JPH06122633A - Antibody having immunoactivity on streptococcus mutans and dental caries preventive - Google Patents

Abstract

PURPOSE:To provide an antibody high in dental caries-preventive activity leading to excellent dental caries-preventive effect, also greatly high in safety. CONSTITUTION:The objective antibody can be obtained by immunizing mammals or birds with an antigen, a fragment obtained by nicking a protein antigen having sticking activity to teeth surface and produced from Streptococcus mutans. The fragment consists of a polypeptide containing part or the whole of an amino acid sequence from the 478th amino acid (aspartic acid) to the 1000th amino acid (glutamine).

Description

Detailed Description of the Invention

[0001]

The present invention relates to an antibody having immunological activity against Streptococcus mutans (hereinafter referred to as S. mutans) of oral streptococcus which is a pathogenic bacterium that induces dental caries, and the antibody as an active ingredient. The present invention relates to an agent for preventing dental caries.

[0002]

BACKGROUND OF THE INVENTION Streptococcus mutans group (Strept)
Ococcus mutans group) is known to be a bacterium that causes caries in humans and animals. Of this group, S. cerevisiae, which belongs to serotype c, is deeply involved in caries in humans. mutans.
S. Mutans adsorb to pellicle formed on the human tooth surface. The pellicle refers to a coating on the tooth surface that is composed of proteins and the like in saliva. After adsorbing to the pellicle, this bacterium is firmly fixed by a polysaccharide synthesized by an extracellular enzyme called glucosyltransferase,
Acid causes tooth enamel demineralization.

S. An important factor in the adsorption of mutans on the pellicle is a protein antigen called fimbriae with a molecular weight of about 190,000, which is present on the surface layer of this bacterium
Although referred to by various names such as c, I / II, B, P1, IF, in this specification, PAc (Protein anti) is used.
gen serototype c). Further, as a factor on the pellicle side, S. cerevisiae contained in saliva. mutan
The involvement of the s aggregation factor has been suggested.

Antibodies obtained from animals immunized with PAc were used to transform S. It has been previously reported that the attachment of mutans is blocked. However, when the antibody obtained by such a method is used as a caries preventive agent, the most important point is which part of PAc will be the effective antibody. However, this point has not been clarified until now.

That is, when an antibody against PAc is used as a caries-preventing antibody, it is necessary to efficiently obtain an effective antibody, but when immunized with PAc itself, a large amount of an antibody that is not involved in efficacy is produced. The problem arises that This is not preferable in terms of effectiveness and safety.

[0006] Therefore, an antibody having such a disadvantage and a higher caries-preventing effect is desired.

[0007]

Means and Actions for Solving the Problems As a result of extensive studies to meet the above-mentioned needs, the present inventor has found that among the fragments obtained by cleaving PAc, amino acids 478 (aspartic acid) to 1000 ( The fragment containing glutamine) is PA
The antibody obtained by immunizing mammals and birds with the polypeptide of this fragment as an antigen was found to be the salivary adhesion region of S. c. S. mutans, specifically showing immunoreactivity against S. mutans, The inventors have found that they have a sufficient inhibitory effect on the attachment of mutans to the tooth surface and give a very excellent effect as an active ingredient for preventing dental caries, and thus completed the present invention.

Accordingly, the present invention provides Streptococcus
A fragment obtained by cleaving a protein antigen having an action of adhering to a tooth surface, which is obtained from mutans, wherein the fragment is a polypeptide containing a part or all of amino acids 478 (aspartic acid) to 1000 (glutamine). And obtained by immunizing this with mammals or birds,
Provided are an antibody having an immunological activity against Streptococcus mutans, and a caries preventive agent containing the antibody as an active ingredient. The above amino acid numbers include signal peptides.

The present invention will be described in more detail below. The antibody of the present invention comprises amino acid 478 of the PAc fragment.
No. (aspartic acid) to No. 1000 (glutamine), or an immunoglobulin obtained by immunizing a mammal or a bird with a polypeptide containing part or all of the glutamine as an antigen.

Here, such a polypeptide can be produced by a method of obtaining a protein expressed by fragmenting PAc using a known genetic engineering technique.

This method will be described in more detail. m
tans chromosomal DNA, PCR (Polyme
Rase chain reaction) method
The Ac gene fragment is amplified. The primer DNA used in the PCR method is synthesized with an appropriate length by a DNA synthesizer based on the already known base sequence of the pac gene. that time,
It is desirable to add an appropriate restriction enzyme recognition sequence to facilitate cloning into a vector plasmid.

[0012] As the plasmid vector, an expression vector which is generally commercially available can be used, but it must be designed so that it can be expressed as a fusion protein with a protein that can be selectively bound to a certain chromatographic carrier so as to allow affinity purification. Designed is desirable. Examples of proteins to be fused include APTG (p-Amino
phenyl-β-D-thiogalactopyr
β-galactosidase having an anoside) as a ligand, chloramphenicol acetyltransferase having chloramphenicol as a ligand, protein-A having IgG as a ligand, and maltose binding protein (MBA) having maltose as a ligand. Further, purification by cation exchange by addition of polyarginine and purification of Ni ²⁺ metal chelate by addition of polyhistidine are also possible. Furthermore, if a sequence encoding a cleavage site by a protease is inserted upstream or downstream of these proteins to be fused, only the target protein can be released.

The pac gene fragment can be cloned by cutting the amplified fragment and the vector DNA with an appropriate restriction enzyme, and then ligating with ligase. Introduction of the recombinant DNA into Escherichia coli is carried out by a conventional method, and it can be confirmed that the desired plasmid is a plasmid by digestion with a restriction enzyme. It is more reliable if the cloned gene fragment is expressed in E. coli and confirmed by Western blotting using an antibody against PAc.

Purification of the expressed PAc fragment protein depends on the type of vector. Even in the case of a vector system that does not have the function of facilitating purification, P
Affinity chromatography using an antibody against Ac is possible. Furthermore, classical methods such as ion exchange chromatography, gel filtration and salting out that have been conventionally used can also be used.

In the above method, Escherichia coli is used as a host, but it goes without saying that a host-vector system such as Bacillus subtilis, yeast, animals and plants can also be used.

In the present invention, the c-type S. mutan
s, the g-type S. Sobrinus also has a surface protein antigen having almost the same amino acid sequence, and a similar region is expected to be an attachment site.

The antibody of the present invention is a mammal (horse, cow, sheep, goat, etc.) immunized with the above-mentioned polypeptide as an antigen.
Alternatively, it can be obtained from serum, milk, egg, egg yolk, etc. of birds, especially poultry (chicken etc.), and in this case, known methods can be adopted as the immunization method and the like.

Specifically, as a method for immunizing a mammal, a usual method such as subcutaneous injection, intramuscular injection, intraperitoneal administration or the like, or a method such as nasal drop or eye drop can be used. The dose of the antigen may be appropriately selected so that the desired antibody titer can be obtained and the animal is not adversely affected. If necessary, for example, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FI
A), an adjuvant such as aluminum hydroxide may be used in combination with the antigen.

For example, in small animals, the antigen is 1 to 500 μg.
As a protein, a large animal is immunized with 0.1 to 1 mg once every 2 to 5 weeks for 3 to 5 times by a conventional method. In the case of rabbits, the antigen solution (100 to 20 as protein)
(0 μg / ml) mixed with an equal amount of Freund's complete adjuvant was administered 3 to 5 times every 2 to 5 weeks, and 10 to 14 days after the final immunization, blood was collected from the animals by a conventional method,
A plasma containing immunoglobulins is obtained. This can be used as it is, or further purified by a conventional method and used as antiserum or immunoglobulin.

On the other hand, chickens to be used for immunization are not particularly limited, but eggs such as white leghorns can be used from the viewpoint of antibody productivity. As an immunization method with an antigen, a usual method such as subcutaneous injection or intraperitoneal administration, or a method such as nasal drop or eye drop can be used.
The dose of the antigen may be appropriately selected so that the desired antibody titer is obtained and the chicken is not adversely affected. Usually, an antibody that specifically reacts with the administered antigen is obtained in chicken eggs (yolk) within a few weeks after the initial immunization. If necessary, an adjuvant such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), or aluminum hydroxide may be used in combination with the antigen. The anti-PAc fragment antibody of the present invention can be prepared from an egg collected from a chicken that has passed one month or more after immunization.

The antibody titer in egg yolk can be measured by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay or the like. By these methods, the transition of the antibody titer can be traced by measuring the antibody titer at intervals of about 2 weeks after immunization. For the activity of the purified antibody, the cell aggregation method is also used. Regarding the persistence of antibody titer, the antibody titer is high for about 3 months. When a decrease in antibody titer is observed after immunization, the antibody titer can be increased by appropriately performing booster immunization at appropriate intervals.

The anti-PAc fragment antibody of the present invention can be obtained by, for example, extracting and separating immunoglobulin contained in egg yolk of the chicken immunized as described above. As the extraction / separation method, for example, a precipitation method using a polysaccharide or polyethylene glycol, an extraction method using ethanol, propanol, or chloroform, and various methods that can be used to extract / separate commonly used immunoglobulins can be used. .

The anti-PA of the present invention obtained as described above
The c-fragment antibody is S. S. mutans, which specifically reacts with the cell surface protein (PAc) present in S. mutans, mutan
It has an immunological activity against s. That is, S. mutan
It has the effect of inhibiting the adhesion of S. s to the tooth surface, and the S. It is possible to prevent mutans from colonizing the oral cavity and prevent caries.

Therefore, the antibody of the present invention is preferably used as an active ingredient of a caries preventive agent, and the caries preventive agent of the present invention contains the above-mentioned anti-PAc fragment antibody and is administered to the oral cavity. It is prepared in various forms depending on the form. For example, when used as a mouthwash, toothpaste, etc., the anti-PAc of the present invention should be prepared when these ingredients are prepared.
It suffices to include an effective amount of the fragment antibody. The amount of the anti-PAc fragment antibody to be incorporated into the caries preventive agent may be appropriately selected according to the dose according to the mode of administration, for example, bacterial cell aggregation value 1
0.0001 to 10% by weight of an antibody of 000 aggregation units or more
It can be a degree.

In the caries preventive agent of the present invention, a purified antibody may be used as the above antibody, or serum, milk, egg or yolk of mammals or birds immunized with the above antigen may be used as it is. Good.

Further, the caries preventive agent of the present invention is prepared in the form of mouthwash, toothpaste, and various other oral compositions as described above, and these ingredients depend on the type of composition. Known components may be blended.

[0027]

INDUSTRIAL APPLICABILITY The antibody of the present invention has a high caries-preventing effect, and a caries-preventing agent containing this antibody as an active ingredient has an excellent caries-preventing effect and is extremely safe.

[0028]

EXAMPLES The present invention will be described more specifically below with reference to reference examples and examples, but the present invention is not limited to the following examples.

Reference Example S. Mutans Chromosome Preparation mutans MT8148 strain 20 mM D, L
-37 ° C in Todd-Hebit medium supplemented with threonine
It was cultured for 18 hours. After collecting cells by centrifugation, TE (10 mM
(Tris.Cl, 1 mM EDTA) buffer and washed with the same buffer containing 20% glucose.
Add 8 mg / ml lysozyme to the bacterial suspension and mix at 37 ° C for 3
The mixture was allowed to act for 0 minutes, and then 0.1 mg / ml N-acetylmuramidase SG was added to lyse the cells at 50 ° C. for 30 minutes. 0.3 mg / ml RNase in the lysate,
0.3 mg / ml Pronase E was allowed to act for 30 minutes at 37 ° C. Then, 2% N-lauryl sarcosine Na salt was added, and after 30 minutes at 37 ° C., centrifugation was performed to obtain a chromosome. This was further extracted with an extraction solvent of phenol-chloroform 1: 1 and dialyzed against TE buffer for purification.

Cloning of pac gene fragment The region shown in FIG. 1 of the pac gene was amplified by PCR.
At that time, the restriction enzyme Kpn was added to the 5'end of the primer DNA.
It was synthesized to have I and SalI recognition sequences. A commercially available pAX plasmid was used as the vector DNA. The numbers in the figure show the base numbers of the pac gene containing the signal sequence, and the numbers in parentheses show the amino acid sequence numbers.

K the pac gene fragment and vector DNA
Digested with pnI and SalI. Next, both fragments were ligated with ligase, and E. coli NM5
Introduced into 22 strains. A plasmid was extracted from the obtained transformant and re-cut with KpnI and SalI to confirm that the length of the fragment inserted into the vector was as expected.

Expression and Purification of pac Gene Fragment A plasmid having pAX with the inserted pac fragment was LB
After culturing in the medium, when the OD ₆₀₀ reaches about 0.5, IPTG, which is an inducer of the lactose promoter, is added to 0.1.
˜0.2 mM was added, and the mixture was cultured at 25 ° C. for 18 hours. After collecting the cells, the cells were washed with an appropriate buffer and suspended in Tris buffer containing 1 mM EDTA. After destroying the cells with ultrasonic waves,
The supernatant was obtained by centrifugation. Expression is by SDS-PAGE of supernatant
And the antibody against PAc were confirmed by Western blotting.

The expressed fusion protein of β-galactosidase and PAc fragment is APTG (p-Aminophenyl-) which is a derivative of the substrate of β-galactosidase.
β-thiogalactopyranoside) was adsorbed on the affinity chromatography in which the carrier was bound, and then eluted by raising the pH to 10.0. Furthermore, pAX is located downstream of β-galactosidase-
It was designed to have the sequence Ile-Glu-Gly-Arg-, and the fusion protein was treated with endoprotease Xa to release the PAc fragment, and the PAc fragment was purified by passing through the APTG column again. .

Interaction between PAc fragment and salivary component The PAc fragment obtained by the above method was treated with carbonate buffer (pH
It was diluted with 9.6) and adsorbed on an ELISA plate.
Phosphate buffer containing 1 mM CaCl ₂ (pH 7.
After washing in 2), whole saliva collected from human was added. Similarly, after washing with a phosphate buffer, P labeled with biotin
Ac protein was added. After washing in the same manner, streptavidin labeled with alkaline phosphatase was added. Then, after washing, a chromogenic substrate was added and OD ₄₀₅ was measured. The results of examining the salivary adhesiveness of PAc fragments by the above method are shown in FIG.

From the results of FIG. 2, amino acids 478 to 10
It was confirmed that the PAc fragment containing No. 00 has the same salivary component adhesiveness as PAc.

[Example 1] (Preparation of hen egg antibody against PAc fragment) β-galactosidase and PAc (47) obtained in Reference Example
8-1000) fusion protein to endoprotease Xa
Processed in. After confirming the release of the PAc fragment by SDS-PAGE, PAc (478 to 1000) was purified by passing through the APTG column again to obtain the flow-through fraction.

1 m of the antigen obtained by the above preparation method
0.5 ml of a liquid containing g / ml and 0.5 ml of FIA (Freund's incomplete adjuvant) were mixed at a ratio of 1: 1,
It was a W / O type emulsion. 0.5 ml each of the obtained emulsion was injected into the left and right pectoral muscles of the chicken, and after performing the initial immunization, the water-soluble fraction (WSF) antibody titer obtained from the collected eggs was measured according to the following method, As a result of examining the transition, 10 ml of a liquid containing 80 units ml (1 mg / ml) of the hen egg antibody was obtained.

Method of collecting antibody from egg To the yolk separated from the egg, an equal amount of water was added, and an equal amount of a suspension of 0.5% λ carrageenan was added, and after stirring, 80
A centrifugal supernatant was obtained at 00 rpm for 20 minutes, and this was used as an antibody-containing fraction (WSF). The method for measuring the titer of WSF is as follows.

Method for measuring antibody titer (1) Suspension of microbial cells One to two colonies on TYC agar were inoculated into 200 ml of BHI broth, cultured anaerobically at 37 ° C. for 18 hours, and then collected. Wash 3 times with physiological saline containing 1% bovine serum albumin (hereinafter referred to as BSA saline). The washed cells are suspended in BSA saline and OD
Adjust to _550nm 1.0. The cell suspension should be prepared before use. (2) Sample antibody Dilution ratio of 20, 30, 50, 70, 90 to 2 each
Dilute ⁿ times. (3) Operation method 50 μl of diluted antibody and 50 μl of cell suspension were mixed on a microplate, stirred, and then at 37 ° C. for 3 hours, then at 4 ° C.
Then let stand overnight. (4) Judgment of bacterial cell aggregation After standing overnight, the bottom of the microplate is observed, and when the bacterial cell spot does not appear, it is determined to be aggregation positive, and when even a few spots are observed, it is determined to be aggregation negative.

[Example 2] (Method for preparing antiserum, breast milk and antibody) PAc purified in the same manner as in Example 1 (478 to 1000)
Was prepared at a concentration of 50 to 2000 μg / ml, mixed with an equal amount of Freund's incomplete adjuvant, and 0.3 ml per kg of body weight was subcutaneously applied to the back of pregnant goats, rabbits, cows and horses.
And then immunized three times with a mixture of an antigen and Freund's incomplete adjuvant every 2 to 4 weeks, and colostrum was collected after delivery. After the immunization, blood was collected and coagulated, and the centrifugation supernatant was used as a sample.

Next, an equal amount of 0.9% saline was added to the above colostrum sample, and the mixture was centrifuged at 12000 rpm for 60 minutes. Then, the upper layer fat and precipitate were removed, and an intermediate liquid component was collected. Concentrated hydrochloric acid was added to adjust the pH to 4. 50 more
After centrifuging at 00 rpm for 30 minutes, the supernatant was neutralized with trishydroxyaminomethane, ammonium sulfate was added to 75% saturation, and the obtained precipitate was collected,
This was dialyzed against a phosphate buffer to obtain an antibody component (internal solution) of breast milk. Further, ammonium sulfate was added to the above antiserum to make it 50% saturated, and the obtained precipitate was dialyzed against a phosphate buffer to obtain an antiserum antibody (internal solution).

As a result of measuring the antibody titer in the same manner as in Example 1,
A liquid containing about 280 units / ml (1 mg / ml) of serum antibody and a liquid containing 20 units / ml (1 mg / ml) of colostral antibody were obtained.

[Example 3] (S. mutan by antibody)
The effect of suppressing the adhesion of s to the smooth surface) S. mutans1044
After smearing 9 and anaerobically culturing for 18 hours, single colonies were collected from the plate and anaerobically cultivated in 4 ml of BHI medium for 18 hours. After that, 100 μl of 200 μl of culture solution
BHI medium was inoculated and anaerobically cultured for 18 hours. The bacterial cells were collected by centrifugation, washed 3 times with a 50 mM phosphate buffer solution, and then adjusted to a turbidity of 2.0 with the same buffer solution (prepared before use).

Next, the following experimental solution was tilted at about 60 degrees and incubated at 37 ° C. for 18 hours under aerobic conditions. Then, after washing 3 times with 4 ml of distilled water, 3 ml of distilled water was added, ultrasonic treatment was performed, turbidity was measured at OD _{550 nm} , and the adhesion inhibition rate was evaluated from the following formula. The results are shown in Table 1. Experimental solution preparation Bacterial solution 1.5 ml 10% sucrose solution 0.3 ml 50 mM phosphate buffer (pH 6.8) 0.9 ml 30 aggregation unit antibody solution 0.3 ml (or 50 mM phosphate buffer (pH 6.8))

[0045]

[Equation 1]

[0046]

[Table 1]

Example 4 (Caries Suppressing Effect of Anti-PAc Fragment Antibody) A caries suppression experiment by hamsters was carried out using three kinds of antisera prepared using rabbits. Hamsters start experiment 3
Eight groups of week-old males were used as the diets for the caries-inducing diet Diet2.
000, and the infectious strain is S. mutans 10449 was used. The experimental period was 3 weeks after the bacterial infection was completed, during which time 0.2 times of antiserum prepared into 3 units of agglutination units was used twice a day.
ml was dropped into the oral cavity of the hamster, and the tooth row was brushed for each of 20 strokes. The determination of caries is performed by the method of Kize (Keyes, PH: J. dent. Re).
s. , 23: 439-444, 1944). The results are shown in Table 2.

[0048]

[Table 2]

From the results of Examples 3 and 4, the anti-PA of the present invention was obtained.
The c-fragment antibody is anti-PAc antibody or anti-S. It is recognized that a caries inhibitory effect superior to that of the mutans whole bacterium antibody is given.

Example 5 The following is a prescription example of a caries preventive agent. (1) Dentifrice Dicalcium phosphate dihydrate 50.0% Sorbit 10.0% Glycerin 10.0% Carrageenan 1.0% Sodium lauryl sulphate 1.0% Fragrance 1.0% Saccharin 0.1% Ethanol 2.0% Dextranase 0.02% Anti-PAc fragment goat milk antibody 0.2% Water remaining 100.0%

(2) Dentifrice Silicic anhydride 30.0% Glycerin 30.0% Sorbit 20.0% Carboxymethyl cellulose 1.0% Sodium lauryl sulfate 1.2% Fragrance 1.0% Saccharin 0.1% Ethanol 2.0% Sodium fluoride 0.1% Anti-PAc fragment horse serum antibody 0.1% Water remaining 100.0%

(3) Dentifrice Aluminum hydroxide 45.0% Sorbit 20.0% Carrageenan 0.5% Carboxymethyl cellulose 1.0% Lauryl diethanolamide 1.0% Sucrose monolaurate 2.0% Perfume 1. 0% Saccharin 0.1% Anti-PAc fragment milk antibody 0.3% Water 100.0%

(4) Dentifrice Dicalcium phosphate 45.0% Carboxymethylcellulose 1.0% Carrageenan 0.5% Solbit 35.0% Propylene glycol 3.0% N-lauroylmethyl taurine sodium 0.5% Gelatin 1. 0% Ethyl paraoxybenzoate 0.2% Sodium saccharin 0.1% Fragrance 1.0% Sodium monofluorophosphate 0.7% Anti-PAc fragment chicken egg antibody 0.4% Water 100.0% remaining

(5) Dentifrice Aluminum hydroxide 40.0% Carboxymethylcellulose sodium 1.0% Carrageenan 0.5% Solbit 35.0% Propylene glycol 3.0% N-myristoylmethyl taurine sodium 0.5% Peptide 1 0.0% Methyl paraoxybenzoate 0.2% Sodium saccharin 0.1% Fragrance 1.0% Anti-PAc fragment sheep serum antibody 0.5% Water 100.0%

(6) Dentifrice 20.0% Silicic Acid Sodium Carboxymethyl Cellulose 1.0% Sorbit 50.0% Polyethylene Glycol 5.0% N-Palmitoyl Methyltaurine Sodium 0.5% Casein 1.0% Paraoxybenzoic Acid Sodium acid 0.2% Saccharin sodium 0.1% Fragrance 1.0% Sodium fluoride 0.2% Anti-PAc fragment chicken egg antibody 0.3% Water remaining 100.0%

(7) Mouthwash Ethanol 20.0% Perfume 1.0% Saccharin 0.05% Lauryl diethanolamide 0.3% Chlorhexidine gluconate 0.01% Anti-PAc fragment horse serum antibody 0.1% Water 100.0% remaining

(8) Mouthwash Solbit 10.0% Ethanol 20.0% N-Myristoylmethyltaurine sodium 0.5% Sucrose stearate 1.0% Peptide 0.5% Methyl paraoxybenzoate 0.1 % Stevioside 0.1% Fragrance 0.5% Dextranase 0.2% Sodium fluoride 0.2% Anti-PAc fragment chicken egg antibody 0.2% Water remaining 100.0%

(9) Mouthwash Solbit 10.0% Ethanol 20.0% N-stearoylmethyltaurine sodium 0.5% POE (20) Sorbitan monooleate 1.0% Collagen 0.5% Methyl paraoxybenzoate 0 1% Saccharin sodium 0.1% Fragrance 0.5% Anti-PAc fragment milk antibody 0.4% Water 100.0%

(10) Tablets Gum arabic 6.0% Glucose 72.0% Gelatin 3.0% Perfume 0.2% l-Menthol 0.1% Spearmint oil 0.1% Sodium ascorbate 0.1% Anti-PAc Fragmented sheep milk antibody 0.1% water 100.0% remaining

(11) Gum Gum base 43.9% Calcium carbonate 2.0% Water candy 15.0% Sugar 30.0% Sucrose palmitate 1.0% Fructose 4.0% Aldose 3.0% Fragrance 1.0 % Anti-PAc fragment chicken egg antibody 0.1% 100.0%

(12) Ice cream Cream (50% defatted rate) 16.84% Non-fat condensed milk without sugar 24.24% Sugar 11.25% Corn syrup 4.65% Stabilizer 0.35% Milk containing anti-PAc fragment antibody 37.67% Egg yolk containing anti-PAc fragment antibody 5.00% 100.0%

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a pac gene fragment region used in a reference example.

FIG. 2 is a graph showing the results of saliva adhesion in the same region.

[Procedure amendment]

[Submission date] November 17, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction content]

For example, in small animals, the antigen is 1 to 500 μg.
(As protein), for large animals 1 to 10 mg
Immunization is performed 3 to 5 times every 2 to 5 weeks by a conventional method. In the case of rabbits, the antigen solution (100 to 2 as protein)
000 μg / ml) mixed with an equal amount of Freund's complete adjuvant was administered 3 to 5 times every 2 to 5 weeks,
On the 10th to 14th day after the final immunization, blood is collected from the animal by a conventional method to obtain plasma containing immunoglobulin. This can be used as it is, or further purified by a conventional method and used as antiserum or immunoglobulin.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0039

[Correction method] Change

[Correction content]

Method for measuring antibody titer (1) Suspension of bacterial cells S. cerevisiae on TYC agar 200 ml of BHI broth was inoculated with 1-2 mutans 10449 colonies and cultured anaerobically at 37 ° C. for 18 hours, and then collected to 0.1
A physiological saline solution containing bovine serum albumin (hereinafter referred to as BS
Wash it three times with A). Washed cells are BS
A. Float in _saline and adjust to OD _550nm 1.0. The cell suspension should be prepared before use. (2) Sample antibody Dilution ratio of 20, 30, 50, 70, 90 to 2 each
Dilute ^n- fold. (3) Operation method 50 μl of diluted antibody and 50 μl of cell suspension were mixed on a microplate, stirred, and then at 37 ° C. for 3 hours, then at 4 ° C.
Then let stand overnight. (4) Judgment of bacterial cell aggregation After standing overnight, the bottom of the microplate is observed, and when the bacterial cell spot does not appear, it is determined to be aggregation positive, and when even a few spots are observed, it is determined to be aggregation negative.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0046

[Correction method] Change

[Correction content]

[0046]

[Table 1]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location A61K 39/00 ACK G 9284-4C 39/09 9284-4C

Claims (2)

[Claims] 1. A fragment obtained by cleaving a protein antigen having an action of adhering to a tooth surface, obtained from Streptococcus mutans, which fragment is part of amino acids 478 (aspartic acid) to 1000 (glutamine) or An antibody having immunological activity against Streptococcus mutans, which is obtained by immunizing a mammal or a bird with a polypeptide containing the whole as an antigen. 2. A caries preventive agent comprising the antibody according to claim 1 as an active ingredient.