JPS608226A - Low-molecular protein - Google Patents

Abstract

NEW MATERIAL:A low-molecular protein [molecular weight: 16,000+ or -1,500; isoelectric point: pH 3.8+ or -0.3; 3mg protein/ml solution of 0.02 M tris-hydrochloride buffer solution (7.8pH) is colorless and transparent, addition of >=70v/v% acetone, etc. to the solution produces preciputate; aqueous solution: weakly acidic; showing color reaction of peptide bond and amino acid in biuret reaction, Folin- Lowry reaction, ninhydrin reaction after hydrolysis with hydrochloric acid; ratio of amino acid composition shown by the table I ; arrangement of amino acid shown by the table II]. USE:An antitumor agent. PREPARATION:Blood is collected from an animal fed for test, prepared serum or plasma is subjected to process of salting-out with ammonium sulfate, gel filtration, hydroxy apatite chromatography, Pharmacia EPLC mono-Q column chromatography, chromatography of salting-out and dissolution of ammonium sulfate, and gel filtration, low-molecular protein is collected, separated, and purified.

Description

[Detailed description of the invention] The present invention relates to a novel low molecular weight protein.

Carswell et al.
We discovered that when endotoxin was administered to mice sensitized with 6tteGu6rin, BCG> on the 14th day, a factor that was cytotoxic to L-cells was produced in the serum 2 hours later. More necrosis factor (Tun+or necrosisf
actor, TNF) (Pro
c, Nat.

Acad, Set, USA, Volume 72, Page 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.
(Proe, Nat.

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

(1976). Then M athew
et al. administered BCG to rabbits, and 2 weeks later, endotoxin was administered to produce TNF, which was purified, and the molecular weight was 39,000 by polyacrylamide gel electrophoresis (Polyacryl-amide gel electrophoresis).
e gel electrophorests, p to G
E) reported that it was 67,000 (Br, J
, Cancer.

Volume 42, page 416, 1980]. Furthermore, Haranaka et al.
baCteri Ulacnes) and endotoxin 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, 1982).
Year〕.

In addition to the above, there are many reports on the existence of physiologically active substances (TNF) that have cytotoxic properties against L-cells.
Even if we look at its molecular weight, the 2 of Kull et al.
25000 [J, 1mmunol, .

126, p. 1279, 1980] to 39,000 by Mateius et al. and Haranaka et al., and it cannot be said that enough single 1111M has been produced yet, and there is not enough quantity to investigate its properties. The current situation is that it has not been obtained.

The present inventors have also carried out extensive research in order to discover the substance of the above-mentioned substance that is cytotoxic to a single cell. As a result, we were able to detect a single low-molecular protein that had never been reported before.
We have successfully developed this compound and discovered that it has anticancer effects, leading to the present invention.

The novel low molecular weight protein of the present invention is characterized by having the following properties.

(1) Gel gel using molecular weight A6 biogel A1.5I11"if
Molecular weight biogel (3iogel) A1.51+ (manufactured by Bio-Rad, USA) by method 4 was added to a column (16X10001
1Pharmacia, Sweden), filled with 0.04
Ml1s monosalt m/0, 1% F T' shi) L
tN2 sodium sulfate (SO8)/4M urea 10. IMN
a Using a CQ (pH 7,8) buffer, add 200 μg (protein amount) of the substance of the present invention, perform gel filtration, and use a standard molecular weight kit (manufactured by Pharmacia, Sweden) from the elution position of the sample. Molecular weight was calculated using the curve. The above protein amount is based on Bradford (B
radford) method (A nal, 8 ioc
Hem., Vol. 72, p. 248, 1976], and the same applies hereinafter.

The results obtained are shown in FIG. (1) in the diagram
is bovine serum albumin (molecule ff167000), (2
) Gt egg white 7) ttlmi><molecular m43000), (3) is chymotrypsinogen A (molecular weight 25000
), (4) acupuncture bonuclease (molecular weight 13,700)
and (a) is the substance of the present invention. From FIG. 1, it can be seen that the substance (a) of the present invention elutes after chymotrypsinogen A (3), and its molecular weight is approximately 17,200.

B. Molecule m by gel filtration method using TSK Gel G3000SW A TSK Gel G3000SW (manufactured by Toyo Soda) column was connected to a Pharmacia FPLC system (manufactured by Pharmacia, Sweden), and 0.1% 5DS 10.1M sodium phosphate ( Gel filtration was performed by adding 100 μg (protein amount) of the substance sample of the present invention using a buffer solution of DH7.0), and these were eluted using a standard molecular bunch kit for high performance liquid chromatography (manufactured by Oriental Yeast Co., Ltd.). The molecular weight of the sample of the substance of the present invention was calculated from the pattern. The elution pattern obtained by high-performance liquid chromatography is shown in FIG. 2, and the molecular weight distribution based on the elution time of O-mardography is shown in FIG. 3, respectively. In each figure, (5) represents glutamate dehydrogenase (molecular weight: 290,000, not shown in Figure 2), (6) represents enolase (molecular weight: 67,000), (
7) is adenylate kinase (molecule 12132000), (8) is cytochrome C (molecular weight 12300), (
a) shows the substances of the present invention, respectively. From each figure, the substance of the present invention <a> is eluted before cytochrome C(8), and its molecular weight is calculated to be about 15,300.

C', Molecular weight by electrophoresis using SDS/polyacrylamide gel. 5DS- 5 μg (protein amount) of the substance sample of the present invention was applied to a polyacrylamide gel, electrophoresis was performed for 7 hours using 4011A, and the electrophoresis pattern (Figure 24) was recorded using a standard molecular weight kit (manufactured by Oriental Yeast Co., Ltd.). ,
A molecular weight curve (Fig. 5) was created from this, and the molecular weight of the sample was calculated from the drawing. In Figures 4 and 5 (a)
(b) Gt cytochrome C7ff1 body (molecular weight 86100), (b) Gt cytochrome C7ff1 body (molecular weight 738)
00), (C) is cytochrome C pentamer (molecule 116
1500), (d) is cytochrome 0411 body (molecule 8149200), (e) is cytochrome C3ff1
(molecular weight 36,900), (f) is cytochrome C2
ff1 form (molecular weight 24,600), and <a) represent cytochrome C5 amount l-mer (molecule ff1l 2300>). Also, (S) is the substance of the present invention. From Fig. 5, the substance of the present invention (S) is shown. , the molecular weight is calculated to be about 16,700.

(2) Isoelectric point Isoelectric point measurement device (manufactured by Bio-Rad, USA) and Amholine (Aml) holine > polyacrylamide play 1- (pH 3.5 to 9.5) (manufactured by LKB, USA) were used. Then, 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 the gel, and heated at 10 W.
The electrophoresis was carried out for about 2 hours at a constant power, and the electrophoresis was terminated when the current became constant. The gel was cut out at 1 mm intervals, eluted with a buffer solution, and subjected to measurement of activity against 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 pH 3.8±0.3.

(3) Measurement of ultraviolet absorption Double beam spectrophotometer UV-300 (Shimadzu @)
using 0.02M Tris-HCl 10, IM Na
The ultraviolet absorption of a sample of the substance of the present invention dissolved in CG (1)87.8) was measured. The results are shown in FIG. From the figure, the maximum value is 277 n1ll, and the minimum value is 250 nll.
Met.

(4) Solubility, color and properties Sample of the substance of the present invention was reduced to 3110 protein It/II Q concentration.
．． 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 70 V/V% or more, precipitation occurs.

Furthermore, the 3n+g protein ffl/IIIQ aqueous solution of the substance of the present invention exhibits weak acidity.

(5) Color reactions Regarding the Biuret reaction, the Folin-Lowry reaction method, and the ninhydrin reaction after hydrochloric acid hydrolysis, the 9-color reactions of peptide bonds and amino acids are all positive.

(6) Amino Acid Composition Ratio A sample of the substance of the present invention was hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours (under reduced pressure) and then analyzed using an amino acid analyzer (Dionex 500, Dionex Corporation). As a result, the substance of the present invention is glycine (Gly).
It was confirmed that each amino acid was contained in the following ratios based on the following.

Gosei amino acid ratio (molar ratio) Gly 1.00 (standard) AS+1 and/or ASnl, 10±0.06Thr
0.45±0.02 3er O, 90±0.05 QILI and/or Qlnl, 82±0.09Pro
0.84±0.04 Ala 1.11±0.06 Cys O,20±0.02 Vat O,86±0.04 Met O,18±0.01) Ic O,38±0.02 Ll 1 .73+0.09 TVr O,64±0.03 Phe O,34±0.02 His O,28±0.01 Lys O,44±0.02 ArtJ 0.46±0.02 (7) Amino acid sequence book The amino acid sequence of the invention substance sample was analyzed using an amino acid sequencer (Beckman sequencer).
Analysis was performed using de189 (M, manufactured by Beckman; each amino acid was identified by reversed-phase high performance liquid chromatography). As a result, it was confirmed that 17 amino acids from the amino terminal side were arranged as shown below.

H-A la -Leu-8er-Asp-Lys-
Pro-L eu -A Ia -His -V al
-V at -A la -A sn -Pro
-G In -V al -G lu - Furthermore, the low molecular weight protein of the present invention is characterized in that it has the following physiological activity.

(a) Cytotoxic effect on L-cells The method of Casewc! et al. and the method of Kloster Oaard (Mo1., Imm, Vol. 17, p. 613, 1)
The L-cell membrane cell effect of the substance of the present invention was evaluated in accordance with (1980). That is, 250 units/m Q
This L-cell suspension was incubated hot at a humidity of 2 x 105 cells/mQ in Eagles Minimal Essential Medium (MEM) medium containing 125 μJ/mQ of streptomycin and 0.11 IIQ of each L-cell suspension.
and sample name of the substance of the present invention diluted to an appropriate degree of haze: 0.1111
Q was placed in each well of a 96-well microplate (Costar, USA) and cultured at 37° C. for 48 hours in air containing 5% carbon dioxide. The cultured cells are stained with neutral red, and the number of viable cells is determined colorimetrically by Titertic Multiscan (manufactured by Flow Laboratories, USA). The activity is - The power to kill 50% of the cells is taken 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 8.99x107 units/mg protein.

(b) Female A-the/Ll-1-v (lyleth A
-sarcoma) Antitumor effect from tumor-bearing mice
After transplantation into the mouse abdomen, the tumor size was 78 mm in diameter.
From the tail vein of a mouse that had grown to ~8II1m, 2.5X10'
0.21 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 over 50% from the middle of the transplanted brain surface) ((14): Significant hemorrhagic necrosis ( The central part 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 Dose evaluation (r+o protein/mouse) -10-1+'1- (physiological saline) 6000 55.6 1 3 2 0 111.2 0 2 3 1 278.0 0 1 3 2 556 .0 1.12.11 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 in the body of a mammal by administering a substance having an immunostimulating effect to the mammal, and then administering an endotoxin derived from Gram-hopping bacteria or a lectin derived from a plant, basically according to a known method. produced in More specifically, for example, the method of Carswell et al.
N at, Δcad, 3 ci,.

LJSA, Vol. 72, p. 3136e, 1975], a substance having an immunostimulatory effect is first administered to the mammalian animal. Examples of mammalian animals include mice, rats, guinea pigs, rabbits, etc., but are not particularly limited thereto. As the substance having an immunostimulatory effect, various known substances can be used. A specific example is Bacillus carmetui Guerin (BCG).
, Corynebacterium halbum (G orynebac
terium garvai), Propionibacterium acnes (Propioni-bactertu)
macnes), Mycobacterium butyricum
), ] Linebacterium goo 7 m, mL rosum (COrynQbaOterium granulos
um), Streptococcus virogines (Stre
ptococcus pyrogenes), Plasmodium (Plasmodium), etc.
Zymosan, Nocardia asteroides
s), Listeria monocytogenes (Lyst
eria monocytogenes), glucan, 1tBB1 membrane skeleton (cell
lallskeleton), dextran sulfate (d
Examples thereof include: extratransulfate), muramyl-dipepHde, Krestin (manufactured by Kenbetsu Kogyo Co., Ltd.), and the like. Administration of these substances having an immunostimulatory effect is generally carried out by intravenous or intraperitoneal injection. The dosage is determined appropriately, but
Usually, it is preferable to set it as the range of about 1-1000+no/kg.

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

Typical examples include lipopolysaccharide derived from Bacillus Taiho, Pseudomonas aeruginosa, Salmonella typhi, etc., jack bean lectin (concanavalin A, ConA), soybean lectin (SBA), red bean lectin (PH8).
etc. can be exemplified.

These administrations are usually preferably administered by intravenous injection. The dosage is not particularly limited, but is usually 10 μg to 110 ml.
It is generally selected from a range of 1/kg. Approximately 1.5 to 3 hours after administration of the endotoxin 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. That is, blood is collected from a test animal according to a conventional method, and the properties of the substance contained in the obtained serum or plasma are used to perform physicochemical or biochemical methods such as salting out, chromatography, and electrophoresis. , extraction method, centrifugation method, and dialysis method alone or in appropriate combination. More specifically, it is carried out by subjecting the blood semen or plasma (hereinafter referred to as crude solution) to the following step.

(1) Ammonium sulfate salting out (2) Gel i filtration (3) Hydroxyapatite chromatography (4) Pharmacia FPLC Mono Q Column chromatography (5) Ammonium sulfate salting out 0-dissolution mudgraphy (6) Glu filtration Below, these The details of the process will be explained.

Purification step 1 Ammonium sulfate (hereinafter referred to as "ammonium sulfate") is added to the crude solution to mPs the 50 to 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.

Next, centrifugation is performed at 10,000 rpm for 10 to 30 minutes using a refrigerated centrifugal force 11ffl (manufactured by Hitachi, Ltd.) to collect a precipitate having physiological activity. At this stage, the activity recovery rate (according to the cytotoxic activity measurement method for L-mlta) is 80-100%, and the degree of purification is 10-2.
It is 0 times.

Purification step 2 The precipitate obtained in step 1 was dissolved in 0.02M l-lis-hydrochloric acid (
pH7,8)10. Suspend in IM Na CQ buffer. 2 to 8 M of urea is added to this suspension, and the insoluble material is subjected to refrigerated centrifugation at 10,000 rpm for 10 to 30 minutes to obtain a clear supernatant having physiological activity, which is subjected to gel filtration. As the carrier for gel filtration, Ultrogel AOA44°54 (manufactured by LKB, USA) or Biogel A1.5m (manufactured by Bio-Rad, USA) is used.

The gel filtration fraction was measured for cytotoxic activity against cells as described above, and the active fraction was collected and subjected to ultrafiltration 11Y.
Ultrafiltration and concentration are performed using an ultrafiltration device TCP-10 (manufactured by Amicon, USA) equipped with M10 (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 The physiologically active fraction obtained in step 2 was pumped using a dialysis tube (Hani Chemical Co., Ltd. 1IlI) and 50 to 100 times the amount of 0.02M phosphoric acid I! Dialyze overnight against a buffer solution (pH 6,8) at 4° C., changing the external solution several times. The dialysate was heated at 10,000 revolutions/min in a refrigerated centrifuge (4°C).
Centrifuge for 20-30 minutes to obtain a clear supernatant.

Next, this supernatant liquid was adsorbed on hydroxyapatite (manufactured by Nippon Chemical Co., Ltd.), and dissolved in a 0.02M to 0.5M phosphoric acid grade buffer <o1-t6.8> according to the continuous haze gradient method or stepwise. The bioactive fraction is eluted at increasing concentrations. The obtained physiologically active fraction is concentrated by ultrafiltration concentration or ammonium sulfate salting out. The recovery rate of the bioactive fraction by this method is 40-80%, and the degree of N production is about 4-10 times higher.

Purification step 4 Put the physiologically active fraction a supply solution obtained in purification step 3 into a dialysis tube and add it to 50 to 100 times the amount of dilute phosphoric acid or Tris buffer (pH 7.0 to 8.0). Te, 4
Dialyze overnight at °C. This dialysate was processed using Pharmacia FPLC.
The bioactive substance is adsorbed onto a MonoQ column (manufactured by Pharmacia, Sweden), and the physiologically active substance is eluted by continuously increasing the buffer concentration or NaC+lK by 1 degree. The activity recovery rate in this process is 70-90%, and the ti production degree is about 3-90%.
Increases by 6 times.

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.

A column was packed with Toyovar 5W50, 55, or 60 (all manufactured by Toyo Soda), and Pharmacia FPL
Using the C system (manufactured by Pharmacia, Sweden),
The physiologically active fraction is salted out with ammonium sulfate in the 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 30 to 70%, and the N production degree is about 3 to 4 times higher.

Purification Step 6 The active fraction obtained in Purification Step 5 is concentrated and applied to a column (16Xi000 mm) packed with Bio-Gel A1.51 or Ultrogel AOA44 or 54 to perform gel filtration. Alternatively, gel filtration is performed using a Toyo Soda high-speed liquid filter column G2000SW or G3000SW (manufactured by Toyo Soda Co., Ltd.). The activity recovery rate in this step is 25-75%, and the degree of purification is approximately 3-6 times higher.

The activity recovery rate through purification steps 1 to 6 is 5 to 30%, and the degree of purification is about 2.7×10′ to 7.8×10 5 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 cells in vitro and an antitumor effect in vivo, and also has antitumor effects on cancer cells as shown in the pharmacological test examples below. It also exhibits cytotoxic or cell-killing effects on Methulama cells, and has low toxicity.

Pharmacological Test Examples Cytotoxic or Cytocidal Effect (a) Cytotoxic Effect on Human Cancer Cells Human Burkitt's lymphoma-derived strain Raji (J.

Nat, Cancer Institute, vol. 37, 5.
47, 1966), human gastric cancer (small ring cell carcinoma)-derived strain KatO-I [1(Jl)n, J, EX$1.
Med, vol. 48, p. 61, 1978) and human pharyngeal carcinoma-derived strain K B (Cancer Res, vol. 48, p. 61, 1978).
Vol., p. 1017, 1958], the cell-killing effect of the substance of the present invention on each cell was evaluated. That is, human Burkitt's lymphoma-derived cell line and human gastric cancer-derived cell line were
RP containing 0 units/IQ of penicillin, 125 μQ/mQ of streptomycin and 10% inactivated live fetal serum.
The cell line was adjusted to 2×10” cells/m Q with M11640 medium. In addition, the cell line derived from human pharyngeal carcinoma was adjusted to 250 units/I
2 x 1 using Eagles Minimal Essential Medium containing Q penicillin, 125 μQ/m Q streptomycin and 10% inactivated bovine serum.
The cells were adjusted to 0.05 IW cells/m Q.

Each of the above cell preparation solutions o-i+nQ and each f! The substance of the present invention diluted to a concentration of 0.1119 was placed in each well of a 96-well microphone plate, and the mixture was incubated in air containing 5% carbon dioxide for 3
The cells were cultured at 7°C for 48 hours.

After 48 hours of culture, the cells were stained with Trivan blue, and the number of viable cells was calculated under a microscope using a Birkerturk counter F (manufactured by Elma Obteil Works, Japan).

As a result, the concentration of the substance of the present invention that inhibits the proliferation of various cells by 50% is 8.5% for human Burkitt's lymphoma-derived cells. Or+o protein amount/m 9 or more, 27 for human gastric cancer-derived cell lines, Ong? J White fA/rAQJX, 11.0no proteinffi/for human n pharyngeal cancer-derived cells.
It was more than mQ.

(b) Cytotoxic effect on Metulama cells using the method of Nelson et al. (Naturc.

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

Cancer Inst, vol. 51, p. 1417,
1973] evaluated the cytotoxic effect on cells. That is, 5 x 104 Metulama A-375 cells were cultured using Eagle's medium containing glutamine, non-essential amino acids, penicillin, streptomycin, and 10% inactivated tallow serum.
A suspension of cells/IIQ was prepared.

This cell suspension solution 111IQ and the sample ffi solution 11nQ obtained by appropriately diluting the substance of the present invention were added to a 3.5 cm diameter tube.
- and cultured at 37°C in air containing 5% carbon dioxide.

On the third day of culture, the cells were stained with trivan blue in the same manner as in (a) above, and the number of viable cells was calculated using a Birkertürk H1 abacus under a microscope. As a result, the amount required for the substance of the present invention to inhibit the proliferation of Metsurama A-375i111 cells by 50% was 140q protein amount/IQ or more.

Pharmacological Test Example ■ Acute Toxicity Using 10 male and female 8-week-old ddY mice, the substance of the present invention was intravenously administered at a rate of 3.7 mQ protein 1/ka.
Acute toxicity was investigated.

As a result, no deaths were observed, and LDo was 3.7 mg/k.
o or more was confirmed. Further, during the i-monitoring period, no obvious symptoms of intoxication 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 types of 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 and administered by various administration routes depending on the form. Examples of the formulation include liquid solutions, suspensions, and milk solutions, 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 depends on the degree of the disease, the patient's age, sex, etc., but is usually about 1.85 to 18.5μ0 in terms of protein amount.
/kM/day is preferably divided into 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 Zealand White or Japanese White female rabbit
2.0 to 3.0 Jl), formalin-killed Corynebacterium parvum (Corynebacterium parvum)
ruvam, Wellcome Ltd., UK) 70IIl
A was injected intravenously. Nine days after the injection, 100 μQ of ribopolyi natucalide (E. coli O55:85, manufactured by Difco, USA) was injected through the ear vein, and 2 hours later, whole blood was collected from the heart. The collected blood is rotated at 5000 rpm for 20 minutes.
The serum was separated by centrifugation for a minute. 100 by this operation
Serum 75301Q was obtained from a feathered rabbit with a titer of 15900 units/IQ.

As shown in Table 2, the production n of this physiologically active substance in New Zealand White and Japanese White rabbits was higher in Japanese White rabbits.

Table 2: Activity measured by cytotoxicity assay method against one cell (units/rabbit) New Zealand White 1.07X108 Japan White 1.31X10θ 472g of ammonium sulfate was added to the serum 1000IIIQ, dissolved, and left overnight at 4°C to determine the activity. The material precipitated.

Centrifuge the precipitate at ioooo revolutions/min for 30 minutes (4°C
), collect the precipitate, and add about 100 mQ of 0.02M Tris-HCl buffer 10.1M Na CQ (EI H7,8
). The activity recovery rate at this stage was 98%, and the degree of purification was 18 times higher.

Then, 36.04 for the precipitated suspension 100+119
Add urea () and dissolve it.I ooooo times @
Refrigerated centrifugation (4°C) was performed for 30 min at /min to remove insoluble materials. 50 mQ of this supernatant was taken and gel-o-filtrated (column size 5
0x1000100O, the activity of each elution fraction was determined by the activity measurement method against L-cells, and the active fractions were 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.

The active fraction was then concentrated using an ultrafiltration concentrator TCF-10 equipped with an Amicon ultrafiltration membrane YMIO. Put this lagoon supply liquid into a dialysis tube and add 50 times the amount of O-02M.
Dialysis was performed at 4°C against phosphate buffer (pH 6, 8). After exchanging the external solution three times every 3 hours, the cells were incubated in the same buffer at 4° C. overnight. A hydroxyapatite gel column (Nice 26
x400mi). Flow rate 5.0ffl 97 hours,
After thorough washing with the same buffer under the conditions of 3IIIQ/fraction, 500ml of 0.02M phosphate buffer (pH 6,8) was added.
Q and 0.5M phosphate buffer (pH 6,8) 500
mQ 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 the activity was adsorbed, and the adsorbed active fraction was eluted between phosphate buffer concentrations of 0.15M and 0.3M.

Collect the active fraction and use Amicon ultrafiltration concentrator TCP-1.
Ultrafiltration and concentration were performed at 4°C using 0. 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 amount of 0.02M Tris-HCl (
1) H7, 8) 10. For IMNa CQ buffer,
Dialysis was performed overnight at 4°C. The dialysis solution was applied to a Pharmacia Mono Q column buffered with the same buffer solution, and the active substance was adsorbed under the conditions of 1 m+1/min and ILN Q1 fractionation. After washing thoroughly with the same buffer, 0.02M Tris-HCl (
pH7,8)/1. OM Na (continuously using l
'The JaCQm degree was increased and the adsorbed active fraction was eluted.

The active fraction was concentrated using an ultrafiltration device equipped with an Amicon ultrafiltration membrane YMIO. 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 degree of purification was 1. lX1
It was 0' times.

Then, the above 111M was added to 0.1M Tris-HCl (D
Toyo Pearl 5W60 equilibrated with H7,8)/70% saturated ammonium sulfate (column size 10X10X500, flow rate 1111 Q/min, 2IIIQZ fractions) and salted out in the column. 1)H
Elution FB separation was carried out using the buffer solution of 7, 8) and continuously decreasing the ammonium sulfate concentration.

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 62%, and the degree of purification was 4 times higher. Activity recovery rate throughout the entire process is 21.5%, purity level is 4.4
It was 0x10' times.

Next, the above concentrate was diluted with 0.1M sodium phosphate (D
H7,0) 10.2M Na CQ10.1%SDS!
l! Toyo Soda G3000S equilibrated with TI liquid
Using a W column (7.5 x 600 mm), flow rate 1 mQ/
Glu filtration was performed on the Q1 fraction. With this method, the activity recovery rate was 26%, and the degree of purification was increased by 5.3 times. The activity recovery rate throughout the entire process was 5.6%, and the degree of purification was 2.33×10 5 times.

The specific activity of the low molecular weight protein having this physiological activity is 9.
24 x 107 units/m (J protein).

Example 2 New Zealand White female rabbit (weight 2.0-3.
0ko) was injected with 150 mg of formalin-killed Mycobacterium butyricum (manufactured by Difco, USA) through the ear vein. 9 days after injection, 100 μg of Noriboboli Satucharide (Bacillus Bacillus 055:85, manufactured by Difco, USA)
was injected into the ear vein, and 2 hours later, whole blood was collected from the heart. The collected blood was centrifuged at 5000 rpm for 20 minutes,
Serum was separated. By this procedure, 7410 m9 of serum with a titer of 5160 units/mQ was obtained from 100 rabbits.
was gotten.

After adding 472 g of ammonium sulfate to the serum 1000111Q, it was well dissolved and left at 4°C overnight. 1000 ammonium sulfate salting out solution
The precipitate was centrifuged at 0 revolutions/min for 30 minutes at 4°C, and the precipitate was mixed with 0.02M of approximately 100111Q.
Tris-HCl 10. IM Na CQ mild buffer (DH7,
8>. The activity recovery rate at this stage was 97%, and the degree of vX production was 20 times higher.

Then 100 ml of the precipitate suspension (36 o4a per l)
Add and dissolve urea at a rate of 30 at 10,000 rpm.
Cooling centrifugation for 11 minutes! (4°C) to remove insoluble matter. Take 50IQ of this supernatant and add 0.02M Tris-
Ultrogel AcA34 (column size 50X) equilibrated with hydrochloric acid (pH 7, 8) 10.5M Na CQ buffer.
1000 mm) at a flow rate of 40 mQ/hour and 7 mQ/fraction, and the active fraction was collected. The activity recovery rate by this method was 92%, and the degree of purification was 28 times higher. The activity recovery rate throughout all stages was 89.2%, and the fit degree was approximately 56.
It was 0 times.

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 get 50g! 0.0 of I amount
Dialysis was performed at 4°C against 2M phosphate buffer (EIH6,8). After exchanging the external solution 3 times every 3 hours, the same M
Dialyzed overnight in Wfi solution. This dialysis activity fraction was prepared in advance in 0.02M phosphorus 111 buffer solution (pH 6,8
) equilibrated with a column of hydroxyapatite gel (
Size 26 x 400 + am) at a flow rate of 5.011191 hours and a fraction of 3 mQ/2. The adsorbed active fraction was mixed with 0.02M phosphate buffer M (pH 6,8) and 0.5
M Phosphorus 1M1ii (D H6,8) and 'ir: were used and eluted by 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 degree of purification was 5.6 x 103 times.

The active fraction was then concentrated using an ultrafilter, placed in a dialysis tube, and mixed with 50 times m of 0.02M Tris-HCl (p
H7,8) Dialysis was performed overnight at 4°C against 10.1M NaCQWi'Wi solution. The dialyzed active fraction was adsorbed onto a Pharmacia MonoQ column which had been M-blocked with the same TIA buffer under conditions of 1111Q/min and 1111Q/fraction. Next, after washing thoroughly with the same buffer, 0.02M I-Lis-HCl (pH 7,8>/1.OM NaCy%)
The adsorbed active fraction was eluted by continuously increasing the temperature of N8098 degrees using a solution. The activity category is Amicon
It was concentrated using an Ei condenser. The activity recovery rate by this method was 62%, and the degree of purification 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 0.1M Tris-HCl (pH
7,8>/Toyovar 5W60 (column size 10X500+m) equilibrated with 70% saturated ammonium sulfate buffer, flow rate 1111T! /min and 2mQ for 1 min, and salted out in the column. Then 0.1M Tris-HCl (pH
Dissolved fractions were carried out using the buffer solution of 7, 8) and continuously decreasing the ammonium sulfate concentration. The active fraction was collected and condensed using an ultrafiltration concentrator. The activity recovery rate at this stage was 34%, and the degree of purification was 4.7 times higher. The activity recovery rate throughout the entire process was 12.8%, and the purity was 1.05×10
It was 5 times more.

Next, the above concentrated solution was diluted with 0.1M sulfurium phosphate (p
H7,0) Using a Toyo Soda G3000SW column (7,5 x 600 mm) equilibrated with 10-2M Na CQ 10.1% SDS buffer, flow rate 1 m Q/min,
Gel filtration was performed on the il Q1 fraction. The activity recovery rate at this stage was 62%, and the degree of purification was 6.3 times higher.

The activity recovery rate throughout the entire process was 7.9%, and the degree of purification was 6.62×10 5 times.

In addition, main student 1! l! The specific activity of the active low molecular weight protein was 9.19 x 10' units/11g protein.

Example 3 New Zealand white type raw V-gi (weight 2.0-3.
Krestin (manufactured by Kureha Chemical Industry Co., Ltd.) 10
1 g of OII was injected through the ear vein. 1oo 9 days after injection
tta lipopolysaccharide (E. coli 055:B5,
(manufactured by Difco, USA) was injected into the ear vein, and 2 hours later, whole blood was collected from the heart. 5,000 rotations of blood-collected white waves/
Serum was separated by centrifugation for 20 minutes. This procedure yielded 7000 IIIQ serum with a titer of 6200 units/m Q from 100 rabbits.

The serum 1000111! After adding and dissolving 472 g of ammonium sulfate, the mixture was left at 4° C. overnight to precipitate the active substance.

The precipitate was centrifuged at 10,000 revolutions/min for 30 minutes at 4°C, the precipitate was collected again, and 10. IMNa CQ (D H7,8
). The activity recovery rate at this stage was 98%, and the degree of purification was 18 times higher.

Next, urea was added at a ratio of 36.04 a to 1100 mQ of the precipitate suspension to dissolve it. Rotate this 10,000 times/
Centrifugation was performed at 4° C. for 30 minutes to remove insoluble matter.

This supernatant liquid 50111! Take 0.02M Tris-
Gel t filtration (
Column Quiz 50x1000mm) L, L-Cell activity was determined using the two-cell activity measurement method, and the activity categories were collected.

The activity recovery rate by this method was 95%, and the degree of purification was 30 times higher. The activity recovery rate throughout all stages was 93.1%.
The degree of I production was approximately 540 times.

Then, an Amicon ultrafiltration membrane YM10 was attached and the active fraction was concentrated using an ultrafiltration concentrator TCP-10. Pour this concentrated solution into a dialysis tube and add 50 times ff1 (7)
Dialysis was performed against 0.02M')>m buffer (pH 6, 8). After exchanging the external solution three times every 3 hours, the device was incubated in the same buffer at 4° C. overnight. This dialyzed active fraction was applied to a hydroxyapatite gel column (size 26 x 400 mm) equilibrated with 0.02M phosphate M buffer (pl-16,8) in advance. Flow rate 5.011111
/ hour, under the conditions of 3mQ1 fractionation, and after thorough washing with the same buffer, 0.02M phosphate buffer (pH 6,8) 500
mQ and 0.5M phosphate buffer (pH 6,8) 500
The adsorbed material was eluted by continuously increasing the concentration gradient using mQ. In this method, no activity was observed in the non-adsorbed fraction, all the activity was adsorbed, and the adsorbed active fraction was eluted between 0.15M and 0.3M in 8 degrees phosphate buffer.

Collect the active fraction and use Amicon ultrafiltration concentrator TCP-j.
Ultrafiltration and concentration were performed using O at 4°C. 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 N production degree was 5.4 x 10' times.

Next, the concentrated solution of the active fraction was put into a dialysis tube and diluted with 0.02M Tris-HCl (pH 7,8
) Dialysis was performed overnight at 4° C. against 10.1 M Na CQ mild buffer. The dialysis solution was applied to a Pharmacia Mono Q column buffered with the same mw solution, and 1197 minutes, 111 Q
The active fraction was adsorbed under the conditions of /fractionation. After washing thoroughly with the same buffer, 0.02M Tris-HCl/1. OM
Continuously N using NaC (!Buffer (1) 87.8)
aC9! The active fraction adsorbed by increasing the concentration of u1 was eluted.

The active fraction was concentrated using an ultrafiltration device equipped with an Amicon ultrafiltration IPJYMIO. The activity recovery rate by this method was 80%, and the degree of purification was increased by 5.5 times. The activity recovery rate throughout the whole process was 41.7%, and the purity was 2.97.
It was X10A times.

Next, the above concentrated solution was diluted with 0.1M Tris-HCl/70%
It was applied to Toyovar 5W60 (column size 10X10X500) equilibrated with saturated ammonium sulfate CD H7,8) buffer at flow rates of both 1R19/min and 2alQ/min, and salted out in the column. Then, 0.1M Tris-HCl ( pH7,
Elution was carried out using the M solution of 8) while continuously decreasing the ammonium sulfate concentration. The active fraction was collected and concentrated using an ultrafiltration concentrator equipped with an Amicon ultrafiltration membrane YMIO. The activity recovery rate at this stage was 64%, and the purity level was 3.
．． It was 9 times more. The activity recovery rate throughout the entire process was 26.
7%, and the degree of purification was 1.16×10 5 times.

Next, the above concentrated solution was diluted with 0.1M sodium phosphate 10
．． 2M Na CQ 10.1% SO8 buffer (pH 7
Toyo Soda G3000SW equilibrated with ,0)
(column size 7.5 x 600 mm), flow rate 1 m
Gel filtration was performed for 97 minutes at 1 m Q/fraction. The activity recovery rate at this stage was 28%, and the degree of purification increased by 5.0 times. The activity recovery rate throughout the entire process was 7.5%, and the degree of purification was 5.8×10 5 times.

The specific activity of the low molecular weight protein having this physiological activity is 8.
99 x 10'm position/mg protein amount.

Example 4 New Zealand White female V-gi (weight 2.0-3
．． 100 o of formalin-killed Corynebacterium bulbum (manufactured by Wellcome, UK) was injected into the ear vein. Nine days after the injection, 10a+o of funcanavalin A (manufactured by Wako II Kodo Co., Ltd.) was injected through the ear vein, and 2 hours later, whole blood was collected from the heart. The collected blood was centrifuged at 5000 rpm for 20 minutes at 4°C to separate serum. By this operation, 3780 units/m Q from 100 rabbits
Serum 7640IQ was obtained with a titer of .

After adding 472Q ammonium sulfate to the serum 1000IIIQ, it was well dissolved and left at 4°C overnight. 1000 ammonium sulfate salting out solution
The precipitate was centrifuged at 0 rpm for 30 minutes at 1 m (4°C), collected, and added with approximately 100 mQ of 0.02 M Tris-HCl.
It was suspended in IM Na GQM liquid C1) H7,8). The activity recovery rate at this stage was 99%, and the degree of purification was 20 times higher.

Then, 36.04 g for 100+z+ of the precipitated suspension
After adding and dissolving urea, the mixture was cooled and centrifuged at 10,000 rpm (4° C.) for 30 minutes to remove insoluble matter. Take 50mQ of this supernatant and add 0.02M Tris-HCl (pH 7,
8) Glu filtration using Ultrogel ΔCΔ54 equilibrated with 10.5M NaCl2 slow solution (column size 50X1)
000n+m, flow rate 40IIIQ/hr, 7 [lQ/fraction) and the elution fractions were collected. The activity recovery rate by this method is 9
At 5%, fi'J1 degree 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 ultrafilter concentrator TCF-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 at 4°C against phosphate buffer (tlH6,8). After exchanging the external solution three times every 3 hours, the cells were incubated in the same buffer at 4° C. overnight. This dialysis active fraction was transferred to a hydroxyapatite gel column (size 2) equilibrated with 0.02M phosphate buffer (pH 6,8).
6x400mm) at a flow rate of 5.0m91, and adsorbed in 3mQ1 fraction. The adsorbed active fraction was mixed with 0.02M phosphate M buffer (pH 6,8) and 0.5M phosphate buffer (pH 6,8).
The adsorbed substances can be removed by continuously increasing the concentration using pH 6, 8). 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 purity was 8.0x.
It was 103 times more.

The active fraction was then concentrated using an ultrafilter, placed in a dialysis tube, and mixed with 50 times m of 0.02M Tris-HCl (p
H7, 8) 10. using IM Na CQ buffer, 4
Dialysis was performed overnight at °C. This dialyzed active fraction was transferred to a Pharmacia MonoQ column equilibrated with the same buffer solution.
Adsorption was carried out under both conditions of 97 minutes and 1111 Q/min. After washing thoroughly with the same buffer, 0.02M Tris-HCl (p
H7,8)/1, OM Na CQ mild buffer was used to continuously increase the NaCQ intensity, and the adsorbed active fraction was eluted. 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. Activity recovery rate throughout the entire process is 45
．． 2%, and the degree of purification was 4.2X10' times.

Next, the concentrate was reduced to 0. IMt-Lis-HCl (pH
7,8>/Toyovar 5W60 (column size 10X5001111) equilibrated with 70% saturated ammonium sulfate buffer
11) at a flow rate of 1+11Q/min in 2mQ1 fraction, and salted out in the column. Then 0.1M Tris-HCl (
Dissolved fractions were performed using a buffer solution (pH 7, 8) and continuously lowering the ammonium sulfate concentration.

The active fraction was collected and concentrated using an ultrafiltration condenser.

The activity recovery rate by this method was 58%, and the purity was 3.
．． It was twice that amount. The activity recovery rate throughout the entire process was 26.
At 2%, the Vi strength was 1.4×10 5 times.

Next, the above concentrated solution was diluted with 0.1M 1-lium phosphate (
pH7,0) 10.2M Na CQ10.1%SD
Toyo Soda G3000SW buffered with S buffer solution
Using a column (7.5 x 600 mm), flow rate 1rn 9
Gel filtration was performed for 7 minutes, 1 m Q/fraction. With this method, the activity recovery rate was 44%, and the degree of purification was 5.7 times higher. The activity recovery rate throughout the entire process was 11.5%, and the degree of purification was 7.8×10 5 times.

The specific activity of the low molecular weight protein having this physiological activity is 9.
It was 3 x 107 units/11g protein.

Example 5 ddYl mice (body weight 25-300) were treated with formalin-killed Corynebacterium bulbum (manufactured by Wellcome Inc.,
UK) In+o was administered intraperitoneally, and 9 days after the injection, Ribopoli Tucaride (Pseudomonas aeruginosa; manufactured by Difco, USA) was administered.
10 μg was injected into the tail vein, and 2 hours later, the eyeball was removed and blood was collected from the orbital vein. 5000 yen of the collected blood
Serum was separated by centrifugation at revolutions/min for 20 minutes. By this operation, 56100 units/m from 100 mice
Serum 90.5mQ was obtained with a titer of Q.

After adding and dissolving 47.2 (+) ammonium sulfate to the serum 1001119, it was left to stand 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 about 20 mQ of 0.02M Tris-HCl buffer 10. Suspended in IM Na CQ (pH 7,8). The activity recovery rate at this stage was 98%, and the degree of purification was 21 times higher.

Then, the Shen F! 7.2 g of urea was added to and dissolved in 20rnQ of the suspension. 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. ffi on top of this? Take f210+11Q and 0.02M Tris-m? ! Gel 3 filtration (column size 26 x 1000 mm) was carried out using Ultrogel Δc A54 equilibrated with 10.5M NaCa buffer, followed by elution and fractionation. The activity recovery rate by this method was 90%, and the degree of purification was 32 times higher. The activity recovery rate throughout all steps was 88.2%, and vX'
The S degree was approximately 672 times higher.

The active fraction was then concentrated using an Amicon ultrafiltration condenser TCF-10. Add this concentrated solution to 0.02M phosphoric acid M
Dialysis was performed overnight at 4°C against a buffer solution (pH 6-8). This dialysis activity fraction was dissolved in 0.02M phosphate buffer (pH
The mixture was applied to a hydroxyl abatai 1-gel column (g size 26X40001111) equilibrated with 6,8).

After washing thoroughly with the same buffer under the conditions of 3 mQ/fraction at a flow rate of 5.0 + nQZ, 0.02 M phosphate buffer (pl
-16,8) 500 m Q and 0.5 M phosphate buffer (p 1-16.8) 500 m Q were used to build up a continuous near-a gradient to elute the adsorbed material. Collect the active fraction and use Amicon ultrafiltration concentrator TCP-10.
Ultrafiltration concentration was performed at 4°C. The activity recovery rate by this method was 40%, 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.
It was 03x103 times.

Next, the active fraction obtained by the above method was mixed with 0.02M Tris-HCl (pH 7,8) for 10. Dialysis was performed overnight at 4°C against IM NaCQWi buffer. The dialysis solution was applied to a Pharmacia Mono Q column buffered with the same buffer, and
The substance was adsorbed under the conditions of mQ/min and 1 mQ/fraction. After washing thoroughly with the same buffer, 0.02M+-Lith-salt M (pH 7,8)/1. OM Na CQtl
The adsorbed active fraction was eluted by continuously raising the Na098°C using i buffer. The active fraction was concentrated by ultrafiltration. The activity recovery rate by this method was 75%, and the degree of purification was five times higher. Activity recovery rate throughout the entire process is 26.5
%, and the degree of purification was 2.02×10L times.

Next, the above concentrate was diluted with 0.1M Tris-HCl (pH
7,8) / Toyo Pearl 5W60 equilibrated with 70% saturated ammonium sulfate (applied to Columnize 10X10X500 at a flow rate of 111Q/min, 2m (1/fraction) and salted out in the column. Then, 0.1M Tris-HCl (p)17.8)
Elution was carried out using a buffer solution of 100% and decreasing the ammonium sulfate depth continuously.

The active fraction was concentrated using an ultra-iPA filter. The activity recovery rate by this method was 47%, and the purity was 4 times higher. Activity recovery rate throughout the entire process was 12.4%
And the tilj manufacturing degree was 8.08×10' times.

Next, the above am solution was mixed with 0.1M sodium phosphate (1)
H7,0) 10.2M Na C (+10.1%5D
Toyo Soda G3000SW equilibrated with SI solution
(column size 7.5 x 600 mm), flow rate 1
Gel filtration was performed at 111 Q/min, 1 m Q/fraction. The activity recovery rate in this method was 31%, and the purity was 4.
It has increased twice. The activity recovery rate throughout the entire process was 3.8%, and the degree of purification was 3.23×10 5 times.

The specific activity of the low molecular weight protein having this physiological activity is 9.
04 x 107 units/III (J protein).

Example 6 Zymosan A was administered to ddYI mice (weight 25-30 g).
(manufactured by Sigma, USA) 2.0 aa was administered intraperitoneally,
Nine days after the injection, 10 Mg of endotoxin (E. coli riboborisatucalide) was injected into the tail vein, and 2 hours later, the eyeballs were removed and blood was collected from the orbital venous plexus. The collected blood was centrifuged at 5000 rpm for 20 minutes to separate serum. This procedure yielded serum 96IIIQ with a titer of 4800 units/m Q from 100 mice.

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

Centrifuge the precipitate at 10,000 times/min for 30 minutes (4℃)
Collect the precipitate and add about 20 mQ of 0.02M Tris-hydrochloric acid grade C solution 10. The activity recovery rate at this stage was 97%, and the degree of purification was 17 times higher.

Next, 7.2 g of urea was added to and dissolved in the precipitated IFJ suspension 20w1Q. 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 10IIIQ of the above synovial fluid and add 0.02M Tris-5
a Gel filtration (column size 26X) using Ultrogel AcA34 equilibrated with 10.5M Na CQ mild buffer.
1000 mm) and elution fractionation. 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 phosphorus buffer (pH 6,8) at 4°C overnight. This dialysis active fraction was mixed with 0.02M phosphoric acid li box solution (T
)H6,8) was applied to a hydroxyapatite gel column (size 26 x 400 mm). flow rate 5
．． After washing thoroughly with the same buffer under the conditions of 3 mQ/fraction at Om 97 hours, 0.02 M phosphate M buffer (pH 6,
8) 500mQ and 0.5M phosphate buffer (pH 6,
8) Using 500 mA, the concentration gradient was continuously raised to elute the adsorbed material. The active fractions were collected and subjected to ultrafiltration concentration at 4° C. using Amicon ultra-i filtration concentration device TCP-10. The activity recovery rate by this method was 43%, and the degree of purification was 6.8 times higher. The activity recovery rate throughout the entire process was 38.3%, and the degree of purification was 4.39xlO' times.

Next, the active fraction obtained by the above method was mixed with 0.02M Tris-HCl (1) 87.8) 10. Dialysis was performed overnight at 4°C in a buffered IMNaCQ buffer solution. The dialysis solution was applied to a Pharmacia MonoQ column buffered with the same buffer solution, and 11
The substance was adsorbed under the conditions of IIQ/min and 1 mQ/fraction. After washing thoroughly with the same buffer, 0.02M Tris-1MM (p 1-17.8)/1. OM Na C
The adsorbed active fraction was eluted by continuously increasing the NaCQffi concentration using a QFli interim solution. 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, add the above concentrate to 0.1M! - Liss-hydrochloric acid (pH
7,8)/70% saturated ammonium sulfate (column size 10 x 500 mm) at a flow rate of 1 m for 91 minutes and 2111Q/fraction, and salted out in the column. Then 0.1M Tris-HCl (DH7,8)
Elution was carried out using a buffer solution of 20% by decreasing the ammonium sulfate concentration continuously.

The active fraction was separated using an ultrafiltration concentrator. The activity recovery rate by this method was 43%, and the degree of purification was 3.2 times higher. The activity recovery rate throughout the entire process was 12.7%, m
The degree of production was 6.2×10′.

The decoy feed was then diluted with 0.1 M sodium phosphate (IT
H7,0) 10.2M Na C1i! 10.1%S
Toyo Soda-G3000S equilibrated with DS buffer
Glu filtration was performed using W (column size 7.5 x 600 mm) at a flow rate of 111Q/min and 11IIQ/fraction. The activity recovery rate in this method was 28%, and the precision increased by 5.4 times. The activity recovery rate throughout the entire process is 3
．． 6%, and the degree of purification was 3.35X105 times.

The specific activity of the low molecular weight protein having this physiological activity is 9.
It was 44 x 10' units/ma protein.

Example 7 Formalin-killed Corynebacterium parvum (manufactured by Wellcome, UK) 10+11 (] was administered intraperitoneally to female Wistar rats (body weight 200-250 g), and 9 days after the injection, 250 μg of endotoxin (bacillus Bacillus lipopolysaccharide) was administered. was injected into the tail vein. Two hours after endotoxin administration, blood was collected from the abdominal vena cava under ether anesthesia. The collected blood was centrifuged at 5,000 rpm for 20 minutes to separate serum. , 100
Serum aoe+nQ with a titer of 1500 units/III Q was obtained from 1 rat.

After adding and dissolving 47.2++ ammonium sulfate to the serum 100+11 (1!), 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)
Then, collect the precipitate and add about 20 mQ of 0.02M IJ Soot IF [buffer solution 10. IM Na GQ (pH 7,8) was heated. The activity recovery rate at this stage was 94%, and 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 10,000 rpm for 30 minutes to remove insoluble substances. Take 110all of this supernatant and add 10.0ml of 0.02M Tris-salt.
Gel i filtration (column size 26X1) using Ultrogel AcA34 equilibrated with 5M Na CQtli buffer.
000 mm) L, elution was fractionated. The activity recovery rate by this method was 90%, and the degree of purification was 47 times higher. The activity recovery rate through all steps was 84.6%, and the degree of purification was about 987 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) 86.8) overnight at 4°C.

This dialysis active fraction was dissolved in 0.02M phosphate buffer (D H
It was applied to a hydroxyapatite gel column (size 26×4001111) equilibrated with 6,8). Flow rate 5.
0111! /hour, 3112/fraction after thorough washing with the same buffer, 0.02M phosphoric acid*W solution (pH 6,
8>500IQ and 0.5M phosphate buffer (pH 6,
8) Using 500 mA, the concentration gradient was continuously raised to elute the adsorbed material. The active fractions were collected and ultrafiltrated and concentrated using an Amicon ultrafiltration concentrator [TCF-10] at 4°C. 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 mixed with 0.02M To'J.Su salt r12 (pH 7,8) 10, IM NaC.
Dialysis was performed overnight at 4°C against Q buffer. The dialysis solution was applied to a Pharmacia MonoQ column buffered with the same M buffer, and the substance was adsorbed under the conditions of 1fflQ/min and 1filQ/fraction. Then, after washing thoroughly with the same buffer,
．． 02M 1~Lis-hydrochloric acid (pH 7,8)/1. OM
The adsorbed active fraction was eluted by increasing the concentration of NaC(!) using NaCQ buffer. The active fraction was concentrated by ultrafiltration. The activity recovery rate with this method was 81%.
The degree of purification was increased by 8.1 times. The activity recovery rate throughout the entire process was 43.9%, and the VI production degree was 5.83X.
It was 10' times larger.

Next, the above concentrate was diluted with 0.1M Tris-HCl (EI
Toyo Pearl 5W60 (column size 10 x 500 mg+) equilibrated with H7,8)/70% saturated ammonium sulfate,
It was applied at a flow rate of 1 mQ/min and 2 mQ/fraction, and salted out in the column. Then 0.1M Tris-HCl (pH 7,8)
Elution was carried out using a buffer solution of 20% by decreasing the ammonium sulfate concentration continuously.

The active fraction was concentrated using an ultrafiltration concentrator. The activity recovery rate by this method was 38%, f! The degree of production was 5.1 times higher. The activity recovery rate throughout the entire process was 16.7%, and the N production degree was 2.97X10' times.

Next, the above concentrate was diluted with 0.1M sodium phosphate (p
H7,0) 10.2M Na CQ10.1%5DSi
Toyo Soda G3000SW equilibrated with l box liquid (
Using a column size of 7.5 x 600 mm, the flow rate was 41Q.
Gel i filtration was performed at 1111 Q/min, 1111 Q/min. The activity recovery rate in this method was 39%, and the purity was 6.1.
It has increased twice. The activity recovery rate throughout the entire process was 6.5%, and the degree of purification was 1.81×10” times.

The specific activity of the low molecular weight protein having this physiological activity is 9.
05 x 10' units/+11111 proteins.

[Brief explanation of drawings]

FIG. 1 is a molecular weight distribution map obtained by measuring the molecular weight of the substance of the present invention by the gel casting method using Biogel A1.5n+. Figure 2 is Toyo Soda TSK Gel G3000SW
This is an elution pattern of the substance of the present invention obtained by a gel filtration method using . FIG. 3 is a molecular weight distribution diagram of the substance of the present invention based on 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 molecule M distribution diagram of the substance of the present invention, which is drawn from FIG. 4. FIG. 6 is an ultraviolet absorption spectrum analysis diagram of the substance of the present invention. (The above) --1 "I' Figure 3 A Gen T To-Gin tjA czu〉) 'its lto 121 Figure 5 sum") ゛Uniformity degree 6121 5 good night (r' + + η)

Claims (1)

[Claims] ■ A low-molecular protein having the following physical and chemical properties and structural characteristics. a) Molecular mouth: 16000±1500 b) Isoelectric point: pH3.8±0.3 0) 0. The maximum ultraviolet absorption value in IM Na CQ and 0.02M Tris-HCl buffer (pH 7,8) was 27.
7nI11. d) 3ma protein fjk/, mQ of 0.02M Tris-HCl buffer (pH
7,8) The I solution is colorless and transparent, and precipitation occurs when acetone or ethanol is added to the solution at 70 V/V% or more.e) The aqueous solution is weakly acidic.f) Biuret reaction, Folin-Lowry reaction method, and hydrochloric acid hydrolysis. g) Amino acid composition ratio: The amino acid composition ratio (molar ratio) analyzed with an amino acid analyzer after hydrolysis with 6N hydrochloric acid at 110°C under reduced pressure for 24 hours is as follows: Gly 1.00 AS+) and/or ASrll, 10±0.06Thr, where Gly is 1.00.
0.45±0.02 Ser O, 90±0.05 Glu and/or Qlnl, 82±0.09Pro 0
．． 84±0.04 Ala 1.11±0.06 Cys O,20±0.02 Val O,86±0.04 Met O,18±0.01 11e O,38±0.02 Leu 1.73± 0.09 Tyr O, 64±0.03 Phe O, 34±0.02 His O, 28±0.01 LIS O, 44±0.02 Arc 0.46±0.02 h) Amino acid sequence By amino acid sequencer The 17 amino acids from the amino terminal side have the following sequence. H-8Ia-L eu-Ser-A sD-L
ys-Pro・-Leu-Ala-His-
Val-Vat-ΔIa-ASn-Pro-
G In -V at -G Iu-