JP2593147B2 - Medium composition for protective antigen-producing bacteria - Google Patents

Description

[Technical field to which the present invention belongs] The present invention relates to a medium for an infection-protecting antigen-producing bacterium for producing a large amount of a bacterial component and a biologically active substance of various bacteria, particularly an infection-protecting antigen. Composition.

[Prior Art] As a method for producing a purified sedimented pertussis vaccine as a component vaccine, for example, Japanese Patent Publication No. 57-5203 is already known. This vaccine uses an HA fraction containing F-HA and LPA-HA as a main infection-protecting antigen, has almost no side effects, and has an excellent prophylactic effect.
It has already been put to practical use.

For the production of this practical vaccine, one-phase bacteria pertussis is inoculated into an appropriate medium, cultivated at about 35 ° C. for 5 days, the culture is centrifuged, and ammonium sulfate is added to the supernatant. The resulting precipitate was centrifuged at 10,000 rpm for 30 minutes and separated. The precipitate was extracted with a buffer containing sodium chloride, and the extracted fraction was subjected to a sucrose density gradient by a conventional method. Centrifugal crest HA by centrifugation
The fractions are collected and detoxified with formalin to form a vaccine.

Then, if desired, diphtheria toxoid and tetanus toxoid are added thereto, and aluminum adjuvant treatment is further performed, if necessary, and a stabilizer such as gelatin and glucose is added thereto to obtain a diphtheria / tetanus mixed vaccine by precipitation and purification.

However, this method has a particular difficulty in culturing, and large-scale culturing is impossible, and mass production of vaccines is difficult. That is, in this known method for producing a purified sedimented pertussis purified vaccine, a liquid medium is placed in a small container such as a roux bottle.
It is placed in a lying position and cultivated at about 35 ° C. for about 5 days in a lying position, and requires a very small scale and a long period of time.

Generally, for mass culture of microorganisms, a stirring culture method using a liquid medium is often employed. The growth of H. pertussis can be achieved to a certain extent by shaking culture in a liquid medium, but it is said that the production of the HA fraction is extremely low (Arai, H & Munoz, JJ, Infect Immun. 25,7
64-767, 1979).

This suggests that at least one of the components of the purified vaccine is difficult to mass produce. Therefore,
Although this pertussis vaccine is an epoch-making vaccine, its production has to rely on stationary culture which requires a long time on a small scale, and there is a need for an improved production method.

Recently, it promoted the growth of phase I pertussis and
Search for additives capable of promoting the production of cyclodextrin and its derivatives, especially methylated β-cyclodextrin (2,6-di (O-methyl) -β-cyclodextrin, hereinafter methylated β-CD ) Was added to the Steiner-Scholte liquid medium (St. pertussis I).
ainer, DW & Scholte, MJ: J.Gen.Microbiol.63,211
-220,1971) to promote bacterial growth and LPD-HA production in agitated cultures, and to increase L in the culture broth.
It has been reported that it also contributes to the stability of PF-HA (see Suzuki et al., 29th Toxin Symposium Proceedings, 1-51982).

However, these additives are expensive and not readily available. Further, when such a method is applied to production of an antigen for protecting infection of B. pertussis on an industrial scale using a fermenter having a scale of 101 or more, it cannot be inferred from the knowledge based on the conventional stirring culture at all. The result was obtained. That is, in shaking culture and stirring culture system with constant stirring conditions, the number of bacteria may be increased, but LPF
-I knew that the production of HA was not enough.

Further, in a medium composition for culturing Bordetella bacteria, a cyclodextrin or a derivative thereof and one or more etherified derivatives of a D-glucose polymer having a molecular weight of at least 2,000 are mixed and contained in a culture medium. Thus, a medium composition for culturing Bordetella bacteria is known. However, even in this medium composition, as described above, there is a problem that the additive substance cyclodextrin is expensive and not easily available.

[Problems to be solved by the invention]

The present inventors promoted the growth of B. pertussis I phase bacteria, and protected the HA fraction (LPF-HA: Leucocytosis-
Promoting the production of Facter Hemagglutinin) and continued to search for inexpensive and readily available additives. As a result, only metrose (trade name of methylcellulose and hydroxypropylmethylcellulose) was used for normal pertussis bacteria. It was found that, in addition to a nutrient medium, the growth of bacteria and the production of LPF-HA and FHA were promoted by shaking culture or stirring culture, and LPF-HA and FHA were also extremely stable in the culture medium.

Furthermore, when only metrose is added, it promotes the growth of Bordetella, Staphylococcus, Streptococcus, Bacteroides, Actinomyces, and other bacteria in submerged culture under the appropriate medium and culture conditions. In addition, it was found that the corresponding antigen production was promoted and the produced antigen was stable even in the culture solution.

The present invention has been completed based on the above findings.

[Means for solving the problem]

The present invention provides a method for producing an infection-protecting antigen, comprising culturing an infection-protecting antigen-producing bacterium in a medium to accumulate the infection-protecting antigen, and collecting the infection-protecting antigen from the culture, wherein cyclodextrin or a derivative thereof is used. This is a medium composition for producing an infection-protecting antigen, which is obtained by adding one or more water-soluble cellulose ether derivatives alone to a nutrient medium for producing an infection-protecting antigen that is not included.

Accordingly, it is an object of the present invention to provide an infection control antigen capable of promoting the growth of infection-protecting antigen-producing bacteria and promoting the production of infection-protecting antigens, thereby enabling industrial production of the infection-protecting antigens in large quantities. It is another object of the present invention to provide a medium composition for producing a protective antigen.

Examples of the infection-protecting antigen-producing bacteria used in the present invention include bacteria producing pertussis infection-protecting antigens (LPF-HA, F-HA fraction), other genus Bordetella, Actinomyces viscosus, and the like. Bacteroide gingivalis (Bacteroide)
S. gingivallis) and bacteria producing infectious defense antigens (microbial cell surface pilus components) belonging to the genus Streptococcus such as Streptococcus mutans.

When the above-mentioned infection-protecting antigen-producing bacteria are cultured in a submerged condition with a well-known liquid medium adapted to each, an appropriate amount of a water-soluble cellulose ether derivative is added to a liquid nutrient medium not containing cyclodextrin or a derivative thereof. Culture is performed with addition.

Examples of liquid cultures that do not contain known cyclodextrins or derivatives thereof include B. pertussis I phase bacterium, Bordejung medium, modified Stener-Scholte medium, Actinomyces viscosas, Streptococcus mutans, TTY medium, and Bacteroides.・ Gingivalis is TCS
A liquid medium such as a medium can be used.

Examples of the water-soluble cellulose ether derivative added to the liquid medium include metrose (trade names of methylcellulose and hydroxypropylmethylcellulose).

This type of metrose differs in the amount of methoxy group, the amount of hydroxypropoxy group, the degree of substitution, the viscosity, the thermal gelation temperature, and other properties depending on the cultivar. The metroose SM type (methylcellulose) contains three types of glucose in one glucose residue contained in cellulose. OH groups, of which about 2 are substituted with methoxy groups, and Metroose SH type (hydroxypropyl methylcellulose) has about 2 OH groups substituted with methoxy groups in the same manner, Some of them are substituted with hydroxypropoxy groups instead of methoxy groups.

In general, the SH type has a smaller number of methoxy groups to raise the thermal gelation temperature than the SM type, and a small amount of a hydroxypropoxy group is introduced to supplement the hydrophilicity, but the methoxy group accounts for 84 to 93% of the total substituents. And retains the properties of methylcellulose. These varieties of the metrose type include methylcellulose SM type, hydroxypropylmethylcellulose 60SH type, 65SH type, and 90SH type, and varieties having different viscosities (viscosity 2%, 20 ° C., 15 to 30,000).

The above-mentioned water-soluble cellulose ether derivatives can be used alone or in combination of two or more. The ratio of addition to the liquid medium not containing cyclodextrin or a derivative thereof is not particularly limited, but is usually appropriately selected and added in the range of 0.001 to 10 g, preferably 0.05 to 1 g per liquid medium.

The growth temperature of the above-mentioned infection-protecting antigen-producing bacteria can be in the range of 20 to 37 ° C, preferably 23 to 37 ° C. Medium pH
Although there is no significant difference in the growth rate in the range of 6.0 to 9.0, the culture is preferably performed in the range of pH 6.5 to 8.5.

In the above submerged culture, when performing aerobic culture, it is preferable to carry out aeration and stirring while maintaining the range of dissolved oxygen in the range of 0.5 to 10 ppm. The amount of aeration varies depending on the type of infection-protecting antigen-producing bacterium and the culturing conditions, but is performed in the range of 0.5 to 101 / min, preferably about 51 / min. When performing anaerobic culture, for example, nitrogen gas 90%, hydrogen gas 5%, carbon dioxide gas 5%
Is performed by replacing with a mixed gas such as

The number of rotations for stirring during the submerged culture is in the range of 50 to 1100 rpm, preferably about 750 rpm. In the above-described deep culture, the presence or absence of the defoaming treatment affects the number of proliferating bacteria in the culture solution and the yield of the amount of antigen, and therefore, is usually performed under the defoaming treatment. As the defoaming treatment, a mechanical defoaming means, a chemical defoaming agent such as ADEKANOL (manufactured by Asahi Denka) 0.00
It is performed by adding about 1% or by combining them, but it is preferable to use a chemical defoaming agent.

Thus, the infection-protecting antigen-producing bacterium is cultured, and the culturing time is such that the infection-protecting antigen-producing bacterium is produced maximally during the logarithmic growth phase or the stationary phase of the bacterial growth phase. Wait and decide.

A well-known method can be used to collect an infection-protecting antigen from the culture using the thus obtained culture medium for an infection-protecting antigen-producing bacterium of the present invention.

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

Example 1 Hydroxypropyl methylcellulose 60SH50 0.01%
The added Stainer-Schorte modified medium 150 ml / Sakaguchi flask, pH 7.6 medium was inoculated to a culture medium of B. pertussis Higashihama 0.2bil / ml, and cultured at 35 ° C for 48 hours at a shaking speed of 130 rpm./min. Was. As a result, as shown in FIG. 1, the amount of the proliferating bacteria reached 104 bil / ml in 40 hours and 118 bil / ml in 43 hours by adding hydroxypropylmethylcellulose, and no decrease was observed even after 48 hours. ELISA after 48 hours of antigen amount
U / ml also showed good results with HP316, PT323 and FHA29.

Example 2 Hydroxypropyl methylcellulose 60SH50 was added to a modified culture medium (shown in Table 1) of hydroxypropylmethylcellulose 60SH50 to a final concentration of 0.01% in a culture tank (50 l, Novoparillias), and 50 l of a medium adjusted to pH 7.8 was added. prepared, inoculated with Borodejangu medium passages pertussis bacteria Higashihama lines so as to 0.2bil / ml, rpm 750R.Pm, vent 5 ml / min, inner pressure 0.3 kg / cm ²
, A 0.001% antifoaming agent (ADEKANOL, manufactured by Asahi Denka) was added, and the cells were cultured at 35 ° C for 43 hours.

Table 1 Stainer-Scholte modified medium components (g /
l) Sodium glutamate 10 l-Proline 0.2 Sodium chloride 1 Potassium dihydrogen phosphate 0.5 Potassium chloride 0.2 Magnesium chloride 0.1 Calcium chloride 0.026 Trishydroxyaminomethane 6 Casamino acid 2.5 Soluble starch 3 * 1-Cistine hydrochloride 0.04 * Ferrous sulfate 0.01 * Ascorbic acid 0.5 * Niacin 0.04 * Glutathione 0.2 * Filter sterilized and mix before use.

* Hydroxypropyl methylcellulose is dissolved in warm water and mixed separately before use.

After each culture time, the proliferating bacteria and the antigen titer were measured by the hemagglutination test and the ELISA method as described above. The results are shown in FIG. As is clear from FIG. 2, the amount of the proliferating bacteria increased to 80 bil / ml after 30 hours, and increased to 104 bil / ml around 40 hours, and thereafter, no decrease was observed.

The HA value was ²⁶ after 30 hours, and no decrease was observed thereafter. ELISA U / ml at the time of collection after 42 hours: HP420, PTH45
0, FHA1907 and good results were obtained.

Example 3 In a culture tank, 50 l of a culture medium adjusted to pH 7.8 in which hydroxypropylmethylcellulose 60SH50 was added to a final concentration of 0.015% in a TTY medium shown in Table 2 below was added to Actinomyces, Viscosis K-TL + strain ( Microfabrication Laboratories No. 411
No.) and a defoamer (ADEKANOL, Asahi Denka) 0.001% in aeration and agitation culture system with a rotation speed of 150 rpm and aeration of 1 / min.
, And cultured at 36 ° C. for 48 hours.

Extraction of the cell surface antigen (pilus structure) was attempted from the culture by the following treatment. That is, the culture was precipitated by adding ammonium sulfate to 33% saturation, and the precipitate was separated by centrifugation (12,000 g, 15 minutes). This was separated from 0.05 M Tris-HCl buffered 1 M NaCl.
(PH7.6) Suspended in 5 liters of liquid and sonicated (20KHg, 5 minutes)
Thereafter, the components extracted by stirring for further 72 hours were treated by sulfuric acid fractionation, Sephacryl S, 300 gel filtration fractionation, and sucrose density gradient centrifugation (5 to 30%).

Pili components were recovered in fractions with a sugar concentration of 11-20%. The recovery from the normal submerged culture was about 30 μg / ml of protein N, but when the medium of the present invention was used, it was about 4 times that of 125 μg / ml.
μg / ml of the desired pilus component was recovered.

Table 2 Contents of TTY medium components (g / l) Tryptocase peptone (BBL) 15 Tryptospeptone (DIFCO) 4 Toast extract (DIFCO) 4 Sodium chloride 2 Sodium carbonate 2 Dipotassium phosphate 2 Monopotassium phosphate 5 Glucose 10 Hydroxypropyl methylcellulose is dissolved in warm water, sterilized, and added and mixed before use.

Example 4 Streptococcus mutans strain K-DP (FERM BP-317) was cultured according to the method described in Example 3, and treated in the same manner to extract a cell surface antigen. The cell surface antigen was recovered by fractionation at a sugar concentration of 10 to 11% by sucrose density gradient ultracentrifugation. From normal submerged culture, protein N amount 28-30μ
g / ml, but in the culture method using the medium of the present invention.
The yield was 53 to 59 μg / ml, which was about 2-3 times the yield. And other Streptococcus mutans d, e, f and g
It was similarly recovered from the culture of the mold. These antimicrobial surface antigens were confirmed by a sedimentation reaction in the gel.

Example 5 In a culture vessel, hydroxypropyl methylcellulose 60SH50 was added to a TCS modified medium shown in Table 3 below at a final concentration of 0.015.
% Of the medium, which was adjusted to pH 7.4, sterilized, and sufficiently replaced with a mixed gas (90% nitrogen gas, 5% hydrogen gas, 5% carbon dioxide gas), followed by Bacteroides gingivalis K- Bg-m ₁ strain (Microtechnical Laboratories No. 410) was inoculated at 0.1 bil / ml, and aeration and agitation culture system with a rotation speed of 150 rpm and a mixed gas of 1 / min was added with an antifoaming agent. And cultured at 36 ° C. for 48 hours.

After each culture time, the enrichment amount and HA were measured by a hemagglutination test. The results are shown in FIG. The growth rate reached 200 bil / ml after 20 hours.

Is a HA titer 2 ⁴ about in normal submerged culture, HA titer in culture with media of the present invention exhibit 2 ⁸ After 20 hours,
Subsequent reduction of the HA value was not observed, and good results were obtained.

Table 3 Modified TCS medium component content (g / l) a) Tryptocase peptone (BBL) 34 Phyton peptone (BBL) 6 Yeast extract (Difco) 6 Dissolve in 200 ml of purified water and dialyze in 800 ml of purified water Use the dialyzed external solution.

b) Sodium chloride 5 dipotassium phosphate 2.5 glucose 2.5 hemin 1 ml (dissolve 5 mg of hemin in 1 ml of 0.1 N NaOH) Menadione (menadione dissolves 0.5 mg in 0.5 ml of ethanol and add 0.5 ml of purified water) pH7 .4 Dissolve hydroxypropylmethylcellulose 60SH50 in warm water, sterilize it separately, add and mix before use.

[Brief description of the drawings]

FIG. 1 is a curve showing the bacterial concentration of B. pertussis when hydroxypropyl methylcellulose is added to the medium and when hydroxypropyl methylcellulose is not added. FIG. 2 is a curve showing the relationship between the bacterial concentration and the HA titer when culturing B. pertussis. FIG. 3 is a curve showing the cell concentration and HA titer relationship when cultured Bacteroides gingivalis K-Bg-m ₁ strain.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12R 1:21) (C12P 21/00 C12R 1:01) (C12P 21/00 C12R 1:04) (C12P 21/00 C12R 1:46)

Claims (5)

(57) [Claims] 1. A method for producing an infection-protecting antigen, comprising culturing an infection-protecting antigen-producing bacterium in a medium to accumulate the infection-protecting antigen, and collecting the infection-protecting antigen from the culture, wherein cyclodextrin or a derivative thereof is used. A medium composition for an infection-protecting antigen-producing bacterium, which comprises adding one or more water-soluble cellulose ether derivatives alone to a nutrient medium composition for an infection-protecting antigen-producing bacterium not containing 2. The method according to claim 1, wherein the infection-protecting antigen-producing bacterium is B. pertussis, another Bordetella bacterium, Actinomyces viscosus, Bacteroides gingivalis or Streptococcus mutans. A medium composition for infection-protecting antigen-producing bacteria. 3. The medium composition according to claim 1, wherein the water-soluble cellulose ether derivative is methylcellulose or hydroxypropylmethylcellulose. 4. The medium composition for an infection-protecting antigen-producing bacterium according to claim 1, wherein the medium for an infection-protecting antigen-producing bacterium is a liquid medium. 5. The medium composition for an infection-protecting antigen-producing bacterium according to claim 1, wherein the liquid medium comprises a water-soluble cellulose ether derivative at a ratio of 0.001 to 1 g per liquid medium.