JPH0368522A - Production of vaccine of dental caries - Google Patents

Abstract

PURPOSE:To obtain an antigen for vaccine of dental caries in high productivity by a simple purification operation by a variant of Streptococcus mutans GS-5 strain having a chromosome gene integrated with a manifestation gene of protein antigen produced on the surface layer of cell of Streptococcus mutans. CONSTITUTION:A manifestation gene of a protein antigen having especially 170,000-220,000 molecular weight measured by SDS polyacrylamide gel electrophoresis, produced on the surface layer or outside of cell of Streptococcus mutans, streptococcus in the oral cavity is transduced to a chromosome gene of Streptococcus mutans GS-5 strain (FERM P-2346) by using shuttle vector pSA3, pVA981, etc., to prepare GS-5 (K-3) strain (FERM P-2437), the variant is cultured, the antigen is extracellularly produced and the antigen is collected from the supernatant liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a caries vaccine and a novel Streptococcus mutans GS-5 mutant strain.

Streptococcus mutans group (Str
E. tococcus mutans group) is the most important causative agent of dental caries in humans and animals.

The S. mutans group is divided into 7 groups based on the properties of their DNA.
Type C mutans, which are classified into bacterial species and are most closely related to human dental caries, have various antigenic substances on their surface, such as serotype-specific polysaccharide antigens, lipoteichoic acid, and protein antigens.

Among these bacterial cell surface substances, protein antigens with a molecular weight of about 190,000 in particular are fine fimbriae-like structures called fimbriae (of which about 20,000 are sugar chains). This protein antigen is called by various names such as ■/II, B, PL, IF, etc., and is not unified. In this specification, this protein antigen is referred to as PAc (Hrotein antigen 5erotype).
A) - Thus, the S. mutans protein antigen is currently attracting the most attention as a caries vaccine, and Japanese Patent Publication No. 56-501364 describes anti-Ml/U that exhibits specific properties as a caries vaccine. It has been reported to be used as a vaccine. Further, Japanese Patent Application Laid-Open No. 164518/1983 discloses a vaccine for preventing dental caries using the surface layer of a microorganism of the genus Streptococcus or an extract thereof as an antigen.

However, when extracting PAc from S.mutans, there was a practical problem as only a few cells could be recovered even if S.+nutans was cultured in 1 (Ml medium).Also, the concentration of PAc in the medium was low. Therefore, labor and time were required for purification work to remove impurities.

The present inventors investigated the PAc expression gene of S. mutans (
5 target genes), Sedan (E. coli), Mutual, m1ller
i (a type of Streptococcus) to produce rPAc, but since the gene was inserted into a different bacterial species, no increase in the amount of production was observed, nor was it released outside the bacterial cells. Note that rP A c refers to P A c (recombinant PA c) made by a genetic recombinant.
c), and is a protein that is structurally substantially identical to PAc.

Furthermore, in order to clarify the biological role of PAc, the present inventors connected the pac gene to a plasmid, a shuttle vector, that can be propagated both in E. coli and streptococci.
This is expressed as S, mutans G S, which does not express PAc.
-5 strains were reported (Japan Dental Review, Ja
n,, 1989. Nn555), (/Kashi, this 05
In the -5 mutant strain, after the Pu gene was introduced into the bacterial cells, the plasmid fell out of the 5 recombinant bacteria, resulting in the failure to produce PAc, and there were also problems with the uniformity of the product.

The present invention provides a method for producing a caries vaccine that improves the production amount of PAc and simplifies the purification process, and a method for producing a caries vaccine that makes it possible to improve the production amount of PAc and simplify the purification process, as well as a method for producing a caries vaccine that makes it possible to improve the production amount of PAc and simplify the purification process, as well as to provide a method for producing a caries vaccine that makes it possible to improve the production amount of PAc and simplify purification work, as well as to provide a method for producing a caries vaccine that makes it possible to improve the production amount of PAc and simplify the purification process, as well as a method for producing a caries vaccine that makes it possible to improve the production amount of PAc and to simplify the purification process.
This provides an S-5 mutant strain.

Streptococcus mutans The method for producing the caries vaccine of the present invention is based on the protein antigen (PAc) expression gene (L
target gene), Streptococcus mutans 05
It is characterized by culturing a mutant strain that has been integrated into the chromosomal gene of the -5 strain.

In addition, the novel strain of the present invention is created by genetic engineering, and is a strain of Streptococcus mutans that has been transformed with the Pu gene integrated into the chromosome and has increased PAc production ability.
mutans) OS-5 (K-3) [Feikoken Joyori No. 2437].

The present invention will be explained in more detail below.

In the present invention, the host bacteria include S, mutans G S
-5 strain was used, while the donor bacterium was P A
Any strain belonging to S. mutans that can produce c can be used, and strains such as cln serotype, e serotype, and f serotype are preferred. In this way, by using the host bacterium and the donor bacterium as belonging to the same bacterial species, structural changes in the produced rPAc can be suppressed as much as possible.

S. mutans GS-5 is a known S. mutans that does not produce PAc, and is stored in various research institutions such as universities, but in consideration of the convenience of third party acquisition, etc. Streptoc No. 2436 (Streptoc)
occus mutans GS-5).

As a donor bacterium, Streptococcus mutans N
CT C10449, Streptococcus mutans MT811, Streptococcus mutans In
gbritt, Streptococcus mutans MT
6. Streptococcus mutans C67 Streptococcus mutans MT118, Streptococcus mutans C67-1, Streptococcus mutans
Streptococcus mutans OMZ70, Streptococcus mutans MT6801. Streptococcus mutans JCI-5, Streptococcus mutans A
T CC33477, Streptococcus mutans 5EI1. Streptococcus mutans OM
Z 175, Streptococcus mutans MT
557, Streptococcus mutans GS, Streptococcus mutans PK-3, etc. are used.

By using such a donor bacterium and introducing the gene encoding PAc into the chromosome of Streptococcus mutans GS-5 strain according to a conventional method, the Streptococcus mutans GS-5 (K
-3) can be obtained. Here, the vectors (plasmids carrying PAc) include shuttle vector PSA3, pV
A981, etc., and restriction enzymes for cutting include Shadow υH1, Plow■, Shika■, Carver R1゜Carver T141. Hi
Mochi ■, Hitto I, Kagebe I, etc. are used.

In the present invention, SDS polyacrylamide electrophoresis (S
The molecular weight as measured by DS-PAGE is approximately 170,000 to 2.
As a gene encoding 20,000 protein antigens (PAc), for example, the start codon (ATG) of the base sequence shown below is used.
) to the stop codon (TGA), or a portion thereof encoding a portion substantially including 1,565 amino acids (molecular weight approximately 170,000). Furthermore, a base sequence in which a part of the base sequence is replaced with another base or a part of the base sequence is deleted, and which encodes a protein having substantially the same antigenicity as PAc, can also be used.

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In the GS-5 (K-3) strain of the present invention, the Totan gene is GS
Since it has been integrated into the chromosome of the -5 strain, the Todan gene is easily shed and the rP Ac production ability is high, and rP Ac is produced outside the bacterial cells.

The location of the 匡 gene in the chromosomal DNA of a bacterial strain can be confirmed by Southern blot method, etc., by collecting such a strain and culturing it.

r P A c can be obtained efficiently. Furthermore, since the rPAc content in the culture medium is very high, the purification procedure is greatly simplified. The resulting rPAc can be used as a caries vaccine in various forms.

S of the present invention, +++utans GS-5 (K-
3) Cultivation of the strain can be carried out, for example, as follows.

37 g of plain heart infusion medium is dissolved in IQ distilled water and sterilized at 121° C. for 20 minutes, then 1 mfl of 5 mg/m Q erythromycin, which has been previously filter-sterilized, is added and mixed. 10 each of the same culture solution
mfl was dispensed into a 24mA sterile test tube, and
S-5 (K-3) strain was inoculated, and the culture solution was incubated at 37°C for 18
Culture for an hour and prepare a seed culture. 100m of the seed culture solution
Q is added to a 2Q Sakaro flask containing medium IQ with the same composition, and cultured at 37°C for 18 hours to obtain a microbial liquid. The microbial solution was centrifuged for 15 minutes using a 10,000 OX machine to obtain a supernatant.

In addition, purification methods from the culture supernatant include dextran-based, agarose-based gel filtration methods, AE, DEAE, QA, etc.
Ion exchange method using E, CM, SP, phosphor, etc.
Hydrophobic chromatography using octyl Sepharose, phenyl Sepharose, etc., affinity chromatography, etc. are used.

The resulting caries vaccine is administered orally, subcutaneously, or the like. The dosage is suitably about 10 μg to 50 mg, preferably 10 μg to 10 mg,
is optimal.

Streptococcus mutans GS-5 (K-3)
The mycological properties of [Feikoken Joyori No. 2437] are as follows. This mycological property and classification method is described by Berge
y's Manual of SystematicB
Acteriology, Vol. 2 (1986).

A. Morphological properties When cultured in plain heart infusion medium at 37°C for 2 days, the following morphological characteristics were observed.

1) Cell shape and size: Cocci, 0.5-0.75 μm, forming chains.

2) Polymorphism: None.

3) Motility: None.

4) Spores: None.

5) Durham staining: Positive.

6) Anti-acidity: Negative.

B. Culture properties 1) Plain heart infusion agar plate culture: Grows at pH 7.0 to 7.4, forming round, oblate, gold-rimmed colonies. The surface of the colony is smooth and glossy, and yellowish-white or pale brown.

2) Plain heart infusion agar slant culture: Grows in a spread shape at pH 7.0 to 7.4, glossy yellow-white or light brown color.

3) Plain heart infusion liquid culture: p) Grows at 17.0 to 7.4, but does not form a bacterial membrane and forms a uniform turbidity.

4) Meat juice gelatin puncture culture: Does not liquefy.

5) Ridmus Milk: It grows well, produces acid, and curdles milk. harden

C1 physiological properties Nitrate reduction: negative.

Denitrification reaction: negative.

MP test: positive.

VP test: positive.

Indole production: negative.

Hydrogen sulfide formation: negative.

7) Starch hydrolysis: Negative.

8) Use of citric acid: 1 (not used in Oser's medium and Christensen's medium).

9) Utilization of inorganic nitrogen sources: Utilizes nitrate and ammonium salts do not have.

Pigment production: #i property.

Urease: Negative.

Oxidase: negative.

Catalase: negative.

Growth range 33°C to 38°C (37°C is the best).

Grows at pH 6.0 to 7.4.

Attitude towards oxygen: Facultatively anaerobic.

0-F test: positive.

Acid and gas production from sugars: D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose,
Acid is produced from lactose, trehalose, D-sorbitol, and D-mannite, but no gas is produced. L-arabinose, D-xylose, inodite, glycerin, and starch do not produce acids or gases.

D. Other properties 1) Decomposition of arginine: Negative.

2) Hemolytic: γ-hemolysis.

3) Liquefied sodium resistance: Resistant to 4% sodium chloride.

4) Other properties recognized as necessary Hydrolyzes 22 scrine and resists bacitracin at 2 units/mn. Inulin, D
- Raffinose produces acid from D-melibiose.

Synthesize glucan from sucrose.

To summarize the above properties, acid is produced from inulin, D-raffinose, D-melibiose, and D-sorbitol,
Due to the fact that it is a bacitracin-resistant Durham-positive streptococcus that does not hydrolyze arginine, 5tre toc
occus mutans (Streptococcus mutans).

According to the present invention, by culturing the Streptococcus mutans GS-5 mutant strain in which the Totan gene has been integrated into the chromosomal gene, rP can be stably produced at a high production amount.
A c can be obtained, and the purification operation is simple. As one such genetically modified bacterium, Streptococcus mutans GS-5 (K-3) has been published by Fermentation Re5ear, Agency of Industrial Science and Technology, as Fermentation Re5ear No. 2437.
ch In5titut).

(1) Introduction of this gene into 0S-5 strain 5tre tococcus mutans N CT
Plasmid pPC77 carrying C10449g and shuttle vector PSA3 were digested with restriction enzyme BamHI and s
Cut each at pH 1. Cleaved pSA3 and p
PC77 was ligated using T4 DNA ligase, and the resulting plasmid was introduced into E. coli strain 88101 (National Institute of Genetics, archive number: ME8568) by the transformation method, resulting in chloramphenicol resistance and ampicillin resistance. Sensitive and tetracycline sensitive clones were selected.

Recombinant plasmids were extracted from the clones that were confirmed to be producing PA and c by colony immunoblotting and Western blotting, and their sizes were confirmed by agarose electrophoresis. It had a 16.4 kb recombinant plasmid. One of these plasmids was named K, and this plasmid was electroporated to produce P A c non-producing S, mut
Ans GS 5 strain was introduced and transformed. The obtained PAc-producing strain was named QS-5 (K-X).

A plurality of O3-5 (K-X) strains were obtained through this operation, but each strain had a different ability to produce P Ac. Among these, strain GS-5 (K-3) has an excellent ability to produce PAc.
The desired integration site was confirmed for the strain GS-5 (K-9), which produces PAc and is not superior to the previous strain.

(2) Confirmation of the integration site of the Todan gene The 08-5 strain and the GS-5 (K-3) strain obtained by transformation were lysed with lysozyme.

Furthermore, the plasmid and chromosomal DNA were separated. When the separated plasmid fractions were subjected to agarose electrophoresis, as shown in Figure 1, GS-5 strain and GS-5(K
-3) No plasmids were present in this fraction of the strain. On the other hand, plasmid was observed in the fraction of GS-5 (K-9) strain.

Further, after cutting the chromosomal DNA with EcoRI, Southern blot analysis was performed using a conventional method.

When the BamHI fragment of the erythromycin resistance gene was used as a probe, as shown in Figure 2,
Chromosomal DNA of GS-5 strain and GS-5(K-9) strain
showed no reactivity, whereas O3-5(K-3
) strain showed reactivity.

These findings confirmed that in the as-5 (K-3) strain, plasmidl 8M1 was integrated into the chromosomal DNA of the GS-5 strain as a result of homologous recombination. On the other hand, P
Although the GS-5 (K-9) strain produces Ac, the OS-5 (K-3) strain has no ability to produce Ac, and the GS-5 (K-9) strain has the L target gene in the plasmid, and the L target gene exists in the chromosome. The gene could not be found.

For Southern plot analysis, see E, M, 5outhern (1975): Det.
Ection of specific 5equanc
es among DNA fragmentssep
arated by gel electrophor
esis J, Mol.

Biol., 98, 503-517.

(3) Culture and recombining
TT PAc) purified S, mutans GS-5 (K-3) strain
After culturing in external dialysis solution of Y broth or BHI broth,
The culture supernatant was salted out with ammonium sulfate at 60% saturation, the precipitate was dissolved in 50 mM Tris-HCl buffer pH 7.5, dialyzed, and this specimen was applied to a DEAE-cellulose column equilibrated with the same buffer. Flow. After flowing 2 bed volumes of the same buffer solution into the column, perform NaCl concentration gradient elution from 0 mol to 0.5 mol. As a result, NaCl concentration is 0.2 mol.
r P A c elutes around the molar range. This elution fraction can be used as it is as a purified sample of rPAc. G
In the S-5 (K-3) strain, since the rPAc content in the culture supernatant is extremely high, the purification procedure is greatly simplified in this way.

These rPAcs are used as caries vaccines in various forms.

In addition, in the same manner, NCT C10449 strain, GS-
5 strains and GS-5 (K-9) strain were cultured, PAc produced in the culture supernatant was purified, and its amount was measured by the Lowry method based on the protein amount, and the relative values are shown in the table below. It is shown in l. It can be seen that the GS-5 (K-3) strain of the present invention significantly increases the production amount.

(Margin below) (4) Immunization experiment using rPAc The composition shown in Table 2 was dissolved in PBS, and 0.1 mQ of this was intraperitoneally injected into a group of 5 mice at IO day intervals.
Immunized twice. Seven days after the second administration, blood was collected from the mice, and the blood antibody titer against rPAc was measured by enzyme-linked immunosorbent assay (ELISA). Table 2 shows the results.
Shown below. The antibody titer was expressed as the reciprocal of the dilution of the serum that showed reactivity by ELISA when the obtained serum was diluted 1710 times and then further diluted one-fold.

[Brief explanation of drawings]

FIG. 1 is a diagram showing agarose electrophoresis of plasmid fractions. Figure 2 shows “Psm erythromycin sin gene BamH
FIG. 2 is a diagram showing the results of Southern blot analysis of chromosomal DNA using an I fragment probe. Figure 1 34 Agarose electrophoresis of plasmid fraction Lane 1 Niblasmid ρSMI (circular plasmid) Lane 'l: Plasmid fraction of GS-53 Lane 3:
Plasmid fraction lane 4 of GS-5 (K~3) building
: GS-5 ('ni-9) strain Kabrasmid fraction 2 32 pgQ dilithromycin resistance gene 5as + HI fragment O-be chromosome DIJA Sazanop mouth-7) - Aggregate lane 1: GS-5a dormant chromosome BemiFiI fragment of DNA v-72: GS-5 (GS-5 (K-3) a^ Moe R1 fragment Lane 3: Momo Rr fragment of GS-5 (Ni-9) clothing chromosome ONA

Claims (1)

[Scope of Claims] 1. The expression gene of a protein antigen produced on the bacterial cell surface layer or outside the bacterial cell of oral streptococcus Streptococcus mutans is expressed in Streptococcus mutans GS-
A method for producing a caries vaccine, which comprises producing the above-mentioned antigen by culturing a mutant strain that has been integrated into the chromosomal gene of five strains. 2. The protein antigen has a molecular weight of approximately 170,000 to 22,000 as determined by SDS polyacrylamide gel electrophoresis.
2. The manufacturing method according to claim 1, wherein the method is 1,000,000. 3. The mutant strain is Streptococcus mutans GS-5 (K-
3) The production method according to claim 1 or 2, which is a strain. 4. Streptococcus mutans GS-5 (K), which has an expression gene for a protein antigen produced on the bacterial surface or outside the bacterial cell of oral streptococcus Streptococcus mutans, is integrated into the chromosome and has the ability to produce the protein antigen. -3).