JPH0665295A - Immunosuppressive substance and fractionation method thereof - Google Patents

Abstract

(57) [Summary] [Purpose] To discover and isolate novel immunosuppressive substances from animals other than cattle and mice, which are known to have substances with immunological activity in the salivary glands. [Structure] Obtained from rat submandibular gland, in the form of white powder,
The molecular weight measured by SDS-10% polyacrylamide disk electrophoresis and Sephadex G-100 gel filtration was in the range of 15-45 kD (kilodalton), and the isoelectric point was pH 4.5-6. Within the range of 0.5, soluble in water and saline, insoluble in acetone,
The ninhydrin reaction, biuret reaction, miron reaction, Sakaguchi reaction, Morish reaction, anthron reaction, and Elson-Morgan reaction are all positive, and the partition coefficient (Kav value)
Is within the range of about 0.25 to 0.85.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel immunosuppressive substance obtained from rat submandibular gland and a method for fractionating the same.

[0002]

BACKGROUND OF THE INVENTION Mammalian salivary glands, especially parotid glands, contain salivary gland hormones, which act on mesenchymal tissues, particularly hard tissue growth promoting action, serum calcium lowering action, and reticuloendothelial system. It has a physiological action such as a stimulating action and a leukocyte increasing action, and is widely used as a medicine.

In 1974, Aonuma et al. Isolated a salivary gland hormone subunit having a molecular weight of 45,000 and having salivary gland hormone activity derived from bovine parotid gland [Folio. E
ndocrinol. Jpn. , 50 , 1468 (19
74)]]. Subsequently, Aonuma et al. Enzymatically decomposed the salivary gland hormone subunit in 1977 to separate FrAA-1 having a leukocytosis effect and FrH-1 having a human calcium-lowering effect and determined the structures thereof [Che.
m. Pharm. Bull. , 28 , 417 (198
0) and JP-B-57-35958].

[0004] In 1983, Ishizaka et al. Stimulated lymphocytes to stimulate salivary gland hormone derived from bovine parotid gland or its subunit and FrAA-1 in mice and humans to nonspecifically enhance antibody productivity. It has been found to have an effect [Immunopharmacolog
y, 6 , 133 (1983)].

[0005] In addition, delayed hypersensitivity in the salivary glands of mice.
y) It is known that there are factors that influence the response (M. Hiramatsu et al., Imm.
unology, 1979, 37, 869), and in recent years, studies on substances having immunosuppressive activity in mouse submandibular gland extracts have been carried out [Hamuro et al., 1990 Summary of Clinical Immunology Society, D80; Murakami et al. , Proc. Of the 64th General Meeting of Japanese Bacteriology, F-III-14; Hamuro et al., Journal of Japanese Bacteriology,
42 (2), 1992, etc.].

[0006]

DISCLOSURE OF THE INVENTION The object of the present invention is to find a novel immunosuppressive substance from animals other than cattle and mice, which are known to have substances having an immunological activity in salivary glands, To isolate.

[0007]

Based on the above report, the present inventors have examined whether or not a substance having an immunological activity is present in the salivary glands of various animals such as pigs, rats, guinea pigs, rabbits as well as bovines and mice. Was examined.

The salivary glands of various animals are divided into the parotid gland and the submandibular gland, and the immunoreactivity of each is measured by the PBA activity test.

[0009] The PBA activity test is a test method by which it is possible to know whether or not a substance (sample) has an immune activity by measuring its activating activity on polyclonal B cells. B cells in the spleen are non-specifically activated to a certain type of antigen to induce the production of IgM antibody, and a substance to be examined whether or not they have immunoreactivity is added to the B cell. It is a method of determining the presence or absence of immunoreactivity depending on whether or not the production of is increased. That is, after the extracted mouse spleen cells and the sample solution were mixed and cultured at 37 ° C., protein A-coated sheep red blood cells, guinea pig complement, and anti-IgM serum were added and mixed, and further 0.5% After adding this mixture on agar, it culture | cultivates at 37 degreeC and the number of the hemolytic plaque (plaque) formed is measured. The number of plaques is compared with the number of plaques on agar treated in the same manner without adding the sample, and the presence or absence of immunoreactivity (including both suppression and activation) of the sample is determined.

The PBA activity test was carried out by centrifuging (18000 rpm, 10 minutes) an extract of water or water containing salts of the parotid gland and submandibular gland of each animal, and the resulting supernatant was adjusted to pH 4 with 1N hydrochloric acid. The pH was adjusted to 0.5, the formed precipitate was removed, and the solution was adjusted to pH 7.0 with a 1N sodium hydroxide solution.

The results are shown in Table 1.

[0012]

[Table 1]

Rats, mice, which are generally taxonomically related,
It is presumed that an immunologically active substance having the same property may be present in the salivary glands of guinea pigs, rabbits and the like, but it is clear from the results in Table 1 above that this estimation does not hold.

The present inventors individually examined immunologically active substances obtained from various animals. As a result, as is clear from Table 1, it was confirmed that a substance having an action of nonspecifically suppressing antibody production was present in the rat submandibular gland, and intensive studies were conducted on its separation and purification. The present invention has been completed by finding a method capable of fractionating, clarifying its physicochemical properties, and completing the present invention.

The immunosuppressive substance of the present invention (hereinafter abbreviated as "the substance of the present invention") is a protein having the following physicochemical properties.

(1) Properties: white powder (2) Molecular weight: SDS-10% polyacrylamide The molecular weight measured by the disc electrophoresis method and Sephadex G-100 gel filtration method is in the range of 15 to 45 kD (kilodalton). It is inside. (3) Isoelectric point: pH is in the range of 4.5 to 6.5. (4) Solubility: Soluble in water and physiological saline, insoluble in acetone. (5) Color reaction: ninhydrin reaction, positive biuret reaction, positive miron reaction, positive Sakaguchi reaction, positive Morish reaction, positive anthron reaction, positive Elson-Morgan reaction, positive (6) partition coefficient (Kav value): about 0.25 to 0.85 It is inside. The partition coefficient (Kav value) is the partition coefficient between the gel layer and the liquid layer in gel filtration, and is calculated by the following formula. Kav = (Ve-Vo) / (Vt-Vo) Vt = total volume of gel bed Ve = amount of eluent Vo = amount of solvent outside gel particle

Sephadex is used as a gel filtration material.
G-100 (Pharmacia Fine Chem
icals) and 0.2 M Tris buffer, pH 7.4, was used as the eluent.

The substance of the present invention was prepared by removing the precipitate formed by centrifuging the extract of rat submandibular gland water or water containing salt and adjusting the pH of the resulting supernatant to about 4.5. The solution is subjected to molecular sieve chromatography and the molecular weight is 15-45.
It is obtained by a fractionation method comprising a step of collecting a kD fraction.

Here, the water containing salt is about 0.1M.
(About 1.0% by weight) sodium chloride, potassium chloride,
Examples thereof include ammonium sulfate and sodium sulfate.

The substance of the present invention comprises a step of subjecting a fraction having a molecular weight of 15 to 45 kD to affinity chromatography using an aprotinin-affinity chromatography column to collect a fraction which is not adsorbed on the column. Is further fractionated.

Of the substances of the present invention, both the fraction not adsorbed on the aprotinin-affinity chromatography column and the fraction adsorbed have immunosuppressive activity, but the fraction not adsorbed on the column has protease activity. It differs from the fraction adsorbed on the column in that it is not.

The method of fractionating the substance of the present invention will be described in detail below. A homogenate of the rat submandibular gland was extracted with water, and the resulting water extract was centrifuged (usually 5000-2).
0000 rpm, 5 to 40 minutes, preferably 15000
˜18,000 rpm, 20 to 30 minutes) and separate the supernatant. Acids such as hydrochloric acid and acetic acid are added to the supernatant to adjust the pH to about 4.5, and the mixture is centrifuged again (usually 5000 to 200).
00 rpm, 5 to 40 minutes, preferably 15000 to 1
8000 rpm, 20-30 minutes) to remove the formed precipitate. The solution after removing the precipitate is subjected to molecular sieve chromatography to obtain a fraction having a molecular weight of 15 to 45 kD.

The molecular sieving operation by the molecular sieving chromatography is carried out by filling the column with gel particles such as dextran and polyacrylamide. Examples of the dextran gel that can be used in the present invention include Sepha
dex G-75, G-100, G-200 and Se
phacryl S-200 (PharmaciaFi
ne Chemicals), as polyacrylamide gel, for example, Bio-Gel P-60 and P
-100 (Bio-Rad Laboratories
Manufactured). As the packed column for molecular sieve, for example, TSK gel G3000 SW, G20
00 SW (manufactured by Tosoh Corporation), Asahipak GS-3
10, GS-510 (manufactured by Asahi Kasei) and the like. As the buffer solution that can be used for development, it is preferable to use the buffer solution used for equilibrating the gel particles, and for example, 0.2 M Tris buffer solution (pH 7.4) and the like can be mentioned. The molecular sieving operation can be carried out by a method known per se, and the above-mentioned molecular sieving operations can be performed alone or in combination with each other to easily obtain a target fraction having a molecular weight of 15 to 45 kD.

Molecular weight 1 which is the substance of the present invention obtained above
The 5-45 kD fraction can be stored in the form of a solution, but it is preferably frozen and / or dried for storage. Drying can be performed by a usual method such as reduced pressure drying, freeze drying, and acetone drying.

The fraction having a molecular weight of 15 to 45 kD, which is the substance of the present invention, can be further fractionated by subjecting it to affinity chromatography with an aprotinin-affinity chromatography carrier shown below.

As described above, the substance of the present invention having a molecular weight of 15 to 45 kD obtained by the molecular sieving operation is further subjected to affinity chromatography with an aprotinin-affinity chromatography column to collect a fraction which is not adsorbed on the column. Examples of the aprotinin-affinity chromatography carrier used for affinity chromatography include aprotinin-agarose resin, aprotinin-acrylic resin and the like. Prior to carrying out affinity chromatography, the aprotinin-affinity chromatographic carrier described above is equilibrated with a buffer. As the buffer solution, various conventionally used buffer solutions can be used, and examples thereof include 0.02M Tris buffer solution (pH 8.0).

The equilibrated aprotinin-affinity chromatographic carrier is packed in a column, and then the substance-containing solution of the present invention obtained by the above-mentioned molecular sieving operation is added to the column so that it is not adsorbed to the aprotinin-affinity column using a buffer solution. Elute the fractions. Then, the eluate containing this fraction is desalted by dialysis or the like and then freeze-dried to obtain a fraction which is not adsorbed on the aprotinin-affinity column. The fraction adsorbed on the aprotinin-affinity column is eluted and collected using an acetate buffer (pH 4.0) containing sodium chloride.

The thus-obtained substance of the present invention (including both the substance obtained by molecular sieve chromatography and the substance obtained by subjecting the same to further affinity chromatography) may, if necessary, itself. Adsorption chromatography with known anion exchanger, isoelectric point fractionation by electrophoresis, isoelectric point fractionation with anion exchanger, chromatography using porous glass, chromatography using hydrophobic adsorbent, reverse phase It can be further purified by subjecting it to high performance liquid chromatography or the like.

The method by isoelectric focusing by electrophoresis is as follows:
A stable pH gradient is formed beforehand in a free solution or an appropriate gel by applying current, and the pH gradient is
The substance of the present invention is allowed to migrate to the same pH layer as its isoelectric point,
In this method, the target substance is concentrated in the pH layer and then extracted from the concentrated fraction. As the free solution or gel, a density gradient solution of sucrose or a polyacrylamide gel can be used. In order to concentrate the substance of the present invention in the pH layer, it takes about 48 hours in the case of the free solution and about 40 hours in the case of the gel. It is necessary to energize for 6 hours. The substance of the present invention has a pH of 4.5 to
It can be obtained from within 6.5 layers.

The chromatographic method using porous glass is carried out by setting the aqueous solution containing the substance of the present invention to acidic, preferably pH 4.5 to 6.5, and then applying CPG-10 (C
PG, INC. (Manufactured by K.K.) and then an amino acid such as glycine or proline is added to the solution to elute the substance of the present invention with an aqueous solution having an alkaline property, preferably pH 7.5 or higher.

The method by chromatography using a hydrophobic adsorbent is, for example, as a hydrophobic adsorbent, butyl-Sepharose CL-6B, octyl-Sepharose CL-.
6B or phenyl-Sepharose CL-6B (Pha
Rmcia Fine Chemicals, etc.) is packed in a column, the substance of the present invention is added to a solution containing about 10 to 20% of an inorganic salt such as ammonium sulfate, sodium chloride, etc., and the mixture is passed through the column for purification. .

As a method of isoelectric point fractionation using an anion exchanger, there can be mentioned a method of using chromatofocusing for fractionating an aqueous solution of protein by utilizing the difference in isoelectric point of protein. Examples of ion exchangers used for chromatofocusing include PBE94 anion exchanger (Pharmacia Fine Chemica).
ls) and the like. The anion exchanger can be packed in a column, the liquid containing the substance of the present invention can be added, and the substance of the present invention can be eluted in a further purified form using the eluent. As the eluent, for example, polybuffer 74 (Pharma)
Cia Fine Chemicals) and the like are used.

The immunosuppressive substance of the present invention thus obtained is useful as an immunosuppressive agent for collagen disease, rheumatic disease, organ transplantation and the like.

The substance of the present invention can be administered as such a therapeutic agent by either oral administration or parenteral administration. The substance of the present invention can be prepared into various dosage forms, for example, oral preparations (tablets, granules, powders, capsules, etc.), transmucosal preparations (suppositories, nasal preparations, etc.), injections (solvents, frozen preparations). Desiccant etc.). Examples of auxiliary agents that can be used in the preparation include sodium chloride, glycine, lactose, mannitol, sorbitol, sucrose, starch, dextran, gelatin and the like.

When the substance of the present invention is used as a clinical therapeutic agent, the effective dose depends on the type of disease, the route of administration, the severity of symptoms,
It should be appropriately changed depending on the age of the patient, etc., but generally it can be used at about 0.01 to 10 mg / kg per day.

[0036]

EXAMPLES The present invention will be further described below with reference to examples.

Example 1 (Example of Fractionation of Substance of the Present Invention) 100 g of submandibular gland of SD rat (7 to 10 weeks old) was homogenized and extracted with 400 ml of water. The obtained water extract was centrifuged at 18000 rpm for 20 minutes to separate the supernatant. Add 1.0 N hydrochloric acid to the supernatant to adjust the pH of the supernatant.
Was adjusted to 4.5, a precipitate was formed and a further 18000
After centrifugation at rpm for 20 minutes, the supernatant was separated. Next, 1200 ml of acetone was added to the separated supernatant liquid (hereinafter referred to as “separated liquid”), and the immunoreactive fraction was concentrated and collected as an acetone precipitate.

The acetone precipitate of the obtained immunoreactive fraction was dissolved in 60 ml of 0.2 M Tris buffer (pH 7.4), and Sephadex G-10 equilibrated with the same buffer.
0 column (Pharmacia Fine Chemi
cals). Elution was performed with the same buffer, and a solution of a fraction containing the substance of the present invention having a molecular weight of 15 to 45 kD was recovered. The collected fraction solution was desalted by dialysis, and then freeze-dried to obtain 3.0 g of the fraction (A) of the substance of the present invention (yield: 3
%) Was obtained.

Obtained fraction having immunosuppressive activity (A)
The results of measuring the physicochemical properties of are shown below.

(1) Properties: white powder (2) Molecular weight: 15 to 45 kD (3) Isoelectric point: pH 4.5 to 6.5 (4) Solubility: Soluble in water and physiological saline, insoluble in acetone . (5) Color reaction: ninhydrin reaction, biuret reaction,
Milon reaction, Sakaguchi reaction, Morish reaction, Anthron reaction, and Elson-Morgan reaction are all positive. (6) Partition coefficient (Kav value): Approximately 0.25 to 0.85 (Note that among the measuring methods of the above physicochemical properties (1) to (6), the measuring method described in this specification is This method is used, and the standard method is used if not mentioned.)

Next, the fraction (A) obtained in the above step was dissolved in 20 ml of 0.02M Tris buffer (pH 8.0) and applied to an aprotinin-agarose column (manufactured by Sigma) equilibrated with the same buffer. Was added. Elution was performed with the same buffer solution, and the solution of the fraction not adsorbed on the column was collected. The collected fraction solution was desalted by dialysis and then lyophilized to obtain 1.2 g (yield: 1.2%) of the fraction (B) of the substance of the present invention which was not adsorbed on the aprotinin-agarose column. It was

Obtained fraction having immunosuppressive activity (B)
As a result of measuring the physicochemical properties of the fraction (B), the fraction (B) had the same physicochemical properties as the fraction (A).

The fraction (B) obtained in the above step was dissolved in 20 ml of an imidazole buffer (pH 7.0) and equilibrated with the same PEG94 anion exchanger column (Ph).
armacaine Fine Chemicals). Poly buffer 74 buffer (pH 4.0)
(Pharmacia Fine Chemicals
The product having the immunosuppressive activity was collected. The immunosuppressive activity was determined by the PBA activity test method (the same applies hereinafter). An ammonium sulfate aqueous solution was added to the collected fraction to dissolve it, and the ammonium sulfate concentration of this solution was adjusted to 10% by weight. Butyl-Sepharose CL equilibrated with a phosphate buffer (pH 7.0) containing 10% ammonium sulfate.
-B column (Pharmacia Fine Chem
icals), the column was washed with the same buffer, and then eluted with a gradient from 10% ammonium sulfate to water (concentration gradient) to collect a solution containing a fraction having immunosuppressive activity. A solution of the obtained fraction having immunosuppressive activity is desalted by dialysis and then lyophilized to give 600 mg of a fraction (C) having immunosuppressive activity (yield: 0.6
%) Was obtained.

The physicochemical properties of the obtained fraction (C) were measured. As a result, the fraction (C) had the same physicochemical properties as the fraction (B).

Example 2 Of the substance of the present invention obtained in the same manner as in Example 1, 1.0 g of the freeze-dried product of the fraction (B) which was not adsorbed on the aprotinin-agarose column was added to 0.02 M Tris buffer (pH 6.5). )
It was dissolved in 20 ml. The obtained solution was equilibrated with the same buffer solution as a DEAE-Sephadex column (Pharm).
Acaine Fine Chemicals), the column was washed with the same buffer, and a solution of a fraction having immunosuppressive activity, which was eluted without adsorbing to the column, was collected.
The immunosuppressive activity was determined by the PBA activity test method (the same applies hereinafter). The solution of the obtained fraction having immunosuppressive activity was desalted by dialysis and then concentrated by freeze-drying. Then, this concentrated solution was added to a TSK gel ODS-120T column (manufactured by Tosoh Corporation) for high performance liquid chromatography, and a linear gradient from 10% acetonitrile containing 0.1% TFA to 50% acetonitrile containing 0.1% TFA was added. (Linear concentration gradient) Elution was performed. The solution of the fraction having immunosuppressive activity was collected, concentrated under reduced pressure,
The obtained concentrated solution is desalted by dialysis and then freeze-dried,
Fraction having immunosuppressive activity (D) 300 mg (yield:
0.3%) was obtained.

As a result of measuring the physicochemical properties of the obtained fraction (D), the fraction (D) had the same physicochemical properties as the fraction (B).

An immunosuppressive activity test was carried out in Example 3 below using the fraction (D) having immunosuppressive activity obtained here.

Example 3 (Test example of immunosuppressive activity of the substance of the present invention) The immunosuppressive activity of the substance of the present invention was measured by IgM,
It was performed by measuring the number of cells producing three types of antibodies, IgG and IgA.

That is, DBA / 2 mice aged 6 to 8 weeks were dislocated and killed, and the spleens were excised. A mixture of the spleen cell suspension prepared from the extracted spleen and the substance of the present invention (fraction (D)) obtained by the method described in Example 2 above was incubated at 37 ° C. for 3 days.
Cultured for ~ 5 days. The number of antibody-producing cells in the cultured spleen cell suspension was measured by E. Gronowieg, A .; Co
utinho, Eur. J. Immunol. , 6 , 5
88 (1976). That is, the cultured spleen cell suspension was incubated with protein A-coated sheep red blood cells, guinea pig complement and anti-Ig subclass antiserum at 37 ° C. for 4 hours. Further, after adding this mixture on 0.5% agar, the mixture was incubated at 37 ° C., the number of hemolytic spots that appeared was measured, and the number of hemolytic plaque forming cells per 10 ⁶ viable spleen cells (PFC) was measured. Was calculated. The measurement results are shown in Table 2.

[0050]

[Table 2]

From the results shown in Table 2, it is clear that the substance of the present invention has immunosuppressive activity.

[0052]

Industrial Applicability According to the present invention, it was found that a novel immunosuppressive substance exists in the rat submandibular gland, and this immunosuppressive substance can be fractionated.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] August 20, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0004

[Correction method] Change

[Correction contents]

Further, in 1983, Ishizaka et al., Enhances the non-specific antibody production by stimulated lymphocytes in mice and humans salivary gland hormones or subunits and FrAA-1 thereof from bovine parotid It has been found to have an effect [Immunopharmacolog
y, 6, 133 (1983)].

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction contents]

The PBA activity test is a test method by which it is possible to know whether or not a substance (sample) has an immunological activity by measuring its activating activity on polyclonal B cells. B cells in the spleen are non-specifically activated to a certain type of antigen to induce the production of IgM antibody, and a substance to be examined whether or not they have immunoreactivity is added to the B cell. It is a method of determining the presence or absence of immunoreactivity depending on whether or not the production of is increased. That is, the extracted mouse spleen cells and the sample solution were mixed and cultured at 37 ° C., and then the cultured cells were mixed with protein A-coated sheep red blood cells, guinea pig complement and anti-IgM serum,
Further, after adding this mixture on 0.5% agar, 3
The cells are cultured at 7 ° C and the number of hemolytic plaques (plaques) formed is measured. The number of plaques is compared with the number of plaques on agar treated in the same manner without adding the sample, and the presence or absence of immunoreactivity (including both suppression and activation) of the sample is determined.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Correction contents]

The PBA activity test was carried out by centrifuging (18000 rpm, 10 minutes) an extract of water or water containing salts of the parotid gland and submandibular gland of each animal.
Was carried out using a solution adjusted to pH 7.0 with a 0.1N sodium hydroxide solution.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0012

[Correction method] Change

[Correction contents]

[0012]

[Table 1]

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction contents]

Here, the water containing salt is about 0.1M.
(About 0.6 % by weight) sodium chloride, potassium chloride,
Examples thereof include ammonium sulfate and sodium sulfate.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0031

[Correction method] Change

[Correction contents]

The method by chromatography using a hydrophobic adsorbent is, for example, butyl-sepharose 4B , octyl-sepharose CL- 4B or phenyl-sepharose CL- 4B (Pharma) as the hydrophobic adsorbent.
Cia Fine Chemicals) and the like are packed in a column, the substance of the present invention is added to a solution containing about 10 to 20% of an inorganic salt such as ammonium sulfate and sodium chloride, and the mixture is passed through the column for purification. .

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0043

[Correction method] Change

[Correction contents]

The fraction (B) obtained in the above step was dissolved in 20 ml of an imidazole buffer (pH 7.0) and equilibrated with the same PEG94 anion exchanger column (Ph).
armacaine Fine Chemicals). Poly buffer 74 buffer (pH 4.0)
(Pharmacia Fine Chemicals
The product having the immunosuppressive activity was collected. The immunosuppressive activity was determined by the PBA activity test method (the same applies hereinafter). An ammonium sulfate aqueous solution was added to the collected fraction to dissolve it, and the ammonium sulfate concentration of this solution was adjusted to 10% by weight. Butyl-Sepharose 4B equilibrated with a phosphate buffer (pH 7.0) containing 10% ammonium sulfate.
Column (Pharmacia Fine Chemical
Als) and washed the column with the same buffer,
A gradient (concentration gradient) from 10% ammonium sulfate to water was eluted, and a solution containing a fraction having immunosuppressive activity was collected. The obtained solution of the fraction having immunosuppressive activity was desalted by dialysis and then lyophilized to obtain 600 mg of a fraction (C) having immunosuppressive activity (yield: 0.6%).

[Procedure Amendment 8]

[Document name to be amended] Statement

[Name of item to be corrected] 0045

[Correction method] Change

[Correction contents]

Example 2 Of the substance of the present invention obtained in the same manner as in Example 1, 1.0 g of the freeze-dried product of the fraction (B) which was not adsorbed on the aprotinin-agarose column was added to 0.02 M Tris buffer (pH 6.5). )
It was dissolved in 20 ml. The obtained solution was equilibrated with the same buffer solution as a DEAE-Sephadex column (Pharm).
Acaine Fine Chemicals), the column was washed with the same buffer, and a solution of a fraction having immunosuppressive activity, which was eluted without adsorbing to the column, was collected.
The immunosuppressive activity was determined by the PBA activity test method (the same applies hereinafter). The solution of the obtained fraction having immunosuppressive activity was desalted by dialysis and then concentrated by freeze-drying. Next, this concentrated solution was added to a TSK gel ODS-120T column (manufactured by Tosoh Corporation) for high performance liquid chromatography, and a linear gradient from 20 % acetonitrile containing 0.1% TFA to 80 % acetonitrile containing 0.1% TFA was added. (Linear concentration gradient) Elution was performed. The solution of the fraction having immunosuppressive activity was collected, concentrated under reduced pressure,
The obtained concentrated solution is desalted by dialysis and then freeze-dried,
Fraction having immunosuppressive activity (D) 300 mg (yield:
0.3%) was obtained.

[Procedure Amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0049

[Correction method] Change

[Correction contents]

That is, DBA / 2 mice aged 6 to 8 weeks were dislocated and killed, and the spleens were excised. A mixture of the spleen cell suspension prepared from the extracted spleen and the substance of the present invention (fraction (D)) obtained by the method described in Example 2 above was incubated at 37 ° C. for 3 days.
Cultured for ~ 5 days. The number of antibody-producing cells in the cultured spleen cell suspension was measured by E. Gronowieg, A .; Co
utinho, Eur. J. Immunol. , 6, 5
88 (1976). That is, 0 spleen cells suspension cultured protein A-coated sheep red blood cells, guinea pig complement and anti-Ig classes antiserum co.
After added pressure on 5% agar, incubated for 4 hours at 37 ° C., and measuring the number of hemolysis plaques appearing, 10 per ^six numbers spleen cells hemolytic plaque forming cells number (PFC) was calculated. The measurement results are shown in Table 2.

Claims (3)

[Claims] 1. An immunosuppressive substance which is obtained from the rat submandibular gland and has the following physicochemical properties. (1) Property: White powder (2) Molecular weight: SDS-10% Polyacrylamide The molecular weight measured by the disk electrophoresis method and the Sephadex G-100 gel filtration method is within the range of 15 to 45 kD (kilodalton). is there. (3) Isoelectric point: pH is in the range of 4.5 to 6.5. (4) Solubility: Soluble in water and physiological saline, insoluble in acetone. (5) Color reaction: ninhydrin reaction, positive biuret reaction, positive miron reaction, positive Sakaguchi reaction, positive Morish reaction, positive anthron reaction, positive Elson-Morgan reaction, positive (6) partition coefficient (Kav value): about 0.25 to 0.85 It is inside. The partition coefficient (Kav value) is the partition coefficient between the gel layer and the liquid layer in gel filtration, and is calculated by the following formula. Kav = (Ve-Vo) / (Vt-Vo) Vt = total volume of gel bed Ve = amount of eluent Vo = amount of solvent outside gel particle 2. An extract of rat submandibular gland water or water containing salt is centrifuged, and the resulting supernatant liquid has a pH of about 4.
5. A method for fractionating an immunosuppressive substance, which comprises a step of adjusting the solution to 5 and removing the generated precipitate, and subjecting the solution to molecular sieve chromatography to collect a fraction having a molecular weight of 15 to 45 kD. 3. The method according to claim 2, further comprising the step of subjecting a fraction having a molecular weight of 15 to 45 kD to affinity chromatography using an aprotinin-affinity chromatography column, and collecting a fraction which is not adsorbed on the column. the method of.