JP6372844B2 - Peptide derived from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and antiallergic composition containing the same - Google Patents

Description

  The present invention relates to a peptide having an antiallergic effect. The peptide of the present invention is derived from a portion of glyceraldehyde 3-phosphate dehydrogenase having an antiallergic effect. The peptides provided by the present invention are useful in the fields of pharmaceuticals, foods and cosmetics.

  In recent years, the number of patients with allergic symptoms such as hay fever and atopic dermatitis has increased rapidly around the world. In particular, in Japan, one in three people has allergic symptoms, which is a social problem. However, the pathogenesis of allergies has not been fully elucidated, and the development of appropriate treatment means has been difficult. In fact, treatment results have been improved by introducing steroids with anti-inflammatory effects in patients with severe symptoms, but these treatments are palliative treatments that suppress the inflammatory response and are essential for allergy treatment. No definitive treatment has been established. In addition, since steroid drugs have a structure similar to that of steroid hormones in the living body, side effects such as disorder of hormone balance have been reported, and the dosage period is often limited.

  On the other hand, functional foods are expected to have health-promoting functions such as disease prevention and symptom relief by incorporating them into daily eating habits, so the market is expanding with the spread of health-consciousness. In addition, functional foods are not originally intended to be fundamentally treated like pharmaceuticals, but recent research suggests that eating together with specific ingredients may have the same effect as pharmaceuticals. There is great expectation as a disease treatment.

  By the way, allergies such as hay fever, atopic dermatitis and bronchial asthma are medically classified as type I allergies. Many previous studies have reported that IgE antibodies play an important role in the development of this type I allergy. Specifically, first, allergens such as pollen and house dust that have entered the body cross-link IgE bound to a high affinity IgE receptor (FcεRI) present on the cell membrane of mast cells or basophils. Aggregate. This causes mast cells and basophils to degranulate and release chemical mediators such as histamine, serotonin, and leukotriene. Ultimately, these substances cause smooth muscle contraction, mucosal edema, and increased vascular permeability, resulting in allergic symptoms. Therefore, reducing the amount of IgE leads to suppression of allergic symptoms.

  The inventors of the present application have established a culture system that reproduces the allergic environment in vitro by culturing the peripheral blood lymphocytes of healthy individuals in a culture solution to which various immunostimulants and antigens have been added. And glyceraldehyde-3-phosphate dehydrogenase (hereinafter sometimes referred to as “GAPDH”) in strawberry components has been found to have an effect of reducing the production of IgE (patent document) 1 and Non-Patent Documents 1 and 2). This GAPDH is known as a main enzyme constituting a glycolytic system, and is present in large amounts in animal cells, plant cells, and microorganisms. Therefore, the application of GAPDH to drugs, foods and cosmetics having antiallergic action is extremely realistic, and rapid development is strongly desired.

International Publication No. WO2012 / 144501

J. et al. Immunol. Methods 233 (2000) pp. 33-40 Cytotechnology 64 (2012) pp. 309-314

  As described above, application of GAPDH to pharmaceuticals, foods, and cosmetics is useful for the treatment, prevention, and alleviation of allergies. However, GAPDH is a protein having a higher-order structure, and thus its function may be inactivated by denaturation or the like. was there. GAPDH is known as an enzyme involved in the metabolism of organisms, but since the amino acid sequence differs depending on the species, there is a concern that the effect of suppressing IgE production may be lost in those derived from a particular species.

  On the other hand, the present inventors have recently found that GAPDH has an IgE production inhibitory effect not only from strawberries, but also from rabbit muscles and those derived from lactic acid bacteria. It has been found that the storage area suppresses IgE production.

  Therefore, it came to the idea that GAPDH having an antiallergic effect could be efficiently identified by identifying an amino acid sequence that suppresses IgE production in these GAPDH.

  In addition, new antiallergic components can be identified by screening peptides and proteins containing the specified amino acid sequence as an index.

  Then, the inventors of the present application have disclosed a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2, or 6 in GAPDH, a peptide containing the amino acid sequence, and an antiallergic composition containing these peptides as active ingredients. Ascertaining to suppress production, the present invention has been achieved.

  In order to solve the above-mentioned problems, the present inventors first purified a fraction that suppresses IgE production from GAPDH fragmented by trypsin treatment. As a result of purification using reverse phase column chromatography, it was found that the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 containing the nicotinamide adenine dinucleotide binding region of GAPDH suppresses IgE production.

  Furthermore, when the influence on the IgE production of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 to 6 synthesized based on the amino acid sequence of SEQ ID NO: 1 was confirmed, the amino acid sequence represented by SEQ ID NO: 2 and 6 was Was found to suppress IgE production. In addition, when the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NO: 3 to 5 is added in combination, IgE is more than when the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is added alone. It was revealed that production was strongly suppressed.

That is, the present invention is as follows.
[1] The peptide of any one of (i) to (iii) below
(i) a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, 2 or 6;
(ii) a fragment peptide of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein comprising the amino acid sequence represented by any of SEQ ID NOs: 7 to 9, represented by SEQ ID NO: 1, 2 or 6 A peptide comprising an amino acid sequence.
[2] The peptide of [1], which has IgE production inhibitory activity.
[3] A composition comprising the peptide of [1] or [2].
[4] The composition according to [3], which is an antiallergic agent.
[5] The composition according to [3], which is a food composition.
[6] The composition according to [3], which is a cosmetic composition.
[7] A peptide composition comprising the following peptide (a) and at least one peptide of (b) to (d):
(a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a fragment peptide of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein consisting of the amino acid sequence represented by any of SEQ ID NOs: 7 to 9 A peptide comprising the amino acid sequence represented by SEQ ID NO: 2;
(b) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 or a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein fragment peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 7 to 9 A peptide comprising the amino acid sequence represented by SEQ ID NO: 3;
(c) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4, or a fragment peptide of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein consisting of the amino acid sequence represented by any of SEQ ID NOs: 7 to 9. A peptide comprising the amino acid sequence represented by SEQ ID NO: 4;
(d) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, or a fragment peptide of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein consisting of the amino acid sequence represented by any of SEQ ID NOs: 7 to 9. A peptide comprising the amino acid sequence represented by SEQ ID NO: 5.
[8] The peptide-containing composition according to [7], which has IgE production inhibitory activity.
[9] The peptide composition according to [8], which is an antiallergic agent.
[10] The peptide-containing composition according to [8], which is a food composition.
[11] The peptide composition according to [8], which is a cosmetic composition.

  According to the present invention, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2 or 6, a peptide containing the amino acid sequence, and an antiallergic composition containing these peptides suppresses IgE production. It is useful as an antiallergic agent (pharmaceutical), food composition, and cosmetic composition for the purpose of treating, improving and preventing type I allergic symptoms such as atopic dermatitis and bronchial asthma. A composition comprising a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, or a peptide comprising the amino acid sequence and a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 to 5, or a peptide comprising the amino acid sequence Is useful as a composition having more excellent antiallergic action.

It is the graph which showed the result which confirmed the effect of the IgE production suppression effect of the refinement | purification fraction of a GAPDH fragment | piece. It is the graph which confirmed the effect of the IgE production inhibitory effect of the peptide which consists of an amino acid sequence represented by sequence number 2-5 (FIG. 2A-D). It is the graph which confirmed the effect of the IgE production inhibitory effect of the peptide which consists of an amino acid sequence represented by sequence number 6. It is the graph which showed the effect of the IgE production inhibitory effect at the time of combining the peptide which consists of an amino acid sequence represented by sequence number 2-5.

  The present invention is a fragment peptide of the GAPDH protein, which is a fragment peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 6. Preferably, it is a fragment peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2, or 6, and is a peptide having an IgE production inhibitory action alone. The fragment peptide is a GAPDH fragment peptide purified from rabbit muscle. The above-mentioned fragment peptide can be obtained by excising from GAPDH purified from rabbit muscle by a known method such as proteolytic enzyme treatment. Furthermore, it can also synthesize based on amino acid sequence information, and can also be obtained by a chemical synthesis method such as Fmoc method or a method such as recombinant expression. The amino acid constituting the peptide of the present invention may be L-form or D-form, but is preferably L-form.

  Moreover, this invention is a peptide containing the amino acid sequence represented by either of sequence number 1-6. Preferably, it is a peptide having an IgE production inhibitory action comprising the amino acid sequence represented by SEQ ID NO: 1, 2, or 6. Examples of such a peptide include GAPDH protein fragment peptides, which have an IgE production inhibitory action comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6, preferably SEQ ID NOs: 1, 2 Or the peptide which has an IgE production inhibitory effect containing the amino acid sequence represented by 6. A peptide consisting of 4 amino acids represented by IGRL (SEQ ID NO: 6) has an inhibitory effect on IgE production and comprises an amino acid sequence represented by SEQ ID NO: 1 or 2 including IGRL (SEQ ID NO: 6) Also has an inhibitory effect on IgE production. Accordingly, a peptide fragment of the GAPDH protein and containing at least the amino acid sequence represented by IGRL (SEQ ID NO: 6) also has an IgE production inhibitory action. SEQ ID NOs: 7 to 9 show the full-length amino acid sequences of GAPDH consisting of 334 amino acids derived from rabbit (Oryctolagus cuniculus) muscle. In the DNA encoding the amino acid sequence represented by SEQ ID NO: 7, the base encoding the 124th amino acid has a mutation and is gcc or tcc. For this reason, the 124th amino acid is Ala or Ser. SEQ ID NO: 8 represents an amino acid sequence in which the 124th amino acid is Ala, and SEQ ID NO: 9 represents an amino acid sequence in which the 124th amino acid is Ser. A partial peptide of the amino acid sequence represented by SEQ ID NO: 7 as a fragment peptide of the GAPDH protein, which has an IgE production inhibitory action comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 6 A peptide consisting of a sequence can be mentioned. A peptide fragment of the GAPDH protein, comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6, has an amino acid length of 5 to 333, preferably 5 to 200, more preferably 5 to 100, and particularly preferably Is 5-39. In these peptides, the amino acid sequence represented by IGRL (SEQ ID NO: 6) may be contained at the N-terminus or at other portions. It has been reported that GAPDH protein suppresses IgE production (International Publication No. WO2012 / 144501), and the present invention clarifies a site having IgE production inhibitory activity in GAPDH protein. Therefore, any of the GAPDH protein fragment peptides containing the amino acid sequence (SEQ ID NO: 1, 2 or 6) of the fragment peptide having IgE production inhibitory activity has IgE production inhibitory activity. In the present invention, the GAPDH protein fragment peptide does not include the GAPDH protein consisting of the full-length amino acid sequence of GAPDH.

  Further, as described above, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 alone has an inhibitory effect on IgE production, but is represented by the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NOs: 3-5. A peptide mixture obtained by combining one, two or three of the peptides consisting of the amino acid sequence exhibits a much stronger IgE production inhibitory effect than the peptide alone consisting of the amino acid sequence represented by SEQ ID NO: 2. As a combination of a peptide mixture obtained by combining a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 to 5, a combination of SEQ ID NO: 2 and SEQ ID NO: 3, Combination of SEQ ID NO: 4, Combination of SEQ ID NO: 2 and SEQ ID NO: 5, Combination of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, Combination of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5, SEQ ID NO: 2 and SEQ ID NO: 4 and SEQ ID NO: 5 as well as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The above combination of the peptide comprising the amino acid sequence represented by SEQ ID NO: 2 and the peptide comprising the amino acid sequence represented by SEQ ID NO: 3 to 5 also has an IgE production inhibitory action, and as such a combination, a fragment peptide of GAPDH protein A combination of a peptide containing the amino acid sequence represented by SEQ ID NO: 2 and a peptide containing the amino acid sequence represented by SEQ ID NOs: 3 to 5 can be mentioned. A peptide mixture obtained by combining a peptide comprising the amino acid sequence represented by SEQ ID NO: 2 and a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 to 5, or a peptide comprising the amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NO: 3 The peptide mixture which combined the peptide containing the amino acid sequence represented by -5 is called a peptide compounding composition. Specifically, for example, a blended composition of peptides containing at least one of the following peptides (a) and (b) to (d) is exemplified.

(a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a fragment peptide of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein consisting of the amino acid sequence represented by any of SEQ ID NOs: 7 to 9 A peptide comprising the amino acid sequence represented by SEQ ID NO: 2;
(b) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 or a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein fragment peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 7 to 9 A peptide comprising the amino acid sequence represented by SEQ ID NO: 3;
(c) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4, or a fragment peptide of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein consisting of the amino acid sequence represented by any of SEQ ID NOs: 7 to 9. A peptide comprising the amino acid sequence represented by SEQ ID NO: 4;
(d) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, or a fragment peptide of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein consisting of the amino acid sequence represented by any of SEQ ID NOs: 7 to 9. A peptide comprising the amino acid sequence represented by SEQ ID NO: 5.

  The peptide of the present invention has a deletion or substitution of at least one, preferably one or several amino acids in the amino acid sequence, as long as the peptide comprising the amino acid sequence or a peptide containing the amino acid sequence has an IgE production inhibitory action. Mutations such as addition may occur. It is suggested that substitution of amino acids having similar properties such as charge and polarity will not lose the function even if a large number of amino acids are substituted.

  For example, at least one, preferably one or several (for example, 1 to 10, more preferably 1 to 5, particularly preferably 1 or 2) amino acids of the amino acid sequence represented by SEQ ID NO: 1 have been deleted. At least 1, preferably 1 or several (for example, 1 to 9, more preferably 1 to 5, particularly preferably 1 or 2) amino acids are added to the amino acid sequence represented by SEQ ID NO: 1. Or at least 1, preferably 1 or several (for example, 1 to 9, more preferably 1 to 5, particularly preferably 1 or 2) of the amino acid sequence represented by SEQ ID NO: 1. An amino acid may be substituted with another amino acid. In addition, at least one, preferably one or several (for example, 1 to 3, preferably 1 or 2) amino acids of the amino acid sequence represented by SEQ ID NOs: 2 to 6 may be deleted. At least 1, preferably 1 or several (for example, 1 to 5, preferably 1 or 2) amino acids may be added to the amino acid sequence represented by 2 to 6, or At least one, preferably one or several (eg, 1 to 5, preferably 1 or 2) amino acids in the amino acid sequence represented may be substituted with other amino acids.

  As an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 to 6, the amino acid sequence represented by SEQ ID NO: 2, and BLAST (Basic Local Alignment At least 85% when calculated using the Search Tool at the National Center for Biological Information) (for example, default or default parameters), preferably Those having a sequence identity of 90% or more, more preferably 95% or more, and particularly preferably 97% or more.

  A peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequences represented by SEQ ID NOs: 1 to 6 has the amino acid sequences represented by SEQ ID NOs: 1 to 6, respectively. It is substantially the same as the peptide it has.

  The peptides of the present invention also include pharmacologically acceptable salts of the peptides. Peptide salts include both organic and inorganic salts, such as sodium, potassium, calcium, magnesium, ammonium, hydrochloride, sulfate, nitrate, and organic acid salts (acetate, trifluoroacetate, citric acid). Acid, maleate, malate, oxalate, lactate, succinate, fumarate, propionate, formate, benzoate, picrate, benzenesulfonate, etc.) It is done. The peptide of the present invention or a salt thereof may be a hydrate or an anhydrate, and may be a solvate or an unsolvate.

  Furthermore, this invention is a composition containing said peptide. The composition is an antiallergic composition for suppressing IgE production containing the above peptide as an active ingredient. The composition includes a fragment peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 6, or a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 6, preferably The fragment peptide which consists of an amino acid sequence represented by sequence number 1, 2, or 6 or the peptide containing the amino acid sequence represented by sequence number 1, 2, or 6 is contained. A fragment peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2, or 6 or a plurality of peptides including the amino acid sequence represented by SEQ ID NO: 1, 2, or 6 may be included. Further, the composition includes a peptide mixture obtained by combining a peptide comprising the amino acid sequence represented by SEQ ID NO: 2 and a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 to 5, or the amino acid represented by SEQ ID NO: 2 The peptide mixture which combined the peptide containing the sequence and the peptide containing the amino acid sequence represented by sequence number 3-5 may be contained. Whether the peptide has an IgE production inhibitory effect may be determined by, for example, adding the peptide to an in vitro allergy-onset model cell culture system and examining whether IgE production is inhibited. As an in vitro allergic model cell culture system, muramyl dipeptide (MDP), interleukin 4 (IL-4), interleukin 6 (IL-6), cedar pollen antigen (Cryj1), etc. are added to human peripheral blood lymphocytes. A system to which allergen is added can be mentioned. IgE production can be measured by an immunological detection method such as ELISA.

  The composition for suppressing IgE production containing the peptide of the present invention can suppress IgE production in vivo by being administered to a living body, suppress IgE-dependent type I allergy, and depend on IgE. Can prevent or treat type I allergic diseases. Moreover, the composition for suppressing IgE production containing the peptide of this invention can improve the symptom of an IgE-dependent type I allergic disease by administering to a biological body. Examples of IgE-dependent type I allergic diseases include hay fever, atopic dermatitis, bronchial asthma, allergic rhinitis, urticaria, food allergies and the like. Here, administration includes administration as a pharmaceutical, intake of a food composition, and application of a cosmetic composition.

  The composition can be used, for example, as a pharmaceutical composition, a food composition or a feed composition, or a cosmetic composition.

  As a pharmaceutical composition, it can be used as an IgE production inhibitor or antiallergic agent for suppressing allergy, preventing or treating allergic diseases, and improving symptoms of allergic diseases. The pharmaceutical composition may be administered to a subject. Examples of the subject include not only humans but also pet animals such as dogs and cats and livestock such as cows, horses, pigs, and sheep.

  The pharmaceutical composition containing the peptide of the present invention as an active ingredient may contain a pharmaceutically acceptable carrier or diluent. Carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose solution, buffered saline, and the like.

  The administration route includes oral administration or parenteral administration such as cutaneous, subcutaneous, intramuscular and intravenous, with oral administration being preferred. Administration forms can be administered in various forms, and include capsules, tablets, granules, syrups, fine granules, sprays, emulsions, suppositories, injections, ointments and the like. Various pharmaceutical additives can be added to the pharmaceutical composition, and examples thereof include sorbitol, gelatin, lactose, glucose, starch, and citric acid, which are generally used as pharmaceutical pharmaceutical additives.

The dosage of the pharmaceutical composition containing the peptide of the present invention as an active ingredient varies depending on symptoms, age, body weight, etc., but usually, oral administration is about 0.01 mg to 50 g per day for an adult. It can be administered once or divided into several times. Per kilogram of body weight is about 0.002 mg to 1 g per day. In addition, the amount applied to the skin when administered to the skin is 1 ng to 500 μg / cm ² , preferably 0.01 to 50 μg / cm ² , more preferably 0.1 to 10 μg / cm ² .

  As a food composition, it can be used as a food composition for suppressing allergies, preventing or treating allergic diseases, and improving symptoms of allergic diseases. The food composition of the present invention contains the peptide of the present invention having an IgE production inhibitory action or an antiallergic agent containing the peptide. The food composition of the present invention includes health food, food for specified health use, nutritional functional food, health supplement food and the like. The food for specified health is a food that is ingested for the purpose of specific health in the diet, and indicates that the purpose of the health can be expected by the intake. These foods can be labeled as being used for the above purposes. Examples of such a display include a display indicating that it is used for suppressing allergy, a display indicating that it is used for improving allergic symptoms, and the like. Food includes beverages. Furthermore, the food composition also includes a feed composition, for example, suppressing allergies in pet animals such as dogs and cats and domestic animals such as cows, horses, pigs and sheep, preventing or treating allergic diseases, and symptoms of allergic diseases It can be used as a feed composition for improving.

  When used as a food composition, the peptide of the present invention may be used in a solid, liquid, or gel food. Examples of food include bread, noodles, prepared dishes, processed meat foods (eg, ham, sausage, etc.), processed fishery products, seasonings (eg, dressing, etc.), dairy products, confectionery (eg, ice cream, chocolate, candy). , Biscuits, jelly, etc.), soup, beverages (eg, soft drinks, etc.).

  The cosmetic composition can be used as a cosmetic composition for suppressing allergy, preventing or treating allergic diseases, and improving symptoms of allergic diseases. The cosmetic composition of the present invention contains the peptide of the present invention having an IgE production inhibitory action or an antiallergic agent containing the peptide.

  Cosmetics include, for example, basic cosmetics such as lotions, emulsions, creams, oils, packs, makeup cosmetics such as foundations, blushers and lipsticks, as well as cleansers such as facial cleansers, cleansings, body cleansers, bathing agents. Etc. may be used in any form.

  Examples of the present invention are illustrated below, but the present invention is not limited to the following examples.

Example 1
<Identification of GAPDH's IgE Production Inhibitory Activity>
[Separation of lymphocytes]
In order to obtain human peripheral lymphocytes, healthy human peripheral blood was separated by density gradient centrifugation. First, peripheral blood is collected from a healthy person in a vacuum tube containing heparin, and 5 ml of peripheral blood is collected on 4 ml of blood separation agent (Ficoll; GE Healthcare) previously dispensed into a 15 ml centrifuge tube. The blood was overlaid and centrifuged at 400 g for 30 min. Then, the uppermost plasma layer was obtained and stored at -20 ° C. Next, the lymphocytes between the plasma layer and the Ficoll layer were collected, washed with a basic synthetic medium ERDF (Kyokuto), and centrifuged and washed three times under conditions of 400 g and 5 min. The obtained lymphocytes were stored frozen at −85 ° C., and after thawing, the lymphocytes were washed with ERDF medium and used.

[In vitro allergy development model cell culture system]
2.0 × 10 ⁶ cells / ml of human peripheral blood lymphocytes removed in vitro in ERDF medium containing 5% fetal bovine serum (FBS; Hyclone) and 10% human plasma and muramyl dipeptide as an immunostimulant (MDP; Sigma) at 10 μg / ml, interleukin-4, -6 (IL-4, -6; R & D) at 10 ng / ml, and cedar pollen antigen (Cryj1; Hayashibara) at a final concentration of 100 ng / ml An in vitro allergy onset model cell culture system was used. The main function of the added immunostimulant is adjuvant activity that enhances the immune response to the antigen by MDP, IgE class switch induction by IL-4, and B cell differentiation induction by IL-6. 200 ml each of human peripheral blood lymphocytes supplemented with these immunostimulators and Cryj1 were dispensed into 96-well plates and cultured in a 5% CO ₂ incubator at 37 ° C. for 10 days to induce IgE antibody production. The human plasma and human peripheral blood lymphocytes contained in this culture system were the same individuals.

[Purification of active fraction]
Rabbit muscle-derived GAPDH (Sigma) was adjusted to 1 mg / ml with phosphate physiological saline (PBS). To this, porcine pancreatic trypsin (Sigma) was added to a concentration of 10 μg / ml, and the mixture was allowed to stand at 37 ° C. for 12 hours. Thereafter, a solution containing a peptide fragment of 10 kDa or less was obtained using an ultrafiltration column (Millipore). This was fractionated using a reverse phase liquid chromatography column SOURCE 15RPC ST 4.6 / 100 (Amersham bioscience), fractions 1 to 5 were collected, and the solvent was replaced with PBS to give a final concentration of 1% (v / V) was added to the in vitro allergy onset model cell culture system to examine the antiallergic effect.

[Measurement of IgE antibody production by enzyme antibody method]
The production amount of IgE antibody in the culture supernatant was measured using an enzyme-linked immunosorbent assay (ELISA). A 96-well immunoplate was diluted 2,000 times with goat anti-human IgE antibody (Biosource) with a carbonate buffer, and dispensed at 100 μl / well and plate-coated. After leaving at 37 ° C. for 1 hour, in order to prevent non-specific reaction, a solution (1% BSA / PBS) of bovine serum albumin (BSA; ICN) diluted to 1% in PBS was dispensed at 300 μl / well. , Blocking. Then, the culture supernatant containing IgE antibody to be quantified was diluted to 1/50 with 1% BSA / PBS and dispensed at 50 μl / well. Similarly, 50 μl / well of a standard solution serially diluted from 1 μg / ml to 1/3 to ^1/8 μg / ml was dispensed and allowed to stand at 37 ° C. for 1 hour. Thereafter, biotin-labeled goat anti-human IgE antibody (Biosource) was diluted 2,000 times with 1% BSA / PBS, dispensed at 100 μl / well, and allowed to stand at 37 ° C. for 1 hour. Thereafter, streptavidin labeled with horseradish peroxidase (Funakoshi) was diluted 1,000 times with 1% BSA / PBS solution, dispensed at 100 μl / well, and allowed to stand at 37 ° C. for 1 hour. As a coloring solution, 0.006% H ₂ O ₂ -0.2 M citrate buffer (pH 4.0), 6 mg / ml ABTS- (NH ₄ ) ₂ (Wako), and ultrapure water were each 10: 1: 9. The solution mixed at a rate of 100 μl / well was dispensed, and after 30 minutes, the absorbance was measured at a wavelength of 414 nm. Between each reaction, PBS was washed 3 times with a solution of polyoxyethylene (20) sorbitan monolaurate (Tween 20; Wako) diluted to 0.05% concentration.

  FIG. 1 is a graph showing the results of examining the effect of fractions 1 to 5 of the GAPDH fragment fractionated by liquid chromatography on IgE production. In FIG. 1, “None” is a control to which no GAPDH fragment is added. From this result, since Fraction 1 suppressed IgE production in particular, it was considered that an amino acid sequence having an action of suppressing IgE production in GAPDH is contained in this fraction. Then, when the amino acid sequence of the peptide contained in this fraction was analyzed, it was found that it had an amino acid sequence represented by IGR from the N-terminal side. Further, mass spectrometry revealed that the molecular weight of this peptide was 4,372.7. Based on these conditions, the amino acid sequence of rabbit GAPDH was examined using a protein database, and as a result, it was found that the peptide that suppresses IgE production was IGRLVTRAAFNSNGKVDVVAINDPFFIDLHYMVYMFQYDST (SEQ ID NO: 1).

(Example 2)
[Confirmation of synthetic peptide activity]
Based on the amino acid sequence represented by SEQ ID NO: 1, from the amino acid sequence represented by IGRLVTRAAF (SEQ ID NO: 2), AAFNSGKVDV (SEQ ID NO: 3), VDVAVAINDPFI (SEQ ID NO: 4), or YMVYMFQYDST (SEQ ID NO: 5) The peptide was synthesized and added to an in vitro allergy model cell culture system to examine its antiallergic effect. 2A to 2D are graphs showing the results of examining the influence of the peptide consisting of the amino acid sequences represented by SEQ ID NOs: 2 to 5 on IgE production. From this result, it was found that IgE production was suppressed when a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was added. Therefore, it was found that the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 has an antiallergic effect.

  Further, IGRL (SEQ ID NO: 6) was synthesized based on the amino acid sequence represented by SEQ ID NO: 2 and added to the in vitro allergy-onset model cell culture system to examine the antiallergic effect. FIG. 3 is a graph showing the results of examining the effect of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 on IgE production. As shown in FIG. 3, IgE production was suppressed in a concentration-dependent manner. From this result, it was clarified that the peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 suppresses IgE production and found to have an antiallergic effect.

  Furthermore, the antiallergic effect was also examined when all peptides consisting of the amino acid sequences represented by SEQ ID NOs: 2 to 5 were combined and added to the in vitro allergy model cell culture system. FIG. 4 is a graph showing the results of examining the IgE production inhibitory effect when the peptides consisting of the amino acid sequences represented by SEQ ID NOs: 2 to 5 are added in combination. From this result, when the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 was added alone, the inhibition rate of IgE production was 37-49%, whereas the amino acid represented by SEQ ID NO: 2-5 It was found to be 63 to 100% when peptides composed of sequences were added in combination. Therefore, the combination of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 to 5 is used alone as the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. It was found to have a higher antiallergic effect than.

  According to the present invention, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2 or 6, a peptide containing the amino acid sequence, and an antiallergic composition containing these peptides suppress anti-EgE production. It can be provided as an allergic agent (pharmaceutical), food, and cosmetics. Further, a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, or a peptide comprising the amino acid sequence and a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 to 5, or a peptide comprising the amino acid sequence are used in combination. Thus, it is possible to provide an antiallergic agent (pharmaceutical), food composition, and cosmetic composition having a higher antiallergic effect.

Claims (11)

Peptides consisting of the amino acid sequence depicted in SEQ ID NO: 1 or 2.   The peptide of Claim 1 which has IgE production inhibitory activity.   A composition comprising the peptide according to claim 1 or 2.   The composition according to claim 3, which is an antiallergic agent.   The composition according to claim 3, which is a food composition.   The composition according to claim 3, which is a cosmetic composition. The following (a) ~ containing peptide of (d), the peptide of the blend composition:
(a) peptides consisting of the amino acid sequence of SEQ ID NO: 2;
(b) peptides consisting of the amino acid sequence represented by SEQ ID NO: 3;
(c) peptides consisting of the amino acid sequence of SEQ ID NO: 4;
(d) peptides consisting of the amino acid sequence represented by SEQ ID NO: 5.   The peptide-containing composition according to claim 7, which has IgE production inhibitory activity.   The peptide combination composition according to claim 8, which is an antiallergic agent.   The peptide combination composition according to claim 8, which is a food composition.   The peptide combination composition according to claim 8, which is a cosmetic composition.

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