Antigen polypeptide

Abstract

To provide antigen polypeptides derived from periodontal disease bacteria, having high reactivity with antibodies against periodontal disease bacteria.SOLUTION: The present invention discloses a polypeptide having a GST tag added to the N-terminal side of a polypeptide of (A) or (B) shown below, while not having the tag added to the C terminal side: (A) a polypeptide composed of a specific amino acid sequence; and (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, etc.

Background Art

[0002] Periodontal disease is an inflammatory disease caused by the infection of periodontal pathogenic bacteria with periodontal tissues. Generally, the diagnosis of periodontal disease is performed by comprehensively considering the results of periodontal pocket examination, palpation / bleeding examination, mobility examination, X-ray examination, etc. However, since these examination methods are based on the experience and skills of dentists, etc., there is a possibility of 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, etc. (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 periodontal pathogenic bacterium antigen polypeptide having high reactivity with an antibody against a periodontal pathogenic bacterium. [Means for solving the problem]

[0006] The inventors discovered that adding a tag to the N-terminus of a periodontal pathogen antigen polypeptide improves its reactivity with antibodies against periodontal pathogens, and further refined the process.

[0007] This disclosure includes, for example, the following subjects: Section 1. A polypeptide having a structure in which a GST tag is attached to the N-terminus and no tag is attached to the C-terminus, as described in (A) or (B) below. (A) Polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1 (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 Sequence ID No. 1, and which is antigenic. Section 2. A polypeptide having a structure in which a GST tag is attached to the N-terminus and no tag is attached to the C-terminus, as described in (C) or (D) below. (C) Polypeptide consisting of the amino acid sequence shown in Sequence ID No. 2 (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 Sequence ID No. 2, and which is antigenic. Section 3. A polypeptide having a structure in which a GST tag is attached to the N-terminus and no tag is attached to the C-terminus, as described in (E) or (F) below. (E) Polypeptide consisting of the amino acid sequence shown in Sequence ID No. 3 (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 Sequence ID No. 3, and which is antigenic. Section 4. A polypeptide having a structure in which a GST tag is attached to the N-terminus and no tag is attached to the C-terminus, as described in (G) or (H) below. (G) Polypeptide consisting of the amino acid sequence shown in Sequence ID No. 4 (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 Sequence ID No. 4, and which is antigenic. Section 5. A polypeptide having a structure in which a GST tag is attached to the N-terminus and no tag is attached to the C-terminus, as described in (I) or (J) below. (I) Polypeptide consisting of the amino acid sequence shown in Sequence ID No. 5 (J) 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 Sequence ID No. 5, and which is antigenic. Section 6. A polypeptide having the structure of the polypeptide (K) below, with a GST tag attached to the N-terminus and no tag attached to the C-terminus. (K) A polypeptide having the amino acid sequence from position 580 to 860 of the amino acid sequence shown in Sequence ID No. 1, and possessing antigenicity. Section 7. A polypeptide having the structure of the polypeptide (L) below, with a GST tag attached to the N-terminus and no tag attached to the C-terminus. (L) A polypeptide having the amino acid sequence from position 698 to 718 of the amino acid sequence shown in Sequence ID No. 1, and possessing antigenicity. Section 8. A reagent for measuring the antibody titer of periodontal pathogens, comprising a polypeptide described in any of items 1 to 7. Section 9. A kit for measuring the antibody titer of periodontal pathogens, including the reagent for measuring antibody titers of periodontal pathogens described in item 8. Section 10. This includes contacting a biological sample with a polypeptide described in any of items 1 to 7. A method for measuring antibody titers against periodontal pathogens in biological samples. [Effects of the Invention]

[0008] There is provided a periodontal pathogen antigen polypeptide that has a high reactivity with an antibody against a periodontal pathogen.

Brief Description of the Drawings

[0009] [Figure 1] The results of Western blot are shown. [Figure 2] The results of dot blot when using positive serum or negative serum are shown. [Figure 3] The measurement results of ELISA are shown. [Figure 4] The measurement results of ELISA when using positive serum or negative serum are shown.

Modes for Carrying Out the Invention

[0010] Hereinafter, each embodiment included in the present disclosure will be described in more detail.

[0011] The present disclosure includes 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)".

[0012] 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)".

[0013] 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)". 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: 1.

[0014] Furthermore, this disclosure also includes polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in (D)Sequence ID 2. In this specification, such polypeptides may be referred to as "(D) polypeptides".

[0015] Furthermore, this disclosure also includes polypeptides comprising the amino acid sequence shown in (E)Sequence ID 3. In this specification, such polypeptides may be referred to as "(E) polypeptides." The amino acid sequence shown in Sequence ID No. 3 corresponds to amino acid sequences 580 to 860 of the amino acid sequence shown in Sequence ID No. 1.

[0016] Furthermore, this disclosure also includes polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in (F)Sequence ID 3. In this specification, such polypeptides may be referred to as "(F) polypeptides".

[0017] Furthermore, this disclosure also includes polypeptides comprising the amino acid sequence shown in (G)SEQ ID NO: 4. In this specification, such polypeptides may be referred to as "(G) polypeptides." The amino acid sequence shown in Sequence ID No. 4 corresponds to amino acid sequences 404 to 860 of the amino acid sequence shown in Sequence ID No. 1.

[0018] Furthermore, this disclosure also includes polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in (H)Sequence ID 4. In this specification, such polypeptides may be referred to as "(H) polypeptides".

[0019] Furthermore, this disclosure also includes the polypeptide comprising the amino acid sequence shown in (I) Sequence ID No. 5. In this specification, such polypeptide may be referred to as "(I) polypeptide". The amino acid sequence shown in Sequence ID No. 5 corresponds to amino acid sequences 24 through 860 of the amino acid sequence shown in Sequence ID No. 1.

[0020] Furthermore, this disclosure also includes polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in (J)SEQ ID NO: 5. In this specification, such polypeptides may be referred to as "(J) polypeptides".

[0021] Furthermore, this disclosure also includes polypeptides having the amino acid sequence from positions 580 to 860 of the amino acid sequence shown in (K)Sequence ID 1. In this specification, such polypeptides may be referred to as "(K) polypeptides".

[0022] Furthermore, this disclosure also includes polypeptides having the amino acid sequence from positions 698 to 718 of the amino acid sequence shown in (L)Sequence ID 1. In this specification, such polypeptides may be referred to as "(L) polypeptides".

[0023] The polypeptide of (B) 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. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence shown in SEQ ID NO: 1 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.

[0024] Techniques for introducing mutations such as deletions, substitutions, or additions of amino acids into specific amino acid sequences are publicly known in the art and can be performed using any method. For example, these can be done using restriction enzyme therapy, treatment with exonucleases or DNA ligases, site-directed mutagenesis, random mutagenesis, etc.

[0025] The polypeptide of (B) may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity with the amino acid sequence shown in Sequence 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.

[0026] The identity of amino acid sequences can be calculated using the default parameters of the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / .

[0027] The polypeptide of (D) 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. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence shown in SEQ ID NO: 2 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.

[0028] The polypeptide of (D) may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity with the amino acid sequence shown in Sequence 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.

[0029] Furthermore, polypeptide (D) may be a polypeptide that does not have 226 or more amino acids added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 2. In other words, if polypeptide (D) has amino acid additions to the N-terminus of the amino acid sequence shown in SEQ ID NO: 2, it may be a polypeptide with 1 to 225 such added amino acids. The upper or lower limit of the number of such 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.

[0030] The polypeptide of (F) 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: 3. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence shown in SEQ ID NO: 3 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.

[0031] The polypeptide of (F) may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity with the amino acid sequence shown in Sequence ID No. 3. 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.

[0032] The polypeptide (H) 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: 4. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence shown in SEQ ID NO: 4 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) may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity with the amino acid sequence shown in SEQ ID NO: 4. 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] The polypeptide of (J) 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: 5. The upper limit of the number of deleted, substituted, or added amino acids in the amino acid sequence shown in SEQ ID NO: 5 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.

[0035] The polypeptide of (J) may be, for example, a polypeptide consisting of an amino acid sequence that shows 85% or more identity with the amino acid sequence shown in SEQ ID NO: 5. 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.

[0036] Furthermore, polypeptide (J) may be a polypeptide that does not have 23 or more amino acids added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 5. In other words, if polypeptide (J) has amino acid additions to the N-terminus of the amino acid sequence shown in SEQ ID NO: 5, then polypeptide (J) may have 1 to 22 such added amino acids. The upper or lower limit of the number of such 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.

[0037] The polypeptide (K) is a polypeptide having the amino acid sequence from position 580 to 860 of the amino acid sequence shown in SEQ ID NO: 1. The polypeptide (K) may have one or more amino acids added to the N-terminus or C-terminus of the amino acid sequence from position 580 to 860 of the amino acid sequence shown in SEQ ID NO: 1. The upper limit of the number of added amino acids may be, for example, 600, 550, 500, 450, 400, 350, 300, 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.

[0038] Polypeptide (L) is a polypeptide having the amino acid sequence from position 698 to 718 of the amino acid sequence shown in SEQ ID NO: 1. Polypeptide (L) may have one or more amino acids added to the N-terminus or C-terminus of the amino acid sequence from position 698 to 718 of the amino acid sequence shown in SEQ ID NO: 1. The upper limit of the number of added amino acids may be, for example, 700, 650, 600, 550, 500, 450, 400, 350, 300, 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.

[0039] Polypeptides (A), (B), (C), (D), (E), (F), (G), (H), (I), and (J) are preferably polypeptides in which the number of deleted, substituted, or added amino acids in the amino acid sequence corresponding to positions 580 to 860 of the amino acid sequence shown in Sequence ID No. 1 is between 0 and 50. The upper or lower limit of the range may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49. More specifically, for example, it may be between 1 and 49. Furthermore, polypeptides (A), (B), (C), (D), (E), (F), (G), (H), (I), and (J) may have a conserved amino acid sequence corresponding to positions 580 to 860 of the amino acid sequence shown in Sequence ID No. 1.

[0040] Polypeptides (A), (B), (C), (D), (E), (F), (G), (H), (I), and (J) are preferably polypeptides in which the number of deleted, substituted, or added amino acids in the amino acid sequence corresponding to positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 1 is between 0 and 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, for example, 1 to 8. Furthermore, polypeptides (A), (B), (C), (D), (E), (F), (G), (H), (I), and (J) may also have the amino acid sequence corresponding to positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 1 preserved.

[0041] The polypeptides of this disclosure are polypeptides having a structure in which a GST tag is attached to the N-terminus of polypeptides (A), (B), (C), (D), (E), (F), (G), (H), (I), (J), (K), and (L), but no tag is attached to the C-terminus. In this specification, these polypeptides may be collectively referred to as "the polypeptides of this disclosure."

[0042] There are no particular limitations on the method of adding tags; conventional methods and reaction conditions can be employed.

[0043] Polypeptides (B), (D), (F), (H), (J), (K), and (L) are preferably antigenic.

[0044] In this specification, "having antigenicity" means that it reacts with antibodies against periodontal pathogens. Examples of antibodies include immunoglobulin antibodies such as IgG antibodies, IgM antibodies, and IgA antibodies, and it is sufficient for the substance to react with any of these immunoglobulin antibodies. Among these, IgG antibodies are preferred.

[0045] Antigenicity can be evaluated by the ELISA method. More specifically, as shown in the examples described later, if the polypeptide whose antigenicity is to be evaluated is used as the antigen, and the positive serum of a periodontal disease patient containing antibodies against periodontal pathogens is used as the primary antibody, then the polypeptide is judged to have antigenicity if these antigen-antibody reactions are detected by the ELISA method.

[0046] The polypeptides of this disclosure can be produced using general chemical synthesis methods for proteins (e.g., liquid-phase and solid-phase methods) based on the amino acid sequence information shown in SEQ ID NOs: 1, 2, 3, 4, or 5. Alternatively, the polypeptides described above can be produced by genetic engineering techniques using the polynucleotides encoding them.

[0047] The polypeptides of this disclosure may further have a labeling substance. Examples of labeling substances include horseradish-derived peroxidase (HRP), alkaline phosphatase (ALP), β-D-galactosidase, biotin, gold colloid, and latex beads.

[0048] This disclosure also includes reagents for measuring periodontal pathogen antibody titers, which contain the polypeptides of this disclosure. In this specification, such reagents may be referred to as "the reagents of this disclosure."

[0049] The reagents of this disclosure may contain other components in addition to the polypeptides of this disclosure. Other components include solvents such as physiological saline, PBS, phosphate buffer, Tris buffer, borate buffer, and Good Buffer; protein component stabilizers such as casein, skim milk, bovine serum albumin (BSA), gelatin, blood proteins or plant proteins as active ingredients, normal animal blood components such as those from mice, rabbits, goats, and fetal bovines, and normal human serum components; various preservatives; surfactants; and enzyme activators such as metal salts such as zinc and magnesium.

[0050] This disclosure also includes a periodontal pathogen antibody titer measurement kit, which includes a reagent for measuring periodontal pathogen antibody titers containing the polypeptide of this disclosure. In this specification, such kit may be referred to as the "Kit of this Disclosure."

[0051] The kit may include, as necessary, other reagents, equipment, etc., in addition to the reagents disclosed herein. Examples include blocking solution, washing solution, antibody containing a labeling substance, substrate solution (chromogenic, fluorescent, or luminescent), etc. The kit may also include components (such as membranes) containing blocking solution, washing solution, antibody containing a labeling substance, substrate solution (chromogenic, fluorescent, or luminescent), etc., in addition to the reagents disclosed herein.

[0052] This disclosure also includes a method for measuring antibody titers against periodontal pathogens in a biological sample, which includes contacting the biological sample with the polypeptide of this disclosure. In this specification, such measurement method may be referred to as the "measurement method of this disclosure."

[0053] The biological sample is not particularly limited and may include, for example, a sample that may contain antibodies against periodontal pathogens (e.g., IgG antibodies, IgA antibodies, IgM antibodies, etc.). From the viewpoint of ease of collection and handling, blood, saliva, gingival crevicular fluid, feces, tears, nasal swabs, or amniotic fluid are preferred as biological samples. In addition, organs such as the liver can also be used as samples that may contain antibodies against periodontal pathogens. When blood is used as a biological sample, plasma or serum is more preferred, and serum is even more preferred. When plasma is used as a biological sample, it may be plasma separated by filter paper or a plasma separator. There are no particular restrictions on the method or conditions for storing the biological sample collected from the subject, and it can be done according to conventional methods.

[0054] The subjects from which biological samples are collected are not limited to humans, but are not particularly restricted to any organism that may be susceptible to periodontal pathogen infection, and may also be non-human mammals. Humans are not particularly restricted; they may be healthy individuals, individuals suffering from periodontal disease (periodontal disease patients), or individuals suspected of having periodontal disease. Individuals with or suspected of having periodontal disease or diseases associated with periodontal pathogens (e.g., rheumatoid arthritis, heart disease, arteriosclerotic disease, diabetes, dementia, non-alcoholic fatty liver disease (NASH / NAFLD), obesity, premature birth, etc.) may also be included. Examples of non-human mammals include mammals kept as pets, livestock, or laboratory animals. Examples include dogs, cats, monkeys, cattle, horses, sheep, goats, pigs, rabbits, mice, rats, camels, and llamas.

[0055] There are no particular restrictions on the method of collecting biological samples from the subject, and it is acceptable to follow conventional methods. Furthermore, the collected biological samples may be used as is, or they may be freeze-dried and stored, and then the freeze-dried material may be dissolved in a suitable solvent as described later before use, or the collected biological samples may be frozen and stored as is, or dissolved in a suitable solvent as described later and then frozen and thawed before use.

[0056] Examples of solvents used to dissolve biological samples include physiological saline, PBS, phosphate buffer, Tris buffer, borate buffer, and Good Buffer. These solvents may also contain casein, skim milk, bovine serum albumin (BSA), gelatin, blood proteins, or plant proteins as active ingredients; protein component stabilizers such as blood components from normal animals like mice, rabbits, goats, and fetal bovines, or components from normal human serum; various preservatives; surfactants; and enzyme activators such as metal salts like zinc and magnesium.

[0057] According to the measurement method of this disclosure, by bringing the polypeptide of this disclosure into contact with a biological sample, antibodies against periodontal pathogens present in the biological sample (e.g., IgG antibodies, IgA antibodies, IgM antibodies, etc.) can be detected using an antigen-antibody reaction with the polypeptide of this disclosure.

[0058] The measurement method of the present disclosure may further include a step of detecting an antigen-antibody reaction product (a complex comprising the antibody and the polypeptide of the present disclosure) between an antibody against periodontal pathogens present in a biological sample and the polypeptide of the present disclosure, which is generated by contacting the biological sample with the polypeptide of the present disclosure.

[0059] The method for detecting the antigen-antibody reaction product is not particularly limited, and conventional methods and reaction conditions can be employed. For example, a method may involve contacting the antigen-antibody reaction product with an anti-immunoglobulin antibody containing a labeling substance (e.g., an anti-IgG antibody) and detecting the labeling substance, or, if the polypeptide of this disclosure contains a labeling substance, a method for detecting the labeling substance.

[0060] The measurement method disclosed herein may optionally include, for example, a step of immobilizing the antigen onto a solid phase, a washing step, a blocking step, a substrate reaction step, etc. Conventional methods and reaction conditions can be employed for these steps.

[0061] The measurement methods disclosed herein preferably utilize immunological techniques such as ELISA, dot blotting, and immunochromatography.

[0062] In this specification, the term "comprising" includes both "consisting essentially of" and "consisting of." Furthermore, this disclosure encompasses all any combination of the constituent elements described herein.

[0063] Furthermore, the various characteristics (properties, structure, function, etc.) described in each embodiment of this disclosure above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein. [Examples]

[0064] The contents of this disclosure will be specifically explained using the following experimental examples. However, this disclosure is not limited to these examples. Unless otherwise specified below, the experiments were conducted under atmospheric pressure and room temperature conditions. Unless otherwise specified, "%" means "weight %".

[0065] Experimental Example 1: SDS-PAGE and Western Blot 35N proteins (SEQ ID NO: 1) with GST tags at either the N-terminus or C-terminus were attached to SDS-PAGE. Two proteins with different tagging sites were adjusted to the same concentration, denatured by heat, and then SDS-PAGE was performed using two polyacrylamide gels. The samples were transferred to membranes and Western blotting was performed with anti-GST antibody and positive serum, respectively. The transferred membranes were blocked with TBS containing 5% skim milk. Antibody solutions (anti-GST polyclonal antibody (Bethyl) or positive serum (serum from periodontal disease patients)) diluted 2,500-5,000 times with TBS containing 3% skim milk were added, and the mixture was reacted at room temperature for 2 hours. After washing three times with TBS containing 0.05% (v / v) Tween20, HRP-conjugated goat anti-rabbit IgG antibody reaction solution (MILLIPORE) or HRP-conjugated goat anti-human IgG antibody reaction solution, diluted 5,000-fold with TBS containing 3% skim milk, was added and reacted at room temperature for 1 hour. After three washes, 4-Methyl-1 Naphtol color development solution was added and reacted at room temperature for 30 minutes. Images of the membrane were scanned using ImageQuant LAS4000 mini (GE Healthcare), and the signal values ​​of each band were determined. Figure 1 shows the relative values ​​for each band, with the band intensity of the 35N protein with a GST tag at the N-terminus set to 100.

[0066] As shown in Figure 1, although the reactivity was similar when using anti-GST antibodies, when using positive serum (serum from periodontal disease patients), the 35N protein with the GST tag at the N-terminus showed higher reactivity compared to the 35N protein with the GST tag at the C-terminus.

[0067] Experimental Example 2: Dot Blot 35N proteins with either a His tag or a GST tag at the N-terminus were attached to dot blots. The amount of 35N (GST tagged) protein was 50 ng, and the amount of 35N (His tagged) protein was prepared to be the same molar quantity. After dot blotting, the samples were shaken with blocking solution (Takara Western BloT blocking buffer), and then shaken again with TBS containing Tween20 (0.05% (v / v)). A 5,000-fold diluted positive serum (serum from periodontal disease patients) or negative serum (serum from healthy individuals) was added, and the samples were shaken overnight at 4°C. After washing with TBS containing Tween20 (0.05% (v / v)), an 80,000-fold diluted HRP-conjugated goat anti-human IgG antibody reaction solution (MILLIPORE) was added, and the samples were reacted at room temperature for 1 hour. After washing with TBS containing Tween20 (0.05% (v / v)), Pierce ECL substrate (Thermo Fisher), an HRP detection reagent, was added. Images of the membrane were scanned using ImageQuant LAS4000 mini (GE Healthcare), and the signal values ​​of the dots were determined. Two types of serum from different sources were used for positive and negative sera. Band intensities are shown in Figure 2.

[0068] As shown in Figure 2, in all cases using positive serum samples, the reactivity was found to be higher when using GST tags compared to when using His tags.

[0069] Experimental Example 3: ELISA The following four types of antigen proteins were subjected to ELISA. The antigen proteins used were polypeptides consisting of the amino acid sequences shown in each sequence number, specifically those with a GST tag at the N-terminus and no tag at the C-terminus (No. 9, 11, 13, 15) and those with a GST tag at the N-terminus and an SBP tag at the C-terminus (No. 10, 12, 14, 16). No. 9, 10 (Sequence ID 2) (corresponding to amino acids 227-860 in 35N) No. 11, 12 (Sequence ID 3) (corresponding to amino acids 580-860 in 35N) No. 13, 14 (Sequence No. 4) (corresponding to amino acids 404-860 in 35N) No. 15, 16 (Sequence ID 5) (corresponding to amino acids 24-860 in 35N)

[0070] The amount of antigen protein was prepared to be equivalent in moles to that obtained when 35N was prepared at 0.5 ng / 100 μl.

[0071] A 96-well plate with glutathione immobilized was washed three times with PBS containing Tween20 (0.1% (v / v)), then 100 μl of the prepared protein solution was added and the plate was reacted at room temperature for 1 hour. After washing three times with PBS containing Tween20 (0.1%), 100 μl of positive serum diluted 620-fold was added and the plate was reacted at room temperature for 1 hour. After washing three times with PBS containing Tween20 (0.1% (v / v)), 100 ml of horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody reaction solution (MILLIPORE), diluted 2000-fold, was added and the plate was reacted at room temperature. After washing three times with PBS containing Tween20 (0.1% (v / v)), 150 μl of the chromogenic substrate ABTS was added and the plate was reacted at room temperature, after which the absorbance at 405 nm was measured using a plate reader. The results are shown in Figure 3.

[0072] As shown in Figure 3, regardless of which antigen protein was used, it was found that the reactivity with positive serum was higher when the antigen protein had a GST tag at the N-terminus and no tag at the C-terminus compared to the antigen protein with a GST tag at the N-terminus and an SBP tag at the C-terminus.

[0073] Experimental Example 4: ELISA Three antigen proteins were attached to an ELISA using the same method as in Experimental Example 3. All antigen proteins used had a GST tag at the N-terminus but no tag at the C-terminus. 35N (Sequence ID 1) No. 4 (Sequence ID 6) (corresponding to amino acids 31-697 in 35N) No. 18 (Sequence ID 7) (corresponding to amino acids 719-860 in 35N) Furthermore, instead of positive serum (serum from periodontal disease patients), negative serum (serum from healthy individuals) was used and subjected to the same ELISA procedure, and the absorbance was measured. The results are shown in Figure 4.

[0074] Protein No. 4 (SEQ ID NO: 6) showed low reactivity with positive serum, while protein No. 18 (SEQ ID NO: 7) reacted with negative serum as well. These findings suggest that among the sequences common to proteins No. 7 and No. 8 (580-860 in 35N (SEQ ID NO: 1)), the epitope is particularly found in sequences 698-718 in 35N.

Claims

1. A polypeptide having a structure in which a GST tag is added to the N-terminal side of the following polypeptide (A) or (B) and no tag is added to the C-terminal side: (A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (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.

2. A polypeptide having a structure in which a GST tag is added to the N-terminal side of the following polypeptide (C) or (D) and no tag is added to the C-terminal side: (C) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (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.

3. A polypeptide having a structure in which a GST tag is added to the N-terminal side of the following polypeptide (E) or (F) but no tag is added to the C-terminal side. (E) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (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: 3, and having antigenicity.

4. A polypeptide having a structure in which a GST tag is added to the N-terminal side of the following polypeptide (G) or (H) and no tag is added to the C-terminal side. (G) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 (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: 4, and having antigenicity.

5. A polypeptide having a structure in which a GST tag is added to the N-terminal side of the following polypeptide (I) or (J) and no tag is added to the C-terminal side: (I) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 (J) 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: 5, and having antigenicity.

6. A polypeptide having a structure in which a GST tag is added to the N-terminal side of the following polypeptide (K) but no tag is added to the C-terminal side. (K) A polypeptide having an amino acid sequence from positions 580 to 860 of the amino acid sequence shown in SEQ ID NO: 1 and having antigenicity.

7. A polypeptide having a structure in which a GST tag is added to the N-terminal side of the following polypeptide (L) but no tag is added to the C-terminal side. (L) A polypeptide having the amino acid sequence of positions 698 to 718 of the amino acid sequence shown in SEQ ID NO: 1 and having antigenicity.

8. A reagent for measuring antibody titers to periodontal pathogens, comprising the polypeptide according to any one of claims 1 to 7.

9. A kit for measuring a periodontal pathogen antibody titer, comprising the reagent for measuring a periodontal pathogen antibody titer according to claim 8.

10. contacting a biological sample with the polypeptide of any one of claims 1 to 7. A method for measuring the antibody titer against periodontal pathogens in the biological sample.

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