JP2982078B2 - Canine epidermal growth factor, anti-inflammatory agent or cosmetic containing the canine epidermal growth factor as an active ingredient, and method for producing canine epidermal growth factor cEGF - Google Patents

Description

The present invention relates to a canine epidermal growth factor having a novel structure, an anti-inflammatory agent or a cosmetic comprising the canine epidermal growth factor as an active ingredient, and the canine. The present invention relates to a method for producing epidermal growth factor.

[Conventional technology]

In 1972, Cohen et al. Used mouse epidermal growth factor (mEGF) as a polypeptide that promotes eyelid cleavage and incisor eruption in neonatal mice from male submandibular glands. And its structure was determined (J. Biol. Chem., 247, 7612-762).
1 (1972)].

Subsequently, in 1975, H. Gregory introduced β-protein as a polypeptide having gastric acid secretion inhibitory activity from human urine.
Urogastrone (human EGF) was isolated and its structure determined [Nature, 257, 325-327 (1975)].

In 1985, Simpson et al. (RJSimpson) et al. Succeeded in isolating a polypeptide consisting of 48 amino acid residues from rat submandibular gland using the binding to the EGF receptor as an index, and modified the primary structure of rat EGF. First reported (Eur.
J. Biocherm., 153, 629-637 (1985)]. The report of Simpson et al. Also describes the primary structure of guinea pig EGF. Each of the above reported polypeptides consists of 48 to 53 amino acid residues and has three intramolecular disulfide bonds, and the homology of these amino acid sequences is as high as 70%. I have.

It is known that each of the above-mentioned polypeptides has a physiological function as a growth factor for each seed organism, which is a source of each, for example, mouse EGF promotes eyelid cleavage in newborn mice And is known to have an incisor eruption promoting action, but human E
It has not been fully elucidated what biological activities other than EGF have on the human body.

In addition, EGF is ubiquitous in mammals, and it is roughly understood that EGF is a polypeptide that plays an important role in the growth of various cells constituting the animal with respect to the animal species in which it exists. However, the actual state of EGF in animals of individual species has been scarcely elucidated, and only five EGF whose structure has been elucidated to date have been human, mouse, rat, rabbit and guinea pig.

[Problems to be solved by the invention]

In addition to EGF known so far, if a new EGF having utility can be found among various EGFs present in many mammals, it is highly practical in various fields such as medicine.

An object of the present invention is to provide a canine epithelial cell growth factor having a novel amino acid sequence which is different from polypeptides constituting EGF such as mouse, human, rat, rabbit and the like which are conventionally known.

Another object of the present invention is to provide an anti-inflammatory agent or cosmetic comprising the novel dog epidermal growth factor as an active ingredient.

It is another object of the present invention to provide a method for producing the novel canine epithelial cell growth factor easily, with high purity and in large quantities.

[Means for Solving the Invention]

The present invention focuses on dogs from various mammals and examines dog epithelial cell growth factor (Canine EGF, hereinafter referred to as “cEGF”).
The above-mentioned object has been achieved by focusing on the biological activity of the present invention, elucidating its structure, examining the usefulness of its pharmaceuticals and cosmetics, and examining its practical production method.

According to the present invention, there are provided cEGF having an amino acid primary sequence represented by the following formula (I), and an anti-inflammatory agent and cosmetic containing the cEGF as an active ingredient. (Hereinafter, in formula (I), X
The polypeptide when the coefficient m = 1 is referred to as “cEGF1”, and the polypeptide when the coefficient m = 0 as X is “cEGF2”
That. ) Formula (I) [N-terminal side] [C terminal side] Abbreviations relating to amino acids, the acid bases, and the like in the above description and the following description are in accordance with the provisions of IUPAC and IUB or conventional symbols in the art. An example is as follows.

Ala… Alanine, Arg… Arginine Asn… Asparagine, Asp… Aspartic acid Cys… Cistine, Cln… Glutamine Glu… Glutamic acid, Gly… Glycine His… Histidine, Ile… Isoleucine Leu… Leucine, Lys… Lysine Met… Methionine, Phe… Phenylalanine Pro ... Proline, Ser ... Serine Trp ... Tryptophan, Tyr ... Tyrosine Val ... Valine In the polypeptide of the present invention (hereinafter referred to as "cEGF"),
cEGF1 has a primary amino acid sequence when the coefficient m of X in the above formula (I) is m = 1, and cEGF2 has a primary amino acid sequence when the coefficient m of X in the above formula (I) is m = 0, and has the above amino acid composition. It is characterized by its novel structure and composition, which is not found in any known substances. It can be effectively used in various fields, as described later, by utilizing its biological activity. .

Hereinafter, the method for producing cEGF of the present invention will be described in detail.
GF can be produced from a tissue such as dog urine or submandibular gland according to a usual extraction method. Further, the cEGF of the present invention can be produced by a general peptide chemical synthesis method based on the amino acid sequence of the above formula (I), and can also be produced by a gene recombination technique.

If the above-mentioned extraction method is explained first, this can be carried out according to a usual method. The details are as shown in Examples below.

The isolation and purification of cEGF obtained according to the various methods described above is performed according to the method described above.
A composition containing cEGF, for example, from dog urine, etc., according to a general operation, a hydrophobic chromatography carrier, a gel filtration chromatography carrier, a reversed-phase high-performance liquid chromatography carrier, a cation exchange chromatography carrier,
It can be carried out by using several types of column chromatography carriers having different fractionation principles such as anion exchange chromatography carriers.

Confirmation of the thus obtained cEGF of the present invention can be performed by a technique such as a radio receptor assay (RRA) utilizing the binding of cEGF to EGF receptors present on various cell surfaces.

In addition, it was confirmed that the cEGF was purified to a high degree of purity, for example, by a single peak by high performance liquid chromatography (HPLC), and by Coomassie brilliant blue staining by polyacrylamide gel electrophoresis (PAGE). It can be easily performed using one band as an index.

Purified cEGF1 and cEG obtained as described above
The structure of F2 can be determined by means similar to ordinary means for analyzing the structure of polypeptides or proteins, for example, by measuring the amino composition using an amino acid analyzer or determining the amino acid sequence using a protein sequencer.

In addition, the SS binding site as found in the EGF molecule as found in other EGFs is obtained by subjecting purified cEGF to oxygen digestion using thermolysin, chymotrypsin or pepsin, followed by cation exchange column carrier and anion exchange. Separation into a fragment containing only one cysteine by separation by HPLC using a column carrier and reverse phase HPLC can be confirmed by amino acid analysis and sequence analysis.

Further, the biological activity of the cEGF of the present invention can be confirmed by, for example, the following various methods.

Cell growth promoting activity Cultured cells such as BALB / c3T3 or cultured cells such as mature rat hepatocytes were cultured in a medium supplemented with cEGF or human EGF as a control under low serum conditions, and labeled in the culture solution. By adding deoxyuridine, thymidine and the like, the amount of radioisotope incorporated into newly synthesized DNA is measured. The amount of radioisotope incorporated into new DNA is measured in proportion to the amount of radioisotope. In proportion to this radioisotope amount,
It can be seen that new DNA synthesis was performed, that is, cell proliferation was promoted.

Colony-forming activity on soft agar Cells such as NRK49F (rat kidney fibroblast) hardly grow on soft agar medium, but grow in the presence of EGF in the presence of TGFP-β (transforming growth factor type β). To form a colony (JEDeLarco
and GJTadaro.Proc.Natl.Acad.Sci., USA., 75 , 4001-
(1978), ABRobertet ai., Proc. Natl. Acad. Sci., US
A., 77 , 3494-3498 (1980), etc.]. That is, EGF has the above-mentioned soft agar colony forming activity.

Neonatal mouse eyelid cleaving and incisor eruption-promoting activity Neonatal mice are injected subcutaneously with cEGF every 24 hours, and the days at which the eyelids of each test animal open and the incisors appear are recorded.

According to the administration of cEGF, the number of days required for such administration is significantly reduced.

The cEGF of the present invention has a biological activity as described above, which has a pharmacological action since it has the properties of epidermal growth factor, and its composition is particularly useful as a medicine or cosmetic. It is. Pharmaceuticals include gastric acid secretion inhibitor, anti-living agent, gastrointestinal mucosa protective agent, calcium release promoter,
It is useful as an analgesic, an anti-inflammatory, a growth promoter, an intestinal function improver, a therapeutic agent for liver disease, a dermatological agent, a wound treatment enhancer, or a corneal repair agent, an ophthalmic agent such as eye drops, and the like. As cosmetics, it is useful together with various cosmetic bases as basic cosmetics such as lotions and emulsions, and makeup cosmetics such as lipsticks.

The therapeutic agent for liver disease of the present invention comprises the cEGF of the present invention subcutaneously at 10 mg / kg and intravenously at 1 mg / kg (about 1 million of the human blood EGF concentration) for each group of 6 male and female mice and 6 rats. Double and 10
Even if administered, it does not change the general symptoms and has low toxicity. The dose may be determined according to the age, weight, and symptoms of the patient. Orally and parenterally (including injection), it is usually desirable to use about 10 ng to 10 mg as an active ingredient per day for an adult. Further, preferred specific examples for administration are:
A form having a unit dose that can be given by administering the daily dose once or several times a day, for example, a tablet,
It is to use a capsule or the like.

The analgesic using cEGF of the present invention has an effect of suppressing, for example, general pain due to trauma and disease, and its action is based on whether or not the number of writhing caused by acetic acid is suppressed as an index. It can be confirmed based on experiments from the viewpoint of the target. The dose may be appropriately adjusted according to the age and body weight, but it is generally preferable to use 1 μm to 1 mg as an active ingredient per day for an adult parenterally.

The anti-inflammatory effect of the anti-inflammatory agent using the cEGF of the present invention,
1) Inhibitory effect of histamine on vascular permeability of rat skin,
And 2) the effect of acetic acid on the inhibition of peritoneal vascular permeability in mice can be confirmed by experiments on two pharmacological aspects.

 The effect was confirmed by experiments.

Example 1 The effect of the active ingredient of this drug was examined as follows using a carbon tetrachloride hepatic disorder model.

Experiments using acute liver injury model (1) Experimental animals Male ddY mice, constant temperature (23 ± 0.5 ° C), constant room (60 ± 5)
%) After pre-breeding in a room for at least one week, mice weighing 35 to 38 g which seemed to be healthy were selected, and 5 to 8 mice were used as a group in this experiment.

(2) Experimental method The test solution was injected subcutaneously into male ddY mice at 5 ml / kg, and 30 minutes later, 2 ml / kg of 3% carbon tetrachloride was injected subcutaneously. Then, 25 hours later, blood was collected from the orbital venous plexus to prepare serum. This serum was diluted and GOP was purified using Transaminase C II-test (Wako).
And GPT values were measured. These measured values were expressed in Karmen units / ml.

 The test liquid used was as follows.

(B) Physiological saline (containing 0.01% Tween 80) (Control) 5 ml / kg (b) cEGF (dissolved in 0.1 N acetic acid and diluted with the above physiological saline) 10 μg / kg → 5 ml / kg (C) cEGF 100 μg / kg → 5 ml / kg (3) Experimental results According to the experiment, it was confirmed that cEGF has a sufficiently large inhibitory effect on liver damage.

EXPERIMENTAL EXAMPLE 2 Rising Number Inhibitory Effect (1) Experimental animal ddY male mouse constant temperature (35 ± 0.5 ° C), constant humidity (60 ± 5)
%) After pre-breeding in the room for one week, the weight considered healthy
28 to 35 g were used for the experiment as 6 to 7 animals per group.

(2) Experimental method cEGF was subcutaneously administered to mice, and 30 minutes later, 0.6% acetic acid (0.1%) was added.
10 minutes after intraperitoneal administration of
Number of risings observed during 0 minutes (number of unique writhing symptoms)
Was measured.

 The test liquids to be tested are as follows.

(A) Physiological saline (containing 0.01% Tween 80) (Control) 10 ml / kg (b) cEGF (dissolved in 0.1 N acetic acid and diluted with physiological saline) 30 μg / kg → 10 ml / kg (3 ) Experimental results According to the results of the above experiments, it was confirmed that there was a sufficiently large inhibitory effect.

Experimental Example 3 Inhibitory effect of histamine on vascular permeability of rat skin (1) Experimental animals SD male rats were conditioned at constant temperature (23 ± 0.5 ° C) and constant humidity (60 ± 0.5).
%) After pre-breeding for at least one week in a room, rats weighing 210 to 230 g, which seem to be healthy, were used as 4 rats per group in the experiment.

(2) Experimental method The back of the rat was shaved in advance, and the test solution was administered subcutaneously 30 minutes before administration of the inflammatory agent. Then, histamine hydrochloride was used as the inflammatory agent at the midline on the back of the rat. 100 μg / 0.1 ml was injected intradermally and immediately 20 mg / kg of Evans blue was injected intravenously. Fifteen minutes later, the rats were exsanguinated and the skin on the back was removed.
The area (major axis × minor axis) of the dye leakage part was measured. Then
After punching out the above-mentioned dye leaking part with a punch (diameter μm), this was finely cut, and then 0.3% sodium sulfate (Na ₂ SO 4)
₄ ): Extract the pigment using a mixture of acetone (= 3: 7),
After passing through the extract, the absorbance was measured at 620 nm, and the dye concentration was calculated based on a test line prepared in advance to determine the amount of dye leakage per part of dye leakage (μg / part).

 The test liquid used was as follows.

(A) Physiological saline (containing 0.01% Tween 80) (Control) 2 ml / kg (b) cEGF (dissolved in 0.1N acetic acid and diluted with saline) 100 μg / kg → 2 ml / kg (c) ) Indomethacin (suspended in physiological saline containing 0.5% carboxymethylcellulose-sodium.) 20 μg / kg → 2 ml / kg (3) Experimental results According to the results of the above experiments, cEGF has a sufficiently large anti-inflammatory effect. Admitted.

In constructing the above-mentioned various medicines using the cEGF of the present invention, various kinds of commonly used additives can be used, and specific examples of such various kinds of additives are as follows. Can be used.

There are carriers (excipients, binders, diluents, etc.), stabilizers, solubilizers and the like. Carriers include, for example, calcium carbonate, lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives, gelatin, cocoa butter, water, paraffin, oils and the like. In the case of water, paraffin and oils and fats, the medicament of the present invention may be in the form of an emulsion or suspension in addition to using it as a solution.

Administration dosage forms include powders, granules, fine granules, tablets, pills, capsules, troches, suppositories, lotions, injections (for example, dissolving cEGF of the present invention in distilled water for injection or various liquids or Suspension), ointments, cataplasms and the like can be administered. They can be administered orally or parenterally (including by injection) and may be formulated with other drugs as needed.

The content of the cEGF contained in the cosmetic of the present invention is usually 0.001 to 5.0% by weight, preferably 0.005 to 2.5% by weight. If the amount is less than 0.001% by weight, a sufficient effect cannot be obtained. On the other hand, if the amount exceeds 5% by weight, the effect is not enhanced, and it is uneconomical.

In addition, dogs are used as experimental animals in the fields of various biochemistry, pharmacology, etc., and a system for accurately measuring the increase or decrease in the cEGF tissue or body fluid is required. A specific antiserum or a specific monoclonal antibody can be prepared, and an assay system for accurately measuring cEGF using the antiserum or the monoclonal antibody can be created. This cEGF is useful as an antigen therefor. In addition, it is useful as a component of tissue culture media, as is EGF from other animals.

〔Example〕

Hereinafter, examples will be given to explain the present invention in more detail.

The measurement of the EGF activity of cEGF in each example was performed by a radio receptor assay (RRA) below.

Measurement of EGF activity by RRA EGF activity of cEGF was measured by RRA using A431 cells.

A431 cells used are a cell line derived from human epidermoid carcinoma and are known to have many EGF receptors on the cell surface (RNFabricant et.al., Proc.nNatl.Acad.Sc.
i., USA., 74, 565, (1977)].

This RRA is for the ECF receptor on the A431 cell surface.
It utilizes the competitive reaction of the binding between cEGF and human EGF, and was carried out according to the following procedures.

・ Preparation of A431 cells A431 cells were added to 10% fetal calf serum (FCS) DME with the following composition
The cells were cultured in 20 ml of the medium at 37 ° C. for 3 days in the presence of 5% CO ₂ .

DME medium supplemented with 10% FCS Dulbecco's modified Eagle medium 900 ml L-glutamine 0.6 g 10% aqueous sodium bicarbonate solution 10 ml streptomycin 200 mg penicillin 200,000 U FDS 100 ml Thereafter, cells were suspended, and after counting the number of cells, 20 ml of 10 ml
% Neutral formalin solution was added and left at 4 ° C. for 30 minutes.
Next, the plate was washed several times with D-PBS of each composition, dispensed, freeze-dried and stored.

D-PBS Sodium chloride 8.0 g Potassium chloride 0.2 g Disodium phosphate 1.15 g Monopotassium phosphate 0.2 g Total 1 • Measurement method Human EGF was used as a standard. In addition, human IGF was iodinated by the chloramine method to prepare ¹²⁵ I-human EGF. The standard, ¹²⁵ I-human EGF, A431 cells and the measurement sample were all used as a solution or suspension in D-PBS containing 0.1% bovine serum albumin.

First, 0.2 ml of standard or measurement sample and about 300,000 cpm
/ ml of ¹²⁵ I-human EGF was added to the mixture, and then the mixture was added with about 1 million cells / ml of A431 cells (0.2 ml) and left at 25 ° C for 20 hours. Thereafter, 1 ml of D-FBS containing 0.1% bovine serum albumin (BSA) was added, and the mixture was centrifuged at 4 ° C. (3000 rpm /
, 30 minutes) and the supernatant was discarded. Next, the radioactivity of the sediment was measured with a γ-counter, and the EGF activity of the sample was determined as a human EGF conversion value based on a standard curve obtained from the standard.

FIG. 1 shows an example of the standard curve. In the figure, the vertical axis is
¹²⁵ I-human EGF binding ratio to A431 cells [B / Bo (%)]
, And the horizontal axis indicates the amount of human EGF (pg / assay tube), respectively.

Example 1 Isolation and purification of dog EGD Dog urine was collected from 50 pigle dogs. Immediately after collection, solid matter was removed from urine using gauze, and the acetic acid concentration was reduced to 10% (V /
Glacial acetic acid was added to obtain V), and the mixture was stored frozen until the required amount of urine was collected. About 55 of the collected dog urine were melted at room temperature, filtered through a filter paper (No. 2), and then dissolved by adding ammonium sulfate powder to 2M. Next, the urine was adsorbed to a column (9.8 × 27 cm) packed with about 2000 ml of butyltoyopearl 650C (manufactured by Tosoh Corporation) equilibrated with 2M ammonium sulfate. The column was washed with a 0.8 M ammonium sulfate solution 4, then eluted with a 0.4 M ammonium sulfate solution, a 0.2 M ammonium sulfate solution, water, and then a 1: 1 mixture of acetonitrile and a 10% acetic acid solution, and each eluate was pooled. Of these eluates, 1: 1 of acetonitrile and 10% acetic acid solution
EGF activity was added to the mixture, and the amount of cEGF recovered, that is, cEGF activity obtained from dog urine 55 was 1290 μg (converted to recombinant human EGF), and the protein mass was 1130 mg (converted to bovine serum albumin). )Met.

. The cEGF fraction in the urine of 55 minutes obtained by the above butyl Toyopearl 650C column chromatogram was concentrated under reduced pressure at about 30 ° C. using a rotary evaporator NE-1 (manufactured by Tokyo Rika Kikai Co., Ltd.). 10% after dialysis
Glacial acetic acid was added to give (V / V) to obtain 3000 ml of a cEGF-containing sample. The thus obtained sample was equilibrated with a 10% (V / V) acetic acid solution, and SP-Toyopearl 650M (manufactured by Tosoh Corporation) was used for 20 minutes.
It was adsorbed on a column (6 × 10 ml) packed with 0 ml. After washing the column with a 10% (V / V) acetic acid solution, an ammonium acetate buffer (pH 4.5) to which sodium chloride was added stepwise (concentration: 0.1 M, 0.2 M, 0.4 M, 0.6 M, 1.0 M). ) And the respective eluates were pooled. Among these eluates, EGF activity was found in acetate buffer containing 0.4 M and 0.6 M sodium chloride. The fractions showing the EGF activity were combined, dialyzed against water, and freeze-dried.

The lyophilized sample was added to 190 ml of 20 mM phosphate buffer (pH
DE dissolved in 6.5) and equilibrated with the same 2 mM phosphate buffer
The column was adsorbed to a column (2.5 × 4 cm) packed with 20 ml of AE-Toyopearl 650M (manufactured by Tosoh Corporation) and washed with a 20 mM phosphate buffer. As a result, EGF activity was found only in the non-adsorbed fraction.

 This non-adsorbed fraction was dialyzed against water and freeze-dried.

. next. Was dissolved in 3 ml of 20 mM Tris-HCl buffer (pH 7.7), and the same 20 mM Tris-HCl buffer (pH
After applying DEAE-Toyopearl 650S (manufactured by Tosoh Corporation) equilibrated in 7.7) to a column (2.2 × 20 ml) packed with 76 ml, MPLC (manufactured by Waters Corporation, pump: M600 multi-solvent delivery system, detector: type 490) (Ultrasensitive multifunctional detector). For elution, a linear gradient elution in which the sodium chloride concentration was changed from 0 M to 1 M in the same 20 mM Tris-HCl buffer (pH 7.7) at a flow rate of 4 m
At 1 / min, the absorbance at 280 nm was measured, and at the same time, the eluate was examined for EGF activity.

 The measurement results H are shown in FIG.

In the figure, the vertical axis indicates absorbance at 280 nm, and the horizontal axis indicates elution time (minutes). In the figure, the ECF activity measured by RRA for each eluate is shown as hatched bar graphs.

As shown in the figure, the eluted fraction having EGF activity shows two peaks, and hereinafter referred to as A fraction and B fraction in the order of elution.

The A fraction and the B fraction were each dialyzed against water and freeze-dried.

. next. Purification of the fractions A and B obtained in step 2 is proceeded separately. First, the A fraction was dissolved in 2 ml of 50 mM phosphate buffer (pH 6.3), and a TSK gel-ODS-120T column (manufactured by Tosoh Corporation,
HPLC using a liquid chromatography system similar to that of the MPLC described above, using 10 mm (V / V) to 30% (V / V) in 50 mM phosphate buffer (pH 6.3) using an inner diameter of 4.6 mm × 25 mm). Linear gradient elution in which the acetonitrile concentration was changed until V) was performed at a flow rate of 1 ml / min, and fractions having EGF activity were collected. Using a TSK gel-ODS-120T column, the sample was subjected to linear gradient elution in which acetonitrile concentration was changed from 20% (V / V) to 45% (V / V) in 0.05% trifluoroacetic acid at a flow rate of 1 ml / min. HPLC was repeatedly performed. Further, the fraction A was analyzed using the TSK-gel-ODS-120T column under the same conditions as the purification, and it was confirmed that a single peak was obtained.
GF1.

In addition, the TSK gel-ODS was used for the B fraction as well as the A fraction.
50 mM phosphate buffer (pH 6.3) / (1
0% → 30%) acetonitrile linear gradient elution and 0.05% trifluoroacetic acid / (20% → 45%) acetonitrile linear gradient elution were repeated.
cEGF2 was isolated.

In FIG. 3, TSK gel-ODS of fractions A and B
0.05% trifluoroacetic acid using a -120T column / (20%
→ 45%) Acetonitrile linear gradient elution HP
The results of LC are shown.

In the figure, the vertical axis indicates absorbance at 280 nm, and the horizontal axis indicates retention time (minutes). From the figure, it was found that single sharp peaks of cEGF1 and cEGF2 were eluted at retention times of about 16 minutes and about 18 minutes.

In addition, if the summary of each of the above purification steps is shown,
It is as shown in the table. In the table, the total protein amount (μg) was measured by the Lowry method using bovine serum albumin (BSA) as a standard, and the total activity (μg.eq.) was measured by the RRA method.

Measurement of the isoelectric point of cEGF1 and cEGF2 (pI) The measurement of the isoelectric point was performed using Fast Gel IEF3-9 (Phast Gel
Electrophoresis was performed using IEF3-9 (Pharmacia) as a support.

That is, cEGF1 and cEGF2 were applied to Fast Gel IEF3-9, and isoelectric focusing was performed using a Fast System (Pharmacia). After that, it was stained with Coomassie Brilliant Blue R250 and the Pharmacia IEF marker (p
H2.5 to 6.5). As a result, cEGF1 was around 5.0 and c
EGF2 showed a single band around 4.9.

Example 2 Characteristics of cEGF (1) Amino acid composition About 2 μg of cEGF1 and cEGF2 solution (obtained in Example 1) was added to a hard glass sample tube (manufactured by Nidec Rika Glass Co., Ltd .;
× 50mm), placed in a hydrolysis vial (Peace), dried in vacuo, and then 6N hydrochloric acid (containing 1% phenol)
200 μg was placed in the reaction vial, sealed under reduced pressure, and hydrolyzed at 130 ° C. for 4 hours.

After the reaction, 400 µm of 0.02N hydrochloric acid was added to the sample tube, transferred to a sample tube for amino acid analysis, and 250 µm thereof was automatically injected into a Hitachi high-speed amino acid analyzer (manufactured by Hitachi, Ltd.) to analyze the amino acid composition. The detection was performed by the OPA (orthophthalaldehyde) method. Pro (proline), Cys (cystine) and Trp (tryptophan)
Is not detected.

Table 2 below shows the results of calculating the composition ratio assuming that it contained one Phe (phenylalanine).

(2) Analysis of amino acid sequence After reducing carboxamide methylation of cEGF1 and cEGF2, the amino acid sequence was analyzed. Ie each
400 μM of 0.08 M Tris-HCl buffer (pH 8.3) with 5 M guanidine hydrochloride added to EGF solution (30 μ to 50 μ)
Was added, and a DDT solution (20 μm to 30 μm) was added to the cEGF1 sample at 20 μm, and the EGF2 sample was added at 30 μm, followed by reacting in a 50 ° C. water bath for 2 hours to perform reduced carboxamide methylation. Then a 4.45% (w / v) iodoacetamide solution was added to each sample at 40μ for the cEGF1 sample and 60μ for the cEGF2 sample.
After adding and reacting for 30 minutes, the reaction was stopped by adding 50% of a 10% (w / v) trifluoroacetic acid solution. CEGF1 and cEGF2 thus reduced carboxamide methylated
Was analyzed using a 477A / 120A gas phase protein sequencer (Applied Biosystems).

The results are as shown in the above formula (I), and thus the primary structures of the cEGF1 and cEGF2 of the present invention were determined.

(3) Measurement of biological activity (colony forming activity in soft agar) Dulbecco's modified Eagle medium (DME) containing 0.6% agar (Ager Noble, manufactured by Difco) and 5% calf serum was used.
Each 2 ml was dispensed into a 35 mm diameter petri dish and solidified. Separately, DME medium containing 0.3% agar, 5% calf serum, 1 ng / ml human TGF-β and various concentrations of cEGF1, cEGF2 or human EGF
To 3.5 ml, 140 × 7.5 × 10 ⁴ cells / ml of NRK-49K cells were added, and 1 ml of the mixture was poured on the 0.6% agar medium and left to stand for 30 minutes at room temperature to solidify. This was cultured in a carbon dioxide incubator at 37 ° C. under 5% CO ₂ for 2 days.
^Two or more (60 μm diameter) NRK-49F cell colonies were counted under a microscope.

The results obtained are shown in FIG. In the figure, the vertical axis represents the number of colonies 1 × 10 ³ / dish, and the horizontal axis represents the concentration of EGF (n
g / ml). In the figure, ○-○ and ●-● indicate dogs of the present invention.
EGF and △-△ indicate human EGF as a control.

FIG. 4 shows that cEGF1 and cEGF2 of the present invention are human EGF
It is apparent that the compound has a colony forming activity in soft agar in the co-presence of TGF-β, similarly to the above.

Example 3 Lotion Lotion was prepared by uniformly dissolving the following components. The content ratio of each component is% by weight. The same applies to the following examples.

Glycerin 5.0 Ethyl alcohol 5.0 Citric acid 0.1 Sodium citrate 0.1 cEGF 0.05 Methyl parahydroxybenzoate 0.1 Perfume Appropriately purified water 89.6 Example 4 Emulsion The following components A and B were each heated and dissolved at 70-75 ° C, and then dissolved in component A. The component B is added and emulsified, and the component C is added and mixed while cooling, and cooled to 30 ° C. to obtain a desired emulsion.

 A) Stearic acid 5.0 Cetyl alcohol 5.0 Squalane 2.0 Glycerin monostearate 1.3 Sorbitan monooleate 1.5 Polyoxyethylene sorbitan monooleate 0.8 B) Glycerin 6.0 Triethanolamine 0.7 Methyl parahydroxybenzoate 0.1 Purified water 77.6 C) cEGF 0.01 Perfume Appropriate Example 5 Cream A cream was prepared by uniformly mixing the following ingredients.

A) Stearic acid 5.0 Cetyl alcohol 1.0 Liquid paraffin 6.0 Wallisen 5.0 Cerecin wax 5.0 Glycerin monostearate 2.5 Sorbitan monostearate 0.5 B) Propylene glycol 5.0 Potassium hydroxide 0.5 Methyl parahydroxybenzoate 0.1 Purified water 69.4 C) cEGF 0.01 Perfume Appropriate Example 6 Ointment The following ingredients were heated to 60 ° C. and dissolved, then mixed, and the temperature was gradually lowered to obtain an ointment.

Polyethylene glycol 400 40.0 Polyethylene glycol 6000 59.8 Menthol 0.1 Preservative As needed cEGF 0.05 Example 7 Lipstick The following component A is dissolved by heating and mixed uniformly. The component B is added thereto, and the mixture is kneaded and uniformly dispersed using a roll mill. This is melted again and component C is added. After defoaming, it is poured into a mold and then rapidly cooled to obtain the desired lipstick.

A) Castor oil 45.0 Hexadecyl alcohol 25.0 Lanolin 5.0 Beeswax 4.0 Candelilla wax 6.0 Carnauba wax 5.0 Antioxidant Appropriate Preservative Appropriate B) Color base 0.5 C) cEGF 0.01 Perfume Appropriate [Effect of the Invention] The cEGF of the present invention is a novel one. Yes, conventionally
Although it corresponds to a substance constituting cEGF, it has a broad biological activity like mEGF and is useful.

These substances are useful for measurement in an assay system using them as antigens.

In addition, since these substances have various biological activities such as a cell growth promoting activity, a newborn mouse eyelid cleavage promoting effect, and an incisor eruption promoting effect, anti-inflammatory agents utilizing these effects or It can be used as a cosmetic.

According to the production method of the present invention, the cEGF of the present invention can be easily produced by a separation method or a synthesis method.

[Brief description of the drawings]

FIG. 1 is a graph showing a standard curve of the RRA method for measuring EGF. FIG. 2 shows a chromatogram of the dog EGF of the present invention by an ion exchange method using a DEAE-Toyopearl S column, and each fraction.
FIG. 4 is a bar graph showing the result of measuring the EGF activity by the RRA method. FIG. 3 is a chromatogram of cEGF1 and cEGF2 of the present invention by reversed-phase HPLC using an ODS-120T column. FIG. 4 is a graph showing the results of measuring the colony forming activity in soft agar possessed by cEGF1 and cEGF2 of the present invention.

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-2-104293 (JP, A) JP-A-60-258105 (JP, A) Biochem. J. , Vol. 229, no. 3 (1985) p. 611-619 (58) Field surveyed (Int. Cl. ⁶ , DB name) C07K 14/485 CA (STN) REGISTRY (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A canine epithelial cell growth factor having a primary amino acid sequence represented by the following formula (I) and having the following amino acid composition: Formula (I) [N-terminal side] [C terminal side] 2. An anti-inflammatory agent comprising the canine epidermal growth factor according to claim 1 as an active ingredient. 3. A cosmetic comprising the canine epidermal growth factor according to claim 1 as an active ingredient. 4. A method for producing canine epithelial cell growth factor, comprising isolating the canine epidermal growth factor according to claim 1 by extraction and purification from canine urine.