JPH08245702A - High-molecular weight lipopolysaccharide - Google Patents

Abstract

PURPOSE: To obtain a new high-molecular weight lipopolysaccharide(LPS) high in safety and biological activity, thus usable as a medicine, etc. CONSTITUTION: This new high-molecular weight LPS is obtained from a specific kind of microbes and has the following physicochemical properties: (a) molecular weight determined by SDS-PAGE method using a protein marker is 30000-60000, and does not virtually contain fractions having <=10000 molecular weight; (b) hexosamine content determined by Elson-Morgan method is 3-9/molecular weight of 30000; and (c) 2-keto-3-deoxyoctonate content determined by diphenylamine method is 20-30/molecular weight of 30000.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel high molecular weight lipopolysaccharide having high biological activity and safety and having specific physicochemical properties. More specifically, the present invention relates to high molecular weight lipopolysaccharides having significantly improved safety, which are useful for drug development such as immune function activators.

[0002]

2. Description of the Related Art lipopolysaccha
ride. Hereinafter sometimes referred to as LPS) is E. coli,
It is a complex compound consisting of lipids and sugars existing in the outer membrane surrounding the peptidoglycan of the cell wall of Gram-negative bacteria such as Salmonella and Pertussis, and is known as the active component of O antigen and endotoxin [J.M. Hackenbeck (JM Ghuysen and R.
Hakenbeck), "New Comprehensive Biochemistry," Vol. 27, Bacterial Cel (Bacterial Cel)
l Wall), page 18, Elsevea,
1994]. The basic structure of LPS consists of lipid A having a specific lipid, an oligosaccharide called R core covalently bonded to it, and an O-specific polysaccharide (Nikkei Biotech ed., "Nikkei Biotechnology latest term dictionary", Page 431, Nikkei McGraw-Hill, 1985
Year).

The basic structure of lipid A is common to many bacterial species, and the basic skeleton is composed of glucosaminyl and glucosamine with β-1,6 linkages, and phosphates are bound at the C-1 and C-4 'positions, respectively. There are many cases. Each amino group is 3-
Hydroxy fatty acid and hydroxyl group combine several kinds of saturated fatty acid or hydroxy fatty acid to form a unique glycolipid, but the kind of fatty acid is slightly different depending on the bacterial species.
Although a small number of cases have completely different basic skeletons, an example consisting only of 2,3-diamino-2,3-dideoxy-D-glucose has also been reported (Nomori Michido, “Medical Science Dictionary No. 4”).
9 ", page 82, Kodansha, 1984).

The structure of R core is common to most of the bacterial species belonging to it, such as Salmonella, or several partially different structures are known, such as Escherichia coli [J.・ JM Ghuysen and R. Hakenbeck, ed., "New Comprehensive Biochemistry (New Compr
ehensive Biochemistry) ", Volume 27, Bacterial
Bacterial Cell Wall, page 283, Elsevea, 1994]. Generally, heptose and 2-keto-3-deoxyoctonate (which may be hereinafter referred to as KDO) are common constituents to many R cores and are bound to lipid A via KDO. It is also known that LPS lacks either or both by JM Ghsen and Earl Hackenbeck.
uysen and R. Hakenbeck), `` New Comprehensive Biochem
istry) ", Volume 27, Bacterial Cell Wall (B
acterial Cell Wall, pp. 294-295, Elsevea, 1994].

The structure of O-specific polysaccharides is the most diverse among the constituent components, is specific to the bacterial species, and exhibits so-called O-antigen activity. Generally characterized by a repeating structure of oligosaccharides consisting of several types of monosaccharides, but consisting of the same monosaccharide,
Alternatively, a non-repeating structure is also known. The biosynthesis of O-specific polysaccharide is controlled by a gene different from that of R-core, and it is possible to replace O-specific polysaccharide of different bacterial species by conjugation or transduction. Has been applied to [JM Ghuysen an
d R. Hakenbeck), `` New Comprehensive Biochemistr
y) ", Volume 27, Bacterial Cell Wall (Bacte
rial Cell Wall, pp. 265-267, Elsevea, 1994].

[0006] LPS has a wide variety of pharmacological actions, but when, for example, an antigen and LPS are administered simultaneously,
Since the immune response is enhanced, LPS is currently applied as a kind of an adjuvant that enhances the vaccine effect (Hiroshi Honma et al., “Bacterial endotoxin”, page 312, Kodansha, 1973). It has been reported that the molecular weight of LPS varies from several millions to several thousands depending on its association state (Hiruma Homma, “Bacterial endotoxin”,
Pages 217 to 218, Kodansha, 1973), as an LPS having a molecular weight of about 30,000 to 60,000, a molecular weight of 30,000 ± 5,000, a hexosamine number of 45 ± 6 / a molecular weight of 30,000, and a KDO number of 5 ± 1 / molecular weight 30,000 LPS derived from E. coli (JP-A-4-492
45), an Escherichia coli-derived LPS having a molecular weight of 40,000 ± 10,000, a hexosamine number of 45 ± 6 / a molecular weight of 30,000, and a KDO number of 5 ± 1 / a molecular weight of 30,000 (JP-A-6-40937). It has been reported so far.

Regarding the use of LPS, the inventors of the present invention have hitherto disclosed an anti-toxoplasma agent (Japanese Unexamined Patent Publication No.
492459), cholesterol lowering agent (JP-A-4-49243), anti-herpes agent (JP-A-4-4).
9242), an anti-rheumatic agent (JP-A-4-492).
41), antidiabetic agents (JP-A-4-49244), anti-peptic ulcer agents (JP-A-4-49240), immune function activators (JP-A-4-99481, JP-A-6-9481). -141849), oral / transdermal immune function promoter (JP-A-4-187640), analgesic (JP-A-6-40937), growth promoter (JP-A-3-155778), anti Withdrawal agent (JP-A-6-
No. 65092) has been proposed.

However, LPS obtained from microorganisms is usually chemically heterogeneous (a mixture of various LPS having different molecular weights and chemical compositions) due to association, and it is extremely difficult to physicochemically define its properties. However, it was also highly toxic and had limited application as a pharmaceutical (edited by the Japan Tissue Culture Society, "Cell Growth Factor part II", page 121, Asakura Shoten, 1987).

Further, the inventors of the present invention have found that the molecular weight is 3
Although the presence of LPS of about 50,000 to 60,000 is disclosed (JP-A-4-99481), this is a mixture with LPS having a molecular weight of about 5,000 to 6,000 and has a low molecular weight. It contained a considerable amount of fractions.

[0010]

As described above, the previously reported high molecular weight LPS is a heterogeneous mixture containing low molecular weight LPS, and is clinically used as a drug component such as an immune function activator. When used, it was not always satisfactory in terms of safety or drug efficacy.

Therefore, it has been desired to improve the prior art, separate highly purified high molecular weight LPS, and examine its industrial utility. The present invention has been made in view of the above circumstances, and provides a novel high molecular weight LPS that has high safety (that is, low toxicity), excellent biological activity, and is chemically specified. Is intended.

[0012]

The inventors of the present invention have
As a result of intensive studies to solve the above-mentioned problems, as a result of purifying a high molecular weight LPS that is physicochemically specified from the previously reported LPS, this novel high molecular weight LPS
However, they have found that they are extremely safe as compared with conventional LPS and have superior biological activity as compared with conventional LPS, and completed the present invention.

That is, the present invention provides the following physicochemical properties a) to c) obtained from microbial cells, and a) a molecular weight of 30,000 to 60, measured by SDS-PAGE using a protein marker. 000,
Substantially free of a fraction having a molecular weight of 10,000 or less, b) a hexosamine content of 3 to 9 / molecular weight of 30,000 measured by the Elson-Morgan method, and c) 2 measured by the diphenylamine method. -Keto-3-
Deoxyoctonate content 20 to 30 / molecular weight 3
A high molecular weight lipopolysaccharide having a molecular weight of 10,000.

In the present invention, it is also desirable that the microorganism is a Gram-negative bacterium, and that the Gram-negative bacterium is a microorganism belonging to the genus Pantoea. Hereinafter, the present invention will be described in detail. In the following explanation, the percentage display is
Unless stated otherwise, values are by weight.

The high molecular weight LPS of the present invention is a gram-negative microorganism such as a microorganism belonging to the genus Pantoea.
Culture by a conventional method, collect the cells from the medium, a known method from the collected cells, for example, the thermal phenol method [O. Westphal ed., Methodes in Carbohydrate Chemistry (Methods in Carbohydrate
Chemistry), Volume 5, page 83, Academic Press, 1965],
Further, it can be produced by purifying with an anion exchange resin.
That is, the microbial cells are suspended in distilled water, the suspension is added to a mixed solution of distilled water and an equal volume of hot phenol, the mixture is stirred, and then centrifuged to recover an aqueous layer. Is dialyzed to remove phenol, concentrated by the ultrafiltration method to collect a crude LPS fraction, and this fraction is subjected to conventional anion exchange chromatography (for example, mono-Q-sepharose or Q-sepharose). Used) and desalted by a conventional method.

The purified LPS thus obtained is subjected to gel filtration in the presence of a surfactant such as sodium deoxycholate to recover only the fraction containing the high molecular weight LPS, which is mixed. High molecular weight LPS of the present invention, which is highly purified by removing low molecular weight LPS
Can be obtained. The high molecular weight LPS of the present invention produced by the above method has a molecular weight of 30,000 to 60,000 measured by SDS-PAGE using a) protein marker, as shown in Test Example 1 described below.
Substantially free of a fraction having a molecular weight of 10,000 or less, b) a hexosamine content of 3 to 9 / molecular weight of 30,000 measured by the Elson-Morgan method, and c) 2 measured by the diphenylamine method. -Keto-3-
Deoxyoctonate content 20 to 30 / molecular weight 3
It has a physicochemical property of being 10,000.

The novel high molecular weight LPS of the present invention can be used as a drug having an immune function activating action, a veterinary drug, etc., and is used in combination with another known substance having an immune function activating action. You can also Next, a test example will be shown to further explain the high molecular weight LPS of the present invention. Test Example 1 This test was conducted to investigate the physicochemical and biological properties of the high molecular weight LPS of the present invention. 1) Preparation of sample High molecular weight L was measured by the same method as in Example 1 and Reference Example 1.
PS and conventional LPS were prepared respectively. 2) Test method Measurement of molecular weight High-molecular weight LPS and conventional LPS (hereinafter simply referred to as LPS) are dissolved in distilled water to prepare a solution having a concentration of 2 mg / ml, and 10 μg of the solution is used in a 1.5 ml plastic tube. Was weighed. Separately, 180 μl of 10% (w / v) SDS, 45 μl of 5% β-mercaptoethanol, 90 μl of CBB dye solution, 112.5
μl 0.5 M Tris-HCl (pH 6.8) and 22.
10 μl of SDS treatment solution prepared by adding 5 μl of distilled water
Was added to each of the above sample solutions and mixed well, then immersed in a boiling water bath for 5 minutes, and immediately thereafter immersed in ice water for rapid cooling.

10 ml of 10% (w / v) SDS, 1
A migration buffer prepared by dissolving 7.9 g of tricine and 3.03 g of Tris in 1 liter of distilled water was placed in a slab gel electrophoresis tank (Marisol). A 20% polyacrylamide gel was fixed to a migration tank, a sample was put in the sample groove, the voltage was fixed to 50 V for 1 hour, and then fixed to 150 V, and migration was continued until the dye was eluted from the gel.
After the completion of the electrophoresis, silver staining was performed at room temperature using a silver staining kit 161-0443 (manufactured by Bio-Rad) to confirm the behavior. Determination of Hexosamine Content The hexosamine content was determined by Elson-Mor
gan) method (edited by the Japanese Biochemical Society, "Biochemistry Experiment Course", No. 4)
Volume, pp. 377-379, 1st edition, Tokyo Kagaku Doujin Shuppan, 1976). LPS was dissolved in distilled water to prepare a solution having a concentration of 2 mg / ml,
100 μl thereof was weighed into a Spitz with screw cap (made by Iwaki Glass Co., Ltd.), and 100 μl of 8NH
Add Cl and heat at 110 ° C for 16 hours, then add 4NN
The pH was adjusted to 7 by adding about 200 μl of aOH. 100 μl thereof was weighed out, put in another Spitz equipped with a screw cap, 200 μl of the reagent A was added, heated at 105 ° C. for 1.5 hours, and cooled with running water. Then 10
0 μl was taken, 670 μl of 96% ethanol was added, 67 μl of reagent B was further added, the mixture was allowed to stand at room temperature for 1 hour, and the absorbance at 535 nm was measured. As a standard sample for preparing a calibration curve, 0-800 μg / ml N-acetylglucosamine (manufactured by Wako Pure Chemical Industries, Ltd.) was used. Reagent A: a mixture of 75 μl acetylacetone and 2.5 ml 1.25N sodium carbonate. Reagent B: 1.6 g of p-dimethylbenzaldehyde, 3
Mixture of 0 ml concentrated hydrochloric acid and 30 ml 96% ethanol. Quantification of KDO content The KDO content was determined by the diphenylamine method [analytical
Biochemistry (Analytical Biochemistry), 5th
Volume 8, Issue 1, Pages 123-129, 1974]
Was quantified as follows.

500 mg of diphenylamine (manufactured by Wako Pure Chemical Industries), 5 ml of ethanol (manufactured by Wako Pure Chemical Industries), 45 m
1 L of glacial acetic acid (manufactured by Wako Pure Chemical Industries) and 50 ml of concentrated hydrochloric acid (manufactured by Wako Pure Chemical Industries) were mixed to prepare a KDO detection reagent. To 500 μl thereof, 250 μl of an aqueous solution containing each sample at a concentration of 0.50 mg / ml was mixed, heated in a boiling water bath at 100 ° C. for 30 minutes, and then 3 times in constant temperature water (24 to 25 ° C.).
Cool for 0 minutes and spectrophotometer (manufactured by Hitachi Ltd. Model U2
010) to measure the absorbance at 420, 470, 630 and 650 nm (measured values are A420 and A47, respectively).
0, A630, and A650). As a standard sample, 250 μl of an aqueous KDO ammonium salt (manufactured by Sigma) solution having a concentration of 0.5 μmol was used.

From the four types of measured values of the specimen sample and the standard sample, the S value was obtained by the equation (1), and the S values of the specimen sample and the standard sample were designated as S _t and S _s , respectively. Then, the number of moles X of KDO was calculated by the formula (2). S = A420-A470 + A630-A650 (1) X = (0.5 × S _s × LPS 1 mol molecular weight) / (0.5 × S _t × 10 ⁶ ) ・(2) Measurement of limulus activity Limulus activity was invented by Levin in 1968. Limuras test, an endotoxin quantification method using horseshoe crab blood cell extract and a chromogenic synthetic substrate (edited by Ikuo Suzuki,
"Development of Pharmaceuticals Volume 14: Quality Control and Testing Methods of Pharmaceuticals", pp. 227-243, Hirokawa Shoten, 1990
This means that this limulus test is known as an LPS detection method. 3 as a standard product
45 pg / EU of E. coli 0111: B4
Was measured using a Toxicolor system (manufactured by Seikagaku Corporation). 3) Test results Molecular weight The results of molecular weight measurement are as shown in FIG. FIG.
Is a SDS-PAGE electrophoretic pattern, and in the figure, lane 1 is a protein and peptide molecular weight markers [94 kD, 67 kD, 43 kD, 30 kD, 20.
1 kD, 17.2 kD, 14.6 kD, 14.4 kD,
8.24 kD, 6.38 kD, 2.56 kD (Pharmacia)], lanes 2, 3 and 4 were LPS (20 μm).
g, 5 μg and 1.25 μg), lanes 5, 6 and 7 are high molecular weight LPS (20 μg, 5 μg and 1.25).
μg μg), and the vertical axis of the figure shows the molecular weight.

The molecular weight of high molecular weight LPS is as shown in FIG.
It was 30 kDa to 60 kDa. In Lane 5, low molecular weight LPS was not observed at all, although 20 μg of high molecular weight LPS was electrophoresed. From the above results, the high molecular weight LPS of the present invention has a molecular weight of 30,
It was found that the low molecular weight LPS was completely removed. Hexosamine content The high molecular weight LPS of the present invention has a hexosamine number of 6.18.
The molecular weight was 30,000. KDO Content KDO contained in the high molecular weight LPS of the present invention is 25.3.
8 / molecular weight was 30,000. Limulus activity The limulus activity of the high molecular weight LPS of the present invention is 1.45E.
It was U / ng.

Although the type of microorganism and the method of producing the sample were changed and tested, almost the same results were obtained. Test Example 2 This test was conducted to examine the acute toxicity of the high molecular weight LPS of the present invention. (1) Preparation of sample and test method Toxicity of high-molecular-weight LPS prepared by the same method as in Example 1 and LPS prepared by the same method as in Reference Example 1 was measured using 7-week-old C3H / He mice (Japan Charles. (Purchased from River Co.). Each sample was dissolved in physiological saline in a group of 4 mice, 5.0
It was administered intravenously at a rate of 10 or 20 mg / kg.
Live or dead mice were observed 72 hours after administration. (2) Test results The results of this test are shown in Table 1. As is clear from Table 1, in the case of intravenous administration, the high-molecular-weight LPS of the present invention caused death of 3 out of 4 mice at 20 mg / kg, and LD ₅₀ was 18 mg / kg. All died at doses of 10 and 20 mg / kg,
The LD ₅₀ was 6.0-8.6 mg / kg. In addition, although the type of microorganism and the manufacturing method of the sample were changed and tested,
Almost similar results were obtained.

[0023]

[Table 1]

Test Example 3 This test was conducted to examine the toxicity of the high molecular weight LPS of the present invention when administered intradermally. (1) Preparation of sample and test method With Test Example 2 except that the same high molecular weight LPS and LPS as in Test Example 2 were intradermally administered at a dose of 10, 20, 40 or 80 mg / kg per animal. Tested by the same method. (2) Test results The results of this test are shown in Table 2. As is clear from Table 2, when LPS was administered at 40 and 80 mg / kg, 4 out of 4 mice died. On the other hand, in the case of high molecular weight LPS, there were no deaths at the dose of 40 mg / kg and 4 at the dose of 80 mg / kg.
Three of the animals died.

From the results of Preliminary Test Example 2 and Test Example 3, L
The calculation of D ₅₀ is shown in Table 3. As is clear from Table 3, the LD ₅₀ value of high molecular weight LPS was about 2.5 times that of LPS in both intravenous administration and intradermal administration. These results indicate that the difference in the molecular weight of LPS influences the toxicity, and high molecular weight LPS is extremely toxic compared with conventional LPS in both cases of intravenous and intradermal administration. Turned out to be low. The test was performed by changing the type of microorganism and the manufacturing method of the sample, but almost the same result was obtained.

[0026]

[Table 2]

[0027]

[Table 3]

Test Example 4 The biological activity of the high molecular weight LPS of the present invention was examined by using the TNF producing ability having an antitumor effect as an index. 1) Test animals and test method Using three 7-week-old male C3H / He mice (purchased from Charles River Japan, Inc.) in each group, TNF productivity was tested as follows by intradermal abdominal administration.

0.05 ml of physiological saline containing 1, 10 or 100 μg of high molecular weight LPS prepared by the same method as in Example 1 or LPS obtained by the same method as in Reference Example 1
Was injected into the abdominal skin of each mouse, 1.5 hours after that, blood was collected and serum was separated by a conventional method. The TNF activity in each obtained serum was measured as follows by the method based on the toxicity to L929 cells. That is, 5 L929 cells
8 × 10 ⁴ cells in MEM medium containing% fetal bovine serum /
Prepare to a concentration of 100 μl, seed 100 μl into each well of a 96-well flat bottom plate, and incubate at 37 ° C for 2 hours with 5% C
It was cultured in the presence of O ₂ . Then add 1 actinomycin D
Serum sample added at a rate of μg / ml and serially diluted with MEM medium or positive control human TNF-α (manufactured by Asahi Kasei)
50 μl was added to each well, and the cells were further cultured under the same conditions for 18 hours. Remove the medium with an aspirator and use PBS at 37 ℃.
The cells were washed with to completely remove dead cells, and 1% methyl alcohol solution containing 0.1% crystal violet was added to stain living cells. The degree of staining was measured using the absorbance at OD (590 nm) as an index, and T was used as a positive control.
The TNF activity was calculated based on the relationship between the dilution ratio of NF-α and the absorbance. 2) Test results The results are shown in Table 4. T in Table 4
The NF activity is the average value of 3 animals in each group. From this result, it was confirmed that the TNF producing effect of the high molecular weight LPS of the present invention was about twice as high as that of the LPS obtained by the method of Reference Example 1. The test was performed by changing the type of microorganism and the manufacturing method of the sample, but almost the same result was obtained.

[0030]

[Table 4]

Reference Example 1 10 g of tryptone (manufactured by Difco), 5 g of yeast extract (manufactured by Difco), NaCl (manufactured by Wako Pure Chemical Industries, Ltd., special grade)
10 g was added to 1 liter of distilled water, pH was adjusted to 7.5 with NaOH, sterilized by autoclave, and separately sterilized glucose (Wako Pure Chemical Industries, Ltd. special grade) was added at a rate of 0.1%. Medium (hereinafter referred to as L-broth medium) 1
In a 500 ml Sakaguchi flask containing 00 ml, -8
Pantoea agglomerans (Pan) stored at 0 ℃
toea agglomerans) A single colony was isolated from a stock strain and inoculated, shake-cultured at 35 ° C. overnight, and the whole amount was inoculated into a 3-liter Sakaguchi flask containing 1,000 ml of L-broth medium, It culture | cultivated similarly.

Further, the cultivated cells were inoculated into a 10-liter tabletop fermenter (manufactured by Marubishi Bioengine Ltd.) containing 7 liters of L-broth medium, aerobically cultivated under the same conditions, and then collected. Then, about 70 g of wet bacterial cells was collected and stored frozen. About 70 g of the cryopreserved cells was suspended in 500 ml of distilled water, 500 ml of 90% hot phenol was added, and the mixture was stirred at 65 to 70 ° C. for 20 minutes, cooled, and cooled to 10.0.
The water layer was collected by centrifugation at 00G for 20 minutes at 4 ° C.
Repeat the same operation once more for the phenol layer,
The collected two aqueous layers were combined, dialyzed overnight to remove phenol, and the dialyzed solution was ultrafiltered (Advantech.
Made by Toyo. Ultrafiltration was carried out under a nitrogen gas of 2 atm with a molecular weight cut-off membrane of 200,000 using UK-200).

The obtained crude LPS lyophilizate was dissolved in distilled water, sterilized by a filter, added with a buffer, and subjected to anion exchange chromatography (Pharmacia Co .; Q-Sepharose Fast Flow) to give 10 mM Tris. -Pass the sample solution through the column with a buffer containing HCl (pH 7.5) and 10 mM NaCl, and
mM NaCl / 10 mM Tris-HCl (pH 7.5)
The limulus active fraction was eluted with. This eluate was ultrafiltered under the same conditions as above, desalted and concentrated, and freeze-dried to obtain about 300 mg of purified LPS from about 70 g of wet bacterial cells.

Next, the present invention will be described in more detail and concretely by showing examples, but the present invention is not limited to the following examples.

[0035]

【Example】

Example 1 5 mg of 100 mg of purified LPS obtained by the same method as in Reference Example 1 was used.
Dissolved in solubilization buffer [consisting of 3% sodium deoxycholate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 M sodium chloride, 5 mM EDTA-2Na and 20 mM Tris-HCl, pH 8.3] at a concentration of mg / ml. Then, 20 ml of the purified LPS solution was gently overlaid on the top of a Sephacryl S-200HR column (Pharmacia), and the elution buffer [0.25% sodium deoxycholate (Wako Pure Chemical Industries, Ltd.), 0.2M sodium chloride was used. It was composed of 5 mM EDTA and 10 mM Tris-hydrochloric acid, and was eluted with 800 ml (50 hours) at a flow rate of 16 ml / hour with pH 8.3].

Perista Pump PI (Pharmacia)
While controlling the flow rate using the, the obtained eluate was used as a fraction collector (manufactured by Advantech. SF212.
0), discard the first 240 ml (24 fractions) and then 80 ml at 10 ml / fraction
Fraction was fractionated. Phenol / sulfuric acid method (Fukui Sakuzo,
"Quantitative method of reducing sugars, 2nd edition", pp. 50-52, Academic Publishing Center, 1990)
The elution state was examined. From the results of the obtained elution state, LP
Fractions in which S is expected to exist (fractions 30 to 60)
Of each of the fractions 37 to 55, 0.
SDS-PAGE was performed using 5 ml to examine the fractionation pattern of LPS. As a result, fractions 30 to 3
In Fig. 6, only high molecular weight (molecular weight about 30-60 kD) LPS was observed, and both high molecular weight and low molecular weight LPS were observed in fractions 37-44. Therefore, the high molecular weight LPS fractions in fractions 30-36 were analyzed as follows. Was further purified as follows.

The respective fractions were mixed, freeze-dried, suspended in ethanol, the ethanol-soluble deoxycholic acid was removed by centrifugation, and high-molecular-weight LPS was recovered in the insoluble fraction. 2 more ethanol treatments of high molecular weight LPS fraction
Repeat to remove deoxycholic acid, then resuspend in 70% ethanol and centrifuge to remove buffer components,
This operation was repeated three more times, and high molecular weight LPS was collected in an insoluble fraction and freeze-dried to obtain about 47 mg of purified high molecular weight LPS. Example 2 Pantoea agglomerans was cultured in an L-broth medium in the same manner as in Reference Example 1 to obtain about 100 g of wet cells. After washing 50 g of the wet cells with physiological saline, 25
An equal volume of 0.5N suspended in 0 ml cold water and cooled to 4 ° C
After adding a trichloroacetic acid (TCA) aqueous solution and shaking for 3 hours, the mixture was centrifuged at 4 ° C. and 6,000 × g for 30 minutes.
Further, the precipitate was extracted with TCA by the same operation, and the respective supernatants were combined to obtain about 450 ml of supernatant. After adjusting the pH of this supernatant to 6.5, ethanol 9 cooled to -10 ° C was used.
00 ml was added with stirring and left overnight at -4 ° C. Then, after dissolving the precipitate in water and dialysis,
The supernatant was recovered by centrifuging at 00 × g for 30 minutes and freeze-dried to obtain crude LPS.

The obtained crude LPS lyophilizate was dissolved in distilled water, sterilized by a filter, added with a buffer solution, subjected to anion exchange chromatography (Pharmacia, Q-Sepharose Fast Flow), and 10 mM Tris. -Pass the sample solution through the column with a buffer containing HCl (pH 7.5) and 10 mM NaCl, and
mM NaCl / 10 mM Tris-HCl (pH 7.5)
The limulus active fraction was eluted with. This eluate was ultrafiltered under the same conditions as above, desalted and concentrated, and freeze-dried to obtain about 200 mg of purified LPS.

Then, in the same manner as in Example 1, 100 mg of this purified LPS was subjected to gel filtration using a Sephacryl S-200HR column (Pharmacia) in the presence of sodium deoxycholate to obtain a sugar content of each fraction. Is measured
SDS-PAG for fractions expected to have LPS
E was performed to collect a fraction containing only high molecular weight LPS, freeze-dry it, suspend it in ethanol, remove impurities such as deoxycholic acid soluble in ethanol by centrifugation, and collect it in an insoluble fraction. The high molecular weight LPS was freeze-dried to obtain about 51 mg of purified high molecular weight LPS.

[0040]

As described in detail above, according to the present invention, the safety that can be used as a drug is extremely high,
And a novel high molecular weight LPS having high biological activity is provided.

[Brief description of drawings]

FIG. 1 is a high molecular weight LPS and SDS of LPS.
-PAGE electropherogram.

Claims (3)

[Claims] 1. The following a) to c) obtained from microbial cells:
A) the molecular weight measured by SDS-PAGE using a protein marker is 30,000-60,000;
Substantially free of a fraction having a molecular weight of 10,000 or less, b) a hexosamine content of 3 to 9 / molecular weight of 30,000 measured by the Elson-Morgan method, and c) 2 measured by the diphenylamine method. -Keto-3-
Deoxyoctonate content 20 to 30 / molecular weight 3
A high molecular weight lipopolysaccharide having a molecular weight of 10,000. 2. The high molecular weight lipopolysaccharide according to claim 1, wherein the microorganism is a Gram-negative microorganism. 3. The high molecular weight lipopolysaccharide according to claim 1 or 2, wherein the microorganism is a microorganism belonging to the genus Pantoea.