JPS628440B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a biologically active fraction (hereinafter referred to as " The present invention relates to a method for producing an active fraction (hereinafter simply referred to as "active fraction"), and a diabetes treatment and prevention drug, an immunomodulator, or a sympathetic beta receptor blocker containing this active fraction as an active ingredient. The term "biological activity" in the present invention refers to insulin secretion enhancing activity, immune enhancing activity, and sympathetic beta receptor blocking activity (hereinafter simply referred to as A, B, and C activities, respectively). The biological definition of activity is as shown in Examples 1-1, 2-1, and 3-1 below, respectively. The pharmacological action of the active fraction of the present invention is summarized as follows. The active fraction has the amazing pharmacological effect of promoting insulin secretion in mammals whenever necessary and maintaining blood sugar levels at normal levels, and can help prevent abnormally high blood sugar levels. It has been found that when a patient exhibits so-called diabetic symptoms, the blood sugar level returns to normal within a few days by administering this active fraction, and the effect lasts for more than a month. These effects are not present in any currently known antidiabetic drugs, and they do not have the extremely dangerous side effects of hypoglycemia that existing antidiabetic drugs have, and they do not have the same effect as insulin given from outside the body. Regarding the sustainability of the effect, it also shows a much longer persistence,
Therefore, it is extremely useful as a therapeutic and preventive drug for various types of diabetes. The active fraction has the effect of enhancing antibody production and enhancing cell-mediated immunity, and has the surprising pharmacological effect of regulating immune effects even in extremely small amounts. Therefore, it is effective against diseases thought to be caused by abnormalities in immune function. It has been found to be a useful drug for, for example, malignant tumors, aplastic anemia, rheumatoid arthritis, nephritis, Behchiet's disease, myasthenia gravis, delayed-type allergies, etc. The active fraction has the surprising pharmacological effect of almost completely suppressing hyperglycemia induced by epinephrine (adrenaline) in mammals, indicating that this substance has a blocking effect on sympathetic β receptors. There was found. Therefore, it is extremely useful as a therapeutic and preventive drug for various diseases based on abnormalities in sympathetic nerves, such as hypertension such as essential hypertension, angina pectoris, and arrhythmia. The active fraction of the present invention is obtained by culturing microorganisms belonging to the genus Bordetella, known as pathogenic bacteria, in a solid medium or liquid medium, and collecting the biologically active components from the cells or the medium. . Advantageous collection and purification of biologically active components from bacterial cells and cultures can be achieved by one or a combination of solubility methods, chromatographic methods, molecular sieve methods, electrophoretic methods and biological methods. Such,
This can be achieved by many fractional purification methods commonly used in the field, and therefore the present invention is not limited to any particular collection and purification method. However, according to the findings of the present inventors, a column chromatography method has been exemplified as an example of an extremely advantageous collection and purification method for active fractions, and in this case, the culture supernatant of microorganisms can contain hydroxyapatite,
Carboxymethyl cephalose CL-6B, P-acetoxymer kyurianiline-cephalose
6MB, Cephadex G-50, Cephadex G-75, Cephadex G-100, Cephadex G-150, Cephadex G-200, Biogel P-100, Biogel P-150, Biogel P-
200, Concanavalin A-Sepharose 4B, Cepharose 6B, DEAE-Sepharose, Cephacryl S-200, and anti-IAP antibody-Sepharose 4B
It is passed through a column made of packing material such as. Active components are highly selectively adsorbed onto these columns and then eluted with appropriately selected eluents to give active fractions. In particular, hydroxyapatite column, carboxymethylcephalose shown in Example 4 below
A method for preparative purification of active ingredients using a combination of a CL-6B column and a P-acetoxymer kyurianiline-Sepharose 6MB column is extremely effective. Incidentally, after the cultured microorganisms belonging to the genus Bordetella are attenuated and inactivated by an appropriate method such as heating, 50 billion cells can be used for the diabetes treatment described below without any further purification. The vaccine can be used as a medicine because it can sufficiently guarantee the required titer of about 100 to 500 units as a medicine, and the present invention also relates to this vaccine. The active fraction obtained from cultures such as bacterial cells and culture media by one or a combination of the above-mentioned collection and purification methods is dependent on the bacterial strain used in the culture, the culture method, and the collection and purification method, etc. They are all protein-like substances, with a molecular weight of 10,000 to 250,000 as determined by gel filtration, 0 to 25% by weight of carbohydrates, 0 to 25% by weight of lipids, and sometimes nucleic acids (detected by UV absorption at 260 nm). , isoelectric point PH
4 to 190, and its detailed chemical and biological characteristics are as shown in each example below. The Bordetella microorganisms that produce the active fraction of the present invention are well known as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica.
It also includes mutant strains obtained by mutating by various conventional means such as changing the medium composition, irradiating various types of radiation such as ultraviolet rays and X-rays, or applying mutagenic agents. As a culture method, a liquid shaking culture method is preferred in terms of viability and yield, but other methods may also be used. The mycological properties, culture conditions, etc. of microorganisms belonging to the genus Bordetella can be found in Bergy's Manual of Determinative
Bacteriology 8th edition 1974 Baltimore: The Williams &
Willkns CO., J Exp Med. 129: 523-550 (1969), Bacteriology practice summary: 3rd edition, pp. 6 et seq., 1972 (Maruzen)
Co., Ltd.), etc. The active fractions (A), (B), and (C) of the present invention thus obtained are extremely useful as therapeutic and preventive agents for diabetes, immunomodulators, and sympathetic β receptor blockers, and the effective amount for the human body is , as a solid substance depending on the specific activity of the active fraction, in A-active applications, approximately
It ranges from 10 Units/Kg (body weight) to tens of thousands of Units/Kg (body weight), and about a few for B active use.
10 Units/Kg (weight) ~ tens of thousands of Units/Kg (weight),
Approximately 0.01EU/Kg (weight) for C-active applications
It is in the range of several 100 EU/Kg (body weight). For administration to patients, intravenous administration is the most effective for each active use, and other methods include intraperitoneal, intramuscular and subcutaneous administration, direct administration into the gastrointestinal tract, oral administration, intrarectal administration, sublingual administration, intradermal administration, Nasal mucosal, arterial, lymphatic or ductal administration is also effective. Examples of dosage forms for each active use include injections, suppositories, enteric/gastric solutions, sublingual tablets, and inhalants. To give an example of the simplest composition of an injection solution,
10,000 Units of A activity, 9 mg of NaCl and 1 ml of sterile distilled water can be given. The above-mentioned administration methods and dosage forms are applicable to patients regardless of whether they are used in adults, children, the elderly, race, gender, or the like. Furthermore, in this specification, the active fraction hereinafter refers to both an active ingredient-containing liquid and an active ingredient-containing solid obtained by drying this by freeze-drying or the like. For example, it is obvious that an injectable drug solution can be obtained using either of the two as starting materials. It will also be obvious to those skilled in the art that when formulating the drug, any other ingredients that do not impair the activity may be mixed. Next, various physical properties of the active fraction of the present invention will be explained in detail. State of existence and solubility characteristics: After desalination, the powder obtained by freeze-drying is a non-deliquescent white or light brown powder with a concentration of about 3-5 mg/ml.
It is soluble in water at room temperature, forms an insoluble white precipitate in 6NHCl, and dissolves in pyridine, sodium dodecyl sulfate, mercaptoethanol, and cysteine solutions. When cold (4°C), add ammonium sulfate to the solution of purified active substance,
Addition of dry ice, acetone, ethanol, trichloroacetic acid, zinc chloride solution, and other solutions containing metal ions cause cloudiness and precipitation, respectively. water and chloroform or n
- In a butanol mixture, it becomes insoluble and collects at the interface between the two liquids. When an aqueous solution of this active fraction is heated above 80°C, it becomes cloudy. 0.1M phosphate buffer containing 0.5M NaCl (PH
When this active fraction is dissolved in 7.0) and then dialyzed using distilled water as an external solution, it becomes cloudy temporarily, but as the dialysis continues, it is completely redissolved and the cloudiness disappears. In addition, for high concentration solutions, 0.01M acetate buffer (PH4.5)
However, if thoroughly dialyzed, it may become colored pale brown and dissolve. Composition: Protein 40% or more, carbohydrates 25% or less, fat
It is less than 25% by weight, and in some cases, nucleic acids can be detected by UV absorption at 260 nm. The method for measuring each component was based on the following documents. Protein Lowry, OH, NJ Rosebrough, ALFarr,
and RJRandall. J.Biol, Chem. 193 :265 1951 Carbohydrate Phenol-H ₂ SO ₄ method Dubois, M., KAGiles, JK Hamilton, PA
Rebers, and, F.Smith. Anal.Chem. 28 350, 1956 Lipids Total lipids and bound lipids were measured by the method of Marsh and Weinstein by extracting substances into chloroform, chloroform-methanol, and hepetane before and after hydrolysis. JBMarsh and DB Weinstein: J. Lipid Res.
, 7 574, 1966 Amino acid composition and composition ratio of protein components (μM/
100 μM; hydrolyzed with 6NHCl at 110°C for 16 or 24 hours): aspartic acid (Asp) 7.5-7.9, threonine (Thr) 6.8-7.6, serine (Ser) 5.9-7.6, glutamic acid (Glu) 9.7-10.8, proline ( Pro) 5.5
~6.4, glycine (Gly) 8.7-9.6, alanine (Ala) 9.1-10.8, cystine/2 (Cys/2) 1.5
~2.6, valine (Val) 5.6-6.6, methionine (Met) 2.5-3.3, isoleucine (Ile) 3.6-4.1,
Leucine (Leu) 7.5-8.0, Tyrosine (Tyr) 5.1
~6.6, Phenylalanine (Phe) 3.3~3.9, Lysine (Lye) 3.1~4.4, Histidine (His) 1.4~
1.6, and arginine (Arg) 6.1 to 6.6. Isoelectric point PH: The isoelectric point of the active fraction is PH4-10, but most of it is PH7-9, and some have a PH47. Disc electrophoresis pattern: Acrylamide gel (polyacrylamide concentration
7.5%, PH4.3 gel, sample 30μg, current 4mA, 2 hours/1 gel, staining with Amidoblack 10B,
In disk electrophoresis under conditions of decolorization with a 7% acetic acid solution, most of the purified active substance gave a band at a distance (relative to the spacer gel tip) of 2.3±0.2 cm. In addition, some of them were observed to migrate to the anode in the PH8.3 gel. Example 1 Insulin secretion enhancing activity 1-1 Activity measurement method The activity of the active fraction can be measured by the reactivity of animals to various insulin secretion stimulating substances.
Glucose is usually used as the stimulant. Γ Animal used for test Wistar male rat (weight 130-140 g) Γ Test method The active fraction of each potency was dissolved in physiological saline, and 0.2 ml of the solution was injected into the femoral vein under ether anesthesia for 3 days. Afterwards, insulin secretion enhancing activity is measured. The subjects were fasted for 18 to 20 hours before the start of the experiment. The measurement method is to collect 0.1 ml of blood from the tail vein of a rat, and immediately administer 1 ml of 30% glucose solution per 100 g of body weight intraperitoneally.
After 15 minutes, 0.1 ml of blood is drawn again. Insulin secretion enhancing activity is determined from the difference in blood sugar level and blood insulin level before and after glucose administration. Blood sugar levels are measured using the glucose oxidase method, and insulin is measured using the dual antibody method. The intravenous solution used in the following pharmacological tests also has the same composition as above. The methods for measuring blood sugar level and insulin are based on the following documents and Kitts, respectively. Blood sugar level: glucose oxidase method Bergmeyer, H.-U., and Bernet, E.in
“Methods of enzymatic analysis”
Bergmeyer, H.−U., eds, New York.
Academic press P 123 (1963) Glucostat insulin: two-antibody method Morgan, CR, and Razarow, A. Diabetes
12 115 (1963) Insulin Reactor Kit - Dynabot Co., Ltd. First, the ΔI/ΔG values of the active substance (active fraction) administration group and control group rats are determined according to the following formula. △I/△G (μU/mg) = Blood insulin level after glucose administration (μU/ml) - Blood insulin level before glucose administration / Blood sugar level after glucose administration (mg/ml) - Blood sugar level before glucose administration Value (mg/ml) / Blood insulin value after glucose administration (μU/ml) - Blood insulin value before glucose administration (μU/ml) The blood sugar level is used to calculate the activity because the insulin secreted This is because the amount is greatly affected by blood sugar levels. Next, the titer of the active substance is calculated as follows: Unit = (Average △I/△G of rats in the substance-administered group - Mean △I/△G of the control group/Average △I/△G of the control group) x 10
Find it using 0. The specific activity of this substance is the titer unit divided by the weight. 1-2 Summary of pharmacological effects related to A activity This active fraction has an insulin secretion enhancing activity as its main action, and also has an action to improve glucose tolerance, an insulin secretion activity, an action to promote healing of streptozotocin-induced diabetes, and an action to promote the healing of streptozotocin-induced diabetes. It has pharmacologically useful effects in improving glucose tolerance in diabetics. Moreover, these effects persist for weeks to months after a single administration of the active fraction. These activities are
Detailed studies were conducted in rats and dogs, and since exactly the same phenomenon was observed in both species, it is thought that species differences do not affect the pharmacological effects of this substance. This active fraction is thought to be useful mainly as a drug for the treatment of diabetes, but current drug treatments for diabetes include insulin injections and oral administration of hypoglycemic drugs, which are only symptomatic treatments and are almost incurable. I can't help it. Furthermore, patients are required to visit the hospital every day for insulin injections, which is a complication, and when administering hypoglycemic drugs, there is always the risk of an abnormal drop in blood sugar levels. The feature of this active fraction is that it not only has insulin secretion activity, but also increases blood insulin only when blood sugar levels are raised under various conditions (hyperglycemic state, especially during glucose load similar to feeding). This substance has the effect of quickly returning blood sugar levels to normal, and a further advantage of this substance is that its activity persists for several weeks to several months after a single administration. Therefore, in cases where the response of insulin secretion to blood sugar levels has decreased, administration of this active fraction will reactivate insulin secretion. From the above points, this active fraction can be used not only as a therapeutic agent for diabetes, complications of diabetes, and various adult diseases caused by diabetes, but also for pre-diabetic conditions, and for the treatment of diabetic conditions for which there is currently no treatment. It is useful as a prophylactic and therapeutic agent as well as a diagnostic agent for the condition juvenile diabetes. 1-3 Insulin secretion enhancing activity This effect is one of the main effects of this active fraction, and experiments were conducted on rats (male Wistar) and dogs (beagles, male and female). Rats The experimental conditions were the same as those described in the activity assay section, but on the third day of administration of this active fraction, the reactivity to each insulin secretion stimulating substance was compared with the normal group (Table 1).
Glucose administration, which is the most physiological factor, resulted in a significant increase in blood insulin concentration compared to the control group, despite the difference in the route of glucose administration. In addition, the responsiveness to hormone stimulation such as glucagon (1 mg/Kg) and epinephrine (200 μg/Kg) was also significantly increased. Furthermore, enhancement of the insulin secretion activity of tolbutamide (200 mg/Kg) and glibenclamide (2 mg/Kg), which are currently clinically used hypoglycemic drugs, was also observed. From the above findings, it was confirmed that administration of the active fraction significantly enhances the reactivity of living organisms to substances that stimulate insulin secretion.

【table】

[Table] Dogs This active fraction was administered intravenously to dogs, and 3 days later, stimulation with glucagon (25 μg/Kg body weight, intravenous injection) or glucose (0.3 g/Kg body weight, intravenous injection) was performed to induce insulin secretion enhancing activity. Examined. They were fasted for 18 hours before the start of the experiment. The experimental results during glucagon stimulation are shown in Table 2. At 50U (as the active fraction, the same applies hereinafter)/Kg (body weight), a weak enhancement of insulin secretion was observed 5 minutes after glucagon administration compared to the control group, and as the dose increased, insulin secretion was enhanced. The reaction almost reached its maximum at 1000U/Kg. In addition to the above results, a similar enhancement of the insulin secretion effect was observed with glucose (oral administration, intravenous administration) and epinephrine, so the reactivity to insulin secretion stimulants was also shown to be similar in dogs. This is thought to have increased significantly due to the administration of more than 10 minutes.

[Table] 1-4 Effect on improving glucose tolerance after glucose loading Rats and dogs were orally loaded with glucose, and glucose tolerance was determined by measuring the attenuation of blood glucose level and increase in blood insulin level after loading. 18～20
Glucose is 0.5g/g in rats after an hourly fast.
The dose was 100g body weight and 15g/dog for dogs. In rats and dogs treated with this active fraction, the rise in blood glucose was markedly suppressed compared to the control group, while blood insulin levels showed a clear increase, and then blood sugar levels normalized. As a result, the insulin level quickly returned to the value before glucose loading, and no occurrence of hypoglycemia due to excessive intake of insulin was observed (3rd and 4th).
table). Therefore, it was shown that glucose tolerance was significantly improved in animals treated with this active fraction.

【table】

[Table] 1-5 Duration of pharmacological activity Each pharmacological activity of this active fraction appears several hours after administration, reaches its peak on the 3rd to 7th day, and after that, each activity gradually decreases. ), we will discuss the experimental results of the sustainability of the action. Administration of this active fraction in rats 1, 3, 7, 14,
Insulin secretion enhancing activity was examined on days 21, 42 and 60. As a result, sufficient enhancing activity was still shown even on the 42nd day (Table 5). From the above results, it is thought that the effect lasts for several weeks to several months, although the strength of the effect varies depending on the dose.

[Table] Example 2 Immune enhancing activity 2-1 Activity measurement method The B activity of the active fraction can be measured in various experimental systems, but one example is a method using adjuvant activity in a humoral antibody production system. is shown below. Γ Animals used for the test Wistar female rats (weight 200±10g) Wistar male rats (weight 250±20g) Γ Test method Dinitrophenyl, which is a protein obtained from Ascuris suuw with a dinitrophenol group attached to it. Ascaris (DNP-
As an antigen, 1 mg of AS and various active fractions are dissolved in 0.5 ml of physiological saline and injected intradermally into the ankles of the front and rear legs of female rats under ether anesthesia for primary immunization. Also, as a control,
1 mg of DNP-AS is dissolved in 0.5 ml of physiological saline solution containing 10 ¹⁰ dead pertussis bacteria and sensitized in the same manner as described above for primary immunization. DNP on day 5 after primary immunization
- 0.5 ml of physiological saline containing 0.5 mg of AS is injected into the dorsal muscles of rats under ether anesthesia, and then on the third day, the anti-DNP-AS antibody titer in the serum is measured. The measurement method is 0.0ml from the rat tail vein.
The blood was pre-prepared with heparinized saline.
Collect and mix in a test tube containing 0.2ml, and store at 4℃.
After centrifugation at 3000 rpm for 15 minutes, collect the supernatant and use it as a 5-fold diluted serum. Next, the antibody titer in the obtained serum is measured using a passive skin anaphylaxis reaction according to the method of Tada et al.*. The antibody titer in serum is indicated by the highest dilution factor of the serum that shows blue spots of 5 mm or more, and the antibody production enhancing activity titer of each specimen is defined as 1000 units if the activity shows an antibody titer of 2024 times. Specific activity is determined by dividing the titer unit by weight. * Tada, T. and Okumura, KJImmunol., 106 1002. (1971) Dose-response relationship: The dose-response relationship of the purified sample obtained in Example 4 described below is shown below. Dose (ng/rat) Anti-DNP-AS antibody titer * 62 X32 125 X128 250 X1024 500 X4048 1000 500 Units of purified sample with antibody production enhancement activity against red blood cells and 2 x 10 ⁸ sheep red blood cells
0.1 ml of Hank's solution containing 500 ml was injected into the footpad of a C57B/6J mouse, and two weeks after the injection, blood was collected from the tail vein of the mouse to obtain serum, and the hemagglutination antibody titer in the serum was measured. To measure the antibody titer, use 0.1ml of each serum diluted with physiological saline solution.
Add 0.01 ml of 2% sheep red blood cell physiological saline solution, mix well, and observe the agglutination image after incubation at 37°C for 2 hours. value.
As a result, the anti-sheep red blood cell antibody titer in serum when immunized with sheep red blood cells alone was 64 times higher, whereas when immunized with this purified specimen, the antibody titer was 2048 times higher, clearly indicating that the antibody Enhancement of production was observed. Therefore, since this purified preparation clearly shows adjuvant activity in the production of antibodies against sheep red blood cells, it is concluded that it is useful for the treatment of various diseases that cause abnormalities in immune function. Example 3 Sympathetic β-receptor blocking activity The action of the sympathomimetic drug, adrenaline (epinephrine), through β-receptors causes important changes in carbohydrate and lipid metabolism (e.g., increased blood sugar, increased free fatty acids in the blood, etc.) Furthermore, it is known that the results of this change in substance metabolism closely match the β effects on the heart, smooth muscles, and the like. Since the blood sugar rise caused by epinephrine was significantly suppressed in animals administered with this active fraction, it is naturally thought that it has a β-receptor blocking effect. Therefore, the sympathetic β-receptor blocking activity (hereinafter referred to as C activity) was determined by the extent to which the blood sugar elevating effect of epinephrine was suppressed in rats administered with the active fraction. 3-1 Activity measurement method Wistar male rats (weight 130-140g)
Dissolve the active fraction of each titer in physiological saline solution.
0.2 ml was injected into the femoral vein under ether anesthesia, and the C activity was measured the next day. The animals were fasted for 18-20 hours before the start of the experiment. Measurements were taken from the rat tail vein.
Immediately after drawing 0.02ml of blood - Epinephrine
Administer 200 μg/Kg of body weight subcutaneously, and 1 hour later, blood is collected again to measure blood sugar level. The method for measuring blood sugar level was the same as in Example 1. Calculation method for Γ C activity The difference in blood glucose levels before and after administration of epinephrine in rats in the active fraction administration group and in rats in the control group is defined as ΔG _T and ΔG _C , respectively, and the epinephrine hyperglycemia suppression rate in the administration group is calculated using the following formula; Inhibition rate (%) = (ΔG _C - ΔG _T /ΔG _C ) × 100 The rate of blood sugar increase in the active fraction administration group compared to the control group
The C activity attenuated by 50% was determined to be 1 EUnit. Specific activity was expressed as EUnit/μg (dry weight). Dose-Response Relationship The dose-response relationship of the C activity of the active fraction obtained in Example 4 is shown below.

[Table] 3-2 Attenuation of the muscle contraction force-enhancing effect of isoproterenol in the atrium of guinea pigs administered with active fraction The specimen 1EU obtained in Example 4 (based on the above activity measurement method) was administered to the forelimbs of guinea pigs (body weight approximately 350 g). The animal was injected intravenously, and 3 days later, the animal was exsanguinated and the heart was quickly removed. Cut out the right atrium and immediately
Locke's solution (NaCl120mM, KCl5.6mM,
_CaCl2 2.2mM, _MgCl2 2.1mM, NaHCO3 _25mM
The tube was suspended in a Magnes tube containing 10 ml (containing 10 mM glucose and gas replaced with 95% O ₂ -5% CO ₂ ). All atrial specimens were weighted with 0.5 g.
Muscle contraction force was recorded with a transducer. l-isoproterenol (β-agonist)
At 2ng/ml (final concentration), the atrium in the control group was approximately 20±
An increase in contractile force of 1% (n = 5) was observed, whereas in the active fraction-treated guinea pig atrium, the increase was 14 ± 1%.
The effect of isoproterenol was significantly attenuated. In the concurrent presence of propranolol (β-blocker), the effect of isoproterenol was attenuated by half. Therefore, it was confirmed that a β-blocking state similar to that caused by propranolol addition occurred in guinea pigs treated with the active fraction. 3-3 Duration of action The pharmacological activity of the active fraction appears several hours after administration and reaches its peak on days 3 to 14, after which the activity gradually decreases; The inhibition rate was investigated.

[Table] The results of the experiment revealed that C activity lasts for more than 2 months. Example 4 Pertussis I phase bacteria Higashihama strain (Bordetlla pertussis)
Phase I, Tohama Strain) freeze-dried preserved strain (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) in Bordet-Gengou medium (BG medium) containing 20% defibrinated horse blood.
After culturing at 37℃ for 3 days, incubate at 37℃20 on BG slant medium.
After culturing for ~24 hours, 200 ml of semi-synthetic liquid medium supplemented with ion exchange resin (Cohen-Wheeler's modified liquid medium = CW medium) whose composition is shown in Table 6 below was dispensed into 500 ml.
One platinum loop was inoculated into a shaken Kolben, and cultured with shaking at 37°C for 20 to 24 hours. Measure the amount of bacteria in this culture using a spectrophotometer (wavelength: 650 nm).
The amount of bacteria when added is a final concentration of 0.07 to 0.15.
Add ^ion exchange resin CW medium 1 to the shaken Kolben from 2 and add 37
Shaking culture was carried out at ℃ for 48 hours (shaking frequency: 100 to 120 times/min). The obtained 48-hour shaking culture solution was heated at 56°C for 30 minutes, and then centrifuged at 4°C (15,000 rpm) to separate the culture supernatant and bacterial cells.

【table】

[Table] Add distilled water to make the total volume 1000 ml, adjust the pH to 7.2 with 20% NaOH aqueous solution, then add 3 g of anion exchange resin (Diaion SA-20AP, manufactured by Mitsubishi Kasei Corporation) to 121
It was used after autoclaving at ℃ for 15-20 minutes. Ten volumes of the culture supernatant obtained as described above were transferred to a hydroxyapatite column (column size 5 x 2
cm, flow rate 60 ml/hr), then washed with 100 ml of 0.01 M phosphate buffer (PH 6.0), and further flushed with 300 ml of 0.1 M phosphate buffer (PH 7.0). Finally 0.1M
Phosphate buffer (containing 0.5M sodium chloride.PH
7.0) at a flow rate of 15 ml/hr to elute the active substance. The active fraction was concentrated with polyethylene glycol (average molecular weight 20,000) to about 15 ml, and then dialyzed against 2 parts of distilled water for 4 times for a total of 24 hours, and 4 times against 1 part of 0.01M phosphate buffer (PH6.0). Equilibrate the dialysis for a total of 24 hours. Carboxymethyl Sepharose CL-6B (manufactured by Pharmacia Fine Chemicals Co., Ltd., column size: 1.5) was equilibrated with the same buffer solution.
×10cm) column. There was no activity in the substances that were not adsorbed to this column, but we then increased the molar concentration of the phosphate buffer to 0.1M and the pH to 7.0, and added 0.5M sodium chloride, which matched the eluted protein. An active fraction was obtained. This active fraction was concentrated to about 10 ml and then dialyzed against 2 ml of distilled water four times for a total of 24 hours. Furthermore, 0.01M acetate buffer (PH4.5 containing 0.1M sodium chloride, or 0.1M lithium chloride-hydrochloric acid PH4.5) 4 times in total 24
Time dialysis was equilibrated. This solution was equilibrated with the same buffer,
Sepharose 6MB (see the preparation method in the note below, size: 1.2 x 8 cm (flow through the column at a flow rate of 5 ml/hr, wash thoroughly with the same buffer (approximately 200 ml used),
The adsorbed material was eluted with the same buffer supplemented with 0.01M cysteine. Here, the active substance was not contained at all in the non-adsorbed area and was concentrated in the final elution area. Add this active fraction to 2 parts of distilled water 6 times for a total of 48
After time dialysis, 7.3 mg of light brown powder was obtained by freeze-drying. This product gives a single band in disk electrophoresis (using PH4.3 gel) as shown in Figure 1, and has an isoelectric point.
PH showed 7.8±0.5. Its composition is approximately 92% protein, 1.8-5.6% carbohydrates, and 0-2% lipids.
% by weight, and the amino acid composition and composition ratio were as shown in Table 7. In addition, gel filtration method (using biogel p-150, column size: 1.8
×95cm, buffer: 0.01M acetate buffer PH4.5), the molecular weight was estimated to be 51000±4300. The recovery rate of activity, degree of purification, etc. in the above purification steps were as shown in Table 8. Also, SDS electrophoresis (1%
SDS, 1% mercaptoethanol, 4M urea) gave three bands as shown in Figure 2. A activity is
1349U/μg, B activity is 1421U/μg, C activity is
It is 1.3EU/μg and the acute toxicity value LD50 is that of mice.
It was 232 μg/Kg, and 174 μg/Kg for mice. Note Preparation method of P-acetoxymer kyurianiline (PAMA)-Sepharose 6MB: 4 g of CNBr-activated Cepharose 6MB (manufactured by Pharmacia Af In Chemicals) was dissolved ^{in 10 -3} N HCl1
Repeat washing and swelling on a glass filter. Separately, prepare a solution of 172 mg (0.5 mM) of P-acetoxymer kyurianiline dissolved in 50 ml of a 60% dimethylformamide solution of 0.1 M sodium bicarbonate buffer (containing 0.5 M sodium chloride, pH 8.3). Immediately after gel washing and swelling are completed, add this solution and incubate at room temperature (22-25℃) for 2 hours.
Mix by shaking well for some time. After the reaction is complete, wash thoroughly with the same buffer and add 50ml of 1M ethanolamine (PH9.0).
Add to the gel and mix well by shaking at room temperature for 2 hours. The gel was then absorbed through a glass filter.
Wash alternately with 1 part of 0.1M borate buffer (PH8.5) and 1 part of 0.1M acetate buffer (PH4.0), and finally wash with 0.01M acetate buffer (containing 0.1M sodium chloride).
PH4.5) or 0.1M lithium chloride-hydrochloric acid buffer (PH4.5) or 0.1M lithium chloride-hydrochloric acid buffer (PH
4.5) before use. In addition, the gel is treated in advance with the same buffer containing 1% mercaptoethanol, and then thoroughly washed and equilibrated with the same buffer before use.

【table】

[Table] Example 5 Freeze-dried preserved bacteria of pertussis I phase bacteria Higashihama strain (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) were cultured by plowing in the same manner as in Example 4, the cells were separated, and the culture supernatant was prepared. Ten
I got it. A 20% zinc chloride solution was added to this culture supernatant under low temperature until the pH reached 6.0 (final concentration: 1
%), the precipitate was dissolved in phosphate buffer, placed in a dialysis membrane, equilibrated with 0.01M phosphate buffer (PH6.0), and then mixed with carboxymethyl Sepharose CL-6B (hereinafter abbreviated as CM-Sepharose). ). The adsorbed substances were dissolved in 0.1M phosphate buffer (PH) containing 0.5M-NaCl.
7.0). The resulting eluate was divided into two parts.
One of the liquids was filtered through a collodion bag, and the external liquid was dialyzed against distilled water, freeze-dried, and 2M
- After solubilization in 0.1M phosphate buffer containing urea and 0.5M NmCl, gel filtration was performed using Sephadex G-50 to obtain an active fraction. This active fraction was then freeze-dried to obtain 3 mg of a white powder. Molecular weight: 15000±3000 Composition: 90% protein or more, carbohydrates approximately 5% by weight, lipids 3% by weight, disk electrophoresis pattern: PH4.3
Move to the cathode side with the gel. A activity: 500U/μg, LD ₅₀ : 522μg/Kg B activity: 450U/μg, ♀405μg/Kg C activity: 0.22EU/μg The other CM-Sepharose eluate was dialyzed against distilled water, and then Freeze-dry Biogel P-100
An active fraction was obtained by gel filtration, and 9 mg of lyophilized powder was obtained. Molecular weight: 115,000±15,000 Composition: 75% by weight or more of proteins, approximately 10% by weight of carbohydrates, 10% by weight of lipids Disc electrophoresis: Moves to the cathode side in a PH4.3 gel. A activity: 630U/μg B activity: 320U/μg C activity: 0.82EU/μg LD ₅₀ : 〓 432μg/Kg ♀ 350μg/Kg Example 6 Freeze-dried preservation of pertussis I phase bacteria Higashihama strain (Kitasato University Faculty of Pharmaceutical Sciences Microbiology) After culturing (provided by the classroom) in the same manner as in Example 4, the bacterial cells were separated to obtain a culture supernatant. 40 ml of 50% zinc chloride solution was added to the culture supernatant from step 2 in ice water while stirring, and after stirring for 2 hours, the mixture was incubated at 4°C.
Centrifuge at 5,000 rpm for 5 minutes, extract the precipitate twice with 25 ml of 10% sodium phosphate solution, and then centrifuge at 4°C, 8,000 rpm for 20 minutes to obtain a supernatant. The obtained supernatant was dialyzed four times for 24 hours against distilled water 2, concentrated, and further diluted with 0.1M containing 2M urea.
Dialyze 3 times for 24 hours against phosphate buffer (PH8.0) 1. Then, it was passed through a diethylaminoethyl cellulose column (1.2 x 15 cm) equilibrated with the same buffer. In this column, the target active substance was not adsorbed and was obtained in the flow-through fraction. This fraction was dialyzed, concentrated, dialyzed against 0.05M phosphate buffer (PH6.0) containing 2M urea, and equilibrated with the same buffer on a CM-Sepharose column (1.2 x 12.5
cm). There was no activity in the substances that were not adsorbed on this column, but when the molar concentration of the phosphate buffer was then increased to 0.1M and the pH was increased to 7, an active fraction was obtained that corresponded to the eluted protein. It was done. The active fraction obtained here was divided into two. One side was directly passed through a Sephadex G-200 column (1.5 x 50cm) equilibrated with 0.1M phosphate buffer (PH7.0) containing 2M urea, and a single protein peak was obtained, and the activity was determined in this fraction. matched. When the obtained fraction is freeze-dried, 1.4 mg of pale yellow powder is obtained.
Obtained. This product was dialyzed five times for 40 hours against distilled water 2, and then migrated to the cathode side using disk electrophoresis (PH4.3 gel). Composition is over 85% by weight of protein, 7.1% by weight of carbohydrates, 4.9% by weight of lipids, estimated molecular weight by gel filtration method is 145,000±15,000, and isoelectric point PH is 8.0±
It was 0.5. The A activity of this fraction is 803 Units/μ
g, B activity 950U/μg, C activity 0.95EU/μg,
_LD50 : 〓320μg/Kg, ♀232μg/Kg. The other fraction obtained by CM-Sepharose column chromatography was dialyzed against distilled water 1 for 3 times for 12 hours, and after concentration, 0.01M phosphate buffer (PH7.0)
The resultant was dialyzed and passed through a concanavalin A sepharose column (1.2 x 8 cm) that had been equilibrated with the same buffer. Wash thoroughly with the same buffer solution, and add the same buffer solution containing 0.5M sodium chloride (PH
7.0), the desired active fraction was obtained in agreement with the protein peak. After concentration, the active fraction was equilibrated by dialysis against 0.1M phosphate buffer containing 2M urea, and then equilibrated with the same buffer.
-200 column (1.5 x 50 cm). the result,
A single protein peak was obtained, and the activity also coincided with this peak. After dialysis with distilled water 2 for 5 times for 45 hours,
Freeze-drying gave 0.6 mg of pale yellow powder. The molecular weight is estimated to be 95000±15000. This fraction gives a single band in disk electrophoresis on a PH4.3 gel, and has a composition of approximately 95% protein, no detectable carbohydrates, and approximately 3.2% lipids by weight. Isoelectric point PH is 7.8
At ±0.5, the amino acid composition and amino acid composition ratio are 9th.
It was as shown in the table. A activity: 1013U/μg B activity: 1120U/μg C activity: 1.3EU/μg LD ₅₀ : 〓 247μg/Kg ♀ 185μg/Kg

[Table] Example 7 Freeze-dried preserved bacteria of Pertussis I phase bacteria Maeno strain (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) were grown in BG medium for 37 hours.
After culturing for 3 days, incubate at 37°C in BG slant medium at 20-24°C.
Cultured for hours. One platinum loop of this bacterium was added to a charcoal-added semi-synthetic medium (Charcoal-added Cohen) whose composition is shown in Table 10.
Inoculate Wheeler's modified solid medium = CA medium),
After culturing at 37°C for 48 hours, the cells were scraped into a 1% casamino acid solution (casamino acids 1%, NaCl 0.35%, PH 7.2), and a bacterial suspension of 10 bil/ml was prepared by turbidimetry (wavelength: 650 nm). When adding 100 ml of a modified CW liquid medium (composition shown in Table 11) with 0.2% charcoal added to 2 Rubins, the final concentration of bacteria should be 0.07 to 0.15 x 10 ⁹ cells/ml. The prepared bacterial solution was added, and the Rubin was left standing with the Rubin covered so that the culture medium spread widely and thinly over the surface of the Rubin, and static culture was performed at 37°C for 4 days. After culturing, a culture supernatant from which the bacterial cells were separated was obtained. To the obtained supernatant 2, 40 ml of 50% ZnCl ₂ solution was added under low temperature while stirring, and after stirring for about 2 hours, centrifugation was performed at 4°C (5000 r.pm x 5 min), and the precipitate was diluted with 10% Na ₂ HPO. Extract with 50 ml of the ₄ solution (extracted in 3 parts) (stir for 1.5 hours each while cooling on ice), and centrifuge at 4°C (8000 r.
pm x 20 min.) to obtain the supernatant. The obtained supernatant was dialyzed (2 or 4 times, total 24 hours) and concentrated (concentrated to about 5 ml), adjusted to pH 6.0 with dilute acetic acid solution and soaked in ice water for 30 minutes.
After stirring for several minutes, centrifugation at 4℃ (12500r.pm
40min.). Add acetic acid to the supernatant to adjust the pH to 3.5.
The resulting precipitate was centrifuged (12,500 rpm x 40 minutes), and 10 ml of 0.25 M phosphate buffer containing 2 M urea (pH 8.3) was added, and the resulting precipitate was stirred in ice water for 1 hour. Stir at 4°C, centrifuge at 4°C (12500r.pm
24 hours in total). After concentration, 0.05M with 2M urea
Dialyzed against phosphate buffer (PH5.5) (1, 3
(total of 12 hr.), pre-equilibrated with the same buffer.
Pass through CM-Sepharose (column size 1.9 x 7 cm). 0.05M phosphate buffer containing 2M urea (PH
Wash the column with 6.0) and further check the phosphate concentration.
When elution was carried out by raising the pH to 0.1M and 7.0, an activity matching the protein peak was obtained. This active fraction is concentrated by dialysis (2 or 5 times, 48 hr.) and further equilibrated by dialysis (2 or 3 times, 12 hr.) against a 0.1 M phosphate buffer containing 2 M urea, pH 7.0. Next, it was equilibrated with the same buffer. Sephadex G-200 column (2.8
×95cm). A single peak was obtained at a molecular weight of 145,000±15,000 (later determined by running a marker), and the activity coincided with this peak. After dialysis (2 or 5 times, 48 hr.) of this active fraction, it was freeze-dried.
2.1 mg of light yellowish brown powder was obtained. This product was electrophoresed on the cathode side of a PH4.3 gel disk, and its composition was about 64% by weight of protein, about 23.5% by weight of carbohydrates, and about 12.3% by weight of lipids. A Activity: 759U/μg B: 858U/μg C: 0.77EU/μg LD ₅₀ : 289μg/Kg ♀ 200μg/Kg

【table】

【table】

[Table] Example 8 Bacillus pertussis I phase 3779B strain Bordetella pertussis
Phase I, 3779B Strain) 1/10000 volume of 25% thimerosal was added to the 48-hour shaking culture obtained by the same culture method as in Example 4, heated at 56°C for 30 minutes, and then centrifuged at 4°C. (15000 rpm for 30 minutes) to obtain a culture supernatant. Obtained culture supernatant 10
Place in a cellophane tube for dialysis and add 0.5M
Dialysis was performed overnight at 4°C against 0.1M phosphate buffer (PH7.0) containing NaCl. Post-dialysis Diaflo Menplan
PM-30, (permeation molecular weight limit 30000 manufactured by Amicon)
Ultrafiltration was performed using a filtration solution, and the volume was concentrated to approximately 40 ml. Ethanol cooled with dry ice was added dropwise to the external liquid that had passed through the membrane to a final concentration of 40% (V/V), and the precipitate was centrifuged (3000 rpm15).
4° C.). This obtained precipitate
0.1M phosphate buffer (PH7.0) containing 0.5M NaCl2
ml and dialyzed against the aforementioned phosphate buffer overnight at 4°C.
Gel filtration was performed using Sephadex G75 (1.5 x 50 cm) equilibrated with 0.1M phosphate buffer (PH7.0) containing 0.5M NaCl. As a result, the desired active fraction was obtained, which had a molecular weight of 30,000 and corresponded to protein peaks eluting at positions before and after. The obtained active fraction was dialyzed (48 hours against distilled water) and lyophilized to obtain 2.5 mg of white fine powder. The molecular weight as determined by gel filtration is 29000±3000, and the composition of this substance is approximately 75% protein by weight based on the total weight.
It consisted of about 23% by weight of carbohydrates and about 1% by weight of lipids, and no nucleic acids were detected. Disk electrophoresis (PH
43), it migrated toward the cathode side. A activity 250U/
μg, B activity 350U/μg, C activity 0.18EU/μg
LD ₅₀ = 884μg/Kg, ♀707μg/Kg. In addition, the internal solution concentrated by the above ultrafiltration was 0.5M
Gel filtration was performed using Sephadex G-150 (4.9 x 89 cm) equilibrated with 0.1M phosphate buffer (PH7.0) containing NaCl. The obtained active fractions were collected and treated as described above.
Concentrate to 10 ml using a Diaflo membrane, and equilibrate this solution again with the above-mentioned phosphate buffer.
Sephadex G-150 (column size 2.6 x 95cm)
Then, gel filtration was performed again to obtain an active fraction. As a result, an active fraction was obtained that corresponded to a protein peak eluting at a position with a molecular weight of around 65,000, and after dialysis, 4.5 mg of lyophilized powder was obtained. The molecular weight was 65,000±6,000, and the chemical composition consisted of approximately 55% protein, approximately 25% carbohydrate, and approximately 20% lipid based on the total weight, and no nucleic acids were detected. Disk electrophoresis (PH
4.3), it migrated to the cathode side. A activity 300U/μg B activity 360U/μg C activity 0.25EU/μg LD ₅₀ : 660μg/Kg ♀ 541μg/Kg Example 9 Pertussis I phase bacteria Higashihama strain (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) was used as Example 4. The cells were cultured with shaking in the same manner, and the bacterial cells were separated to obtain culture supernatant 5. Adjust the pH of the culture supernatant to 4.0 with 6NHCl. Pre-activated CM Sepharose (manufactured by Pharmacia) was added at a ratio of 5 g/100 ml and stirred for 1 hour using a stirrer. The target fraction is adsorbed to CM-sepharose, so collect the CM cellulose part.
Wash twice with 0.01M phosphate buffer (PH6.0).
Then CM-Sepharose containing 1M NaCl
Suspend in 0.1M phosphate buffer (PH8.0) and stir with a stirrer for about 2 hours to obtain a supernatant. The supernatant liquid was dialyzed against distilled water overnight and freeze-dried. Dissolve the lyophilized powder in 0.01M phosphate buffer,
Pass through a CM-Sepharose column (3 x 15 cm) equilibrated with phosphate buffer and elute with a gradient of phosphate buffer pH, molar concentration, and salt concentration, and the target fraction is 0.5M - 0.1M containing NaCl. Phosphate buffer (PH7.5)
It was obtained as a protein peak eluted at . The obtained fraction was dialyzed against distilled water overnight and then lyophilized.The lyophilized powder was then dissolved in approximately 100 ml of 0.01M phosphate buffer and ultraconcentrated (Amicon, permeation molecular weight limit 30,000, membrane type Diaflo). PM−30)
Activity was observed in the internal and external fluids, so the internal fluid was
Sephadex G-150 (3 x 100 cm) and external solution passed through G50 (2 x 65 cm). As a result, we obtained an activity that matched the protein peak. Fraction 1.9mg obtained from G150
and 0.8 mg of the fraction obtained from G50. The molecular weight of the obtained freeze-dried powder is approximately 71000
±1000, is approximately 12000±3000. The composition is 82% protein, approximately 10% carbohydrates, and approximately 8% lipids, with no detectable nucleic acids. The protein content is 65% by weight, carbohydrates are about 20% by weight, and lipids are about 15% by weight, and no nucleic acids are detected. , a band is observed on the cathode side in disk electrophoresis at pH 4.3. Activity of fraction Activity of fraction A: 400U/μg A: 250U/μg B: 740U/μg B: 450U/μg C: 0.34EU/μg
C 0.15EU/μg LD ₅₀ :〓 732μg/Kg LD ₅₀ 〓 904μg/Kg 〃 :♀ 496μg/Kg ♀ 796μg/Kg Example 10 Pertussis bacteria No. 22 strain (provided by Department of Microbiology, Faculty of Pharmacy, Kitasato University) was used as an example. The cells were cultured in the same manner as in 4, the bacterial cells were separated, and the resulting supernatant was used as the starting material for purification. Acetone was added to supernatant 5 at 0° C. to a final concentration of 60%, and the mixture was stirred and immediately centrifuged at 3000 rpm for 15 minutes. The obtained precipitate was made into acetone powder, which was dissolved in 10 ml of 0.01M phosphate buffer (PH6.0) and mixed with carboxymethylcephadex G-25 (2 x 20
cm, flow rate 40 ml/hr). The activity was concentrated and eluted in the fraction eluted with 0.1M phosphate buffer (PH7.5) containing 0.5M sodium chloride. After collecting and concentrating this fraction, it contains 0.5M sodium chloride.
After dialysis with 0.1M phosphate buffer (PH7.0) 1 for 3 times for 2 days, gel chromatography was performed using Sephadex G-100 (2 x 90cm, flow rate 20ml/hr) equilibrated with the same buffer. Ta. The resulting molecular weight
Obtain an active fraction that matches the protein component showing a peak at around 45,000 positions (45,300 ± 5,500, average of 3 times),
1.2 mg of lyophilized powder was obtained. The chemical composition of the obtained substance is 85% protein by weight and about 12% carbohydrates by weight.
It migrated to the cathode side by polyacrylamide disc electrophoresis (PH4.3). A activity is 453U/
μg, B activity is 300U/μg, C activity is 0.44EU/
μg, and the LD ₅₀ is 420 μg/Kg for mice and 380 μg/Kg for mice. Example 11 A 48-hour shaking culture solution obtained by culturing freeze-dried bacteria of Bordetella pertussis in the same manner as in Example 4 was used.
After heating at 56°C for 30 minutes, the mixture was centrifuged at 15,000 rpm at 4°C for 30 minutes, and the resulting supernatant was used as the starting material for purification. To the supernatant 20, acetone cooled with dry ice was gradually added to the supernatant 20 while stirring under ice-cooling so that the final concentration was 60%.
It was centrifuged for a minute and collected. The obtained precipitate was dissolved and suspended in 10 ml of distilled water, and this was dialyzed against 0.01M phosphate buffer (PH6.0) 3 times for 2 days. After removing the precipitated insoluble matter by centrifugation, the same CM-Sepharose column (2 x 20 cm) equilibrated with buffer solution
The elution separation operation was carried out at a flow rate of 20 ml/hr. The desired active fraction was obtained in agreement with the protein peak eluted with 0.1M phosphate buffer (PH8.0) containing 0.5M sodium chloride. Then,
After concentrating this fraction, it was dialyzed with 0.5M sodium chloride 0.1M phosphate buffer (PH7.0) 5 times for 2 days, and 2 ml of the resulting sample solution was equilibrated with the same buffer. 100 column (1.2 x 80 cm) and gel chromatography was performed at a flow rate of 24 ml/hr. As a result, the desired active substance was obtained, which corresponded to a protein peak eluting at a molecular weight of 55,000±5,000, and 1.16 mg of lyophilized powder was obtained. The obtained active fraction consisted of more than 91% by weight of protein and about 4.1% by weight of carbohydrates, and no lipids or nucleic acids were detected. A activity is 38U/μg, B activity is 25U/μg, C activity is 0.02EU/μg,
LD ₅₀ is mouse〓1560μg/Kg, mouse♀1660μ
g/Kg. Example 12 Freeze-dried preserved bacteria of pertussis bacteria No. 41 (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) were cultured in the same manner as in Example 4. The resulting 48-hour shaking culture was heated at 56°C for 30 minutes and then heated for 15,000 r. The mixture was centrifuged at 4°C for 30 minutes, and the resulting supernatant was used as the starting material for purification. Add ethanol to supernatant 5 at a final concentration of 35% while stirring on ice.
Add the precipitate gradually until 3000r.p.
The mixture was centrifuged and collected at m.0°C for 15 minutes. Then, the precipitate was dissolved in 10 ml of 0.01M phosphate buffer (PH6.0),
Dialyze with the same buffer 1 for 3 times in total for 2 days, remove the precipitated insoluble matter by centrifugation, and then equilibrate with the above buffer solution CM-Sepharose column (2 x 20 cm)
The elution operation was performed at a flow rate of 40 ml/hr. The active fraction is obtained in agreement with the protein peak eluted with 0.1M phosphate buffer (PH7.5) containing 0.5M sodium chloride. After concentrating the obtained active fraction, dialysis was performed for 3 times for 2 days with 0.1M phosphate buffer (PH7.0) containing 0.5M sodium chloride, and 2 ml of the obtained sample was equilibrated with the same buffer. Chromatography was performed using a Sephadex G.-100 column (2 x 90 cm). As a result, the molecular weight
The desired active fraction was obtained according to the protein peak eluting at the position of 36500±4000, and the lyophilized powder was obtained.
Obtained 1.34 mg. The obtained substance is protein 80±2
% by weight, carbohydrates 16±3% by weight, and lipids and nucleic acids are not included. It also migrates toward the cathode side in disk electrophoresis (PH4.3). Furthermore, its A activity is
380U/μg, B activity is 200U/μg, C activity is
0.40EU/μg, and LD ₅₀ is mouse = 560μg/
Kg, mouse ♀580μg/Kg. Example 13 Freeze-dried preserved bacteria of pertussis bacteria Higashihama strain (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) were cultured with shaking in the same manner as in Example 4, and the bacterial bodies were separated and culture supernatant liquid 10 was added (NH ₄ ). ₂ After adding SO ₄ to 90% saturation, leave it overnight at 4°C and collect the precipitate by centrifugation (10,000 rpm for 30 minutes). The precipitate was extracted with a 1M NaCl solution, centrifuged, and the supernatant was dialyzed against distilled water, and further diluted with a 0.01M phosphate buffer containing 2M urea (PH8.0).
After equilibration with
Both active fractions were obtained which were eluted with 0.1M phosphate buffer (PH6.0) containing -NaCl. Therefore, for further purification, after concentrating the non-adsorbed active fraction, Sepharose-6B
Perform gel filtration at , freeze-dry the active fraction,
And 2.1 mg of colored fine powder was obtained. The molecular weight is estimated to be 230,000±20,000 by gel filtration, and a band migrating toward the cathode is observed in electrophoresis (PH4.3 gel). Composition: 70% protein by weight
The content is 7-13% by weight of carbohydrates and 6-10% by weight of fat. A activity is 500U/μg, B activity is 450U/μg,
C activity is 0.71 EU/μg, and LD ₅₀ is mouse
670μg/Kg, mouse ♀580μg/Kg. On the other hand, the active fraction eluted with 0.1M phosphate buffer (PH6.0) containing 2M-urea and 0.5M-NaCl was concentrated and then subjected to gel filtration using Sephadex G-75.
An active fraction that matched the protein peak on the low molecular side was obtained. The active fraction was freeze-dried to obtain 6.5 mg of white fine powder. The molecular weight is 36000±4000, and the composition is protein 90.
Weight% or more carbohydrates 6.5-7.5% by weight, fat 5.3-6.7% by weight. It moves slightly to the anode side in electrophoresis (PH8.3 gel). A activity is 350U/μg, B activity is 480U/μg,
C activity is 0.09EU/μg, and _LD50 is mouse
448μg/Kg, mouse ♀ 340μg/Kg. Example 14 Freeze-dried preserved bacteria of pertussis faiolia Maeno strain (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) were cultured with shaking in the same manner as in Example 4, and then the cells were isolated to obtain a culture supernatant. After adjusting the pH of this culture supernatant 2 to 7.5 with 6NHCl, 2M calcium chloride solution was added until no precipitate was formed (approximately 400 ml), and after stirring for 30 minutes, centrifugation at 4°C (4000 r.pm x 7 min) and precipitate 20ml
After adding water, 30% sulfuric acid was added while stirring to adjust the pH to 5.0. After stirring for 1 hour, centrifugation was performed at 4°C (4000 rpm x 7 minutes) and the supernatant was collected. This supernatant was dialyzed (external solution 2, 5 times, 45 hr) and concentrated (approx.
(concentrated to 10 ml) and thoroughly dialyzed and equilibrated (2
, 5 times, 48 hr.) and (if a precipitate occurs, centrifuge (4000 r.pm x 7 min.)) CM-Sepharose column (1.8 x 17
cm), wash the column with the same buffer, and then increase the pH sequentially. Then, in the final elution area with 0.1M phosphate buffer (PH7.0) containing 2M urea, activity consistent with the protein peak was observed. This active fraction was dialyzed (2 or 3 times, 12 hours) and concentrated, containing 2M urea.
Dialysis equilibration against 0.1M phosphate buffer PH7.0 (2
, 5 times, 48 hr.) and equilibrated with the same buffer solution using a Sephadex G-200 column (column size 2.8
x95cm). The active part is concentrated in a part with an estimated molecular weight of 115,000 (later determined to be 115,800±15,000 by gel filtration).
This fraction was dialyzed (2.5 times, 48 hr.) and then lyophilized to obtain a light brown powder with a pH of 0.8. This material migrated to the cathode side in the disk electrophoresis PH4.3 gel. Chemical composition: 75% protein by weight
Above, carbohydrates are 12.5 to 16.9% by weight, and fat is 6.8 to 7.2%.
It was in weight%. A activity is 600U/μg, B activity is 550U/μg,
C activity is 0.47EU/μg, and _LD50 is mouse
490 μg/Kg, mouse 390 μg/Kg. Example 15 Freeze-dried preserved bacteria of Bordetella pertussis strain No. 22 (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) were subjected to liquid stationary culture in the same manner as in Example 7, and culture supernatant 10 was obtained after centrifugation. This culture supernatant was adjusted to pH 6.0 with 1N HCl and then passed through a hydroquine apatite column. After washing the column with 0.01M phosphate buffer (PH6.0), the adsorbed substances were washed with 0.1M phosphate buffer (PH6.0) containing 0.5M-NaCl.
7.0). The obtained solution containing the active fraction is put into a dialysis membrane and dialyzed against distilled water as an external liquid.
Then, it was added to a CM-Sepharose column equilibrated with 0.01M phosphate buffer (PH6.0).
After washing with 0.01M and 0.05M phosphate buffer (PH6.0), 0.2M phosphate buffer (PH6.0) containing 1M NaCl was added.
An active fraction matching the protein eluted with 8.0) was obtained. For further purification, the obtained active fraction was dialyzed against water and lyophilized, and 4M-urea,
0.1M phosphate buffer containing 0.5M NaCl (PH7.0)
After solubilization, gel filtration was performed using Biogel-P200, and the active fraction was lyophilized to obtain about 11.3 mg of white fine powder. The molecular weight is 95000±11000, and the composition is protein 91±1.0% by weight and carbohydrate 7±0.7%.
Weight% Lipid is 2±1.2% by weight and migrates to the cathode side in disk electrophoresis (PH4.3 gel). A activity is 900U/μg, B activity is 970U/μg,
C activity is 0.99EU/μg, and _LD50 is mouse
260 μg/Kg, mouse 185 μg/Kg. Example 16 A freeze-dried preserved strain of Higashihama strain of pertussis bacteria (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) was incubated in BG medium for 37 hours.
℃, after 3 days incubation, BG slant medium at 37℃ 20-24
After culturing for an hour, one loopful of charcoal-free semi-synthetic solid medium (Cohen-Wheealer's modified solid medium) having the composition shown in Table 7-1 was inoculated into two rubins, and the mixture was kept at 37°C for 48 hours. After culturing, add 100 ml of 0.01 M phosphate buffer (PH6) containing 0.5 M NaCl to the agar medium from which the cells have been removed, freeze and thaw, and centrifuge (15000 rpm x 30 minutes) to obtain a supernatant. The supernatant liquid is passed through a hydroxyapatite column (4 x 3 cm) equilibrated in advance with 0.01M phosphate buffer (PH6). Since the target fraction was adsorbed to hydroxyapatite, it was washed with 0.01M phosphate buffer (PH6.3) and eluted with 0.1M phosphate buffer (PH7.0) containing 0.5M NaCl. The obtained active fraction was freeze-dried, placed in a dialysis membrane with a permeability molecular weight limit of 8000, and dialyzed overnight against distilled water and 0.01M citrate buffer (PH4.0). The solution containing the active fraction after the above dialysis is
P equilibrated with 0.01M citrate buffer (PH4.0)
- Passed through cellulose (manufactured by Selva). The target substance is adsorbed on P-cellulose and sequentially added to citrate buffer.
Raise the pH, molar concentration, and salt concentration step by step,
The target fraction was efficiently eluted with 0.1M citrate buffer (PH6.0). For further purification, the active fraction was concentrated and passed through a Biogel P-150 column (2.2 x 75 cm) that had been equilibrated in advance with 0.1M citrate buffer (PH6.0) containing 1M NaCl. Obtain an active fraction that matches the protein peak at a position with an estimated molecular weight of approximately 70,000±10,000 by gel filtration. The composition of the active fraction (2.1 mg) obtained by this method is approximately 40% by weight of protein, approximately 25% by weight of carbohydrates, and 25% by weight of lipids. In addition, absorption at 260 nm, which is thought to be due to nucleic acids, is also observed, and it is thought that some amount of nucleic acids are contained. In PH4.3 gel disk electrophoresis, a fairly sharp band is observed on the cathode side. The A activity of this substance is approximately 700U/
μg, B activity 600U/μg, C activity 0.63EU/μg
And the mouse LD ₅₀ value is 〓330μg/Kg, ♀204
It was μg/Kg. Example 17 Pertussis faiolia Maeno strain was cultured at 37° C. for 48 hours in the same manner as in Example 4, and then centrifuged to obtain supernatant 10. Add 6N-HCl to the supernatant to adjust the pH to 6.0, then add it to a hydroxyapatite column (2.5 x 4
cm). The adsorbed substances were eluted in the same manner as in Example 4 with 0.1M phosphate buffer (PH7.0) containing 0.5M sodium chloride. After concentrating the solution containing the desired active fraction to about 10 ml, it was dialyzed against distilled water 4 times for 24 hours, and then dialyzed against 0.1M phosphate buffer (PH7.0) containing 2M urea (4 times, 24 hours). Sepharose 6B (2.7 x 70
cm) column. The fraction containing the target substance was concentrated, dialyzed, centrifuged at 15,000 rpm for 30 minutes, and the supernatant was subjected to density gradient isoelectric focusing. The average final concentration of carrier ampholine (PH6-10) was 1%, and electrophoresis was carried out at 4° C. and a constant voltage of 500 V for 30 hours, and 1 ml of each fraction was collected. The target active fraction had a peak centered around PH8.0, and activity was observed in a portion corresponding to the protein portion ranging from PH7.5 to 8.5. The obtained fraction was freeze-dried to obtain 1.18 mg of brownish fine powder. The molecular weight determined by gel filtration method is
65,000±6,000, the composition is over 95% by weight of protein and about 2.5% by weight of carbohydrates, but the lipid content is below the detection limit. Polyacrylamide desk electrophoresis (PH
4.3) showed a sharp band on the cathode side. A activity is 850U/μg, B activity is 800U/μg/C activity is
It is 0.83EU/μg. The acute toxicity value (LD ₅₀ ) in mice is 310 μg/Kg, 240 μg/Kg. Example 18 Freeze-dried preserved bacteria of pertussis bacteria strain 3779B (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) were cultured with shaking in the same manner as in Example 4, and the culture supernatant liquid 10 from which the bacterial cells were isolated was cultured.
After adjusting the pH to 6.0 with 1N hydrochloric acid, pass through a hydroxyapatite column (2 x 4 cm) to 0.01M phosphate buffer (PH6.0) and 0.1M phosphate buffer (PH7.0).
After washing impurities with 0.5M NaCl-containing 0.1M phosphate buffer (PH7.0), an active fraction corresponding to the protein eluted with 0.1M phosphate buffer (PH7.0) was obtained. Since this active fraction still contained a trace amount of impurities, it was then purified using a preparative disk electrophoresis device (manufactured by Canalco). The active fraction was eluted by applying electricity to the separation PH4.3 gel for 10 hours at 3 mA. Furthermore, after dialysis and concentration of this solution, Sephacryl-S-200 (2 x 90 cm) (manufactured by Pharmacia)
An active fraction matching the protein peak was obtained by gel filtration, and 5 mg of light brown powder was obtained by freeze-drying. Molecular weight: 55000±8000 Composition: Protein 90% by weight or more, carbohydrates 7-9% by weight, lipid 1% by weight or less Disk electrophoresis pattern: Single band that migrates to the cathode side in PH4.3 gel Isoelectric point: 7.5 ±0.5 A activity is 900U/μg, B activity is 1050U/μg
g, C activity is 0.95 EU/μg Amino acid composition: Shown in Table 12. The acute toxicity value ( _LD50 ) in mice is 〓293μ
g/Kg, male was 204 μg/Kg.

[Table] Example 19 Pertussis bacteria Higashihama strain was cultured in the same manner as in Example 4, and after centrifugation, precipitated bacterial cells were obtained. The obtained bacterial cells were made into a homogeneous suspension with distilled water, and suspended at 15,000 rpm for 30
Washed by centrifugation for 1 minute. After making the bacterial cells into a homogeneous suspension with distilled water again, nephelometry (wavelength
650 nm) to make a concentrated bacterial solution of 200 bil/ml. 0.1M phosphate buffer containing 0.5M NaCl (PH7.0)
Dilute to make a bacterial solution of 100bil/ml, and add 10ml of this bacterial solution.
The supernatant and the bacterial cells were separated by applying ultrasonic waves (20 KHz) at 4°C for 10 minutes. This supernatant was divided into two halves, and one half was added to a 0.01M phosphate buffer (PH) containing 0.5M NaCl.
Sepharose 6B (3.2 x 90 cm) equilibrated with 7.0)
The other side was passed through CM cellulose (2.0 x 20 cm). The fraction obtained by gel filtration with Sepharose 6B corresponded to a protein peak eluting at a molecular weight of 180,000±20,000, and the desired active fraction was obtained. After dialysis, this active fraction is lyophilized to produce a white powder with a concentration of 0.70%
I got mg. The chemical composition is 50.3-60.2% by weight of protein, 22-28% by weight of carbohydrates, and 17.5-22.5% by weight of lipids.
Absorption thought to be from nucleic acid was observed at 260 nm. In disk electrophoresis (PH8.3), it migrated slightly toward the anode side. A activity is 400U/μg, B activity is 280U/μg, and C activity is 0.45EU/μg.
The acute toxicity value ( _LD50 ) in mice is 〓807μ
g/Kg, ♀694μg/Kg. Meanwhile, preliminarily add 0.01M phosphate buffer (PH)
6.0), the target substance was adsorbed in the fraction passed through the equilibrated CM-Sepharose column, and the target substance was adsorbed sequentially into 0.01M phosphate buffer (PH6.0) and 0.05M phosphate buffer (PH7.0). Wash the column with 0.5M NaCl.
Elution was performed with 0.1M phosphate buffer (PH8.0). The active fractions were collected and concentrated, and Sepharose 6B was equilibrated with 0.1M phosphate buffer (PH7.0) containing 0.5M-NaCl.
(3.2 x 90 cm) was used for gel filtration. As a result, an active fraction was obtained that corresponded to a protein peak eluting with a molecular weight of 160,000±20,000. This active fraction was dialyzed and freeze-dried to obtain 0.28 mg of pale yellowish white powder. The chemical composition was 56-61% by weight of proteins, 15-19% by weight of carbohydrates, and 18-22% by weight of lipids, and absorption at a wavelength of 260 nm, which seemed to be due to nucleic acids, was observed. In disk electrophoresis (PH4.3), migration occurred toward the cathode side. A activity is 550U/μg and B activity is 470U/μg.
μg, C activity is 0.78 EU/μg. The acute toxicity value (LD ₅₀ ) in mice is 706 μg/Kg, ♀
It is 612μg/Kg. Example 20 Freeze-dried preserved bacteria of Pertussis phase Bacterium Sakairi strain and Bordetella pertussis 17903 strain and Bacillus bronchiseptica L-3 strain were respectively cultured in the same manner as in Example 4 and then centrifuged to obtain respective bacterial cells. The obtained bacterial cells were washed once with distilled water, and then transformed into a homogeneous suspension with distilled water again using turbidimetry (wavelength
650nm), B. pertussis is 300 bil/ml, B. parapertussis is 200 bil/ml, and B. bronchiseptica is 200 bil/ml.
ml of concentrated bacterial solution. For each strain, the following operations were performed in the same manner.
Dilute each with 0.1M phosphate buffer (PH7.0) containing 0.5M-NaCl to make a bacterial solution of 100 bil/ml, and add this bacterial solution.
The microbial cells were destroyed by ultrasonication (20 KHz, 10 minutes) of 10 ml at 4°C. After sonication, centrifugation (15000rpm for 30 minutes)
The mixture was separated into a supernatant and a precipitate. This supernatant was divided into two parts, and one part was Sepharose 6B (3.2×
90 cm), and the other side was a CM cellulose column (2.0 x 10
cm). The molecular weight of the product obtained by gel filtration with Sepharose 6B is 180000± for pertussis bacteria.
30,000, 200,000±20,000 for B. parapertussis, and 200,000±30,000 for B. bronchiseptica. The obtained active fractions were dialyzed and then freeze-dried. 0.65 mg of white powder is obtained from pertussis bacteria, and its chemical composition is 45-50% protein, 20-25% carbohydrate, and 23-28% lipid.
Absorption at a wavelength of 260 nm was observed in weight%. A activity is 30U/μg, B activity 1.2U/μg, C activity
It is 0.01EU/μg. The acute toxicity value in mice is 2500 μg/Kg or more for both. 0.47 mg of B. parapertussis was obtained as a white powder, and its chemical composition was 55 to 60% by weight of protein, 18 to 23% by weight of carbohydrates, and 20 to 25% by weight of lipids, and absorption that appeared to be nucleic acid was observed at a wavelength of 260 nm. Ta. A activity is 21U/μg,
The B activity is 35U/μg, and the C activity is 0.01EU/μg. The acute toxicity value (LD ₅₀ ) in mice is 〓
2500μg/Kg, 2400μg/Kg for males. 0.31mg of pale yellow powder of Bacillus bronchiseptica was obtained.
Its chemical composition is 50-55% protein, 20-26% carbohydrate
% by weight, lipids at 18 to 25% by weight, absorption that appeared to be due to nucleic acids was observed at a wavelength of 260 nm. The A activity of this product is 15U/μg, and the B activity is
The C activity was 0.02 EU/μg.
LD ₅₀ is male mouse 1846μg/Kg (weight) female 1605μ
g/Kg (body weight). B. pertussis, B. parapertussis, and B. bronchiseptica all slightly migrated toward the anode side in disk electrophoresis (PH8.3). Meanwhile, prepare 0.01M phosphate buffer (PH) in advance.
In the fraction passed through the CM-Sepharose column equilibrated with 6.0), the target substance is adsorbed and sequentially
Wash the column with 0.01M phosphate buffer (PH6.0) and 0.05M phosphate buffer (PH7.0) containing 0.5M-NaCl.
Elution was performed with 0.1M phosphate buffer (PH8.0). The active fractions were collected and concentrated, and Sepharose 6B was equilibrated with 0.1M phosphate buffer (PH7.0) containing 0.5M-NaCl.
(3.2 x 90 cm) was used for gel filtration. As a result, the molecular weight of the obtained product was 160000± for pertussis bacteria.
30,000, 170,000±20,000 for B. parapertussis, and 190,000±20,000 for B. bronchiseptica. The obtained active fractions were dialyzed and then freeze-dried. From pertussis bacteria, 0.18 mg of pale yellow powder is obtained, and its chemical composition is 50-58% by weight of protein, 17-22% by weight of carbohydrates, 20-26% by weight of lipids, and has a wavelength of 260 nm.
Absorption thought to be due to nucleic acids was observed. A
The activity was 45U/μg, the B activity was 5.2U/μg, and the C activity was 0.03EU/μg. LD50 is male mouse 2500
μg/Kg (body weight) or more, female 2500 μg/Kg (body weight)
That's all. B. parapertussis yields 0.09 mg of pale yellow powder, the chemical composition of which is 55-65% protein by weight;
Carbohydrates 12-18% by weight, lipids 17-22% by weight, wavelength 260
Absorption thought to be due to nucleic acid was observed in mm.
The A activity was 25 U/μg, the B activity was 94 U/μg, and the C activity was 0.04 EU/μg. LD ₅₀ is a male mouse
It was 2500 μg/Kg (body weight) or more, and 2500 μg/Kg (body weight) for females. From B. bronchiseptica, 0.06 mg of pale yellow powder is obtained, and its chemical composition is 53-58% protein, 16-20% carbohydrate, 14-18% lipid, and the wavelength
Absorption thought to be due to nucleic acid was observed at 260 nm. A activity is 20U/μg, B activity is 0U/μg, C
The activity was 0.08 EU/μg. LD ₅₀ is a male mouse
It was 2500 μg/Kg (body weight) or more, and 2500 μg/Kg (body weight) or more for females. In the obtained active fraction, B. pertussis, B. parapertussis, and B. bronchiseptica all migrated toward the cathode side in disk electrophoresis (PH4.3). Example 21 A freeze-dried preserved strain of pertussis bacteria No. 41 (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) was cultured in the same manner as in Example 17 at 37° C. for 48 hours on a charcoal-paste agar medium.
After collecting and washing the bacterial cells, add 10 g of bacterial cells to 2% TritonX-
0.1M Tris containing 100, lmM EDTA, 0.5M NaCl
- The suspension was suspended in 550 ml of HCl buffer (PH8.0), stirred for 5 hours at 4°C using a Teflon homogenizer, and then centrifuged at 40,000 g for 20 minutes, and the supernatant was collected.
Then add 50% zinc chloride solution to 500 ml of the supernatant obtained.
ml, stirred for 2 hours, centrifuged at 5000 rpm, 4°C for 5 minutes, added 25 ml of 10% disodium phosphate solution to the resulting precipitate, stirred for 30 minutes, 8000 rpm, 4°C.
C. for 20 minutes to obtain 28 ml of supernatant, which was dialyzed against distilled water 3 times for 2 days and freeze-dried to obtain 2.3 g of a powder sample. Divide this into several times and add 8% sucrose solution (0.025M phosphate buffer containing 1M NaCl).
PH8.0) Suspended in 0.2 ml, added to the top layer of 6 ml of 10-20% sucrose density gradient layer, and incubated at 40,000 rpm for 17 hours at 4°C.
Sucrose density gradient centrifugation was performed with a (SW-65-Ti-rotor, Petsukuman L4 ultracentrifuge). As a result, the desired active substance was observed only in the first protein peak. Collect this active fraction solution and add it to 10 times the volume of dilute acetic acid solution (PH4.0).
Dialyze for 24 hours and concentrate by lyophilization. This concentrated solution was then subjected to starch zone electrophoresis (starch block 30 x 1.0 x 5.0 cm, 0.1M acetate buffer PH4.0).
Perform electrophoresis in the cathode direction for 10 hours at 4°C, 300V, 10-12mA using a device. After electrophoresis, cut the starch layer into 0.5cm-wide pieces at equal intervals and take them out. Place each section in a test tube.
0.01M phosphate buffer (PH
7.0) Extracted with 1 ml and measured protein and each activity. As a result, the desired activity was found to coincide with the second protein peak from the anode. Furthermore, this active fraction was added to 0.01M acetate buffer (PH
4.0) Gel chromatography was performed on a Biogel P-200 column (1.5 x 75 cm) equilibrated with the same buffer with 1.8 ml of the sample solution after dialysis with 1 for 3 times for 2 days. As a result, the desired active substance was obtained, which corresponded to a protein peak with a molecular weight of 115,000±15,000.
The active fraction was freeze-dried to obtain 2.4 mg of brownish-white fine powder. The active fraction is 75% protein and 6 to 6 carbohydrates.
10% by weight, 12-16% by weight of lipids, and further
A shoulder that appeared to be a nucleic acid was seen at UV 260nm. It migrated to the cathode side by disk electrophoresis (PH4.3), and its isoelectric point was approximately 8.0±0.4. A activity is 750U/μg, B activity is 888/μg, C
The activity was 0.79 EU/μg. LD ₅₀ is a male mouse
It was 410 μg/Kg (body weight) and 360 μg/Kg (body weight) in females. Example 22 10 g of bacterial cells obtained by culturing pertussis faoid bacteria Higashihama strain (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) in the same charcoal-supplemented semi-synthetic solid medium as in Example 16 (37°C, 72 hours)
(wet weight) 1M−NaCl, 0.02M containing 4M urea
Suspend in 100 ml of Tris-HCl buffer (PH8.0), stir with a glass homogenizer at 4°C for 1 hour, centrifuge at 40000G for 20 minutes, and collect the supernatant. While stirring the obtained supernatant in ice water, gradually add cold ethanol to give a final concentration of 40%. After standing in ice water for 1 hour, centrifugation is performed at 0°C, 5000 rpm for 15 minutes to obtain a supernatant and a precipitate. The supernatant was then dialyzed 3 times for 2 days against distilled water 1 and then concentrated, and further dialyzed 3 times for 2 days against 0.01M phosphate buffer (PH6.0) and then treated with carboxymethylcephade equilibrated with the same buffer. Flow rate 20 added to Tux G-50 column (2 x 20 cm)
Elution separation was performed at ml/hr. The desired active fraction was obtained in agreement with the protein peak eluted with 0.5M sodium chloride and 0.1M phosphate buffer (PH8.0). Therefore, after collecting and concentrating this active fraction,
Dialyzed 3 times for 2 days against 0.01M phosphate buffer (PH6.0) 1, added to concanavalin A-Sepharose 4B (1.8 x 15 cm) equilibrated with the same buffer, and eluted and separated at a flow rate of 18 ml/hr. I went there. The protein peak eluted with 0.01M phosphate buffer (PH7.0) containing 0.5M-NaCl and 0.2M-α-methyl-D-mannoside, and the desired active substance was obtained. After concentrating the fraction obtained here
0.01M phosphate buffer containing 0.5M NaCl (PH7.0)
Gel chromatography was performed on a Sephadex G-75 column (1.5 x 80 cm), which was equilibrated with the same buffer. As a result, the molecular weight is 26000
3.0 mg of the desired active fraction was obtained in agreement with the protein peaks located before and after the protein peaks. Composition is protein 85
It consists of about 1% by weight of carbohydrates, about 10% by weight of lipids, and does not contain nucleic acids. A activity is 250U/μg,
B activity is 170U/μg, C activity is 0.23EU/μg, and LD ₅₀ is 1043μg/Kg (body weight) for male mice and female mouse.
It is 980 μg/Kg (body weight). On the other hand, the precipitate obtained from the ethanol fraction was dissolved and suspended in a dilute acetic acid solution (PH4.0), and after dialyzing with the same solution 13 times for 24 hours,
Concentrate by lyophilization. This concentrated solution was then subjected to starch zone electrophoresis (starch block 30x
0.8×5cm, 0.1M acetate buffer PH4.0) apparatus at 4℃,
Electrophoresis was performed at 300V, 10-12mA, towards the cathode for 10 hours. After electrophoresis, the starch layer was cut into 0.5cm widths at equal intervals, taken out, and each section was placed in a test tube with 0.15M−
The protein and each activity were measured after extraction with 1 ml of 0.01M phosphate buffer containing NaCl. As a result, the desired activity was found to correspond to the second protein peak from the anode. Furthermore, this active fraction
Gel chromatography was performed on a Biogel P-200 column (1.5 x 75 cm) equilibrated with the same buffer with 1.8 ml of the sample solution after dialysis with 0.01M acetate buffer 1 for 3 times for 2 days. As a result, the molecular weight is 90000±
2 mg of the desired active substance was obtained in agreement with the protein peak of 10,000. The active fraction contained more than 90% protein, 2-5% carbohydrates, and a shoulder that appeared to be due to nucleic acids was observed in the UV and 260 nm. Moves to the cathode side by disk electrophoresis (PH4.3), and the isoelectric point PH is 8.5
±0.5, A activity is 550U/μg, B activity is
640U/μg, C activity is 0.50EU/μg,
LD ₅₀ is mouse = 520μg/Kg (body weight), ♀474μ
g/Kg (body weight). Example 23 After culturing Higashihama strain of pertussis bacteria in a charcoal-supplemented semi-synthetic solid medium (CA medium) at 37°C for 48 hours according to Example 16, the bacterial bodies were scraped off, and the CA medium from which the bacterial bodies had been removed was frozen and thawed. The mixture was centrifuged at 15,000 rpm for 30 minutes to obtain a supernatant. To this supernatant 10, 1 portion of a 50% polyethylene glycol solution was added at 4° C. while stirring, and after standing for 1 hour, the mixture was centrifuged at 35,000 g for 30 minutes to collect the precipitate.
Add 200 ml of 0.05 M phosphate buffer (PH7.0) containing 1.0 M NaCl to the collected precipitate, make a suspension using a Teflon homogenizer, centrifuge the suspension at 15,000 rpm for 30 minutes, and separate it into a supernatant and a precipitate. Sediment is 1.0M−
Extract 3 times with 60 ml of 0.05 M phosphate buffer (PH7.0) containing NaCl, mix this extract with the supernatant,
ml to 0.2M phosphate buffer (PH8.0) at 4°C.
After overnight dialysis, it was equilibrated with 0.2M phosphate buffer (PH8.0). Starch block (length 50cm, width 10
cm, height 1.5 cm), and electrophoresis was performed at 4°C.
The starch support was cut into 1 cm width and 1.0M−
10ml of 0.05M phosphate buffer (PH8.0) containing NaCl
Each was extracted. As a result, it was divided into three main protein peaks,
The target active substance migrated to the cathode side. 0.2 ml of the collected and concentrated active fractions was layered on a 10-20% sucrose density gradient of 0.05 M phosphate buffer (PH8.0) containing 1.0 M NaCl, and ultracentrifuged to collect the desired active fraction. 0.05M phosphate buffer (PH) containing 1.0M NaCl.
After dialysis against 8.0), sucrose density gradient centrifugation was performed again to collect the target active fraction. After dialysis of this
Freeze-drying yielded 2.00 mg of white powder. The molecular weight determined by gel filtration method is 118000±7500, and the chemical composition is:
Approximately 47% protein, approximately 25% carbohydrates, approximately fat
In disk electrophoresis (PH4.3) at 20% by weight, it migrated toward the cathode side. A activity is 820U/μg, B activity is 970U/μg,
C activity was 0.68 EU/μg. The LD ₅₀ value was 279 μg/Kg for male mice and 204 μg/Kg for females. Example 24 Freeze-dried preserved bacteria of pertussis bacteria strain 3779B (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) were cultured with shaking in the same manner as in Example 4, and the cells were isolated to obtain culture supernatant liquid 20. After adding thimerosal to the culture supernatant to a final concentration of 0.02%, 400 ml of 50% zinc chloride solution was added, and the mixture was stirred overnight at 4°C to precipitate. Collect the precipitate by centrifugation;
After extraction with % disodium phosphate solution, the supernatant was collected by centrifugation, equilibrated with 0.1M Tris buffer (PH10) containing 0.5M NaCl, concentrated to 20ml, and then 6ml
was layered on a cesium chloride density gradient solution, and heated at 4°C.
It was centrifuged at 50000g for 3.5 hours. The density gradient is 0.5M−
6 ml of cesium chloride with densities of 1.5, 1.3, 1.25, and 1.2 in 0.1M Tris buffer (PH10) containing NaCl. The active fraction obtained after centrifugation was diluted with 0.5M−
Dialysis was performed against 0.1M-Tris buffer (PH10) containing NaCl. For further purification, gel filtration was performed using Bio Gel P-200 (2 cm x 90 cm) equilibrated with the above buffer. The effluent was run at a rate of 15 ml/hr at 4°C to obtain an active fraction. The active fraction was lyophilized to give pale yellow powder 1
I got mg. The molecular weight is 90000± from the SDS electrophoresis pattern.
Estimated to be 6,000, the chemical composition was over 98% protein by weight, and no carbohydrates or lipids were detected. In the disk electrophoresis pattern, a sharp band migrating toward the cathode side was observed in the PH4.3 gel. The isoelectric point PH was 9.5±0.5, and the amino acid composition and composition ratio are shown in Table 13. Activity is A activity 960U/μg, B activity 770U/μg
g, C activity is 1.8 EU/μg, and LD ₅₀ is mouse
They were 242μg/Kg and 175μg/Kg.

【table】

[Table] Example 25 Bordetella pertussis strain Higashihama strain (provided by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Kitasato University) was cultured in the same manner as in Example 16 in a semi-synthetic solid medium supplemented with charcoal (cultured at 37°C for 48 hours), and the bacteria collected. Suspend 5 g (wet weight) of the body in 300 ml of distilled water,
After heating to 56℃ for minutes and cooling, add thimerosal to the final concentration.
Add to 0.01% and centrifuge (15000 rpm)
x 30 minutes), then add 4M urea to the collected bacteria.
Suspended in distilled water containing 1M sodium chloride at 20K
The bacterial cells were destroyed using an ultrasonic device (manufactured by Tomy Seiko Co., Ltd.) at Hz for 5 minutes. This suspension was centrifuged (15000rpm)
x 30 minutes) to separate the precipitate, solid ammonium sulfate was added to the supernatant until near saturation, and after stirring at 4°C all day and night, the precipitate was collected by centrifugation (10,000 rpm x 30 minutes).
The obtained precipitate was dissolved in a 1M saline solution containing 4M urea, and the insoluble residue was filtered and discarded, followed by ultracentrifugation (105000G for 1 hour, manufactured by Hitachi) to obtain a supernatant. The solution containing the active fraction was applied to a 5% to 20% sucrose density gradient and ultracentrifuged at 35,000 rpm for 16 hours. The obtained fraction was dialyzed and then freeze-dried to obtain 1.1 mg of brownish fine powder. The molecular weight determined by gel filtration was 95,000±15,000. Chemical composition: approximately 60% protein, 5±2 carbohydrates
Nucleic acids were also detected at approximately 20% by weight and lipids. In addition, a band is observed on the cathode side in disk electrophoresis at PH4.3. Acute toxicity in mice
LD ₅₀ 〓279μg/Kg, ♀204μg/Kg, A activity is 400U/μg, B activity is 542U/μg, C activity is
It was 0.38EU/μg. Example 26 10 ml of a 0.16M sodium chloride solution containing 500 μg of the purified specimen (hereinafter abbreviated as IAP) obtained in Example 4 is mixed with 10 ml of complete Freund's adjuvant to prepare an emulsion. 1 ml of the solution was injected into the footpad and intradermal area of rabbits to immunize them. 1 injection
Once a week for a total of 3 times, and on the 10th day after the final injection
Whole blood was collected from the carotid artery of rabbits in which IAP antibody production was observed and used as anti-IAP antibody rabbit serum. After thoroughly washing the IAP-Sepharose 4B column (1.2 x 8 cm) (see Note 1) with 0.01M phosphate buffer (PH7.4) containing 0.15M-NaCl, the anti-IAP antibody rabbit obtained above was used. Add serum and flow rate 20ml/
hr to bind the contained antibodies to the immunoadsorbent, and then flush with the same buffer to thoroughly wash away serum proteins other than the bound antibodies. Then 3M−
Elute the anti-IAP antibody bound to the adsorbent with a 0.15M-NaCl solution containing NaSCN and collect the antibody-containing fraction.
Dialysis was performed against 10 ^-3 M ^-ammonia water (PH8.3) until SCN ^- ions were no longer detected, and freeze-drying was performed to obtain 150 mg of purified anti-IAP antibody. Purification of IAP using anti-IAP antibody-Sepharose 4B Anti-IAP antibody-Sepharose 4B column (1.8 x 13
cm) (see Note 2) with culture supernatant 2 (6N-HCl).
(adjusted to PH7.0) at a flow rate of 20ml/hr, then 0.1M
-After thorough washing (approximately 200ml) with 0.01M phosphate buffer (PH7.0) containing NaCl, 2mM EDTA-0.15M
−0.1M glycine-HCl buffer containing NaCl (PH
3.0) at a flow rate of 60 ml/hr, and the eluate was immediately
Neutralized with 1M glycine buffer (PH11.5). The active fractions were collected, dialyzed (5 times, 3 times, 48 hours), and lyophilized to obtain 1.3 mg of pale dry powder. Disc electrophoresis gave a single band on a PH4.3 gel, and the composition was more than 98% protein by weight, with no carbohydrates, lipids, or nucleic acids detected. The molecular weight was estimated to be 55000±5000 by gel filtration. The amino acid composition was as listed in Table 14. The A activity is 957 U/μg, the B activity is 1050 U/μg, and the C activity is 1.18 EU/μg, and the acute toxicity (LD ₅₀ ) when intravenously injected into mice is 241 μg/Kg (body weight), ♀
It is 165 μg/Kg (body weight). (Note 1) IAP-Sepharose 4B preparation method CNBr-activated Cepharose 4B (manufactured by Pharmacia Fine Chemicals) 4g was added to 10 ^-3 N HCl1
Repeat washing and swelling on a glass filter. 20 mg of IAP separately purified according to Example 4
is dissolved in 20 ml of 0.1 M sodium bicarbonate buffer (PH8.3) containing 0.5 M NaCl. Immediately after washing and swelling of the gel is completed, add IAP solution and incubate at room temperature (20-25°C). Shake well for 2 hours to mix. After the reaction, aspirate the gel with a glass filter and wash it several times with the same buffer to remove excess IAP.
then 1M ethanolamine (PH9.0) solution
Add 50ml and mix well by shaking at room temperature for 2 hours.
After the reaction, wash with 500 ml of the above buffer several times, then wash with 0.1 M acetate buffer (PH) containing 0.5 M NaCl.
4.0) Wash with 500 ml in several portions (repeat this operation 3 times by changing the buffer solution and PH), and finally wash with working buffer 1 before use. (Storage is between 4°C and 8°C.
℃) (Note 2) Anti-IAP antibody-Sepharose 4B preparation method CNBr-activated Cepharose 4B (manufactured by Pharmacia Fine Chemicals) 10g was dissolved in 10 ^-3 N HCl2
Repeat washing and swelling on a glass filter. Separately add 100 mg of purified anti-IAP antibody to 0.1 M sodium bicarbonate buffer (PH8.3) containing 0.5 M NaCl for 50 min.
ml, and as soon as the washing and swelling of the gel is completed, add the anti-IAP antibody solution and mix well by shaking at room temperature (22-25°C) for 2 hours. After the reaction, the gel was aspirated through a glass filter and washed several times with the same buffer to remove excess anti-IAP antibody.
Then 1M monoethanolamine (PH9.0) solution 100
ml and mix by shaking at room temperature for 2 hours. After the reaction, wash several times with the above buffer, then wash with 0.1M acetate buffer (PH4.0) 1 containing 0.5M NaCl (repeat this operation three times by changing the buffer and pH), and finally Wash with buffer 1 before use. (Storage is 4℃~8℃)

【table】

【table】

【table】 [Brief explanation of the drawing]

1 and 2 are explanatory diagrams of an experiment of Example 4 of the present invention.

Claims (1)

[Claims] 1. Molecular weight by gel filtration method is 10,000 to 250,000,
Protein content is 40% by weight or more by Lowry method, carbohydrate content is 25% by weight or less by phenol/sulfuric acid method, lipid content is 25% by weight or less by Marsh-Weinstein method,
Amino acid composition and composition ratio of protein components (μM/
100μM) is aspartic acid: 7.5-7.9, threonine: 6.8-7.6, serine: 5.9-7.6, glutamic acid: 9.7-10.8, proline: 5.5-6.4, glycine:
8.7-9.6, alanine: 9.1-10.8, cystine/2:
1.5-2.6, valine: 5.6-6.6, methionine: 2.5-
3.3, Isoleucine: 3.6~4.1, Leucine: 7.5~
8.0, Tyrosine: 5.1-6.6, Phenylalanine:
3.3-3.9, Lysine: 3.1-4.4, Histidine: 1.4-
1.6 and arginine: 6.1-6.6, polyacrylamide gel (polyacrylamide concentration: 7.5%, PH
4.3) A protein-like active fraction that gives a single band on the cathode side in electrophoresis and has an isoelectric point of PH4-10 and has insulin secretion enhancing activity, immune enhancing activity, and sympathetic β receptor blocking activity. . 2. Cultivating microorganisms belonging to the genus Bordetella, and treating the culture with a fractional purification method of one or a combination of solubility method, chromatography method, molecular sieve method, electrophoresis method, and biological method. The molecular weight determined by the gel filtration method, which is characterized by
10,000 to 250,000, protein content by Lowry method is 40% or more, carbohydrates are 25% by weight or less by phenol/sulfuric acid method, lipids are 25% by weight or less by Marsh-Weinstein method, amino acid composition and composition ratio of protein components (μM /100μM) is aspartic acid: 7.5
~7.9, Threonine: 6.8~7.6, Serine: 5.9~
7.6, glutamic acid: 9.7-10.8, proline: 5.5-
6.4, glycine: 8.7~9.6, alanine: 9.1~
10.8, cystine/2: 1.5-2.6, valine: 5.6-
6.6, Methionine: 2.5-3.3, Isoleucine: 3.6
~4.1, Leucine: 7.5~8.0, Tyrosine: 5.1~
6.6, Phenylalanine: 3.3-3.9, Lysine: 3.1
~4.4, histidine: 1.4-1.6 and arginine:
6.1 to 6.6, gives a single band on the cathode side in polyacrylamide gel (polyacrylamide concentration: 7.5%, PH4.3) electrophoresis, and has an isoelectric point of PH
A method for producing a protein-like active fraction having an insulin secretion enhancing activity, an immune enhancing activity and a sympathetic β receptor blocking activity showing a value of 4 to 10. 3 Molecular weight by gel filtration method is 10,000 to 250,000,
Protein content is 40% by weight or more by Lowry method, carbohydrate content is 25% by weight or less by phenol/sulfuric acid method, lipid content is 25% by weight or less by Marsh-Weinstein method,
Amino acid composition and composition ratio of protein components (μM/
100μM) is aspartic acid: 7.5-7.9, threonine: 6.8-7.6, serine: 5.9-7.6, glutamic acid: 9.7-10.8, proline: 5.5-6.4, glycine:
8.7-9.6 Alanine: 9.1-10.8, Cystine/2:
1.5-2.6, valine: 5.6-6.6, methionine: 2.5-
3.3, Isoleucine: 3.6~4.1, Leucine: 7.5~
8.0, Tyrosine: 5.1-6.6, Phenylalanine:
3.3-3.9, Lysine: 3.1-4.4, Histidine: 1.4-
1.6 and arginine: 6.1-6.6, polyacrylamide gel (polyacrylamide concentration: 7.5%, PH
4.3) A protein-like active fraction that gives a single band on the cathode side in electrophoresis and has an isoelectric point of PH4-10 and has insulin secretion enhancing activity, immune enhancing activity, and sympathetic β receptor blocking activity. Diabetic treatment or prevention drug containing as an active ingredient. 4 Molecular weight by gel filtration method is 10,000 to 250,000,
Protein content is 40% by weight or more by Lowry method, carbohydrate content is 25% by weight or less by phenol/sulfuric acid method, lipid content is 25% by weight or less by Marsh-Weinstein method,
Amino acid composition and composition ratio of protein components (μM/
100μM) is aspartic acid: 7.5-7.9, threonine: 6.8-7.6, serine: 5.9-7.6, glutamic acid: 9.7-10.8, proline: 5.5-6.4, glycine:
8.7-9.6, alanine: 9.1-10.8, cystine/2:
1.5-2.6, valine: 5.6-6.6, methionine: 2.5-
3.3, Isoleucine: 3.6~4.1, Leucine: 7.5~
8.0, Tyrosine: 5.1-6.6, Phenylalanine:
3.3-3.9, Lysine: 3.1-4.4, Histidine: 1.4-
1.6 and arginine: 6.1-6.6, polyacrylamide gel (polyacrylamide concentration: 7.5%, PH
4.3) A protein-like active fraction that gives a single band on the cathode side in electrophoresis and has an isoelectric point of PH4-10 and has insulin secretion enhancing activity, immune enhancing activity, and sympathetic β receptor blocking activity. An immunomodulator containing as an active ingredient. 5 Molecular weight by gel filtration method is 10,000 to 250,000,
Protein content is 40% by weight or more by the Lowry method, carbohydrates are 25% by weight or less by the phenol/sulfuric acid method, and lipids are 25% by weight or less by the Marsh-Weinstein method.
Amino acid composition and composition ratio of protein components (μM/
100 μM) is aspartic acid: 7.5-7.9, threonine: 6.8-7.6, serine: 5.9-7.6, glutamic acid: 9.7-10.8, proline: 5.5-6.4, glycine:
8.7-9.6, alanine: 9.1-10.8, cystine/2:
1.5-2.6, valine: 5.6-6.6, methionine: 2.5-
3.3, Isoleucine: 3.6~4.1, Leucine: 7.5~
8.0, Tyrosine: 5.1-6.6, Phenylalanine:
3.3-3.9, Lysine: 3.1-4.4, Histidine: 1.4-
1.6 and arginine: 6.1-6.6, polyacrylamide gel (polyacrylamide concentration: 7.5%, PH
4.3) A protein-like active fraction that gives a single band on the cathode side in electrophoresis and has an isoelectric point of PH4-10 and has insulin secretion enhancing activity, immune enhancing activity, and sympathetic β receptor blocking activity. Sympathetic nerve beta receptor blocker containing as an active ingredient.