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WO2010050369A1 - Prophylactic agent for dental caries - Google Patents

Prophylactic agent for dental caries Download PDF

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Publication number
WO2010050369A1
WO2010050369A1 PCT/JP2009/067903 JP2009067903W WO2010050369A1 WO 2010050369 A1 WO2010050369 A1 WO 2010050369A1 JP 2009067903 W JP2009067903 W JP 2009067903W WO 2010050369 A1 WO2010050369 A1 WO 2010050369A1
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WO
WIPO (PCT)
Prior art keywords
lectin
amino acid
acid sequence
acg
3galnac
Prior art date
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Ceased
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PCT/JP2009/067903
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French (fr)
Japanese (ja)
Inventor
正悟 高柴
靖弘 吉田
孝 伊東
幸治 今村
英明 竹内
英治 岡本
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Okayama University NUC
Glyence Co Ltd
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Okayama University NUC
Glyence Co Ltd
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Publication date
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Priority to JP2010535753A priority Critical patent/JP5558362B2/en
Publication of WO2010050369A1 publication Critical patent/WO2010050369A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses

Definitions

  • the present invention relates to a caries preventive agent containing a lectin, in particular, a caries preventive agent containing a lectin having an effect of suppressing adhesion and proliferation to plaques or biofilms in the oral cavity, and a pharmaceutical composition containing the caries preventive agent,
  • the present invention relates to a food composition, a dentifrice and a mouth rinse.
  • the plaque or biofilm grows when saliva-derived biopolymer adheres to the tooth surface to form a pellicle film, and oral bacteria adhere to the pellicle. Therefore, in order to prevent dental caries and periodontal disease, it is necessary to suppress or prevent the adhesion of oral bacteria to the pellicle.
  • Sugar alcohols such as erythritol and xylitol are known to have caries-inhibiting effects, but sugar alcohols do not produce lactic acid that causes caries due to metabolism of oral bacteria, making it difficult to produce caries. Yes, it is not involved in inhibiting or preventing oral bacterial adhesion.
  • sugar chain-binding protein lectin produced by oral bacteria is involved. That is, it is considered that oral bacteria adhere to the pellicle by the sugar chain-specific binding between saliva-derived biopolymers accumulated in the pellicle, that is, polysaccharides or glycoproteins, and the oral bacterial lectin. Then, by administering the lectin that competes with the oral bacterial lectin from the outside into the oral cavity and inhibiting the binding of the oral bacterial lectin to the sugar chain of the pellicle, the adhesion and growth of oral bacteria on the biofilm is suppressed. Alternatively, it can be inhibited to prevent oral bacterial infections such as tooth decay and periodontal disease.
  • Non-Patent Documents 1 to 3 The lectins reported in these documents include plant-derived glucose mannose recognition lectins and algae-derived mucin recognition lectins.
  • mutans bacteria Streptococcus mutans
  • the present invention provides a caries preventive agent characterized by containing a lectin.
  • the lectin includes Fuc ⁇ 1-6GlcNAc, Fuc ⁇ 1-3GalNAc, Gal ⁇ 1-3GalNAc, GlcNAc, Gal ⁇ 1-3GalNAc, Silyal ⁇ 2-3 lactose, Gal ⁇ 1-3Gal1GalGalGal, And at least one sugar chain selected from the group consisting of GalNAc.
  • the lectin comprises (1) AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, and (2) an amino acid sequence described in SEQ ID NOs: 1 to 9.
  • the lectin may recognize sialic acid.
  • the lectin may be selected from the group consisting of ACG, MAH and Jacalin.
  • the lectin may be selected from the group consisting of ABA, ACA, ACG, BPL and RCA-I.
  • the lectin may recognize mannose or N-acetylglucosamine.
  • the lectin may be GRFT.
  • the present invention provides a pharmaceutical composition comprising the caries preventive agent of the present invention.
  • the present invention provides a food composition containing the caries preventive agent of the present invention.
  • the present invention provides a dentifrice containing the caries preventive agent of the present invention.
  • the present invention provides a mouth rinse containing the caries preventive agent of the present invention.
  • the present invention provides a method for preventing caries, comprising the step of administering a caries preventive agent containing a lectin into the oral cavity.
  • the lectin includes Fuc ⁇ 1-6GlcNAc, Fuc ⁇ 1-3GalNAc, Gal ⁇ 1-3GalNAc, GlcNAc, Gal ⁇ 1-3GalNAc, Sialyl ⁇ 2-3lactose, Gal ⁇ 1-3GalNA1GalNAc1, Gial ⁇ 1-3GalNAc, Sialyl-Gal ⁇ 1-3GalNAc, In some cases, at least one sugar chain selected from the group consisting of Gal and GalNAc may be recognized.
  • the lectin comprises (1) AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, and (2) an amino acid sequence described in SEQ ID NOs: 1 to 9.
  • the lectin may recognize sialic acid.
  • the lectin may be selected from the group consisting of ACG, MAH and Jacalin.
  • the lectin may be selected from the group consisting of ABA, ACA, ACG, BPL and RCA-I.
  • the lectin may recognize mannose or N-acetylglucosamine.
  • the lectin may be GRFT.
  • the present invention provides use of a lectin for the manufacture of a pharmaceutical composition for preventing caries.
  • the lectin includes Fuc ⁇ 1-6GlcNAc, Fuc ⁇ 1-3GalNAc, Gal ⁇ 1-3GalNAc, GlcNAc, Gal ⁇ 1-3GalNAc, Sialyl ⁇ 2-3lactose, Gal ⁇ 1-3GalNAc, Sial1Gal -3GalNAc, Sialyl ⁇ 2-3Gal ⁇ 1-3GalNAc, Gal and GalNAc may be recognized at least one kind of sugar chain selected from the group.
  • AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I and (2) SEQ ID NOs: 1 to 9 for use in the manufacture of the pharmaceutical composition for preventing caries of the present invention
  • a protein comprising the amino acid sequence described above, and (3) an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence described in SEQ ID NOs: 1 to 9, and A protein having the same sugar chain recognition specificity as AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, and (4) 80% or more of the amino acid sequence set forth in SEQ ID NOS: 1 to 9 Of the amino acid sequence, and AAL, ABA, ACA, ACG, BPL, GRFT, Jac, respectively.
  • the lectin may recognize sialic acid.
  • the lectin may be selected from the group consisting of ACG, MAH and Jacalin.
  • the lectin may be selected from the group consisting of ABA, ACA, ACG, BPL and RCA-I.
  • the lectin may recognize mannose or N-acetylglucosamine.
  • the lectin may be GRFT.
  • the lectins of the present invention are ACG (Agrocybe cylindracea gallectin) derived from willow matsutake, PSL1 ⁇ (Polyporus squamusus lectinus rapeselpsein, PSLinsensen from P.
  • ACG Agrocybe cylindracea gallectin
  • PSL1 ⁇ Polyporus squamusus lectinus rapeselpsein, PSLinsensen from P.
  • AAL Aleuria aurantia lectin derived from Hilochawantake
  • ABA Agaricus bisporus agglutinin
  • ACA Amaranthus caudatus agglutinin
  • Amaranthus Rasosocinka-derived BPL Bauhinia purpurea electin
  • Jackfruit Jackfruit (Artocarpus integrifolia) -derived Jacalin
  • castor-derived RCA-I Ricinus communis agglutinin-I
  • Hengenta-derived algae magnuminh GRiffithsia sp.-Derived GRFT, but is not limited thereto.
  • the sugar chains recognized by the lectin of the present invention are Sially ⁇ 2-6Gal, Gal ⁇ 1-4GlcNAc, GlcNAc ⁇ 1-2Man, Neu5Ac2-3Gal1-4GlcNAc1-2Man1-6 (Neu5Ac2-3 / 6 (Neu5Ac-2-3Gal1-4) GlcNAc-4 (Neu5Ac2-3 / 6Gal1-4GlcNAc1-2) Man1-3) Man, Man ⁇ 1-6Man, Gal ⁇ 1-3GalNAc, Sialyl-Gal ⁇ 1-3 GalNAc, Fuc ⁇ 1-6GlcNAc, Fuc ⁇ 1-3GalNAG, Gal ⁇ 1-3GalNAG, Sialyl ⁇ 2-3 lactose, Gal ⁇ 1-3GalNAc, Mannose, GlcNAc, Sialyl-Gal ⁇ 1 3GalNAc, Sialyl ⁇ 2-3Gal ⁇ 1-3GalNAc, Gal, including GalNAc, but not limited to.
  • amino acid sequences of AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I are described in SEQ ID NOs: 1 to 9, respectively.
  • the amino acid sequence of the protein of the present invention is one in the amino acid sequence shown in SEQ ID NOs: 1 to 9 on the condition that it has the same sugar chain recognition specificity as the lectin consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 9.
  • Several amino acids may be deleted, substituted or added.
  • the number of amino acids to be deleted, substituted or added may be 10 or 11 or more as long as it is a site other than the domain involved in sugar chain specificity recognition.
  • the number of amino acids deleted, substituted or added may be 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • the homology of amino acid sequences means that the amino acid sequences of the present invention and the amino acid sequence to be compared are aligned so that the number of amino acid residues having the same sequence is the largest, and the sequences match. It is expressed as a percentage of the quotient obtained by dividing the total number of amino acid residues by the total number of amino acid residues of the amino acid sequence of the present invention.
  • the homology of the nucleotide sequence and amino acid sequence of the present invention can be calculated by using the sequence alignment program CLUSTALW well known to those skilled in the art.
  • the homology of the amino acid sequence of the protein of the present invention may be 80% or more, and 85% or more, provided that they have the same sugar chain recognition specificity as a lectin having a 100% homology amino acid sequence.
  • 90% or more is more preferable, and 95% or more is more preferable.
  • stringent conditions refers to Sambrook, J. et al. And Russell, D .; W. , Molecular Cloning A Laboratory Manual 3rd Edition, Cold Spring Harbor Laboratory Press (2001), and the Southern blot method described in the following experimental conditions.
  • a polynucleotide having a nucleotide sequence to be compared is band-formed by agarose electrophoresis and then immobilized on a nitrocellulose filter or other solid phase by capillary action or electrophoresis. Pre-wash with a solution consisting of 6x SSC and 0.2% SDS.
  • a hybridization reaction between a probe obtained by labeling a polynucleotide comprising the nucleotide sequence of the present invention with a radioisotope or other labeling substance and a polynucleotide to be compared immobilized on the solid phase is performed with 6 ⁇ SSC and 0.2. % In SDS solution at 65 ° C overnight. Thereafter, the solid phase was washed twice at 65 ° C. for 30 minutes each in a solution consisting of 1 ⁇ SSC and 0.1% SDS, and 65 ° in a solution consisting of 0.2 ⁇ SSC and 0.1% SDS. C. Wash twice for 30 minutes each. Finally, the amount of the probe remaining on the solid phase is determined by quantifying the labeling substance.
  • hybridization under “stringent conditions” means that the amount of the probe remaining on the solid phase immobilized with the polynucleotide consisting of the nucleotide sequence to be compared is equal to the polynucleotide consisting of the nucleotide sequence of the present invention. It refers to at least 25%, preferably at least 50%, more preferably at least 75% or more of the amount of probe remaining in the immobilized solid phase of the positive control experiment.
  • the sugar chain commonly recognized by the lectins of the present invention is GalNAc or Gal ⁇ 1-3 GalNAc.
  • the glycoprotein mucin contained in saliva is a sugar chain (Tn antigen) in which GalNAc is bound to serine / threonine, or a sugar chain (T antigen) in which Gal ⁇ 1-3 GalNAc is bound. Yes. From the results of the examples of the present specification, it was suggested that the mutans bacteria recognize mucin Tn antigen or T antigen and bind to saliva. In addition to those used in the examples of the present invention, the following are known as lectins that recognize Tn antigen or T antigen.
  • HSL Holothuria scabralectin
  • Hamanai sea cucumber AVL Amaranthus viridis lectin
  • AAltL Aralpus altilis lectin
  • Panca AHA Artocarpus inhabited
  • Species derived from Artocarpus lakocha agglutinin Lactipanca APA (Abrus precatorius agglitinin)
  • Species Touzuki ALL (Amaranthus leucocarpus lectin)
  • Shibamugi LDelL Lobamugi LDelL (Lactarius deliciosus lectin)
  • Species Red-billed Vaginalia VGA (Vicia graminea agg
  • the lectin contained in the dental caries preventive agent of the present invention is a protein purified from an individual, tissue, cell, or part thereof derived from the above-described species, or expressed and purified in various hosts by recombinant DNA technology. Or an individual, tissue, cell or part thereof, or a partially purified preparation of the above-mentioned species, and when administered into the oral cavity, oral bacteria into the intraluminal hard tissue, in particular, In some cases, it has sufficient activity to exert an action of suppressing or inhibiting the adhesion and growth of mutans bacteria. That is, the lectin may be used as an individual, tissue, cell or part of the derived biological species, or a processed product thereof.
  • the pharmaceutical composition of the present invention includes all pharmaceuticals for suppressing or inhibiting the adhesion and proliferation of oral bacteria, particularly mutans bacteria, to the oral hard tissue, and the lectin of the present invention is derived from oral bacteria and saliva. Any other component may be included on the condition that the activity of suppressing or inhibiting the binding with the biopolymer is not substantially impaired.
  • the food composition of the present invention contains any other component, provided that the lectin of the present invention does not substantially impair the activity of inhibiting or inhibiting the binding between oral bacteria and saliva-derived biopolymers. It does not matter.
  • the food composition of the present invention may contain sugar alcohols such as erythritol and xylitol.
  • the dentifrice or mouth rinse of the present invention contains any other components, provided that the lectin of the present invention does not substantially impair the activity of inhibiting or inhibiting the binding between oral bacteria and saliva-derived biopolymers. It may be included.
  • the bar graph which shows the inhibitory effect by the lectin with respect to adhesion and proliferation of a mutans bacterium The microscope picture which shows the growth inhibitory effect of mutans bacteria by a lectin.
  • the bar graph which shows the inhibitory effect by the sialic acid recognition lectin with respect to adhesion and proliferation of a mutans bacterium The bar graph which shows the inhibitory effect by the lectin with respect to the growth of a mutans bacterium on the substrate coated with the saliva from different test subjects.
  • the bar graph which shows the inhibitory effect by the lectin with respect to the growth of a mutans bacterium on the substrate coated with the saliva from a different test subject.
  • the bar graph which shows the inhibitory effect by the lectin with respect to the growth of a mutans bacterium on the substrate coated with the saliva from different test subjects.
  • the bar graph which plotted the suppression index
  • the bar graph which shows interaction with three types of multivalent biotinylated sugar polymers and mutans bacteria.
  • the multiplate was washed with PBS, 5 ⁇ 10 7 mutans bacteria in 100 ⁇ L of PBS were added to each well, and cultured at 37 ° C. overnight.
  • the plate was washed with PBS, 100 ⁇ L of 0.25% glutaraldehyde / PBS was added, and fixed at room temperature for 30 minutes. After washing with PBS, 100 ⁇ L of 2.3% crystal violet (for Gram staining) was added and stained at room temperature for 1 hour. Thereafter, it was washed with running water, 100 ⁇ L of ethanol was added, and the dye was eluted at room temperature for 1 hour.
  • the number of bacteria was quantified by measuring the dye concentration in the ethanol solution of each well at an absorption wavelength of 570 nm using a plate reader.
  • FIG. 1 is a bar graph showing the inhibitory effect of lectins on adhesion and growth of mutans bacteria.
  • the experiment was repeated twice for each identical condition.
  • Wells that were not coated with saliva (saliva ⁇ ) and wells that were not added with mutans (St. mutans ⁇ ) did not show significant absorbance.
  • the absorbance of the well (lectin ⁇ ) that was coated with saliva but not incubated with lectin was 0.10.
  • the absorbance of wells (BSA) incubated with BSA instead of saliva was 0.05, half of lectin ⁇ .
  • the absorbance of wells incubated with MVL and EPL exceeded 0.15, and the absorbance of wells incubated with OAA, Con A, PNA, PHA-L and RCA was approximately 0.10.
  • the absorbance of wells incubated with GRFT, GNA and AAL was between 0.05 and 0.10, whereas the absorbance of wells incubated with ACG and NPL was less than 0.05.
  • FIG. 2 is a table showing the respective conditions of this example, and a photomicrograph showing the results of observing the growth of mutans bacteria under each condition with a microscope.
  • mutans bacteria spread on the substrate surface to form colonies.
  • saliva ⁇ the positive control condition
  • ACG ACG
  • NPL NPL
  • ACG belongs to the sialic acid recognition lectin.
  • FIG. 3 is a bar graph showing the inhibitory effect of sialic acid-recognizing lectins on adhesion and growth of mutans bacteria.
  • the experiment was repeated twice for each same condition.
  • Wells to which no mutans were added did not show significant absorbance.
  • the absorbance of the well (lectin ⁇ ) coated with saliva but not incubated with lectin was 0.45.
  • Table 1 shows the sugar chains recognized by the lectins that were confirmed to have the effect of suppressing adhesion and growth of S. mutans.
  • the cells were washed with PBS, and 5 ⁇ 10 7 mutans bacteria (ATCC 25175) in 100 ⁇ L of PBS were cultured at 37 ° C. overnight.
  • the bacteria were washed with PBS, 100 ⁇ L of 0.25% glutaraldehyde / PBS was added, and fixed at room temperature for 30 minutes. After washing with PBS, 100 ⁇ L of 2.3% crystal violet was added and stained at room temperature for 1 hour. Thereafter, it was washed with running water, 100 ⁇ L of ethanol was added, and the dye was eluted at room temperature for 1 hour.
  • the number of bacteria was quantified by measuring the dye concentration in the ethanol solution of each well at an absorption wavelength of 570 nm using a plate reader.
  • the lectins used were as follows. Natural lectins are AAA, APP, BDA, CA, CAA, CSA, DBA, DSA, ECA, GNA, GS-II, HPA, Jacalin, LAA, LBA, LFA, Lotus, NPA, PNA, PSA manufactured by EY Laboratories.
  • Recombinant lectins are rACG and rGRFT, which were prepared in-house at Glience.
  • FIG. 4-7 is a bar graph showing the inhibitory effect of lectins on the growth of S. mutans on substrates coated with saliva from different subjects. In each case, the experiment was repeated twice for the same conditions.
  • the four bar graphs from the left end of each figure are the results of a control experiment. From the left end, lectin-, mutans bacteria- and saliva- (wells only), lectin-, mutans bacteria + and saliva- (mutans bacteria only) ), Lectin-, mutans bacteria and saliva + (saliva only), lectin-, mutans bacteria + and saliva + (saliva + mutans bacteria (positive control without lectin)). It was.
  • AAL, ABA, ACA, BPL, Jacalin, RCA-I, MAH, ACG and GRFT were found to have an effect of suppressing the growth of mutans bacteria.
  • Table 2 shows the sugar chains recognized by the lectin used in this example.
  • the binding between the synthetic sugar chain adsorbed on the solid phase and the mutans bacterium is measured by the surface plasmon effect, and the mutans bacterium and the sugar chain are specifically identified.
  • the interaction was quantitatively analyzed.
  • HOCH 2 4 CH 2 NH—PAA-biotin (control) Gal ⁇ 1-3GalNAc ⁇ -PAA-biotin (Core1) GlcNAc ⁇ 1-3GalNAc ⁇ -PAA-biotin (Core3) Neu5Ac ⁇ 2-3Gal ⁇ 1-3GalNAc ⁇ -PAA-biotin (Sialyl T)
  • Each of the multivalent biotinylated sugar polymers is diluted to 50 ⁇ g / mL with PBS, loaded onto a Biacore sensor chip (BR-1000-32) at 10 ° C. at a flow rate of 10 ⁇ L / min, and measured at about 1000 RU with Biacore 2000. It was immobilized on the surface of the sensor substrate until the value was reached.
  • FIG. 10 shows the results after adding and rinsing the mutans sample solution to the Core1, Core3, and Sialyl T polymers, and subtracting the measured value after adding the mutans sample solution to the control polymer and rinsing. It is a bar graph which shows the value.
  • the Core1 polymer interacted with mutans bacteria much more strongly than the other polymers.
  • sugar chains containing galactose at the end of sugar chains derived from saliva are involved in adhesion to the substrate of mutans bacteria. Therefore, the caries preventive agent containing the lectin of the present invention and the caries preventive method using the caries preventive agent inhibit the interaction between the sugar chain derived from saliva, in particular, the sugar chain containing galactose at the terminal and the mutans bacterium. I think that.

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Abstract

Disclosed is a prophylactic agent for dental caries, which comprises a lectin which has an activity to prevent or inhibit the binding between a biopolymer derived from saliva from Japanese persons who live on Japanese foods and an oral bacterium, particularly Streptococcus mutans that a bacterium believed to cause dental caries. Specifically disclosed is a prophylactic agent for dental caries, which is characterized by comprising a lectin selected from the group consisting of AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH, RCA-I and the like.  Also disclosed are a pharmaceutical composition, a food composition, a dentifrice and an oral washer, each of which comprises the prophylactic agent for dental caries.

Description

虫歯予防剤Caries prevention agent

　本発明は、レクチンを含む虫歯予防剤、特に、口腔内のプラーク又はバイオフィルムへの付着及び増殖を抑制する効果を有するレクチンを含む虫歯予防剤と、該虫歯予防剤を含む、医薬品組成物、食品組成物、歯磨き剤及び口内洗浄剤とに関する。 The present invention relates to a caries preventive agent containing a lectin, in particular, a caries preventive agent containing a lectin having an effect of suppressing adhesion and proliferation to plaques or biofilms in the oral cavity, and a pharmaceutical composition containing the caries preventive agent, The present invention relates to a food composition, a dentifrice and a mouth rinse.

　口腔細菌は、口腔内のプラーク又はバイオフィルムに付着して増殖し、齲蝕すなわち虫歯や歯槽膿漏その他の歯周病を引き起こす。前記プラーク又はバイオフィルムは、唾液由来の生体高分子が歯の表面に付着してペリクルという皮膜を形成し、該ペリクルに口腔細菌が付着することにより成長する。したがって虫歯や歯周病の予防のためにはペリクルへの口腔細菌の付着を抑制又は阻止する必要がある。エリスリトール、キシリトール等の糖アルコール類には虫歯抑制効果があることが知られているが、糖アルコールは口腔細菌の代謝によりう蝕の原因となる乳酸を産生しないために虫歯が出来にくくなるものであり、口腔細菌の付着の抑制や阻止には関与しない。 Oral bacteria grow on adhering to plaque or biofilm in the oral cavity, causing caries, that is, dental caries, alveolar pyorrhea and other periodontal diseases. The plaque or biofilm grows when saliva-derived biopolymer adheres to the tooth surface to form a pellicle film, and oral bacteria adhere to the pellicle. Therefore, in order to prevent dental caries and periodontal disease, it is necessary to suppress or prevent the adhesion of oral bacteria to the pellicle. Sugar alcohols such as erythritol and xylitol are known to have caries-inhibiting effects, but sugar alcohols do not produce lactic acid that causes caries due to metabolism of oral bacteria, making it difficult to produce caries. Yes, it is not involved in inhibiting or preventing oral bacterial adhesion.

　口腔細菌がペリクルや他の口腔細菌の菌体に付着する際には、口腔細菌の産生する糖鎖結合タンパク質レクチンが関与していると考えられる。つまり、ペリクルに滞積した唾液由来の生体高分子、すなわち、多糖類又は糖タンパク質と口腔細菌レクチンとが糖鎖特異的な結合を行うことにより、ペリクルに口腔細菌が付着すると考えられる。すると、外部から口腔細菌のレクチンと競合するレクチンを口腔内に投与することによって、ペリクルの糖鎖への口腔細菌のレクチンの結合を阻害すれば、口腔細菌のバイオフィルムへの付着及び増殖を抑制又は阻害して、虫歯や歯周病のような口腔細菌による感染症を予防することができる。 When oral bacteria adhere to pellicles and other oral bacteria, it is considered that sugar chain-binding protein lectin produced by oral bacteria is involved. That is, it is considered that oral bacteria adhere to the pellicle by the sugar chain-specific binding between saliva-derived biopolymers accumulated in the pellicle, that is, polysaccharides or glycoproteins, and the oral bacterial lectin. Then, by administering the lectin that competes with the oral bacterial lectin from the outside into the oral cavity and inhibiting the binding of the oral bacterial lectin to the sugar chain of the pellicle, the adhesion and growth of oral bacteria on the biofilm is suppressed. Alternatively, it can be inhibited to prevent oral bacterial infections such as tooth decay and periodontal disease.

　実際にこのような発想に基づいて、口腔細菌とペリクルとの結合を阻害するレクチンが非特許文献１ないし３に報告されている。これらの文献で報告されたレクチンには、植物由来のグルコース・マンノース認識レクチンや、藻類由来のムチン認識レクチンがある。 Based on such an idea, lectins that inhibit the binding between oral bacteria and pellicles are reported in Non-Patent Documents 1 to 3. The lectins reported in these documents include plant-derived glucose mannose recognition lectins and algae-derived mucin recognition lectins.

Ｔｅｉｘｅｉｒａ，　Ｅ．Ｈ．ら、Ｊ　Ａｐｐｌ．　Ｍｉｃｒｏｂｉｏｌ．、１０１：　１１１－６　（２００６）．Teixeira, E.M. H. Et al., J Appl. Microbiol. 101: 111-6 (2006). Ｔｅｉｘｅｉｒａ，　Ｅ．Ｈ．ら、Ｊ　Ａｐｐｌ．　Ｍｉｃｒｏｂｉｏｌ．、１０３：　１００１－６　（２００７）．Teixeira, E.M. H. Et al., J Appl. Microbiol. 103: 1001-6 (2007). Ｉｓｌａｍ，Ｂ．ら、Ｊ　Ａｐｐｌ．　Ｍｉｃｒｏｂｉｏｌ．、１０６：　１６８２－９　（２００９）．Islam, B.I. Et al., J Appl. Microbiol. 106: 1682-9 (2009).

　しかし、Ｃｏｎ　Ａ（コンカナバリンＡ）のようなグルコース・マンノース認識レクチンは、ほ乳類のリンパ球の幼若化反応を誘発することが知られており、免疫系に影響を与えるおそれがある。また、ヒトの唾液成分は人種、食物等によって変化することが知られている。そこで、日本食をとって生活する日本人の唾液由来の生体高分子と口腔細菌、特に、虫歯の原因とされるストレプトコッカス・ミュータンス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｍｕｔａｎｓ、以下、「ミュータンス菌」という。）との結合を抑制又は阻害する活性のあるレクチンを含む虫歯予防剤を開発する必要がある。 However, glucose mannose-recognizing lectins such as Con A (Concanavalin A) are known to induce a blastogenic reaction of mammalian lymphocytes, which may affect the immune system. Further, it is known that human saliva components vary depending on race, food, and the like. Therefore, the combination of biomolecules derived from saliva of Japanese living with Japanese food and oral bacteria, particularly Streptococcus mutans (hereinafter referred to as “mutans bacteria”), which is the cause of dental caries. Therefore, it is necessary to develop a caries preventive agent containing a lectin having an activity of suppressing or inhibiting the above.

　本発明は、レクチンを含むことを特徴とする虫歯予防剤を提供する。 The present invention provides a caries preventive agent characterized by containing a lectin.

　本発明の虫歯予防剤において、前記レクチンはＦｕｃα１－６ＧｌｃＮＡｃ、Ｆｕｃα１－３ＧａｌＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、ＧｌｃＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３ｌａｃｔｏｓｅ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌ－Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３Ｇａｌβ１－３ＧａｌＮＡｃ、Ｇａｌ及びＧａｌＮＡｃからなるグループから選択される少なくとも１種類の糖鎖を認識する場合がある。 In the dental caries preventive agent of the present invention, the lectin includes Fucα1-6GlcNAc, Fucα1-3GalNAc, Galβ1-3GalNAc, GlcNAc, Galβ1-3GalNAc, Silyalα2-3 lactose, Galβ1-3Gal1GalGalGal, And at least one sugar chain selected from the group consisting of GalNAc.

　本発明の虫歯予防剤において、前記レクチンは、（１）ＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと、（２）配列番号１ないし９に記載のアミノ酸配列からなるタンパク質と、（３）配列番号１ないし９に記載のアミノ酸配列に記載のアミノ酸配列に１個若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、（４）配列番号１ないし９に記載のアミノ酸配列と８０％以上の相同性を示すアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、（５）配列番号１ないし９に記載のアミノ酸配列をエンコードするヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチドによってエンコードされるアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、からなるグループから選択される１種類又は２種類以上のタンパク質の場合がある。 In the caries preventive agent of the present invention, the lectin comprises (1) AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, and (2) an amino acid sequence described in SEQ ID NOs: 1 to 9. And (3) an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence described in SEQ ID NOS: 1 to 9, and each of AAL and ABA , ACA, ACG, BPL, GRFT, Jacalin, MAH and a protein having the same sugar chain recognition specificity as RCA-I, and (4) 80% or more homology with the amino acid sequence of SEQ ID NOS: 1 to 9 It consists of an amino acid sequence, and AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MA And a protein having the same sugar chain recognition specificity as RCA-I, and (5) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NOS: 1 to 9 Selected from the group consisting of the amino acid sequences encoded by and having the same sugar chain recognition specificity as AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, respectively. There may be one or more proteins.

　本発明の虫歯予防剤において、前記レクチンは、シアル酸を認識する場合がある。 In the caries preventive agent of the present invention, the lectin may recognize sialic acid.

　本発明の虫歯予防剤において、前記レクチンは、ＡＣＧ、ＭＡＨ及びＪａｃａｌｉｎからなるグループから選択される場合がある。 In the caries preventive agent of the present invention, the lectin may be selected from the group consisting of ACG, MAH and Jacalin.

　本発明の虫歯予防剤において、前記レクチンは、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ及びＲＣＡ－Ｉからなるグループから選択される場合がある。 In the caries preventive agent of the present invention, the lectin may be selected from the group consisting of ABA, ACA, ACG, BPL and RCA-I.

　本発明の虫歯予防剤において、前記レクチンは、マンノース又はＮアセチルグルコサミンを認識する場合がある。 In the caries preventive agent of the present invention, the lectin may recognize mannose or N-acetylglucosamine.

　本発明の虫歯予防剤において、前記レクチンは、ＧＲＦＴの場合がある。 In the caries preventive agent of the present invention, the lectin may be GRFT.

　本発明は、本発明の虫歯予防剤を含む医薬品組成物を提供する。 The present invention provides a pharmaceutical composition comprising the caries preventive agent of the present invention.

　本発明は、本発明の虫歯予防剤を含む食品組成物を提供する。 The present invention provides a food composition containing the caries preventive agent of the present invention.

　本発明は、本発明の虫歯予防剤を含む歯磨き剤を提供する。 The present invention provides a dentifrice containing the caries preventive agent of the present invention.

　本発明は、本発明の虫歯予防剤を含む口内洗浄剤を提供する。 The present invention provides a mouth rinse containing the caries preventive agent of the present invention.

　本発明は、レクチンを含む虫歯予防剤を口腔内に投与するステップを含む、虫歯の予防方法を提供する。 DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for preventing caries, comprising the step of administering a caries preventive agent containing a lectin into the oral cavity.

　本発明の虫歯の予防方法において、前記レクチンは、Ｆｕｃα１－６ＧｌｃＮＡｃ、Ｆｕｃα１－３ＧａｌＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、ＧｌｃＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３ｌａｃｔｏｓｅ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌ－Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３Ｇａｌβ１－３ＧａｌＮＡｃ、Ｇａｌ及びＧａｌＮＡｃからなるグループから選択される少なくとも１種類の糖鎖を認識する場合がある。 In the method for preventing dental caries according to the present invention, the lectin includes Fucα1-6GlcNAc, Fucα1-3GalNAc, Galβ1-3GalNAc, GlcNAc, Galβ1-3GalNAc, Sialylα2-3lactose, Galβ1-3GalNA1GalNAc1, Gialβ1-3GalNAc, Sialyl-Galβ1-3GalNAc, In some cases, at least one sugar chain selected from the group consisting of Gal and GalNAc may be recognized.

　本発明の虫歯の予防方法において、前記レクチンは、（１）ＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと、（２）配列番号１ないし９に記載のアミノ酸配列からなるタンパク質と、（３）配列番号１ないし９に記載のアミノ酸配列に記載のアミノ酸配列に１個若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、（４）配列番号１ないし９に記載のアミノ酸配列と８０％以上の相同性を示すアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、（５）配列番号１ないし９に記載のアミノ酸配列をエンコードするヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチドによってエンコードされるアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、からなるグループから選択される１種類又は２種類以上のタンパク質の場合がある。 In the method for preventing dental caries of the present invention, the lectin comprises (1) AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, and (2) an amino acid sequence described in SEQ ID NOs: 1 to 9. And (3) an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence described in the amino acid sequences of SEQ ID NOS: 1 to 9, and each of which is AAL, A protein having the same sugar chain recognition specificity as ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I; and (4) at least 80% homology with the amino acid sequence of SEQ ID NOS: 1 to 9 Consisting of the amino acid sequences shown, and AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, A protein that hybridizes under stringent conditions with a protein having the same sugar chain recognition specificity as AH and RCA-I and (5) a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NOS: 1 to 9 A protein consisting of an amino acid sequence encoded by nucleotides and having the same sugar chain recognition specificity as AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, respectively. May be one or more proteins.

　本発明の虫歯の予防方法において、前記レクチンはシアル酸を認識する場合がある。 In the method for preventing caries of the present invention, the lectin may recognize sialic acid.

　本発明の虫歯の予防方法において、前記レクチンは、ＡＣＧ、ＭＡＨ及びＪａｃａｌｉｎからなるグループから選択される場合がある。 In the method for preventing caries according to the present invention, the lectin may be selected from the group consisting of ACG, MAH and Jacalin.

　本発明の虫歯の予防方法において、前記レクチンは、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ及びＲＣＡ－Ｉからなるグループから選択される場合がある。 In the method for preventing caries of the present invention, the lectin may be selected from the group consisting of ABA, ACA, ACG, BPL and RCA-I.

　本発明の虫歯の予防方法において、前記レクチンは、マンノース又はＮアセチルグルコサミンを認識する場合がある。 In the method for preventing caries according to the present invention, the lectin may recognize mannose or N-acetylglucosamine.

　本発明の虫歯の予防方法において、前記レクチンは、ＧＲＦＴの場合がある。 In the method for preventing caries according to the present invention, the lectin may be GRFT.

　本発明は、レクチンの虫歯予防用医薬品組成物の製造のための使用を提供する。 The present invention provides use of a lectin for the manufacture of a pharmaceutical composition for preventing caries.

　本発明の虫歯予防用医薬品組成物の製造のための使用において、前記レクチンは、Ｆｕｃα１－６ＧｌｃＮＡｃ、Ｆｕｃα１－３ＧａｌＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、ＧｌｃＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３ｌａｃｔｏｓｅ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌ－Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３Ｇａｌβ１－３ＧａｌＮＡｃ、Ｇａｌ及びＧａｌＮＡｃからなるグループから選択される少なくとも１種類の糖鎖を認識する場合がある。 In the use for producing the pharmaceutical composition for preventing caries of the present invention, the lectin includes Fucα1-6GlcNAc, Fucα1-3GalNAc, Galβ1-3GalNAc, GlcNAc, Galβ1-3GalNAc, Sialylα2-3lactose, Galβ1-3GalNAc, Sial1Gal -3GalNAc, Sialylα2-3Galβ1-3GalNAc, Gal and GalNAc may be recognized at least one kind of sugar chain selected from the group.

　本発明の虫歯予防用医薬品組成物の製造のための使用において、（１）ＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと、（２）配列番号１ないし９に記載のアミノ酸配列からなるタンパク質と、（３）配列番号１ないし９に記載のアミノ酸配列に記載のアミノ酸配列に１個若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、（４）配列番号１ないし９に記載のアミノ酸配列と８０％以上の相同性を示すアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、（５）配列番号１ないし９に記載のアミノ酸配列をエンコードするヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチドによってエンコードされるアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、からなるグループから選択される１種類又は２種類以上のタンパク質の場合がある。 (1) AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, and (2) SEQ ID NOs: 1 to 9 for use in the manufacture of the pharmaceutical composition for preventing caries of the present invention A protein comprising the amino acid sequence described above, and (3) an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence described in SEQ ID NOs: 1 to 9, and A protein having the same sugar chain recognition specificity as AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, and (4) 80% or more of the amino acid sequence set forth in SEQ ID NOS: 1 to 9 Of the amino acid sequence, and AAL, ABA, ACA, ACG, BPL, GRFT, Jac, respectively. Hybridization under stringent conditions with a protein having the same sugar chain recognition specificity as lin, MAH and RCA-I, and a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NOS: 1 to 9 A protein having the same sugar chain recognition specificity as AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I. There may be one or more proteins selected.

　本発明の虫歯予防用医薬品組成物の製造のための使用において、前記レクチンはシアル酸を認識する場合がある。 In the use for producing the pharmaceutical composition for preventing caries of the present invention, the lectin may recognize sialic acid.

　本発明の虫歯予防用医薬品組成物の製造のための使用において、前記レクチンは、ＡＣＧ、ＭＡＨ及びＪａｃａｌｉｎからなるグループから選択される場合がある。 In the use for producing the pharmaceutical composition for preventing caries of the present invention, the lectin may be selected from the group consisting of ACG, MAH and Jacalin.

　本発明の虫歯予防用医薬品組成物の製造のための使用において、前記レクチンは、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ及びＲＣＡ－Ｉからなるグループから選択される場合がある。 In the use for producing the pharmaceutical composition for preventing caries of the present invention, the lectin may be selected from the group consisting of ABA, ACA, ACG, BPL and RCA-I.

　本発明の虫歯予防用医薬品組成物の製造のための使用において、前記レクチンは、マンノース又はＮアセチルグルコサミンを認識する場合がある。 In the use for producing the pharmaceutical composition for preventing caries of the present invention, the lectin may recognize mannose or N-acetylglucosamine.

　本発明の虫歯予防用医薬品組成物の製造のための使用において、前記レクチンはＧＲＦＴの場合がある。 In the use for producing the pharmaceutical composition for preventing caries of the present invention, the lectin may be GRFT.

　本発明のレクチンは、ヤナギマツタケ由来のＡＣＧ（Ａｇｒｏｃｙｂｅ　ｃｙｌｉｎｄｒａｃｅａ　ｇａｌｅｃｔｉｎ）と、アミヒラタケ由来のＰＳＬ１α（Ｐｏｌｙｐｏｒｕｓ　ｓｑｕａｍｏｓｕｓ　ｌｅｃｔｉｎ　１　ａｌｐｈａ）と、ムジナタケ由来のＰＶＬ（Ｐｓａｔｈｙｒｅｌｌａ　ｖｅｌｕｔｉｎａ　ｌｅｃｔｉｎ）と、ラッパズイセン由来のＮＰＬ（Ｎａｒｃｉｓｓｕｓ　ｐｓｅｕｄｏｎａｒｃｉｓｓｕｓ　ｌｅｃｔｉｎ）と、ヒイロチャワンタケ由来のＡＡＬ（Ａｌｅｕｒｉａ　ａｕｒａｎｔｉａ　ｌｅｃｔｉｎ）と、マッシュルーム由来のＡＢＡ（Ａｇａｒｉｃｕｓ　ｂｉｓｐｏｒｕｓ　ａｇｇｌｕｔｉｎｉｎ）と、ヒモゲイトウ由来のＡＣＡ（Ａｍａｒａｎｔｈｕｓ　ｃａｕｄａｔｕｓ　ａｇｇｌｕｔｉｎｉｎ）と、ムラサキソシンカ由来のＢＰＬ（Ｂａｕｈｉｎｉａ　ｐｕｒｐｕｒｅａ　ｌｅｃｔｉｎ）と、ジャックフルーツ（Ａｒｔｏｃａｒｐｕｓ　ｉｎｔｅｇｒｉｆｏｌｉａ）由来のＪａｃａｌｉｎと、ヒマ由来のＲＣＡ－Ｉ（Ｒｉｃｉｎｕｓ　ｃｏｍｍｕｎｉｓ　ａｇｇｌｕｔｉｎｉｎ－Ｉ）と、イヌエンジュ由来のＭＡＨ（Ｍａａｃｋｉａ　ａｍｕｒｅｎｓｉｓ　ｈｅｍａｇｇｌｕｔｉｎｉｎ）と、紅藻（Ｇｒｉｆｆｉｔｈｓｉａ　ｓｐ．）由来のＧＲＦＴとを含むが、これらに限定されない。 The lectins of the present invention are ACG (Agrocybe cylindracea gallectin) derived from willow matsutake, PSL1α (Polyporus squamusus lectinus rapeselpsein, PSLinsensen from P. ), AAL (Aleuria aurantia lectin) derived from Hilochawantake, ABA (Agaricus bisporus agglutinin) derived from mushrooms, ACA (Amaranthus caudatus agglutinin) derived from Amaranthus Rasosocinka-derived BPL (Bauhinia purpurea electin), Jackfruit (Artocarpus integrifolia) -derived Jacalin, castor-derived RCA-I (Ricinus communis agglutinin-I), and Hengenta-derived algae magnuminh (GRiffithsia sp.)-Derived GRFT, but is not limited thereto.

　本発明のレクチンが認識する糖鎖は、Ｓｉａｌｙｌα２－６Ｇａｌ、Ｇａｌβ１－４ＧｌｃＮＡｃ、ＧｌｃＮＡｃβ１－２Ｍａｎ、Ｎｅｕ５Ａｃ２－３Ｇａｌ１－４ＧｌｃＮＡｃ１－２Ｍａｎ１－６（Ｎｅｕ５Ａｃ２－３／６（Ｎｅｕ５Ａｃ－２－３Ｇａｌ１－４）ＧｌｃＮＡｃ１－４（Ｎｅｕ５Ａｃ２－３／６Ｇａｌ１－４ＧｌｃＮＡｃ１－２）Ｍａｎ１－３）Ｍａｎ、Ｍａｎα１－６Ｍａｎ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌ－Ｇａｌβ１－３　ＧａｌＮＡｃ、Ｆｕｃα１－６ＧｌｃＮＡｃ、Ｆｕｃα１－３ＧａｌＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、ＧｌｃＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３ｌａｃｔｏｓｅ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｍａｎｎｏｓｅ、ＧｌｃＮＡｃ、Ｓｉａｌｙｌ－Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３Ｇａｌβ１－３ＧａｌＮＡｃ、Ｇａｌ、ＧａｌＮＡｃを含むが、これらに限定されない。 The sugar chains recognized by the lectin of the present invention are Siallyα2-6Gal, Galβ1-4GlcNAc, GlcNAcβ1-2Man, Neu5Ac2-3Gal1-4GlcNAc1-2Man1-6 (Neu5Ac2-3 / 6 (Neu5Ac-2-3Gal1-4) GlcNAc-4 (Neu5Ac2-3 / 6Gal1-4GlcNAc1-2) Man1-3) Man, Manα1-6Man, Galβ1-3GalNAc, Sialyl-Galβ1-3 GalNAc, Fucα1-6GlcNAc, Fucα1-3GalNAG, Galβ1-3GalNAG, Sialylα2-3 lactose, Galβ1-3GalNAc, Mannose, GlcNAc, Sialyl-Galβ1 3GalNAc, Sialylα2-3Galβ1-3GalNAc, Gal, including GalNAc, but not limited to.

　本発明のレクチンのうち、ＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉのアミノ酸配列は、それぞれ、配列番号１ないし９に記載される。 Among the lectins of the present invention, the amino acid sequences of AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I are described in SEQ ID NOs: 1 to 9, respectively.

　本発明のタンパク質のアミノ酸配列は、配列番号１ないし９に記載のアミノ酸配列からなるレクチンと同じ糖鎖認識特異性を有することを条件として、配列番号１ないし９に記載のアミノ酸配列に１個若しくは数個のアミノ酸が欠失、置換又は付加されたものであってもよい。ここで、前記欠失、置換又は付加されるアミノ酸の数は、糖鎖の特異性認識に関与するドメイン以外の部位であれば１０個又は１１個以上であってもかまわない。前記欠失、置換又は付加されるアミノ酸の数は、１、２、３、４、５、６、７、８又は９の場合がある。 The amino acid sequence of the protein of the present invention is one in the amino acid sequence shown in SEQ ID NOs: 1 to 9 on the condition that it has the same sugar chain recognition specificity as the lectin consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 9. Several amino acids may be deleted, substituted or added. Here, the number of amino acids to be deleted, substituted or added may be 10 or 11 or more as long as it is a site other than the domain involved in sugar chain specificity recognition. The number of amino acids deleted, substituted or added may be 1, 2, 3, 4, 5, 6, 7, 8 or 9.

　本明細書においてアミノ酸配列の相同性は、本発明のアミノ酸配列と、比較対象のアミノ酸配列との間で配列が一致するアミノ酸残基の数が最も多くなるように整列させて、配列が一致するアミノ酸残基の数の合計を本発明のアミノ酸配列のアミノ酸残基の総数で割った商の百分率で表される。本発明のヌクレオチド配列及びアミノ酸配列の相同性は、当業者に周知の配列整列プログラムＣＬＵＳＴＡＬＷを使用することにより算出することができる。 In the present specification, the homology of amino acid sequences means that the amino acid sequences of the present invention and the amino acid sequence to be compared are aligned so that the number of amino acid residues having the same sequence is the largest, and the sequences match. It is expressed as a percentage of the quotient obtained by dividing the total number of amino acid residues by the total number of amino acid residues of the amino acid sequence of the present invention. The homology of the nucleotide sequence and amino acid sequence of the present invention can be calculated by using the sequence alignment program CLUSTALW well known to those skilled in the art.

　本発明のタンパク質のアミノ酸配列の相同性は、それぞれ相同性が１００％のアミノ酸配列からなるレクチンと同じ糖鎖認識特異性を有することを条件として、８０％以上の場合があり、８５％以上が好ましく、９０％以上がより好ましく、９５％以上がさらに好ましい。 The homology of the amino acid sequence of the protein of the present invention may be 80% or more, and 85% or more, provided that they have the same sugar chain recognition specificity as a lectin having a 100% homology amino acid sequence. Preferably, 90% or more is more preferable, and 95% or more is more preferable.

　本明細書において「ストリンジェントな条件」とは、Ｓａｍｂｒｏｏｋ、Ｊ．及びＲｕｓｓｅｌｌ、Ｄ．Ｗ．、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　３ｒｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（２００１）に説明されるサザンブロット法で以下の実験条件で行うことを指す。比較対象のヌクレオチド配列からなるポリヌクレオチドをアガロース電気泳動によりバンドを形成させた上で毛管現象又は電気泳動によりニトロセルロースフィルターその他の固相に不動化する。６×　ＳＳＣ及び０．２％　ＳＤＳからなる溶液で前洗浄する。本発明のヌクレオチド配列からなるポリヌクレオチドを放射性同位元素その他の標識物質で標識したプローブと前記固相に不動化された比較対象のポリヌクレオチドとの間のハイブリダイゼーション反応を６×　ＳＳＣ及び０．２％　ＳＤＳからなる溶液中で６５°Ｃ、終夜行う。その後前記固相を１×　ＳＳＣ及び０．１％　ＳＤＳからなる溶液中で６５°Ｃ、各３０分ずつ２回洗浄し、０．２×　ＳＳＣ及び０．１％　ＳＤＳからなる溶液中で６５°Ｃ、各３０分ずつ２回洗浄する。最後に前記固相に残存するプローブの量を前記標識物質の定量により決定する。本明細書において「ストリンジェントな条件」でハイブリダイゼーションをするとは、比較対象のヌクレオチド配列からなるポリヌクレオチドを不動化した固相に残存するプローブの量が、本発明のヌクレオチド配列からなるポリヌクレオチドを不動化した陽性対照実験の固相に残存するプローブの量の少なくとも２５％、好ましくは少なくとも５０％、より好ましくは少なくとも７５％以上であることを指す。 In this specification, “stringent conditions” refers to Sambrook, J. et al. And Russell, D .; W. , Molecular Cloning A Laboratory Manual 3rd Edition, Cold Spring Harbor Laboratory Press (2001), and the Southern blot method described in the following experimental conditions. A polynucleotide having a nucleotide sequence to be compared is band-formed by agarose electrophoresis and then immobilized on a nitrocellulose filter or other solid phase by capillary action or electrophoresis. Pre-wash with a solution consisting of 6x SSC and 0.2% SDS. A hybridization reaction between a probe obtained by labeling a polynucleotide comprising the nucleotide sequence of the present invention with a radioisotope or other labeling substance and a polynucleotide to be compared immobilized on the solid phase is performed with 6 × SSC and 0.2. % In SDS solution at 65 ° C overnight. Thereafter, the solid phase was washed twice at 65 ° C. for 30 minutes each in a solution consisting of 1 × SSC and 0.1% SDS, and 65 ° in a solution consisting of 0.2 × SSC and 0.1% SDS. C. Wash twice for 30 minutes each. Finally, the amount of the probe remaining on the solid phase is determined by quantifying the labeling substance. In the present specification, hybridization under “stringent conditions” means that the amount of the probe remaining on the solid phase immobilized with the polynucleotide consisting of the nucleotide sequence to be compared is equal to the polynucleotide consisting of the nucleotide sequence of the present invention. It refers to at least 25%, preferably at least 50%, more preferably at least 75% or more of the amount of probe remaining in the immobilized solid phase of the positive control experiment.

　以下の実施例で説明するとおり、本発明のレクチンが共通して認識する糖鎖は、ＧａｌＮＡｃ又はＧａｌ　β　１－３　ＧａｌＮＡｃである。唾液に含まれる糖蛋白質のムチンはセリン／スレオニンにＧａｌＮＡｃが結合した糖鎖（Ｔｎ抗原）か、Ｇａｌ　β　１－３　ＧａｌＮＡｃ　が結合した糖鎖（Ｔ抗原）かが糖鎖の基本骨格となっている。本明細書の実施例の結果から、ミュータンス菌はムチンのＴｎ抗原又はＴ抗原を認識して唾液に結合していることが示唆された。Ｔｎ抗原又はＴ抗原を認識するレクチンには本発明の実施例で用いた他に以下のものが知られている。
ＨＳＬ　（Ｈｏｌｏｔｈｕｒｉａ　ｓｃａｂｒａ　ｌｅｃｔｉｎ）　由来生物種：ハネジナマコ
ＡＶＬ（Ａｍａｒａｎｔｈｕｓ　ｖｉｒｉｄｉｓ　ｌｅｃｔｉｎ）由来生物種：ホナガイヌビ
ＡＡｌｔＬ　（Ａｒｔｏｃａｒｐｕｓ　ａｌｔｉｌｉｓ　ｌｅｃｔｉｎ）由来生物種：パンノキ
ＡＨＡ（Ａｒｔｏｃａｒｐｕｓ　ｈｉｒｓｕｔａ　ａｇｇｌｕｔｉｎｉｎ）由来生物種：インド産パンノキ属樹木
ＡＬＡ（Ａｒｔｏｃａｒｐｕｓ　ｌａｋｏｏｃｈａ　ａｇｇｌｕｔｉｎｉｎ）由来生物種：ラクチパンノキ
ＡＰＡ（Ａｂｒｕｓ　ｐｒｅｃａｔｏｒｉｕｓ　ａｇｇｌｉｔｉｎｉｎ）由来生物種：トウアズキ
ＡＬＬ（Ａｍａｒａｎｔｈｕｓ　ｌｅｕｃｏｃａｒｐｕｓ　ｌｅｃｔｉｎ）由来生物種：ヒユ属植物
ＣＢＬ（Ｃｌａｒｉａｓ　ｂａｔｒａｃｈｕｓ　ｌｅｃｔｉｎ）由来生物種：インド産ナマズ
ＳＳＬ（Ｓａｌｖｉａ　ｓｃｌａｒｅａ　ｌｅｃｔｉｎ）由来植物：クラリセージ
ＡＲＡ　（Ａｇｒｏｐｙｒｕｍ　ｒｅｐｅｎｓ　ａｇｇｌｕｔｉｎｉｎ）由来生物種：シバムギ
ＬＤｅｌＬ　（Ｌａｃｔａｒｉｕｓ　ｄｅｌｉｃｉｏｓｕｓ　ｌｅｃｔｉｎ）由来生物種：チチタケ属食用キノコ
ＬＤｅｔＬ　（Ｌａｃｔａｒｉｕｓ　ｄｅｔｅｒｒｉｍｕｓ　ｌｅｃｔｉｎ）由来生物種：アカハツモドキ
ＶＧＡ　（Ｖｉｃｉａ　ｇｒａｍｉｎｅａ　ａｇｇｌｕｔｉｎｉｎ）由来生物種：ソラマメ属植物
ＭＬＬ　（Ｍｏｌｕｃｃｅｌｌａ　ｌａｅｖｉｓ　ｌｅｃｔｉｎ）由来生物種：カイガラサルビア As will be described in the following examples, the sugar chain commonly recognized by the lectins of the present invention is GalNAc or Gal β 1-3 GalNAc. The glycoprotein mucin contained in saliva is a sugar chain (Tn antigen) in which GalNAc is bound to serine / threonine, or a sugar chain (T antigen) in which Gal β 1-3 GalNAc is bound. Yes. From the results of the examples of the present specification, it was suggested that the mutans bacteria recognize mucin Tn antigen or T antigen and bind to saliva. In addition to those used in the examples of the present invention, the following are known as lectins that recognize Tn antigen or T antigen.
Biological species derived from HSL (Holothuria scabralectin): Hamanai sea cucumber AVL (Amaranthus viridis lectin) -derived species: Red-backed beetle AAltL (Artocarpus altilis lectin) species: Panca AHA (Artocarpus inhabited) Species derived from Artocarpus lakocha agglutinin: Lactipanca APA (Abrus precatorius agglitinin) Species: Touzuki ALL (Amaranthus leucocarpus lectin) Species from the genus Clarus ass Biospecies: Indian catfish SSL (Salvia sclerea lectin) derived plant: Clarisage ARA (Agropyrum repens agglutinin) Species: Shibamugi LDelL (Lactarius deliciosus lectin) Species: Red-billed Vaginalia VGA (Vicia graminea agglutinin) -derived species: Broad bean plant MLL (Moluccella laevis lectin) -derived species: Caesar salvia

　本発明の虫歯予防剤に含まれるレクチンは、以上に説明された由来生物種の個体、組織、細胞又はその一部から精製されたタンパク質か、組換えＤＮＡ技術によってさまざまな宿主で発現され精製されたタンパク質か、あるいは、前記由来生物種の個体、組織、細胞又はその一部か、その部分精製標品かであって、口腔内に投与されると腔内硬組織への口腔細菌、特に、ミュータンス菌の付着及び増殖を抑制又は阻害する作用を発揮するのに十分な活性を有するものかの場合がある。すなわち、前記レクチンは由来生物種の個体、組織、細胞又はその一部そのもの、あるいは、その加工物として利用される場合がある。 The lectin contained in the dental caries preventive agent of the present invention is a protein purified from an individual, tissue, cell, or part thereof derived from the above-described species, or expressed and purified in various hosts by recombinant DNA technology. Or an individual, tissue, cell or part thereof, or a partially purified preparation of the above-mentioned species, and when administered into the oral cavity, oral bacteria into the intraluminal hard tissue, in particular, In some cases, it has sufficient activity to exert an action of suppressing or inhibiting the adhesion and growth of mutans bacteria. That is, the lectin may be used as an individual, tissue, cell or part of the derived biological species, or a processed product thereof.

　本発明の医薬品組成物は、口腔内硬組織への口腔細菌、特に、ミュータンス菌の付着及び増殖を抑制又は阻害するための全ての医薬品を含み、本発明のレクチンが口腔細菌と唾液由来の生体高分子との結合を抑制又は阻害する活性を実質的に損なわないことを条件として、他のいかなる成分を含むものであってもかまわない。 The pharmaceutical composition of the present invention includes all pharmaceuticals for suppressing or inhibiting the adhesion and proliferation of oral bacteria, particularly mutans bacteria, to the oral hard tissue, and the lectin of the present invention is derived from oral bacteria and saliva. Any other component may be included on the condition that the activity of suppressing or inhibiting the binding with the biopolymer is not substantially impaired.

　本発明の食品組成物は、本発明のレクチンが口腔細菌と唾液由来の生体高分子との結合を抑制又は阻害する活性を実質的に損なわないことを条件として、他のいかなる成分を含むものであってもかまわない。本発明の食品組成物は、エリスリトール、キシリトール等の糖アルコール類を含む場合がある。 The food composition of the present invention contains any other component, provided that the lectin of the present invention does not substantially impair the activity of inhibiting or inhibiting the binding between oral bacteria and saliva-derived biopolymers. It does not matter. The food composition of the present invention may contain sugar alcohols such as erythritol and xylitol.

　本発明の歯磨き剤又は口内洗浄剤は、本発明のレクチンが口腔細菌と唾液由来の生体高分子との結合を抑制又は阻害する活性を実質的に損なわないことを条件として、他のいかなる成分を含むものであってもかまわない。 The dentifrice or mouth rinse of the present invention contains any other components, provided that the lectin of the present invention does not substantially impair the activity of inhibiting or inhibiting the binding between oral bacteria and saliva-derived biopolymers. It may be included.

ミュータンス菌の付着及び増殖に対するレクチンによる抑制効果を示す棒グラフ。The bar graph which shows the inhibitory effect by the lectin with respect to adhesion and proliferation of a mutans bacterium. レクチンによるミュータンス菌の増殖抑制効果を示す顕微鏡写真。The microscope picture which shows the growth inhibitory effect of mutans bacteria by a lectin. ミュータンス菌の付着及び増殖に対するシアル酸認識レクチンによる抑制効果を示す棒グラフ。The bar graph which shows the inhibitory effect by the sialic acid recognition lectin with respect to adhesion and proliferation of a mutans bacterium. 異なる被験者由来の唾液でコーティングされた基質上でのミュータンス菌の増殖に対するレクチンによる抑制効果を示す棒グラフ。The bar graph which shows the inhibitory effect by the lectin with respect to the growth of a mutans bacterium on the substrate coated with the saliva from different test subjects. 異なる被験者由来の唾液でコーティングされた基質上でのミュータンス菌の増殖に対するレクチンによる抑制効果を示す棒グラフ。The bar graph which shows the inhibitory effect by the lectin with respect to the growth of a mutans bacterium on the substrate coated with the saliva from a different test subject. 異なる被験者由来の唾液でコーティングされた基質上でのミュータンス菌の増殖に対するレクチンによる抑制効果を示す棒グラフ。The bar graph which shows the inhibitory effect by the lectin with respect to the growth of a mutans bacterium on the substrate coated with the saliva from a different test subject. 異なる被験者由来の唾液でコーティングされた基質上でのミュータンス菌の増殖に対するレクチンによる抑制効果を示す棒グラフ。The bar graph which shows the inhibitory effect by the lectin with respect to the growth of a mutans bacterium on the substrate coated with the saliva from different test subjects. 図４－７の同一条件ごとの吸光度の測定値の平均から算出された同一条件ごとの抑制指数をプロットした棒グラフ。The bar graph which plotted the suppression index | exponent for every same condition computed from the average of the measured value of the light absorbency for every same condition of FIG. 4-7. ３種類のマルチバレントビオチン化糖ポリマーとミュータンス菌との相互作用を示す棒グラフ。The bar graph which shows interaction with three types of multivalent biotinylated sugar polymers and mutans bacteria.

　以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。 The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the appended claims.

　ミュータンス菌の付着及び増殖に対するレクチンによる抑制効果の検討
　マルチプレート（平底、９６穴、ポリスチレン製、Ｎｕｎｃ社　マルチソープ４６７１４０）に歯磨き後２時間のヒト唾液又はＢＳＡ（ウシ血清アルブミン、１００μｇ／ｍＬをＰＢＳに溶解）を１００μＬ添加して、３７℃で２時間コーティングを行った。その後前記マルチプレートをＰＢＳで洗浄し、１００μｇ／ｍＬのレクチンを１００μＬ添加して３７℃で１時間インキュベーションした。用いたレクチンは、ＡＣＧ（Ａｇｒｏｃｙｂｅ　ｃｙｌｉｎｄｒａｃｅａ　ｇａｌｅｃｔｉｎ）、ＯＡＡ（Ｏｓｃｉｌｌａｔｏｒｉａ　ａｇａｒｄｈｉｉ　ａｇｇｌｕｔｉｎｉｎ）、ＭＶＬ（Ｍｉｃｒｏｃｙｓｔｉｓ　ｖｉｒｉｄｉｓ　ｌｅｃｔｉｎ）、ＧＲＦＴ（Ｇｒｉｆｆｉｔｈｓｉｎ）、ＥＰＬ（Ｅｎｔｅｒｏｍｏｒｐｈａ　ｐｒｏｌｉｆｅｒａ　ｌｅｃｔｉｎ）、Ｃｏｎ　Ａ（Ｃｏｎｃａｎａｖａｌｉｎ　Ａ）、ＧＮＡ（Ｇａｌａｎｔｈｕｓ　ｎｉｖａｌｉｓ　ａｇｇｌｕｔｉｎｉｎ）、ＮＰＬ（Ｎａｒｃｉｓｓｕｓ　ｐｓｅｕｄｏｎａｒｃｉｓｓｕｓ　ｌｅｃｔｉｎ）、ＰＮＡ（Ｐｅａｎｕｔ　ａｇｇｌｕｔｉｎｉｎ）、ＡＡＬ（Ａｌｅｕｒｉａ　ａｕｒａｎｔｉａ　Ｌｅｃｔｉｎ）、ＰＨＡ－Ｌ（Ｐｈａｓｅｏｌｕｓ　ｖｕｌｇａｒｉｓ　ｌｅｕｃｏａｇｇｌｕｔｉｎｉｎ）及びＲＣＡ（Ｒｉｃｉｎｕｓ　ｃｏｍｍｕｎｉｓ　ａｇｇｌｕｔｉｎｉｎ）であった。その後前記マルチプレートをＰＢＳで洗浄し、１００μＬのＰＢＳ中の５×１０7個のミュータンス菌を各ウェルに添加して３７℃で終夜培養した。前記プレートをＰＢＳで洗浄し、０．２５％のグルタルアルデヒド／ＰＢＳを１００μＬ添加して室温で３０分固定した。その後ＰＢＳで洗浄し、２．３％のクリスタルバイオレット（グラム染色用）を１００μＬ添加して室温で１時間染色した。その後流水で洗浄し、エタノールを１００μＬ添加して室温で１時間色素を溶出した。菌体数の定量は、プレートリーダーを用いて各ウェルのエタノール液中の色素濃度を吸光波長５７０ｎｍで測定することによって実施された。 Examination of inhibitory effect of lectin on adhesion and growth of S. mutans Human saliva or BSA (bovine serum albumin, 100 μg / mL) 2 hours after brushing on multiplate (flat bottom, 96-well, polystyrene, Nunc Multisoap 467140) 100 μL of the solution (dissolved in PBS) was added, and coating was performed at 37 ° C. for 2 hours. Thereafter, the multiplate was washed with PBS, 100 μL of 100 μg / mL lectin was added, and incubated at 37 ° C. for 1 hour. Lectins used were, ACG (Agrocybe cylindracea galectin), OAA (Oscillatoria agardhii agglutinin), MVL (Microcystis viridis lectin), GRFT (Griffithsin), EPL (Enteromorpha prolifera lectin), Con A (Concanavalin A), GNA (Galanthus nivalis agglutinin ), NPL (Narcissus pseudonarcissus lectin), PNA (Peanut agglutinin), AAL (Aurelia aurantia Lectin), PHA-L (Phaseolus vulgaris leucoag) lutinin) and RCA (Ricinus communis agglutinin). Thereafter, the multiplate was washed with PBS, 5 × 10 7 mutans bacteria in 100 μL of PBS were added to each well, and cultured at 37 ° C. overnight. The plate was washed with PBS, 100 μL of 0.25% glutaraldehyde / PBS was added, and fixed at room temperature for 30 minutes. After washing with PBS, 100 μL of 2.3% crystal violet (for Gram staining) was added and stained at room temperature for 1 hour. Thereafter, it was washed with running water, 100 μL of ethanol was added, and the dye was eluted at room temperature for 1 hour. The number of bacteria was quantified by measuring the dye concentration in the ethanol solution of each well at an absorption wavelength of 570 nm using a plate reader.

　結果
　図１は、ミュータンス菌の付着及び増殖に対するレクチンによる抑制効果を示す棒グラフである。図１では同一条件ごとに２回重複して実験を行った。唾液によるコーティングを行わなかったウェル（ｓａｌｉｖａ－）と、ミュータンス菌を添加しなかったウェル（Ｓｔ．　ｍｕｔａｎｓ－）とは有意な吸光度を示さなかった。唾液によるコーティングを行ったが、レクチンによるインキュベーションを行わなかったウェル（ｌｅｃｔｉｎ－）の吸光度は０．１０であった。唾液のかわりにＢＳＡによるインキュベーションを行ったウェル（ＢＳＡ）の吸光度は、ｌｅｃｔｉｎ－の半分の０．０５であった。レクチンのうち、ＭＶＬ及びＥＰＬによるインキュベーションを行ったウェルの吸光度は０．１５を超えており、ＯＡＡ、Ｃｏｎ　Ａ、ＰＮＡ、ＰＨＡ－Ｌ及びＲＣＡによるインキュベーションを行ったウェルの吸光度はほぼ０．１０であり、ＧＲＦＴ、ＧＮＡ及びＡＡＬによるインキュベーションを行ったウェルの吸光度は０．０５と０．１０の間であったが、ＡＣＧ及びＮＰＬによるインキュベーションを行ったウェルの吸光度は０．０５未満であった。これらの結果から、唾液によるコーティングよるミュータンス菌のウェル基質への付着及び増殖の促進はＢＳＡによって部分的に阻害された。そこで、ミュータンス菌のウェル基質への付着には非特異的な吸着の寄与が無視できないことが明らかになった。しかし、ＡＣＧ及びＮＰＬはミュータンス菌の付着及び増殖をＢＳＡより強力に抑制した。そこで、ＡＣＧ及びＮＰＬはミュータンス菌の付着及び増殖を特異的に阻害する可能性が高いと考えられた。 Results FIG. 1 is a bar graph showing the inhibitory effect of lectins on adhesion and growth of mutans bacteria. In FIG. 1, the experiment was repeated twice for each identical condition. Wells that were not coated with saliva (saliva − ) and wells that were not added with mutans (St. mutans − ) did not show significant absorbance. The absorbance of the well (lectin − ) that was coated with saliva but not incubated with lectin was 0.10. The absorbance of wells (BSA) incubated with BSA instead of saliva was 0.05, half of lectin − . Among the lectins, the absorbance of wells incubated with MVL and EPL exceeded 0.15, and the absorbance of wells incubated with OAA, Con A, PNA, PHA-L and RCA was approximately 0.10. Yes, the absorbance of wells incubated with GRFT, GNA and AAL was between 0.05 and 0.10, whereas the absorbance of wells incubated with ACG and NPL was less than 0.05. From these results, the adhesion of mutans to the well substrate and the promotion of proliferation by coating with saliva were partially inhibited by BSA. Therefore, it became clear that the contribution of non-specific adsorption cannot be ignored for the attachment of mutans bacteria to the well substrate. However, ACG and NPL suppressed adhesion and growth of S. mutans more strongly than BSA. Therefore, it was considered that ACG and NPL are highly likely to specifically inhibit the adhesion and growth of mutans bacteria.

　図２は、本実施例のそれぞれの条件を示す表と、各条件でのミュータンス菌の増殖を顕微鏡で観察した結果を示す顕微鏡写真とである。陽性対照条件（ｌｅｃｔｉｎ－）のウェルでは、ミュータンス菌が基質表面に拡がってコロニーを形成した。しかし、唾液によるコーティングを行わなかった条件（ｓａｌｉｖａ－）と、ＡＣＧによるインキュベーションを行った条件（ＡＣＧ）と、ＮＰＬによるインキュベーションを行った条件（ＮＰＬ）とでは、ミュータンス菌はほとんど基質上にみられなかった。以上の形態学的な観察結果は吸光度による定量的な測定結果（図１）に対応していた。 FIG. 2 is a table showing the respective conditions of this example, and a photomicrograph showing the results of observing the growth of mutans bacteria under each condition with a microscope. In the well of the positive control condition (lectin − ), mutans bacteria spread on the substrate surface to form colonies. However, under the conditions where saliva coating was not performed (saliva − ), conditions where incubation was performed with ACG (ACG), and conditions where incubation was performed with NPL (NPL), mutans bacteria were mostly found on the substrate. I couldn't. The above morphological observation results corresponded to the quantitative measurement results by absorbance (FIG. 1).

　ミュータンス菌の付着及び増殖に対する他のシアル酸認識レクチンによる抑制効果の検討
　実施例１の結果から、レクチンＡＣＧ及びＮＰＬは虫歯菌のプラーク・バイオフィルム形成を抑制する効果が期待できる。これらのうちＡＣＧはシアル酸認識レクチンに属する。そこで、ＡＣＧの他、ＰＳＬ１α（Ｐｏｌｙｐｏｒｕｓ　ｓｑｕａｍｏｓｕｓ　ｌｅｃｔｉｎ　１　ａｌｐｈａ）、ＰＶＬ（Ｐｓａｔｈｙｒｅｌｌａ　ｖｅｌｕｔｉｎａ　ｌｅｃｔｉｎ）、ＭＡＬ（Ｍａａｃｋｉａ　ａｍｕｒｅｎｓｉｓ　ｌｅｕｃｏａｇｇｌｕｔｉｎｉｎ）、ＭＡＨ（Ｍａａｃｋｉａ　ａｍｕｒｅｎｓｉｓ　ｈｅｍａｇｇｌｕｔｉｎｉｎ）とＳＳＡ（Ｓａｍｂｕｃｕｓ　ｓｉｅｂｏｌｄｉａｎａ　ａｇｇｌｕｔｉｎｉｎ）、ＳＮＡ（Ｓａｍｂｕｃｕｓ　ｎｉｇｒａ　ａｇｇｌｕｔｉｎｉｎ）、ＬＦＡ（Ｌｉｍａｘ　ｆｌａｖｕｓ　ａｇｇｌｕｔｉｎｉｎ）及びＷＧＡ（Ｗｈｅａｔ　ｇｅｒｍ　ａｇｇｌｕｔｉｎｉｎ）というシアル酸認識レクチンについて、実施例１と同じ手順でミュータンス菌の付着及び増殖に対する抑制効果を検討した。 Examination of inhibitory effect by other sialic acid recognition lectins on adhesion and growth of mutans bacteria From the results of Example 1, lectins ACG and NPL can be expected to suppress plaque biofilm formation of caries. Of these, ACG belongs to the sialic acid recognition lectin. Therefore, other ACG, PSL1α (Polyporus squamosus lectin 1 alpha), PVL (Psathyrella velutina lectin), MAL (Maackia amurensis leucoagglutinin), MAH (Maackia amurensis hemagglutinin) and SSA (Sambucus sieboldiana agglutinin), SNA (Sambucus nigra agglutinin), With respect to sialic acid recognition lectins such as LFA (Limax flavus agglutinin) and WGA (Wheat germ agglutinin), the inhibitory effect on adhesion and growth of mutans bacteria was examined by the same procedure as in Example 1.

　結果
　図３は、ミュータンス菌の付着及び増殖に対するシアル酸認識レクチンによる抑制効果を示す棒グラフである。図３では同一条件ごとに２回重複して実験を行った。ミュータンス菌を添加しなかったウェル（Ｓｔ．　ｍｕｔａｎｓ－）は有意な吸光度を示さなかった。唾液によるコーティングを行ったが、レクチンによるインキュベーションを行わなかったウェル（ｌｅｃｔｉｎ－）の吸光度は０．４５であった。今回検討されたシアル酸認識レクチンのうち、ＭＡＨ、ＳＳＡ、ＳＮＡ、ＬＦＡ及びＷＧＡはほぼ唾液によるコーティングを行ったが、レクチンによるインキュベーションを行わなかったウェル（ｌｅｃｔｉｎ－）に近い吸光度であり、ミュータンス菌の付着及び増殖を抑制する効果がほとんど認められなかった。しかし、ＰＳＬ１α及びＰＶＬについてはＡＣＧに近い吸光度であり、ミュータンス菌の付着及び増殖を抑制する効果が認められた。 Results FIG. 3 is a bar graph showing the inhibitory effect of sialic acid-recognizing lectins on adhesion and growth of mutans bacteria. In FIG. 3, the experiment was repeated twice for each same condition. Wells to which no mutans were added (St. mutans − ) did not show significant absorbance. The absorbance of the well (lectin − ) coated with saliva but not incubated with lectin was 0.45. Of the sialic acid-recognizing lectins examined this time, MAH, SSA, SNA, LFA and WGA were almost saliva-coated, but the absorbance was close to that of wells (lectin − ) which were not incubated with lectin, and mutans The effect which suppresses adhesion and proliferation of a microbe was hardly recognized. However, for PSL1α and PVL, the absorbance was close to that of ACG, and an effect of suppressing adhesion and growth of mutans bacteria was observed.

　今回ミュータンス菌の付着及び増殖を抑制する効果が認められたレクチンが認識する糖鎖を表１に示す。 Table 1 shows the sugar chains recognized by the lectins that were confirmed to have the effect of suppressing adhesion and growth of S. mutans.

Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001

　拡大スクリーニング
　本実施例では、実施例１又は２とは異なる４名の被験者由来のヒト唾液でコーティングされた基質へのミュータンス菌の増殖に対する７０種類のレクチンの効果を調べた。 Extended Screening In this example, the effect of 70 lectins on the growth of S. mutans on a substrate coated with human saliva from 4 subjects different from Example 1 or 2 was examined.

　マイクロプレート（Ｎｕｎｃ社、マルチソープ４６７１４０）の各ウェルに歯磨き後３時間の唾液１００μＬを添加し、３７°Ｃ、２時間コーティングを行った。その後、前記マイクロプレートをＰＢＳで洗浄し、ＴＢＳ（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ、１５０ｍＭ　ＮａＣｌ、１ｍＭ　ＣａＣｌ２、１ｍＭ　ＭｎＣｌ２）で１００μｇ／ｍＬに希釈されたレクチン又はＢＳＡ１００μＬを各ウェルに添加して、３７°Ｃ、１時間反応させた。その後、ＰＢＳで洗浄し、１００μＬのＰＢＳ中の５×１０７個のミュータンス菌（ＡＴＣＣ２５１７５）を３７°Ｃ、終夜培養した。菌をＰＢＳで洗浄し、０．２５％　グルタルアルデヒド／ＰＢＳを１００μＬ添加して室温で３０分固定した。その後ＰＢＳで洗浄し、２．３％　クリスタルバイオレットを１００μＬ添加して、室温で１時間染色した。その後流水で洗浄し、エタノールを１００μＬ添加して室温で１時間色素を溶出した。菌体数の定量は、プレートリーダーを用いて各ウェルのエタノール液中の色素濃度を吸光波長５７０ｎｍで測定することによって実施された。 100 μL of saliva 3 hours after brushing was added to each well of a microplate (Nunc, Multisoap 467140), and coating was performed at 37 ° C. for 2 hours. Thereafter, the microplate was washed with PBS, and 100 μL of lectin or BSA diluted to 100 μg / mL with TBS (50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MnCl 2 ) was added to each well, and 37 ° C. Reacted for 1 hour. Thereafter, the cells were washed with PBS, and 5 × 10 7 mutans bacteria (ATCC 25175) in 100 μL of PBS were cultured at 37 ° C. overnight. The bacteria were washed with PBS, 100 μL of 0.25% glutaraldehyde / PBS was added, and fixed at room temperature for 30 minutes. After washing with PBS, 100 μL of 2.3% crystal violet was added and stained at room temperature for 1 hour. Thereafter, it was washed with running water, 100 μL of ethanol was added, and the dye was eluted at room temperature for 1 hour. The number of bacteria was quantified by measuring the dye concentration in the ethanol solution of each well at an absorption wavelength of 570 nm using a plate reader.

　用いたレクチンは以下のとおりであった。天然レクチンは、ＥＹ　Ｌａｂｏｒａｔｏｒｉｅｓ社製のＡＡＡ、ＡＰＰ、ＢＤＡ、ＣＡ、ＣＡＡ、ＣＳＡ、ＤＢＡ、ＤＳＡ、ＥＣＡ、ＧＮＡ、ＧＳ－ＩＩ、ＨＰＡ、Ｊａｃａｌｉｎ、ＬＡＡ、ＬＢＡ、ＬＦＡ、Ｌｏｔｕｓ、ＮＰＡ、ＰＮＡ、ＰＳＡ、ＰＴＡ－Ｇａｌ、ＰＴＡ－ＧａｌＮＡｃ、ＰＷＭ、ＳＨＡ、ＳＪＡ、ＳＮＡ、ＳＴＡ、ＴＫＡ、ＴＬ、ＵＤＡ、ＵＥＡ－Ｉ、ＵＥＡ－ＩＩ、ＶＦＡ、ＷＧＡ及びＲＣＡ－Ｉと、Ｖｅｃｔｏｒ　Ｌａｂｏｒａｔｏｒｉｅｓ社製のＡＣＡ、ＢＰＬ、ＥＥＬ、ＧＮＬ、ＤＳＬ－ＩＩ、ＨＨＬ、ＭＡＬ、ＮＰＬ、ＰＨＡ－Ｅ４、ＰＴＬ－Ｉ、ＰＴＬ－ＩＩ、ＳｃＷＧＡ、ＷＦＬ及びＭＡＨと、生化学工業株式会社製のＡＡＬ、ＡＢＡ、Ｃｏｎ　Ａ、ＳＢＡ、ＳＳＡ、ＴＪＡ－Ｉ、ＴＪＡ－ＩＩとであった。組換えレクチンは、ｒＡＣＧ及びｒＧＲＦＴで、これらはグライエンス社内で調製された。 The lectins used were as follows. Natural lectins are AAA, APP, BDA, CA, CAA, CSA, DBA, DSA, ECA, GNA, GS-II, HPA, Jacalin, LAA, LBA, LFA, Lotus, NPA, PNA, PSA manufactured by EY Laboratories. , PTA-Gal, PTA-GalNAc, PWM, SHA, SJA, SNA, STA, TKA, TL, UDA, UEA-I, UEA-II, VFA, WGA and RCA-I, and ACA and BPL made by Vector Laboratories , EEL, GNL, DSL-II, HHL, MAL, NPL, PHA-E4, PTL-I, PTL-II, ScWGA, WFL and MAH, AAL, ABA, Con A, SBA, manufactured by Seikagaku Corporation With SSA, TJA-I, and TJA-II It was. Recombinant lectins are rACG and rGRFT, which were prepared in-house at Glience.

　結果
　図４－７はそれぞれ異なる被験者由来の唾液でコーティングされた基質上でのミュータンス菌の増殖に対するレクチンによる抑制効果を示す棒グラフである。いずれも同一条件ごとに２回重複して実験を行った。各図の左端から４本の棒グラフは対照実験の結果で、左端から順にレクチン－、ミュータンス菌－及び唾液－（ウェルのみ）と、レクチン－、ミュータンス菌＋及び唾液－（ミュータンス菌のみ）と、レクチン－、ミュータンス菌－及び唾液＋（唾液のみ）と、レクチン－、ミュータンス菌＋及び唾液＋（唾液＋ミュータンス菌（レクチン無しの陽性対照））という４種類の条件であった。唾液によるコーティングを行わなかったウェルと、ミュータンス菌を添加しなかったウェルとは、有意なミュータンス菌の増殖を示さなかった。唾液によるコーティングを行ったが、レクチンによるインキュベーションを行わなかったウェルと、唾液のかわりにＢＳＡによるインキュベーションを行ったウェル（ＢＳＡ）とでは、ミュータンス菌の増殖が認められた。図４－７に示すとおり、唾液を適用した被験者ごとにミュータンス菌の増殖を抑制するレクチンの種類及び抑制効果の程度に差が見られた。また、ＢＳＡによるミュータンス菌の増殖抑制効果の程度も、被検者ごとに異なっていた。 Results FIG. 4-7 is a bar graph showing the inhibitory effect of lectins on the growth of S. mutans on substrates coated with saliva from different subjects. In each case, the experiment was repeated twice for the same conditions. The four bar graphs from the left end of each figure are the results of a control experiment. From the left end, lectin-, mutans bacteria- and saliva- (wells only), lectin-, mutans bacteria + and saliva- (mutans bacteria only) ), Lectin-, mutans bacteria and saliva + (saliva only), lectin-, mutans bacteria + and saliva + (saliva + mutans bacteria (positive control without lectin)). It was. Wells that were not coated with saliva and wells to which no mutans were added did not show significant growth of mutans. Proliferation of mutans was observed in wells that were coated with saliva but not incubated with lectin and wells that were incubated with BSA instead of saliva (BSA). As shown in FIG. 4-7, there were differences in the types of lectins that inhibit the growth of S. mutans and the degree of inhibitory effect for each subject who applied saliva. In addition, the degree of the effect of inhibiting the growth of S. mutans by BSA was different for each subject.

　図８は、図４－７の同一条件ごとの吸光度の測定値の平均Ａｘと、同一被験者ごとの唾液によるコーティングを行わなかったウェルの吸光度の測定値の平均Ａｎと、同一被験者ごとの唾液によるコーティングを行ったが、レクチンによるインキュベーションを行わなかったウェルの吸光度の測定値の平均Ａｐとから、同一条件ごとの抑制指数Ｉｘを以下の式を用いて算出してプロットした棒グラフである。
Ｉｘ＝１００×（Ａｘ－Ａｎ）／（Ａｐ－Ａｎ） 8, the average A x of the measured values of absorbance of each of the same conditions in Figure 4-7, the average A n of measurements of absorbance of the wells was not performed coating with saliva per same subject, of each of the same subject were subjected to coating with saliva, the average a p of measurements of absorbance of the wells that did not perform the incubation with lectin, the inhibition index I x of each of the same conditions in a bar graph plotting calculated by using the following formula is there.
I x = 100 × (A x −A n ) / (A p −A n )

　図８に示すとおり、調べたレクチンのうちＡＡＬ、ＡＢＡ、ＡＣＡ、ＢＰＬ、Ｊａｃａｌｉｎ、ＲＣＡ－Ｉ、ＭＡＨ、ＡＣＧ及びＧＲＦＴについては、ミュータンス菌の増殖を抑制する効果が認められた。 As shown in FIG. 8, among the lectins examined, AAL, ABA, ACA, BPL, Jacalin, RCA-I, MAH, ACG and GRFT were found to have an effect of suppressing the growth of mutans bacteria.

　本実施例に用いたレクチンが認識する糖鎖を表２に示す。 Table 2 shows the sugar chains recognized by the lectin used in this example.

Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002

　合成糖鎖とミュータンス菌との相互作用
　本実施例では、固相に吸着された合成糖鎖とミュータンス菌との結合を表面プラズモン効果によって測定して、ミュータンス菌と糖鎖との特異的な相互作用を定量的に解析した。 In this example, the binding between the synthetic sugar chain adsorbed on the solid phase and the mutans bacterium is measured by the surface plasmon effect, and the mutans bacterium and the sugar chain are specifically identified. The interaction was quantitatively analyzed.

　以下の４種類のマルチバレントビオチン化糖ポリマー（ＧｌｙｃｏＴｅｃｈ社）を用いた。
ＨＯＣＨ２（ＨＯＣＨ）４ＣＨ２ＮＨ－ＰＡＡ－ｂｉｏｔｉｎ　（対照）
Ｇａｌβ１－３ＧａｌＮＡｃα－ＰＡＡ－ｂｉｏｔｉｎ　（Ｃｏｒｅ１）
ＧｌｃＮＡｃβ１－３ＧａｌＮＡｃα－ＰＡＡ－ｂｉｏｔｉｎ　（Ｃｏｒｅ３）
Ｎｅｕ５Ａｃα２－３Ｇａｌβ１－３ＧａｌＮＡｃα－ＰＡＡ－ｂｉｏｔｉｎ　（Ｓｉａｌｙｌ　Ｔ） The following four types of multivalent biotinylated sugar polymers (GlycoTech) were used.
HOCH 2 (HOCH) 4 CH 2 NH—PAA-biotin (control)
Galβ1-3GalNAcα-PAA-biotin (Core1)
GlcNAcβ1-3GalNAcα-PAA-biotin (Core3)
Neu5Acα2-3Galβ1-3GalNAcα-PAA-biotin (Sialyl T)

　前記マルチバレントビオチン化糖ポリマーはそれぞれＰＢＳで５０μｇ／ｍＬに希釈され、１０°Ｃのビアコア社製センサーチップ（ＢＲ－１０００－３２）に１０μＬ／分の流速でローディングされ、Ｂｉａｃｏｒｅ２０００で約１０００ＲＵの測定値に達するまでセンサー基板表面に固相化された。 Each of the multivalent biotinylated sugar polymers is diluted to 50 μg / mL with PBS, loaded onto a Biacore sensor chip (BR-1000-32) at 10 ° C. at a flow rate of 10 μL / min, and measured at about 1000 RU with Biacore 2000. It was immobilized on the surface of the sensor substrate until the value was reached.

　ミュータンス菌は、Ｔｏｄｄ－Ｈｅｗｉｔｔ　Ｂｒｏｔｈで３７°Ｃ、終夜培養され、１，０００×ｇ、１０分間の遠心により上清除去後、等量のＰＢＳに懸濁された。同様の操作を２度繰り返して洗浄した後、６６０ｎｍでの吸光度を測定した。測定した吸光度を基に、ミュータンス菌はＰＢＳでＯＤ６６０＝０．０５又は０．１に希釈され、ミュータンス菌サンプル液が調製された。前記サンプル液は、密封され、２５°Ｃで５時間保温された後測定に供された。前記マルチバレントビオチン化糖ポリマーが固相化されたセンサーチップに１０μＬ／分の流速で４０μＬの前記サンプル液が添加され、ＰＢＳを１０μＬ／分の流速で１分間流してセンサー基板表面をリンスした後、Ｂｉａｃｏｒｅ２０００で測定した。図１０は、Ｃｏｒｅ１、Ｃｏｒｅ３及びＳｉａｌｙｌ　Ｔのポリマーにミュータンス菌サンプル液を添加してリンスした後の測定値から、対照ポリマーにミュータンス菌サンプル液を添加してリンスした後の測定値を差し引いた値を示す棒グラフである。 M. mutans was cultured overnight at 37 ° C. in Todd-Hewitt Broth, and the supernatant was removed by centrifugation at 1,000 × g for 10 minutes and then suspended in an equal volume of PBS. The same operation was repeated twice and washed, and then the absorbance at 660 nm was measured. Based on the measured absorbance, the mutans bacterium was diluted with PBS to OD660 = 0.05 or 0.1 to prepare a mutans bacterium sample solution. The sample solution was sealed and kept at 25 ° C. for 5 hours, and then subjected to measurement. After rinsing the sensor substrate surface by adding 40 μL of the sample solution at a flow rate of 10 μL / min to the sensor chip on which the multivalent biotinylated sugar polymer is immobilized, and flowing PBS at a flow rate of 10 μL / min for 1 minute. , Measured with Biacore 2000. FIG. 10 shows the results after adding and rinsing the mutans sample solution to the Core1, Core3, and Sialyl T polymers, and subtracting the measured value after adding the mutans sample solution to the control polymer and rinsing. It is a bar graph which shows the value.

　結果
　図９に示すとおり、Ｃｏｒｅ１ポリマーは他のポリマーより格段に強くミュータンス菌と相互作用した。そこで、唾液由来の糖鎖のうちガラクトースを末端に含む糖鎖はミュータンス菌の基質への付着に関与することが実証された。したがって、本発明のレクチンを含む虫歯予防剤と、該虫歯予防剤を用いる虫歯予防方法とは、唾液由来の糖鎖、特に、ガラクトースを末端に含む糖鎖とミュータンス菌との相互作用を阻害すると考えられる。 Results As shown in FIG. 9, the Core1 polymer interacted with mutans bacteria much more strongly than the other polymers. Thus, it was demonstrated that sugar chains containing galactose at the end of sugar chains derived from saliva are involved in adhesion to the substrate of mutans bacteria. Therefore, the caries preventive agent containing the lectin of the present invention and the caries preventive method using the caries preventive agent inhibit the interaction between the sugar chain derived from saliva, in particular, the sugar chain containing galactose at the terminal and the mutans bacterium. I think that.

Claims (28)

　レクチンを含むことを特徴とする虫歯予防剤。 A caries preventive agent characterized by containing a lectin. 　前記レクチンはＦｕｃα１－６ＧｌｃＮＡｃ、Ｆｕｃα１－３ＧａｌＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、ＧｌｃＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３ｌａｃｔｏｓｅ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌ－Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３Ｇａｌβ１－３ＧａｌＮＡｃ、Ｇａｌ及びＧａｌＮＡｃからなるグループから選択される少なくとも１種類の糖鎖を認識することを特徴とする、請求項１に記載の虫歯予防剤。 The lectin is selected from Fucα1-6GlcNAc, Fucα1-3GalNAc, Galβ1-3GalNAc, GlcNAc, Galβ1-3GalNAc, Sialylα2-3 lactose, Galβ1-3GalNAc, Sialyl-Galβ1-3GalNAc, and Sialylα-3GalNAc, The caries preventive agent according to claim 1, wherein at least one kind of sugar chain is recognized. 　前記レクチンは、
（１）ＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと、
（２）配列番号１ないし９に記載のアミノ酸配列からなるタンパク質と、
（３）配列番号１ないし９に記載のアミノ酸配列に記載のアミノ酸配列に１個若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、
（４）配列番号１ないし９に記載のアミノ酸配列と８０％以上の相同性を示すアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、
（５）配列番号１ないし９に記載のアミノ酸配列をエンコードするヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチドによってエンコードされるアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、
からなるグループから選択される１種類又は２種類以上のタンパク質であることを特徴とする、請求項１に記載の虫歯予防剤。 The lectin is
(1) AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I;
(2) a protein comprising the amino acid sequence of SEQ ID NOS: 1 to 9,
(3) It consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence described in SEQ ID NOs: 1 to 9, and each of AAL, ABA, ACA, and ACG , Proteins having the same sugar chain recognition specificity as BPL, GRFT, Jacalin, MAH and RCA-I;
(4) consisting of an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NOS: 1 to 9, and AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, respectively A protein having the same sugar chain recognition specificity;
(5) consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NOs: 1 to 9, and AAL, ABA, A protein having the same sugar chain recognition specificity as ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I;
The caries preventive agent according to claim 1, which is one or more proteins selected from the group consisting of: 　前記レクチンはシアル酸を認識することを特徴とする、請求項１に記載の虫歯予防剤。 The caries preventive agent according to claim 1, wherein the lectin recognizes sialic acid. 　前記レクチンは、ＡＣＧ、ＭＡＨ及びＪａｃａｌｉｎからなるグループから選択されることを特徴とする、請求項４に記載の虫歯予防剤。 5. The dental caries preventive agent according to claim 4, wherein the lectin is selected from the group consisting of ACG, MAH and Jacalin. 　前記レクチンは、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ及びＲＣＡ－Ｉからなるグループから選択されることを特徴とする、請求項１に記載の虫歯予防剤。 The caries preventive agent according to claim 1, wherein the lectin is selected from the group consisting of ABA, ACA, ACG, BPL and RCA-I. 　前記レクチンは、マンノース又はＮアセチルグルコサミンを認識することを特徴とする、請求項１に記載の虫歯予防剤。 2. The caries preventive agent according to claim 1, wherein the lectin recognizes mannose or N-acetylglucosamine. 　前記レクチンはＧＲＦＴであることを特徴とする、請求項８に記載の虫歯予防剤。 The caries preventive agent according to claim 8, wherein the lectin is GRFT. 　請求項１ないし８のいずれか１つに記載の虫歯予防剤を含むことを特徴とする、医薬品組成物。 A pharmaceutical composition comprising the caries preventive agent according to any one of claims 1 to 8. 　請求項１ないし８のいずれか１つに記載の虫歯予防剤を含むことを特徴とする、食品組成物。 A food composition comprising the caries preventive agent according to any one of claims 1 to 8. 　請求項１ないし８のいずれか１つに記載の虫歯予防剤を含むことを特徴とする、歯磨き剤。 A dentifrice comprising the caries preventive agent according to any one of claims 1 to 8. 　請求項１ないし８のいずれか１つに記載の虫歯予防剤を含むことを特徴とする、口内洗浄剤。 Mouth washing agent characterized by including the caries preventive agent according to any one of claims 1 to 8. 　レクチンを含む虫歯予防剤を口腔内に投与するステップを含むことを特徴とする、虫歯の予防方法。 A method for preventing caries, comprising a step of administering a caries preventive agent containing lectin into the oral cavity. 　前記レクチンはＦｕｃα１－６ＧｌｃＮＡｃ、Ｆｕｃα１－３ＧａｌＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、ＧｌｃＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３ｌａｃｔｏｓｅ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌ－Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３Ｇａｌβ１－３ＧａｌＮＡｃ、Ｇａｌ及びＧａｌＮＡｃからなるグループから選択される少なくとも１種類の糖鎖を認識することを特徴とする、請求項１３に記載の虫歯の予防方法。 The lectin is selected from Fucα1-6GlcNAc, Fucα1-3GalNAc, Galβ1-3GalNAc, GlcNAc, Galβ1-3GalNAc, Sialylα2-3 lactose, Galβ1-3GalNAc, Sialyl-Galβ1-3GalNAc, and Sialylα-3GalNAc, The method for preventing caries according to claim 13, wherein at least one sugar chain is recognized. 　前記レクチンは、
（１）ＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと、
（２）配列番号１ないし９に記載のアミノ酸配列からなるタンパク質と、
（３）配列番号１ないし９に記載のアミノ酸配列に記載のアミノ酸配列に１個若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、
（４）配列番号１ないし９に記載のアミノ酸配列と８０％以上の相同性を示すアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、
（５）配列番号１ないし９に記載のアミノ酸配列をエンコードするヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチドによってエンコードされるアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、
からなるグループから選択される１種類又は２種類以上のタンパク質であることを特徴とする、請求項１３に記載の虫歯の予防方法。 The lectin is
(1) AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I;
(2) a protein comprising the amino acid sequence of SEQ ID NOS: 1 to 9,
(3) It consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence described in SEQ ID NOs: 1 to 9, and each of AAL, ABA, ACA, and ACG , Proteins having the same sugar chain recognition specificity as BPL, GRFT, Jacalin, MAH and RCA-I;
(4) consisting of an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NOS: 1 to 9, and AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, respectively A protein having the same sugar chain recognition specificity;
(5) consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NOs: 1 to 9, and AAL, ABA, A protein having the same sugar chain recognition specificity as ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I;
The method for preventing dental caries according to claim 13, wherein the protein is one or more proteins selected from the group consisting of: 　前記レクチンはシアル酸を認識することを特徴とする、請求項１３に記載の虫歯の予防方法。 The method for preventing caries according to claim 13, wherein the lectin recognizes sialic acid. 　前記レクチンは、ＡＣＧ、ＭＡＨ及びＪａｃａｌｉｎからなるグループから選択されることを特徴とする、請求項１６に記載の虫歯の予防方法。 The method for preventing caries according to claim 16, wherein the lectin is selected from the group consisting of ACG, MAH and Jacalin. 　前記レクチンは、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ及びＲＣＡ－Ｉからなるグループから選択されることを特徴とする、請求項１３に記載の虫歯の予防方法。 The method for preventing caries according to claim 13, wherein the lectin is selected from the group consisting of ABA, ACA, ACG, BPL and RCA-I. 　前記レクチンは、マンノース又はＮアセチルグルコサミンを認識することを特徴とする、請求項１３に記載の虫歯の予防方法。 The method for preventing caries according to claim 13, wherein the lectin recognizes mannose or N-acetylglucosamine. 　前記レクチンは、ＧＲＦＴであることを特徴とする、請求項１９に記載の虫歯の予防方法。 The method for preventing caries according to claim 19, wherein the lectin is GRFT. 　レクチンの虫歯予防用医薬品組成物の製造のための使用。 使用 Use of lectins for the manufacture of pharmaceutical compositions for preventing caries. 　前記レクチンは、Ｆｕｃα１－６ＧｌｃＮＡｃ、Ｆｕｃα１－３ＧａｌＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、ＧｌｃＮＡｃ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３ｌａｃｔｏｓｅ、Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌ－Ｇａｌβ１－３ＧａｌＮＡｃ、Ｓｉａｌｙｌα２－３Ｇａｌβ１－３ＧａｌＮＡｃ、Ｇａｌ及びＧａｌＮＡｃからなるグループから選択される少なくとも１種類の糖鎖を認識することを特徴とする、請求項２１に記載の使用。 The lectin is selected from Fucα1-6GlcNAc, Fucα1-3GalNAc, Galβ1-3GalNAc, GlcNAc, Galβ1-3GalNAc, Sialylα2-3 lactose, Galβ1-3GalNAc, Sialyl-Galβ1-3GalNAc, and SiialβG Use according to claim 21, characterized in that it recognizes at least one sugar chain. 　前記レクチンは、
（１）ＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと、
（２）配列番号１ないし９に記載のアミノ酸配列からなるタンパク質と、
（３）配列番号１ないし９に記載のアミノ酸配列に記載のアミノ酸配列に１個若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、
（４）配列番号１ないし９に記載のアミノ酸配列と８０％以上の相同性を示すアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、
（５）配列番号１ないし９に記載のアミノ酸配列をエンコードするヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチドによってエンコードされるアミノ酸配列からなり、かつ、それぞれＡＡＬ、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ、ＧＲＦＴ、Ｊａｃａｌｉｎ、ＭＡＨ及びＲＣＡ－Ｉと同じ糖鎖認識特異性を有するタンパク質と、
からなるグループから選択される１種類又は２種類以上のタンパク質であることを特徴とする、請求項２１に記載の使用。 The lectin is
(1) AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I;
(2) a protein comprising the amino acid sequence of SEQ ID NOS: 1 to 9,
(3) It consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence described in SEQ ID NOs: 1 to 9, and each of AAL, ABA, ACA, and ACG , Proteins having the same sugar chain recognition specificity as BPL, GRFT, Jacalin, MAH and RCA-I;
(4) consisting of an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NOS: 1 to 9, and AAL, ABA, ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I, respectively A protein having the same sugar chain recognition specificity;
(5) consisting of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NOs: 1 to 9, and AAL, ABA, A protein having the same sugar chain recognition specificity as ACA, ACG, BPL, GRFT, Jacalin, MAH and RCA-I;
Use according to claim 21, characterized in that it is one or more proteins selected from the group consisting of. 　前記レクチンはシアル酸を認識することを特徴とする、請求項２１に記載の使用。 The use according to claim 21, characterized in that the lectin recognizes sialic acid. 　前記レクチンは、ＡＣＧ、ＭＡＨ及びＪａｃａｌｉｎからなるグループから選択されることを特徴とする、請求項２４に記載の使用。 25. Use according to claim 24, characterized in that the lectin is selected from the group consisting of ACG, MAH and Jacalin. 　前記レクチンは、ＡＢＡ、ＡＣＡ、ＡＣＧ、ＢＰＬ及びＲＣＡ－Ｉからなるグループから選択されることを特徴とする、請求項２１に記載の使用。 The use according to claim 21, characterized in that the lectin is selected from the group consisting of ABA, ACA, ACG, BPL and RCA-I. 　前記レクチンは、マンノース又はＮアセチルグルコサミンを認識することを特徴とする、請求項２１に記載の使用。 Use according to claim 21, characterized in that the lectin recognizes mannose or N-acetylglucosamine. 　前記レクチンはＧＲＦＴであることを特徴とする、請求項２７に記載の使用。 28. Use according to claim 27, characterized in that the lectin is GRFT.
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