Composition containing antigen polypeptide and use thereof

Abstract

To provide a composition containing an antigen polypeptide that produces consistent results in antibody titer measurement.SOLUTION: The present invention provides a composition containing an antigen polypeptide that is synthesized through a cell-free or living-cell protein synthesis system, and that consists of an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1. When the antigen polypeptide is subjected to separation by SDS-PAGE, the ratio of the intensity of the 120 kDa band to the intensity of the 25 kDa band is 5.25 to 13.60, and / or the ratio of the intensity of the 120 kDa band to the intensity of the 78 kDa band is 14.69 to 29.41.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a composition containing an antigen polypeptide and its use, etc.

Background Art

[0002] Periodontal disease is an inflammatory disease caused by the infection of periodontal pathogenic bacteria in the 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 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] When the inventors synthesized an antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 using a cell-free or cell-based protein synthesis system, they found that in addition to the antigen polypeptide itself (120 kDa), short polypeptide fragments derived from the antigen polypeptide were included. Depending on the lot, there was a difference in the purity of the antigen polypeptide (120 kDa) in the mixture containing the short polypeptide fragments. When the antibody titer of P. gingivalis was measured using antigen polypeptides with different purities, a difference was observed in the measurement results. Surprisingly, it was found that a more stable measurement result could be obtained when the purity of the antigen polypeptide (120 kDa) was lower. The present disclosure aims to provide a composition containing an antigen polypeptide that can obtain such a stable antibody titer measurement result.

Means for Solving the Problems

[0006] The inventors found that a stable antibody titer measurement result can be obtained when an antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 synthesized by a cell-free or cell-based protein synthesis system contains polypeptides of specific band sizes in a specific ratio, and further improvements were made.

[0007] The present disclosure includes, for example, the subject matters described in the following items. Item 1. A composition containing an antigen polypeptide synthesized by a cell-free or cell-based protein synthesis system, which consists of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, when the antigen polypeptide is separated by SDS-PAGE, the ratio of the intensity of the 120 kDa band to the intensity of the 25 kDa band is 5.25 to 13.60, and / or the ratio of the intensity of the 120 kDa band to the intensity of the 78 kDa band is 14.69 to 29.41. Item 2. Synthesized by a cell-free or cell-based protein synthesis system, A composition containing an antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, when the antigen polypeptide is separated by SDS-PAGE, the ratio of the intensity of the 120 kDa band to the intensity of the 96 kDa band is 10.74 to 18.20, the ratio of the intensity of the 96 kDa band to the intensity of the 25 kDa band is 0.37 to 0.93, and / or the ratio of the intensity of the 96 kDa band to the intensity of the 78 kDa band is 1.27 to 1.77, a composition. Item 3. The composition according to item 1 or 2, wherein the cell-free protein synthesis system is a wheat cell-free protein synthesis system. Item 4. The composition according to item 1 or 2, wherein the in vivo cell protein synthesis system is an E. coli protein synthesis system. Item 5. The composition according to item 1 or 2, which is a reagent for measuring the antibody titer against periodontal pathogens. Item 6. A kit for measuring the antibody titer against periodontal pathogens, comprising the reagent for measuring the antibody titer against periodontal pathogens according to item 5. Item 7. Including contacting a biological sample with the composition according to item 1 or 2, A method for measuring the antibody titer against periodontal pathogens in a biological sample.

Effect of the Invention

[0008] A composition containing an antigen polypeptide capable of obtaining stable antibody titer measurement results is provided.

Modes for Carrying Out the Invention

[0009] Hereinafter, each embodiment included in the present disclosure will be described in more detail. The composition included in the present disclosure contains an antigen polypeptide synthesized by a cell-free or cell-based protein synthesis system and consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1. In this specification, the composition may be referred to as "the composition of the present disclosure".

[0010] The amino acid sequence shown in SEQ ID NO: 1 is an amino acid sequence with a GST tag added to the N-terminus of the amino acid sequence at positions 239 to 1098 of the Arg-gingipain RgpA protein derived from Porphyromonas gingivalis (P.g.). The amino acid sequence at positions 239 to 1098 of the Arg-gingipain RgpA protein derived from P.g. is an antigen polypeptide (has antigenicity). In this specification, "has 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 with the antigen in an antigen-antibody reaction. Among them, IgG antibody is preferred.

[0011] In the antigen polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, the identity with the amino acid sequence shown in SEQ ID NO: 1 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.

[0012] The identity of the amino acid sequences 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 / .

[0013] An antigen polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 may be, for example, an antigen polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added (preferably substituted, more preferably conservatively substituted) in the amino acids shown in SEQ ID NO: 1. The upper limit of the number of deleted, substituted, or added amino acids may be, for example, 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. For example, it may be 1 to 50, it may be 1 to 35, or it may be 1 to 30. In the antigen polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, sequences added in vector design such as restriction enzyme sites may remain before and after each of the Arg-dingipine RgpA protein derived from P.g. and the N-terminal GST tag. As used herein, "conservative substitution" means substituting an amino acid with an amino acid having a side chain with properties similar to those of the side chain of the amino acid. Specific examples of conservative substitutions include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine; substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; substitutions between amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions between amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions between amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; substitutions between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine; and the like. The deleted, substituted, or added amino acids may be, for example, amino acids in the amino acid region encoding the GST tag, or may be amino acids in the amino acid region encoding the RgpA protein (amino acid sequence positions 239 to 1098).

[0014] 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 exonucleases, DNA ligases, etc., site-directed mutagenesis, random mutagenesis, etc.

[0015] Examples of amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 include, for example, the amino acid sequence shown in SEQ ID NO: 2. The amino acid sequence shown in SEQ ID NO: 2 is an amino acid sequence in which a GST tag is added to the N-terminus of the amino acid sequence positions 230 to 1065 of the Arg-gingipain RgpA protein derived from P.g. The amino acid sequence positions 230 to 1065 of the Arg-gingipain RgpA protein derived from P.g. are antigenic polypeptides (having antigenicity).

[0016] Examples of cell-free protein synthesis systems include, for example, a wheat cell-free protein synthesis system, a silkworm cell-free protein synthesis system, etc. Among them, the wheat cell-free protein synthesis system is preferred. Synthesis of the antigen polypeptide by the cell-free protein synthesis system can be carried out according to a conventional method. More specifically, it can be carried out by the method shown in the examples described later. The vector to be used is not particularly limited, and examples include, for example, the pEU vector etc. In the case of the wheat cell-free protein synthesis system, for example, wheat germ extract (preferably WEPRO7240G (Cell-Free Science Co., Ltd.) etc. can be used.

[0017] Examples of in vivo protein synthesis systems include, for example, an E. coli protein synthesis system etc. Among them, the E. coli protein synthesis system is preferred. Synthesis of the antigen polypeptide by a living cell protein synthesis system can be carried out according to a conventional method. More specifically, it can be carried out by the method shown in the examples described later. The vector to be used is not particularly limited, and examples thereof include pEU vectors, pGEX vectors, and the like. In the case of an E. coli protein synthesis system, for example, BL21 or the like can be used.

[0018] When the antigen polypeptide contained in the composition of the present disclosure is separated by SDS-PAGE, the ratio of the intensity of the 120 kDa band to the intensity of the 25 kDa band is 5.25 to 13.60, and / or the ratio of the intensity of the 120 kDa band to the intensity of the 78 kDa band is preferably 14.69 to 29.41. The lower limit of the numerical range of the ratio of the intensity of the 120 kDa band to the intensity of the 25 kDa band may be, for example, 6, 7, or 8. The upper limit of the range may be, for example, 11, 12, or 13. The lower limit of the numerical range of the ratio of the intensity of the 120 kDa band to the intensity of the 78 kDa band may be, for example, 15, 16, 17, 18, or 19. The upper limit of the range may be, for example, 25, 26, 27, 28, or 29.

[0019] When the antigen polypeptide contained in the composition of the present disclosure is separated by SDS-PAGE, the ratio of the intensity of the 120 kDa band to the intensity of the 96 kDa band is 10.74 to 18.20, the ratio of the intensity of the 96 kDa band to the intensity of the 25 kDa band is 0.37 to 0.93, and / or the ratio of the intensity of the 96 kDa band to the intensity of the 78 kDa band is more preferably 1.27 to 1.77. Among the above three, one may be satisfied, two may be satisfied, or all three may be satisfied. The lower limit of the numerical range of the ratio of the intensity of the 120 kDa band to the intensity of the 96 kDa band may be, for example, 11, 12, or 13. The upper limit of the range may be, for example, 16, 17, or 18. The lower limit of the numerical range of the ratio of the intensity of the 96 kDa band to the intensity of the 25 kDa band may be, for example, 0.4, 0.45, or 0.5. The upper limit of the range may be, for example, 0.8, 0.85, or 0.9. The lower limit of the numerical range of the ratio of the intensity of the 96 kDa band to the intensity of the 78 kDa band may be, for example, 1.3, 1.35, or 1.4. The upper limit of the range may be, for example, 1.65, 1.7, or 1.75.

[0020] When the antigen polypeptide contained in the composition of the present disclosure is separated by SDS-PAGE, the ratio of the intensity of the 78 kDa band to the intensity of the 25 kDa band may be, for example, 0.35 to 0.5.

[0021] SDS-PAGE can be performed according to a conventional method. More specifically, it can be performed by the method shown in the examples described later. Examples of the electrophoresis buffer include using 1×TGS and performing electrophoresis at 200 V and 3 A for 30 to 40 minutes. As the gel, gels such as 10%, 12%, 2-15%, 4-15%, or 4-20% gels can be used. Considering calculating the ratio of each band from 120 kDa to 25 kDa, a 2-15%, 4-15%, or 4-20% gradient gel is desirable from the perspective of separation, and a 4-15% gradient gel is more preferable. For example, a Mini-PROTEAN TGX gel (Biorad) can be used. As the molecular weight marker, for example, a molecular weight marker containing bands of 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa (such as Precision Plus Protein Dual Color Standards (Biorad), etc.) can be used. Among them, it is preferable to use a molecular weight marker containing bands of 150, 100, 75, and 25 kDa. The amount of protein to be subjected to electrophoresis needs to be set so that each band can be separated and a band with a small amount of protein can be detected. Therefore, the amount of protein to be electrophoresed per lane is desirably in the range of 350 - 1200 ng, more preferably 500 - 1000 ng, and most preferably 700 - 800 ng. In addition, the molecular weight indicated by each protein band in SDS-PAGE analysis is measured from the relative position to the molecular weight marker. Therefore, although the molecular weight of each protein band cannot be precisely determined, a person skilled in the art can recognize a 25 kDa band, a 78 kDa band, a 96 kDa band, and a 120 kDa band when the antigen polypeptide contained in the composition of the present disclosure is separated by SDS-PAGE.

[0022] As shown in the examples described below, the density of the band is evaluated by acquiring an image of the gel after staining the gel subjected to SDS-PAGE with a CBB staining solution for 1 hour and then decolorizing it for 1 hour and analyzing it with image analysis software. For example, it can be evaluated by ImageQuant TL (manufactured by Cytiva). The ratio of the density of each band is calculated from the ratio (%) of each band when the total density of all bands is set to 100% for each lane.

[0023] When the antigen polypeptide contained in the composition of the present disclosure is separated by SDS-PAGE, the ratio (%) of the 120 kDa band when the total density of all bands is set to 100% may be, for example, 66 - 77%. The upper or lower limit of the range may be, for example, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76%. Specifically, it may be 67 - 76%.

[0024] When the antigen polypeptide contained in the composition of the present disclosure is separated by SDS-PAGE, the ratio (%) of the 96 kDa band when the total density of all bands is set to 100% may be, for example, 3.9 - 6.05%. The upper or lower limit of the range may be, for example, 4, 4.5, 5, 5.5, or 6%. Specifically, it may be 4 - 6%.

[0025] When the antigen polypeptide contained in the composition of the present disclosure is separated by SDS-PAGE, the ratio (%) of the 78 kDa band when the total intensity of all bands is taken as 100% may be, for example, 2.45 to 4.15%. The upper or lower limit of this range may be, for example, 3, 3.5, or 4%. Specifically, it may be 3 to 4%.

[0026] When the antigen polypeptide contained in the composition of the present disclosure is separated by SDS-PAGE, the ratio (%) of the 25 kDa band when the total intensity of all bands is taken as 100% may be, for example, 5.09 to 10.31%. The upper or lower limit of this range may be, for example, 6, 7, 8, 9, or 10%. Specifically, it may be 6 to 10%.

[0027] When the antigen polypeptide contained in the composition of the present disclosure is separated by SDS-PAGE, bands of sizes other than those described above may be included.

[0028] As shown in the examples described later, since the antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 synthesized by a cell-free or in vivo protein synthesis system is a mixture containing polypeptides of a plurality of sizes, the antigen polypeptide contained in the composition of the present disclosure includes, of course, an antigen polypeptide containing a GST tag, and may also include an antigen polypeptide not containing a GST tag.

[0029] The antigen polypeptide contained in the composition 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, chemiluminescent compounds, and the like.

[0030] The total amount of the antigen polypeptide contained in the composition of the present disclosure is not particularly limited, and can be appropriately set, for example, within the limit of 100% by mass. As described above, since the antigen polypeptide contained in the composition of the present disclosure is a mixture containing polypeptides of a plurality of sizes, the "content of the antigen polypeptide contained in the composition of the present disclosure" refers to the total amount of the mixture.

[0031] The composition of the present disclosure contains the above-described antigen polypeptide and may further contain other components. Examples of such other components include pharmacologically or food hygienically acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, surfactants, antioxidants, preservatives, coating agents, colorants, fragrances, pH adjusters, and the like. These components can be used alone or in combination of two or more.

[0032] The composition of the present disclosure can be prepared by a known method or a method easily conceived from a known method. For example, it can be prepared by appropriately mixing the above-described antigen polypeptide and other components as necessary.

[0033] According to the composition of the present disclosure, as shown in the examples described later, stable antibody titer measurement results can be obtained. Furthermore, since there is little variation in the antibody titer measurement values over time, high storage stability is also expected.

[0034] The composition of the present disclosure can be suitably used as a reagent for measuring the antibody titer against periodontal pathogenic bacteria. In this specification, the reagent may be referred to as "the reagent of the present disclosure".

[0035] The reagent of the present disclosure may contain other components in addition to the composition 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 from mice, rabbits, goats, fetal calves, etc., and normal human serum components; various preservatives; surfactants; enzyme activators such as salts of metals such as zinc and magnesium, etc.

[0036] The present disclosure also encompasses a periodontal pathogen antibody titer measurement kit containing the reagent of the present disclosure. In this specification, the kit may be referred to as "the kit of the present disclosure".

[0037] In addition to the reagent of the present disclosure, the kit may optionally contain other reagents, instruments, etc. For example, it may include blocking solution, washing solution, antibody containing a labeling substance, substrate solution (chromogenic, fluorescent, luminescent), etc. Also, 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 (chromogenic, fluorescent, luminescent), etc.

[0038] 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 composition of the present disclosure. In this specification, the measurement method may be referred to as "the measurement method of the present disclosure".

[0039] 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, etc., 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 samples 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.

[0040] 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 can infect, and 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 having a disease associated with 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.) or those suspected of having such a disease may also be used. Examples of non-human mammals include mammals bred 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.

[0041] The method for collecting a biological sample from the subject is not particularly limited and 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 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.

[0042] 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., protein components such as normal human serum components; various preservatives; surfactants; enzyme activators such as salts of metals such as zinc and magnesium, etc.

[0043] According to the measurement method of the present disclosure, by bringing the composition of the present disclosure into contact 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 composition of the present disclosure (more specifically, the antigen polypeptide contained in the composition of the present disclosure).

[0044] 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 antigen polypeptide contained in the composition of the present disclosure) between the antibody against periodontal pathogenic bacteria present in the biological sample generated by bringing the biological sample into contact with the composition of the present disclosure and the composition of the present disclosure (more specifically, the antigen polypeptide contained in the composition of the present disclosure).

[0045] 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 (for example, anti-IgG antibody, etc.) containing a labeling substance and detecting the labeling substance, or when the polypeptide contained in the composition of the present disclosure contains a labeling substance, a method of detecting the labeling substance, etc.

[0046] 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.

[0047] The measurement method of the present disclosure preferably uses immunological methods such as ELISA method, CLEIA method, latex agglutination method, dot blot method, immunochromatography method, etc.

[0048] In addition, in this specification, the term “comprising” includes “consisting essentially of” and “consisting of.” Further, the present disclosure includes all arbitrary combinations of the constituent elements described in this specification.

[0049] Moreover, the various characteristics (properties, structures, functions, etc.) described for each embodiment of the present disclosure above may be combined in any manner when specifying the subject matter encompassed by the present disclosure. That is, the present disclosure includes all subject matters consisting of any combination of the combinable characteristics described in this specification.

Example

[0050] 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 are carried out under atmospheric pressure and normal temperature conditions. Also, unless otherwise specified, “%” means “mass / volume %”.

[0051] Synthesis of an antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 An antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 was synthesized by the following method. Wheat cell-free protein synthesis system The protein automatic large-scale synthesizer Protemist XE was used. A plasmid containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 inserted into the pEU vector (pEU-E01-GST-PS-MCS-N2) was used. Using the plasmid as a template, a transcription reaction was carried out at 37 °C for 20 hours to synthesize mRNA. Using the wheat germ extract WEPRO7240G (Cell Free Science Co., Ltd.), protein synthesis was carried out from the mRNA by the filter-feed method on a 10 ml scale (120 OD / ml) (26 °C, 24 hours). As a pretreatment for purification, the insoluble fraction was removed by centrifuging the synthesis reaction solution, and the supernatant was purified. Purification was carried out manually or using the AKTA explorer 10S System under the condition of 5 - 10 °C. In the case of manual operation, it was bound to glutathione resin (4 ml, 100% slurry), and the synthesized protein was eluted with 4 ml × 6 times of elution buffer. The eluates from the 2nd to 5th times were mixed and concentrated to 5 ml using an Amicon Ultra-15 tube. In the case of the AKTA explorer 10S System, an affinity purification column GSTrap 4B (5 ml column volume; GE Healthcare) was used. The synthesized protein was diluted to 10 OD / ml, bound to GSTrap, then washed with 5 CV of PBS and eluted with 5 CV of elution buffer (50 mM Tris-HCl and 10 mM glutathione (reduced), pH 8.0). The protein concentration after purification was made to be 1 mg / mL or more. The purified protein was stored frozen at -80 °C until use.

[0052] Synthesis of an antigen polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 An antigen polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 was synthesized by the following method. E. coli protein synthesis system In addition to the plasmid in which the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 was inserted into the pEU vector (pEU-E01-GST-PS-MCS-N2), a plasmid in which the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 was inserted into pGEX4T-1 was also constructed. The plasmid was transformed into Escherichia coli (BL21(DE3)pLys). This Escherichia coli was cultured with shaking in 1 mL of LB (Amp) medium at 37°C. After reaching an OD600>1.0, it was cultured with shaking in 1 L of LB (Amp) + 1% Glucose medium until the OD600 reached about 0.6 (5 - 6 hours, 37°C). After culturing, it was cooled on ice, 1 mM IPTG was added, and expression induction was carried out overnight at 18°C. The cultured solution after the expression induction treatment was dispensed, centrifuged (3500 rpm, 10 min., 4°C), the supernatant was removed, and it was suspended in 1% TritonX100 + Protease Inhibitor / PBS, and the extract was recovered by sonication. After centrifugation (13000 rpm, 20 mim., 4°C), the supernatant was recovered and used for purification as the cell extract. The cell extract was purified using a GST column. The cell extract was added to the column to bind the target protein, washed with PBS at pH 7.4, and eluted with 50 mM TrisHCl 30 mM glutathione. It was dialyzed and replaced with PBS (pH 7.4).

[0053] Five lots of antigen polypeptides synthesized by the wheat cell-free protein synthesis system were analyzed by the following method. <SDS-PAGE method> Sample adjustment The protein concentration of the synthetic antigen polypeptide solution was adjusted to a concentration of 200 ng / μl, mixed as follows, and subjected to heat denaturation at 95°C for 5 minutes. Synthetic antigen polypeptide solution 6 μL (diluted with 1×TBS) 2× Sample buffer 7.5 μL 1M DTT 1.5 μL

[0054] Electrophoresis SDS-PAGE was performed on 5 μL of the marker (Precision Plus Protein Dual Color Standards (Biorad)) and 10 μL of each sample. (Electrophoresis buffer: 1×TGS (800 mL), electrophoresis conditions: 200 V, 3 A, 30 - 40 minutes, electrophoresis gel: 4 - 15% Mini-PROTEAN TGX Gel (Biorad), protein amount per lane was 800 ng)

[0055] CBB staining After electrophoresis, the gel was immersed in Milli-Q water and washed about 3 times for 5 minutes each. Subsequently, it was immersed in CBB staining solution (Biorad) for about 1 hour (with shaking) for staining, then the gel was immersed in Milli-Q water and shaken for about 1 hour for decolorization, and analyzed using ImageQuant TL. Table 1 shows the ratio of each band when the total intensity of all bands for each lane was set to 100%. Also, the ratio between each band was calculated from the said content ratio. The results are shown together in Table 1.

[0056] ELISA method A solution for solid phase was prepared (1×Carbonate Buffer pH9.6) so that the protein concentration of the antigen became 1.0, 0.5, 0.1 μg / mL. 50 μL / well of the solid phase reagent was put into an ELISA plate (Maxisorp, Thermo) for solid phase (4°C, O / N). Blocking was performed with a blocking solution (SkimMilk PBS), and after washing, the serum of a Porphyromonas gingivalis-infected person (10,000-fold dilution, PBS + 0.02% Tween20) was put into the well for reaction. After washing, the secondary antibody (HRP-labeled anti-human IgG Fcγ, JacksonLab) was diluted 10,000-fold (0.1% skim milk + 0.02% Tween PBS) and put into the well for reaction. After washing, the substrate was added for color development, and the absorbance OD450 was measured. PBS + 0.02% Tween20 was used as the wash solution. The results are shown in Table 1. Also, for the absorbance, the relative values based on Lot.1 are shown together in Table 1.

[0057]

Table 1

[0058] Similar results were also obtained for the antigen polypeptide synthesized by the Escherichia coli protein synthesis system. From the results of SDS-PAGE, it was confirmed that in any of the synthesis systems, polypeptides shorter than the antigen polypeptide itself (120 kDa) were also contained. It was found that the short polypeptide is a fragment of the antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0059] Normally, even when the purity of the target protein varies between lots, it is possible to adjust the measured values by performing purity correction. However, regarding the antigen polypeptide used this time, it has been found that the reactivity in antibody titer measurement varies depending on each fragment contained. Even if purity correction is performed on the antigen polypeptide itself (120 kDa), the measured values cannot be adjusted. As a result, as shown in Table 1, it was found that in Lots 11 and 14, a measurement value deviation of 10% or more occurred. Also, it was found that the deviation of the measurement values is particularly significant when the amount of the antigen polypeptide used is small and the purity of the antigen polypeptide itself (120 kDa) is high.

[0060] Three lots of the antigen polypeptide synthesized by the wheat cell-free protein synthesis system were immobilized on an ELISA plate, and measurements were taken after 2 weeks. The results are shown in Table 2.

[0061]

Table 2

[0062] As shown in Table 2, it was found that in Lot 11, the change rate of the measurement results after 2 weeks of storage was also large.

Claims

1. A composition containing an antigen polypeptide synthesized by a cell-free or in vivo protein synthesis system, which consists of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, when the antigen polypeptide is separated by SDS-PAGE, the ratio of the intensity of the 120 kDa band to the intensity of the 25 kDa band is 5.25 to 13.60, and / or the ratio of the intensity of the 120 kDa band to the intensity of the 78 kDa band is 14.69 to 29.

41.

2. A composition containing an antigen polypeptide synthesized by a cell-free or in vivo protein synthesis system, which consists of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, when the antigen polypeptide is separated by SDS-PAGE, the ratio of the intensity of the 120 kDa band to the intensity of the 96 kDa band is 10.74 to 18.20, the ratio of the intensity of the 96 kDa band to the intensity of the 25 kDa band is 0.37 to 0.93, and / or the ratio of the intensity of the 96 kDa band to the intensity of the 78 kDa band is 1.27 to 1.

77.

3. The composition according to claim 1 or 2, wherein the cell-free protein synthesis system is a wheat cell-free protein synthesis system.

4. The composition according to claim 1 or 2, wherein the in vivo protein synthesis system is an Escherichia coli protein synthesis system.

5. The composition according to claim 1 or 2, which is a reagent for measuring the antibody titer against periodontal pathogens.

6. A kit for measuring the antibody titer against periodontal pathogens, comprising the reagent for measuring the antibody titer against periodontal pathogens according to claim 5.

7. A method for measuring the antibody titer against periodontal pathogens in a biological sample, comprising contacting the biological sample with the composition according to claim 1 or 2. ​

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