JPH0848695A - Peptides containing sequence of porphyromonas gingivalis type ii pilus protein and their uses - Google Patents

Abstract

PURPOSE:To obtain the subject new peptides for producing a composition for diagnosis of periodontal diseases, preventive vaccine and an antibody for preventing and treating periodontal disease and being a subunit protein fragment constituting type II pilus of Porphyromonas gingivalis. CONSTITUTION:These new peptides are peptides corresponding to a fragment derived from an amino acid sequence of 72KDa subunit protein constituting type II pilus of Porphyromonas gingivalis and comprising a fragment containing continuing 5-10C amino acid residues expressed by formulas I to III, etc., and are useful for producing a composition for diagnosis of periodontal disease, vaccine for preventing periodontal disease and an antibody for preventing and treating periodontal disease. These peptides are obtained by synthesizing by a liquid phase or solid phase synthetic method of peptides from the amino sequence of 72KDa subunit protein fragment derived from Porphyromonas gingivalis 0MZ409 strain.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to Porphyromonas ging iv.
alis) type II pili and peptides corresponding to fragments derived from the amino acid sequence of the 72KD _a subunit protein constituting the type II pili and their use. More specifically, the present invention relates to peptides acting as an antigen that undergoes an antigen-antibody reaction with an antibody against the above 72KD _a subunit protein, and serum of the peptides for periodontal disease patients,
The present invention relates to the detection of specific antibodies and the like in saliva and gingival crevicular fluid and their use as preventive and therapeutic agents for periodontal disease.

[0002]

2. Description of the Related Art Periodontal disease is divided into gingivitis and periodontitis.
Further, periodontitis includes adult periodontitis, localized juvenile periodontitis, etc., but in reality, 90% or more of periodontitis is occupied by adult periodontitis. These periodontal diseases include gingival inflammation, bleeding, drainage, periodontal pocket formation, periodontal ligament destruction,
It is a disease that causes resorption of alveolar bone and shaking and loss of teeth.
And, although various bacteria exist at the focal site of these periodontal diseases, among them, Porphyromonas gingivalis is the main periodontopathic bacterium, especially as the causative bacterium of adult periodontitis. Has been done. A significant increase in the bacterium is observed locally in the lesions of periodontal disease.

At present, a treatment method for periodontal disease has not been completely established, and it is most important to detect the periodontal disease as early as possible, to accurately grasp the pathological condition thereof, and to take appropriate measures. The development of a preventive method by developing a periodontal disease vaccine is awaited.

[0004] First, regarding the diagnosis of periodontal disease, it is expected to provide a diagnostic method for discovering the periodontal disease as early as possible and accurately grasping its pathological condition, but in reality, it is a reliable periodontal disease. Diagnostic agents have not yet been developed. However, dare to list them, attention is focused on periodontopathic bacteria, and various methods for knowing the presence or absence of periodontopathic bacteria and the amount thereof have been proposed as means for diagnosing periodontal disease. .
Further, a method has also been proposed in which a specific antibody against periodontal pathogenic bacteria is measured and used for diagnosis of periodontal disease. Furthermore,
A method by measuring inflammatory products in the gingival crevicular fluid of patients with periodontal disease has also been proposed.

Various methods for knowing the presence or absence of periodontopathic bacteria and the amount of periodontal pathogens include, for example, periodontopathogenicity inhabiting dental plaque (plaque), gingival crevicular fluid and saliva of patients with periodontal disease. A method for detecting periodontopathic bacteria has been performed by culturing bacteria under anaerobic conditions using blood agar medium and examining detailed biochemical properties of various colonies obtained.

Under a microscope, Gram stains of periodontopathic bacteria inhabiting plaque, gingival crevicular fluid and saliva of patients with periodontal disease are observed by gram staining and examined by a dark field microscope. A method for detecting periodontopathic bacteria by combining an antibody against the periodontopathic bacteria and a fluorescent dye has also been performed.

[0007] Further, by using an immunological method such as an enzyme antibody method (ELISA method), plaque of a periodontal patient,
A method for detecting periodontopathic bacteria inhabiting gingival crevicular fluid, saliva and the like with an antibody against each periodontopathic bacterium has been performed.

Further, paying attention to an enzyme produced by periodontopathic bacteria, and using the enzyme activity as an index, the periodontopathogenicity inhabiting dental plaque (plaque), gingival crevicular fluid, saliva and the like of patients with periodontal disease Methods for detecting bacteria have also been practiced.

Recently, attention has been paid to the genes of periodontopathic bacteria, and the DNA probe method is used to detect periodontopathic bacteria which live in plaque, gingival crevicular fluid, saliva and the like of patients with periodontal disease. The method of detecting is also performed.

In addition, a method for measuring inflammatory products is also used as one of the methods for diagnosing periodontal disease.

However, the above-described method for diagnosing periodontal disease, which knows the presence or absence of periodontopathic bacteria and the amount of bacteria, is only for diagnosing the etiology and is not sufficient for grasping the pathological condition of the patient. is there. As a pair with this, as a method for diagnosing periodontal disease to know the response to the periodontopathic bacteria on the patient side as a host, and to grasp the pathological condition of the patient, a specific antibody against periodontopathic bacteria, A method of diagnosing periodontal disease that knows the class and subclass is considered. Of course, it is also important to have a method for diagnosing periodontal disease that knows the presence or absence of periodontopathic bacteria and the amount of it. It is said that it is important to perform an appropriate diagnosis of periodontal disease by combining both methods. There is.

[0012] As one of such diagnostic methods, the specific antibody titer against periodontopathic bacteria in patients with adult periodontitis is determined by EL.
A method for diagnosing periodontal disease in individual patients by combining it with a bacterial test, which is measured by the ISA method, has been proposed (Mouto
n, C.I. , Et al: Infect. Immun . Three
1 , 182-192, 1981. JP-A-61-162
753, JP-A-2-107969).

Recently, not only the specific antibody against periodontal pathogenic bacteria in periodontal disease patients has risen, but also the subclass of the specific antibody changes from IgG1 to IgG4 with the change in the condition of periodontal disease. The method for diagnosing periodontal disease may be effective, and detects antibody-producing cells against periodontopathic bacteria in gingival tissues of patients with adult periodontitis E
Proven by LISPOT method (Ogawa,
T. et al: Clin. Exp. Immunol .
76 , 103-110, 1989. ).

An antigen used for detection is required when investigating an increase in a specific antibody against periodontopathic bacteria in a patient with periodontal disease and further a change in its class or subclass. In the above-mentioned conventional method, the periodontopathic bacteria as a whole or the cell surface components thereof, such as lipopolysaccharides and fimbriae, have been used as antigens. However, it is difficult to obtain a large amount of a pure product by separating and purifying it, although some results can be obtained, and there are many contamination reactions. It is not preferable to examine the increase of the specific antibody against, and further the variation of its class or subclass.

Therefore, the present inventors have investigated the increase in specific antibodies against periodontopathic bacteria in patients with periodontal disease as well as the change in the class or subclass thereof, using Porphyromonas gingivalis rays as an antigen used for detection. Make up hair 4
We found that peptides corresponding to fragments derived from the amino acid sequence of 1KD _a subunit protein were effective and filed a patent application (see Japanese Patent Application No. 5-264140). However, as a result of the recent studies by the inventors, it was found that in Porphyromonas gingivalis, in addition to fimbriae composed of 41KD _a subunit protein (type I fimbriae), 72KD
Pili composed of _a subunit protein (type II pili)
It became clear that there exist. Therefore, when investigating the rise of specific antibodies against periodontopathic bacteria in patients with periodontal disease, and further the variation of their class or subclass, accurate diagnosis of periodontal disease is made by using these two types of pili. It is possible that you can do.

Therefore, in the case of examining the increase in specific antibodies against periodontopathic bacteria of periodontal disease patients and further the variation of their class or subclass, Porphyromonas gingivalis type I pili is used as an antigen for detection. Composing 41
In addition to peptides corresponding to the fragment derived from the amino acid sequence of the KD _a subunit protein, a fragment derived from the amino acid sequence of the 72 KD _a subunit protein that constitutes another type II pilus of Porphyromonas gingivalis It is considered to be more effective if peptides corresponding to can be used.

[0017]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide peptides and peptides that can effectively detect an increase in specific antibodies against periodontopathic bacteria in patients with periodontal disease, as well as changes in their class or subclass. The present invention provides a composition for diagnosing periodontal disease, which comprises the same. Further, an object of the present invention is to provide a periodontal disease preventive vaccine comprising such peptides and an oral cavity for periodontal disease prevention or periodontal disease treatment comprising an antibody obtained by immunizing an animal with these peptides. To provide a composition.

[0018]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have responded to various fragments corresponding to the fragments derived from the amino acid sequence of 72KD _a subunit protein constituting type II pili of Porphyromonas gingivalis. When a synthetic peptide was studied for antigen-antibody reactivity with an antibody against the protein, it was surprisingly found that a peptide containing 5 to 10 amino acid residues has very excellent properties.

Therefore, according to the present invention, Porphyromonas gi ( Porphyromonas gi)
N. givalis ) type II pili of 72K
A peptide corresponding to a fragment derived from the amino acid sequence of the D _a subunit protein, wherein the fragment is selected from the group of fragments containing 5 to 10 consecutive amino acid residues, or a peptide thereof The above problems can be solved by providing a derivative or a salt thereof.

According to a preferred embodiment of the present invention, the fragment has the following amino acid sequence (SEQ ID NO: 1 to SEQ ID NO: 1 in the sequence listing).
20) are provided, wherein said peptide or derivative thereof or a salt thereof is selected from the group of fragments comprising at least 5 consecutive amino acid residues. Ala Gly Asp Gly Gln Asp Gln Ala Asn Pro (SEQ ID NO: 1); Thr Ile Asp Pro Ala Thr His Asn Ala Ile (SEQ ID NO: 2); Thr His Asn Ala Ile Leu Lys Pro Lys Lys (SEQ ID NO: 3); Leu Lys Pro Lys Lys Gly Ile Lys Val Asn (SEQ ID NO: 4); Gly Ile Lys Val Asn Ser Ala Val Gly Lys (SEQ ID NO: 5); Ser Ala Val Gly Lys Thr Val Lys Val Tyr (SEQ ID NO: 6); Asn Val Asn Ala Ala Asp Phe Asp Ala Lys (SEQ ID NO: 7); Glu Leu Ser Thr Gln Ala Gln Ala Leu Gly (SEQ ID NO: 8); Ala Gln Ala Leu Gly Thr Val Ala Asp Gly (SEQ ID NO: 9); Pro Asn Pro Ala Thr Ala Ala Gly Lys Ile (SEQ ID NO: 10); Ala Ala Gly Lys Ile Ala Lys Lys Asn Gly (SEQ ID NO: 11); Arg Ala Met Val Ser Thr Lys Ala Gln Ser (SEQ ID NO: 12); Thr Lys Ala Gln Ser Tyr Glu Ile Lys Ala (SEQ ID NO: 13); His Lys Ser Gly Ala Asn Ala Ala Ser Ser (SEQ ID NO: 14); Ser Met Lys Ser Val Thr Asp Pro Lys Val (SEQ ID NO: 15); Thr Asp Pro Lys Val Gly Gly Val Ala Gly (SEQ ID NO: 16); Gly Gly Val Ala Gly Met Lys Ala His Lys (SEQ ID NO: 17); Met Lys Ala His Lys Tyr Val Lys Gly Lys (SEQ ID NO: 18); His Ile Lys Ser Ile Lys Lys Leu Gly Phe (SEQ ID NO: 19); and Asp Pro Asn Pro Asp Glu Pro Gly Thr Pro (SEQ ID NO: 20).

[0021] According to another embodiment of the present invention, the fragment has the above-mentioned amino acid sequence (SEQ ID NOS: 1 to 20 in the sequence listing).
One or two of the above peptides or derivatives thereof or salts thereof selected from the group of fragments comprising at least 5 consecutive amino acid residues of
Provided is a composition for diagnosing periodontal disease, which comprises one or more species.

According to another aspect of the present invention,
The peptide or its derivative selected from the group of fragments in which the fragment comprises at least 5 consecutive amino acid residues of the above amino acid sequence (see SEQ ID NOS: 1 to 20), or a salt thereof. There is provided a periodontal disease preventive vaccine containing one or more of the above. By using such a vaccine, an antibody against them can protect against infection of Porphyromonas gingivalis. As a result, suppression of production of inflammatory cytokines, inhibition of osteoclast activation, etc. can be expected.

Furthermore, according to another aspect of the present invention, the fragment comprises at least 5 consecutive amino acid residues of the above amino acid sequence (see SEQ ID NOS: 1 to 20). Oral composition for preventing periodontal disease or treating periodontal disease, which comprises an antibody obtained by immunizing an animal with one or more of the above peptides or derivatives thereof selected from the group consisting of Things are offered. By using such a composition, the antibody contained therein can protect against infection of Porphyromonas gingivalis. As a result, suppression of inflammatory cytokine production, inhibition of osteoclast activation, etc. can be expected.

In addition, from the viewpoint that it contributes to more accurate diagnosis of periodontal disease by investigating changes in the class and subclass of antibodies against periodontal pathogenic bacteria, the peptide of the present invention or its derivative or salt thereof. And 41KD constituting the type I pili of Porphyromonas gingivalis
There is also provided a composition for diagnosing periodontal disease, which comprises peptides corresponding to a fragment derived from the _a subunit protein in combination with each other.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION 72 KD _a constituting type II pili of Porphyromonas gingivalis referred to in the present invention
The amino acid sequence of the subunit protein is disclosed in the specification of Japanese Patent Application No. 6-81074 previously filed by the present inventors, and specifically as shown in SEQ ID NO: 31.

Therefore, the peptides of the present invention include 5 to 5 peptides corresponding to the fragments derived from the amino acid sequences.
A peptide comprising 10 amino acid residues,
All that are suitable for the purposes of the present invention are included.
Among these peptides, those having a highly antigenic region when reacted with serum of periodontal disease patients are particularly preferable for the purpose of the present invention, and specific examples thereof include SEQ ID NOS: 1 to 3 above. Mention may be made of peptides of various chain lengths comprising at least 5 consecutive amino acid residues of 20 amino acid sequences.

These peptides having various chain lengths can be used as the antigen of the present invention because they have a minimum of 5 amino acid residues, and those having a maximum of 10 amino acid residues can also be used. Those having the above can generally be advantageously used for reducing non-specific reactivity with impurities contained in a measurement sample other than the target antibody. But,
Unless it causes such non-specific reaction, it may further contain residues including other amino acid residues or fragments derived from type II pili. Therefore, as a concrete example of the peptide of the present invention, the above-mentioned SEQ ID NO: 1 is used.
Contiguous 5, 6 and 7 of ~ 20 amino acid sequences
Mention may be made of peptides comprising 1, 8, 9 or 10 amino acid residues. For example, among those having the above-mentioned 5 amino acid residues, an example of a preferred specific peptide is that shown by SEQ ID NO: 30 in the sequence listing below.

Ala Gly Asp Gly Gln (SEQ ID NO: 30) The derivative according to the present invention refers to the above-mentioned peptide to which a protecting group, a functional group, a spacer, a carrier and the like are bound. More specifically, the N-terminal of the peptide of the present invention is protected with an acetyl group or a urethane group, or the C-terminal is protected with an amide group or an ester group.
It is an example of a derivative having a protecting group. Further, when chemically bonding a peptide directly or indirectly to a solid phase or a carrier, the amino group or carboxyl group of the peptide itself can be used, but in order to utilize a phenol group or a sulfhydryl group, the peptide of the present invention can be used. Those into which tyrosine or cysteine is introduced are also included in the derivative of the present invention. Furthermore, when an antibody binds to the peptide of the present invention, a spacer can be introduced for the purpose of making it less susceptible to steric hindrance, and such a spacer is also included in the above derivatives. Specific examples of such spacers include:
α, ω-diaminoalkane (n = 2 to 12); α-amino-ω-carboxyalkane (n = 2 to 10); 3,3
-Diaminodipropylamine; succinyl-3,3
-Diaminodipropylamine; m-aminophenol conjugated with bisdiazobenzidine; Gly-Gly-Gl
y; Gly-Gly-Tyr; succinyl-1,3-diaminopropan-2-ol; polylysine and the like.

The carrier-bound derivative of the present invention includes
Lysines commonly used with the above spacer, those bound with various polymers or bovine serum albumin, etc., and bovine serum albumin, tetanus toxoid, ovalbumin, key used when a peptide is used as an immunogen.
Whole limpet hemocyanin, thyroglobulin, γ
-Including those to which globulins, polysaccharides and the like are bound.

The salt of the above-mentioned peptide or derivative is hydrochloric acid, sulfuric acid, phosphoric acid or pyrophosphoric acid which has a salt-forming effect with the peptide or its derivative when the peptide itself or its derivative has a primary, secondary or tertiary amino group. Salts with inorganic acids such as acetic acid, lactic acid, palmitic acid, stearic acid, propionic acid, citric acid, tartaric acid, malic acid, ascorbic acid, oxalic acid, methanesulfonic acid, benzenesulfonic acid, p-
Examples thereof include acid addition salts such as salts with organic acids such as toluenesulfonic acid.

On the other hand, when the peptide itself or a derivative thereof has a carboxyl group or a sulfonyl group, alkali metal salts such as sodium salt and potassium salt thereof, alkaline earth metal salts such as calcium salt and magnesium salt, and ammonium salt thereof. And salts such as triethylamine salt.

The peptide of the present invention is Porphyromonas
Can also be obtained by limited proteolysis of 72 kD _a subunit protein constituting the type II pili gingivalis, it is advantageous according to the per se known peptide synthesis methods. Examples of such peptide synthesis methods include chemical synthesis methods [solid phase synthesis method, liquid phase synthesis method (edited by the Chemical Society of Japan, Experimental Chemistry Course 22, Organic Synthesis IV, pages 258-309, etc., reference, 1992]. November 30, published by Maruzen Co., Ltd.)] and genetic engineering techniques.

In this case, MULTI-PIN PEP
TIDE SYNTHESIS KIT (CHIRON
MIMOTOPES PTY LTD: Austra
lia) and Model 9050 Peptides
synthesizer (Millipore corp
oration) and the like.

Derivatives from the peptides thus obtained can be obtained by chemical modification means commonly used in the technical field of peptide chemistry. For example, when the carrier is chemically bonded to the peptide, a method using glutaraldehyde, water-soluble carbodiimide, succinimide or the like can be used.

In order to detect a specific antibody in a patient with periodontal disease, one kind or a combination of two or more kinds of the above-mentioned peptides or derivatives thereof or salts thereof can be used. Known immunological means can be used for this detection, and examples of these include polystyrene plates, glass filters, magnetic particles, latex, etc. as a solid phase, and labeled secondary antibodies with periodontal disease patients. ELISA method, RIA method, fluorescent antibody method, chemiluminescent antibody method, etc. for detecting the specific antibody of A., immunoagglutination method using sheep red blood cells / latex as a solid phase, immunoturbidimetric method using latex as a solid phase, and A Western blotting method using a nitrocellulose membrane or the like as a solid phase can be mentioned. Various well-known methods can be used for immobilizing the peptides, their derivatives and salts thereof on a solid phase, including physically adsorbing methods and chemically immobilizing using glutaraldehyde or the like. To be

In order to immobilize the antibody of periodontal disease patients on a solid phase, an antibody against human antibody can be used, or after being physically adsorbed, various immunological techniques using labeled peptides can be used.

In the composition for diagnosing periodontal disease of the present invention, when used in combination with the peptides of the present invention, as peptides corresponding to the fragments derived from the 41KD _a subunit protein constituting the type I pili, Is a peptide selected from a fragment group consisting of at least 5 consecutive amino acid residues of the amino acid sequences shown in SEQ ID NOs: 21 to 29 disclosed in Japanese Patent Application No. 5-264140 as described above. Alternatively, a derivative thereof or a salt thereof can be mentioned. The term “derivative” or “salt” used herein has the same meaning as described for the peptides related to type II pili.

Glu Asn Ala Thr Lys Val Glu Asp Ile Lys (SEQ ID NO: 21) Glu Val Lys Ala Leu Thr Thr Glu Leu Thr (SEQ ID NO: 22) Ala Glu Asn Gln Glu Ala Ala Gly Leu Ile (SEQ ID NO: 23) Ala Ala Gly Leu Ile Met Thr Ala Glu Pro (SEQ ID NO: 24) Thr Gly Ser Leu Thr Thr Phe Asn Gly Ala (SEQ ID NO: 25) Thr Phe Asn Gly Ala Tyr Thr Pro Ala Asn (SEQ ID NO: 26) Gly Phe Tyr Val Leu Glu Asn Asp Tyr Ser (SEQ ID NO: 27) Ala Asn Gly Gly Thr Ile His Pro Thr Ile (SEQ ID NO: 28) Glu Gly Lys Thr Tyr Tyr Pro Val Leu Val (SEQ ID NO: 29) In this case, each peptide or its derivative as described above. Alternatively, salts thereof may be separately prepared to detect a specific antibody of a periodontal disease patient. However, when the peptide is prepared, it is prepared as one peptide regardless of whether it is mediated by a spacer or the like. Specific antibody of periodontal disease patient by derivatizing like You may detect.

When the peptide of the present invention is used as a periodontal disease preventive vaccine, it can be adsorbed to a carrier protein such as bovine serum albumin or tetanus toxoid. In addition, it can be orally administered with an adjuvant such as aluminum hydroxide, alum, and a synthetic adjuvant such as muramyldipeptide, or can be locally administered parenterally. For parenteral topical administration, for example,
Subcutaneous, intracutaneous, intramuscular, intravenous, nasal, etc. are also acceptable, but periodontal disease is known to have an immune response in a limited part of the gingival region, so that it can also be administered intragingivally. As an adjuvant, aluminum hydroxide gel is preferably added so that the final concentration of aluminum is 0.05 to 0.2 mg / ml. The content of the peptide in the vaccine to be administered once is suitably 0.004-2.5 mg, preferably 0.02-0.6 mg. It is preferable to add 0.01% (w / v) of thimerosal as a preservative.

Further, instead of directly using the above-mentioned peptide as a vaccine, an oral composition containing a specific antibody contained in serum, milk or egg obtained by immunizing bovine, chicken or the like is used for preventing or treating periodontal disease. It can also be used.

As a conventional method for immunizing a mammal with the peptide of the present invention, for example, a complex obtained by binding the peptide to a carrier such as bovine serum albumin or tetanus toxoid is Freund's complete adjuvant or water. It may be administered to an organism together with an adjuvant such as aluminum oxide.

As the mammal to be immunized, goat, sheep, horse, cow and the like are used. When an antiserum or milk containing a specific antibody obtained by immunizing a mammal with a complex of the peptide and a carrier is used, several kinds of antiserum or milk containing the specific antibody may be mixed.

The preparation of an antibody against the above-mentioned peptide by hen's egg is carried out by, for example, adding a complex obtained by binding the peptide to a carrier such as bovine serum albumin or tetanus toxoid to a chicken together with an adjuvant such as Freund's complete adjuvant or aluminum hydroxide. ~ Immunized twice, from 1-2 months later,
The specific antibody may be prepared by collecting eggs, separating the yolk, adding phosphate buffered saline and chloroform and stirring, and then separating the water-soluble portion of the upper layer.

The antibody against the peptide prepared according to the present invention can be formulated as an emulsion, dispersion, paste, gel, or aerosol. Specifically, it can be blended in substances such as toothpaste, chewing gum, mouthwash and mouthwash.

Further, the peptide of the present invention can be used for producing a specific antibody, and such a specific antibody can be used for passive immunization as a means for diagnosing periodontal disease or as a preventive measure against Porphyromonas gingivalis infection.

Furthermore, the peptides of the present invention can be used for the production of polyclonal or monoclonal antibodies. For obtaining the polyclonal antibody, any of commonly used large and small animals may be used. After immunizing the host animal with the peptide, the antibody can be recovered by a known method. On the other hand, in order to obtain a monoclonal antibody, it can be obtained by immunizing a mouse with the peptide and fusing its B lymphocytes with myeloma cells.

[0047]

EXAMPLES The present invention will be specifically described below with reference to examples.

Example 1 MULTI-PIN PEPT
Partial Pep with IDE SYNTHESIS KIT
Cido Synthesis CHIRON MIMOTOPES PTY LTD
(Australia) MULTI-PIN P
EPTIDE SYNTHESIS KIT (Non-
Clearable Type: Code No. 59
3-28201) using, based on the deduced amino acid sequence of type II pili consisting of 72 kD _a subunit protein from P. gingivalis OMZ409 strain (see Japanese Patent Application No. Hei 6-81074), 10 amino acids Peptides consisting of residues were synthesized every five N-terminal side.

First, after immersing the tip of the pin in dimethylformamide (DMF) for 2 minutes, methanol (MeO) was added.
H), shaken 3 times for 10 minutes and washed. After air-drying for 30 minutes, soak in 20% piperidine / DMF for 30 minutes,
Deprotected. Then, after soaking in DMF for 10 minutes, MeO
It was washed by shaking in H for 4 minutes for 2 minutes. After air-drying for 30 minutes, soaking in DMF for 2 minutes, then soaking in each Fmoc-amino acid solution (60 mM) / DMF + 120 mM 1-hydroxybenzotriazole monohydrate (HoBt), reacting at room temperature for 16 hours, and The amino acids were coupled. From the next day, the operation of coupling the next amino acid was repeated in the same manner.

Therefore, when the coupling of up to 10 amino acid residues of interest was completed, it was immersed in DMF for 2 minutes, then shaken in MeOH for 10 minutes 3 times and washed. Three
After air-drying for 0 minutes, it was deprotected by soaking in 20% piperidine / DMF for 30 minutes. It was then soaked in DMF for 10 minutes, then shaken 4 times in MeOH for 2 minutes and washed. 30
After air-drying for a minute, it was immersed in a mixed solution of DMF: acetic anhydride: triethylamine = 50: 5: 1 for 90 minutes, acetylated, and then shaken in MeOH for 2 minutes for washing. After air-drying for 15 minutes, it was immersed in a mixed solution of trifluoroacetic acid: ethanedithiol: thioanisole = 95: 2.5: 2.5 for 60 minutes for deprotection. After air-drying for 10 minutes, ultrasonic cleaning was performed for 15 minutes, and further air-drying was performed for 10 minutes.

Test Example 1 Epitope using serum from patients with periodontal disease
Elucidation of the epitope The epitope was elucidated by the ELISA method using the peptide synthesized in Example 1 and the serum of the periodontitis patient.

First, the tip of the pin to which the peptide is bound is soaked in 2% bovine serum albumin (BSA) / phosphate buffered saline (pH 7.4) + Tween 20 and blocked at room temperature for 1 hour, and then phosphate buffered. Saline (p
(H7.4) + Tween 20 for 5 minutes, and shaken 3 times for washing. Next, as a primary antibody, a type II pilus of Porphyromonas gingivalis was preliminarily subjected to an absorption operation and either an unabsorbed healthy person or periodontal disease patient serum was prepared, and this was used as 2% BSA / It was diluted 10 ⁵ times with phosphate buffered saline (pH 7.4) + Tween 20 and reacted at 4 ° C. for 16 hours. After washing with phosphate buffered saline (pH 7.4) + Tween 20 for 5 minutes by shaking three times, alkaline phosphatase-labeled anti-human IgG antibody (10 ³ -fold dilution) /0.1 was used as a secondary antibody.
% Casein / phosphate buffered saline (pH 7.4) + T
ween 20 was reacted at 37 ° C. for 3 hours. 5 minutes with phosphate buffered saline (pH 7.4) + Tween 20
After washing by shaking 3 times, the mixture was incubated at 37 ° C. in p -nitrophenyl phosphate / diethanolamine buffer.
Were reacted for 3 hours, the absorbance (405 nm) was measured, and the one having a difference in absorbance before and after the absorption operation of 0.2 or more was selected.
In addition, these peptides were used in 0.1M phosphate buffer (p
H7.2) + 1% sodium dodecyl sulfate + 0.1% 2
Ultrasonic cleaning in mercaptoethanol (55 to 65 ° C.) for 10 minutes, shaking with distilled water (60 ° C.) twice for 30 seconds, and further shaking and washing for 30 minutes. Next, Me
Boil in OH for 15 seconds, air dry for 15 minutes, and then
It was subjected to the ISA method.

From the above results, a unique epitope that reacts with the serum of periodontal patients was found. The results are shown in Table 1.

Table 1 Unique epitopes that react with serum of periodontal patients ──────────────────────────── Amino acid sequence Abbreviation ─── ───────────────────────── Ala Gly Asp Gly Gln Asp Gln Ala Asn Pro Peptide 1 Thr Ile Asp Pro Ala Thr His Asn Ala Ile Peptide 2 Thr His Asn Ala Ile Leu Lys Pro Lys Lys Peptide 3 Leu Lys Pro Lys Lys Gly Ile Lys Val Asn Peptide 4 Gly Ile Lys Val Asn Ser Ala Val Gly Lys Peptide 5 Ser Ala Val Gly Lys Thr Val Lys Val Tyr Peptide 6 Asn Val Asn Ala Ala Asp Phe Asp Ala Lys Peptide 7 Glu Leu Ser Thr Gln Ala Gln Ala Leu Gly Peptide 8 Ala Gln Ala Leu Gly Thr Val Ala Asp Gly Peptide 9 Pro Asn Pro Ala Thr Ala Ala Gly Lys Ile Peptide 10 Ala Ala Gly Lys Ile Ala Lys Lys Asn Gly Peptide 11 Arg Ala Met Val Ser Thr Lys Ala Gln Ser Peptide 12 Thr Lys Ala Gln Ser Tyr Glu Ile Lys Ala peptide 13 His Lys Ser Gly Ala Asn Ala Ala Ser Ser peptide 14 Ser Met Lys Ser Val Thr Asp Pro Lys Val peptide 15 Thr Asp Pro Lys Val Gly Gly Val Ala Gly peptide 16 Gly Gly Val Ala Gly Met Lys Ala His Lys peptide 17 Met Lys Ala His Lys Tyr Val Lys Gly Lys Peptide 18 His Ile Lys Ser Ile Lys Lys Leu Gly Phe Peptide 19 Asp Pro Asn Pro Asp Glu Pro Gly Thr Pro Peptide 20 ────────────── ─────────────── Example 2 Peptide Synthesis Test Using Peptide Synthesizer The unique epitope found in Example 1 that reacts with the serum of periodontal disease patients was analyzed by B. Merrifield ( JA
m. Chem. Soc . 85 , 2149-2154, 1
963. ) According to the solid-phase method of), Model 9050
Peptidesynthesizer (Millip
ore corporation).
Purification was performed by reverse phase HPLC on Capcell Pak C.
Using an 18SG120 (1.5 × 15 cm: Shiseido) column, the peptide fraction of the main peak was collected by a gradient of acetonitrile concentration (6 to 60%). The purified peptide was analyzed by quantitative amino acid analysis to determine its composition and M
odel 6400/6600 Protein se
The amino acid sequences were confirmed by automatic Edman degradation using a quencer (Japan Millipore), respectively.

Test Example 2 Blood of periodontal disease patient using peptide
1. Determination of specific antibody of Sein 1 mg of each peptide synthesized in Example 2 was dissolved in 1 ml of 0.1 M phosphate buffer (pH 6.0) to prepare sulfated SMCC.
5 mg (manufactured by Pierce) was added, and the mixture was incubated at 30 ° C. for 30 minutes for maleimidation and then subjected to Sephadex G-10 column chromatography to collect the first elution fraction. In addition, 7 mg of bovine serum albumin (BSA: manufactured by Wako Pure Chemical Industries, Ltd.) was added to 0.1 M phosphate buffer (pH 6.0).
It was dissolved in 1 ml of +10 mM EDTA, mixed with the above maleimidated peptide, and reacted at 30 ° C. for 1 hour. The resulting peptide-modified BSA was subjected to Sephadex G-50 column chromatography, and the first elution fractions were collected and used as an antigen solution for plate preparation.

On the other hand, as a control, 6 kinds of peptides were synthesized as in Example 2, and an antigen solution for plate preparation was prepared in the same manner.

These antigen solutions were diluted with 0.1 M carbonate buffer (pH 9.6) and 150 mM NaCl, and diluted to 100
ml. 100 μl / well was added to a polystyrene microtiter plate (Immunoplate; Maxisorp type: Nunc), and the mixture was allowed to stand overnight at 4 ° C., and then an automatic microtiter washing machine (hereinafter referred to as a washing machine; manufactured by Bio-Rad) Each well was washed 3 times with 200 μl of phosphate buffered saline (pH 7.4). Phosphate buffered saline containing 0.1% gelatin (manufactured by Nitta Gelatin Co., Ltd.) (p
H7.4) was added at 250 μl / well and left at 4 ° C. overnight to block the plate. The blocking solution was removed by suction, the plate was dried by applying a vacuum dryer, put in an aluminum bag, sealed, and stored at 4 ° C. for use in the following measurements.

Periodontal disease patients (n = 3) and healthy subjects (n =)
Using the serum of 3) as a sample, each serum was diluted 100-fold with phosphate buffered saline (pH 7.4) containing 10% bovine serum. 100 μl of this diluted solution on each antigen coated plate
Add and incubate at 37 ° C. for 1 hour. Using a washing machine, 200 μl of phosphate buffered saline (pH 7.4)
After washing three times with + Tween 20, a commercially available peroxidase-labeled anti-human IgG antibody (diluted 10 ³ ) /0.1% casein / phosphate buffered saline (p) was used as a secondary antibody.
H7.4) + Tween 20 (100 μl) and add 3 again
Incubated at 7 ° C for 1 hour. After washing with a washing machine in the same manner, 100 μl of an o -phenylenediamine solution was added and incubated at 37 ° C. for 30 minutes, 100 μl of 0.1N sulfuric acid was added to stop the reaction, and a reaction well without a sample was used as a control. Absorbance (405 nm) was measured with a reader (manufactured by Bio-Rad). Table 2 shows the measurement results using various antigen plates. In addition, the measured value is shown by the average absorbance of the periodontal disease patient group and the healthy subject group, each of 3 samples.

Among the peptides used, those having an amino acid sequence shown in Table 1 showed specific reactivity with the periodontal disease patient group. Therefore, by using these peptides as an antigen, it is possible to detect the anti-Porphyromonas gingivalis pili antibody contained in the serum of periodontal disease patients, and it was found to be useful for screening periodontal disease patients. did.

Table 2 Antibody titer measurement of periodontal disease patient group serum and healthy subject group serum using various peptide-coated plates ──────────────────────── ──────────── Coat peptide type Periodontitis patient group Healthy group ──────────────────────────── --Peptide of the present invention Peptide 1 0.199 0.005 Peptide 2 0.224 0.009 Peptide 3 0.335 0.011 Peptide 4 0.342 0.010 Peptide 5 0.317 0 .008 Peptide 6 0.194 0.006 Peptide 7 0.233 0.008 Peptide 8 0.206 0.004 Peptide 9 0.172 0.003 Peptide 10 0.238 0.006 Peptide 11 0.267 0.002 Peptide 12 .265 0.009 peptide 13 0.239 0.001 peptide 14 0.278 0.003 peptide 15 0.329 0.005 peptide 16 0.168 0.001 peptide 17 0.315 0.008 peptide 18 0.411 0.006 Peptide 19 0.243 0.005 Peptide 20 0.223 0.009 Comparative peptide Glu Ile Ala Ala Gly Ser Val Leu Ala Thr 0.005 0.001 Tyr Asp Tyr Ala Gly Leu Trp Glu Asp His 0. 003 0.013 Leu Trp Glu Asp His Asn Thr Asn Glu Ala 0.011 0.012 Gln Asp Val Thr Gly Glu Leu Ala Asn Ala 0.011 0.011 Phe Val Gly Glu Asn Gly Gln Phe Tyr Val 0.001 0 001 Asp Gln Asp Thr Phe Met Ser Val Glu Val 0.006 0.002 ───────────────────────────────── ─── Trial Experimental Example 3 Periodontal disease using peptides derived from two types of fimbriae
Measurement of specific antibody in patient serum Peptides 1-2 derived from type I pili described in Test Example 2 above
By using the peptides shown in the peptide 21 in Table 3 of the peptide 21-29 in derived the 41 kD _a subunit protein constituting the type I pili peptides 10 and Porphyromonas gingivalis in 0, the The specific antibody of the periodontal disease patient serum was measured.

Table 3 Specific epitopes that react with serum of periodontal patients ──────────────────────────── Amino acid sequence Abbreviation ─── ───────────────────────── Glu Asn Ala Thr Lys Val Glu Asp Ile Lys Peptide 21 Glu Val Lys Ala Leu Thr Thr Glu Leu Thr Peptide 22 Ala Glu Asn Gln Glu Ala Ala Gly Leu Ile Peptide 23 Ala Ala Gly Leu Ile Met Thr Ala Glu Pro Peptide 24 Thr Gly Ser Leu Thr Thr Phe Asn Gly Ala Peptide 25 Thr Phe Asn Gly Ala Tyr Thr Pro Ala Asn Peptide 26 Gly Phe Tyr Val Leu Glu Asn Asp Tyr Ser Peptide 27 Ala Asn Gly Gly Thr Ile His Pro Thr Ile Peptide 28 Glu Gly Lys Thr Tyr Tyr Pro Val Leu Val Peptide 29 ─────────────────── ─────────── 0.1 or by mixing the plate preparation antigen solution described in Test Example 2 Carbonate buffer (pH 9.6), 0.99
Diluted with mM NaCl to 100 ml. 100 μl / well was added to a polystyrene microtiter plate (Immunoplate; Maxisorp type: Nunc), and the mixture was allowed to stand overnight at 4 ° C., and then an automatic microtiter washing machine (hereinafter referred to as a washing machine; manufactured by Bio-Rad) Each well was washed 3 times with 200 μl of phosphate buffered saline (pH 7.4). 0.1% gelatin (manufactured by Nitta Gelatin Co., Ltd.)
Phosphate buffered saline (pH 7.4) containing
1 / well was added, and the plate was blocked by standing overnight at 4 ° C. Remove the blocking solution by suction, apply a vacuum dryer to dry the plate, put it in an aluminum bag and seal it.
It was stored at ℃ and used for the following measurements.

Serum of a periodontal patient was used as a sample, and 1 of each serum was used.
Phosphate buffered saline containing 0% bovine serum (pH 7.
4) diluted 100 times. 100 μl of this diluted solution was added to each antigen-coated plate and incubated at 37 ° C. for 1 hour. After washing three times with 200 μl of phosphate buffered saline (pH 7.4) + Tween 20 using a washing machine, commercially available peroxidase-labeled human I as a secondary antibody
gG antibody (10 ³ culture dilution) /0.1% casein / phosphate buffered saline (pH 7.4) + Tween 20 10
0 μl was added, and the mixture was again incubated at 37 ° C. for 1 hour.
After washing with a washing machine in the same manner, 100 μl of an o -phenylenediamine solution was added and incubated at 37 ° C. for 30 minutes, 100 μl of 0.1N sulfuric acid was added to stop the reaction, and a reaction well without a sample was used as a control. Absorbance (405 nm) was measured with a reader (manufactured by Bio-Rad). Table 4 shows the measurement results using various antigen plates. The measured value is indicated by the absorbance of the patient with periodontal disease.

The periodontal disease patient serum used showed a reaction with either or both of the type II pili-derived peptide 10 and the type I pili-derived peptide 21 described in Test Example 2 above. When the peptides were mixed, specific reactivity was observed in the serum of all periodontal patients. Therefore,
It was found that the use of each of these two types of pili-derived peptides as an antigen enables more effective screening of periodontal disease patients.

Table 4 Measurement of specific antibody of periodontal disease patient serum using two kinds of peptides derived from fimbriae ──────────────────────── ──────────── Patient type II pili derived mixture of type I pili derived peptide 10 peptide 21 ───────────────────── ─────────────── 1 0.290 0.946 1.080 2 0.376 0.720 0.937 3 0.394 0.142 0.401 4 0.336 0 .148 0.398 5 0.168 0.870 0.913 6 0.221 0.784 0.855 7 0.144 0.815 0.798 8 0.285 0.120 0.326 9 0.345 0 .462 0.610 10 0.064 0.294 0.304 11 0.180 .446 0.575 12 0.098 0.234 0.289 13 0.336 0.465 0.676 14 0.058 0.805 0.792 15 0.286 0.270 0.471 16 0.279 0 278 0.462 17 0.357 0.232 0.513 18 0.146 0.770 0.806 ─────────────────────────── ──────────

[0065]

INDUSTRIAL APPLICABILITY According to the present invention, there are provided peptides corresponding to specific fragments derived from the type II pilus amino acid sequence of Porphyromonas gingivalis.
These peptides are useful for diagnosing and preventing periodontal disease, and according to the present invention, a composition that can be used for diagnosing a specific periodontal disease comprising the peptides and is effective for preventing periodontal disease. Also provided are a vaccine having the same, and an oral composition useful for preventing or treating periodontal disease, which comprises an antiserum obtained by immunizing an animal with such peptides and a monoclonal antibody.

[0066]

[Table 1]

[0067]

[Table 2]

[0068]

[Table 3]

[0069]

[Table 4]

[0070]

[Table 5]

[0071]

[Table 6]

[0072]

[Table 7]

[0073]

[Table 8]

[0074]

[Table 9]

[0075]

[Table 10]

[0076]

[Table 11]

[0077]

[Table 12]

[0078]

[Table 13]

[0079]

[Table 14]

[0080]

[Table 15]

[0081]

[Table 16]

[0082]

[Table 17]

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication location A61K 39/395 DC 07K 14/195 G01N 33/50 G (72) Inventor Tomohiko Ogawa 1 Okamachi Kita, Toyonaka City, Osaka Prefecture −1-8 (72) Inventor Keiko Yamada 10-2 Makishinata-cho, Ogaki City, Gifu Prefecture 72 (72) Inventor Shuzaburo Kitakuni 8-55 Wakabadai, Kani City, Gifu Prefecture Inventor Go Hasegawa Aso Ward, Kawasaki City, Kanagawa Prefecture 1-9-26 Katahira (72) Masanori Fukui 1333-45 Uchikoshi, Himeji City, Hyogo Prefecture (72) Hideharu Mori 1188 Shimochikari, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture

Claims (7)

[Claims] 1. Porphyromonas gingivalis ( Po
rphyromonas gingivalis) a type II pili peptides corresponding to fragments derived from the amino acid sequence of the 72 kD _a subunit protein constituting the, from the fragment group comprising 5-10 amino acid residues wherein the fragment is continuous A peptide characterized by being selected, or a derivative thereof or a salt thereof. 2. The fragment is SEQ ID NO: 1 in the sequence listing.
At least 5 contiguous amino acid sequences represented by
The peptide, the derivative thereof, or the salt thereof according to claim 1, which is selected from the group of fragments containing one amino acid residue. 3. A composition for diagnosing periodontal disease, which comprises one or more of the peptide according to claim 2 or a derivative thereof or a salt thereof. 4. A peptide corresponding to the peptide according to claim 2 or a derivative thereof or a salt thereof, and a fragment corresponding to a fragment derived from a 41KD _a subunit protein constituting type I pili of Porphyromonas gingivalis. Peptides selected from the fragment group consisting of at least 5 consecutive amino acid residues of the amino acid sequences shown in SEQ ID NOs: 21 to 29 of the sequence listing, or a derivative thereof or a salt thereof, is used alone or in combination of two or more. A composition for diagnosing periodontal disease comprising as a combination. 5. A periodontal disease-preventing vaccine comprising, as a main component, one or more of the peptide according to claim 2 or a derivative thereof or a salt thereof. 6. The periodontal disease preventive vaccine according to claim 5, wherein the vaccine is for intragingival administration. 7. An oral cavity for preventing periodontal disease or treating periodontal disease, which comprises an antibody obtained by immunizing an animal with one or more of the peptide according to claim 2 or a derivative thereof or a salt thereof. Composition.