KR100646313B1 - A Peptide or salt thereof for treatment allergy and pharmaceutical formulation having it - Google Patents

Abstract

The present invention relates to a peptide for preventing and treating allergy and a salt thereof, and provides a peptide or salt thereof comprising at least one amino acid sequence selected from the group consisting of the following amino acid sequences,

(a) RVSSCRGRNHIV;

(b) ETIGARWVRIE;

(c) PHVMRGGEYSGA;

(d) LVAHVGAGGVL; And

(e) LSYLLWRSRLP

Provided is a pharmaceutical composition for preventing and treating allergic diseases containing the peptide or a salt thereof as an active ingredient.

Peptides discovered in the present invention has a property that binds to the serum of human allergic patients, exhibits an allergic inhibitory effect in cell lines and animal experiments can be useful in the preparation for allergy prevention or treatment.

Allergic diseases, peptides, prevention, treatment, pharmaceutical composition

Description

Peptides or salts thereof for preventing and treating allergic diseases and pharmaceutical compositions containing the same

1 is a schematic diagram showing a recombinant plasmid for expressing a random peptide library used in the present invention in E. coli, and its expression,

Figure 2 is a schematic diagram of biopanning showing the discovery process of peptides that bind to the serum of allergic patients by reacting the serum of the allergic patient with a random peptide library in the present invention,

Figure 3 is the present invention confirms the presence of the peptide by PCR after extracting a plasmid from a bacterium encoding a peptide binding to the serum of allergy patients showing positive reaction to egg white (a) and house dust mite (b) in the present invention Yeongdong Pictures,

Figure 4 is an electrophoretic picture confirming the insertion by inserting a PCR product containing a gene encoding a peptide that binds to the serum of an allergic patient in the T vector, and then by cutting with EcoRI restriction enzyme,

5 is a Western blot photograph verifying the expression of the peptide- thioredoxin fusion protein by tryptophan in the present invention,

6 is a result graph of ELISA showing binding to the serum of an allergic patient in E. coli containing peptides discovered in the present invention,

7 is a graph showing the secretion effect of beta hexosaminidase by antigen stimulation (b-hexosaminidase),

8 is a graph showing the relationship between signaling molecules required for secretion of beta hexoseminidayes,

9 is a view showing the secretion inhibitory effect of beta hexose aminidayes by the peptide of the present invention.

The present invention relates to peptides and salts for allergy prophylaxis and treatment, and more particularly, at least one peptide selected from a group consisting of five peptides and salts thereof extracted using an artificial 12-mer random peptide library as an active ingredient. The present invention relates to a pharmaceutical composition for preventing and treating allergy.

Conventional methods for treating allergy include allergen avoidance therapy, antiallergic drug use, immunotherapy for specific allergens, and the like. The most widely used allergy medications include antihistamines and corticosteroids.

However, a large number of drugs have been developed over the past few years, which only alleviate the symptoms, and there are currently no countermeasures against the root cause of allergy. As a result, the development of a new concept of allergy treatment is urgently being developed.

And currently under development or conventional treatment for allergies are as follows.

(1) Specific allergen immunotherapy

Traditional immunotherapy is a method of injecting allergens into patients with allergies while increasing their amount over a long period of time, which has been effective in treating allergic rhinitis and is effective in allergic asthma. By changing the T cell response from Th2 type to Th1, allergy patients can alleviate symptoms and induce allergen-specific Ig G response.

However, the immunotherapy can alleviate the symptoms of the patient, but there is a problem that the underlying treatment is not.

(2) Cytokine Therapy

Interleukin 4 (IL-4) plays a very important role in the production of IgE antibodies, and has been recognized as a target protein for allergy treatment. According to animal models, antibodies to IL4 receptors are known to inhibit various allergic diseases. Among them, STAT6 (signal transducer and activator of transcription 6) -deficient mice showed a deficiency in signaling by IL-4. Based on these results, development of therapeutic agents targeting STAT6 has been attempted. .

In addition, the development of therapeutic agents targeting cytokine (cytokines) such as IL-5, IL-13 and IL-9 has been attempted.

(3) Vaccine

Infections by viruses or bacteria often have the effect of suppressing allergic reactions by inducing a Th1 immune response. With this in mind, a vaccine that induces a Th1 response may have an allergic effect. It is expected.

In the case of Mycobacterium tuberculosis, it may play a role in reducing the susceptibility to allergens by inducing Th1 responses such as IFN-gamma and IL-12. For example, BCG inoculation inhibits allergens.

In addition, there are several reports that DNA vaccines induce a Th1 response, and thus allergic drugs have been developed. For example, beta-galactosidase genes have been reported to induce Th1 responses, and genes encoding Der p5, a type of house dust mite allergen, inhibit IgE production and inhibit histamine secretion. . In animal studies, CpG oligodeoxynucleotides (CpG oligodeoxynucleotides) induce Th1 responses, while CpG-ODN stimulates NK (natural killer) and DC (dendritic cells). Stimulated DCs (dendritic cells) induce expression of costimulatory molecules such as MHC class II, CD80, and CD86 and mediate Th1 responses such as IL-1, IL-6, IL-12, TNF-alpha, etc. It is known to exhibit an effect of increasing their expression.

The fact that CpG-ODN actually suppresses allergens in humans requires a lot of research, but current in vitro studies show that human lymphocytes show a similar response to CpG-ODN in rats. Known CpG- ODN is expected to be developed as an allergy treatment.

(4) anti-IgE antibody treatment

The most effective allergy treatment is to neutralize IgE antibodies, and to neutralize IgE, it is effective to prepare antibodies to specific sites of IgE that bind to receptors.

Anti IgE-antibodies have been produced that do not cause anaphylactic shock, and these antibodies recognize IgE but do not cause histamine secretion and are expected to be developed as a very effective allergy treatment in the future.

To date, a number of humanized antibodies for the treatment of allergies have been made using complementarity determining region (CDR) transplantation methods. These antibodies have been treated by selectively inhibiting IgE production.

(5) peptide immunotherapy

This method utilizes peptide fragments that inhibit CD4 T cell responses induced by allergens.

Allergen extracts (Allergen extracts) is likely to cause an irritable shock reaction mediated by Ig E, but the use of peptide fragments are known to not cause such problems.

For example, studies have reported that peptides derived from cat allergen Fel d alleviate allergic symptoms by reducing the expression level of Th2 (T-helper 2) cytokines, but long peptides have side effects. Recently, therapies using relatively short peptides (16 or 17-mer amino acids) have been actively developed.

Attempts have been made to treat allergies by interfering with the binding of the IgE antibody to the receptors of the RBL2H3 cell line using a mast cell degranulating peptide. ought.

Angelica Buku et al. (Peptides 22, 1993-1998, 2001) or Anna Erdei (Immunology letters 92, 39-42, 2004,), etc., used peptides derived from complement, By inhibiting the activation of apoptotic cells, the peptides (peptides derived from complement) have shown potential as an allergy treatment.

Iwata A et al. Have demonstrated that z-VAD-Fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) peptide, a universal caspase inhibitor that inhibits suicide, inhibits T cell activation in animal models. By inhibiting asthma (J. Immunol. 2003 170 (6): 3386-3391).

As described above, while researches on substances that suppress the induction of allergic diseases are actively conducted, researches on substances that cause allergies are also progressing simultaneously as follows.

(1) Research Status on the Discovery of Allergenic Proteins

Research into the diagnosis and treatment of allergic diseases has focused on the discovery of proteins present in several foods that cause allergies. Mao JL, et al., Dermatophagoides , a house dust mite Proteins from the farinae were subjected to 2D-PAGE (two-dimensional protein electrophoresis) and then reacted with the sera of allergic patients with house dust mites to induce Ig E antibody responses, designated Der f 4,5,6. New allergens of the species were identified (J Allergy Clin. Immunol. 102: 631-636, 1998).

Cashew nuts are known as foods that cause contact, systemic, or atopic dermatitis. They are used to find new proteins that cause allergies. F (Wang F) et al. Extracted the whole RNA from the cache fruit to prepare the cDNA library, and then expressed it in Escherichia coli to produce a recombinant cDNA expression library and reacted with the serum of a patient with cache allergy to react positively. By selecting the visible plaques and uncovering their sequencing, we discovered a novel protein that causes cache allergy (2002, J. Allergy Clin. Immunol. 110: 160-166).

However, the new protein is 50 Kd in size and is named Ana o1 (Anacardium occidental), which is found in 50% of patients with cache allergies, but has a 25% positive rate even when resistant to cache. It is difficult to see them as suitable markers of diagnosis.

The allergy to sesame seeds is increasing in number due to the rapid growth of the fast food industry. Beyer K et al. Segregated proteins from sesame seeds to identify proteins that cause allergy to sesame. Electrophoresis was performed and these proteins were transferred to nitrocellulose membrane, and the proteins that reacted with serum of sesame allergy patients were identified.

In addition, Western blots of two-dimensional electrophoresis revealed 10 IgE-antibody-binding proteins with sizes of 78,52, 45,34 and 29 Kd. Among them, 75% of sesame allergy patients Was found to be positive. In addition, as a result of sequencing the protein, it was found to be 7S vicilin-type globulin (J. Allergy Clin. Immunol. 110: 154-159, 2002).

Allergy to peanuts is a typical allergic disease affecting children and adults. Many studies have been conducted to discover proteins that cause peanut allergy. Pons L., et al. Have found that oleosin is a protein that causes peanut allergy, and recombinant oleosine is known to react not only with peanuts but also with serum of soy allergy patients (Allergy 72: 88-). 93, 2002).

In addition, Quirce S., et al. Identified trypsin inhibitors as soy allergens (J. Allergy Clin. Immunol. 109 (1): 178, 2002), Ogawa A ( Ogawa A) et al. Discovered an allergen-producing protein that reacts with the serum of a patient with soybean-derived atopic dermatitis. These are a type of glycoprotein and show results of 60Kd, 30Kd and 28Kd. (J. Nutri Sci. Vitaminol. 46 (6): 271-279, 2000) and Polroninier P., et al. Have discovered allergen-producing proteins in almonds (Prunus dulcis). It was found to be albumin and coglutin gamma (2002 Int. Arch. Allergy Immunol. 128 (2): 97-104).

In addition, Ou K et al. Isolated proteins from the nest of Collocalia spp., And then carried out 2D-PAGE to transfer these proteins to the nitrocellulose membrane and reacted with the serum of the patient. Proteins with a positive response were identified.

Tove, et al., Discovered allergen-producing proteins in house dust mites through phage display technique and named them Ld 5,7,13 (Eur J. Biochem. 268: 287-294). , 2001).

These allergen-producing proteins discovered by the 2D-PAGE can be used as a useful material for the development of diagnostics and therapeutics by utilizing a very important research data on allergic diseases.

Accordingly, the present inventors have discovered a number of peptides that bind to the serum of various allergic patients, and tried to develop a pharmaceutical composition for the prevention and treatment of allergies by verifying the function of the discovered peptides, more specifically positive for egg whites, mites, etc. To detect a plurality of peptides that bind to the serum of the allergic patient responding, five novel peptides among the many discovered peptides proved that the anti-allergic reaction was completed the invention.

Accordingly, it is an object of the present invention to provide at least one peptide selected from a group consisting of a plurality of peptides having a short amino acid sequence that binds to the serum of an allergic patient that is positive for an allergen and salts thereof.

 Another object of the present invention is to provide a pharmaceutical composition containing the peptide or a salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a peptide or salt thereof comprising at least one amino acid sequence selected from the group consisting of the following amino acid sequences.

(a) RVSSCRGRNHIV;

(b) ETIGARWVRIE;

(c) PHVMRGGEYSGA;

(d) LVAHVGAGGVL; And

(e) LSYLLWRSRLP

The present invention also provides a pharmaceutical composition for the prevention and treatment of allergic diseases, which comprises the above peptide or a salt thereof as an active ingredient in order to achieve another object.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

 In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

The present invention finds a peptide that binds to the serum of an allergic patient who shows a positive reaction in egg white, house dust mite, etc. in a random peptide library developed on the surface of E coli , and through the biological verification A novel peptide having anti-allergic properties and a pharmaceutically acceptable salt thereof are determined, and a pharmaceutical composition for allergy prevention and treatment containing the peptide and its salt as an active ingredient.

In addition, the present invention is a random peptide library of 12-mer (12 mer) expressed on the surface of E. coli in order to discover a novel protein that causes allergy, serum (egg white, house dust mite of allergy patients) And a large number of peptides that react with each other, and then develop the peptides as anti-allergic agents.

At this time, since the random peptide library used in the present invention is a peptide produced by artificial random random 12-mer, it is a novel peptide showing a lot of difference in the amino acid sequence from allergic serum binding peptides derived from conventional natural proteins.

In addition, since the present invention does not discover peptides from natural substances, it is possible to infinitely develop peptides by artificially limiting the size of peptides that specifically bind to allergic serum binding peptides as well as the surface of other proteins.

In the present invention, the random peptide library (dodeca peptides) is thioredoxin (bioredning) through a known biopanning method (biopanning; Sidhu Phage display in pharmaceutical biotechnology, Curr Opin biotech, 11, 610-616, 2000). Thioredoxin itself, which is present in the active site of the allele, is expressed at the end of the bacterial pili using a recombinant phagemid inserted into the insoluble site of the flagellin gene. Facilitate the discovery of peptides that react to

In addition, the peptide library was reacted with serum of an allergic patient who showed positive reaction to egg whites and house dust mites through five biopanning methods, and peptides reacted with serum were eluted with an acid (pH 2.2) buffer and eluted. The colonized solution is neutralized and plated on a solid selection medium for bacteria, and colonies are formed to obtain a large amount of genes encoding peptides that react with the allergy by PCR.

In addition, the obtained PCR product is inserted into a T vector and transformed into Escherichia coli according to a known method, and the plasmid DNA of the transformed Escherichia coli is also extracted according to a known method to determine the DNA base sequence serum of an allergic patient. The amino acid sequence of the peptide which binds to is determined.

In the present invention, a total of 42 peptides were discovered by the above method, and the following known methods were used to verify the efficacy of the discovered peptides.

(1) enzyme immunoassay

First, reactivity with the serum of an allergic patient using a bacterium containing a gene encoding peptides is verified by ELISA (enzyme immunoassay) method.

(2) Rat Basophils Cell Line

In addition, the rat basophils cell line (RBL2H3), which is well known as a model of the allergic reaction, is used to verify the antiallergic function of the peptides discovered in the present invention.

This RBL2H3 cell line has receptors of IgE antibody on its surface, and when the receptors are complexed with each other by stimulation by antigen, beta hexosaminidase contained in the particles as the particles in RBL2H3 cells burst. When the secretion of the enzyme increases rapidly, an allergic reaction occurs, and the inflow of calcium ions increases during the allergic reaction, thereby activating serine / tyrosine kinase (Syk), a type of tyrosine kinase. This is because it is known that kinase activity such as mitogen activated protein kinase (MAPK) occurs in series.

In the present invention, as a result of investigating whether the peptides bound to the serum of allergic patients discovered inhibit the secretion of beta hexosaminidase, the above-mentioned five peptide concentrations among the 42 peptides It has been demonstrated to inhibit the secretion of the enzyme dependently.

(3) naive cell lines

Mast cells are present in tissues such as lungs, skin and small intestine and are closely related to allergic reactions. Receptors of IgE antibodies are present on the surface of the visceral cells, and upon stimulation by the antigen, these receptors form a complex and particles in the vitrec cells are secreted. At this time, beta hexosaminidase contained in the particle is secreted and mediators such as leukotriene (leukotriene) and histamine (histamine) are secreted.

Using this property, in the present invention, to investigate whether the five peptides excavated inhibit the secretion of these mediators, pulmonary tissues of guinea pigs were isolated and isolated lungs according to a known method. The tissues were treated with enzymes such as collagenase, elastase, and the like to purify the viable cells with a discrete density gradient using percoll, and then purify the viable cells to ovalbumin. After sensitization with a polyclonal IgG antibody that reacts, stimulation with Ovalbumin was conducted, the stimulation by Ovalbumin induces the release of histamine, but the pretreatment of the peptide found in the present invention induces the histamine The phenomenon is reduced.

In addition, in the present invention, the study on the signaling molecule involved in the secretion of beta hexosaminidase (b-hexosaminidase) was also performed in parallel, the production of free radicals in the secretion of beta hexosaminidase (β-hexosaminidase) It was found that this is accompanied, and when the function of signaling molecules such as ERK, PI3 kinase and the like is inhibited, it was found that the secretion of the enzyme is also inhibited.

In conclusion, the peptides bound to the serum of allergy patients discovered in the present invention concentration-dependently decreases allergic marker beta hexoseminidayes, and also inhibits the release of histamine, an allergic agent.

In addition, the five peptides discovered in the present invention have a similar inhibitory effect of beta hexoseminidayes to ketotifen fumarate, a conventional anti-allergic drug.

Therefore, the present invention can be developed as a medical composition for the prevention and treatment of allergy by applying these peptides.

At this time, the peptide found in the peptide is used by itself or in the form of pharmaceutically acceptable acid addition salts or metal complexes, for example, salts such as zinc and iron.

More specifically, the acid addition salt is preferably hydrogen chloride, hydrogen bromide, sulfate, phosphate, maleate, acet salt, citralate, benzoate, succinate, dried salt, ascorbate, tartalate.

In addition, the composition containing the peptide and salt thereof discovered in the present invention as an active ingredient is conventionally diluted according to the administration method, dosage form, and therapeutic purpose by mixing the active ingredient with a pharmaceutically acceptable carrier or diluting it. It is preferable to enclose it in the carrier of a container form.

When the carrier is used as a diluent, oral administration using at least one carrier selected from the group consisting of saline, buffer, dextrose, water, glycerol, Ringer's solution, lactose, sucrose, calcium silicate, methyl cellulose and ethanol And for parenteral administration in the form of powders, granules, injections, syrups, solutions, tablets, suppositories, pesaries, ointments, creams or aerosols. However, the carrier of the present invention is not limited to the above carrier.

In this case, parenteral administration refers to the administration of the drug through rectal, intravenous, peritoneal, muscle, arterial, transdermal, nasal, inhalation, in addition to oral.

In addition, the formulation may further include fillers, anti-coagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, and the like, and may be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

In addition, the dosage of the present invention can be adjusted according to the condition of the patient, the route of administration and the dosage form, and can be used within the range of not being desensitized by the body depending on the symptoms, but is usually an effective amount of the peptide preparations. 10-5,000 mg / kg is administered continuously or intermittently per day.

 Meanwhile, the amino acid sequences used in the present invention are abbreviated as follows according to the IUPAC-IUB nomenclature.

Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D

Cysteine: C, glutamic acid: E, glutamine: Q, glycine: G

Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M

Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W

Tyrosine: Y, Valine: V

In addition, the method of synthesizing the novel peptides discovered in the present invention is known solid-phase synthesis technique (Kaumaya. P. et al., Synthetic peptide vaccine: Misconceptions and problem, strategies and prospects Innovation and Perspectives in solide Phase Synthesis, Mayflower, 279 to 292, 1994), but any method may be used as long as the peptide can be synthesized.

At this time, it is preferable to use aminomethyl resin, methylbenzhydrylamine resin, aminomethylphenoxymethyl resin, and derivatives thereof as an insoluble resin having an amino group used in the synthesis of a novel peptide.

The protective amino acids include tos (tosyl), NOa (nitro), Mtr (4-methoxy-2,3,6-trimethylbenzenesulfonyl) and Pmc (2,2,5) which are arginine protecting groups for guanitino groups. , 7,8-pentamethylchloman-6-sulfonyl), Boc (t-butyloxycarbonyl), Fmoc (9-fluoroenylmethyloxycarbonyl), which is an amino acid protecting group for amino acids, and mer Bzl (benzyl), MBzl (4-methoxybenzyl), Acm (acetaminomethyl), etc. which are protective groups for capto groups can be used.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples, and it will be apparent to those skilled in the art that various changes or modifications can be made within the spirit and scope of the present invention.

Example 1 Diagnosis and Pathological Characteristics of Allergic Serum

In this example, the serum used to discover anti-allergic peptides was prepared to determine their diagnostic and pathological characteristics.

First, the serum of 8 allergic patients was diagnosed and divided by patients, and then the pathological characteristics of the serum divided by patients were shown in Table 1 below for atopic dermatitis patients and Table 2 below for asthma patients.

At this time, the response to egg white indicates that the level of Ig E antibody in serum against egg white is usually 3 or more, and it can not be excluded that it reacts to other foods due to the nature of food allergy patients. In allergic patients, it was usually analyzed that they did not have an allergic reaction to food (see Table 2).

number age Inspection code test results number age Inspection code test results 21 Egg white 45.2 Egg white 101 One bean 101 D.F. 0.49 milk 101 Total IgE 1156 D.F. 4.13 D.P. 0.74 Total IgE 3041 wheat 58.9 D.P. 3.94 buckwheat 16.4 wheat 30.1 peanut 16.9 buckwheat 4.64 28 One bean 58.4 peanut 101 milk 2.08 22 One bean 48.8 D.F. 42.8 Egg white 101 Egg white 101 milk 0.5 Total IgE 6910 Total IgE 944 D.P. 71.6 D.P. 0 wheat 101 wheat 36.2 buckwheat 14.4 buckwheat 10.8 peanut 69.3 peanut 83.6 tomato 32.3 D.F. 0 corn 101 26 One milk 29.5 potato 57.1 bean 18.8

number age Inspection code test results number age Inspection code test results 12 4 Total IgE 539 grass 0 D.P. 60.4 tree 0 D.F. 101 weed 0 grass 0 17 6 D.F. 101 tree 0 D.P. 56.7 weed 0 milk 0.81 14 6 D.F. 101 Total IgE 1144 D.P. 101 bean 0 milk 0.59 grass 0 Total IgE 1235 tree 0 Egg white 1.27 weed 0 buckwheat 0 19 5 D.F. 76.2 bean 0 5 D.P. 16.1 Egg yolk 0 5 Total IgE 220

And the serum which mixed the serum of the said lifetime was used for the next biopanning experiment.

Example 2 Biopanning

This example attempts to discover peptides that react with the serum of allergy patients from random peptide libraries using known biopanning methods.

First, mouse anti-Human Ig E antibody (Fc receptor-specific) was applied to 100 μl of sepharose beads containing mouse Ig G (immunoglobulin G) antibodies. 10 μl of the antibody) was added to 400 μl of PBS buffer (containing 1% BSA) and allowed to react for 1 hour in a locker. After the reaction, PBS buffer solution (500 μl) was added and mixed well, and centrifuged at 4 ° C. 2000 rpm for 5 minutes, the supernatant was discarded and washed with PBS buffer solution.

In addition, 500 μl of blocking buffer solution (1X PBS, 0.05% Tween 20, 2% BSA) was added and reacted in a rocker for 1 hour, washed with PBS buffer solution, and then allergic patient serum including serum of several patients. 200 μl of the solution was reacted in a rocker for 1 hour, and then washed with PBS buffer. In addition, 100 μl of a random peptide library of 12-mers (12-mer peptides) was added thereto, and reacted in a rocker for 1 hour, washed once with distilled water and PBS buffer, respectively, and then with a washing solution (5mM Tris-Cl ( pH7.5), 150mM NaCl, 0.1% Tween 20) and washed five times, and finally washed once with distilled distilled water and PBS buffer solution.

Then, elute 100 μl of elution buffer (0.1 M HCl, 0.1% BSA pH2.2) to elute the peptides bound to the serum of the allergic patient, and neutralize the eluted eluate with neutralizing solution (2 M Tris base). And smeared in E. coli solid medium (LB / Amp) (see FIGS. 1 and 2).

And the colony formed in the solid medium was used in Example 3 below.

Example 3 Colony PCR (Polymerase Chain Reaction) and T-Vector Cloning. Gene expression, sequencing

In this embodiment, the colony obtained in Example 2 is a bacterium expressing peptide-thioredoxin fusion DNA, and the colony is PCR-recognized to know the presence and sequence of peptides bound to the serum of allergy patient. Now.

(1) PCR

First, the collected colonies were released in 1 ml of distilled water, heat treated (for 5 minutes at 95 ° C.), and then 1 μl of the collected colonies was used as a DNA template to perform PCR reaction. In this case, the PCR reaction was performed at 94 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 1 minute, and the PCR reaction was set to 30 cycles. And the base sequence of the primer used for PCR reaction was as follows.

Forward primer: 5'-ATTCACCTGACTGACGAC-3 '

Reverse primer: 5'-CCCTGATATTCGTCAGCG-3 '

In addition, the composition of the mixture used for PCR reaction was as follows.

Reaction mixture: 50 µl, DNA template: 1 µl, 10 X PCR buffer: 5 µl,

100 mM dNTPs: 5 μl, forward primer: 1 μl, reverse primer: 1 μl

Sterile distilled water: 46 μl, Taq polymerase: 1 μl

Then, 1 μl in 50 μl of the PCR reaction product was loaded onto a 2% agarose gel and subjected to electrophoresis.

As a result, as shown in Figure 3, 177 bp product gene sequence (36 bp) encoding a peptide that reacts with the serum of allergic patients positive for egg white (a) and house dust mite (b) It was found to include.

This is because it is developed on the surface of the bacteria and inserted into the insoluble part of the thioredoxin gene to amplify other parts besides the gene encoding the peptide.

(2) T-vector cloning

Then, the band separated by electrophoresis was cut to elute DNA according to a known method, the eluted DNA was inserted into a T vector, and the T vector containing the inserted DNA was cut with EcoRI, a restriction enzyme, to insert DNA. As a result of confirming that this is normally done, as shown in FIG.

At this time, the reaction of inserting the gene encoding the peptide into the T vector was performed as follows.

T-Vector cloning; 1 μl of ligation buffer A, 1 μl of ligation buffer, 2 μl of yT & A cloning vector, 1-3 μl of PCR product, and 1 μl of T4 DNA ligase After reaction at room temperature for 20 minutes, transformant was transformed into competent cells of E. coli DH5α ( E- coli DH5α), and then 100 μM of IPTG 100 μl and X-gal (50 mg / mL) were placed 20 μl. The plate was plated on LB / Ampicillin agar, incubated for 16 hours, and white colonies were selected.

In addition, the plasmid DNA was extracted from the colony according to a known method to confirm whether the peptide was reacted with the serum of the allergic patient, and then the nucleotide sequence was confirmed.

The base sequence of the gene encoding the peptides inserted into the T-vector was confirmed using an automated ABI377 sequencer, and the primer used was 5'-ATTCACCTGACTGACGAC-3 '.

(3) western blot

In addition, in order to see the expression of genes encoding the peptides discovered in the present excavation, first, the bacteria containing the gene encoding the peptides were cultured in the medium with or without tryptophan. This is based on the action of E. coli tryptophan inhibitory operon based on the recombinant plasmid shown in FIG.

In addition, Western blot of the protein expressed in the cultured E. coli was confirmed as follows whether the expression of the peptide.

After incubating the bacteria containing the genes encoding the peptides for about 16 hours in RM medium, 40 μl of this was put in 2 ml of IMC medium containing tryptophan (100 μl / ml) to induce gene expression.

Then, centrifugation of the expression-induced bacterial solution for 20 seconds at 15,000 rpm to obtain a precipitate, put the known sample buffer solution in the precipitate and boil for 5 minutes, the protein dissolved in the sample buffer solution 10% SDS-polyacryl The amide gel was loaded and transferred to the nitrocellulose membrane. The protein transferred to the membrane is stained according to a known method (ponceau S staining method) to check whether the protein is delivered, and then the blocking solution (TBS-T, 3% BSA) is added to the membrane for 1 hour at room temperature, followed by washing. The solution was washed four times for 5 minutes and washed for a total of 20 minutes. Subsequently, an anti-thioredoxin antibody (1: 5,000 dilution) was added to the membrane, reacted for 1 hour, washed by the method described above, and a secondary antibody (anti Ig G-alkaline phosphatase, 1: 10,000 dilution) to which alkaline phosphatase was attached. ) Was added and reacted for 1 hour, followed by washing according to the method described above. After washing, 132 µl of NBT (nitroblue tetrazolium) and 66 µl BCIP (bromochloro indolyl phosphate) were added to alkaline phosphate buffer to confirm the color reaction. By doing so, it was possible to know whether the peptide was expressed.

As a result, as shown in Figure 5, the genes encoding the peptides were found to be induced by tryptophan in its peptide form (70kd) fused with thioredoxin.

In this case, the + sign in Fig. 5 shows the treatment of tryptophan, and the-sign shows no tryptophan.

In addition, peptides that bind to the serum of allergy patients for egg white and peptides that bind to the serum of allergy patients for dust mites in the amino acid sequences of the peptides discovered by the above-described method are shown in Tables 3 and 4, respectively.

Peptide name Amino acid sequence One QQWKRIPAGSAA 2 IGNVSGVINGMG 3 CGLEGRDLHVCQ 4 RLIWISKREPDS 5 AGRNAYMGKKCA 6 PSLACSGNRGW 7 QFARDYGTRLV 8 RARSGLSGAS 9 IRCRDFAAIGHQ 10 PWYQAFLVRGPG 11 WIRVKAYGWE 12 RRRPPSGHTGR 13 PKYCGVWCLGEV 14 SILKQTAGWR 15 RVVRYDADFWI 16 KVSGRGARKEAK 17 AVKGGAECASN 18 GRRLSTSGPGL 19 VGEGCVGRNA 20 GKADCTTSGTV 21 NMWGHLMGRLA 22 VLVLLLLEPRT 23 DMAGVRGSSQR 24 DTTTPLRGAVG 25 LSYLLWRSRLP 26 LVAHVGAGGVL 27 GFWCRRSGLVGV

Peptide name Amino acid sequence 28 PFPIRDGVQGP 29 LERVSPRSRLE 30 TSTIHPATGRR 31 VRGEIRNSIIQ 32 GTSRPCAPTLG 33 RVSSCRGRTHIV 34 RVSWCSGRTHIV 35 RVSSCRGRNHIV 36 ACKSSGGNGRVV 37 NSGQLNELALDH 38 PARLSGWYSR 39 ETIAGRWVRIE 40 TGRLLFHTDPVA 41 AVRASDQGIH 42 PHVMRGGEYSGA

Example 4 Enzyme Immunoassay (ELISA)

This example was to verify whether the peptides discovered in the above example actually binds to the serum of an allergic patient through enzyme immunoassay.

First, 2 ml of RM base medium was inoculated with colonies containing genes encoding peptides that bind to the serum of allergy patients obtained through biopanning and incubated at 37 ° C. for 12 hours, followed by tryptophan at a concentration of 20 μl / ml. 40 μl of the cells grown in the RM base medium was added to the IMC-induced medium, and cultured at 37 ° C. for about 16 hours.

After incubation, 1 X ^{10 9} cells were placed in an Eppendorf tube and centrifuged at 13,000 rpm for 30 seconds. After centrifugation, the supernatant was discarded and 1 ml of PBS buffer was added to the sediment, followed by voltexing. 10 μl each patient's serum was added and the reaction was performed in a rocker for 25 minutes. After the reaction, the supernatant was discarded by centrifuging at 13,000 rpm for 1 minute, and 1 ml of PBS buffer solution was added to the pellet, followed by vortexing. Subsequently, 1 ml of PBS buffer was added to the sediment again, 0.5 μl of biotin-conjugated-mouse anti human Ig E antibody was added, and the reaction was performed by a rocker for 25 minutes and centrifuged. .

The supernatant was discarded, and 1 ml of PBS buffer solution was added to the sediment again, and 0.5 µl of streptavidin-horse radish peroxidase (HRP) was added thereto, followed by 25 minutes of reaction with a rocker, followed by centrifugation. The supernatant was discarded and the PBS buffer solution was added back to the sediment, and each sample was dispensed into a 96 well plate, and the TMB solution (100 µl) was added to observe the color change.

The reaction was stopped using 2M phosphoric acid and absorbance was measured at 450 nm.

At this time, the criterion of the positive response to the allergic reaction is that the O.D. (optical density) value is the average value of the normal person + 2 S.D. Defined as exceeding (standard deviation).

In the present invention, the egg white positive allergy patient was defined as the level of the egg white specific IgE antibody is usually 3 or more, and the same criterion was also applied to dust mites and beans.

As a result, as shown in Figure 6, the serum used in the biopanning of the present invention are sera of allergy patients who showed positive reactions to egg white, house dust mite, etc., in the case of egg white allergy patients, antigen-specific IgE level is usually 3 or more Serum from allergic patients who were positive for house dust mites also showed antigen-specific IgE levels of 3 or more. Of the following seven peptides, EIGARWVRIE, RVSSCRGRNHIV, and PHVMRGGEYSGA are peptides that bind to the serum of allergy patients positive for house dust mite, and others bind to the serum of allergy patients positive for egg white.

Therefore, the peptides discovered through the biopanning of the present invention was found to react with the serum of allergy patients.

Example 4 Beta-hexosaminidase Secretion Analysis

In this embodiment, peptides whose amino acid sequences are determined through biopanning are synthesized by an automatic peptide synthesizer to determine whether to inhibit the secretion of beta hexosaminidase, a labeling enzyme of allergy. It was carried out according to the method.

In addition, the anti-allergic ketotifen fumrate was pretreated before antigen stimulation to investigate whether heximominides inhibited secretion.

To determine the secretion inhibitory effect of beta hexoseminidayes was performed as follows.

First, 2% BSA solution was reacted in a 96-well plate for 2 hours to coat each well, and 2 × 10 ⁵ cell lines (RBL2H3) were dispensed for each well.

The next day, anti-DNP IgE antibody (Anti DNP Ig E antiboy; 100 ng / ml) was sensitized to RBL2H3 cell line for 12-16 hours. After sensitization, the medium was sucked and washed once with PBS buffer, and Tyrode's buffer (Tyrode's buffer; 119 mM NaCl, 4.74 mM KCl, 2.54 mM CaCl2, 1.19 mM MgSO4, 1.19 mM KH2PO4, 10 mM HEPES, 100 μl of 5 mM glucose, 0.1% BSA, pH7.3) was added and incubated for 15 minutes. After incubation, 100 μl of peptide was added and incubated for 15 minutes, treated with DNP-HSA (100 ng / ml), and incubated for 1 hour. After incubation, 80 µl of the supernatant was added to another 96 well plate, and the same volume of substrate solution (0.2M citrate, 1 mM 4-methylumberliferyl-N-acetyl-bD-glucosamine, pH4.5) was added and reacted for 1 hour. The remaining cells during the reaction were scraped with a scraper and placed in a eppendorf tube, treated with lysis buffer (1% Triton X-100 in 1X PBS), centrifuged at 12,000 g at 4 ° C for 5 minutes, and the supernatant. Was added to a new 96-well plate, 100 μl of a stop solution (sodium bicarbonate pH 10.0) was added to stop the substrate reaction, and the reaction was performed for 5 minutes, and the absorbance was measured at 405 nm.

As shown in Figure 7, the RBL2H3 cell line (rat basophils, 10 ⁵ / well) used in the present invention was sensitized with anti-IgE antibody (100 ng / ml) specific for DNP (Dinitrophenol) After stimulation with various concentrations of antigen (DNP-HSA; 0.1, 1, 10, 100ng / ml), the level of secretion of beta hexosaminidase was measured. It was proportional to the concentration of, and the antigen showed the maximum effect at the concentration of 100 ng / ㎖.

In the present invention, it was confirmed by ElISA (enzyme immunoassay) that the Ig E antibody and the antigen DNP-HSA (DNP-dinitrophenyl-human serum albumin) bind to each other (data not shown).

8 is an experiment for identifying signal transduction molecules required for secretion of beta hexosminiminides by antigen stimulation. First, after sensitizing RBL2H3 cells with anti IgE antibody (100 ng / ml) for 16 hours. After treatment with various signal transduction inhibitors (LY294002, PD98059, N-acetyl-L-cystein,) hourly, incubate for 15 minutes with the antigen (DNP-HSA 100ng / ㎖), beta hexosaminidayes (b- hexosaminidase) secretion was measured according to known methods.

At this time, N-acetyl L-cystein inhibits the production of free radicals, PD98059 inhibits the phosphorylation of extracellular regulated kinase (ERK), a kind of kinase, and LY294002 also inhibits the function of PI3 kinase, a kind of kinase ( 8).

As shown in FIG. 8, the RBL2H3 cell line requires a signaling material such as ERK and PI3 kinase to secrete beta hexosminiday, and was closely related to the generation of active oxygen. In particular, ERK is a kinase that plays an important role in cell growth, differentiation, etc., and PI3 kinase is also expected to be able to expand into the region by playing an important role in cell growth, ringing, and angiogenesis.

At this time, the concentration of the antigen was used fixed to 100 ng / ㎖.

In addition, FIG. 9 illustrates the effect of the peptide discovered in the present invention on the secretion of beta hexosaminidase by antigen stimulation in an experiment using the RBL2H3 cell line. 100 ng / ml) for 16 hours and then pretreated with ketotifen fumarate (antiallergic drug) or peptide for 15 minutes. After stimulation with antigen (DNP-HSA 100 ng / ml) for 15 minutes, the level of beta hexosaminidase secretion was measured according to a known method.

As shown in FIG. 9, ketotifen fumarate (100 μM) was found to inhibit beta hexosaminidase secretion as expected.

At this time, the amino acid sequence of the peptide used in FIG. 9 was as follows.

RVV: RVVRYDADFWI

RVSS: RVSSCRGRNHIV

ETI: ETIGARWVRIE

PHV: PHVMRGGEYSGA

LVA: LVAHVGAGGVL

GFW: GFWCRRSGLVGV

TDG: TDGVTYTNDCL

LSY: LSYLLWRSRLP

In addition, the five peptides (RVSS, ETI, PHV, LVA, LSY) of the peptides apparently inhibit the secretion of beta hexosaminidase by antigen stimulation, it is known that the existing anti-allergic drug The effect was similar to that of totifen fumarate.

In this case, * indicates statistical significance. In the present invention, at least three independent experiments were performed, and statistical significance is indicated by an asterisk (*). Statistical processing was performed using student's T test.

In addition, the five peptides showed the maximum effect at 100 ° C (data not shown).

[Example 5] Isolation of hepatocytes from elucidation and measurement of histamine secretion

Histamine (molecular weight: 111) is a substance produced by decarboxylation of the amino acid histidine and is widely distributed in mammalian tissues. Histamine is a substance that mediates immediate immune hypersensitivity reactions. Excess in basophils and secreted by allergens, allergens can be identified through the amount of histamine secreted from basophils.

Using such a known method, the anti-allergic effect of the peptides discovered in the present invention was measured by measuring histamine secretion using an animal model.

First, in the present invention, the clarification (Hartley albmno female guinea pig) was used and the weight was about 200-250g. And separation of the plaque cells from the elucidation was performed according to a known method as follows (Ro et al. Journal of Pharamcology and Experimental Therapeutics 292 (1) 114-121, 2000).

Lung tissues were isolated from 10 elucidations according to known methods, with elucidation causing concussion without death.

Lung tissue obtained from the elucidation was purified with 50 ml of Tirod buffer (137 nM NaCl, 0.36 nM NaH2PO4, 2.6nM KCl, 1nM CaCl2, 1.5nM MgCl2, 119nM NaHCO3, 5.5nM glucose, 1g / l gelatin, pH7.4) After perfusion, the airways and blood vessels were removed. The lung tissue is then chopped with McWlain tissue chopper, and the chopped lung tissue is treated three times with collagenase (125 U / g tissue) and elastase (5 U / g tissue), respectively. It was set as 15,15,25 minutes. The cells constituting the lung tissue are separated out. The cells are filtered through a nytex network (100 μm), and then washed with a T-load buffer.

Then, the cells were poured onto a percoll solution (density, 1.045 g / mL), centrifuged at 800 g for 20 minutes, and the pellets containing the astigmatism cells were suspended in an xl tyrod buffer solution. Purification was through a discrete percol density gradient.

At this time, centrifugation was carried out at 800 g for 20 minutes through a density gradient, and then cut bands corresponding to the density of the apoptotic cells and washed with a T-load buffer to prepare the astigmatism cells.

In addition, 1 ml per 10 ⁶ cells of anti-ovalbumin antibody was added to purified plaque cells according to the above method, and then reacted for 2 hours at 37 ° C., and then the plaque cells were washed with a T-load buffer solution. And stimulated with antigen (0.1 μg / ml ovalbumin) for 10 minutes and the reaction was stopped.

Then, the supernatant was collected by centrifugation to measure the amount of histamine secreted by the antigen.

At this time, histamine secretion was measured using an automatic fluorescence spectrometer. As a result, it was found that the peptides of ETIGARWVRIE and RVVRYDAFWI showed histamine secretion inhibitory effect in the elucidation model.

Amount released (ng) Secretion degree (%) Suppression degree (%) Natural secretion (ng) 43.27 3.19 Total secretion (ng) 1451.96 Ovalbimin) 489.63 31.54 DMSO 468.22 30.01 4.82 RVSSCRGRNHIV 474.36 30.45 3.44 LVAHVGAGGVL 467.82 29.98 4.91 ETIGARWVRIE 400 25.16 20.21 PHVMRGGEYSGA 469.79 30.12 4.47 RVVRYDAD FWI 329.42 20.14 36.13

At this time, when the degree of inhibition of secretion exceeds 20%, the degree of inhibition was considered significant, and the measured value was performed three times, indicating that the average value was calculated.

As described above, the five peptides discovered in the present invention showed that five peptides inhibited the secretion of beta hexoseminidayes using the RBL2H3 cell line, but only two of the peptides in the experiment using elucidation. It was shown that histamine secretion was suppressed because the antibodies and antigens used to induce an allergic reaction in this example are different from those used in the RBL2H3 cell line.

However, the five peptides discovered in the present invention are novel peptides having sequences that are completely different from the amino acid sequences of conventional anti-allergic peptides, and their effects are not significantly different from those of conventional formulations. It is expected to be able to develop into pharmaceutical compositions.

In addition, the information of the five peptides discovered in the present invention are briefly as follows.

Information on Peptide No. 25:

(i) sequence characteristics:

a) length of sequence: 11

b) type of sequence: amino acid

c) form: linear

(ii) Type of sequence: peptide

(Iii) amino acid sequence: 1 LSYLLWRSRLP 11

Information for Peptide No. 26:

(i) sequence characteristics:

a) length of sequence: 11

b) type of sequence: amino acid

c) form: linear

(ii) Type of sequence: peptide

(Iii) amino acid sequence: 1 LVAHVGAGGVL 11

Information for Peptide No. 35:

(i) sequence characteristics:

a) length of sequence: 12

b) type of sequence: amino acid

c) form: linear

(ii) Type of sequence: peptide

(Iii) amino acid sequence: 1 RVSSCRGRNHIV 12

Information on Peptide No. 39:

(i) sequence characteristics

a) length of sequence: 11

b) type of sequence: amino acid

c) form: linear

(ii) Type of sequence: peptide

(Iii) amino acid sequence: 1 ETIGARWVRIE 11

Information of peptide number 42:

(i) sequence characteristics:

a) length of sequence: 12

b) type of sequence: amino acid

c) form: linear

(ii) Type of sequence: peptide

(Iii) amino acid sequence: 1 PHVMRGGEYSGA 12

As described above, the peptides discovered in the present invention have the property of binding to the serum of human allergic patients, and exhibit an allergic inhibitory effect in cell lines and animal experiments, and thus can be usefully used in the preparation for preventing or treating allergies. .

In addition, the peptides discovered in the present invention can be used for allergy diagnosis when used in a mixed form.

Claims (4)

Peptide or salt thereof consisting of at least one amino acid sequence selected from the group consisting of the following amino acid sequences. (a) RVSSCRGRNHIV; (b) ETIGARWVRIE; (c) PHVMRGGEYSGA; (d) LVAHVGAGGVL; And (e) LSYLLWRSRLP A pharmaceutical composition for preventing and treating allergic diseases, comprising the peptide of claim 1 or a salt thereof as an active ingredient. The method of claim 2, The allergic disease is asthma, atopic dermatitis, urticaria or allergic rhinitis disease, characterized in that the allergic prevention and treatment pharmaceutical composition. The method of claim 2 or 3, A pharmaceutical composition for preventing and treating allergies, the composition further comprising a stabilizer selected from the group consisting of proteins, sugars, buffers and mixtures thereof.

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