardiaceae, Cucurbitaceae, Musaceae, Rutaceae, and Rosaceae. Examples of the vegetables include fruits belonging to the families solanaceae, cucurbitaceae, and lauraceae. The nuts (seed fruits) include, for example, nuts (seed fruits) belonging to the family Anacardiaceae, Rosaceae, Juglandaceae. The edible grass includes, for example, edible grass belonging to the family of compositae. Examples of the grains include grains belonging to the families gramineae and polygonaceae.
Without limitation, the fruit of the actinidiaceae family comprises kiwi fruit (Actinidia deliciosa). Fruits of the family ananatidae comprise pineapple (Ananas comosus). The Anacardiaceae family includes nuts (seed fruits) such as cashew nuts (cashew nuts) in addition to fruits such as mango (Mangifera indica). Cucurbitaceae includes fruits such as melons (Cucumis melo) and vegetables such as cucumbers (Cucumis sativus). The fruit of Musaceae family comprises banana (Musaacacetinate). The fruit of the family Rutaceae comprises orange (Citrus sinensis). Rosaceae family includes fruits such as peach, strawberry, apple, pear, loquat, etc., and nuts (seed fruits) such as almond. In one embodiment, the Rosaceae family comprises apple (Malusdormestica) and almond (Prunus dulcis).
The vegetables of Solanaceae include, but are not limited to, eggplant (Solanum melongena), tomato (Solanum lycopersicum). The vegetables of the family lauraceae comprise avocado (Persea americana). The nuts (seed fruits) include cashew nuts (Anacardium occidentale) of the family Anacardiaceae and almond nuts (Prunus dulcis) of the family Rosaceae, but not limited thereto. The nuts (seed fruits) of Juglandaceae family comprise Juglans regia (Juglans regia).
The edible grass of the Compositae family includes, but is not limited to, Artemisia argyi (Artemisia indica var. maximowiczii or Artemisia indica). Cereals of the Gramineae family comprise wheat (Triticum aestivum). The grains of Polygonaceae family include, for example, buckwheat (Fagopyrum esculentum) of Fagopyrum genus of Polygonaceae family.
The type of meat is not particularly limited. In one embodiment, the food comprises meat of birds (chicken, duck, etc.), pork, beef, mutton, etc. In one form, the meat is bird meat. In one embodiment, the meat is chicken (Gallus gallous).
The source of the milk (milk) is not particularly limited. In one embodiment, the milk is derived from cow, goat, sheep, or the like. In one embodiment, the milk is cow's milk (Bos Taurus). The dairy product is processed product of milk. Including, without limitation, butter, whipped cream, cheese, yogurt, ice cream.
Parasites are for example, but not limited to, ectoparasites of the family heterotopicaceae. The parasites of the family of the species ectoparasites comprise the species Anisakis simplex.
In one embodiment, the allergen is any of the following. Shrimp, hairy crab (Erimacrus issenbeckii), snow crab (Chionoecetes optilio), blue crab (Portugulus trituberculatus), Allaska king crab (Paralitdes camtschanicus), Pacific Fish (Todarodes pacificus), Octopus vulgaris (Octopus vulgares), Oriental blue Fin tuna (Thunnus orientalis), Scomber japonicus (Scomber japonicus), Gadus macrocephala (Gadus macrocephalus), Philippine clam (Ruditapes philippinarum), Kiwi berry (Actinidia deliciosa), Pear (Ananasus), mango (Mangifera indica), Anacardia (Anacardia), Cucumis sativus (Cucumis), Muscovitum (Citrus sinensis), Muscovitum), orange (Citrus sinensis), orange peel (Citrus sinensis), Citrus sinensis (Citrus sinensis), Citrus sinensis (C. and/or Citrus sinensis), Citrus sinensis (Citrus sinensis), Citrus sinensis (C. and/or Citrus sinensis), Citru, Cow's milk (Bos Taurus), Anisakis simplex (Anisakis simplex).
In the present specification, the shrimp means a shrimp belonging to the order of the Pandales eutropha (Eucarida). All species of the order of the ten-podales (order of the shrimps) except the order of the short-tailed sub-order (alias: crab sub-Brachyura) and the order of the heterotrophe (sub-Anomura) belong to the order of the ten-podales. The shrimp belonging to the order Prodales can be a shrimp belonging to, for example, the family Penaeidae, Procamidae, Acrophoridae, Glabropsidae, Caprifoliaceae, Euphausiaceae, Krill. The shrimp belonging to the family Penaeidae may be, for example, Penaeus vannamei, Penaeus monodon, Penaeus japonicus; the shrimp belonging to the family of Penaeidae may be, for example, Penaeus monodon, Pandalus vannamei; the shrimp belonging to the family crayfish may be, for example, Lobster (Lobster). The shrimp belonging to the family ceratophyllidae may be, for example, a ceratophyllous shrimp; the shrimp belonging to the family vitronecidae may be, for example, white shrimp; the shrimps belonging to the family of the; the shrimp belonging to the family krillaceae may be, for example, krill. The shrimp to which the present invention is directed may be any of the above-mentioned shrimps. Preferably, the shrimp to which the present invention is directed is a shrimp belonging to family Penaeidae (family Panaeeidae).
In this specification, shrimp allergy means: it has an allergic reaction state in which proteins and the like contained in shrimp are used as antigens. Shrimp allergy means an allergy that can occur when contacted with or ingested by an antigen contained in shrimp. The allergy occurring when food is ingested is often referred to as food allergy in particular. The shrimp allergy may also be a food allergy.
In the present specification, an antigen refers to a substance that causes an allergic reaction. When the protein is contained in a raw material such as a food material, the protein is also referred to as an allergen component. The antigen is preferably a protein.
In the present specification, a protein is a molecule having a structure in which natural amino acids are linked by peptide bonds. The number of amino acids contained in the protein is not particularly limited. In the present specification, the term "polypeptide" also means: a molecule having a structure in which natural amino acids are linked by peptide bonds. The number of amino acids contained in the polypeptide is not particularly limited. "polypeptide" is a concept that includes "protein". In addition, a polypeptide in which about 2 to 50 amino acids are linked by peptide bonds is sometimes referred to as a peptide.
When an optical isomer may exist in an amino acid, the amino acid represents an L-form unless otherwise specified. The labeling method of the amino acid sequence of the protein, polypeptide or peptide used in the present specification is as follows: amino acids are indicated by single letter symbols based on standard usage and labeling methods customary in the art, with the left direction being the amino terminal direction and the right direction being the carboxy terminal direction. In the one-letter symbols of amino acids, X represents: any substance having an amino group and a carboxyl group that can bind to amino acids at both ends may be used, and in particular, any of 20 kinds of natural amino acids may be used.
The alanine scanning method (or "alanine glycine scanning method") is a method in which a mutant is prepared by mutating one residue in a protein to alanine (when the original amino acid is alanine, the mutant is mutated to glycine), and a residue site that is important for the structure and function of the protein is specifically identified. Even if the amino acid residue is mutated to alanine (when the original amino acid is alanine, the amino acid residue is mutated to glycine), when the binding to the IgE antibody of the patient remains, the binding to the IgE antibody is not important, and the binding remains even if the amino acid residue is changed to another amino acid. Binding to an IgE antibody means that the binding and reaction of the subject's epitope to the IgE antibody is detected. In the sequence listing of the present invention, the residue represented by X is an amino acid residue at a site where the binding to the IgE antibody of an allergic patient remains even if the residue is substituted with alanine (glycine when the original amino acid is alanine) by the alanine scanning shown in example 4. It is known to those skilled in the art that such a site can be highly modified to retain a high ability to bind to the IgE antibody even when substituted with any other amino acid. That is, the amino acid residue may be replaced with any amino acid residue other than alanine, instead of alanine or glycine.
Binding and maintenance of IgE to an antigen (epitope) is important for the subsequent allergic reaction, and the binding and maintenance are responsible for charge, hydrophobic bond, hydrogen bond, and aromatic interaction of the epitope. The fact that the amino acids are bound and maintained even if they are lost by the substitution into alanine or glycine means that: the amino acid is not critical.
Definition of antigens
The proteins contained in the shrimp were subjected to two-dimensional electrophoresis under the following conditions to limit the antigens of shrimp allergy.
The first dimension electrophoresis is performed by using a gel having a gel length of 5 to 10cm and a pH of 3 to 10, and a gel having a pH gradient with respect to the electrophoresis direction, wherein when the total length of the gel is 1, the gel length up to pH5 is a, the gel length of 5 to 7 is b, and the gel length of at least pH7 is 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, as the gel for isoelectric point electrophoresis, specifically, using IPG gel Immobiline Drystrip (pH3 to 10NL) manufactured by GE healthcare bioscience (hereinafter, abbreviated as GE). IPGphor manufactured by GE was used as the electrophoresis apparatus. The upper limit of the value of the current of the electrophoresis apparatus was set to 75 μ a per 1 gel, and the voltage program was set as follows: (1) a constant voltage step (current change width of 5 μ a for 30 minutes of migration before completion of the step) of increasing the voltage to 750Vhr at a constant voltage of 300V; (2) gradually increasing the voltage from 300Vhr to 1000V; (3) further, the voltage was gradually increased from 4500Vhr to 5000V; (4) thereafter, isoelectric point electrophoresis was performed in the first dimension at a constant voltage of 5000V until the total voltage of Vhr became 12000.
The second dimension electrophoresis uses: specifically, SDS-PAGE was performed using NuPAGE 4-12% Bris-Tris Gels IPG well mini 1mm manufactured by Life technologies, wherein the gel concentration at the base end in the running direction was set to 3-6% and the gel concentration at the tip side in the running direction was set to be higher than the gel concentration at the base end in the running direction. The electrophoresis apparatus used was XCell SureLock Mini-Cell manufactured by Life technologies. As the running buffer, 50mM MOPS, 50mM Tris base, 0.1% (w/V) SDS, 1mM EDTA were used, and running was performed at a constant voltage of 200V for about 45 minutes.
As a result, it was found that when proteins of shrimps (Litopenaeus vannamei, Penaeus monodon and Marsupenaeus japonicus) were subjected to two-dimensional electrophoresis under the above conditions, the antigen of the following
Antigens
(1)Antigen of
As a result of sequence identification of the
In addition, for
Accordingly, in the present application, the antigen of
(1A-a) a protein comprising an amino acid sequence wherein 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 2, 45 or 87.
(1A-b) a protein comprising an amino acid sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence represented by SEQ ID NO. 2, 45, or 87.
(1A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO.1, 44 or 86 in which 1 or more nucleotides are deleted, substituted, inserted or added.
(1A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO.1, 44, or 86.
(1A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO.1, 44, or 86; or
(1B) The protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 2 to 43, 45 to 85, or 87 to 115, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 50 or all of the amino acid sequences. More preferably: the protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 2 to 5, 7 to 13, 16 to 24 and 26 to 43, SEQ ID Nos. 45, 46, 48, 49, 51 to 55, 58 to 66 and 68 to 85, or SEQ ID Nos. 87 to 90 and 92 to 115, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45 or all of the sequences of the amino acid sequence. Wherein the amino acid sequence represented by any one of SEQ ID Nos. 2 to 43, 45 to 85, and 87 to 115 is optionally deleted, substituted, inserted, or added with 1 or more amino acids.
The proteins (1A-a) to (1A-e) and (1B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", the protein appears as a spot having a molecular weight of 80kDa to 260kDa, preferably a molecular weight of about 90kDa to 230kDa, more preferably a molecular weight of 100kDa to 200kDa, and an isoelectric point of 3.0 to 7.0, preferably 4.0 to 6.5, and even more preferably 5.0 to 6.0.
(2)Antigen of
As a result of sequence identification by mass spectrometry for the
In addition, for
Accordingly, in the present application, the antigen of
(2A-a) a protein comprising an amino acid sequence wherein 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 117, 141 or 146;
(2A-b) a protein comprising an amino acid sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence represented by SEQ ID NO. 117, 141, or 146.
(2A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 116, 140 or 145 in which 1 or more nucleotides are deleted, substituted, inserted or added.
(2A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 116, 140, or 145.
(2A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 116, 140, or 145.
(2B) The protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 117 to 139, 141 to 144, or 146 to 159, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or all of the amino acid sequences. More preferably: the protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 117 to 126, 128 to 132, 136, 138 and 139, 141 to 144, or 146 to 159, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or all of the amino acid sequences. Wherein the amino acid sequence represented by any one of SEQ ID Nos. 117 to 139, 141 to 144, and 146 to 159 is optionally deleted, substituted, inserted, or added with 1 or more amino acids.
The proteins (2A-a) to (2A-e) and (2B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", the protein appears as a spot having a molecular weight of 50kDa to 160kDa, preferably a molecular weight of about 55kDa to 150kDa, more preferably a molecular weight of 60kDa to 130kDa, and an isoelectric point of 4.5 to 10.0, preferably 5.0 to 9.5, and even more preferably 5.5 to 9.0.
(3) Antigen of
As a result of sequence identification by mass spectrometry for
In addition, for
Accordingly, in the present application, the antigen of
(3A-a) a protein comprising an amino acid sequence of SEQ ID NO. 161, 179 or 230 in which 1 or more amino acids have been deleted, substituted, inserted or added.
(3A-b) a protein comprising an amino acid sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence represented by SEQ ID NO. 161, 179, or 230.
(3A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 160, 178 or 229 in which 1 or more nucleotides are deleted, substituted, inserted or added.
(3A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 160, 178 or 229.
(3A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO. 160, 178 or 229.
(3B) The protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO. 161-177, SEQ ID NO. 179-228, and SEQ ID NO. 230-274, and preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45 or all of the amino acid sequences. More preferably: the protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO. 161, 162 and 164-177, SEQ ID NO. 179-181, 183-186, 188-190, 192-212, 214-216 and 218-228, SEQ ID NO. 230-233, 235-240, 242, 244-258, 260-262 and 264-274, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or all of the amino acid sequences. Wherein the amino acid sequence represented by any one of SEQ ID Nos. 161 to 177, 179 to 228, and 230 to 274 is optionally deleted, substituted, inserted, or added with 1 or more amino acids.
The proteins (3A-a) to (3A-e) and (3B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", the protein appears as a spot having a molecular weight of 80kDa to 260kDa, preferably a molecular weight of about 90kDa to 230kDa, more preferably a molecular weight of 100kDa to 200kDa, and an isoelectric point of 3.0 to 7.0, preferably 4.0 to 6.5, and even more preferably 5.0 to 6.0.
(4)Antigen of
As a result of sequence identification by mass spectrometry for the
In addition, for the
Accordingly, in the present application, the antigen of the
(4A-a) a protein comprising the amino acid sequence of SEQ ID NO. 276, 300 or 306 in which 1 or more amino acids are deleted, substituted, inserted or added.
(4A-b) a protein comprising an amino acid sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence represented by SEQ ID NO. 276, 300, or 306.
(4A-c) a protein comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 275, 299 or 305 in which 1 or more nucleotides are deleted, substituted, inserted or added.
(4A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 275, 299, or 305.
(4A-e) a protein comprising an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions with a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO. 275, 299 or 305.
(4B) The protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 276 to 298, 300 to 304, or 306 to 320, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or all of the amino acid sequences. More preferably: the protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 276 to 293 and 295 to 298, SEQ ID Nos. 300 to 304, or SEQ ID Nos. 306, 307, and 309 to 319, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or all of the amino acid sequences. Wherein the amino acid sequence represented by any one of SEQ ID Nos. 276 to 298, 300 to 304, and 306 to 320 is optionally deleted, substituted, inserted, or added with 1 or more amino acids.
The proteins (4A-a) to (4A-e) and (4B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", the protein appears as a spot having a molecular weight of 50kDa to 160kDa, preferably a molecular weight of about 55kDa to 150kDa, more preferably a molecular weight of 60kDa to 130kDa, and an isoelectric point of 4.5 to 10.0, preferably 5.0 to 9.5, and even more preferably 5.5 to 9.0.
(5)Antigen of spot 5
As a result of sequence identification of the spot 5 by mass spectrometry, the amino acid sequences of SEQ ID Nos. 321 to 336 were detected for Penaeus vannamei, 337 to 360 were detected for Penaeus monodon, and 363 to 379 were detected for Penaeus japonicus.
In addition, for spot 5, the mass spectrum data obtained by the mass spectrometer was compared with the protein data of NCBI and analyzed. As a result, it was identified as glycogen phosphorylase derived from Japanese Pacific shrimp (amino acid sequence: SEQ ID NO: 362, base sequence encoding the same: SEQ ID NO: 361) as SEQ ID NO. 363 to 379. Moreover, the glycogen phosphorylase derived from Japanese Pacific shrimp was identified as SEQ ID Nos. 321 to 336 and 337 to 360. That is, since proteins having high homology are detected in Penaeus vannamei and Penaeus monodon, glycogen phosphorylase or a homologue thereof can be judged as an antigen of shrimp.
Accordingly, in the present application, the antigen of spot 5 is any of the following (5A-a) to (5A-e) and (5B).
(5A-a) a protein comprising the amino acid sequence of SEQ ID NO. 362 in which 1 or more amino acids are deleted, substituted, inserted, or added.
(5A-b) a protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence represented by SEQ ID NO. 362.
(5A-c) a protein comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 361 in which 1 or more nucleotides are deleted, substituted, inserted or added.
(5A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 361.
(5A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO. 361.
(5B) The protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 321 to 336, 337 to 360, or 362 to 379, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or all of the amino acid sequences. More preferably: the protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 322 to 326, 329 to 330 and 332 to 336, SEQ ID Nos. 338 to 343, 345, 347 to 349 and 352 to 360, and SEQ ID Nos. 362 to 367, 369, 371 and 373 to 379, and preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or all of the amino acid sequences. Wherein the amino acid sequence represented by any one of SEQ ID Nos. 321 to 336, 337 to 360, and 362 to 379 is optionally deleted, substituted, inserted, or added with 1 or more amino acids.
The proteins (5A-a) to (5A-e) and (5B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above item "restriction of antigen", the protein appears as a spot having a molecular weight of 70kDa to 160kDa, preferably a molecular weight of about 75kDa to 140kDa, more preferably a molecular weight of 80kDa to 130kDa, and an isoelectric point of 5.0 to 9.0, preferably 5.5 to 8.5, and even more preferably 6.0 to 8.0.
(6)Antigen of
As a result of sequence identification of the
In addition, for the
Accordingly, in the present application, the antigen of
(6A-a) a protein comprising an amino acid sequence wherein 1 or more amino acids are deleted, substituted, inserted or added in SEQ ID NO. 381, 399 or 414.
(6A-b) a protein comprising an amino acid sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence represented by SEQ ID NO. 381, 399, or 414.
(6A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 380, 398 or 413 with deletion, substitution, insertion or addition of 1 or more nucleotides.
(6A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 380, 398, or 413.
(6A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 380, 398 or 413.
(6B) The protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 381 to 397, 399 to 412, and 414 to 419, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or all of the amino acid sequences. More preferably: the protein comprising at least one amino acid sequence selected from the group consisting of sequence numbers 381 to 384 and 386 to 397, sequence numbers 399 to 402, 404 and 407 to 412, or sequence numbers 414 to 416, 418 and 419, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of the amino acid sequences. Wherein the amino acid sequence represented by any one of sequence numbers 381 to 397, 399 to 412 and 414 to 419 is optionally deleted, substituted, inserted or added with 1 or more amino acids.
The proteins (6A-a) to (6A-e) and (6B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", a protein appears as a spot having a molecular weight of 50kDa to 110kDa, preferably a molecular weight of about 55kDa to 100kDa, more preferably a molecular weight of 60kDa to 90kDa, and an isoelectric point of 4.0 to 7.0, preferably 4.5 to 6.5, and even more preferably 5.0 to 6.0.
(7)Antigen of spot 7
And as for the spot 7, the sequence identification result is carried out by mass spectrometry, and the amino acid sequences of the sequence numbers 422 to 435 and 436 to 441 are detected for the penaeus vannamei and the penaeus monodon.
In addition, for the spot 7, the mass spectrum data obtained by the mass spectrometer was compared with the protein data of NCBI and analyzed. As a result, the sequence numbers 422 to 435 were identified as
Accordingly, in the present application, the antigen of spot 7 is any of the following (7A-a) to (7A-e) and (7B).
(7A-a) a protein comprising the amino acid sequence of SEQ ID NO. 421 in which 1 or more amino acids are deleted, substituted, inserted, or added.
(7A-b) a protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence represented by SEQ ID NO. 421.
(7A-c) a protein comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 420 in which 1 or more nucleotides are deleted, substituted, inserted, or added.
(7A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 420.
(7A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO. 420.
(7B) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 421 to 435 or 436 to 441, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of the amino acid sequences. Wherein the amino acid sequence represented by any one of SEQ ID Nos. 421 to 435 and 436 to 441 is optionally deleted, substituted, inserted, or added with 1 or more amino acids.
The proteins (7A-a) to (7A-e) and (7B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", a protein appears as a spot having a molecular weight of 45kDa to 80kDa, preferably a molecular weight of about 50kDa to 75kDa, more preferably a molecular weight of 55kDa to 70kDa, and an isoelectric point of 4.5 to 9.0, preferably 5.0 to 8.5, and even more preferably 5.5 to 8.0.
(8)Antigen of spot 8
As a result of sequence identification of the spot 8 by mass spectrometry, amino acid sequences of SEQ ID Nos. 442 to 453 were detected for Penaeus vannamei, 456 to 479 for Penaeus monodon, and 482 to 484 for Penaeus japonicus were detected.
In addition, for the spot 8, the mass spectrum data obtained by the mass spectrometer was compared with the protein data of NCBI and analyzed. As a result, the sequences of SEQ ID Nos. 456 to 479 were identified as phosphopyruvate hydratase derived from Penaeus monodon (amino acid sequence: SEQ ID NO: 455, base sequence encoding the same: SEQ ID NO: 454). In addition, the sequence numbers 442-453 were also identified as the phosphopyruvate hydratase derived from Penaeus monodon. That is, since a protein having high homology is detected in Penaeus vannamei, phosphopyruvate hydratase or a homologue thereof can be judged to be an antigen of Penaeus vannamei. The sequences No. 482 to 484 were identified as phosphopyruvate hydratase derived from Penaeus japonicus (amino acid sequence: SEQ ID NO: 481, nucleotide sequence encoding the same: SEQ ID NO: 480).
Therefore, in the present application, the antigen of spot 8 is any of the following (8A-a) to (8A-e) and (8B).
(8A-a) a protein comprising the amino acid sequence of SEQ ID NO. 455 or 481 in which 1 or more amino acids are deleted, substituted, inserted, or added.
(8A-b) a protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more, with the amino acid sequence represented by SEQ ID NO. 455 or 481.
(8A-c) a protein comprising an amino acid sequence encoded by the base sequence of SEQ ID NO. 454 or 480 in which 1 or more nucleotides are deleted, substituted, inserted, or added.
(8A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 454 or 480.
(8A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO. 454 or 480.
(8B) The protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 442 to 453, 455 to 479, and 481 to 484, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, and 20 or all of the amino acid sequences. More preferably: the protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 442-450, 452, and 453, SEQ ID Nos. 455-459, 463-469, 471-473, and 476-479, or SEQ ID Nos. 481-484, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or all of the amino acid sequences. Wherein the amino acid sequence represented by any one of SEQ ID NOS.442-453, SEQ ID NOS.455-479 and SEQ ID NOS.481-484 is optionally deleted, substituted, inserted, or added with 1 or more amino acids.
The proteins (8A-a) to (8A-e) and (8B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", the protein appears as a spot having a molecular weight of 35kDa to 80kDa, preferably a molecular weight of around 40kDa to 75kDa, more preferably a molecular weight of 45kDa to 70kDa, and an isoelectric point of 4.0 to 8.0, preferably 4.5 to 7.5, and even more preferably 5.0 to 7.0.
(9)Antigen of spot 9
As a result of sequence identification of the spot 9 by mass spectrometry, amino acid sequences of SEQ ID Nos. 487 to 490 were detected for Penaeus vannamei, 491 to 497 were detected for Penaeus monodon, and 498 to 518 were detected for Penaeus japonicus.
As a result of comparison and analysis of the mass spectrum data obtained by the mass spectrometer with the protein data of NCBI for the spot 9, a mitochondrial ATP synthetase subunit α precursor derived from Penaeus vannamei (amino acid sequence: SEQ ID NO: 486, base sequence encoding the same: SEQ ID NO: 485) was identified for SEQ ID Nos. 487 to 490, and further, a mitochondrial ATP synthetase subunit α precursor derived from Penaeus vannamei, that is, a protein having high homology was detected in Penaeus vannamei and Penaeus japonicus, and therefore, it could be judged that the mitochondrial ATP synthetase subunit α precursor or a homologue thereof was an antigen of shrimp.
Therefore, in the present application, the antigen of the spot 9 is any of the following (9A-a) to (9A-e) and (9B).
(9A-a) a protein comprising an amino acid sequence of SEQ ID NO. 486 in which 1 or more amino acids have been deleted, substituted, inserted or added.
(9A-b) a protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence represented by SEQ ID NO. 486.
(9A-c) a protein comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 485 in which 1 or more nucleotides are deleted, substituted, inserted, or added.
(9A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 485.
(9A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO. 485.
(9B) The protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 486-490, 491-497, and 498-518, and preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, and 20 or all of the amino acid sequences. More preferably: the protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 486-490, 491-497, 498-501, 503-507, 509, and 512-518, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or all of the amino acid sequences. Wherein the amino acid sequence represented by any one of SEQ ID Nos. 486-490, 491-497 and 498-518 is optionally deleted, substituted, inserted or added with 1 or more amino acids.
The proteins (9A-a) to (9A-e) and (9B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", the protein appears as a spot having a molecular weight of 40kDa to 70kDa, preferably a molecular weight of about 45kDa to 65kDa, more preferably a molecular weight of 50kDa to 60kDa, and an isoelectric point of 5.0 to 10.0, preferably 5.5 to 9.5, and even more preferably 6.0 to 9.0.
(10)Antigen of
As a result of sequence identification of the
In addition, the
Accordingly, in the present application, the antigen of the
(10A-a) a protein comprising the amino acid sequence of SEQ ID NO. 520 in which 1 or more amino acids have been deleted, substituted, inserted, or added.
(10A-b) a protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence represented by SEQ ID NO. 520.
(10A-c) a protein having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 519 wherein 1 or more nucleotides are deleted, substituted, inserted or added.
(10A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 519.
(10A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO. 519.
(10B) The protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOS 520 to 527, SEQ ID NOS 528 to 532, and SEQ ID NOS 533 to 539, and preferably comprises at least 2, 3, 4, 5, 6 or all of the amino acid sequences. More preferably: the protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID Nos. 520 to 525 and 527, 528 to 530 and 532, or 533 to 537 and 539, preferably a protein comprising at least 2, 3, 4, 5 or all of the amino acid sequences. Wherein the amino acid sequence represented by any one of SEQ ID NOS 520 to 527, 528 to 532, and 533 to 539 is optionally deleted, substituted, inserted, or added with 1 or more amino acids.
The proteins (10A-a) to (10A-e) and (10B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", the protein appears as a spot having a molecular weight of 10kDa to 50kDa, preferably a molecular weight of around 15kDa to 40kDa, more preferably a molecular weight of 20kDa to 40kDa, and an isoelectric point of 7.0 to 11.0, preferably 7.5 to 10.5, and even more preferably 8.0 to 10.0.
(11)Antigen of spot 11
As a result of sequence identification of the spot 11 by mass spectrometry, the amino acid sequences of SEQ ID Nos. 542 to 547 were detected for Penaeus vannamei, 550 to 553 were detected for Penaeus monodon, and 554 to 557 were detected for Penaeus japonicus.
In addition, the spot 11 was analyzed by comparing the mass spectrum data obtained by the mass spectrometer with the protein data of NCBI. As a result, it was identified as cyclophilin A derived from Penaeus vannamei (amino acid sequence: SEQ ID NO: 541, base sequence encoding it: SEQ ID NO: 540) in SEQ ID NO. 542-547. In addition, the sequence No. 554-557 is also determined to be cyclophilin A derived from the Penaeus vannamei. That is, since a protein having high homology is detected in Japanese Pacific shrimp, cyclophilin A or a homologue thereof can be judged as an antigen of shrimp. Further, each of SEQ ID Nos. 550 to 553 was identified as a Penaeus monodon-derived cyclophilin A (amino acid sequence: SEQ ID NO: 549, base sequence encoding the same: SEQ ID NO: 548).
Accordingly, in the present application, the antigen of the spot 11 is any of the following (11A-a) to (11A-e) and (11B).
(11A-a) a protein comprising the amino acid sequence of SEQ ID NO. 541 or 549 in which 1 or more amino acids have been deleted, substituted, inserted, or added.
(11A-b) a protein comprising an amino acid sequence having an identity of 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more, to the amino acid sequence represented by SEQ ID NO. 541 or 549.
(11A-c) a protein comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 540 or 548 wherein 1 or more nucleotides are deleted, substituted, inserted or added.
(11A-d) a protein comprising an amino acid sequence encoded by a base sequence having 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence represented by SEQ ID NO. 540 or 548.
(11A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a base sequence complementary to the base sequence represented by SEQ ID NO. 540 or 548.
(11B) The protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO. 541-547, SEQ ID NO. 549-553, or SEQ ID NO. 554-557, and preferably comprises at least 2, 3, 4, 5 or all of the amino acid sequences. More preferably: the protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO. 541-543, 545 and 546, SEQ ID NO. 549-553, or SEQ ID NO. 554-556, and preferably comprises at least 2, 3 or all of the amino acid sequences. Wherein the amino acid sequence shown in any one of SEQ ID Nos. 541-547, 549-553, and 554-557 is optionally deleted, substituted, inserted, or added with 1 or more amino acids.
The proteins (11A-a) to (11A-e) and (11B) may be: in the gel obtained by two-dimensional electrophoresis under the conditions described in the above "restriction of antigen", the protein appears as a spot having a molecular weight of 10kDa to 30kDa, preferably a molecular weight of about 13kDa to 25kDa, more preferably a molecular weight of 15kDa to 20kDa, and an isoelectric point of 7.0 to 11.0, preferably 7.5 to 10.5, and even more preferably 8.0 to 10.0.
The proteins as antigens in the above (1) to (11) and the polypeptides (E1) to (E50) described later further include: the amino acid residue of the protein or polypeptide is modified by phosphorylation, sugar chain modification, aminoacylation, ring opening, deamination, etc.
Preferably, the proteins as antigens of the above (1) to (11) and the polypeptides of (E1) to (E50) described later are antigens of allergy.
In the present specification, the case where "1 or more amino acids are deleted, substituted, inserted or added" in an amino acid sequence means: an amino acid sequence obtained by deleting 1 or more amino acids, substituting another amino acid, inserting another amino acid, and/or adding another amino acid in the target amino acid sequence. "multiple amino acids" refers to: but not limited to, 200 or less, 100 or less, 50 or less, 30 or less, 20 or less, 15 or less, 12 or less, 10 or less, 8 or less, 6 or less, 4 or less, and 3 or less amino acids. Alternatively, multiple amino acids refer to: 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1% of the amino acids relative to the entire length of the amino acid sequence.
Among the above, the substitution is preferably a conservative substitution. Conservative substitutions are those in which a particular amino acid residue is replaced with a residue having similar physicochemical characteristics; any substitution may be made as long as the characteristics relating to the structure of the original sequence are not substantially changed, and for example, any substitution may be made as long as the substitution of an amino acid does not destroy the helix present in the original sequence or destroy other types of secondary structures that are the characteristics of the original sequence. Hereinafter, conservative substitutions of amino acid residues are exemplified by the classification of substitutable residues, but the substitutable amino acid residues are not limited to the ones described below.
Group A: leucine, isoleucine, valine, alanine, methionine
Group B: aspartic acid and glutamic acid
Group C: asparagine and glutamine
Group D: lysine and arginine
Group E: serine and threonine
And F group: phenylalanine, tyrosine
In the case of non-conservative substitutions, one member of the above categories may be replaced with another member of the category. For example, the B, D, E group amino acids may be replaced with other groups of amino acids in order to exclude unexpected sugar chain modifications. Alternatively, cysteine may be deleted or substituted with another amino acid in order to prevent the protein having a three-dimensional structure from being folded. Alternatively, in order to maintain the balance between hydrophilicity and hydrophobicity or to facilitate synthesis, the amino acids may be replaced by considering the hydropathic index (j.kyte and r.doolittle, j.mol.biol., vol.157, p.105-132,1982) of the amino acids as an index of hydrophobicity and hydrophilicity in order to increase the degree of hydrophilicity.
Alternatively, substitution with an amino acid having less steric hindrance than the original amino acid, for example, substitution from group F to group A, B, C, D, E; substitution from an amino acid having a charge to an amino acid having no charge, for example, substitution from group B to group C, may be performed. This improves the binding to IgE antibodies.
In the present specification, the identity% of 2 amino acid sequences can be determined by visual inspection and mathematical calculation. Alternatively, the% identity may be determined using a computer program. Examples of such computer programs include: BLAST and ClustalW, etc. In particular, various conditions (parameters) for identity search using the BLAST program are publicly available in accordance with the website of NCBI, DNA Data Bank of Japan (DDBJ) and the like described in Altschul et al (Nucl. acids. Res.,25, p.3389-3402,1997) (BLAST handbook, NCB/NLM/NIH Bethesda, Md 20894; Altschul et al). The determination may be performed using programs such as genetic information processing software GENETYX ver.7(GENETYX), DINASIS Pro (Hitachi Soft, Ltd.), Vector NTI (Infomax), and the like.
In the present specification, the case where "1 or more nucleotides are deleted, substituted, inserted or added" in the base sequence refers to: a nucleotide sequence obtained by deleting 1 or more nucleotides, replacing with another nucleotide, inserting another nucleotide, and/or adding another nucleotide to the target nucleotide sequence. By "plurality of nucleotides" is meant: non-limitingly, within 600, within 300, within 150, within 100, within 50, within 30, within 20, within 15, within 12, within 10, within 8, within 6, within 4, within 3 nucleotides. Alternatively, a plurality of nucleotides means: 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1% nucleotides over the entire length of the base sequence. It is preferable that no frame shift is generated in the sequence encoding amino acids by deletion, substitution, insertion or addition of the above-mentioned nucleotides.
In the present specification, the identity% of 2 base sequences can be determined by visual inspection and mathematical calculation. Alternatively, the% identity may be determined using a computer program. Examples of such a sequence comparison computer program include: BLASTN programs available from the website https of the national library of medicine,// blast.ncbi.nlm.nih.gov/blast.cgi (Altschul et al (1990) J.mol.biol.215: 403-10): version 2.2.7 or WU-BLAST2.0 algorithm, etc. The following internet website may be used for the setting of Default parameters (Default Parameter) for the WU-BLAST2.0 standard: http:// blast.
"under stringent conditions" in the present specification means that hybridization is carried out under conditions of moderate or high stringency. Specifically, the moderately stringent conditions can be easily determined by a person skilled in the art having ordinary skill based on the length of the DNA, for example. Basic conditions are as described in Sambrook et al, Molecular Cloning: A Laboratory Manual, 3 rd edition, chapters 6-7, Cold Spring Harbor Laboratory Press, 2001. Preferably, moderately stringent conditions include the following hybridization conditions: 1 XSSC to 6 XSSC, at 42 ℃ to 55 ℃; more preferably 1 XSSC to 3 XSSC, at 45 ℃ to 50 ℃; most preferably 2 XSSC at 50 ℃. In the case where formamide is contained in the hybridization solution at a concentration of, for example, about 50%, a temperature 5 to 15 ℃ lower than the above temperature can be used. The washing conditions include: 0.5 XSSC to 6 XSSC, 40 ℃ to 60 ℃. In hybridization and washing, 0.05% to 0.2%, preferably about 0.1% SDS is usually added. The conditions of high stringency can also be easily determined by those skilled in the art, for example, based on the length of DNA. Typically, as highly stringent (high stringency) conditions, hybridization and/or washing at higher temperatures and/or lower salt concentrations than moderately stringent conditions are included. For example, as the hybridization conditions, there can be mentioned: 0.1 XSSC to 2 XSSC, at 55 ℃ to 65 ℃; more preferably 0.1 XSSC to 1 XSSC, 60 ℃ to 65 ℃; most preferably 0.2 XSSC at 63 ℃. Examples of the washing conditions include: 0.2 XSSC to 2 XSSC, 50 ℃ to 68 ℃, more preferably 0.2 XSSC, 60 ℃ to 65 ℃.
The antigen can also be obtained by isolation/purification from shrimp by combining protein purification methods well known to those skilled in the art. Alternatively, the antigen may be obtained by expressing the antigen as a recombinant protein by a gene recombination technique known to those skilled in the art, and then isolating/purifying by a protein purification method known to those skilled in the art.
Examples of the method for purifying a protein include: methods utilizing solubility such as salting out and solvent precipitation; methods utilizing differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods using charge, such as ion exchange chromatography and hydroxyapatite chromatography; methods utilizing specific affinity such as affinity chromatography; methods utilizing the difference in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and isoelectric point electrophoresis, etc.
The preparation of proteins using gene recombination techniques can be carried out as follows: an expression vector comprising a nucleic acid encoding an antigen is prepared, the expression vector is introduced into a suitable host cell by gene introduction or transformation, the host cell is cultured under conditions suitable for expression of the recombinant protein, and then the recombinant protein expressed in the host cell is recovered.
A "vector" is a nucleic acid that can be used to introduce a nucleic acid linked thereto into a host cell; an "expression vector" is a vector capable of directing the expression of a protein encoded by a nucleic acid into which the vector is introduced. Examples of the vector include plasmid vectors, viral vectors and the like. The skilled person can select an expression vector suitable for recombinant protein expression depending on the kind of host cell used.
The "host cell" is a cell into which a gene has been introduced or transformed by a vector. The host cell can be appropriately selected by those skilled in the art according to the vector to be used. The host cell may be derived from a prokaryote such as e. When a prokaryotic cell such as E.coli is used as a host, the antigen of the present invention preferably contains an N-terminal methionine residue in order to facilitate expression of a recombinant protein in the prokaryotic cell. The N-terminal methionine may also be cleaved from the recombinant protein after expression. Alternatively, it may be a unicellular eukaryote such as yeast; eukaryotic cells such as plant cells and animal cells (e.g., human cells, simian cells, hamster cells, rat cells, mouse cells, and insect cells), and silkworms.
The gene transfer or transformation of the expression vector into a host cell can be carried out by a method known to those skilled in the art as appropriate. In addition, those skilled in the art will appropriately select conditions suitable for expression of the recombinant protein depending on the kind of the host cell to culture the host cell, thereby enabling expression of the recombinant protein. Then, the host cells expressing the recombinant protein are homogenized, and the resulting homogenized mixture is purified into the above-mentioned proteins by a suitable combination of methods, whereby the antigen expressed as the recombinant protein can be isolated/purified. The antigen may be prepared by introducing the expression vector, the synthetic double-stranded DNA, or the mRNA transcribed therefrom into a cell-free protein synthesis system, expressing the DNA, and isolating and purifying the expressed protein.
Diagnostic kit/diagnostic method (1)
The present invention provides a method for providing an index for diagnosing allergy in a subject shrimp, comprising the steps of:
(i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing an IgE antibody;
(ii) detecting binding of an IgE antibody in a sample obtained from the subject to the antigen;
(iii) providing an indication that the subject is a shrimp allergy upon detection of binding of IgE antibodies of the subject to the antigen;
here, the antigen is at least one of the proteins defined as the antigens of (1) to (11) above.
In the present specification, "diagnosis" includes individual "detection" including a possibility, in addition to a diagnosis (confirmed) usually made by a doctor.
The sample obtained from the subject refers to a solution containing IgE antibodies collected from the subject. Such a solution contains, for example, blood, saliva, sputum, nasal discharge, urine, sweat, and tears. The sample obtained from the subject may be subjected to pretreatment for increasing the concentration of IgE antibodies in the sample before contacting with the antigen. Examples of the pretreatment of the sample include obtaining serum or plasma from blood. In addition, the Fab part, which is a part binding to the antigen, can be further purified. In a particularly preferred embodiment, the step (i) is performed by contacting an antigen with IgE antibodies in serum obtained from the subject.
The IgE antibody may be the IgE antibody itself, or a mast cell to which the IgE antibody binds.
The contact between the sample obtained from the subject and the antigen and the detection of the binding thereof can be carried out by a known method. As such a method, for example, a method using: ELISA (Enzyme-linked immunosorbent Assay), sandwich immunoassay (sandwich immunoassays), Western immunoblotting, immunoprecipitation, and immunochromatography. These methods are all methods in which an antigen is brought into contact with and bound to an IgE antibody of interest, and the antigen is allowed to act on a secondary antibody obtained by enzymatically labeling the IgE antibody specifically bound to the antigen, and a substrate for an enzyme (usually a color-developing or luminescent reagent) is added to detect a product of the enzymatic reaction, thereby detecting the binding of the antigen to the IgE antibody of interest. Or a method of detecting a fluorescently labeled secondary antibody. Alternatively, the detection may be performed by a measurement method such as Surface Plasmon Resonance (SPR) which can evaluate the binding of the antigen to the IgE antibody. A mixture of multiple antigen-specific IgE antibodies is also possible.
The antigen may be in a state in which the isolated antigen is immobilized on a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, or the like can be used in the steps (i) and (ii); in the step (i), the sample obtained from the subject is brought into contact with the surface to which the antigen is immobilized. The isolated antigen can be obtained by isolation/purification of shrimp by combining protein purification methods well known to those skilled in the art, or can also be prepared by gene recombination techniques. In addition, the carrier may be attached with an antibody.
The antigen may be immobilized on a carrier. In this case, in the steps (i) and (ii), the presence of the antigen to which the antibody is bound can be confirmed by laser light using a flow cytometer or the like. Examples thereof include Basophil Activation Test (BAT). Further, a Histamine Release Test (HRT) may be mentioned in which an antigen is brought into further contact with blood cells in a sample to confirm whether histamine is released or not.
Alternatively, the antigen may be detected by western blotting by state transfer separated by two-dimensional electrophoresis. A method for separating a protein sample by two-dimensional electrophoresis, which comprises performing isoelectric point electrophoresis in a first dimension and performing SDS-PAGE in a second dimension. In this case, the conditions for two-dimensional electrophoresis are not particularly limited as long as the antigen of the present invention can be separated. For example, the conditions of two-dimensional electrophoresis described in the above "definition of antigen" item can be used. Alternatively, the electrophoresis conditions may be set as described in
(A) as the first-dimension isoelectric point electrophoresis gel, when the gel length is within the range of 5-10 cm, the pH range of the gel is 3-10, the gel length from the pH gradient of the gel in the electrophoresis direction to the pH5 is a, the gel length of the pH 5-7 is b, and the gel length of the pH7 or more is c, the first-dimension isoelectric point electrophoresis gel satisfies the relationship of "a < b" and "b > c";
(B) in the case of (A), when the total length of the gel is 1, a is in the range of 0.15 to 0.3, b is in the range of 0.4 to 0.7, and c is in the range of 0.15 to 0.3;
(C) in the first-dimension isoelectric point electrophoresis, a constant voltage step is performed by applying a constant voltage having a value in the range of 100V to 600V to 1 gel containing a subject, and a voltage increase step is started after the range of a change in electrophoresis width of 5 μ A for 30 minutes per electrophoresis;
(D) in the case of (C), the final voltage in the voltage increasing step is set to be in the range of 3000V to 6000V;
(E) the gel length of the first-dimension isoelectric point electrophoresis gel in the long-side direction is 5-10 cm, and the gel concentration of the second-dimension electrophoresis gel at the base end in the electrophoresis direction is 3-6%; and
(F) in the case of (E), the gel concentration at the forward end portion of the second dimensional electrophoresis gel in the electrophoresis direction is set to be higher than the gel concentration at the proximal end portion in the electrophoresis direction.
The antigens (1) to (11) are antigens that specifically bind to IgE antibodies of shrimp allergy patients. Thus, detection of binding of IgE antibodies to the antigen in the subject provides an indication that the subject is shrimp allergic.
The present invention also provides a diagnostic kit for shrimp allergy, which comprises at least one of the antigens (1) to (11) described above. The diagnostic kit of the present invention can be applied to the above-described method for providing an index for diagnosing shrimp allergy or the diagnostic method described below. The diagnostic kit of the present invention may further comprise, in addition to at least one of the antigens of (1) to (11), an enzyme-labeled anti-IgE antibody and a chromogenic substrate or a luminescent substrate which is a substrate for the enzyme. In addition, fluorescently labeled anti-IgE antibodies can also be used. The diagnostic kit of the present invention may be provided in a state where the antigen is immobilized on a carrier. The diagnostic kit of the present invention may also be provided with instructions for the steps used for diagnosis, a method kit comprising the instructions.
In another aspect, the diagnostic kit described above comprises: a companion diagnostic medicine for shrimp allergy. Companion diagnostic agents refer to substances used for the following purposes: the reactivity of a pharmaceutical is investigated for specifying a patient expected to have a pharmaceutical effect, specifying a patient at risk of severe side effects of a pharmaceutical, or optimizing a treatment using a pharmaceutical. Here, optimization of treatment includes, for example: determination of the volume of administration, judgment of discontinuation of administration, and determination of what allergen component to use to confirm whether or not immune tolerance is obtained.
The present invention also provides a composition for diagnosing shrimp allergy, which comprises at least one of the antigens (1) to (11) described above. The diagnostic composition of the present invention can be used in the diagnostic method described below. The diagnostic composition of the present invention may further contain pharmaceutically acceptable carriers and additives usually used together with the antigen of the present invention, as required.
In one aspect, the present invention is a method for diagnosing allergy in a subject shrimp, comprising the steps of:
(i) contacting a sample obtained from a subject with an antigen;
(ii) detecting binding of an IgE antibody in a sample obtained from the subject to the antigen;
(iii) determining that the subject is a shrimp allergy when the IgE antibody of the subject is detected to be combined with the antigen;
here, the antigen is at least one of the proteins defined as the antigens of (1) to (11) above. Wherein the steps (i) and (ii) are performed in accordance with the description of the steps providing a method for diagnosing an allergic reaction index in shrimp.
In another embodiment, the present invention provides a method for diagnosing shrimp allergy in a subject, comprising administering at least one of the antigens (1) to (11) above to the subject. The method may also be performed in the form of a skin test characterized by the application of an antigen to the skin. The skin test included the following format: a prick test (cock test) in which the skin is slightly scratched without bleeding after applying the diagnostic composition to the skin, soaked with an antigen, and then the skin reaction is observed; scratch test (scratch test) to gently scratch the skin and observe the reaction on the basis of the application of the diagnostic composition; a Patch test (Patch test) in which a diagnostic composition in the form of cream, ointment, or the like is applied to the skin and the reaction is observed; intradermal testing of responses to intradermal administration of antigen; and the like. When a skin reaction such as swelling occurs in the skin to which the antigen moiety has been applied, it is diagnosed that the subject has shrimp allergy. Here, the amount of the antigen to be applied to the skin may be, for example, 100 μ g or less per 1 time.
In the diagnosis of allergy, tolerance tests are often performed for the purpose of limiting antigens. At least one of the antigens (1) to (11) above can be used as an active ingredient in a tolerance test for diagnosing shrimp allergy. Here, the antigen protein used in the tolerance test may be a protein expressed and purified, or may be a protein expressed in a raw material or processed product, such as pollen rice in which a gene of a cedar pollen antigen is converted in rice and the antigen protein is expressed in rice.
In one embodiment, the diagnostic composition and the diagnostic kit can be used for a prick test, a scratch test, a skin spot test, an intradermal test, and the like.
In another embodiment, the present invention provides at least one of the antigens (1) to (11) used for the diagnosis of shrimp allergy. Here, the antigen may be provided by mixing at least one of the above-mentioned antigens (1) to (11) with a known antigen.
In still another embodiment, the present invention provides a use of at least one of the antigens (1) to (11) above for preparing a composition for diagnosing shrimp allergy.
Pharmaceutical compositions/methods of treatment (1)
The present invention provides a pharmaceutical composition comprising at least one of the antigens (1) to (11) above.
In one embodiment, the pharmaceutical composition described above is used to treat shrimp allergy. In the present specification, "treatment of allergy" is intended to increase the amount of an antigen that does not cause an allergy even when taken into the body, and is ultimately intended to achieve a state in which the allergy does not cause an allergy (remission) after a normal amount of the antigen is taken.
The present invention also provides a method of treating shrimp allergy comprising: at least one of the antigens (1) to (11) above is administered to a patient in need of treatment of shrimp allergy.
In another embodiment, the present invention provides at least one of the antigens (1) to (11) described above for use in the treatment of shrimp allergy. In still another embodiment, the present invention provides a use of at least one of the antigens (1) to (11) above for producing a therapeutic agent for shrimp allergy.
In the treatment of allergies, desensitization treatments aimed at inducing immune tolerance by administering an antigen to the patient are often performed. At least one of the antigens (1) to (11) above can be used as an active ingredient in a desensitization therapy for treating shrimp allergy. Here, the antigen protein used in desensitization therapy may be a protein expressed and purified, or may be a protein expressed in raw materials/processed products, such as pollen rice in which a gene for converting a cedar pollen antigen in rice is used and the antigen protein is expressed in rice.
The pharmaceutical composition of the present invention can be administered by a usual administration route. Typical routes of administration include, for example: oral, sublingual, transdermal, intradermal, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, intrarectal administration.
The pharmaceutical composition of the present invention may be prepared by adding a pharmaceutically acceptable adjuvant, excipient, or various additives (e.g., a stabilizer, a solubilizer, an opacifying agent, a buffer, a storage agent, a colorant, etc.) to the antigen of the present invention by a conventional method, if necessary. The dosage form of the pharmaceutical composition may be appropriately selected by those skilled in the art according to the administration route. It may also be, for example: tablet, capsule, syrup, sublingual tablet, injection, nasal spray, ointment, solution, cream, lotion, suppository, etc. The dose, frequency of administration and/or period of administration of the pharmaceutical composition of the present invention can be appropriately selected by a physician according to the characteristics of the patient such as the route of administration, symptoms, age, body weight, and the like. For example, in the case of an adult, the administration amount may be 100. mu.g or less per 1 time. The dosing interval may be, for example, 1 time per day, 1 time per week, 2 times per month, or about 1 time in 3 months. The period of administration may be, for example, weeks to years. The administration period may be a period in which the amount of the drug to be administered is increased in a gradient manner.
Detecting composition (1)
The present invention provides a detection composition comprising an antibody against at least one of the antigens (1) to (11) above.
The antibody can be produced by a conventional method. For example, a mammal such as a rabbit is immunized with the antigens (1) to (11) described above. The antibody can be an Ig antibody, a polyclonal antibody, a monoclonal antibody, or an antigen binding fragment thereof (e.g., Fab, F (ab')2、Fab’)。
In the above-mentioned detection composition, the antibody may be provided in a form bound to a carrier. The carrier is not particularly limited as long as it is a carrier that can be used for detection of binding of an antibody to an antigen. Any vector known to those skilled in the art may be used.
Examples of the method for detecting the presence or absence of an antigen include the following methods.
And a method in which a detection composition containing the prepared Ig antibody is brought into contact with a sample obtained from a raw material, a processed product, or the like, and binding of the Ig antibody to an antigen in the sample is detected by, for example, an ELISA method, and when binding of the Ig antibody to the antigen is detected, it is determined that the target raw material, processed product, or the like contains the antigen.
A method of detecting an antigen contained in a material or a processed product by wrapping the material or the processed product with a filter paper or the like and reacting the wrapped material or the processed product with an antibody solution.
In another embodiment of the present invention, a detection composition comprising an antigen for determining the presence or absence of shrimp allergy in a subject, the detection composition comprising a primer having a nucleotide sequence complementary to at least a part of the nucleotide sequence represented by seq id No.1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540, or 548 is further provided. The primer includes, for example, but not limited to: a nucleotide sequence complementary to at least a part of the 3' -end portion or the central portion of at least one sequence of the nucleotide sequences represented by SEQ ID Nos. 1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540 or 548, preferably 12 residues, 15 nucleotides, 20 nucleotides or 25 nucleotides. Particularly, in the case of mRNA, a complementary primer of poly (A) tail is present. In a preferred embodiment, the detection composition comprising the primer further comprises a primer comprising a nucleotide sequence, preferably a nucleotide sequence consisting of 12 nucleotides, 15 nucleotides, 20 nucleotides, and 25 nucleotides, at the 5' -end portion of at least one of the nucleotide sequences represented by SEQ ID Nos. 1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540, and 548.
For example, the presence or absence of an antigen can be determined by amplifying cDNA using a complementary primer by PCR (Polymerase Chain Reaction) including RT-PCR (Reverse Transcription-Polymerase Chain Reaction) using DNA or mRNA obtained from shrimp as a template, and comparing the sequence of the amplified cDNA with the
In one embodiment, the detection composition can be used to detect whether or not an antigen is contained in a subject such as a food material (shrimp) or a food production line. The above-mentioned detection composition can be used by manufacturers for quality inspection of products before production line and shipment; the present invention can also be used by a human taste tester to check whether or not a target material/processed product contains an antigen.
Judgment method of existence of antigen (1)
The present invention includes a method for determining the presence or absence of an antigen, in which an antibody against at least one of the antigens (1) to (11) is brought into contact with a raw material/processed product (including a liquid), thereby determining the presence or absence of the antigens (1) to (11) in a target substance.
The raw material can be food material, cosmetic material, medicinal material, etc. The processed product may be edible processed product, or cosmetic, medicinal product, etc.
The antibody, the method for producing the antibody, the method for contacting the antibody with the raw material/processed product, the binding of the antibody to the antigen, and the like are the same as those described above in the "detection composition (1)".
Allergen-removed food and the like (1)
The present invention provides a shrimp or a shrimp-processed product characterized by having at least one of the antigens (1) to (11) removed or reduced.
There is no limitation on the method for removing or reducing the antigen of the present invention in shrimp or a shrimp-processed product. The removal or reduction of the antigen is not limited to the removal or reduction of the antigen of the present invention, and any method may be used.
For example, shrimp from which the antigen of the invention has been removed or reduced can be transgenic to produce shrimp in which the expression of the antigen of the invention has been altered.
Transgenic technology can use the technicians in this field known methods of any. For example, Oishi, et al (Scientific Reports, Vol.6, Article number: 23980,2016, doi:10.1038/srep23980) describes the application of CRISPER/Cas9 to chicken primordial germ cells as a genome editing technique to obtain individuals with Ovomucoid (Ovomucoid) gene deletions. The antigen-removed shrimp of the present invention can also be obtained by the same method.
Alternatively, the antigen-removed or reduced shrimp of the present invention may be obtained by artificial insemination of shrimp not containing or containing a small amount of antigen. Artificial mating of shrimps can be carried out by a conventional method such as the method of "クルマエビ people's aquatic research and education institution aquatic engineering research institute" (the "クルマエビ people's mature, the egg と skill man" (aocun zhuo shui teng xi) jezui zui water-fired fish ( zhengxinggun, 2014).
The processed shrimp product from which the antigen of the present invention has been removed or reduced may be a processed shrimp product from which the antigen of the present invention has been removed or reduced. When general shrimp is used as a raw material, the antigen-removing or antigen-reducing treatment of the present invention is performed before or after the production of a processed shrimp product. In the case of processed shrimp products using normal shrimp as a raw material, the method for removing or reducing the antigen in the present invention includes: a method of removing protein components in raw materials/processed products by high-pressure treatment, elution with neutral salt solution, high-temperature steam, etc.; hydrolysis, denaturation, and amino acid denaturation (chemical modification/removal of side chains, etc.) by heat treatment and acid treatment.
Method (1) for producing allergen-removed processed product
The present invention is a method for producing a shrimp or a shrimp-processed product from which an antigen has been removed or reduced, comprising the steps of: the antigen was confirmed to be removed or reduced during the production of the processed product,
here, the antigen is at least one of the antigens of (1) to (11) above.
In the production of the shrimp or shrimp-processed product from which the antigen has been removed or reduced, the step of confirming the removal or reduction of the antigen can be performed by confirming whether or not the antigen is contained by the method described in the above "detector (1)".
The shrimp or processed shrimp product from which the antigen has been removed or reduced can be produced by the method described in the above-mentioned "allergen-removed food or the like".
Epitopes of antigens
As shown in examples 1 to 3, epitopes and amino acids within the epitopes that are important for binding to IgE antibodies of allergy patients are defined for the defined antigens and arginine kinase (arginine kinase) as shown in example 4.
The present invention provides a polypeptide comprising an amino acid sequence that specifically binds to an IgE antibody of an allergy patient, wherein the polypeptide comprises each of the amino acid sequences of SEQ ID NO. 558-984 described in Table 3 below, or each of the amino acid sequences (E1) to (E50). (E1) Each of (E) to (E50) is an amino acid sequence (hereinafter, sometimes referred to as "epitope") that binds to an IgE antibody derived from a protein described in "origin" in Table 3.
Myosin
Myosin
Glycogen phosphorylase sources: (E12) , (E13)
Sources of hemocyanin subunit L1: (E14) (E15), (E44)
Sources of pyruvate kinase 3: (E16) (E17), (E45)
Sources of phosphopyruvate hydratase: (E18) (E46), (E47)
The precursor of the subunit α of the mitochondrial ATP synthetase is derived from (E19), (E20)
Troponin I source: (E21) (E48), (E49)
Cyclophilin a sources: (E22) (E23), (E50)
Sources of arginine kinase: (E24) , (E25)
The polypeptide comprising the amino acid sequences of (E1) to (E50) can also be produced by chemical synthesis such as solid phase synthesis of peptides. Alternatively, the epitope-containing polypeptide may be obtained by expression as a recombinant polypeptide by a gene recombination technique known to those skilled in the art, followed by isolation/production by a protein production method known to those skilled in the art. The polypeptides may be linked by combining two or more kinds, or may be linked by repeating 1 kind of epitope. In this case, generally, the binding property to the Ig antibody is improved.
The length of the polypeptide comprising the amino acid sequences of (E1) to (E50) is not particularly limited. In a preferred embodiment, the length of the polypeptide including the amino acid sequences of (E1) to (E50) is 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. In the case where the polypeptide is a polypeptide in which one or more of the above-mentioned amino acid sequences (E1) to (E50) are repeated 1 or 2 or more times, in a preferred embodiment, the length of the amino acid sequence portion is 1000 amino acids or less, 750 amino acids or less, 500 amino acids or less, 250 amino acids or less, 100 amino acids or less, 75 amino acids or less, 50 amino acids or less, 30 amino acids or less, 15 amino acids or less, 10 amino acids or less, or 5 amino acids or less. The length of the polypeptide is preferably the number of amino acid residues described in the above-mentioned preferred embodiment is the total length of the sequences before and after (excluding) the spacer.
Each of 50 sequences of sequence numbers 558, 566, 582, 586, 594, 599, 614, 624, 634, 640, 654, 671, 672, 678, 681, 686, 691, 697, 704, 705, 708, 717, 725, 729, 741, 750, 752, 765, 770, 778, 788, 793, 810, 817, 826, 833, 847, 858, 865, 879, 881, 892, 908, 912, 915, 917, 928, 935, 939 and 943 described in sequence number 558-984 described in table 3 and "common 15 residue sequence" described in "common 15 residue sequence" of table 3 was a common 15 amino acid residue sequence identified as an epitope to which an antibody IgE binds among epitopes of (E1) - (E50) by overlap-based epitope mapping. In one embodiment of the present invention, the polypeptide comprises or consists of these amino acid sequences. The epitope sequence contained in the polypeptide of the present invention may be the whole of 15 amino acid residues of the common epitope, or a part thereof. The epitope sequence is 4 amino acid residues or more, 5 amino acid residues or more, 6 amino acid residues or more, 7 amino acid residues or more, 8 amino acid residues or more, 9 amino acid residues or more, 10 amino acid residues or more, 11 amino acid residues or more, 12 amino acid residues or more, 13 amino acid residues or more, 14 amino acid residues or more.
In the examples of the present specification, for example, in a polypeptide composed of 4 amino acid residues in many cases (for example, SEQ ID NOS: 587, 593, 595, 636, 655, 662, 663, 679, 707, 726, 727, 794, 880, etc.), cross-reactivity of an epitope is confirmed. Furthermore, it is often confirmed that the epitope cross-reactivity is present at 5 amino acid residues or more. Thus, it is useful as an epitope sequence for detecting cross-reactivity with various antigens as long as at least 4 amino acid residues are present.
Thus, a polypeptide comprising or consisting of an amino acid sequence other than the "common 15-residue sequence" of sequence No. 558,566, 582,586, 594,599, 614,624, 634, 640,654, 671, 672,678, 681, 686, 691, 697, 704,705, 708,717, 725,729, 741, 750, 752,765, 770,778, 788, 793, 810, 817,826, 833,847, 858,865, 879,881, 892,908, 912,915, 917,928, 935,939 and 943 is also encompassed by the present invention.
In Table 3, the "preferred" sequence is a shorter partial sequence that can function as an epitope in the "common 15 residue sequence". "Critical" sequences refer to sequences of "common 15 residue sequence" or "critical" sequences that are considered to be of particular importance. The amino acid sequence denoted by "X" in the "key" sequence is an amino acid residue for which it was confirmed by alanine glycine scan that the binding property to the IgE antibody is retained even when the amino acid residue is changed to an arbitrary alanine (glycine in the case where the original amino acid residue is alanine). Thus, X is any amino acid residue, preferably alanine (or glycine). Note that the sequence represented by "X" is not included in the "critical" sequence, and means that: it was not found that the alanine glycine scan confirmed the remaining amino acid residues that bound to the IgE antibody.
Regarding the epitope of (E1), the amino acid residues represented by X in sequence nos. 560, 562, 564, and 565 are amino acid residues that remain even when the binding property to an IgE antibody is changed. Thus, in sequence No. 558-565, 1 or more of the amino acid residues corresponding to the amino acid residues at
The number of optionally substituted amino acid residues is not particularly limited, but is preferably 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. The same applies to (E2) to (E50).
Information about the (E1) - (E50) epitopes is summarized in table 1.
[ Table 1]
The sequences shown in the right columns of FIGS. 1 to 114 of Table 3, i.e., the "synthetic sequences" and/or the "sequence numbers" are those for which epitope cross-reaction was confirmed in example 5. "epitope cross-reactivity was confirmed" means that the IgE antibodies in the serum of each allergy patient showed higher binding than the IgE antibodies in the serum of non-allergy subjects (specifically, greater than "1" in fig. 7 to 12). Thus, in one form, the polypeptide of the invention comprises: polypeptides comprising or consisting of these amino acid sequences. In one embodiment, the IgE antibody in the serum of each allergic patient exhibits a high binding property of 1.05-fold or more, 1.10-fold or more, 1.15-fold or more, 1.20-fold or more, or 1.25-fold or more to the polypeptide of the present invention, as compared with the IgE antibody in the serum of a non-allergic subject.
In the present specification, the "polypeptide comprising the amino acid sequences of (E1) to (E50)" includes: any of the polypeptides comprising or consisting of the amino acid sequences of SEQ ID NO. 558 and 984 and the forms in which the amino acid residues are substituted as described above. "comprising the respective amino acid sequences of sequence No. 558-984" means: any other amino acid sequence may be included within the range that does not affect the binding of the amino acid sequences of 558. sup. 984 (including the above-described substitution pattern thereof) to the IgE antibody (that is, the functions thereof as an epitope).
Diagnostic kit/diagnostic method (2)
The present invention provides a method for providing an index for diagnosing allergy in a subject, comprising the steps of:
(i) contacting a sample obtained from a subject with an antigen, wherein the sample is a solution containing an IgE antibody;
(ii) detecting binding of an IgE antibody in a sample obtained from the subject to the antigen;
(iii) providing an indication that the subject is allergic upon detection of binding of IgE antibodies to the antigen in the subject;
here, the antigen is at least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) described above, or 2 or more of the polypeptides comprising the amino acid sequences of (E1) to (E50) described above linked with or without a spacer.
Hereinafter, in the present specification, a polypeptide including at least one of the polypeptides having the amino acid sequences of (E1) to (E50) described above, or a polypeptide in which 2 or more of the polypeptides having the amino acid sequences of (E1) to (E50) described above are linked with or without a spacer may be referred to as an "antigen including (E1) to (E50) described above. The type of the spacer is not particularly limited, and a substance generally used by those skilled in the art can be used to link a plurality of peptides. The spacer may be a polypeptide such as a hydrocarbon chain such as Acp (6) -OH, or an amino acid chain.
When the polypeptides comprising the amino acid sequences of (E1) to (E50) are linked with or without a spacer, the number of the polypeptides to be linked is not particularly limited. In one embodiment, the number is 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, or 15 or more. In one embodiment, the number is 30 or less, 20 or less, 15 or less, 10 or less, 8 or less, 6 or less, 5 or less, 3 or less, or 2 or less.
The polypeptides comprising the amino acid sequences of (E1) to (E50) may be repeatedly linked to each other by the same polypeptide or by different polypeptides. In this way, when a plurality of polypeptides comprising the amino acid sequences of (E1) to (E50) are linked, the methods, kits, and compositions of the present invention can be applied as the polypeptide of the present invention.
The sample obtained from the subject is as described in the above-mentioned item "diagnostic kit/diagnostic method (1)".
The contact between the sample obtained from the subject and the polypeptide and the detection of the binding thereof can be carried out by a known method described in the item of the above "diagnostic kit/diagnostic method (1)", for example: ELISA (Enzyme-Linked immunosorbent Assay), sandwich immunoassay (sandwich immunoassays), Western immunoblotting, immunoprecipitation, immunochromatography, and the like.
The polypeptide comprising the amino acid sequences of (E1) to (E50) may be immobilized on a carrier. In this case, ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance can be used in the steps (i) and (ii); the step (i) may be performed by bringing the polypeptide comprising the amino acid sequences of (E1) to (E50) into contact with the sample obtained as the object on the immobilized surface. Alternatively, the binding to the polypeptide comprising the amino acid sequences of (E1) to (E50) may be detected by the above-described method using a substance in which the target IgE antibody is immobilized on a carrier. For binding to a carrier, or for providing a carrier and a spacer, or for facilitating contact of the polypeptide with an antibody, a spacer, biotin, or the like may be added to the N-terminus or C-terminus of the polypeptide. In the case of binding to biotin, avidin is preferably present in the carrier.
The polypeptides comprising the amino acid sequences of (E1) to (E50) may be in a state in which they are not immobilized on a carrier. In this case, in the steps (i) and (ii), the presence of the polypeptide having the amino acid sequences of (E1) to (E50) to which the IgE antibody is bound can be confirmed by laser light using a flow cytometer or the like. Examples of such a method include: basophil Activation Test (BAT) as a method for detecting surface antigen CD203c appearing upon basophil activation by contacting a polypeptide comprising the amino acid sequences of (E1) to (E50) above. Further, there is also included a Histamine Release Test (HRT) in which whether or not histamine is released is confirmed by further contacting a polypeptide comprising the amino acid sequences of (E1) to (E50) with blood cells in a sample.
The polypeptide comprising the amino acid sequences of (E1) to (E50) is an antigen that specifically binds to an IgE antibody of an allergic patient. Thus, detection of binding of IgE antibodies to the antigen in a subject involves crossability, providing an indication that the subject is allergic. In the synthesis of polypeptides comprising the amino acid sequences of (E1) to (E50), sequences may be added before and after the epitope to increase the sequence length, for example, to facilitate the synthesis using E.coli. In this case, when the binding of the IgE antibody of the subject to the amino acid sequences of (E1) to (E50) is detected, crossability is included, and the subject is an indicator of allergic reaction. Therefore, any substance may be added before or after the amino acid sequences of (E1) to (E50) as epitopes.
The present invention also provides a diagnostic kit for allergy, which comprises at least one polypeptide comprising the amino acid sequences of (E1) to (E50) above. The diagnostic kit of the present invention can be used in the above-described method for providing an indicator for diagnosing allergy or the below-described diagnostic method. The diagnostic kit of the present invention may further comprise an enzyme-labeled anti-IgE antibody and a chromogenic substrate or a luminescent substrate which is a substrate for the enzyme, in addition to at least one of the polypeptides having the amino acid sequences of (E1) to (E50) described above. In addition, fluorescently labeled anti-IgE antibodies can also be used. The diagnostic kit of the present invention may be provided in a state in which polypeptides comprising the amino acid sequences of (E1) to (E50) described above are immobilized on a carrier. The diagnostic kit of the present invention may also be provided with instructions for the steps used for diagnosis, a method kit comprising the instructions.
In another aspect, the diagnostic kit described above comprises: a companion diagnostic drug for allergy. Companion diagnostic agents refer to substances used for the following purposes: the reactivity of a pharmaceutical is investigated for the purpose of limiting patients expected to have a pharmaceutical effect, limiting patients at risk of severe side effects of a pharmaceutical, or optimizing therapy using a pharmaceutical. Here, optimization of treatment includes, for example: determination of the volume of administration, judgment of discontinuation of administration, and determination of what allergen component to use to confirm whether or not immune tolerance is obtained.
The present invention also provides a diagnostic composition for allergy, which comprises at least one polypeptide comprising the amino acid sequences of (E1) to (E50) above. The diagnostic composition of the present invention can be used in the diagnostic method described below. The diagnostic composition of the present invention may further contain pharmaceutically acceptable carriers and additives usually used together with the polypeptide of the present invention, as required.
In one aspect, the present invention is a method for diagnosing allergy in a subject, comprising the steps of:
(i) contacting a sample obtained from a subject with an antigen;
(ii) detecting binding of an IgE antibody in a sample obtained from the subject to the antigen;
(iii) determining that the subject is allergic when the IgE antibody of the subject is detected to be combined with the antigen;
here, the antigen is at least one of the polypeptides defined as the polypeptides comprising the amino acid sequences of the above (E1) to (E50). Wherein each step (i) and (ii) is performed in accordance with the description of each step for providing a method for diagnosing an allergy index.
In another embodiment, the present invention provides a method for diagnosing allergy in a subject, which comprises administering to the subject at least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) above. The method may also be carried out in the form of a skin test characterized by applying to the skin a polypeptide comprising the amino acid sequences of (E1) to (E50) above. The skin test included the following format: a prick test (cock test) in which the skin is soaked with a polypeptide comprising the amino acid sequences of (E1) to (E50) and then the skin reaction is observed after applying the diagnostic composition to the skin and lightly scratching the skin to a non-bleeding degree; scratch test (scratch test) to gently scratch the skin and observe the reaction on the basis of the application of the diagnostic composition; a Patch test (Patch test) in which a diagnostic composition in the form of cream, ointment, or the like is applied to the skin and the reaction is observed; an intradermal test in which a reaction is observed after intradermal administration of a polypeptide comprising the amino acid sequences of the above (E1) to (E50); and the like. When a skin reaction such as swelling occurs in the skin to which the polypeptide portion including the amino acid sequences of (E1) to (E50) is applied, it is diagnosed that the subject has an allergic reaction. Here, the amount of the above-mentioned polypeptide to be applied to the skin may be, for example, 100. mu.g or less per 1 application.
In the diagnosis of allergy, oral tolerance tests are often performed for the purpose of limiting antigen uptake and verifying the degree of symptoms. At least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) above can be used as an active ingredient in an oral tolerance test for diagnosing allergy. Here, the polypeptide used in the oral tolerance test may be a polypeptide that is expressed and purified, or may be a polypeptide that is expressed in a raw material or processed product, such as pollen rice that is obtained by converting a cedar pollen antigen in rice and expressing the polypeptide in rice.
In one embodiment, the diagnostic composition and the diagnostic kit can be used for a prick test, a scratch test, a skin spot test, an intradermal test, and the like.
In another embodiment, the present invention provides at least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) described above, which are used for the diagnosis of allergy.
In still another embodiment, the present invention provides a use of at least one polypeptide comprising the amino acid sequences of (E1) to (E50) above for the preparation of a diagnostic drug for allergy.
In this item, the allergy to be diagnosed is an allergy against a polypeptide comprising the amino acid sequences of (E1) to (E50) described above. That is, the diagnosis of allergy including the detection and diagnosis index of allergy can be performed not only for allergy to a single polypeptide including the amino acid sequences of (E1) to (E50) but also for allergy including crossability.
Pharmaceutical compositions/methods of treatment (2)
The present invention provides a pharmaceutical composition comprising at least one of the polypeptides having the amino acid sequences of (E1) to (E50) above. In one embodiment, the pharmaceutical composition described above is used to treat allergy. The term "treatment of allergy" is intended to increase the limit amount of a polypeptide which does not cause an allergy even when ingested into the body, and is ultimately intended to achieve a state in which the allergy does not cause an allergy (remission) after the usual amount of the polypeptide is ingested.
The present invention also provides a method of treating allergy comprising: at least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) above is administered to a patient in need of treatment for allergy.
In another embodiment, the present invention provides at least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) described above for use in the treatment of allergy. In still another embodiment, the present invention provides a use of at least one polypeptide comprising the amino acid sequences of (E1) to (E50) above for the preparation of a diagnostic drug for allergy.
In the treatment of allergies, desensitization treatments aimed at inducing immune tolerance by administering an antigen to the patient are often performed. At least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) above can be used as an active ingredient in a desensitization therapy for treating allergy. Here, the antigen used in desensitization therapy may be a polypeptide expressed and purified, or may be a substance expressed in a raw material or processed product, such as pollen rice.
The route of administration, the dose, the number of times of administration and/or the administration interval of the pharmaceutical composition of the present invention, other components contained in the pharmaceutical composition, and the dosage form can be the same as those described in the above "pharmaceutical composition/therapeutic method (1)". The amount of the polypeptide having the amino acid sequences of (E1) to (E50) used may be 100. mu.g or less per 1 time, for example, in the case of an adult.
In this item, the allergy to be treated is an allergy against a polypeptide comprising the amino acid sequences of (E1) to (E50) described above. That is, the treatment of allergy is not only for allergy to a single polypeptide comprising the amino acid sequences of (E1) to (E50) but also for allergy including crossovers.
Detecting composition (2)
The present invention provides a detection composition containing an antibody against at least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) described above.
The antibody can be produced by a conventional method. For example, a mammal such as a rabbit is immunized with a polypeptide comprising the amino acid sequences of (E1) to (E50) above. The antibody can be an Ig antibody, a polyclonal antibody, a monoclonal antibody, or an antigen binding fragment thereof (e.g., Fab, F (ab')2、Fab’)。
In the above-mentioned detection composition, the antibody may be provided in a form bound to a carrier. The vector is not particularly limited as long as it is a vector that can be used for detecting binding of an antibody to a polypeptide comprising the amino acid sequences of (E1) to (E50). Any vector known to those skilled in the art may be used. Further, the antibody against the polypeptide comprising the amino acid sequences of (E1) to (E50) is preferably an antibody against a polypeptide having an amino acid sequence in which the epitope described in the above "epitope of antigen" and an important amino acid are the same. Thus, a detection composition capable of detecting crosslinkage can be prepared.
Examples of the method for examining whether or not a polypeptide comprising the amino acid sequences of (E1) to (E50) is contained include the following methods.
A method of bringing the detection composition containing the prepared antibody into contact with a sample obtained from a raw material, a processed product, or the like, detecting the binding of the antibody to the polypeptide containing the amino acid sequences of (E1) to (E50) in the sample by, for example, an ELISA method, and determining that the target raw material, processed product, or the like contains the polypeptide containing the amino acid sequence when the binding of the antibody to the polypeptide containing the amino acid sequences of (E1) to (E50) is detected. (in the "method for detecting the presence or absence of a polypeptide", a method for determining that a polypeptide having an amino acid sequence of (E1) to (E50) is removed or reduced when the binding between the antibody and the polypeptide is reduced.)
A method of detecting the polypeptides contained in the coating material or processed product such as filter paper, which comprises the amino acid sequences of (E1) to (E50) described above, by reacting the coating material or processed product with an antibody solution.
In another embodiment of the present invention, there is provided a detection composition for determining the presence or absence of an allergic reaction in a subject, comprising a polypeptide having an amino acid sequence identical to an epitope and important amino acids and comprising the amino acid sequences of (E1) to (E50) above. The primer is designed, for example, to contain a part of the nucleotide sequence of the nucleic acid encoding the amino acid sequences defined in (E1) to (E50) or the complementary strand thereof, without limitation. Alternatively, the above primer is designed to: in the nucleic acid encoding a protein comprising a polypeptide having an amino acid sequence in which an epitope which is an amino acid sequence defined in the above (E1) to (E50) and an important amino acid are identical, a nucleotide sequence encoding a region on the upstream side of a polypeptide portion having an amino acid sequence in which the epitope and the important amino acid are identical, or a nucleotide sequence encoding a complementary strand of a region on the downstream side of a polypeptide portion having an amino acid sequence in which the epitope and the important amino acid are identical. Examples of such primers include: a primer of at least a part of at least one of the base sequences represented by SEQ ID Nos. 1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540 or 548 and/or a primer of a part of a sequence complementary to at least one of the base sequences represented by SEQ ID Nos. 1, 44, 86, 116, 140, 145, 160, 178, 229, 275, 299, 305, 361, 380, 398, 413, 420, 454, 480, 485, 519, 540 or 548. Here, the positions of epitopes in the full-length sequence of the antigen are as defined in table 3 of example 4 below. Particularly, in the case of mRNA, a complementary primer of poly (A) tail may be included.
For example, the presence or absence of the antigen containing the above (E1) to (E50) can be determined by amplifying a DNA by PCR (Polymerase Chain Reaction) including RT-PCR using a DNA or mRNA obtained from a sample as a template and using the above primers, and determining whether or not the amplified DNA sequence contains a nucleic acid encoding the amino acid sequence defined in the above (E1) to (E50). As a method for amplifying mRNA by PCR, RACE method and the like can be exemplified. In the case where one of the possible 3 amino acid sequences encoding open reading frames in the amplified DNA comprises the amino acid sequences defined in the above-mentioned (E1) to (E50), it is judged to have an antigen. When the DNA was not amplified, it was judged that the DNA had no antigen.
In one embodiment, the detection composition can be used for detecting whether or not a polypeptide comprising the amino acid sequences of (E1) to (E50) is contained in a raw material or a target such as a processed product production line. The raw material can be food material, cosmetic material, medicinal material, etc. The processed product may be edible processed product, or cosmetic, medicinal product, etc. The detection composition can be used for searching biological species which can be contained as raw materials, and can be used for quality inspection of products in production lines and before delivery by manufacturers; it can also be used by the taster or the user himself to check whether the subject material/processed product contains an antigen.
Method for determining presence or absence of polypeptide (2)
The present invention encompasses determining whether or not a polypeptide comprising the amino acid sequences of (E1) to (E50) is present in a raw material/processed product. The method comprises detecting a polypeptide having the whole or part of the amino acid sequence of the polypeptide comprising the amino acid sequences of (E1) to (E50) described above in the raw material/processed product.
In one embodiment, the method of the present invention includes the steps of: the presence or absence of a polypeptide having an amino acid sequence of (E1) to (E50) in a substance to be determined, which is obtained by bringing an antibody against at least one of the polypeptides having an amino acid sequence of (E1) to (E50) into contact with a raw material or a processed product (including a liquid).
The antibody, the method for producing the antibody defined in the raw material/processed product, the method for bringing the antibody into contact with the raw material/processed product, the binding of the antibody to the antigen, and the like are the same as those described in the "detection composition (2)".
Alternatively, the method for determining the presence or absence of the antigen may further comprise the following steps: the part of the epitope of the polypeptide comprising the amino acid sequences of (E1) to (E50) described above contained in the antigen was detected. The "part of the epitope" is preferably 4 amino acid residues or more, 6 amino acid residues or more, or 8 amino acid residues or more. Detection of a portion of an epitope can be performed by a known method for detecting a specific amino acid sequence of a portion of a polypeptide. The following methods and the like can be considered, for example: proteins of a target material, a processed product, or the like (for example, a food material) are cleaved with a digestive enzyme used for an antigen removal treatment, and are separated by HPLC or the like, thereby determining whether or not a peak of an arbitrary epitope peptide is reduced by the antigen removal treatment. Alternatively, the presence or absence of the polypeptide having the amino acid sequence of (E1) to (E50) above in the target substance of the antigen can be determined by using an antibody that recognizes a portion of the polypeptide having the amino acid sequence of (E1) to (E50) above.
Allergen-removing materials and the like (2)
The present invention provides a raw material or a processed product characterized by removing or reducing at least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) described above.
The method for removing or reducing the antigen of the present invention from the raw material or the processed product is not limited. The removal or reduction of the antigen is not limited to the removal or reduction of the polypeptide having the amino acid sequence of (E1) to (E50), and may be carried out by any method, for example, the method described in the above item "allergen-removed food or the like" may be used.
The removal or reduction of at least one of the polypeptides comprising the amino acid sequences of the above (E1) to (E50) may be achieved by removing or reducing the entire amino acid sequence; alternatively, the cleavage or removal may be carried out from the antigen protein in the amino acid sequence portions of (E1) to (E50) described above. "removed" includes deletion and alteration of all or a part of the sequence portions defined in (E1) to (E50) above.
For example, a raw material from which a polypeptide comprising the amino acid sequences of the above-mentioned (E1) to (E50) is removed or reduced may be prepared by using a transgenic technique, and a raw material in which the expression of a polypeptide comprising the amino acid sequences of the above-mentioned (E1) to (E50) is abolished may be prepared. Transgenic knockout techniques can use any of the methods known to those skilled in the art.
The processed product from which the polypeptide having the amino acid sequence of (E1) to (E50) is removed or reduced may be a processed product from which the polypeptide having the amino acid sequence of (E1) to (E50) is removed or reduced, such as milk powder using a protein digest as a raw material. In the case of using a usual raw material, a treatment for removing or reducing the polypeptide comprising the amino acid sequences of (E1) to (E50) described above is performed before, during or after the production of the processed product. The term "preparation of processed product" means: a processed food (e.g., a shrimp processed product such as a fried shrimp) is prepared from, for example, a food material (e.g., a shrimp).
In the processed product using a usual raw material, as a method for removing or reducing the polypeptide comprising the amino acid sequence of the above-mentioned (E1) to (E50), the method described in the above-mentioned "allergen-removed food or the like" may be used. As a method for cleaving a polypeptide comprising the amino acid sequences of (E1) to (E50), a method of cleaving with a specific digestive enzyme can be mentioned.
Method (2) for producing allergen-removed processed product
The present invention is a method for producing a processed product from which at least one of the polypeptides having the amino acid sequences (E1) to (E50) described above has been removed or reduced, the method comprising the steps of: it was confirmed that the antigen was removed or reduced in the process of producing the processed product.
In this production method, removal or reduction of the polypeptide having the amino acid sequences of (E1) to (E50) means that: at least one of polypeptides comprising the amino acid sequences of (E1) to (E50) above is removed or reduced; alternatively, the antigen may be one in which the sequence portions defined in (E1) to (E50) are cleaved or removed.
In the production of the processed product, there is no particular limitation on the method for confirming removal or reduction of the polypeptide, and any method capable of detecting at least one of the polypeptides comprising the amino acid sequences of (E1) to (E50) described above may be used. For example, the presence or absence of a polypeptide in a processed product can be confirmed by the binding property between a sample containing a material produced in the process of producing the processed product and an antibody against at least one of the polypeptides having the amino acid sequences of (E1) to (E50). Details of such a method are as described in the above-mentioned item "diagnostic kit/diagnostic method (2)". That is, in the above-mentioned production method, the "target IgE antibody" in the item of the "diagnostic kit/diagnostic method (2)" may be replaced with "an antibody against at least one of the polypeptides having the amino acid sequences of the above-mentioned (E1) to (E50)", and the "antigen" and "polypeptide" in the item of the "diagnostic kit/diagnostic method (2)" may be replaced with "a sample containing a material generated in the production process of a processed product", and the method described in the item of the "diagnostic kit/diagnostic method (2)" may be used to confirm that the antigen is removed or reduced in the production process of a processed product. In addition, the test composition described in the item of the above "test composition (2)" may also be used.
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