Antigen polypeptide

Abstract

To provide antigen polypeptides derived from periodontal disease bacteria having improved stability.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, etc.

Background Art

[0002] Periodontal disease is an inflammatory disease caused by the infection of periodontal pathogens 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, etc., 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 pathogens as an index of the infection degree or severity of periodontal disease has been reported. In the method developed by the present inventors, a polypeptide having a specific primary structure isolated from Porphyromonas gingivalis, a periodontal pathogen, or a modified polypeptide thereof (periodontal pathogen antigen polypeptide) is used, whereby 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 periodontal pathogen antigen polypeptide with improved stability.

Means for Solving the Problems

[0006] The inventors have found that a polypeptide having a specific amino acid sequence has excellent stability and have further improved it.

[0007] This disclosure includes, for example, the subject matter described in the following clauses. Clause 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. Clause 2. The polypeptide according to clause 1, wherein the polypeptide of (B) 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: 1. Clause 3. (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. Clause 4. The polypeptide according to clause 3, 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. Clause 5. Furthermore, a polypeptide according to any one of clauses 1 to 4, including a tag. Clause 6. A reagent for measuring the antibody titer against periodontal pathogenic bacteria, comprising the polypeptide according to any one of clauses 1 to 5. Clause 7. A kit for measuring the antibody titer against periodontal pathogenic bacteria, comprising the reagent for measuring the antibody titer against periodontal pathogenic bacteria according to clause 6. Clause 8. A method for measuring the antibody titer against periodontal pathogenic bacteria in a biological sample, comprising contacting the biological sample with the polypeptide according to any one of clauses 1 to 5. A method for measuring the antibody titer against periodontal pathogenic bacteria in a biological sample.

Advantages of the Invention

[0008] An antigen polypeptide with excellent stability is provided.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Modes 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 24 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 encompasses 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 present specification, the polypeptide may be referred to as "the polypeptide of (D) of the present disclosure".

[0014] In the present 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, and the polypeptide of (D) of the present disclosure may be collectively referred to as "the polypeptide of the present disclosure".

[0015] 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.

[0016] Techniques for adding mutations such as deletion, substitution, or addition of amino acids 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.

[0017] 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.

[0018] 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 / .

[0019] Moreover, the polypeptide of (B) in 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: 1. In other words, when the polypeptide of (B) 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: 1, the number of the added amino acids may be a polypeptide of 1 to 22. 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21. More specifically, for example, it may be 2 to 21.

[0020] The polypeptide of (D) in 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.

[0021] The polypeptide of (D) in the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence showing 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.

[0022] In addition, the polypeptide of (D) in the present disclosure may be a polypeptide having no 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 added amino acids may be 1 to 225. 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.

[0023] 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.

[0024] The polypeptide of (B) and the polypeptide of (D) in the present disclosure preferably have antigenicity.

[0025] 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 it may react with any of the immunoglobulin antibodies in an antigen-antibody reaction. Among them, IgG antibody is preferred.

[0026] Antigenicity can be evaluated by the ELISA method. More specifically, as shown in the examples described later, when a polypeptide for evaluating antigenicity is used as an antigen and 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.

[0027] 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 or SEQ ID NO: 2. Further, the above-mentioned polypeptide can be produced by genetic engineering techniques using a polynucleotide encoding the polypeptide.

[0028] The polypeptide of the present disclosure may further contain a tag. Examples of tags 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.

[0029] The tag may be bound to the N-terminal side of the polypeptide of the present disclosure or the C-terminal side. Among them, it is preferable that the tag is bound to the N-terminal side of the polypeptide of the present disclosure. Preferred polypeptides containing a tag include, for example, polypeptides further containing a tag on the N-terminal side or C-terminal side of the polypeptides (A), (B), (C), and (D) of the present disclosure.

[0030] The method for adding a tag is not particularly limited, and conventional methods and reaction conditions can be adopted.

[0031] The polypeptide of the present disclosure may further have a labeling substance. Examples of labeling substances include horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-D-galactosidase, biotin, gold colloid, latex beads, and the like.

[0032] The polypeptide of the present disclosure preferably has improved stability as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3. As shown in the examples described later, the stability is evaluated by the change over time of the absorbance measured by the ELISA method. The polypeptide of the present disclosure preferably has a suppressed decrease in absorbance over time as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.

[0033] 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".

[0034] 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 protein or plant protein as active ingredients, normal animal blood components such as mouse, rabbit, goat, fetal bovine, and normal human serum components; various preservatives; surfactants; enzyme activators such as salts of metals such as zinc and magnesium.

[0035] The present disclosure also includes a kit for measuring the antibody titer of periodontal pathogenic bacteria containing a reagent for measuring the antibody titer of periodontal pathogenic bacteria containing the polypeptide of the present disclosure. In this specification, the kit may be referred to as "the kit of the present disclosure".

[0036] In addition to the reagent of the present disclosure, the kit may optionally contain other reagents, instruments, etc. For example, a blocking solution, a washing solution, an antibody containing a labeling substance, a substrate solution (chromogenic, fluorescent, luminescent), etc. may be mentioned. Further, the kit may include a member (such as a membrane) containing a blocking solution, a washing solution, an antibody containing a labeling substance, a substrate solution (chromogenic, fluorescent, luminescent), etc. in addition to the reagent of the present disclosure.

[0037] The present disclosure also includes a method for measuring the antibody titer against periodontal pathogenic bacteria in a biological sample, which includes contacting the biological sample with the polypeptide of the present disclosure. In the present specification, the measurement method may be referred to as "the measurement method of the present disclosure".

[0038] The biological sample is not particularly limited, and examples thereof include samples that may contain antibodies against periodontal pathogenic bacteria (for example, IgG antibody, IgA antibody, IgM antibody, etc.). From the viewpoints of ease of collection and handling, etc., as the biological sample, blood, saliva, gingival crevicular fluid, feces, tears, nasal discharge, or amniotic fluid is preferable. Also, as a sample that may contain antibodies against periodontal pathogenic bacteria, organs such as the liver can also be used. When using blood as the biological sample, plasma or serum is more preferable, and serum is even more preferable. When using plasma as the biological sample, it may be plasma separated by a filter paper, a plasma separator, or the like. Note that the method, conditions, etc. for storing the biological sample collected from the subject are not particularly limited and can be carried out according to conventional methods.

[0039] The subject from whom the biological sample is collected is not particularly limited to humans, and may be any living body in which infection with periodontal pathogenic bacteria can occur, and may be a non-human mammal. Humans are not particularly limited, and may be healthy individuals, humans suffering from periodontal disease (periodontal disease patients), or humans suspected of suffering from periodontal disease. Humans having or suspected of having a disease related to periodontal disease or periodontal pathogenic bacteria (for example, rheumatoid arthritis, heart disease, atherosclerotic disease, diabetes, dementia, non-alcoholic fatty liver (NASH / NAFLD), obesity, premature birth, etc.) 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.

[0040] The method for collecting a biological sample from a subject is not particularly limited and may be performed according to a conventional method. Also, the collected biological sample may be used as it is, or may be freeze-dried and stored, and then the freeze-dried product may be dissolved in an appropriate solvent described below and used. Alternatively, the collected biological sample may be stored frozen as it is, or after being dissolved in an appropriate solvent described below and then stored frozen, and thawed and used at the time of use.

[0041] 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 casein, skim milk, bovine serum albumin (BSA), gelatin, blood proteins or plant proteins as active ingredients, protein component stabilizers such as normal animal blood components of mice, rabbits, goats, fetal calves, etc., normal human serum components, etc.; various preservatives; surfactants; enzyme activators such as salts of metals such as zinc and magnesium.

[0042] According to the measurement method of the present disclosure, by contacting the polypeptide of the present disclosure with a biological sample, antibodies against periodontal pathogenic bacteria 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.

[0043] 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 periodontal pathogenic bacteria 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.

[0044] 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 a method of detecting the labeling substance when the polypeptide of the present disclosure contains a labeling substance, etc. can be mentioned.

[0045] The measurement method of the present disclosure may include, if necessary, for example, a step of immobilizing an antigen on a solid phase, a washing step, a blocking step, a substrate reaction step, and the like. Conventional methods and reaction conditions can be employed for these steps.

[0046] For the measurement method of the present disclosure, immunological techniques such as ELISA, dot blot method, immunochromatography method, etc. are preferably used.

[0047] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of". Further, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0048] Moreover, regarding the various characteristics (properties, structures, functions, etc.) described for each of the above-described embodiments of the present disclosure, they may be combined in any manner when specifying the subject matter encompassed by the present disclosure. That is, the present disclosure encompasses all subject matters consisting of any combination of the combinable characteristics described in this specification.

Examples

[0049] The content of the present disclosure will be specifically described using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, the experiments are conducted under atmospheric pressure and normal temperature conditions. Also, unless otherwise specified, "%" means "% by volume".

[0050] ELISA The following three types of antigen proteins were subjected to ELISA. Note that for each antigen protein, those with a GST tag added to the N-terminal side were used. 35N (SEQ ID NO: 3) No. 15 (SEQ ID NO: 1) (corresponding to amino acids 24 to 860 in 35N) No. 7 (corresponding to amino acids 227 to 860 in SEQ ID NO: 2) (at 35N)

[0051] The amount of the antigen protein was prepared such that 35N was 0.25 or 0.5 ng / 100 μl, and the other proteins were prepared to have the same number of moles as 35N.

[0052] A 96-well plate immobilized with glutathione for protein immobilization was washed three times with PBS containing Tween20 (0.05 - 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 (0.05 - 0.1%), 100 μl of positive serum (serum from periodontal disease patients) diluted 500 - 620 times was added and reacted at room temperature for 1 hour. After washing three times with PBS containing Tween20 (0.05 - 0.1%), 100 μl of a horseradish peroxidase (hereinafter referred to as HRP)-conjugated goat anti-human IgG antibody reaction solution (MILLIPORE) diluted 1000 - 2000 times was added and reacted at room temperature. After washing three times with PBS containing Tween20 (0.05 - 0.1%), 150 μl of the chromogenic substrate ABTS was added and reacted at room temperature, and then the absorbance at 405 nm was measured using a plate reader. In addition, at the timing of each measurement in STEP2_6, STEP2_7, and STEP3_11, the measurements were carried out under the same conditions of the concentrations of the antigen and reagents. The absorbance measurement was made with the first measurement as the initial value and measured three times in tests from the initial stage to two years (STEP2_6, STEP2_7, STEP3_11). The results are shown in FIGS. 1 and 2. In FIGS. 1 and 2, the ratio of the absorbance of 35N when the absorbance of No. 15 or No. 7 in STEP2_6, STEP2_7, and STEP3_11 was taken as 1 is shown.

[0053] During the measurements in STEP2_6, STEP2_7, and STEP3_11, the antigen protein was subjected to freeze-thaw cycles, and it was found that 35N had a greater decrease in reactivity with the increase in storage period and the number of freeze-thaw cycles compared to No. 15 and No. 7.

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 50 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1 and reacting with an antibody against a periodontal pathogen in an antigen-antibody reaction, and having no addition of 23 or more amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1, and the decrease in absorbance measured by the ELISA method over time is suppressed compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

3.

2. The polypeptide according to claim 1, wherein the polypeptide of (B) has no addition of amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO:

1.

3. (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 1 to 50 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2 and reacting with an antibody against a periodontal pathogen in an antigen-antibody reaction, and having no addition of 50 or more amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2, and the decrease in absorbance measured by the ELISA method over time is suppressed compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

3.

4. The polypeptide according to claim 3, wherein the polypeptide of (D) has no addition of amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO:

2.

5. Furthermore, the polypeptide according to any one of claims 1 to 4, including a tag.

6. A reagent for measuring the antibody titer against a periodontal pathogen, comprising the polypeptide according to any one of claims 1 to 5.

7. 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 6.

8. 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 5.

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