Antigen polypeptide

Abstract

To provide novel antigen polypeptides.SOLUTION: The present invention discloses (A) a polypeptide composed of a specific amino acid sequence, or (B) a polypeptide having an amino acid sequence where the amino acid sequence of (A) has one or more amino acids deleted, substituted, or added, and having antigenicity.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to antigen polypeptides and their application technologies and the like.

Background Art

[0002] Periodontal disease is an inflammatory disease caused by the infection of periodontal pathogenic bacteria in periodontal tissues. Generally, the diagnosis of periodontal disease is performed by comprehensively considering the results of periodontal pocket examination, palpation and bleeding examination, mobility examination, X-ray examination, etc. However, since these examination methods are based on the experience and skills of dentists and the like, there may be differences in the examination results and diagnosis results, and there is a problem of lacking objectivity. Therefore, the development of a method for objectively diagnosing periodontal disease is desired.

[0003] As a method for objectively diagnosing periodontal disease, for example, a method using the IgG antibody titer in serum against periodontal pathogenic bacteria as an index of the infection degree or severity of periodontal disease has been reported. In the method developed by the present inventors, by using a polypeptide having a specific primary structure isolated from Porphyromonas gingivalis, which is a periodontal pathogenic bacterium, or a modified polypeptide thereof (periodontal pathogenic bacterium antigen polypeptide), periodontal diseases of a wide range of patients having various immune types can be examined with high accuracy, and high-speed processing can be performed by an automated device or the like (Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present disclosure is to provide a novel antigen polypeptide.

Means for Solving the Problems

[0006] The inventors have found that a polypeptide having a specific amino acid sequence has antigenicity (reacting with an antibody against a periodontal pathogen in an antigen-antibody reaction), and have further made improvements.

[0007] This disclosure includes, for example, the subject matter described in the following items. Item 1. (A) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or (B) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1 and having antigenicity. Item 2. (C) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or (D) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2 and having antigenicity. Item 3. The polypeptide according to Item 2, wherein the polypeptide of (D) does not have an addition of 226 or more amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2. Item 4. (E) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10, or (F) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 10 and having antigenicity. Item 5. The polypeptide according to Item 4, wherein the polypeptide of (F) does not have an addition of 23 or more amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 10. Item 6. (G) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, or (H) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 11 and having antigenicity. Item 7. The polypeptide according to item 1, wherein the polypeptide of (B) has 0 to 9 amino acids deleted, substituted, or added in the amino acid sequence at positions 119 to 139 of the amino acid sequence shown in SEQ ID NO: 1. Item 8. The polypeptide according to item 2 or 3, wherein the polypeptide of (D) has 0 to 9 amino acids deleted, substituted, or added in the amino acid sequence at positions 472 to 492 of the amino acid sequence shown in SEQ ID NO: 2. Item 9. The polypeptide according to item 4 or 5, wherein the polypeptide of (F) has 0 to 9 amino acids deleted, substituted, or added in the amino acid sequence at positions 675 to 695 of the amino acid sequence shown in SEQ ID NO: 10. Item 10. The polypeptide according to item 6, wherein the polypeptide of (H) has 0 to 9 amino acids deleted, substituted, or added in the amino acid sequence at positions 295 to 315 of the amino acid sequence shown in SEQ ID NO: 11. Item 11. Furthermore, the polypeptide according to any one of items 1 to 10, which contains a tag. Item 12. A reagent for measuring the antibody titer of periodontal pathogenic bacteria, which contains the polypeptide according to any one of items 1 to 11. Item 13. A kit for measuring the antibody titer of periodontal pathogenic bacteria, which contains the reagent for measuring the antibody titer of periodontal pathogenic bacteria according to item 12. Item 14. A method for measuring the antibody titer against periodontal pathogenic bacteria in a biological sample, which includes contacting the biological sample with the polypeptide according to any one of items 1 to 11. A method for measuring the antibody titer against periodontal pathogenic bacteria in a biological sample.

Advantages of the Invention

[0008] A novel antigen polypeptide is provided.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0010] Hereinafter, each embodiment included in the present disclosure will be described in more detail. The polypeptide included in the present disclosure is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. In this specification, the polypeptide may be referred to as "the polypeptide of (A) of the present disclosure". The amino acid sequence shown in SEQ ID NO: 1 corresponds to the amino acid sequence at positions 580 to 860 of the amino acid sequence shown in SEQ ID NO: 3.

[0011] In addition, the present disclosure also includes a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1. In this specification, the polypeptide may be referred to as "the polypeptide of (B) of the present disclosure".

[0012] In addition, the present disclosure also includes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2. In this specification, the polypeptide may be referred to as "the polypeptide of (C) of the present disclosure". The amino acid sequence shown in SEQ ID NO: 2 corresponds to the amino acid sequence at positions 227 to 860 of the amino acid sequence shown in SEQ ID NO: 3.

[0013] In addition, the present disclosure also includes a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2. In this specification, the polypeptide may be referred to as "the polypeptide of (D) of the present disclosure".

[0014] In addition, the present disclosure also includes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10. In this specification, the polypeptide may be referred to as "the polypeptide of (E) of the present disclosure". The amino acid sequence shown in SEQ ID NO: 10 corresponds to the amino acid sequence at positions 24 to 860 of the amino acid sequence shown in SEQ ID NO: 3.

[0015] In addition, the present disclosure also includes a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 10. In this specification, the polypeptide may be referred to as "the polypeptide of (F) of the present disclosure".

[0016] In addition, the present disclosure also includes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11. In this specification, the polypeptide may be referred to as "the polypeptide of (G) of the present disclosure". The amino acid sequence shown in SEQ ID NO: 11 corresponds to the amino acid sequence at positions 404 to 860 of the amino acid sequence shown in SEQ ID NO: 3.

[0017] In addition, the present disclosure also includes a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 11. In this specification, the polypeptide may be referred to as "the polypeptide of (H) of the present disclosure".

[0018] In this specification, the polypeptide of (A) of the present disclosure, the polypeptide of (B) of the present disclosure, the polypeptide of (C) of the present disclosure, the polypeptide of (D) of the present disclosure, the polypeptide of (E) of the present disclosure, the polypeptide of (F) of the present disclosure, the polypeptide of (G) of the present disclosure, and the polypeptide of (H) of the present disclosure may be collectively referred to as "the polypeptide of the present disclosure".

[0019] The polypeptide of (B) of the present disclosure is a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1. In the amino acid sequence shown in SEQ ID NO: 1, the upper limit of the number of amino acids deleted, substituted, or added may be, for example, 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0020] Techniques for adding mutations such as amino acid deletions, substitutions, or additions to a specific amino acid sequence are known in the art and can be performed using any method. For example, it can be performed using restriction enzyme treatment, treatment with exonuclease, DNA ligase, etc., site-directed mutagenesis, random mutagenesis, etc.

[0021] The polypeptide of (B) of the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 1. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0022] The identity of the amino acid sequence can be calculated by using the default parameters in the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) of the United States, http: / / www.ncbi.nlm.nih.gov / BLAST / .

[0023] As shown in the examples described below, it is predicted that the amino acid sequence at positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 3 is likely to contain an epitope. From this, the polypeptide of (B) of the present disclosure preferably has 0 to 9 amino acids deleted, substituted, or added in the amino acid sequence corresponding to the amino acid sequence at positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 3, which is the amino acid sequence at positions 119 to 139 of the amino acid sequence shown in SEQ ID NO: 1. The upper or lower limit of this range may be, for example, 1, 2, 3, 4, 5, 6, 7, or 8. More specifically, it may be 1 to 8. Further, in the polypeptide of (B) of the present disclosure, the amino acid sequence at positions 119 to 139 of the amino acid sequence shown in SEQ ID NO: 1 may be conserved.

[0024] The polypeptide of (D) of the present disclosure is a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2. In the amino acid sequence shown in SEQ ID NO: 2, the upper limit of the number of amino acids deleted, substituted, or added may be, for example, 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0025] The polypeptide of (D) of the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 2. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0026] In addition, the polypeptide of (D) in the present disclosure may be a polypeptide that does not have an addition of 226 or more amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2. In other words, when the polypeptide of (D) in the present disclosure has an addition of amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2, the number of the added amino acids may be 1 to 225. The upper or lower limit of the number of the amino acids may be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220. More specifically, for example, it may be 2 to 220.

[0027] As shown in the examples described below, it is predicted that the amino acid sequence at positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 3 is likely to contain an epitope. From this, the polypeptide of (D) in the present disclosure preferably has 0 to 9 amino acids deleted, substituted, or added in the amino acid sequence corresponding to positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 3, which is the amino acid sequence at positions 472 to 492 of the amino acid sequence shown in SEQ ID NO: 2. The upper or lower limit of the range may be, for example, 1, 2, 3, 4, 5, 6, 7, or 8. More specifically, it may be 1 to 8. In addition, the amino acid sequence at positions 472 to 492 of the amino acid sequence shown in SEQ ID NO: 2 of the polypeptide of (D) in the present disclosure may be conserved.

[0028] The polypeptide (F) of the present disclosure is a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 10. In the amino acid sequence shown in SEQ ID NO: 10, the upper limit of the number of amino acids deleted, substituted, or added may be, for example, 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0029] The polypeptide (F) of the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 10. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0030] In addition, the polypeptide (F) of the present disclosure may be a polypeptide having no addition of 23 or more amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 10. In other words, when the polypeptide (F) of the present disclosure has an addition of amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 10, the number of the added amino acids may be a polypeptide of 1 to 22. The upper limit or lower limit of the number of the amino acids may be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. More specifically, for example, it may be 2 to 21.

[0031] As shown in the examples described below, it is predicted that the amino acid sequence at positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 3 is likely to contain an epitope. From this, the polypeptide of (F) of the present disclosure is preferably a polypeptide in which the number of amino acids deleted, substituted, or added in the amino acid sequence corresponding to the amino acid sequence at positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 3, i.e., the amino acid sequence at positions 675 to 695 of the amino acid sequence shown in SEQ ID NO: 10, is 0 to 9. The upper or lower limit of this range may be, for example, 1, 2, 3, 4, 5, 6, 7, or 8. More specifically, it may be 1 to 8. Further, in the polypeptide of (F) of the present disclosure, the amino acid sequence at positions 675 to 695 of the amino acid sequence shown in SEQ ID NO: 10 may be conserved.

[0032] The polypeptide of (H) of the present disclosure is a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 11. In the amino acid sequence shown in SEQ ID NO: 11, the upper limit of the number of amino acids deleted, substituted, or added may be, for example, 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0033] The polypeptide of (H) of the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 11. The identity may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0034] As shown in the examples described below, it is predicted that the epitope is likely to be included in the amino acid sequence at positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 3. From this, the polypeptide of (H) of the present disclosure is preferably a polypeptide in which the number of deleted, substituted, or added amino acids in the amino acid sequence at positions 295 to 315 of the amino acid sequence shown in SEQ ID NO: 11, which corresponds to the amino acid sequence at positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 3, is 0 to 9. The upper or lower limit of the range may be, for example, 1, 2, 3, 4, 5, 6, 7, or 8. More specifically, it may be 1 to 8. Further, in the polypeptide of (H) of the present disclosure, the amino acid sequence at positions 295 to 315 of the amino acid sequence shown in SEQ ID NO: 11 may be conserved.

[0035] The polypeptide of the present disclosure preferably does not include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3. The polypeptide of the present disclosure preferably does not include a polypeptide containing the amino acid sequence shown in SEQ ID NO: 3.

[0036] The polypeptide of (B), the polypeptide of (D), the polypeptide of (F), and the polypeptide of (H) of the present disclosure preferably have antigenicity.

[0037] In the present specification, "having antigenicity" means reacting with an antibody against a periodontal pathogen in an antigen-antibody reaction. Examples of the antibody include immunoglobulin antibodies such as IgG antibody, IgM antibody, and IgA antibody, and any of the immunoglobulin antibodies may react in an antigen-antibody reaction. Among them, IgG antibody is preferred.

[0038] Antigenicity can be evaluated by the ELISA method. More specifically, as shown in the examples described below, when a polypeptide for evaluating antigenicity is used as an antigen and a positive serum of a periodontal disease patient containing an antibody against a periodontal pathogen is used as a primary antibody, and these antigen-antibody reactions are detected by the ELISA method, the polypeptide is determined to have antigenicity.

[0039] The polypeptide of the present disclosure can be produced using general protein chemical synthesis methods (e.g., liquid phase method and solid phase method) based on the amino acid sequence information shown in SEQ ID NO: 1, 2, 10, or 11. Further, the above-described polypeptide can be produced by genetic engineering techniques using a polynucleotide encoding the polypeptide.

[0040] The polypeptide of the present disclosure may further contain a tag. Examples of the tag include peptide tags such as His tag and SBP (Streptavidin Binding Peptide) tag; polypeptide tags such as GST (Glutathione-S-transferase) tag and MBP (Maltose Binding Protein) tag. Among them, polypeptide tags are preferred, and GST tags are more preferred.

[0041] The tag may be bound to the N-terminal side or the C-terminal side of the polypeptide of the present disclosure. Among them, it is preferable that the tag is bound to the N-terminal side of the polypeptide of the present disclosure. Preferable polypeptides containing a tag include, for example, polypeptides further containing a tag on the N-terminal side or the C-terminal side of the polypeptides (A), (B), (C), (D), (E), (F), (G), and (H) of the present disclosure.

[0042] The method for adding a tag is not particularly limited, and conventional methods and reaction conditions can be adopted.

[0043] The polypeptide of the present disclosure may further have a labeling substance. Examples of the labeling substance include horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-D-galactosidase, biotin, gold colloid, latex beads, and the like.

[0044] The present disclosure also includes a reagent for measuring the antibody titer of periodontal pathogenic bacteria containing the polypeptide of the present disclosure. In this specification, the reagent may be referred to as "the reagent of the present disclosure".

[0045] The reagent of the present disclosure may contain other components in addition to the polypeptide of the present disclosure. Examples of other components include solvents such as physiological saline, PBS, phosphate buffer, Tris buffer, borate buffer, and Good Buffer; protein stabilizers such as those containing casein, skim milk, bovine serum albumin (BSA), gelatin, blood proteins or plant proteins as active ingredients, normal animal blood components such as those of mice, rabbits, goats, and fetal calves, and normal human serum components; various preservatives; surfactants; enzyme activators such as salts of metals such as zinc and magnesium.

[0046] The present disclosure also encompasses a periodontal pathogen antibody titer measurement kit containing a reagent for measuring the periodontal pathogen antibody titer containing the polypeptide of the present disclosure. In this specification, the kit may be referred to as "the kit of the present disclosure".

[0047] In addition to the reagent of the present disclosure, the kit may optionally contain other reagents, instruments, etc. For example, it includes blocking solution, washing solution, antibody containing a labeling substance, substrate solution (color development / fluorescence / luminescence), etc. Further, the kit may include a member (such as a membrane) containing, in addition to the reagent of the present disclosure, a blocking solution, a washing solution, an antibody containing a labeling substance, a substrate solution (color development / fluorescence / luminescence), etc.

[0048] The present disclosure also encompasses a method for measuring the antibody titer against periodontal pathogens in a biological sample, which includes contacting the biological sample with the polypeptide of the present disclosure. In this specification, the measurement method may be referred to as "the measurement method of the present disclosure".

[0049] The biological sample is not particularly limited, and examples include samples that may contain antibodies against periodontal pathogens (such as IgG antibody, IgA antibody, IgM antibody, etc.). From the viewpoints of ease of collection and handling, the biological sample is preferably blood, saliva, gingival crevicular fluid, feces, tears, nasal mucus, or amniotic fluid. Also, organs such as the liver can be used as a sample that may contain antibodies against periodontal pathogens. When using blood as a biological sample, plasma or serum is more preferred, and serum is even more preferred. When using plasma as a biological sample, it may be plasma separated by filter paper, a plasma separator, or the like. Note that there are no particular restrictions on the method, conditions, etc. for storing the biological sample collected from the subject, and it can be carried out according to a conventional method.

[0050] The subject from which the biological sample is collected is not particularly limited as long as it is a living body in which periodontal pathogenic bacteria infection can occur, and it may be a non-human mammal. There are no particular restrictions on humans, and they may be healthy individuals, humans suffering from periodontal disease (periodontal disease patients), or humans suspected of suffering from periodontal disease. Humans with a disease associated with periodontal disease or periodontal pathogenic bacteria (for example, rheumatoid arthritis, heart disease, atherosclerotic disease, diabetes, dementia, non-alcoholic fatty liver disease (NASH / NAFLD), obesity, premature birth, etc.) or those suspected of having such a disease may also be used. Examples of non-human mammals include mammals raised as pets, livestock, experimental animals, etc. For example, dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, etc. can be mentioned.

[0051] The method for collecting the biological sample from the subject is not particularly limited, and it may be carried out according to a conventional method. Also, the collected biological sample may be used as it is, or after being freeze-dried and stored, the freeze-dried product may be dissolved in an appropriate solvent described later and used. Alternatively, the collected biological sample may be stored frozen as it is, or after being dissolved in an appropriate solvent described later and then stored frozen, it may be thawed and used at the time of use.

[0052] Examples of the solvent for dissolving the biological sample include physiological saline, PBS, phosphate buffer, Tris buffer, borate buffer, Good Buffer, etc. These solvents may contain protein stabilizers such as those containing casein, skim milk, bovine serum albumin (BSA), gelatin, blood protein or plant protein as active ingredients, normal animal blood components such as mouse, rabbit, goat, fetal bovine, etc., normal human serum components, etc.; various preservatives; surfactants; enzyme activators such as salts of metals such as zinc and magnesium, etc.

[0053] According to the measurement method of the present disclosure, by contacting the polypeptide of the present disclosure with a biological sample, an antibody against a periodontal pathogen present in the biological sample (for example, IgG antibody, IgA antibody, IgM antibody, etc.) can be detected by utilizing the antigen-antibody reaction with the polypeptide of the present disclosure.

[0054] The measurement method of the present disclosure may further include a step of detecting an antigen-antibody reaction product (a complex containing an antibody and the polypeptide of the present disclosure) between the antibody against the periodontal pathogen present in the biological sample generated by contacting the biological sample with the polypeptide of the present disclosure and the polypeptide of the present disclosure.

[0055] The method for detecting the antigen-antibody reaction product is not particularly limited, and conventional methods and reaction conditions can be adopted. For example, a method of contacting the antigen-antibody reaction product with an anti-immunoglobulin antibody containing a labeling substance (for example, anti-IgG antibody, etc.) and detecting the labeling substance, or when the polypeptide of the present disclosure contains a labeling substance, a method of detecting the labeling substance, etc. can be mentioned.

[0056] The measurement method of the present disclosure may, if necessary, include, for example, a step of immobilizing the antigen on a solid phase, a washing step, a blocking step, a substrate reaction step, etc. These can adopt conventional methods and reaction conditions.

[0057] For the measurement method of the present disclosure, immunological techniques such as ELISA method, dot blot method, immunochromatography method, etc. are preferably used.

[0058] In addition, as used herein, the term "comprising" includes "consisting essentially of” and "consisting of." Further, the present disclosure encompasses any combination of the components described herein.

[0059] Moreover, the various characteristics (properties, structures, functions, etc.) described for each embodiment of the present disclosure above may be combined in any manner in identifying the subject matter encompassed by the present disclosure. That is, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

Example

[0060] The content of the present disclosure will be specifically described using the following experimental examples. However, the present disclosure is not limited thereto in any way. In the following, unless otherwise specified, the experiments were conducted under atmospheric pressure and normal temperature conditions. Also, unless otherwise specified, “%” means “volume %”.

[0061] Experimental Example 1: Dot Blot The following eight kinds of antigen proteins were subjected to dot blot. Note that all of the antigen proteins used had a GST tag added to the N-terminal side. 35N (SEQ ID NO: 3) No. 2 (SEQ ID NO: 4) (corresponding to amino acids 1 to 226 in 35N) No. 3 (SEQ ID NO: 5) (corresponding to amino acids 1 to 579 in 35N) No. 4 (SEQ ID NO: 6) (corresponding to amino acids 31 to 697 in 35N) No. 5 (SEQ ID NO: 7) (corresponding to amino acids 227 to 579 in 35N) No. 6 (SEQ ID NO: 8) (corresponding to amino acids 227 to 697 in 35N) No. 7 (corresponding to amino acids 227 to 860 in SEQ ID NO: 2) No. 8 (corresponding to amino acids 580 to 860 in SEQ ID NO: 1)

[0062] The amount of the protein of 35N (GST tag) was set to 50 ng, and other proteins were prepared to have the same molar number as 35N (GST tag). After dot blotting on the membrane, it was blocked with Takara Western BloT blocking buffer. A positive serum (serum from a periodontal disease patient) diluted 5,000-fold was added and reacted overnight at 4°C. After washing with TBS containing Tween20 (0.05%), an HRP-conjugated goat anti-human IgG antibody reaction solution (MILLIPORE) diluted 80,000-fold was added and reacted at room temperature for 1 hour. After washing with TBS containing Tween20 (0.05%), Pierce ECL substrate of HRP detection reagent (Thermo Fisher) was added. The image of the membrane was scanned using ImageQuant LAS4000 mini (GE Healthcare), and the signal value of the dot was determined. The results are shown in FIG. 1.

[0063] As shown in FIG. 1, it was found that when the proteins of No. 7 and No. 8 were used, the reactivity with the positive serum was high. Also, it was suggested that the epitope was included in the sequence common to the proteins of No. 7 and No. 8 (580 to 860 in 35N (SEQ ID NO: 3)).

[0064] Experimental Example 2: ELISA The following four kinds of antigen proteins were subjected to ELISA. Note that all antigen proteins having a GST tag on the N-terminal side were used. 35N (SEQ ID NO: 3) No. 4 (SEQ ID NO: 6) (corresponding to amino acids 31 to 697 in 35N) No. 7 (SEQ ID NO: 2) (corresponding to amino acids 227 to 860 in 35N) No. 8 (SEQ ID NO: 1) (corresponding to amino acids 580 to 860 in 35N)

[0065] The amount of the antigen protein was prepared as 0.5 ng / 100 μl of 35N, and the other proteins were prepared to have the same number of moles as 35N.

[0066] The 96-well plate immobilized with glutathione was washed three times with PBS containing Tween20 (0.1%), then 100 μl of the prepared protein solution was added and reacted at room temperature for 1 hour. After washing three times with PBS containing Tween20 (0.1%), 100 μl of the positive serum diluted 620-fold was added and reacted at room temperature for 1 hour. After washing three times with PBS containing Tween20 (0.1%), 100 μl of the horseradish peroxidase (hereinafter referred to as HRP)-conjugated goat anti-human IgG antibody reaction solution (MILLIPORE) diluted 2000-fold was added and reacted at room temperature. After washing three times with PBS containing Tween20 (0.1%), 150 μl of the chromogenic substrate ABTS was added and reacted at room temperature, and then measured at 405 nm using a plate reader. The value obtained by subtracting the value of ABTS alone as the background was used as the absorbance. The results are shown in Figure 2.

[0067] As shown in Figure 2, similar to the evaluation by dot blot, it was found that when using the proteins of No. 7 and No. 8, the reactivity with the positive serum was high.

[0068] Experimental Example 3: ELISA In the same manner as in Experimental Example 2, the following three kinds of antigen proteins were subjected to ELISA. Note that for all antigen proteins, those with a GST tag added to the N-terminal side were used. 35N (SEQ ID NO: 3) No. 4 (SEQ ID NO: 6) (corresponding to amino acids 31 to 697 in 35N) No. 18 (SEQ ID NO: 9) (corresponding to amino acids 719 to 860 in 35N) Also, instead of the positive serum (serum of periodontal disease patients), the negative serum (serum of healthy individuals) was used, and similarly subjected to ELISA, and the absorbance was measured. The results are shown in Figure 3.

[0069] The protein of No. 4 (SEQ ID NO: 6) had low reactivity with the positive serum, and it was suggested that the protein of No. 18 (SEQ ID NO: 9) also reacted with the negative serum. From the above, among the sequences common to the proteins of No. 7 and No. 8 (580 - 860 in 35N (SEQ ID NO: 3)), it was suggested that the epitope was contained particularly in the 698 - 718 sequence of 35N.

[0070] Experimental Example 4: ELISA By the same method as in Experimental Example 2, the following five kinds of antigen proteins were subjected to ELISA. In addition, for all antigen proteins, those with a GST tag added to the N-terminal side were used. 35N (SEQ ID NO: 3) No. 7 (SEQ ID NO: 2) (corresponding to amino acids 227 - 860 in 35N) No. 8 (SEQ ID NO: 1) (corresponding to amino acids 580 - 860 in 35N) No. 13 (SEQ ID NO: 11) (corresponding to amino acids 404 - 860 in 35N) No. 15 (SEQ ID NO: 10) (corresponding to amino acids 24 - 860 in 35N)

[0071] The absorbance was as follows respectively. 35N (SEQ ID NO: 3): 0.224 No. 7 (SEQ ID NO: 2): 0.161 No. 8 (SEQ ID NO: 1): 0.191 No. 13 (SEQ ID NO: 11): 0.174 No. 15 (SEQ ID NO: 10): 0.663

[0072] Similar to the proteins of No. 7 and No. 8, it was also confirmed that when using the proteins of No. 13 and No. 15, it was possible to measure the antibody titer against periodontal pathogenic bacteria.

Claims

1. (A)A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or (B)A polypeptide having an amino acid sequence in which 1 to 25 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1 and which reacts with an antibody against a periodontal pathogen in an antigen-antibody reaction, wherein the number of amino acids deleted, substituted, or added in the amino acid sequence at positions 119 to 139 of the amino acid sequence shown in SEQ ID NO: 1 is 0 to 1.

2. (G)A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, or (H)A polypeptide having an amino acid sequence in which 1 to 45 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 11 and which reacts with an antibody against a periodontal pathogen in an antigen-antibody reaction, wherein the number of amino acids deleted, substituted, or added in the amino acid sequence at positions 295 to 315 of the amino acid sequence shown in SEQ ID NO: 11 is 0 to 1.

3. Furthermore, the polypeptide according to claim 1 or 2, further comprising a tag.

4. A reagent for measuring the antibody titer against a periodontal pathogen, comprising the polypeptide according to any one of claims 1 to 3.

5. A kit for measuring the antibody titer against a periodontal pathogen, comprising the reagent for measuring the antibody titer against a periodontal pathogen according to claim 4.

6. A method for measuring the antibody titer against a periodontal pathogen in a biological sample, comprising contacting the biological sample with the polypeptide according to any one of claims 1 to 3.

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