WO2010107120A1 - Antibody and anti-periodontal disease composition containing antibody - Google Patents

Abstract

Provided are an antibody which is effective in inhibiting the formation of a biofilm and removing the same and has a high effect against periodontal bacteria; and an anti-periodontal disease composition which contains said antibody. A chicken egg antibody, which is obtained from an avian egg having been immunized with an antigen that is a culture obtained by mixed-culturing two or more kinds of periodontal bacteria, is used as an anti-periodontal disease composition. The periodontal bacteria preferably comprise two or more kinds of bacteria selected from among Porphyromonas gingivalis, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Tannerella forsythensis, Treponema denticola, Prevotella intermedia and Streptococcus gordonii.

Description

Antibody and anti-periodontal disease composition containing antibody

The present invention relates to an antibody and an anti-periodontal disease composition containing the antibody, and in particular, a chicken egg antibody that suppresses the formation of a biofilm that becomes a lesion of periodontal disease bacteria and reduces the periodontal disease risk, and the chicken egg antibody. It is related with the anti-periodontal disease composition containing.

Periodontal disease is a national disease affecting approximately 80% of adults in Japan, and not only directly causes tooth loss, but also systemic diseases such as heart disease, cerebrovascular disease, pneumonia, lifestyle-related diseases, etc. A strong relationship with is clear.

Periodontal disease is an oral infection caused by periodontal disease bacteria attaching to and growing on the tooth surface and gingiva to form a biofilm. Biofilms are said to form and mature as follows.

First, organic matter (pellicle) in saliva adheres to the tooth surface, and Streptococcus mitis ( Streptococcus oralis ), Streptococcus gordonii ( Streptococcus gordonii ), Streptococcus sanguis ( Streprococcus sanguis ), etc. will start to form colonies. When colonies of these early colonized bacteria are formed on the tooth surface, Fusobacterium nucleatum colonizes and proliferates using the surface protein of these bacteria as a receptor, causing coaggregation. Furthermore, periodontal pathogens such as Porphyromonas gingivalis , Prevotella intermedia , Actinobacillus actinomycetemcomitans , Tannerella forsythesis, Tannerella forsythe · Dentikora (Treponema denticola) late colonizers such is coaggregation, biofilm through the production of polysaccharides are formed. Thus, the balance of the normal bacterial flora in the oral cavity is lost, and a plurality of periodontal pathogens interact with each other to form a biofilm (lesion) having periodontal pathogenicity.

Conventionally, for the prevention and improvement of periodontal disease, it has been proposed to produce an antibody against the above-mentioned periodontal disease bacteria and use it. For example, Patent Document 1 discloses ciliates or capsules of oral bacteria selected from Bacteroides gingivalis, Bacteroides intermedius, Actinomyces viscosus, Haemophilus actinomycetemcomitans, Fusobacterium nucleatum and Aiconella collodense. An oral composition characterized by containing an egg, egg yolk or a water-soluble yolk fraction obtained from a poultry immunized with an antigen as an antigen.

Patent Document 2 contains an antibody obtained by immunizing an animal with a synthetic peptide corresponding to a fragment derived from the amino acid sequence constituting the cilia of Actinobacillus actinomycetemcomitans as an antigen. The composition for oral cavity characterized by this is disclosed.

Patent Document 3 discloses a peptide corresponding to a fragment derived from the amino acid sequence of the 72 KDa subunit protein constituting the type II pili of Porphyromonas gingivalis , wherein 5 to 10 consecutive fragments are present. Periodontal disease preventive or periodontal comprising an antibody obtained by immunizing an animal with a peptide or derivative thereof or a salt thereof selected from a fragment group comprising the amino acid residues of An oral composition for treating disease is disclosed.

Patent Document 4 discloses an oral composition characterized by containing an antibody obtained by immunizing an animal with a polysaccharide derived from the surface of a bacterial body causing periodontal disease as an antigen.

Patent Document 5 includes a composition for oral formation for inhibiting biofilm formation or promoting biofilm removal containing sugar alcohol and / or amino acid, and Patent Document 6 includes a histidine kinase inhibitor selected from ofloxacin and closantel. An oral biofilm characterized in that it is contained at a concentration of 0.0001 to 10% by mass with respect to the whole product, and contains a phenolic fungicide, a surfactant, and one or more of alcohols. Inhibitory composition, Patent Document 7, discloses a coaggregation inhibitor of oral bacteria containing glycine or alanine as an active ingredient.

JP-A-10-152425 JP-A-9-52822 Japanese Patent Laid-Open No. 8-48695 JP-A-6-40871 JP 2005-29484 A JP 2005-187377 A JP 2005-53851 A

However, antibodies obtained using conventional components of periodontal disease bacteria as antigens have the advantage that they can sterilize and remove specific bacteria without affecting other oral resident bacteria. Since it is necessary to act, once the biofilm is formed, it is difficult to act on the bacteria in the biofilm, and a sufficient prevention / improvement effect cannot be expected.

On the other hand, when the drug is used, there is a problem that not only periodontal disease bacteria but also other useful oral resident bacteria are sterilized.

In addition, periodontal pathogens in biofilms are known to interact with multiple types of periodontal pathogens, but little is known about the antigenic changes of periodontal pathogens. No antibody has been developed that focuses on the antigenic changes.

Therefore, an object of the present invention is to provide biofilm formation inhibition and removal thereof, an antibody having a high effect on periodontal disease bacteria, and an anti-periodontal disease composition containing the same.

The present inventors can pay attention to the interaction of multiple types of periodontal pathogens in biofilms, and obtain a more effective antibody by using a mixed culture of multiple types of periodontal pathogens as an antigen. The present inventors have found that this can be done and have completed the present invention.

That is, the antibody of the present invention is obtained from a bird egg immunized with a culture obtained by culturing a mixture of two or more kinds of periodontal disease bacteria as an antigen.

In the above invention, the culture is preferably a biofilm-like deposit produced when two or more kinds of periodontal disease bacteria are mixed and cultured.

In addition, the periodontal disease bacteria may be Porphyromonas gingivalis , Fusobacterium nucleatum , Actinobacillus actinomycetemcomitans , Tanner forsythen, Tanner forsythen · Dentikora (Treponema denticola), Prevotella intermedia (Prevotella intermedia), is preferably 2 or more kinds of bacteria selected from Streptococcus gordonii (Streptococcus gordonii).

In particular, the two or more types of periodontal disease bacteria are two or more types of periodontal disease bacteria having different aggregation states depending on whether they are cultured alone or in a mixed culture.

Specifically, the two or more types of periodontal disease bacteria include Fusobacterium nucleatum , Porphyromonas gingivalis , Actinobacillus actinomycetemcomitans , Prevotella interferi Media (Prevotella intermedia), is preferably 1 or more periodontal bacteria selected from four Tan'nera Four Shi Ten cis (Tannerella forsythensis). There is a possibility that the surface layer of periodontal pathogens may interact with each other between Fusobacterium nucleatum and the above four kinds of periodontal pathogens, MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Sept. 2002, p. It can be expected that the aggregation state changes in the mixed culture compared to the single culture, and that the antibody may change when the mixed culture is used as an antigen. Compared with single culture, the aggregation state of the mixed culture changes due to the interaction between the periodontal disease bacteria surface layers. The inventors of the present application, in particular, use a mixed culture of P. gingivalis and F. nucleatum as an antigen, so that a very high biofilm formation inhibition rate and coaggregates are obtained. It was found that the adhesion deterrence rate was realized.

Furthermore, the inventors of the present application have conducted intensive studies on the antigen preparation method, immunization method, combination of periodontal disease bacteria to be mixed and cultured. As a result, it was revealed that antibodies with higher effectiveness can be obtained. In particular, the results suggesting that there is no particular limitation on the combination of periodontal disease bacteria to be mixed and cultured were obtained.

The anti-periodontal disease composition according to the present invention is characterized by containing the antibody as an active ingredient.

According to the present invention, by using a culture obtained by mixing and cultivating two or more kinds of periodontal disease bacteria as an antigen, it is possible to provide a more effective antibody than conventional antibodies using a single fungus. . And the anti-periodontal disease composition with a high anti-periodontal disease effect can be provided by using this antibody.

It is image data which shows the mode of each antigen. It is image data which shows the electron micrograph of each antigen. It is a figure which shows the result of having measured the biofilm formation inhibition rate. It is a figure which shows the result of having measured the adhesion inhibitory rate of a co-aggregate. It is a figure which shows the result of having analyzed the protein composition of each antigen by SDS-PAGE. It is a figure which shows the measured value of ELISA with respect to the standard strain and clinical isolate of Porphyromonas gingivalis. It is a figure which shows the measured value of ELISA with respect to the standard strain and clinical isolate of Fusobacterium nucleatum. It is a figure which shows the result of having measured the biofilm formation inhibition rate. It is a figure which shows the result of a co-aggregation suppression test.

In the present invention, periodontopathic bacteria used for the preparation of antigens are Porphyromonas gingivalis , Fusobacterium nucleatum , Actinobacillus actinomycetemcomitans , Tenneraforsi cis (Tannerella forsythensis), Treponema Dentikora (Treponema denticola), Prevotella intermedia (Prevotella intermedia), is preferably 2 or more kinds of bacteria selected from Streptococcus gordonii (Streptococcus gordonii). In the following description and figures, Porphyromonas gingivalis ( P. gingivalis ) is referred to as "Gingivalis" or "Pg", Fusobacterium nucleatum ( F. nucleatum ) is referred to as "Nucleatum" or "Fn", Actinobacillus -Actinomycetemcomitans ( Actinobacillus actinomycetemcomitans ) may be referred to as "actinomycetemcomitans" or "Aa".

These strains can be obtained from the American Type Culture Collection (ATCC) and the RIKEN BioResource Center (BRC).

Hereinafter, the antigen preparation method, immunization method, etc. will be described. In the present invention, the biofilm-like adherent means an insoluble matter having adherence composed of microbial cells, polysaccharides, proteins, etc., produced when two or more kinds of periodontal disease bacteria are mixed and cultured.

The mixed culture conditions of the above periodontal disease bacteria are those under which suitable growth and coaggregation can be confirmed when two or more selected periodontal disease bacteria are mixed and cultured (usually anaerobic conditions at about 37 ° C. and about 24-72). For example, the following method can be exemplified.

1) For example, after culturing the above-mentioned two types of periodontal disease bacteria using BHI broth or TSB medium under anaerobic conditions at about 37 ° C for about 24 to 72 hours, the turbidity (OD600) of each culture solution is measured. In addition, each bacterial cell culture solution is mixed at a predetermined ratio (volume). A part of this mixed solution is added to a fresh medium, and statically cultured at 37 ° C. for 24-72 hours under anaerobic conditions.

After mixed culture of periodontal disease bacteria, the cells are inactivated by a method such as adding formalin to the culture solution according to a conventional method, and after centrifugation and washing with physiological saline (or PBS), an appropriate amount of physiological saline Suspend in water or PBS. An antigen sample can be prepared by homogenizing this suspension by homomixing and sonication.

2) Dispense the cell mixture prepared in the same manner as in 1) above into a cell culture dish (plate), and incubate at 37 ° C for 24 hours under anaerobic conditions. After removing the supernatant so as not to break the biofilm-like deposits formed on the bottom of the dish, a new medium is added, and the culture is statically cultured again at 37 ° C. for 24 hours under anaerobic conditions. In addition, it turns out from the result of an Example that a biofilm-like deposit | attachment is formed very favorable when two types, Gingivalis bacterium and Nucleatum bacterium are used.

Collect the biofilm-like deposit formed on the bottom of the cell culture dish using a scraper, discard the supernatant by centrifugation, wash with PBS, physiological saline, etc. Add an appropriate amount of saline and suspend. By sonicating this suspension in ice-cold water for 5 to 15 minutes and homogenizing it, an antigen preparation can be prepared. In this case, inactivation treatment may be performed using formalin as in the case of 1) above.

In the above, it is preferable to count the number of bacteria by microscopic observation under Gram staining of a part of the suspension before homogenization such as sonication to confirm the bacteria number ratio. For example, when two types of periodontal disease bacteria are used, the bacterial count ratio of the two bacteria is preferably 1: 1 to 100: 1, and more preferably 1: 1 to 30: 1.

The antigen preparation prepared as described above may be stored frozen or lyophilized until use for immunization.

The chicken used for immunization is not particularly limited, but it is particularly preferable to use egg species such as white leghorn, Rhode Island Red, lateral primus rock, New Hampshire from the viewpoint of mass production of antibodies. .

The immunization method is not particularly limited as long as it can immunize animals such as chickens, such as subcutaneous injection, intramuscular injection, and intraperitoneal administration.
The inoculation amount of the antigen may be appropriately selected so that the desired antibody titer is obtained and does not adversely affect animals such as chickens. Usually, the amount of protein is 0.01 to 10 mg per immunization preferable. Moreover, you may immunize together with adjuvants, such as FCA (Freund's complete adjuvant) and FIA (Freund's incomplete adjuvant), as needed.

Usually, after the first immunization, booster immunization is recommended every 1 to 4 months for the purpose of maintaining antibody titer. If necessary, the antigen may be repeatedly inoculated about 3 to 5 times once a week after the first immunization to before the booster immunization.

The antibody titer in chicken eggs can be measured using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, microtiter method, etc., and the antibody titer is measured by measuring the antibody titer at intervals of about 2 weeks after immunization. The transition of the price can be tracked. Usually, when one month or more has passed since the first immunization, a desired specific antibody having a sufficient antibody titer is transferred and accumulated in the yolk of a chicken egg.

The egg yolk antibody of the present invention can be obtained as egg yolk liquid or powdered egg yolk powder by separating egg yolk from chicken eggs immunized as described above. Further, immunoglobulin (IgY) may be extracted and purified from egg yolk by a known method.

Preparation of egg yolk antibody from egg yolk may be performed according to known methods used for immunoglobulin extraction and separation, such as dextran sulfate and polyethylene glycol (PEG), agar, carrageenan, farseleran, pectin, xanthan gum, alginic acid, Methods for precipitating lipoproteins using alginates, alginic acid derivatives, etc., and separating and purifying them from the supernatant (for example, Journal of Immunological Methods, 46, P63-68 (1981), Immunological
Communication, 9 (5), P475-493 (1980), JP-A-63-215699, JP-A-64-38098), extraction methods using propanol, chloroform and the like. For example, in the case of use in the field of food, etc., it is preferable from the viewpoint of safety to the human body to use those recognized as food natural additives such as carrageenan, xanthan gum, pectin and the like.

For example, egg yolk fluid is egg yolk water-soluble protein powder obtained by pulverizing egg yolk water-soluble protein using carrageenan or the like, or egg yolk water-soluble protein is ion-exchange chromatography, hydrophobic chromatography, affinity It can be prepared in various forms such as a purified chicken egg antibody purified by a known protein purification method such as chromatography, gel filtration, sodium sulfate salting out, ammonium chloride salting out.

The purity of the egg antibody of the various prepared samples obtained as described above is calculated based on the weight of the egg egg antibody relative to the weight of the powder. In the form of egg yolk powder, usually 1 to 2% of the egg antibody, and in the form of egg yolk water-soluble protein powder It is usually 8-12%, and in the form of purified chicken egg antibody, it is usually 95% or more.

In the present invention, egg yolk juice, egg yolk powder, egg yolk water-soluble protein powder, purified antibody, etc. containing the above-described chicken egg antibody can be used as an anti-periodontal disease composition as they are. The dosage form can be appropriately selected from powder / granular form, liquid form, capsule form, tablet form, jelly form and the like.

The anti-periodontal disease composition of the present invention comprises a tooth color remover, a halitosis preventive agent, a caries preventive agent such as fluorine, a medicinal component such as an anti-enzyme preventive agent, an excipient, an emulsifier, a saccharide, and a vitamin as necessary. Other ingredients such as flavours, perfumes and the like can be included.

The anti-periodontal disease composition of the present invention includes toothpastes such as toothpaste, toothpaste and liquid toothpaste, dental materials, mouthwash, intraoral pasta, gingival massage cream, gargle tablets, troches, chewing gum, canned beverages, etc. Not only oral materials but also foods such as chocolate, candy, yogurt, cheese, ice cream, prepared milk, baby food, pet food and the like. In addition, when mix | blending with the above products, in order to improve stability of the anti-periodontal disease composition of this invention, you may mix | blend and encapsulate.

For example, in the case of toothpaste, a polishing agent such as calcium carbonate, calcium hydrogen phosphate, calcium pyrophosphate, insoluble sodium metaphosphate, alumina, anhydrous silicic acid, humectant such as glycerin, sorbit, propylene glycol, sodium laurate sulfate, lauroyl sarcosine A foaming agent such as sodium and soap powder, a binder such as sodium carboxymethylcellulose and carrageenan, and other components such as perfume ingredients, sweeteners, preservatives and colorants may be mixed with water and produced according to a conventional method.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

(1) Preparation of antigen As periodontal disease bacteria, Porphyromonas gingivalis ( P. gingivalis ) and Fusobacterium nucleatum ( F. nucleatum ) were used. For Gindivarius, 33277 strains distributed from ATCC were used, and for Nukuretam, 8532 strains distributed from RIKEN BRC were used.

The gingivalis and nucleatum were each cultured in BHI broth according to a conventional method. Adjust the turbidity (OD600) of each culture to 1.0, mix with Gindivarius culture: Nucleatum culture = 19: 1 (volume), and add 1/100 volume of this mixture to the new BHI broth The cells were then statically cultured at 37 ° C. for 48 hours under anaerobic conditions.

Formalin was added to the obtained mixed culture solution to 0.5% (v / v), and the cells were left at 37 ° C. for 24 hours to inactivate the cells. Next, the mixed culture solution was centrifuged to collect the insoluble fraction, washed three times with physiological saline, and then the insoluble fraction was dispersed in physiological saline and then homogenized. This solution was subjected to microscopic observation under Gram staining to determine the number of bacteria and appropriately diluted to obtain antigen G3 (Gingivalis: 5.0 × 10 ⁹ cells / mL, Nucleatum: 1.3 × 10 ⁹ cells / mL).

As a comparative sample, gingivalis was cultivated and treated alone in the same manner as described above, and the bacterial concentration was adjusted to be equivalent to the number of mixed culture antigen G3 (5.0 × 10 ⁹ cells / mL). Got.

Also, in the same manner as described above, the nucleatum is cultured and treated alone, and the bacterial concentration is adjusted to be the same as that of the mixed culture antigen G3 (1.3 × 10 ⁹ cells / mL) to obtain the antigen G2. It was.

In addition, the above antigens G1 and G2 are mixed and adjusted to be equivalent to the number of the above mixed culture antigen G3 (Gingivalis: 5.0 × 10 ⁹ cells / mL, Nucleatum: 1.3 × 10 ⁹ cells / mL) And antigen G4 was obtained.

FIG. 1 is image data showing the state of each antigen obtained as described above. No. 1 is Gimbalis alone (G1), No. 2 is Nucleatum alone (G2), No. 3 is a mixed culture of both (G3), No. 4 is a single culture of Gimbalis and Nucleatum (G4) It is a thing. Each image shows a state after each antigen is dispersed by ultrasonic treatment and allowed to stand for 30 minutes. It is clearly observed that a change in the state of the antigen is caused by the mixed culture.

FIG. 2 is image data showing an electron micrograph of each antigen. Each image data was imaged using an electron microscope (manufactured by Hitachi High-Technologies Corporation, TM-1000). No. 1 is Gimbalis alone (G1), No. 2 is Nucleatum alone (G2), No. 3 is a mixed culture of both (G3), No. 4 is a single culture of Gimbalis and Nucleatum (G4) It is a thing. Aggregation between Gimbalis and Nucleatum is clearly different between No. 4 in which each single culture is mixed and No. 3 which is a mixed culture. It can be presumed that the cohesiveness is different from the mixture of the single culture solution when mixed culture is performed due to the interaction between the surface layers of both bacteria.

(2) Immunization of laying hens with antigens Antigens G1 to G4 prepared above are mixed in equal amounts with Freund's complete adjuvant, and 1 ml each is injected into the thigh muscle of a hen (Boris Brown) for the first immunization. went. Similarly, the second immunization was performed 9 weeks after the first immunization. In this example, the change in antibody titer was followed by ELISA, and eggs at the stage where the antibody titer was sufficiently increased were collected and used for the preparation of each antibody.

(3) Purification of antibody (IgY) from egg yolk Eggs 13 weeks after the first immunization were collected from chickens immunized with each antigen. Divided into egg yolk and egg white, an equal amount of water was added to the egg yolk, a suspension of 0.15% λ-carrageenan was added, the mixture was stirred and centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. Further, fractional precipitation with ammonium sulfate was repeated three times, dialyzed with a 0.15 M NaCl aqueous solution, and then filtered through a 0.45 μm filter, and the filtrate was collected to obtain each antibody. Hereinafter, antibodies obtained using the antigens G1 to G4 are referred to as P1 to P4.

As a control, IgY (control IgY) prepared in the same manner from unimmunized chicken eggs was used.

(4) Measurement of biofilm formation inhibition rate In each hole of a collagen-coated polystyrene 96-well plate (IWAKI, flat-bottom type), a TSB culture solution containing 2 strains of gingivalis and nucleatum is dispensed. Subsequently, antibodies P1 to P4 and control IgY were added at predetermined concentrations, respectively, and cultured at 30 ° C. for 24 hours.

After that, the plate is lightly washed four times with distilled water, the biofilm formed on the plate is stained with crystal violet (hereinafter referred to as CV), ethanol is added, and the dye is extracted from the colored biofilm. The extract was measured for absorbance (OD570) using a spectrophotometer. Then, the absorbance value of the positive control to which no chicken egg antibody was added was the biofilm formation inhibition rate of 0%, the absorbance value of the negative control to which neither the chicken egg antibody nor periodontal bacteria were added was the biofilm formation inhibition rate of 100%, The biofilm formation inhibition rate was determined from the absorbance value. The results are shown in FIG.

From FIG. 3, antibody P3 obtained using antigen G3 obtained by mixing and culturing two bacteria, especially antibodies P1 and P2 obtained using a single fungus as an antigen at a low concentration (2 μg / mL) It can also be seen that the biofilm formation inhibition rate is higher than that of antibody P4 obtained using a mixture of these antigens.

(5) Measurement of the amount of coaggregate adhering to the collagen-coated plate Dispensing PBS containing 2 strains of gingivalis and nucleatum into each well of a collagen-coated 96-well plate (IWAKI, flat bottom type) Subsequently, antibodies P1 to P4 and control IgY were respectively added at predetermined concentrations and allowed to stand at 37 ° C. for 3 hours. Then wash the plate 4 times with distilled water, stain the bacteria attached on the plate with CV, add ethanol, extract the dye from the colored biofilm, and use the spectrophotometer to extract the extract , Measured by absorbance (OD570). Then, the absorbance value of the positive control with no chicken egg antibody added is 0%, and the absorbance value of the negative control with no egg antibody or periodontal disease bacteria added is 100%. The deterrence rate was calculated. The results are shown in FIG.

From Fig. 4, antibody P3 obtained using antigen G3 obtained by mixing and culturing two bacteria is obtained using antibodies P1 and P2 obtained using a single bacterium as an antigen, or a mixture of these antigens. It can be seen that the coaggregation adhesion inhibition rate to the collagen-coated plate is higher than that of the obtained antibody P4.

From the above results, the antibody P3 obtained using the antigen obtained by mixing and culturing the two bacteria effectively suppresses adhesion of the coaggregate to the collagen, which is the initial stage of biofilm formation, It can be seen that the biofilm formation itself is also effectively suppressed. As shown in Fig. 1 and Fig. 2, the anti-biofilm formation inhibition rate and the co-aggregate adhesion inhibition rate of the antibody obtained due to the different aggregation properties of at least two periodontal disease bacteria in the mixed culture antigen solution If periodontitis is improved, the periodontal disease bacteria that can be used are not limited to two bacteria, Gindivarius bacteria and Nucleatum bacteria, and the possibility that other periodontal disease bacteria may be used can be predicted.

Hereinafter, the results of further studies conducted by the inventors of the present application for the purpose of more effective biofilm formation inhibition will be described.

(1) Preparation of antigen (mixed culture method)
Pg bacteria and Fn bacteria were cultured according to a conventional method using TSB medium. Each culture solution was added to and mixed with an appropriate amount of TSB medium so that the turbidity (OD600) of each culture solution was 0.1 to prepare a mixed solution of Pg bacteria and Fn bacteria. 10 ml of this mixed solution was dispensed into 8 dishes for cell culture (diameter 10 cm) and left to stand at 37 ° C. for 24 hours under anaerobic conditions. Carefully remove the supernatant so as not to destroy the biofilm-like bacterial adherence formed on the bottom of the dish, add 10 ml each of fresh TSB medium, and leave again at 37 ° C for 24 hours under anaerobic conditions. Cultured.

The biofilm-like bacterial cell adhering mixture cultured in the cell culture dish was collected using a scraper, the supernatant was discarded by centrifugation, washed twice with PBS, and finally 20 ml of PBS. After adding and suspending, the suspension is sonicated in ice-cold water for 10 minutes (Sonics & Materials Inc., Ultrasonic processor, Model: VC130,
Amplitude: 50%, Timer: 10 min, Pulser: 5 sec), and antigen G9 was obtained.

In addition, when the number of bacteria was counted by microscopic observation under Gram staining of the above suspension, they were Fn bacteria: 3.6 × 10 ⁸ cells / mL and Pg bacteria: 7.6 × 10 ⁹ cells / mL.

In addition, the following antigens were prepared as comparative samples.
Antigen G5 was obtained in the same manner as antigen G1 of Example 1 except that TSB medium was used and sonication was performed as an inactivation treatment.

Antigen G6 was obtained in the same manner as antigen G2 of Example 1, except that TSB medium was used and sonication was performed as an inactivation treatment.

Antigen G7 was obtained in the same manner as antigen G3 of Example 1 except that TSB medium was used and sonication was performed as an inactivation treatment.

Antigen G8 was obtained in the same manner as antigen G4 in Example 1, except that TSB medium was used and sonication was performed as an inactivation treatment.

For the antigens G5, G6, and G9 obtained above, the protein composition of the antigen was analyzed by SDS-PAGE. The results are shown in FIG.

FIG. 5 shows that in the case of antigen G9, peaks appear around 30 KDa and 28 KDa, and the protein composition is clearly different from antigen G6 (F.n alone) and antigen G5 (P.g alone).

(2) Immunization of antigens to laying hens Using antigens G7 to G9, immunization was performed in the same manner as in Example 1 (in this example, 3 additional immunizations), and the antibody titer was monitored by ELISA. The eggs at the stage where the antibody titer was sufficiently raised were collected and used for the preparation of each antibody.

(3) Purification of antibody (IgY) from egg yolk Antibodies were obtained from eggs obtained from chickens immunized with each antigen G7 to G9 in the same manner as in Example 1. Hereinafter, antibodies obtained using the antigens G7 to G9 are referred to as P7 to P9.

(4) Clinical isolate crossover test (ELISA)
A “clinical isolate” is a strain that is actually isolated from the oral cavity of a human and is said to be a strain with high biofilm-forming ability. Therefore, it can be said that an actual practical effect can be confirmed if the effectiveness of the antibody studied by the present inventors can be confirmed against clinical isolates.

In this example, it is isolated from a periodontal disease patient according to a conventional method, Real Time
Clinical isolates whose genera / species were confirmed by PCR were used.

That is, 11 strains (ST1, TO1, YM4, KS1, KS2, TK3, TK4, KO1, YH1, CT2, and AT2) were used as clinical isolates of P. g. Two types (ATCC33277, W83) were used as standard strains of P. g.

In addition, 11 strains (S-10, # 2, # 20, KO1, TS1, IY1, ST1, KS1, OM1, YE1, YK1) were used as clinical isolates of the genus Fusobacterium . S10, # 2 and # 20 are Fn bacteria, and KO1 and later are clinical isolates identified up to the genus Fusobacterium . Two types (ATCC10953, ATCC25586) were used as standard strains of Fn bacteria.

ST1, S-10, etc. of clinical isolates of Pg and Fusobacterium are control numbers and do not specify the structure of the strain.

The following preparation was performed to prepare an antigen solution used for ELISA.
The clinical isolates and standard strains used were anaerobically cultured (37 ° C.) using a TSB liquid medium containing 5 μg / mL hemin and 1 μg / mL vitamin K3.

The culture broth was collected by centrifugation and washed 3 times with PBS. Thereafter, the bacteria were suspended in 0.1 M carbonate buffer (pH 9.6) containing a protease inhibitor and stored at −80 ° C. until disrupted. The thawed bacterial suspension was subjected to ultrasonic treatment (30 seconds × 10 times, on ice) to obtain a bacterial cell disruption solution. Centrifuge supernatant of the disrupted solution is diluted with 0.1 M carbonate buffer (pH 9.6) containing protease inhibitors so that the protein concentration is 500 μg / mL, and then the antigen solution for solid phase (clinical isolate / Standard strain) was prepared and stored at -30 ° C.

Next, a specific measurement method of ELISA will be described.
The test was performed at a protein concentration of 50 μg / mL and an antibody concentration of 5 μg / mL in the antigen solution for immobilization.

First, 100 μL of an immobilized antigen solution was added to a 96-well plate, and allowed to stand overnight at 8 ° C. to immobilize the antigen on the plate. Next, 150 μL of 1% BSA-containing PBS (blocking solution) was poured into each well, allowed to stand at room temperature for 1 hour for blocking treatment, and then washed 3 times with 0.05% Tween20-containing PBS (PBST). It was.

The antibodies P7 to P9 were each dissolved in a blocking solution and then centrifuged, and 100 μL of each supernatant was poured into each well as a primary antibody solution and incubated at 37 ° C. for 1 hour to perform the primary antibody treatment. . Thereafter, each well was washed 3 times with PBST and transferred to secondary antibody treatment.

As a secondary antibody, anti-chicken IgY [IgG] (H + L) (AP label, ZYMED LABORATORIES) was used. 100 μL of a solution obtained by diluting the secondary antibody with a blocking solution 2,000-fold was poured into each well and incubated at 37 ° C. for 1 hour. After washing each well 3 times with PBST, 100 μL of coloring solution (1 mg / mL Disodium p-nitrophenyl phosphate)
Hexahydrate in 10% diethanolamine-HCl buffer (pH 9.8)) was poured into each well. After incubating at room temperature for about 15 minutes, the reaction was stopped by adding 50 μL of 5N NaOH and subjected to absorbance measurement (405 nm) using a plate reader.

First, FIG. 6 shows the results of applying the ELISA method to the standard strain and clinical isolate of P. g. In FIG. 6, gray indicates antibody P8, black indicates antibody P9, and white indicates antibody P7. The high affinity of antibodies P7 and P9 for various clinical isolates compared to antibody P8 indicates the usefulness of the antibody obtained in the present invention.

Next, FIG. 7 shows the result of applying the ELISA method to the standard strain and clinical isolate of the genus Fusobacterium . In FIG. 7, gray indicates antibody P8, black indicates antibody P9, and white indicates antibody P7. Similar to Pg bacteria, the high affinity of antibodies P7 and P9 for various clinical isolates compared to antibody P8 indicates the usefulness of the antibody obtained in the present invention.

(5) Measurement of biofilm formation inhibition rate The bifilm formation inhibition test was performed in the same manner as in Example 1 (4) to determine the biofilm formation inhibition rate. The results are shown in FIG.

From FIG. 8, the antibody P9 obtained by using the antigen G9 obtained by mixing and culturing the two bacteria on the cell culture dish is compared with the antibody P8 obtained by using the antigen G8 mixed with the single bacteria. It can be seen that the biofilm formation inhibition rate is high.

Finally, as another periodontal disease fungus, the case where an antigen mixed and cultured for the above-mentioned two types of bacteria, nucleatum (F.n) and actinomycetemcomitans (A.a) is described.

(1) Preparation of antigen (mixed culture method)
Aa bacteria and Fn bacteria were each cultured in a conventional manner using TSB medium. Add each culture solution to TSB medium and mix it so that the turbidity (OD600) of the Aa bacteria culture solution is 0.06 and the turbidity (OD600) of the Fn culture solution is 0.04. A liquid was prepared. This mixture is allowed to stand at 37 ° C for 24 hours under anaerobic conditions, and then the cells are collected by centrifugation. The supernatant is discarded, washed twice with PBS, and finally 20 mL of PBS is added. After suspension, this suspension was subjected to sonication in the same manner as in Example 2 to obtain antigen G11.

In addition, when the number of bacteria was counted by microscopic observation under Gram staining of the above suspension, they were Aa bacteria: 3.4 × 10 ⁸ cells / mL and Fn bacteria: 6.4 × 10 ⁷ cells / mL.

In addition, as a comparative sample, the A.a bacterium was cultured and treated alone in the same manner as described above to obtain the antigen G10.

Further, in the same manner as described above, A.a bacteria and F.n bacteria were cultured and treated independently to obtain antigen G12.

(2) Immunization of laying hens with antigen Antigen G10 to G12 are used for immunization in the same manner as in Example 2 (2), and the change of antibody titer is followed by ELISA method. Eggs at the stage of rising were collected and used for the preparation of each antibody.

(3) Purification of antibody (IgY) from egg yolk Antibodies were obtained from eggs obtained from chickens immunized with the respective antigens G10 to G12 in the same manner as in Example 1. Hereinafter, antibodies obtained using the antigens G10 to G12 are referred to as P10 to P12.

(4) Coaggregation inhibition test of periodontal disease bacteria Aa bacteria and Fn bacteria cultured using TSB medium as usual were washed twice with PBS, and turbidity (OD 600
nm) is suspended in a co-aggregation buffer (1 mM Tris-HCl pH 8.0, 0.1 mM CaCl ₂ , 0.1 mM MgCl ₂ , 0.15 M NaCl, 0.02% NaN ₃ ) so that Aa bacteria = 1.0 and Fn bacteria = 1.0. It became cloudy.

To this suspension, the P10 to P12 antibodies and the unimmunized control antibody were added at 100 μg / ml, respectively, and 200 μl was dispensed into each well of a 96-well flat bottom transparent plate.

The plate was shaken for 30 seconds for the first 30 seconds using a 96-well microphone plate reader (ARVO MX, Perkin Elmer) with shaking function, and then turbid for a total of 30 times every 1 minute and 20 seconds (40 minutes in total). Degree (OD630) was measured. When coaggregation of periodontal disease bacteria occurs, the turbidity (OD630) decreases, so the coaggregation inhibitory effect was measured by observing the decrease in turbidity (OD630). The results are shown in FIG.

In FIG. 9, the antibody P11 obtained using the mixed culture antigen (G11) of Aa bacteria and Fn bacteria is the antibody P10 obtained using the Aa bacteria single antigen (G10), and a mixture of Aa bacteria and Fn bacteria. Compared to antibody P12 obtained using the antigen (G12), the turbidity (OD630) decreased more slowly and was found to have a high ability to inhibit coaggregation. This suggests the possibility that antibodies obtained using an antigen obtained by mixing and cultivating two or more kinds of other periodontal disease bacteria may exhibit a high ability to inhibit biofilm formation.

The present invention can be applied to, for example, an anti-periodontal disease composition excellent in the effect of suppressing the formation of a biofilm that becomes a lesion of periodontal disease.

Claims (10)

1. An antibody characterized by being obtained from a bird egg immunized with a culture obtained by mixing and cultivating two or more kinds of periodontal disease bacteria as an antigen.
2. 2. The antibody according to claim 1, wherein the culture is a biofilm-like deposit produced when two or more kinds of periodontal disease bacteria are mixed and cultured.
3. The periodontal pathogens are Porphyromonas gingivalis , Fusobacterium nucleatum , Actinobacillus actinomycetemcomitans , Tannerella forsythensy , and Tannerella forsythensydensy. (Treponema denticola), Prevotella intermedia (Prevotella intermedia), antibody of claim 1 or 2 which is 2 or more kinds of bacteria selected from Streptococcus gordonii (Streptococcus gordonii).
4. The two or more types of periodontal disease bacteria are two or more types of periodontal disease bacteria having different aggregation states depending on whether they are cultured alone or in a mixed culture. An antibody according to any one of the above.
5. The two or more types of periodontal disease bacteria are Fusobacterium nucleatum , Porphyromonas gingivalis , Actinobacillus actinomycetemcomitans , Prevotella intermedia ( Prevoterra intermedia ) The antibody according to any one of claims 1 to 4, wherein the antibody is one or more periodontal pathogens selected from four kinds of Tannerella forsythensis .
6. The aggregation state is changed in the mixed culture as compared with the single culture by interacting with the surface layers of the two or more kinds of periodontal disease bacteria. An antibody according to any one of the above.
7. The antibody according to any one of claims 1 to 6, wherein the two kinds of periodontal disease bacteria are Porphyromonas gingivalis ( P. gingivalis ) and Fusobacterium nucleatum ( F. nucleatum ).
8. The two or more kinds of periodontal disease bacteria are two kinds of Fusobacterium nucleatum ( F. nucleatum ) and Actinobacillus actinomycetemcomitans ( Actinobacillus actinomycetemcomitans ). The antibody according to any one of the above.
9. The antibody according to any one of claims 1 to 8, wherein the bacterial cell used as an antigen is inactivated by sonication.
10. An anti-periodontal disease composition comprising the antibody according to any one of claims 1 to 9 as an active ingredient.

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