JPS59118716A - Low molecular protein having carcinostatic effect - Google Patents

Abstract

NEW MATERIAL:A low molecular protein having the following properties. Molecular weight, 29,000+ or -2,500; isoelectric point, 4.1+ or -0.3 pH; peak of UV absorption in 0.02M tris-HCl buffer solution added with 0.1M NaCl (7.8 pH), 276nm, the lowest point of the absorption, 250nm; the solution obtained by dissolving 3mg of the protein in 1ml of 0.02M tris-HCl buffer solution (7.8 pH) is colorless and transparent; addition of >=60V/V% of acetone or ethanol to the solution causes precipitation; the aqueous solution is weakly acidic; color reactions of peptide bond and amino acid, positive to biuret reaction and Folin-Lawry reaction, etc. USE:A carcinostatic agent exhibiting direct cytotoxicity to cultured mouse L-cell in vitro, and antitumor activity to a female mouse affected with A sarcoma in vivo. PROCESS:A substance having immunoactivating activity is administered to a mammal, then an endotoxin originated from Gram-negative bacteria or lectin originated from vegetable is administered thereto to induce and produce the objective low molecular protein.

Description

[Detailed description of the invention] TECHNICAL FIELD The present invention relates to a low molecular weight protein that has anticancer activity.

Carswell et al.
We found that when endotoxin was administered to mice sensitized with etteQuer+n, BCG on the 14th day, a factor that was cytotoxic to L-aWi was produced in the serum 2 hours later. Necrosis factor
sfactor, TNF) (P
roc, Nat.

Acad, Sci, USA, vol. 72, p. 3666.

1975]. Green et al. partially purified the above substance by fractional precipitation with ammonium sulfate, gel filtration, etc., and found that the molecular weight of the TNF was about 150,000.
(PrOc, Nat.

Acad, Sci., USA, vol. 73, p. 381.

1976]. The [7
) administered BCG to rabbits and 2 weeks later administered endo1-xin to produce and purify TNF.
The molecular weight by gel filtration method is 39,000, and the molecular weight by polyacrylamide gel electrophoresis method (Polyacryl-am
ide gel electrophoresis, P
reported by AGE) to be 6.7000 (Br, J, Cancer.

Volume 42, page 416, 1980]. Furthermore, Haranaka et al.
Bacterium acnes) and endotoxin were used to produce TNF in mice and rabbits, and the molecular weight was reported to be 39,000 by gel filtration and PAGE (Nippon Clinical, Vol. 40, p. 1872, 198
2 years].

In addition to the above, the existence of many physiologically active substances (TNF) that are cytotoxic to L-cells has been reported.
Considering its molecular weight, Kull et al.'s 22
5000 (J, IIIlmunol, .

126, p. 1279, 1980] to 39,000 by Mateius et al. The current situation is that it has not been obtained.

The present inventors have also conducted extensive research in order to determine the substance that is cytotoxic to the L-1 cells. As a result, we succeeded in isolating and purifying a low-molecular-weight protein that had never been reported before, and discovered that this protein has an anticancer effect, leading to the present invention.

The novel low-molecular-weight protein of the present invention is induced and produced by administering a substance with an immunostimulatory effect to a mammalian animal, and then administering an endotoxin derived from Durham-negative bacteria or a lectin derived from a plant, and has the following characteristics. It is characterized by

(1) Molecular weight A, molecular weight Biogel (3iogel) A 1,5m (
Column (16x100 manufactured by Bio-Rad, USA)
0mm, manufactured by Pharmacia, Sweden).
．． 04M+-Lis-hydrochloric acid 10.1% dodecyl sulfate (SDS)/4M urea 10.1MNa CQ
Using the buffer solution of ([I H7,8), sample 2 of the substance of the present invention
00u (J (protein amount)) was added, gel filtration was performed, and the molecular ends were calculated from the elution position of the sample using a standard curve obtained from the standard molecule Kira]- (manufactured by Pharmacia, Sweden). The above protein amount is Bradford ([
3radford) method (Anal, Bioch
Em., Vol. 72, p. 248, 1976), and the same applies hereinafter.

The results obtained are as shown in FIG. (1) in the diagram
is raw serum albumin (molecule fi67000), (2)
is ovalbumin (molecule ff143000), (3)
is chymotrypsinogen A (molecular weight 25,000), (
4) Shows acupuncture bonuclease (molecular weight 13,700),
(S) is the substance of the present invention. From FIG. 1, the present invention W(s
) is found to elute before chymotrypsinogen A (3), and its molecular weight is approximately 26,500.

8. Molecular weight by Gluton filtration method using TSK Gel G3000SW A TSK Glu G 3000 SW (manufactured by Toyo Soda) column was connected to a Pharmacia FPLC system (manufactured by Pharmacia, Sweden), and a 0.1% 5O810,1M'
) > lst-1) Add 100 μΩ (protein amount) of the substance sample of the present invention using a buffer solution of rum (pH 7.0), perform gel filtration, and prepare a standard molecular flea kit for high performance liquid chroma l-. (manufactured by Oriental Yeast Co., Ltd.), and the molecular weight of the substance sample of the present invention was calculated from these elution patterns. Figure 2 shows the elution pattern obtained by high-performance liquid chromatography, and Figure 3 shows the molecular weight distribution determined from m1 at the time of elution in the chromatography. (6) is enolase (molecular weight 67,000), and (7) is adenylate kinase (molecular weight 67,000).
32000), and 8) are Ti1 to Chromium C (molecular weight 1
2300>, (S> represents the substance of the present invention, respectively. From each figure, the substance (S) of the present invention is adenylate kinase (7)
The molecular weight is calculated to be about 31,200.

C, Electric body W using SDS/polyacrylamide gel
Sodium phosphate/S'DS (pH 7, 2>
Then, 5 μg (protein amount) of the substance sample of the present invention was applied to a 5DS-polyacrylamide gel, electrophoresis was performed at 40 mA for 7 hours, and the standard molecular weight was exceeded [-<Oriental Yeast Co., Ltd.!]. ! The electrophoresis pattern (Fig. 4) was recorded using a microelectrophoresis system, a double molecular weight curve (Fig. 5) was created, and the molecular weight of the sample was calculated from the internuclear distance. In Figures 4 and 5, (a) is di1-chrome C heptamer (molecular weight 86
100), (b) is cytochrome C6 monomer (molecular weight 73,800), (c) is cytochrome C pentamer (molecular weight is 61,500), (d) is cytochrome C1ft.
body (molecular weight 49200), (e') is cytochrome C3ff! (f) shows cytochrome C2M form (molecular weight 24600), and <(1) shows cytochrome C1ft (molecular weight 12300). Further, (S> is the substance of the present invention. From FIG. 5, the molecular weight of the substance (S) of the present invention is calculated to be about 30,000.

(2) Isoelectric focusing device (manufactured by Bio-Rad, USA) and Amphor 1ne polyacrylamide plate (p-83.5 to 9.5) (manufactured by LKB, USA) The isoelectric point of the substance of the present invention was measured using a standard isoelectric point measurement marker kit (manufactured by Pharmacia, Sweden). That is, 5 μg (protein amount) of the substance of the present invention was absorbed onto a piece of filter paper, placed on a gel, and
The electrophoresis was started at a constant W power for about 2 hours, and the electrophoresis was terminated when the current became constant. The gel was cut out at intervals of 1 ml, eluted with a buffer solution, and subjected to measurement of activity against L-[cells]. The isoelectric point was calculated based on the isoelectric point marker. As a result, the isoelectric point of the substance of the present invention was calculated to be DH4.1±0.3.

(3) Ultraviolet absorption constant double beam spectrophotometer UV-300 (Shimadzu)
using 0.02M t-Lis-HCl 10.1~I
The ultraviolet absorption of a sample of the substance of the present invention dissolved in Na CQ (pH 7,8) was measured. The results are shown in Figure 6. The maximum value is 276 μm and the minimum value is 2501 m from the internuclear distance.
Met.

(4) Solubility, color, and properties A sample of the substance of the present invention was adjusted to a concentration of 3 mg protein/'1llQ by 0.00%.
The solution dissolved in 02M-Tris-HCl buffer (pH 7,8) is clear and colorless.

When acetone or ethanol is added to the solution in an amount of 60 V/V% or more, precipitation occurs.

In addition, the 3 mg protein ffi/m Q aqueous solution of the substance of the present invention is
Shows weak acidity.

(5) Color reaction Biuret reaction, Folin-Lowry reaction method, and ninhydrin reaction after hydrochloric acid hydrolysis The color reaction of peptide bonds and amino acids are all positive.

Moreover, the low molecular weight protein 9 of the present invention is characterized in that it has the following physiological activities.

(a) Cytotoxic effect on L-cells The method of Casewell et al.
) method (Mo1., I mm, vol. 17, 613
The L-cell-killing effect of the substance of the present invention was evaluated according to the method described in J. J. P., 1980). That is, 250 units of cells/
2 x 10 cells/m in Eagles Minimal Essential Medium (MEM) medium containing re Q of penicillin and 125 μJ/mQ of streptomycin.
This L-cell suspension was suspended at a concentration of 0.1
111Q and sample name Q of the substance of the present invention diluted to an appropriate concentration,
i+nQ was placed in each well of a 96-well microplate (Costar, USA) and cultured for 48 hours at 37°C in air containing 5% carbon dioxide. The cultured cells are neutralized and stained with neutral red, and the number of viable cells is determined colorimetrically using Titertic Multiscan (Flow Laboratories, USA).

The power to kill 50% of active leprosy cells is defined as 1 unit, and this is multiplied by the dilution factor of the sample.

As a result, the cytotoxicity of the substance of the present invention to L-cells was
It was more than 9.99×107 units/n+o protein.

(b) Female A-monkey]-? (Meth A-sa
BALB/C
8 implanted into mouse abdominal skin, 78 days later the tumor size was 7 in diameter.
From the tail vein of a mouse that had reached ~ Bmm, the cytotoxic effect measurement method (a > 2.5 x 10' on one cell) was performed.
0.21 RQ of a sample of the substance of the present invention diluted to ~2.5×10 5 units/mQ was injected, and 48 hours later, the antitumor effect was determined according to the following criteria according to the method of Carlwell et al.

<-): No change +): Faint hemorrhagic necrosis -(+): Moderate hemorrhagic necrosis (necrosis extending from the center of the graft layer surface to more than 50%) (44)): Marked hemorrhagic necrosis ( (The center of the transplanted cancer was severely necrotic, with only a small amount of surrounding cancer tissue remaining.) The results obtained are shown in Table 1 below.

Table 1 Dosage Evaluation
Price now Shoto Etchibun λ1 - 2 - Earth 1 Earth 1'' - 1 (Physiological saline) 6000 48.5 1 3 2 0 - 97.0 0 2 3 1 242.5 0 1 2 2 Engineering Hong 85. O(He023-) Next, a method for obtaining the novel low molecular weight protein of the present invention will be described.

The substance of the present invention can be produced into the body of a mammal by administering a substance having an immunostimulatory effect to the mammal and then administering an endotoxin derived from Durham-negative bacteria or a lectin derived from a plant, basically according to a known method. produced. Yo°
In detail, for example, the method of Rikisu Iru et al.
c, Nat, A cad, sci.

USA, Vol. 72, p. 3666, 1975], a substance having an immunostimulatory effect is first administered to a mammal. Examples of mammals include mice, rats, guinea pigs, rabbits, etc., but are not particularly limited thereto. Various known substances can be used as the substance that exerts an immunostimulatory effect. Specific examples include Bacillus carmetui Guerin (BCG), Corynebacterium parvum (Corynebacterium parvum),
un+ parvam), Propionobacterium
acnes (propioni-bacterium)
acnes), Mycobacterium butyricum (M
ycobacterium butyricum )
, :] Corynebacterium granulosum (Cory
nebacter; umgranulosum), Streptococcus virogines
Cus pyrooenes), Plasmodium, etc., as well as Zymosan, Nocardia asteroldes, and Listeria monocytogenes.
monocytogenes), glucan (glu
can), cell membrane skeleton
on), dextran sulfur m (dextran sulphate)
lfate), muramyl dipeptide (RIura
Iyl-dipeptide), Krestin (manufactured by Kenbetsu Kogyo Co., Ltd.), and the like. These immunostimulatory substances are generally administered by intravenous or intraperitoneal injection. The dosage is determined appropriately, but is usually 1 to 10
It is preferable to cover the range of about 00 tI1g/kg.

In the water washing, endotoxin derived from Durham-negative bacteria or lectin derived from plants is then administered to the test animal 7 to 14 days after administration of the substance having an immunostimulatory effect. As the endotoxin and lectin, any of various known endotoxins can be used.

Typical examples include lipopolysacrid derived from Escherichia coli, Salmonella typhi, etc., jack bean lectin (concanavalin A, ConΔ), soybean lectin (SBAJ), red bean lectin (P)-4
A) etc. can be exemplified.

These administrations are usually preferably administered by intravenous injection. The dosage is not particularly limited, but is usually about 10μΩ to 10mM.
It is generally selected from the kg range. Approximately 1.5 to 3 hours after administration of the Endo-1 hexin or lectin, the low molecular weight protein of the present invention having the desired anticancer effect is produced in the serum or plasma of the test animal.

Collection, separation and purification of the substance of the present invention is carried out according to conventional methods. In other words, the test animals are subjected to facial measurements according to conventional methods.
Taking advantage of the properties of the substance to be dissolved in the obtained serum or plasma, it can be carried out according to physicochemical or biochemical means, such as salting out, chromatography, electrophoresis, extraction,
This can be carried out by centrifugation, dialysis, etc. alone or in an appropriate combination. More specifically, the above serum or plasma (
These are hereinafter referred to as crude solutions) to the next step.

(1) Ammonium sulfate salting out (2) Gel filtration (3) Hydroxyapatite chromatography (4) Pharmacia FPLC Mono Q column chromatography (5) Ammonium sulfate salting out dissolution chromatography (6) Gel filtration Below are details of these steps. Explain.

Purification Step 1 Ammonium sulfate (hereinafter referred to as "ammonium sulfate") is added to the crude solution to prepare a 50-80% saturated solution. This solution is left in a cold room (4°C) for 2 hours to overnight to sufficiently precipitate the physiologically active low molecular weight protein.

Then, using a refrigerated centrifuge (manufactured by Hitachi),
Centrifugation is performed at 000 rpm for 10 to 30 minutes to collect the bioactive precipitate. Activity recovery rate at this stage (
(according to the cytotoxic activity assay method for single cells described above) is 8
It is 0 to 100%, and the degree of purification is 10 to 20 times higher.

Purification step 2 The precipitate obtained in step 1 was mixed with 0.02M) Lith-Salt F
II (pH 7, 8) 10. IM NaC (!) buffer. 2-8 M urea is added to this suspension, and the insoluble material is subjected to refrigerated centrifugation at 10,000 rpm for 10-30 minutes to obtain a clear biologically active supernatant. Obtain synovial fluid;
This is subjected to gel filtration. As carriers for gel filtration, Ultrogel ACA44゜54 (manufactured by LKB, USA),
Or Biogel A1.5n+ (manufactured by Bio-Rad,
America) etc. are used.

The gel filtration fraction was measured for cytotoxic activity against one cell as described above, and the active fraction was collected and filtered using an ultrafiltration membrane YM.
Ultra tr perconcentration is performed using an ultrafiltration membrane @TcF-10 (manufactured by Amicon, USA) equipped with IO (manufactured by Amicon, USA).

However, the concentration may be carried out by 50 to 80% saturated ammonium sulfate precipitation. The activity recovery rate by this method is 80-100%, and the degree of purification is 20-50 times higher.

Purification Step 3 Using a dialysis tube (manufactured by Gyudon Chemical Co., Ltd.), the concentrated solution of the physiologically active fraction obtained in Step 2 was mixed with 50 to 100 ml of 0.02M phosphoric acid solution (pH 6,8). against 4
Dialyze overnight at <RTIgt;oC,</RTI> changing the external solution several times. The dialysate was centrifuged for 20 to 30 minutes at F100OO rotations/min in a cooled centrifuge F1 machine (4°C'), and the clear F supernatant was collected.Then, this supernatant liquid was mixed with hydroxyapatite (manufactured by Nippon Chemical Co., Ltd.). Adsorb, 0.02M-0.5M
The bioactive fraction is eluted with IJ acid buffer (pH 6,8) according to a continuous concentration gradient method or by increasing the concentration stepwise. The obtained physiologically active fraction is concentrated by ultrafiltration concentration or ammonium sulfate salting out. The recovery rate of bioactive fraction by this method is 40-80%, and the degree of purification is about 4-10%.
It's double.

Purification step 4 Place the concentrated solution of the physiologically active fraction obtained in purification step 3 into an induction tube, and add it to dilute phosphoric acid at a concentration of 50 to 100 times or Tris buffer (pH 7.0 to 8.0). Te, 4
Dialyze overnight at °C. This dialysate was processed using Pharmacia FPLC.
The biologically active substance is adsorbed onto a MonoQ column (manufactured by Pharmacia, Sweden), and the buffer concentration or Na098°C is continuously increased to elute the physiologically active substance. The activity recovery rate in this step is 60-90%, and the degree of purification is about 3-6 times higher.

Purification Step 5 The active fraction in Purification Step 4 is subjected to ultrafiltration concentration and subjected to ammonium sulfate salting out dissolution column chromatography.

1--Yopearl 5W50.55 or 60 (all manufactured by Toyo Soda) was packed in a column, and Pharmacia FP
Using an LG system (manufactured by Pharmacia, Sweden), the physiologically active fraction is salted out with ammonium sulfate in a column, and then the ammonium sulfate concentration is continuously lowered to dissolve and fractionate the active fraction. The activity recovery rate at this stage is 15-40%, and the degree of purification is approximately 3-5%.
It's double.

Purification Step 6 The active fraction obtained in Purification Step 5 is concentrated and applied to a column (16 x 1000 mm) packed with Bio-Gel A 1.5m or Ultrogel ACA 44 or 54 for gel filtration. Alternatively, gel filtration is performed using a Toyo Soda high performance liquid chromatography column G2000SW or G3000SW (manufactured by Toyo Soda Co., Ltd.). The activity recovery rate in this step is 7 to 20%, and the degree of purification is approximately 3 to 6 times higher.

The activity recovery rate through purification steps 1 to 6 is 3 to 15%, and the degree of purification is approximately 2×10 5 to 1.0×10 6 times.

The results of measuring the characteristics of the physiologically active low-molecular protein thus obtained are as described above.

In this way, the low molecular weight protein of the present invention is obtained. As mentioned above, the obtained substance of the present invention has a direct cytotoxic effect on L-cells in vitro and an antitumor effect in vivo. It also exhibits cytotoxic or cell-killing effects on melanoma cells, and has low toxicity.

Pharmacological test example ■ Cytotoxic or cytocidal effect (a) Human Burkitt's lymphoma-derived strain Raji (J.

Nat. Cancer Inst., vol. 37, 5.
p. 47, 1966], a strain derived from human gastric cancer (marked cell carcinoma) KatO-1tl [Jpn, J, EXt),
Med, vol. 48, p. 61, 1978] and the human gonophagopharyngeal carcinoma-derived cell line KB (Chancer Res, vol. 18, p. 1017, 1958), the cell killing effect of the substance of the present invention was evaluated. That is, the to1-Burkitt's lymphoma-derived cell line and the human gastric cancer-derived cell line were
The cells were adjusted to 2×10581 cells/+n Q with RPM11640 medium containing 0 units/ffi Q of penicillin, 125 μg/mQ of streptomycin, and 10% inactivated tallow serum. In addition, human throat cancer-derived cell lines were treated with 250 units/mQ of penicillin and 125 μLJ/mQ of s[-
The cells were adjusted to 2×10 5 cells/m Q using Eagles Minimal Essential Medium containing levtomycin and 10% inactivated bovine serum.

The above-mentioned cell preparation solutions Q and imQ and 0.1111 (!) of the present invention substance diluted to 3 degrees were placed in each well of a 96-well microplate, and then placed in air containing 5% carbon dioxide.
The cells were cultured at 37°C for 48 hours.

After 48 hours of culture, the cells were stained with trypan blue, and the number of viable cells was calculated under a microscope using a Birkerturk counting board (manufactured by Elsen Optical Works, Japan). As a result, the concentration of the substance of the present invention that inhibits the proliferation of various cells by 50% is 7% for human Burkitt's lymphoma-derived cells.
．． 0 no protein/mQ or more, 23.6 no protein amount/mQ or more for human gastric cancer-derived cells, 9.6 ng protein/III 9 or more for crushed pharyngeal cancer-derived cells .

(b) Cytotoxic effect on melanoma cells The method of Helson et al. (Nature).

258, p. 731, 1975], melanoma A-375 (J, Natl.) of the substance of the present invention.

Qancer In5t, vol. 51, p. 1417, 1
[973] evaluated the cytotoxic effect on cells. That is, a suspension of melanoma A-375 cells (5 x 10' cells/mQ) was prepared using Eagle's medium containing glutamine, non-essential amino acids, penicillin, streptomycin, and 10% unworked tallow serum.

1 mQ of each of the cell p, the measured liquid, and 1 mQ of a sample solution prepared by appropriately diluting the substance of the present invention were placed in a 3.5 cm diameter petri dish, and cultured at 37° C. in air containing 5% charcoal gas.

On the 3rd day of J8 cultivation, the cells were stained with trypan blue in the same manner as in (a) above, and the number of viable cells was counted under a microscope using a Gorgalturk abacus. As a result, the amount of protein required for the substance of the present invention to suppress the proliferation of Methulama A-375 cells by 50% was 12 no protein/mQ or more.

Drug Implant Test Example (2) Acute Toxicity The substance of the present invention was intravenously administered to 10 male and 8-week-old ddY mice at a rate of 3.2 mg protein fi/kg to examine acute toxicity.

As a result, no deaths were observed, and the LDo was 3.211 (+
/ kg or more. In addition, 1. During the observation period, no obvious symptoms of poisoning that were considered to be caused by the substance of the present invention were observed.

As described above, the substance of the present invention exhibits cytotoxic or cell-killing effects on various cells, and has low toxicity, making it useful as an antitumor agent.

When the substance of the present invention is used as an antitumor agent, it is prepared into various forms containing an effective amount thereof and administered by various administration routes depending on the form. Examples of the formulation include liquid solutions, suspensions, and emulsions, which are generally administered intravenously, subcutaneously, or intramuscularly. They can also be presented as dry products which can be made into liquid form by addition of suitable carriers before use. The dose of the antitumor agent varies depending on the degree of disease, age, sex, etc. of the patient, but usually the amount of protein is approximately 1.61 to 16.1μ (
], / kgZ day is preferably administered in one to several doses.

Examples will be shown below to describe the present invention more specifically.
The present invention is not limited to these examples.

Example 1 New Cilantropo wide or Japanese white n rabbit (body =
2. o~3.0ka), formalin-killed Corynebacterium parvum (Corynebacterium ρa)
ruvam (manufactured by Wellcome, UK) 70 mg was injected through the ear vein. Note #J After 9 days, 100 μg of Bobolisatucalide (E. coli 055:85, manufactured by Difco, USA) was injected into the ear vein, and 2 hours later, the pig was exposed completely naked. Transfer the blood sampled surface fluid at 5000 rR/min 2
Centrifugation was performed for 0 minutes, and the serum was removed. 10 by this operation
7530 mQ of serum with a titer of 15900 units/'mQ was obtained from 0 rabbits.

As shown in Table 2, the production of this physiologically active substance (9) in New Cilantropoide and Japanese white rabbits was higher in Japanese white rabbits.

Table 2 - Activity by assay of cytotoxic activity against cells Japanese white type 1, - Support ■ Otsuyu Q6 --- 472 fJ in 1000 m9 of serum (7) Sulfur safety ff1
After decomposition of FJO1, the mixture was left at 4°C overnight to precipitate the active substance.

Centrifuge the precipitate at 10,000 rpm for 30 minutes (4°C)
The precipitate was collected and diluted with about 100 mQ(7)0.02M tris-13 acid MF[10,'1M NaCQ(ρ1-17
．． 8. ). The activity recovery rate at this stage is 98
%, the refinement was 18 times higher.

Then, the precipitation suspension 100111 (!36.0
4q of urea was added and dissolved. Rotate this 10,000 times/
Refrigerated centrifugation (4° C.) was performed for 30 minutes to remove insoluble materials. Take 5㎜Q of this supernatant and filter it using Ultrogel Ac△54 equilibrated with 0.02M Tris-f3!10.5M N8091m solution (column size 50 x 1000 mm). The activity was investigated using a measuring method and the activity classification was collected. The activity recovery rate by this method was 92%, and the degree of purification was 33 times higher. The activity recovery rate through all steps was 90.2%, and the degree of purification was about 600 times higher.

Next, an Amicon ultrafiltration membrane YM10 was attached (, - The active fraction was concentrated using an ultrafiltration concentrator TCP-10. This concentrated solution was poured into a dialysis tube, and 50 times the amount of
．． 02M phosphate buffer (pH 6,8) at 4°C
Dialysis was performed. After exchanging the external solution 3 times for every 3 cycles, the cells were incubated in the same buffer at 4° C. overnight. This dialysis activity fraction was preliminarily mixed with 0.02M phosphate buffer ([I H6,
It was applied to a hydroxyapatite gel column (size 26 x 400 mm) equilibrated at 8>. Flow rate 5.0Il
At the time of lQZ, after washing thoroughly with the same buffer under the conditions of 3mQ1 fraction, 0.02M phosphate buffer (pH 6,8>5
00m Q and 0.5M Lin Fl! i Slow rain liquid (p
500 m 9 of H6,8) was used to elute the adsorbed material using a continuously rising concentration gradient. In this method, no activity was observed in the non-adsorbed fraction, all □ activity was adsorbed, and the adsorbed active fraction was found in phosphate buffer with concentrations ranging from 0.15M to 0.
It eluted between 3M.

Collect active categories, Amicon Limited! Filtration concentrator TCP-
Ultrafiltration and concentration were carried out at 4°C using 10. The activity recovery rate by this method was 48%, and the degree of purification was 4.5 times higher. The activity recovery rate throughout the entire process was 43.3%, and the degree of purification was 2.7×10 3 times.

Next, the concentrated solution of the active fraction obtained by the above method was put into a dialysis tube, and 50 times the volume of 0.02M Tris-salt M was added.
(p 1-17.8) Dialysis was performed overnight at 4°C against 10.1M Na CQ buffer. The dialysis solution was applied to a Pharmacia Mono Q column made into M% with the same buffer solution, and 1 m
The active substance was adsorbed under the conditions of Q/min and 1111Q/fraction. Next, after thorough washing with the same buffer solution, the adsorbed active fraction was eluted by continuously increasing NaC by 115 degrees using 0.02M 1-Lis-salt M (pH 7, 8)/1.0M Na CQ.

The active fraction was concentrated using an ultrafiltration device equipped with an Amicon ultrafiltration membrane YM10. The activity recovery rate by this method was 80%, and the degree of purification was increased by 4 times. The activity recovery rate throughout the entire process was 34.6%, and the purity was 1.1X1.
．． It was O' times.

Next, the concentrated solution was diluted with 0.1M t-lis-hydrochloric acid (pH
7,8) /Toyo Pearl 5W60 (column size 10 x 500 mm) equilibrated with 70% saturated ammonium sulfate at a flow rate of 1
mQ/min, 2111 <! / fraction and salted out in the column. Then O, 1M Tris-HCl (pH 7, 8)
Elution was carried out using a mild solution ii'i of 20% by decreasing the ammonium sulfate concentration continuously.

The active fraction was collected and concentrated using an ultrafiltration concentrator equipped with an Amicon ultrafiltration membrane YMIO. The activity recovery rate by this method was 19%, and the degree of purification was 3.7 times higher.

The activity recovery rate throughout the entire process was 6.6%, and the purity was 3.
It was 96×10' times.

Next, the above concentrate was diluted with 0.1M sodium phosphate (p
H7,0>10.2M Na CQ10.1%SDS
Toyo Soda-G3000SW1 equilibrated with buffer solution
7A (7,5X600mm)! using, flow rate 1+nQ/
Gel filtration was performed at 1111Q/fraction. With this method, the activity recovery rate was 9.0%, and the degree of purification was increased by 6.5 times. The activity recovery rate throughout the entire process was 0.6%,
The degree of purification was 2.57x105 times.

The specific activity of the low molecular weight protein having this physiological activity is 1.
02X108 units/ma? J white matter.

Example 2 - New Zealand White female rabbit (weight 2.0-3.
．． 0ka), 150mA of formalin-killed Bacterium butyricum (manufactured by Difco, USA) was injected through the ear vein. Nine days after the injection, 100 μg of polysaccharide (E. coli 055: B5, manufactured by Difco, USA) was injected into the ear vein, and 2 hours later, the whole animal was harvested from the heart. The collected blood was centrifuged at 5000 rpm for 20 minutes, and the serum was separated. By this procedure, serum 741 with a titer of 5160 units/mQ was obtained from 100 rabbits.
0ffIQ was obtained.

After adding 472 g of ammonium sulfate to 1000 m+1 of the serum, it was well dissolved and left overnight at 4°C. 10,000 ammonium sulfate salting out solution
Rotations per minute, 30 minutes of refrigerated centrifugation! ! (4°C), collect the precipitate, and add about 100 mQ of 0.02Ml
Salt Fi10.1M Na CQ loose part (pH7,8)
suspended in. The activity recovery rate at this stage was 97%, and the degree of purification was 20 times higher.

Then, 36.0 for the precipitated suspension 100111!2
4! Add urea at a ratio of It and dissolve it, then rotate at 10,000 rpm
Refrigerated centrifugation (4°C) was performed for 30 minutes to remove insoluble matter. Take 50mQ of this supernatant and add 0.02Mt
-Squirrel-Fj, M (p)-17,8) 10.5M
Ultrogel AcA3 equilibrated with Na CQ buffer
4 (using column size 50×100100O and flow rate 40
111G! The active fraction was collected by gel filtration at 7 mQ for 1 minute. The activity recovery rate by this method was 92%, and the degree of purification was 28 times higher. Activity recovery rate throughout all stages is 8
At 9.2%, the degree of purification was approximately 560 times higher.

The active fraction was then concentrated using an ultrafiltration concentrator TCP-10 equipped with an Amicon ultrafiltration membrane YMIO. Pour this concentrated solution into a dialysis tube and add 50@ volume of 0.02M.
Dialysis was performed at 4°C against phosphate M equilibration (DI-16,8). After exchanging the external solution three times every 3 hours, the mixture was dialyzed in the same buffer for one night. This dialysis active fraction was transferred to a hydroxyapatite gel column (size 2) equilibrated with 0.02M phosphate buffer (pH 6,8).
6x400n+m>, flow rate 5.0m91, 3111Q
/2 fraction was adsorbed. The adsorbed activity category is 0.02
M Phosphoric acid loose! Li solution (pH 6,8) and 0.5M
Elution was performed using a phosphate buffer (pH 6,8) using a continuously increasing concentration gradient. The activity recovery rate in this method was 68%, and the degree of purification was 10 times higher.

The activity recovery rate throughout the entire process was 60.7%, and the purity level was 5.
, 6×103 times.

Next, the active fraction was concentrated using an ultrafilter, put into a dialysis tube, and mixed with 50 times Mo0.02M Tris-HCl (p
H7,8)/'O1IMNa(:, for Q buffer,
Dialysis was performed overnight at 4°C. This dialyzed active fraction is
1 m on a Pharmacia Mono Q column buffered with the same buffer.
Adsorption was carried out under the conditions of 91 minutes and 1 m Q1 fraction. After washing thoroughly with the same buffer, the adsorbed active fraction was eluted by continuously increasing NaGQt by 4 degrees using 0.02 M t-Lis-HCl (DH7,8>/1.OM Na CQ buffer). The active fraction was concentrated using an Amicon ultrafiltration concentrator.The activity recovery by this method was 62%, and the purity was increased by 4 times.

The activity recovery rate throughout the entire process was 37.6%, and the degree of purification was 2.24×10' times.

Next, the above concentrate was diluted with 0.1. M tris-mm(r
+ t-17,8) / Toyo Pearl 5W60 equilibrated with 70% saturated sulfuric acid solution (column size 1° x 50
0ml1ll) at a flow rate of 1m for 97 minutes in 2mQ1 fractions and boiled down in the column. Then, using a slow Wii solution of O, 1M Tris-hydrochloric acid (tlHγ, 8), and continuously decreasing the ammonium sulfate concentration, soluble fractionation was performed. The active fraction was collected and concentrated using an ultrafiltration concentrator. The activity recovery rate at this stage was 2096, and the degree of purification was 4.9 times higher. The activity recovery rate throughout the entire process was 7.5%, and the degree of purification was 1.10×10 5 times.

Next, the above concentrate was diluted with 0.1M sodium phosphate (p
H7,0>10.2M Na CQ10.1%SO8
Toyo Soda-03000S W equilibrated with buffer solution
Using a column (7.5 x 600 m'i), flow rate 1 m 9
Gel filtration was performed for 7 minutes at 1 mQ/fraction. The activity recovery rate at this stage was 11%, and the degree of purification was 6.7 times higher. The activity recovery rate throughout the entire process was 0.8%, and the degree of purification was 7.35×10 5 times.

The specific activity of the low-molecular protein that determines this physiological activity is 1.
02 x 108 units/11g protein.

Example 3 New Zealand Why] - female rabbit (weight 2.0-3
．． 0kg) and Krestin (manufactured by Kureha Chemical Industry Co., Ltd.) 1
00ma was injected through the ear vein. 1100 9 days after injection
U lipopolysaccharide (E. coli 055:B5, manufactured by Difco, USA) was injected through the ear vein, and 2 hours later, whole blood was collected from the heart. The collected white fluid was centrifuged at 5000 rpm for 20 minutes to separate serum. With this operation,
Serum 7000 IllQ with a titer of 6200 units/mQ was obtained from 100 rabbits.

472 g of ammonium sulfate was added to and dissolved in the serum 1000IIIQ, and the mixture was left at 4° C. overnight to precipitate the active substance.

The precipitate was centrifuged at 10,000 rpm for 30 minutes at 4°C, the precipitate was collected again, and 0.02M l of approximately 100 mQ was added.
-Lis-hydrochloric acid buffer 10. IMNa CQ (+1 H
7, 8). The activity recovery rate at this stage is 98
%, the degree of purification was 18 times higher.

Then, the precipitate suspension 1001! 36.0 for l++
Urea was added and dissolved at a ratio of 4 ().
Centrifugation was performed at 4° C. for 30 minutes at 0 rpm to remove insoluble matter. Take this supernatant liquid 50111Q and add 0.02M
t-Lis-salt M10.5MNa CQ (1) H7,
8) Gel j filtration using Ultrogel AcA34 equilibrated with buffer (column size 50 x 1000 mm) L, L
-The activity was investigated by the activity measurement method on cells, and the activity fraction was collected.

The activity recovery rate by this method was 95%, and the degree of purification was 30 times higher. The activity recovery rate through all steps was 93.1%, and the degree of purification was about 540 times higher.

The active fraction was then concentrated using an ultrafiltration concentrator TCP-10 equipped with an Amicon ultrafiltration membrane YM10. Pour this concentrated solution into a dialysis tube and add 50 times the volume to 0.02M.
Dialysis was performed against phosphate buffer 1 (pH 6,8). After exchanging the external solution three times every 3 hours,
℃, - night. This dialysis activity classification is set in advance to 0.
Hydroxyapatite gel column (size 26 x 40) equilibrated with 02M phosphate buffer (pH 6,8)
0111111). Flow rate 5.0mG! / hour, 3
After thorough washing with the same buffer under the conditions of mQ7 fractionation, 0.
02M') M buffer (+1 H6,8/500
mQ and 0.5M phosphorus M buffer (pi-16,8)5
00WIQ was used to elute the adsorbed material by continuously increasing the concentration gradient. In this method, no activity was observed in the non-adsorbed section, all the activity was adsorbed, and the adsorbed active section had a phosphoric acid concentration of 0.15M to 0.3.
It was eluted at the gate of M.

Collect the active fraction and use Amicon ultrafiltration concentrator CP-1.
Ultrafiltration and concentration were performed at 4°C using 0. The activity recovery rate in this method was 56%, and the degree of purification was 10 times higher. The activity recovery rate throughout the entire process was 52.1%, and the degree of purification was 5.4×10 3 times.

Next, the concentrated solution of the active fraction was put into a dialysis tube, and 50 times the amount of 0.02 M l-lys-FA acid (pH
7,8) For 10.1M Na CQ buffer, 4
Dialysis was performed overnight at °C. The dialysis solution was applied to a Pharmacia Nanano Q column buffered with the same buffer solution, and
1! The active fraction was adsorbed under IIQ/fraction conditions. After washing thoroughly with the same buffer, 0.02M Tris-HCl/
1. OM NaCG! The adsorbed active fraction was eluted by successively increasing NacQ concentration using buffer (p, 87.8).

The active fraction was concentrated using an ultrafiltration device equipped with an Amicon ultrafiltration membrane YM10. The activity recovery rate by this method was 80%, and the degree of purification was increased by 5.5 times. Activity recovery rate throughout the entire process is 41.7%, purity is 2.97X1
It was 0' times.

Next, the above concentrated solution was diluted with 0.1M t-lis-hydrochloric acid/70%
Toyo Pearl 5W60 (column size 1° x 5001Wm) equilibrated with saturated ammonium sulfate (pt-17,8) buffer
), the flow rate is 1 mG! /min in 2 mQ1 fractions and salted out in the column. Then 0.1M Tris-HCl (pH 7)
, 8), and elution was carried out by continuously decreasing the ammonium sulfate concentration. The active fraction was collected and concentrated using an ultrafiltration concentrator equipped with an Amicon ultrafiltration membrane YM10. The activity recovery rate at this stage was 19%, and the degree of purification was 3.7 times higher. The active yield throughout the entire process is 7.
9%, and the degree of purification was 1.10×10 5 times.

Then, the concentrated solution was mixed with 0.1M sodium phosphate 10.2M Na (110, 1% SDS buffer (p
Toyo Soda G3000 equilibrated with H7,0)
Gel I filtration was performed using SW (column size 7.5 x 600 mm) at a flow rate of 1 mG/min and 1111 (!/fraction).The activity recovery rate at this stage was 10%, and the degree of purification was 5.9 times. The activity recovery rate throughout the entire process was 0.
．． 8%, and the degree of M production was 6.48×105 times.

The specific activity of low molecular weight white matter, which determines this physiological activity, is 1.
0OX108 units y'mg protein.

Example 4 New Zealand White female rabbit (weight 2.0-3
．． 0 kg) was injected with 100 mg of formalin-killed Corynebacterium parvum (manufactured by Wellcome, UK) through the ear vein. Nine days after the injection, 1 On+o concanavalin A (manufactured by Wako Pure Chemical Industries, Ltd.) was injected through the ear vein, and 2 hours later, whole blood was collected from the heart. Rotate the collected blood 5000 times/
Serum was separated by centrifugation at 4° C. for 20 minutes. By this operation, 3780 units/m from 100 rabbits
7640 mQ of serum with a titer of Q was obtained.

After adding 472 g of ammonium sulfate to 10,100 O of the serum, it was well dissolved and left overnight at 4°C. The ammonium sulfate salt precipitation solution was subjected to refrigerated centrifugation at 10,000 rpm (4°C) for 30 minutes, the precipitate a was collected, and the precipitate was collected and mixed with about 100 m Q o, 02 M Tris-salt m10. IM
7.8).

The activity recovery rate at this stage was 99%, and the degree of purification was 20 times higher.

Then, 36.04 (+
(7) After adding urea and dissolving it, rotate at 1oooo rotation/min for 30
The mixture was cooled and centrifuged (4°C) for a minute to remove insoluble materials. This supernatant 50n+G! Take 0.02M 1-'J sous salt 1
Gel filtration using Ultrogel ACA54 equilibrated with 1 (pH 7,8>10.5M NaCQ buffer) (column size 50 x 11000 in, flow rate 40 mQ/hour, 7 m'
Q/fraction) and the elution fractions were collected. The activity recovery rate by this method was 95%, and the degree of purification was 41 times higher. The activity recovery rate through all steps was 94.1%, and the degree of purification was about 820 times higher.

The active fraction was then concentrated using an ultrafiltration concentrator TCr-10 equipped with an Amicon ultrafiltration membrane YMIO. Pour this concentrated solution into a dialysis tube and add 501M (7) 0.0
2M') m buffer (pH 16,8) at 4°C.
Dialysis was performed. After exchanging the external fluid three times every three verses,
In the same buffer, 4? C1 - Fatigue device. This dialysis activity fraction was prepared in advance in 0.02M phosphate buffer (pH 6,
8) A flow rate of 5. OmQ/hour, 3
Adsorption was carried out in mQ/fraction. The adsorbed activity category is 0.0
The adsorbed substances were eluted using a 2M phosphate M interim solution (pH 6,8) and a 0.5M phosphate buffer (pH 6,8) by continuously increasing the concentration. The activity recovery rate by this method was 65%, and the degree of purification was 9.8 times higher. The activity recovery rate throughout the entire process was 61.1%, and the degree of purification was 8.0×10 3 times.

Next, the active fraction was concentrated using an ultrafilter, put into a dialysis tube, and added with 50 times the amount of 0.02M Tris-HCl (D
7.8) 10. Dialysis was performed overnight at 4° C. using IM Na CQM interim solution 4. This dialyzed active fraction was transferred to a Pharmacia Nanano Q column equilibrated with the same buffer for 11 hours.
Adsorption was carried out under the conditions of 11 Q/min and 1 m Q1 fraction. After washing thoroughly with the same buffer, the adsorbed active fraction was eluted by continuously increasing the NaCQ concentration using 0.02M 1-Lis-HCl (pH 7, 8)/1.0M NaCQ buffer. The active fraction was concentrated using an Amicon ultrafiltration concentrator. The activity recovery rate by this method was 74%, and the degree of purification was 5.3 times higher. The activity recovery rate throughout the entire process was 45.2%, and the degree of purification was 4.2X10' times.

Next, the above concentrate was diluted with 0.1M Tris-HCl (p,
87.8>/Toyo Pearl 5W60 equilibrated with 70% saturated ammonium sulfate buffer (column size 10X10X500
was applied at a flow rate of 1 mQ/min, 2IIIQ/fraction, and salted out in the column. Then 0.1M1~lith-hydrochloric acid (1
) I-17,8> Buffer solution was used to continuously lower ammonium sulfate tl to perform soluble fractionation.

The active fraction was collected and subjected to ms using an ultrafiltration concentrator.

The activity recovery rate by this method was 18%, and the purity was 3.
．． It was 8 times more. Activity recovery rate throughout the entire process is 8.1
%, the degree of VI production was 1.62X105 times.

Next, the above concentrated solution was mixed with 0.1M sodium phosphate (
t+ H7,0) 10.2M Na CQ10.1%
Toyo Soda G30 equilibrated with 5DSXiti liquid
Glut filtration was performed using a 00SW column (7,5 x 600 + n + n) at a flow rate of 1 + 11 (1/min, 1 m (1/fraction). With this method, the activity recovery rate was 7%, and the degree of purification was 5.3 times. So 1 [.Activity recovery rate throughout the entire process is 0
．． At 6%, the accuracy of V was 8.5×10 5 times.

The specific activity of the low molecular weight protein having this physiological activity is 1.
It was 0OX108 units/rno protein.

Example 5 ddY female mice (body weight 25-30q) were injected with formalin-killed bacteria Corybata terium Barbum (manufactured by Wellcome Inc.,
Lipopolysaccharide (green me; manufactured by Difco, USA) was administered intraperitoneally 9 days after sexual intercourse.
10μ9 was injected into the tail vein, and 2 hours later, the eyeballs were removed and blood was collected from the orbital venous plexus. 5000 yen of the collected blood
Serum was separated by centrifugation at revolutions/min for 20 minutes. By this operation, from 100 mice! 56100 units/m
Serum 90.6mQ with a titer of 9 was obtained.

After adding and dissolving 47.2o ammonium sulfate to 100mQ of the serum,
The active substance was precipitated by standing overnight at °C.

Centrifuge the precipitate at 10,000 rpm for 30 minutes (4°C)
The precipitate was collected and diluted with Q(pl-17
, 8). Activity recovery rate at this stage is 98%
The degree of purification was 21 times higher.

Next, 7.2 g of urea was added to and dissolved in the precipitate suspension 20IIIQ. This is done at 10,000 revolutions/minute for 30
The mixture was cooled and centrifuged (4°C) for a minute to remove insoluble materials. Take 10+11Q of this upper synovial fluid and add 0.02M Tris-jM
Glue was filtered (column size 25 x 1000 mm) using Ultrogel ACA54 equilibrated with a 10.5 M Na CQ slow drop solution (column size 25 x 1000 mm). The activity recovery rate by this method was 90%, and the degree of purification was 32 times higher. The activity recovery rate through all steps was 88.2%, and the degree of purification was about 672 times higher.

The active fraction was then fused using an Amicon ultrafiltration concentrator T' CF-10. This concentrated solution was dialyzed against 0.02M phosphate buffer (pt+6.8) at 4°C overnight. This dialysis activity classification is 0,02M I) N WJ
Hf! (pH 6,8) (7 Equilibrated hydroxyapatite gel column (size 26Xb) After thorough washing with the same buffer under the conditions of 3mQ1 fraction during QZ, 0.02M phosphate buffer (1 )) 16.8>
500m Q and U0.5M') > Acid buffer stop (p l
-16,8) 500 mA was used to continuously raise the concentration gradient to elute the adsorbed material. The active fractions were collected and ultrafiltrated and concentrated at 4°C using an Amicon ultrafiltration concentrator TCP-10. The activity recovery rate by this method is 4
0%, and the degree of purification was 6 times higher. The activity recovery rate throughout the entire process was 35.3%, and the purity was 4.03X1.
It was 03 times.

Next, the active fraction obtained by the above method was added to 0.02 Mt!
, 1S-HCl (+1H7,8) 10.1M NaCC1 buffer solution was dialyzed overnight at 4°C. Passed through a Pharmacia Nanano Q column buffered with the same M equilibration. The substance was adsorbed under conditions of 1+119/min and 111Q/fraction. After washing thoroughly with the same buffer, 0.02
Mt-lis-salt! (p)-17,8)/1. OM
NaCQlliM was used to continuously increase the Na CQ solubility and elute the adsorbed active fraction. The active fraction was concentrated by ultrafiltration. The activity recovery rate by this method was 75%, and the degree of purification was increased by 5 times. The activity recovery rate throughout the entire process was 26.5%, and the degree of purification was 2.02 x 104 times.

Next, the above concentrate was diluted with 0.1M Tris-HCl (p
It was applied to Toyovar 5W60 (column size 10 x 500 mm) equilibrated with H7,8)/70% saturated ammonium sulfate at a flow rate im of 91 minutes in 2 mQ1 fractions, and salted out in the column. Then, elution was carried out using a buffer of 0.1 M Tris-HCl (pH 7, 8) while continuously decreasing the ammonium sulfate concentration.

The active fraction was concentrated using an ultrafiltration concentrator. The activity recovery rate by this method was 17%, and the degree of purification was 3.2 times higher. The activity recovery rate throughout the entire process was 4.5%, and the degree of purification was 6.5X10' times.

Next, the above concentrated solution was mixed with O, 1M sodium phosphate (11
H7,0) 10.2M Na C1/, 0.1%SD
Toyo Soda G3000SW equilibrated with S buffer
(column size 7.5 x 600 mm), flow rate 1 m
Glu filtration was performed with Q/min, Iln Q/fraction. The activity recovery rate with this method was 7%, and the degree of purification was increased by 5.5 times. The activity recovery rate throughout the entire process was 0.3%, and the degree of purification was 3.5.7×10 5 times.

The specific activity of the low molecular weight protein having this physiological activity is 9.
It was 99x107 units/111g1 white matter.

Example 6 Zymosan A was administered to ddYI mice (weight 25-30 g).
(manufactured by Sigma, USA) 2.0 mo was administered intraperitoneally,
Nine days after the injection, endotoxin (E. coli lipopolysaccharide >10 μQ) was injected into the tail vein, and 2 hours later, the eyeballs were removed and blood was collected from the orbital vein layer.
Serum was separated by centrifugation at 000 rpm for 20 minutes. Through this operation, 4800 units/unit were obtained from 100 mice.
96 mG1 of serum with a titer of It Q was obtained.

After adding and dissolving 47.2a of ammonium sulfate to 100mQ of the serum, 4
The active substance was precipitated by standing overnight at °C.

Centrifuge the precipitate at 10,000 rpm for 30 minutes (4°C)
Then, collect the precipitate and add about 20mQ (Do, 02M Tris-HCl buffer 10.1M NaCQ (pH 7,
8). The activity recovery rate at this stage was 97%, and the degree of purification was 17 times higher.

Next, 7.29 ml of urea was added to 20 ml Q of the 0 ml precipitate to dissolve it. This is 30 revolutions per minute.
The mixture was cooled and centrifuged (4°C) for a minute to remove insoluble materials. Take 10 mQ of this arsenal serum solution and add 0.02 M t-li),
Gel 3 filtration (column size 2.6×1000111111) was performed using Ultrogel AcA34 equilibrated with -JgMlo, 5M Na CQ, and elution fractionation was performed. The activity recovery rate by this method was 92%, and the degree of purification was 38 times higher. The activity recovery rate through all stages was 89.2%, and the degree of purification was about 646 times higher.

The active fraction was then concentrated using an Amicon ultrafiltration concentrator TCP-10. This concentrated solution was dialyzed against 0.02M phosphate buffer (1) H6', 8) at 4°C overnight. This dialysis active fraction was applied to a hydroxyapatite gel column (size 26 x 400 ml++) equilibrated with 0.02 M phosphate buffer (pH 6,8) at a flow rate of 5. Qm Q/old, 3 mQ/fraction conditions. After thoroughly washing with the same buffer, add 0.02M phosphate M buffer (p
H16,8) 500 IRQ and 0.5M phosphate buffer (pH 6,8>50C) + Q were used to elute the adsorbed material using a continuously increasing concentration gradient. The active fraction was collected and filtered using Amicon ultrafiltration concentrator TCP-10.
Ultrafiltration concentration was performed at °C. The activity recovery rate by this method was 43%, and the degree of purification was 6.8 times higher. Activity recovery rate throughout the entire process was 38.3%, purity level was 4.39
It was X1'03 times.

Next, the active fraction obtained by the above method was dialyzed overnight at 4°C against 0.02M Tris-HCl (pH 7,8) 10.1M NaCQ buffer. The dialysis solution was applied to a Pharmacia MonoQ column buffered with the same buffer, and im
The substance was adsorbed under the conditions of Q/min and 1111Q/fraction. Next, after washing thoroughly with the same buffer, 0.02M Tris-HCl (pH 7,8)/1. OM Na CQ
Viij solution was used to continuously increase the NaCQ concentration to elute the adsorbed active fraction. The active fraction was concentrated by ultrafiltration. The activity recovery rate by this method was 77%, and the degree of purification was increased by 4.4 times. The activity recovery rate throughout the entire process was 29.5%, and the degree of purification was 1.93×10′ times.

Next, the above concentrated solution was mixed with 0.1M Tris-HCl ([)
Toyo Pearl 5W60 (column size 10X500n+n+) equilibrated with H7,8)/70% saturated ammonium sulfate,
Flow rate 1 m 91 minutes, 2 m (!/ fraction) was applied and salted out in the column. Then, 0.1 M Tris-salt 1. acid (pl-
Elution was carried out using the buffer solution of 17, 8) and continuously decreasing the ammonium sulfate concentration.

The active fraction was concentrated using an ultrafiltration concentrator. The activity recovery rate by this method was 15%, and the degree of purification was 4.1 times higher. The activity recovery rate throughout the entire process was 4.4%, and the degree of purification was 7.91×10A times.

Next, the above concentrate was diluted with 0.1M sodium phosphate (p
H7,0) 10.2M Na CQ10.1%SDS
Toyo Soda G3000SW equilibrated with slow ladle solution
(column size 7.5 x 600 mm), flow rate 1 m
Gel filtration was performed at Q/min, IIIQ/fraction. The activity recovery rate with this method was 6%, and the degree of purification was increased by 4.8 times. The activity recovery rate throughout the entire process was 0.27%, and the degree of purification was 3.8x105 times.

The specific activity of the low-molecular protein that determines this physiological activity is 1.
0IXIO8 units/m (J protein.

Example 7 Female Wistar rats (body weight 200-250 g) were intraperitoneally administered with 10 mo of formalin-killed Corynebacterium bulbum (manufactured by Wellcome, UK), and 9 days after the injection, endotoxin (E. coli lipopolysaccharide) 2
50 μQ was injected into the tail vein. Two hours after endotoxin administration, blood was collected from the abdominal superior vena cava under ether anesthesia. The collected blood was centrifuged at 5000 rpm for 20 minutes to separate serum. By this procedure, serum 306 with a titer of 1500 units/IIQ was obtained from 100 rats.
III'''Q was obtained.

After adding and dissolving 47.2 (] of ammonium sulfate to 100 IIQ of the serum, the mixture was left at 4° C. overnight to precipitate the active substance.

Centrifuge the precipitate at 10,000 rpm for 30 minutes (4°C)
Collect the precipitate and add 0.02M Tris-1 of about 20 rivers.
3M W interim solution/'0.1M NaCQ ([I H7
, 8>. Activity recovery rate at this stage is 94%
The degree of purification was 21 times higher.

Next, 7.2 g of urea was added to and dissolved in 20 mQ of the precipitate suspension. This was subjected to refrigerated centrifugation (4° C.) at io'ooo revolutions/min for 30 minutes to remove insoluble substances.

Take 10 m of this supernatant (!) and 0.02 Mh
+a/0, gel filtration (
Column size 25 x 1000 mm) L, elution fractionation. The activity recovery rate by this method was 90%, and the purity was 47%.
It was double that. Activity recovery rate throughout all stages is 84.6%
The degree of purification was approximately 987 times higher.

The active fraction was then concentrated using an Amicon ultrafiltration concentrator TCP-10. This concentrate was dialyzed against 0.02M phosphate buffer (FI H6,8) overnight at 4°C. This dialysis activity fraction was dissolved in 0.02M phosphate buffer (
It was applied to a hydroxyapatite gel column (size 26×400111 m) equilibrated at pH 6,8>. After thorough washing with the same buffer under the conditions of flow rate 5.0111Q/hour and 3mQ1 fraction, 0.02M phosphate buffer (+
)H6,8)500111i2 and 0.5M phosphate buffer (p)-16,8>500mQ were used to elute the adsorbed material using a continuously increasing concentration gradient. The active fractions were collected and ultrafiltrated and concentrated at 4°C using an Amicon ultrafiltration concentrator TCP-10. The activity recovery rate by this method was 64%, and the degree of purification was 7.3 times higher.

The activity recovery rate throughout the entire process was 54.1%, and the degree of purification was 7.2×10 3 times.

Next, the active fraction obtained by the above method was added to 0.02M
I-lith-salt M (p 1-(7,8>10.1M
Dialysis was performed overnight at 4°C against NaCQ buffer.

The dialysis solution was applied to a Pharmacia Mono Q column buffered with the same buffer solution, and the substance was adsorbed under the conditions of 1 fHQ/min and 1 ffl Q/fraction. After washing thoroughly with the same buffer, 0.02M )-Lith-salt m (pH 7,8)/
1. NaCQ continuously using OM NaCQ buffer
The concentration was increased to elute the adsorbed active fraction. The active fraction was concentrated by ultrafiltration. The activity recovery rate by this method was 81%, and the degree of purification was increased by 8.1 times. The activity recovery rate throughout the entire process was 43.9%, and the purity was 5.8.
It was 3X10' times.

Next, the above concentrate was diluted with 0.1Ml-Lis-HCl (pH
7,8) / Toyovar 5W60 (column size 10 x 500111 m) equilibrated with 70% saturated ammonium sulfate was applied at a flow rate of 1 m, 91 minutes, 210 G!/fraction, and salted out in the column. Then, O, 1 M Tris-hydrochloric acid (1)87.
Elution was carried out using the buffer solution of 8) and continuously lowering the ammonium sulfate a content.

The active fraction was concentrated using an ultrafiltration concentrator. The activity recovery rate by this method was 11%, and the degree of purification was 5.4 times higher. The activity recovery rate throughout the entire process was 4.83%,
The degree of purification was 3.14X105 times higher.

Next, the above concentrate was diluted with 0.1M sodium phosphate (D
H7,0>10.2M Na CQ10.1%SDS
Toyo Soda G3000SW equilibrated with buffer solution
(column size 7.5X600++++++),
Gel filtration was performed at a flow rate of 1 mQ, "min, and 1 mQ1 fraction. The activity recovery rate with this method was 9%, and the degree of purification was 6.
．． It has increased five times. Activity recovery rate throughout the entire process is 0.4
3%, and the degree of purification was 2.04×108 times.

The specific activity of the low molecular weight protein having this physiological activity is 1.
02 x 108 units/mgl white matter.

[Brief explanation of the drawing]

FIG. 1 is a molecular weight distribution map obtained by measuring the molecular weight of the substance of the present invention by gel filtration using Biogel A1.5m. FIG. 2 shows the elution pattern of the substance of the present invention by the gel tr filtration method using Toyo Soda TSK Gel G3000SW. FIG. 3 is a molecular weight distribution diagram of the substance of the present invention determined from the elution time of each standard protein shown in FIG. FIG. 4 is an electropherogram of the substance of the present invention obtained by polyacrylamide gel electrophoresis. FIG. 5 is a molecular weight distribution map of the substance of the present invention determined from FIG. 4. FIG. 6 is an ultraviolet absorption spectrum analysis diagram of the substance of the present invention. (The above) Agent: Eiji Saegusa, Patent Attorney Figure 3 Maintenance time (minutes) Figure 4 Figure 5 Ai #j Mobility Figure 6 Motion (nm)

Claims (1)

[Scope of Claims] ■ A low-molecular protein having the following properties that is induced and produced by administering to a mammal an immunostimulatory agent and then administering an endotoxin derived from Durham-negative bacteria or a lectin derived from a plant. . a) Molecule fi: 29000±2500b) Isoelectric point:
I) 84.1±0.3 c) The maximum ultraviolet absorption value in 0.1M Na CQ plus 0.02M Tris-HCl buffer (p) 47.8) is 2
76r+Ill, the minimum value is around 250 nmd)
3mg protein/mQ 0.02Ml-Lis-HCl buffer (pH 7,8> The solution is colorless and transparent, and if acetone or ethanol is added to the solution at 60V/V% or more, a precipitate will form.e) The aqueous solution is weak. Shows acidity f) Shows color reaction of peptide bonds and amino acids for Biuret reaction, Folin-Lowry reaction method and ninhydrin reaction after hydrolysis with hydrochloric acid g) Direct cytotoxic effect in vitro on cultured mouse L-cells and h) has an antitumor effect in vivo against female A sarcoma tumor-bearing mice.