WO2007072576A1

WO2007072576A1 - Method for production of strain capable of expressing m toxin derived from helicobacter pylori in large quantity

Info

Publication number: WO2007072576A1
Application number: PCT/JP2005/024010
Authority: WO
Inventors: Hiroyuki Ohno
Original assignee: Fourier Inc.
Priority date: 2005-12-21
Filing date: 2005-12-21
Publication date: 2007-06-28

Abstract

Disclosed are a method for producing M toxin derived from Helicobacter pylori in a large quantity by culturing Helicobacter pylori for a long period of time to give the M toxin in a large quantity and a culture medium for use in the method. A brain heart infusion medium for use in the long-term culture of Helicobacter pylori which is free from the blood serum, comprises 0.1 to 1% of 2,6-di-o-methylcyclodextrin and has a pH value ranging from 7.45 to 7.65.

Description

TECHNICAL FIELD Field of the Invention

The present invention relates to a method for cultivating Helicopacter pylori over a long period of time in a serum-free medium and a method for producing a large amount of cytotoxic protein (M protein) by culturing Helicopacter pylori over a long period of time in a serum-free medium. About. Background art

Many gastritis, gastric ulcers or gastric cancer have been thought to be due to Helicobacter pylori. As described above, it is estimated by many researchers that Helicopacter pylori secretes a factor that directly inhibits gastric mucosal cells in the gastric environment of the living body, and in view of the importance of the disease, Although the entire sequence of the gene was confirmed in 1996, the culture conditions that were still difficult even with the use of serum, and the separation and purification conditions and the unestablished evaluation system have hindered the isolation and identification of putative toxins. Under these conditions, the present inventor previously found and isolated a responsible protein responsible for gastritis associated with Helicobacter pylori infection, gastric ulcer, gastric cancer, etc. (W0 03/048 199) It was a toxin (mucous layer devasting toxin) that caused irreversible cell death not only to gastric epithelial cells but also to a wide range of warm-blooded cells including immune system cells. Disclosure of the invention

An object of the present invention is to provide a method for culturing a large amount of Helicobacter pylori over a long period of time to produce a large amount of Helicobacter pylori M toxin and a medium therefor.

The present inventor has found that M toxin derived from Helicobacter pylori has cancer cell cytotoxicity and can be used as a therapeutic drug for fixed cancer. In addition, compounds that promote the cytotoxic activity of M toxins can be used as drugs such as cancer therapeutics, while compounds that inhibit the function of M toxins can be used as drugs for the treatment and prevention of gastritis and gastric ulcers. Screening these compounds with M-toxin I found a method.

However, when isolating and purifying M toxin from Helicobacter pylori and purifying it, in the case of the conventional culture method in which 5% to 15% of serum is added, Helicopacter pylori is M The toxin was not produced with sufficient activity. In addition, when attempting to produce recombinant M toxin using host cells into which DNA encoding M toxin has been introduced, the host cell can hardly be present due to the cytotoxic activity of M toxin, making it difficult to produce M toxin. It was. Therefore, the present inventor has studied the culture conditions in detail in order to complete a mass culture method of Helicobacter pylori that produces M toxin. As a result, stable and large-scale expression of M-toxin protein is achieved by culturing for a long time using a serum-free Brain Heart Infusion agar medium adjusted to a pH range of 7.45 to 7.65. Helicobacter pylori was established, and it was shown that a large amount of M toxin could be purified and isolated from the Helicobacter pylori, and the present invention was completed.

That is, the present invention is as follows.

[1] Brain Heart Infusion for long-term culture of Helicobacter pylori at pH 7.45 to 7.65, containing 0.1% to 1% 2,6di-O-methylcyclodextrin without serum Culture medium.

[2] Serum-free Helicobacter pylori using brain heart infusion medium at pH 7.45 to 7.65, containing 0.1% to 1% 2,6di-O-methylcyclodextrin, without serum Incubate in

[3] The method of culturing Helicobacter pylori over a long period of time of 1 month or more [2] The method of culturing Helicobacter pylori without serum.

[4] A method for producing a Helicobacter pylori M toxin protein, comprising culturing Helicobacter pylori by the method of [2] or [3], and extracting the M toxin protein from the Helicobacter pylori.

[5] Serum-free Helicobacter pylori using serum-free brain heart infusion medium containing 0.1% to 1% 2,6di-〇-methylcyclodextrin, pH 7.45 to 7.65 A method of culturing and establishing a Helicobacter pylori strain that stably expresses the Helicobacter pylori M toxin protein. [6] A Helicobacter pylori strain that is established by the method of [5] and stably expresses the Helicobacter pylori M toxin protein under serum-free culture conditions.

[7] The Helicobacter pylori strain established by the method of [5] is cultured under serum-free conditions, and the Helicobacter pylori M toxin protein is produced by extracting the M toxin protein from the cultured Helicobacter pylori how to.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing the morphological changes of HeLa cells after 24 hours with the supernatant of Helicopacter pylori and the extract of bacterial cells when serum is used and without serum. Fig. 2 is a photograph showing morphological changes of HeLa cells after 6 hours and 12 hours of 1. Omg / mL bacterial cell extract by serum or serum-free culture method.

FIG. 3 shows the cytotoxic activity of Helicobacter pylori by serum-free culture or serum culture. A shows the cytotoxic activity of the culture supernatant, and B shows the cytotoxic activity of the bacterial cell extract.

FIG. 4 is a graph showing the time course of the cytotoxic activity of anion-exchange chromatography fractions of Helicobacter pylori cell extracts in serum-free culture. A shows the results at the start of culture, B after about 50 passages from the start of culture, and C after about 100 passages from the start of culture.

FIG. 5 shows the results of analysis of the cytotoxic activity of the bacterial cell extract by serum-free culture. A is the cation exchange chromatographic fraction of the cell extract after about 100 passages from the start of serum-free culture, and B is the limit of the cell extract after about 100 passages from the start of serum-free culture. The results of the filtration chromatography fraction are shown.

FIG. 6 is a graph showing the cytotoxicity of partially purified cell extracts by anion exchange chromatography to various cell lines. BEST MODE FOR CARRYING OUT THE INVENTION

Cultivation of Helicobacter pylori [Helicobacter / · /) by the method of the present invention is carried out under serum-free conditions. As a base medium, a Bra in Heart Infus ion medium is used. Brain Heart Infusion medium includes semi-solid agar and liquid broth. Agar medium is preferred. A commercially available brain heart infusion agar medium may be used. In addition, a commercially available Brain Heart Infusion John broth medium

It can also be prepared by adding 1 to several percent agar.

Add 2,6-di-O-methylcyclodextrin (2,6-di-0-me thy 卜 / 3-cyc lodext rin) to the above medium. The addition amount is 0, 1% to 10% (w / w), preferably 0.5% to 5% (w / w).

Further, the pH of the medium is adjusted to 7 to 7.7, preferably 7.45 to 7.65, more preferably 7.50 to 7.60. PH may be adjusted using an acid solution such as HC 1 or a basic solution such as NaOH.

Helicobacter pylori bacterial strains cultured by the method of the present invention include, for example, ATCC49503 (60190), NCTC 11637, NCTC 1 1916, DT 61A, NCTC 1 1639, R85-13 6P, R85-13-12F, R85 -13-11P, T81213- NTB, J99, 4, U2- L 85D08, MC903, MC123, Tx30a, 26695, UA 1182, etc. Helicobacter pylori can be obtained from established isolates (for example, from Amer i can Type Culture Collection) or can be isolated and cultured from clinical specimens.

The M toxin produced by these bacterial strains has the amino acid sequence represented by SEQ ID NO: 1. The base sequence of DNA encoding M toxin is represented by SEQ ID NO: 2. M toxin has a molecular weight of 37 kDa to 45 kDa.

Cultivation is carried out for several days, preferably 7-10 days, in a brain heart infusion medium containing Helicobacter pylori first containing animal serum and adjusting the pH to the above range. Animal serum used in this case is not limited, and rabbit serum, horse serum, etc. may be used, and rabbit embryo serum is particularly preferable. Next, transfer the Helicobacter pylori to brain heart infusion medium containing 2, 6 di-O-methylcyclodextrin and pH adjusted to the above range. At this time, at the beginning of the culture, set the 2,6di-0-methylcyclodextrin concentration in the medium high, and gradually decrease the 2,6di-0-methylcyclodextrin concentration in the medium as the culture continues. It is preferable to do. For example, at the start of culture, the concentration of 2,6di-0-methylcyclodextrin in the medium should be 5% or more, and every few days, for example every 2-4 days, 2, 6di-0 Transfer to a medium with reduced rhodextrin concentration. The pace at which the 2,6di-0-methylcyclodextrin concentration is lowered can be set as appropriate. For example, it may be lowered by 0.5 to several percent every 2 to 4 days. Finally, it may be transferred to brain heart infusion medium containing serum supplemented with 0.1 to 1% 2,6 di-O-methylcyclodextrin. Subsequently, the culture is continued by transferring to a brain-free infusion medium containing no serum supplemented with 0.1 to 1% 2,6di-O-methylcyclodextrin. At this point, Helicobacter pylori may be collected by centrifugation or the like and stored frozen.

According to the method of the present invention, the Helicobacter pylori is subcultured for 2 weeks or more, preferably 1 month or more, more preferably 3 months or more, more preferably 6 months or more, and particularly preferably 1 year or more. be able to. In the meantime, the bacteria grow and continue to express M toxin, so that a large amount of Helicobacter pylori can be cultured and a large amount of M toxin can be obtained.

The present invention includes a method for establishing a Helicobacter pylori strain that stably expresses a large amount of M toxin over a long period of time, and also stably expresses a large amount of the M toxin thus established under serum-free culture conditions. The resulting Helicobacter pylori strain is included.

M toxin can be purified from the culture supernatant of Helicobacter pylori. In addition, Helicopacter pylori cells can be purified by centrifuging the proteins in the cells by ultrasonic disruption or the like. Extraction can be performed from the culture supernatant or bacterial cells by ammonium sulfate precipitation, and the extract can be purified and isolated by combining chromatography such as ion exchange chromatography and hydrophobic chromatography.

The cytotoxic activity of M toxin can be measured using a mammalian cell line. Mammalian cell lines include normal cells including mammalian blood cells or various warm-blooded animal cancer cells (eg, endometrial cancer, endometrial tumor, breast cancer, gastric cancer, liver cancer, knee cancer) Gallbladder cancer, colorectal cancer, prostate cancer, lung cancer, kidney cancer, neuroblastoma, bladder cancer, malignant melanoma, tongue cancer, gingival cancer, mouse fibroblasts, mixed monkey kidney cells, rat liver cancer, etc.) Used. For example, HeLa cells, AZ521 cells, HLF cells, Caco2 cells, HL60 cells, etc. may be used. At this time, the helicopterapi cultured by the method of the present invention. Oral culture supernatant, bacterial cell extract, or purified M toxin can be added to the above cell culture and continued for a certain period of time to determine whether cell damage is observed.

Cell damage can be determined by directly observing cell changes under a microscope and counting them using a hemocytometer, etc., by detecting changes in potassium, hemoglobin, etc. that elute into the solution from inside the cell due to cell death. It can be performed by measuring the number of cells using tetrazolium salt, etc., by measuring the number of remaining living cells using a radiolabeled substance, or by confirming cell death by inducing cell apoptosis. .

Specifically, cell damage can be determined by, for example, cell morphology. For example, the vacuole disappears and the cell becomes circular or the outline of the cell is destroyed and fragmented. It is also possible to determine whether or not a cell has been damaged by examining cell survival. Cell survival is, for example, 1) A method in which cell changes are observed directly under a microscope and counted using a hemocytometer. 2) Changes in the amounts of potassium, hemoglobin, and various enzymes that elute into the solution from inside the cell due to cell death. 3) Method of counting the number of viable cells remaining after reaction using tetrazolium salt, etc., 4) Method of counting the number of viable cells remaining using radiolabeled substance, 5) By inducing apoptosis of cells Examples include a method for confirming cell death.

The M toxin produced by the method of the present invention has cytotoxic activity against cancer cells and can be used as a cancer therapeutic agent. In addition, a compound that promotes or suppresses the cytotoxic activity of M toxin produced by the method of the present invention can also be used as a medicament. For example, a compound that suppresses the cytotoxic activity of M toxin can be gastritis, gastric ulcer, stomach It can be used as a therapeutic or prophylactic agent for cancer and the like. For example, as described in the W003 / 048199 international pamphlet, a compound that can be used as a medicine and that promotes or suppresses the cytotoxic activity of M toxin can be screened. Examples of test compounds used for screening include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. These compounds are novel compounds. It may be a known compound. In order to carry out the above screening method, when measuring the cytotoxic activity of the M toxin protein of Helicopactor, the test compound To determine whether the cytotoxic activity of M toxin is promoted or suppressed. For example, when WST-1 tetrazolium and lactate dehydrogenase release assay is performed and the test compound is added, it is increased by about 20% or more, preferably 30% or more, more preferably about 50% or more, compared with the case where no test compound is added. As a compound that promotes the cytotoxic activity of M toxin, a compound that inhibits the cytotoxic activity of M toxin by a test compound that inhibits about 20% or more, preferably 30% or more, more preferably about 50% or more Can be selected.

When a compound obtained by screening using M toxin is used as the above-mentioned therapeutic / preventive agent, it can be carried out according to known means. For example, a compound that promotes or suppresses the cytotoxic activity of M toxin can be formulated into tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like. The preparations obtained in this way are safe and low toxic. For example, humans or warm-blooded animals (eg mice, rats, rabbits, hidges, bushes, bushes, horses, birds, cats, dogs) , Monkeys, etc.). The dose of the compound or a salt thereof varies depending on its action, target disease, administration subject, administration route, etc. For example, a compound that promotes the function of M toxin as a tissue regeneration agent after excision of diseased tissue is used. When administered orally, generally for adults (with a body weight of 60 kg), the compound is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg is administered. In the case of other animals, an amount converted per 60 kg can be administered. The present invention will be specifically described by the following examples, but the present invention is not limited to these examples. Material

Helicopacter pylori 60190 was obtained from American Type Collection. Brain Heart Infusion was obtained from DIFCO LABORATORIES, and 2,6-di-〇-methyl cyclodextrin was obtained from Wako Pure Chemical. Culture conditions and preparation of bacterial extracts

Helicopacter pylori 60190 (ATCC49503) was used as a purification resource. The bacteria were cultured in brain heart infusion containing 5% bovine serum for 7 to 10 days, and then transferred to a medium containing 26 di-0-methylcyclodextrin in order of low concentration. For example, transfer to 5%, 2%, 1%, or 0.5%, with concentrations lowered every 2 to 4 days, and then pass from 100 to 120 times every 3 to 4 days while maintaining a stable state. Cultured. The bacterial cells are transferred to a brain heart infusion solution containing 0.5% 2,6di-0-methylcyclodextrin and no serum, and at 37 in 5% C0 ₂ microaerobic conditions. Cultured with shaking for 16 hours.

The pH of the brain heart infusion medium used was adjusted to pH 7.50-7.60 using hydrochloric acid or sodium hydroxide.

Bacterial cells were centrifuged at 12000 g, 20 min, 4 conditions and suspended in 10 μl / L Tris-HCl buffer. Bacterial cells were washed once, sonicated, and stored at -80 overnight. The ultrasonically disrupted bacterial cells were freeze-thawed and centrifuged again at 4 000 g for 60 minutes at 4. The top layer fraction was stored at -80 until next use. Cell damage caused by cell activity

HeLa cells cultured in Eagles minimal synthetic medium containing 10% bovine serum were seeded in 96-well plates at a concentration of 10,000 cells per well. After 24 hours, the cells are washed twice with phosphate buffer, and 10; ti L is continuously added to a minimal synthetic medium containing 90 L of 10% bovine serum so that there is a total lOO ^ L per well. Fractions or fractions of serial dilution series were added. These cells were cultured at 37 ° (: 24 hours, and cell viability was assessed with WST-1 using Ce 1 Counting kits (D0J IND0 Laboratories). Purification of cytotoxic factors

The protein in the cell extract was recovered as a precipitate in a 70% ammonium sulfate solution. After centrifugation at 12000 g for 20 minutes, the pellet was resuspended in 1 Oimno 1 / L squirrel hydrochloric acid buffer at pH 7.7 and dialyzed against the same buffer. Anion exchange chromatography was performed using DEAE Sephacel (Amersham Pharmacia Biotech UK Ltd.) in 10 ol / L Tris-HCl buffer pH 7.7. Cation exchange chromatography was performed using CM Sepharose Fast Flow (Amersham Pharmacia Biotech UK Ltd.) in 1 Ommol Tris-HCl buffer ρΗ5 · 8. The proteins were performed with 0 to 0.3 mol / L and 0 to 0.1 mol / L NaCl, respectively, using the same buffer. Gel filtration chromatography is performed at 0.25 mL / min in a buffer containing 10 歷 ol / L Tris and 0.15 mol / L NaCl using Superose 12 HR 10/30 col umn (Pharmacia). It was. result

Morphological changes of HeLa cells

Helicobacter pylori 60190 was cultured for about 2 years with and without serum. A method using 0.5% 2,6di-〇-methylcyclodextrin without using serum is not preferable for bacteria, but rather easily causes autolysis during the culture process. In this study, a serum-free culture method was used in Brain Heart Infusion (DIFC0) and 0.5% 2,6di-〇-methylcyclodextrin, without serum and cultured in agar for about 2 years. The conventional method was defined as using brain heart infusion and 7% serum and not 2,6di-0-methylcyclodextrin. Bacterial supernatants and cell extracts that have been cultured in serum-free culture after more than about 100 passages show more distinctive changes compared to those using serum-based culture methods. As shown. After 24 hours, the number of cells both decreased, and the cells exposed to the serum-free culture supernatant had no vacuoles, and the round shape or outline was broken into small fragments that were exposed to the cell extract of serum-free culture. The ones shown are oval or spindle shaped. In contrast, the serum and serum extract groups of the supernatant and bacterial cell extract had little change, and had several vacuoles similar to the previously reported morphological changes (Fig. 1).

Figure 1 shows the morphological changes of HeLa cells after 24 hours with Helicobacter pylori supernatant and bacterial cell extract, with and without serum. Healer cell Is sown 5000 per hole. The concentration of serum and serum-free culture supernatants was 2. lmg / ml, and the concentration of each cell extract was 0.5 mg / nil. As shown in Figure 1A, both the healer cells exposed to the supernatant from the serum culture and those exposed to the bacterial cell extract did not show any significant changes, but were slightly different compared to the target group. Small vacuoles were observed. In addition, as shown in Fig. 1B, HeLa cells exposed to the serum-free culture supernatant showed a rounded or broken outline with a significant decrease in cell number and were exposed to the cell extract. Showed an elliptical or fusiform morphology with decreasing cell numbers. The scale bar in the photo is 200 m.

It was observed that the supernatant of the bacteria cultured in serum-free and the cells exposed to the cell extract did not form vacuoles, but passed through an elliptical or spindle-shaped form into circular or circular fragments. . Next, it was examined whether the same change would occur when heLa cells exposed to the bacterial cell extract were used at higher concentrations. It was confirmed that the cells exposed to the supernatant and the bacterial cell extract eventually reached a circular or contracted form after passing through an ellipse or spindle after 24 hours. A comparison of morphological changes in HeLa cells exposed to bacterial cell extracts from serum culture and serum-free culture for about 2 years (Fig. 2) suggests that both contain the same cytotoxic factors.

Fig. 2 shows the morphological changes of HeLa cells by serum or serum-free culture method 1. Omg / mL cell extract. HeLa cells exposed to cell-free extracts from serum-free culture showed special morphological changes after 12 hours. As shown in Figure 2A, HeLa cells exposed to serum culture cell extracts showed no significant change at 6 and 12 hours after addition, but a slight decrease in cell number was observed. . In addition, as shown in Fig. 2B, HeLa cells exposed to cell-free extracts from serum-free culture did not become vacuolated at 6 and 12 hours after addition, and they were destroyed after passing through an oval or spindle shape. A circular or circular piece with a contoured shape is shown. The scale bar in the photo is 200 m.

For conventional quantification of cytotoxic factors in Helicobacter pylori, a method has been used to measure the amount of neutral red taken up into the vacuoles of sample-treated cells. This is because the culture supernatant of this bacterium causes vacuolation of the cells, but also because it causes little deterioration of the cells to be evaluated and marked phenomenon of the number of cells. So Therefore, the direct live cell evaluation method was not used to search for the cytotoxic factor of Helicobacter pylori. However, the present inventor has found that this live cell evaluation method is usefully used in place of the neutral red uptake method due to the strong cytotoxic activity of the supernatant and the cell extract from the serum-free culture method. Cytotoxic activity by live cell evaluation method

Cytotoxic activity is determined by the activity of viable HeLa cells using WST-1 tetrazolium and lactate dehydrogenase-release assembly. This live cell evaluation system can confirm the strong cytotoxic activity against healer cells. Both the supernatant and the cell extract obtained by the serum-free culture method had a greater cytotoxic activity than that obtained by the serum culture. In particular, the cell extract from serum-free culture had a much stronger cytotoxic activity than that from serum culture (Fig. 3).

Figure 3 shows the evaluation system for the viable cell activity of the supernatant and bacterial cell extract at the start of culture of Helicopacter pylori in serum-free culture and after about 100 passages after the start of culture. Figure 3A shows that the serum-free supernatant for a long period of time shows a dose-dependent decrease curve of live cell activity, with each point representing the mean and variance from three independent experiments. As shown in FIG. 3B, the cell extract cultured for a long period of time without serum shows a larger dose-dependent decrease curve of the viable cell activity. Each point represents the mean and variance from three independent experiments.

Since the live cell evaluation method used in this example measured intracellular enzymes in dead or destroyed cells, this cytotoxic activity was considered to be irreversible. After 6 hours and 12 hours after the addition, the supernatant and the bacterial cell extract were replaced with a control solution and observed for another 24 hours, but no reversibility was observed. Purification according to passage time

Next, the increasing cytotoxic activity of the bacterial cell extract according to the passage time in serum-free culture was examined using live cell activity and ion exchange chromatography. Cell extracts at the start of serum-free culture are approximately 0.1 mol / L NaCl and 0.2 mol / L NaCl by gradient elution. However, each showed two decreases in viable cell activity. The cell extract 6 months after the start of culture (approximately 50 passages) showed a more obvious decrease in viable cell activity at the same concentration of the NaCl linear gradient. On the other hand, after about one year in serum-free culture, the cell extract had a large decrease at 0.2 mol / L NaC 1 and a small decrease at 0.2 mol / L NaC 1. Indicated by amount. Since the buffer used by this potential cytotoxic agent was pH 7.7, the isoelectric point of the toxin was found to be 7.7 or less (Figure 4).

Figure 4 shows the results of anion exchange column analysis of bacterial cell extracts from serum-free culture. The viable cell activity representing the cytotoxic activity is measured as the viable cell activity at an absorbance of 415 nm using the WST-1 method. The protein concentration in the column eluate is obtained by measuring the absorbance at 280 mn. Fig. 4A shows the result of anion exchange chromatography of the cell extract at the start of serum-free culture. Protein is eluted with a linear gradient from 0 to 0.3 mol / L NaCl. Two decreased viable cell activities have been shown (arrows). Fig. 4B shows the results of anion exchange chromatography of the cell extract after about 50 passages (after about 6 months) from the start of serum-free culture. The protein is a straight line from 0 to 0.3 mol / L NaCl. It is eluted with a gradient. Two more obvious small decreases are shown (arrows). Fig. 4C shows the results of anion exchange chromatography of the bacterial cell extract after about 100 passages (about 1 year) after the start of serum-free culture. The protein is a straight line from 0 to 0.3 IDO1 / L NaCl. It is eluted with a gradient. One small and large decrease is shown at 0. lmol / L NaCl and 0.2 mol / L NaCl (arrows). Purification of M toxin by cation exchange chromatography and ultrafiltration chromatography

Furthermore, microbial cell extracts about 1 year after the start of serum-free culture (after about 100 passages) were examined using cation exchange chromatography and ultrafiltration chromatography. Cation exchange chromatography showed one obvious decrease in the vicinity of 0.3 mol / L NaCl of gradient elution. This suggests that the isoelectric point is pH 5.8 or higher due to the use of a pH 5.8 buffer as a possible cytotoxic factor. Ultrafiltration chromatography shows a sharp decrease around 37 kDa to 45 kDa. This is a vacuolating toxin This is different from the molecular size of 87 kDa.

Fig. 5 shows the results of cell extract analysis by serum-free culture. Viable cell activity, which represents the activity of cytotoxic factors, is measured at an absorbance of 415 nm using the WST-1 method. The protein concentration in the column eluate is determined by measuring the elution fraction at an absorbance of 280 nm. Figure 5A shows the results of cation exchange chromatography of cell extracts after about 100 passages (about 1 year) after the start of serum-free culture, and the cell extract proteins range from 0 to 0. It is eluted with a step gradient of 5 mol / L NaCl. Fig. 5B shows the results of ultrafiltration chromatography of the cell extract after about 100 passages (about 1 year) from the start of serum-free culture. Cytotoxicity for each human cell

These bacterial cell extracts, partially purified by anion exchange chromatography, are HeLa cells and other human cells of AZ521 (gastric cancer cells), HLF (hepatoma cells), Caco2 (colon cancer cells), HL60 (leukemia cells). Showed clear toxicity.

Figure 6 shows the cytotoxicity results of partially purified bacterial cell extracts by anion exchange chromatography at a concentration of 28 _g / mL. Cytotoxicity is measured in a human cell system, which includes HeLa cells, AZ521 (gastric cancer cells), HLF (hepatoma cells),

Caco2 (colorectal cancer cells) and HL60 (leukemia cells) were used. Each bar represents the mean + deviation value of these three independent experiments. Industrial applicability

As shown in the Examples, by using the brain heart infusion medium of the present invention, it is possible to culture for a long period of one month or longer without killing Helicobacter pylori. In addition, a large amount of M toxin protein can be produced from Helicobacter pylori that has been cultured and proliferated for a long time.

All publications cited in this specification are hereby incorporated by reference in their entirety. Further, it is easily understood by those skilled in the art that various modifications and changes of the present invention are possible without departing from the scope of the technical idea and the invention described in the appended claims. Let's go. The present invention provides such modifications and And is intended to encompass changes.

Claims

The scope of the claims

1. Brain herb for long-term culture of Helicobacter pylori at pH 7.45-7.65 without serum and containing 0.1% to 1% 2,6 di-〇-methylcyclodextrin Toinfusion medium.

2. Serum free from Helicobacter pylori using brain heart infusion medium with pH 7.45-7.65, containing 0.1% to 1% 2,6di-O-methylcyclodextrin. A method of culturing with serum.

3. The method of culturing Helicobacter pylori according to claim 2, wherein the Helicobacter pylori is cultured over a long period of one month or more.

4. A method for producing Helicobacter monopylori M toxin protein, comprising culturing Helicobacter pylori by the method according to claim 2 or 3, and extracting M toxin protein from H. pylori to the culture.

5. Serum free of Helicobacter pylori using Brain Heart Infusion medium with pH 7.45-7.65 containing 0.1% -1% 2,6 di-O-methylcyclodextrin without serum. A method of establishing a Helicobacter pylori strain that stably expresses Helicobacter pylori M toxin protein by culturing in serum-free.

6. A Helicobacter pylori strain that is established by the method according to claim 5 and stably expresses the Helicopacter pylori M toxin protein under serum-free culture conditions.

7. Culturing Helicopacter pylori strain established by the method of claim 5 under serum-free conditions, and extracting M toxin protein from the cultured Helicopacter pylori M toxin A method for producing a protein.