KR101492236B1 - Composition for preventing or treating asthma comprising Mutant protease allergen - Google Patents

Abstract

The present invention relates to a composition for preventing and treating asthma using a mutant protein allergen, and more particularly, to a composition for preventing or treating asthma induced by a fungal protease allergen A mutant protein allergen derived from Aspergillus oryzae , a gene encoding the mutant protein allergen, and a method for producing a mutant protein allergen.
The mutant protease allergen in which the protease activity of the present invention is removed induces immunotolerance against allergens and exhibits an inflammation-suppressing effect by allergens, so that it can be effectively used as a composition for preventing or treating asthma.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating asthma including mutant protease allergens,

The present invention relates to a composition for preventing and treating asthma using a mutant protein allergen, and more particularly, to a composition for preventing or treating asthma induced by a fungal protease allergen Aspergillus oryzae , a gene encoding the mutant protein allergen, and a method for producing the mutant protein allergen.

Asthma is a complex genetic disorder caused by the interaction of genetic and environmental factors in patients. In recent years, the prevalence of bronchial asthma has been increasing steadily as the number of causative allergens has increased due to the westernization of Korean people's lifestyles (Kim YK et al . al ., Clin Exp Allergy , 32: 1706,2002). Recent developments in immunology and molecular genetics have led to an increased understanding of the pathophysiology of bronchial asthma and the development of new therapies. In particular, steroids or bronchodilators, which are used as conventional therapeutic agents, should be used for a long time as a medicament for the control and prevention of symptoms, not the fundamental healing of diseases, and the economic burden of individuals and society is significant.

The immunological development mechanism of allergic diseases is mainly described as "hygiene hypothesis" (imbalance of Th1 / Th2 response). Th1 (type of helper cells Tianhe-I) response and Th2 (type of T helper cells Tianhe-2) response normally maintain their balance while antagonizing each other. However, allergic diseases including bronchial asthma have decreased Th1 response , And the imbalance of the immune system in which the Th2 response is exacerbated (Mosmann TR. Et al ., J Immunol ., 136: 2348, 1986).

There are several factors that determine the differentiation of Th1 and Th2 cells, including the interaction of antigen presenting cells with T cells, the amount of exposed antigens, and the secretion of cytokines, among which cytokines include Th1 / Th2 It plays a key role in determining response. A representative cytokine that increases the Th2 response is IL-4, and IL-12 is known as a cytokine that increases the Th1 response. IL-3, IL-4 and IL-5 secretion from Th2 cells was increased in bronchoalveolar lavage fluid of patients with asthma compared with normal subjects (Robinson DS. Et al ., N Engl J Med , 326: 298, 1992), indicating higher levels of IL-5 compared to other lung diseases (Walker C. et al ., Am J Respir Crit Care Med . , 150: 1038,1994).

Aspergillus , a representative allergen with protease activity, oryzae), and Aspergillus fumigatus (Aspergillus fumigatus- derived protease allergen was found to increase the production of inflammatory and mucous secretions, resulting in the development of asthma. However, when allergen without protease activity is administered, asthma develops did (Kheradmand et al Journal of Immunology, 169:. 5911, 2002), in the case of no inhibition of protease activity of the allergens that cause asthma or protease activity allergen induced expression of the MHC molecule is a co- IL-5 and IL-13, which are cytokines specific for Th2 cells, because they do not induce the proliferation of T cells because they do not induce the expression of stimulatory molecules (Lamhamedi- Cherradi et al . Journal of Immunology , 180: 6000, 2008).

Accordingly, the present inventors have made intensive efforts to develop a preventive and therapeutic agent for allergic asthma, and as a result, they have found that a mutant protein allergen inactivating the protease activity forms immune tolerance to allergens and inhibits the onset of asthma, Thereby completing the present invention.

It is an object of the present invention to provide a method for producing a mutant protein allergen from which protease activity has been eliminated, a gene encoding the mutant protein allergen, and a mutant protein allergen.

In order to achieve the above object, the present invention provides a mutant protein allergen represented by the amino acid sequence of SEQ ID NO: 4.

The present invention also provides a recombinant microorganism having mutagenic protein allergen producing ability, which is characterized in that a gene encoding a mutant protein allergen, a recombinant vector containing the gene, and the gene or a recombinant vector are introduced into a host cell .

The present invention also provides a method for producing a mutant protein allergen, comprising: (a) culturing the recombinant microorganism having mutant protein allergen producing ability to produce a mutant protein allergen; And (b) recovering the resulting mutant protein allergen. The present invention also provides a method for producing a mutant protein allergen.

The present invention also provides a composition for preventing or treating asthma containing the mutant protein allergen.

The mutant protease allergen in which the protease activity of the present invention is removed induces immunotolerance against allergens and exhibits an inflammation-suppressing effect by allergens, so that it can be effectively used as a composition for preventing or treating asthma.

1 is a graph showing the activity of an inactive protein allergen.
FIG. 2 is a graph showing the expression of immunoregulatory T cells and FoxP3 induced in lung tissue when ovalbumin was injected.
FIG. 3 is a schematic diagram for immunostimulatory use of an inactive protein allergen. FIG.
FIG. 4 is a graph showing the expression of immunoregulatory T cells and FoxP3 according to injection of an inactive protein allergen (A: physiological saline injection-negative control, B: saline-induced asthma induction-positive control, C: Induction of asthma after injection of heat (heat denaturation), and induction of asthma after injection of inactive protein (treatment with protease inhibitor).
FIG. 5 is a graph showing the inhibitory effect of asthma on induction of immune tolerance (A: lung inflammation reaction; B: degree of mucus production).
FIG. 6 is a graph showing the inflammation inhibition effect induced by induction of immunity (A: physiological saline infusion-negative control group, B: asthma induction-positive control group after injection of physiological saline, C: asthma after inactivated protease Induction, D; inactivation (treatment with protease inhibitor) injection).
FIG. 7 is a diagram showing a purification scheme of the produced mutant protein allergen. FIG.
Figure 8 is a graph showing the inhibitory effect of the invention of asthma according to the injection of the mutant protein allergen (A, airway hyper-responsiveness (AHR), B, mucus production, C, bronchoalveolar lavage cell, D, bronchoalveolar lavage (BAL) eosinophilia).
9 is a graph showing the expression of Treg cells and effect T cells upon injection of a mutant protein allergen (A: physiological saline injection (negative control), B: positive control (wild type AP), C: Heat allergen (heat AP) injection, D; mutant protein allergen (mutant AP) injection).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention does not induce the proliferation of T cells when administering an allergen without protease activity and does not produce cytokines such as IL-4, IL-5 and IL-13 specific to Th2 cells, Studies that do not cause inflammation and asthma (Kheradmand et al . Journal of Immunology, 169: 5911,2002; Lamhamedi-Cherradi et al . Inactivated protease allergen from which protease activity has been abolished induces immune tolerance to allergen, based on the results of the present study (Journal of Immunology, 180: 6000, 2008), confirming the possibility of being used as a composition for prevention and treatment of asthma And a mutant protease allergen with the protease activity removed was prepared.

The term "inflammatory, allergic or asthmatic" of the present invention is intended to encompass allergic, allergic, allergic or allergic diseases, including acute inflammation, chronic inflammation, bronchitis, bronchial asthma, atopy, allergic rhinitis, allergic asthma or allergic dermatitis, preferably acute inflammation, Rhinitis, allergic asthma, more preferably bronchial asthma, atopic or allergic rhinitis.

In one embodiment of the present invention, in order to prepare a composition for the prevention and treatment of allergic asthma induced by fungal protease allergen, a representative fungus-derived protease allergen, Aspergillus oryzae were heated at 95 캜 for 30 minutes or treated with a serine protease inhibitor to remove protease activity. As a result of the specific activity of the allergen from which the protease activity was removed, when the protease allergen was heated at 95 ° C for 30 minutes or when the serine protease inhibitor was treated, (Fig. 1).

To demonstrate the importance of protease activity of allergen causing asthma, OVA (chicken egg ovalbumin), a typical allergen without protease activity, was injected into the nose of the mouse without systemic sensitization, The presence of induction was confirmed. As a result, it was confirmed that immune tolerance was formed by inducing immunoregulatory T cells expressing FoxP3, rather than allergen-specific T cells (Fig. 2).

Immunoregulatory T cells developing in the thymus function to inhibit T cell proliferation and autoimmune response, and immunoregulatory T cells express a transcription factor called FoxP3 (forkhead box p3) to maintain their function have. Immunoregulatory cells expressing FoxP3 protein are known to play an important role in preventing excessive inflammation and immune response. In the present invention, immunoregulatory T cells are induced by injecting an inactive protein allergen through the respiratory tract, (See FIG. 3). The present invention also provides a composition for preventing and treating asthma.

In order to confirm the induction of immune tolerance and the asthma suppression effect of the inactivated protease allergen prepared according to the present invention, the mice were injected into the nose and then immunostimulatory induction was confirmed. As a result, the allergen with protease activity did not induce the expression of FoxP3, but the inactive protein allergen confirmed the expression of FoxP3 together with the induction of immunoregulatory T cells (Fig. 4). In addition, as a result of measurement of degree of lung / airway inflammation and mucus production and secretion degree, immunological tolerance induced mice were inoculated with an inactive protein allergen, and even in the case of allergen having protease activity, (BAL) was stained with Giemsa to measure the number of eosinophils. In order to analyze the extent of inflammation, the number of eosinophils was measured by inoculation of an inactivated protease It was confirmed that the inflammatory reaction was suppressed only in the group to which the allergen was administered (Fig. 6). This demonstrates that the allergen eliminating the protease activity has the effect of inhibiting the disease by inducing immune tolerance rather than inducing the disease by immune induction.

In one aspect, the present invention relates to a mutant protease allergen represented by the amino acid sequence of SEQ ID NO: 4.

In the present invention, the protein allergen is a yeast mold ( Aspergillus oryzae . < / RTI >

In the present invention, the mutant protein allergen may be characterized in that the protease activity is removed.

In another aspect, the present invention relates to a gene encoding a mutant protein allergen.

In the present invention, the gene may be characterized by being represented by the nucleotide sequence of SEQ ID NO: 3.

In another aspect, the present invention relates to a recombinant vector comprising a gene encoding a mutant protein allergen.

In the present invention, "vector" means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. Transfection was carried out as described in Sambrook, et . al ., supra , using the calcium chloride method described in section 1.82. Alternatively, electroporation (Neumann, et . al ., EMBO J. , 1: 841, 1982], and can also be used for transformation of these cells.

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

In another aspect, the present invention relates to a recombinant microorganism having a mutant protein allergen-producing ability, wherein a gene encoding a mutant protein allergen or a recombinant vector containing the gene is introduced into a host cell .

The recombinant microorganism according to the present invention can be prepared by inserting the gene on a chromosome of a microorganism according to a conventional method, or introducing the recombinant vector onto a plasmid of a microorganism.

In the present invention, the host cell may be Escherichia coli coli ).

In the present invention, the host cell may be selected from the group consisting of Agrobacterium , Aspergillus , Acetobacter , Aminobacter , Agromonas , Acidphilium , Bulleromyces , Bullera , Brevundimonas , Cryptococcus , Chionosphaera , Candida , Cerinosterus , Escherichia , Exisophiala in, Exobasidium in, Fellomyces in, Filobasidium genus, Geotrichum genus, Graphiola genus, Gluconobacter genus, Kockovaella in, Curtzmanomyces in, Lalaria in, Leucospoidium genus, Legionella genus, Psedozyma in, Paracoccus genus, Petromyc genus, Rhodotorula genus, Rhodosporidium genus, Rhizomonas in, Rhodobium in, Rhodoplanes genus Rhodopseudomonas genus Rhodobacter genus Sporobolomyces, A Spridobolus genus Saitoella genus Schizosaccharomyces genus Sphingomonas genus Sporotrichum genus, Sympodiomycopsis in, Sterigmatosporidium in, Tapharina genus Tremella genus, Trichosporon genus, Tilletiaria in, Tilletia genus, Tolyposporium in, Tilletiposis in, Ustilago genus, Udenlomyce in, Xanthophilomyces in, Xanthobacter in , Paecilomyces sp., Acremonium sp., Hyhomon sp., Rhizobium sp. Escherichia coli ).

In another embodiment of the present invention, a mutant protein allergen was produced to produce an allergen free of protease activity for the development of a vaccine for the prevention and treatment of asthma. A recombinant vector (pET-21a-wild AP) was constructed by introducing the gene of protein allergen derived from Nuruk Fungi (SEQ ID NO: 1) into the pET-21a vector and then amplifying the inserted protein allergen Mutations were induced in the genes.

In the case of serine protease, aspartic acid (D), histidine (H) and serine (S) residues are commonly present on the enzyme active site The aspartic acid and serine which are important for the dual protease activity are replaced by alanine (A), histidine is replaced with arginine (R) to induce a mutation (SEQ ID NO: 3), and a mutant protein allergen gene A recombinant vector (pET-21a-mutant AP) was prepared and then Escherichia coli coil , BL21) to prepare recombinant microorganisms.

The mutation of the protein means a protein having a sequence having a different amino acid sequence from a wild type and at least one amino acid residue. The mutant protein allergen is a protein having an amino acid sequence of a wild type protease allergen and at least one amino acid residue deleted , Insertions, non-conservative or conservative substitutions, or combinations thereof (H. Neurath, RL Hill, The Proteins, Academic Press, New York , 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

(A) culturing a recombinant microorganism having mutant protein allergen-producing ability to produce a mutant protein allergen; And (b) recovering the resulting mutant protein allergen.

In the present invention, the mutant protein allergen-producing recombinant microorganism was cultured to produce a mutant protein allergen (SEQ ID NO: 4), and the produced mutant protein allergen was purified by passing through a His-tag adsorption column (Fig. 7).

In another aspect, the present invention relates to a composition for preventing and treating asthma containing a mutant protein allergen.

In the present invention, the mutant protein allergen may be characterized by inducing immunoregulatory T cells to form immune tolerance to allergens.

In the present invention, in order to confirm the asthmatic inhibitory effect of the mutant protein allergen produced by the method of the present invention, the mutant protease allergen of the present invention was found to be an inactive protein allergen (Heat -inactivated AP), and showed similar protease activity to the control (water) (Table 2).

As a result of confirming the induction of immune tolerance and the asthma suppression effect of the mutant protein allergen prepared in the present invention, in the case of airway hyper-responsiveness (AHR) which is the main symptom of asthma, mutant protein allergen One group showed lower airway hyperresponsiveness than the group treated with the inactive protein allergen, and it was confirmed that they showed similar or low airway hyperresponsiveness to the negative control group (FIG. 8A). 8B), total cell (FIG. 8C) and eosinophilia (FIG. 8D) in bronchoalveolar lavage (BAL) in the immunostimulatory group induced by administration of the mutant protein allergen Was found to inhibit the inflammatory reaction to a degree similar to that of the group to which the inactive protein allergen was administered and it was confirmed that the mutant protein allergen of the present invention in which the protease activity was eliminated did not cause the disease caused by the immune induction, And it was confirmed that there was an effect of inhibiting disease.

In the present invention, in order to examine the mechanism of reduction of asthmatic symptoms by the mutant protein allergen, activation of T cells and production of T reg cells in the lung tissue of each mouse were examined. As a result, a positive control (wild type AP) Treg cells and effector T cells were significantly increased in one group, whereas Treg increased in the group injected with heat-inactivated AP and mutant protein allergen (mutant AP), whereas effector T cells were positive Compared with the control group (Fig. 9). The decrease in effector T cells, which are allergen-specific T cells, and the increase in Treg cells expressing FoxP3 means that immunity tolerance is formed. Therefore, the mutant protein allergen of the present invention is injected through the respiratory tract to induce immune tolerance It can be used as a composition for preventing and treating asthma.

The composition for preventing and treating asthma containing the mutant protein allergen of the present invention may contain 0.1 to 50% by weight of the mutant protein allergen based on the total weight of the composition, May contain one or more components.

The composition for preventing and treating asthma containing the mutant protein allergen according to the present invention may further comprise a pharmaceutically acceptable carrier. In the case of oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment and a flavoring agent may be used. , A stabilizer, and the like. In case of topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used. Formulations of the compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like in the case of oral administration, and in the case of injections, unit dosage ampoules or a plurality of dosage forms. In addition, the anti-cancer composition may typically comprise a surfactant that facilitates migration through the membrane. Such surfactants are those derived from steroids or cationic lipids such as N- [1- (2,3-dioloyl) propyl-N, N, N-trimethylammonium chloride (DOTMA), or cholesterol hemi- , Phosphatidylglycerol, and the like.

A composition for preventing and treating asthma containing a mutant protein allergen can be administered in a pharmaceutically effective amount for the prevention and treatment of asthma. The composition can be administered to a patient in accordance with the age, body weight, symptom characteristics and degree of the patient, The route of administration, the route of administration, the route of administration, the route of administration, the route of administration, the route of administration, the route of administration, the route of administration, The composition of the present invention may be administered together with the above-described pharmacological or physiological components or may be administered sequentially, or may be administered in combination with additional conventional therapeutic agents, and administered sequentially or concurrently with conventional therapeutic agents. Such administration may be single or multiple administrations. It is important to take into account all of the above factors and to administer the amount in which the maximal effect can be obtained in a minimal amount without side effects and can be easily determined by a person skilled in the art and the daily dose is 0.0001 based on the amount of the mutant protein allergen To 100 mg / kg, preferably 0.001 to 10 mg / kg, and can be administered 1 to 6 times a day. The methods may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In the present invention, 'administering' means introducing a predetermined substance into a subject by any suitable method, so that the administration route of the composition comprising the antibody of the present invention is administered through any conventional route so long as it can reach the target tissue . But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal. However, at the time of oral administration, since the protein is digested, it is preferable to formulate the oral composition so as to coat the active agent or protect it from decomposition at the top.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Inactive Protocols  Allergen ( protease allergen ) Produce

In order to prepare a composition for the prevention and treatment of allergic asthma induced by fungal protease allergen, a representative fungus-derived protease allergen, Aspergillus oryzae- derived protease activity was removed.

The protease activity was removed by heating the protease allergen derived from Nuruk Fungi at 95 ° C for 30 minutes or by treating with a serine protease inhibitor. To confirm the removal of protease activity, a protease assay kit (Product code: PF 0100, Sigma-Aldrich, USA) using a fluorogenic substrate (Casein-FITC) capable of measuring serine protease- USA) as follows.

20 μl of FITC-casein and 20 μl of incubation buffer were added to 10 μl of each sample (allergen and inactive protein allergen with protease activity) and control (trypsin), and incubated at 37 ° C. for 60 minutes Then, 150 μl of 0.6N TCA buffer was added to the sample and the control, respectively, and reacted at 37 ° C for 30 minutes. After centrifugation at 10,000 g for 10 minutes, 200 μl of each sample was transferred to a 96-well plate, and fluorescence values were measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. At this time. After centrifugation, the allergen with protease activity is based on the principle that the substrate is cut and FITC color is detected.

As a result, when the protease allergen was heated at 95 캜 for 30 minutes or treated with a serine protease inhibitor, it was confirmed that all of the protease activity was lost (Fig. 1).

Protocols  Inactive allergen ( chicken egg ovalbumin , OVA ) For immunostimulatory ( immune tolerance ) formation

To demonstrate the importance of protease activity, chicken egg ovalbumin (OVA), a representative allergen without protease activity, was injected into the nasal cavity of the mouse without systemic sensitization and the presence of induction of immune tolerance was confirmed .

Immune tolerance was induced by injecting five albumin allergens without protease activity through the nose of mice at intervals of 4 days (0 day, 4 days, 8 days, 12 days, and 16 days) For confirmation, the presence or absence of production of immunoregulatory T cells (T reg) and the expression of FoxP3 were examined. The mouse lung tissue was excised and mixed with antibodies labeled with CD4-FITC and FoxP3-APC, followed by analysis using a fluorescence activated cell sorter (FACS).

As a result, it was confirmed that immunoregulatory T cells expressing FoxP3, which is not an allergen-specific T cell, were induced and immune tolerance to allergens was formed (FIG. 2), which shows that immune protein allergen is injected through the respiratory tract And induce tolerance, thereby providing a possibility of being used as a composition for prevention and treatment of asthma (FIG. 3).

Identification of allergen-specific immunostimulatory effects against asthma

Experiments were carried out in the same manner as in Example 2 to confirm the induction of immunotolerance and the asthma suppression effect of the two kinds of inactive protein allergens prepared in Example 1.

5 μl of the inactive protein and 45 μl of ovalbumin were mixed and injected five times at 4-day intervals through the nose of the mice (0, 4, 8, 12 and 16 days) to induce immunotolerance . After that, 5 알 of allergen with protease activity and 45 의 of ovalbumin were mixed and injected through the nose of the mouse 5 times at intervals of 4 days to induce asthma. As the positive control group, when inducing immune tolerance, the mice were injected with physiological saline, which is not an inert protein allergen, to induce asthma by injecting allergen with protease activity after inducing immune tolerance. Negative control group was saline salin) did not induce both immune tolerance and asthma.

Thereafter, mouse lung tissues were excised and cultured with antibodies labeled with CD4-FITC and FoxP3-APC. FACS analysis was performed to determine whether or not immunoregulatory T cells (Treg) were produced and expression of FoxP3 Respectively.

The most common symptoms of asthma are airway constriction caused by airway hyperresponsiveness, chronic lung / airway inflammation based on eosinophils, excessive mucus production and secretion, and high potency of antigen-specific immunoglobulin E (IgE) antibodies . Therefore, in this experiment, lung / airway inflammation was measured by BAL differential cell count of bronchoalveolar lavage (BAL) and mucus production and secretion by ELISA method.

As a result, it was confirmed that the allergen with protease activity did not induce the expression of FoxP3, but that the inactive protein allergen induces immunoregulatory T cells together with the expression of FoxP3 (FIG. 4).

In addition, as a result of measurement of degree of lung / airway inflammation and mucus production and secretion degree, immunological tolerance induced mice were treated with an inactive protein allergen and treated with allergen having protease activity, (BAL) was stained with Giemsa to measure the number of eosinophils. In order to analyze the extent of inflammation, the number of eosinophils was measured by inoculation of an inactivated protease It was confirmed that the inflammatory reaction was suppressed only in the group to which the allergen was administered (Fig. 6). This demonstrates that the allergen eliminating the protease activity has the effect of inhibiting the disease by inducing immune tolerance rather than inducing the disease by immune induction.

Mutation Protocols  Production and production of allergens

In order to develop a vaccine for the prevention and treatment of asthma, a mutant protein allergen was produced to produce an allergen free of protease activity.

A recombinant vector (pET-21a-wild AP) was constructed by introducing the gene of protein allergen derived from Nuruk Fungi (SEQ ID NO: 1) into the pET-21a vector and then amplifying the inserted protein allergen Mutations were induced in the genes.

The nucleotide sequence of the oligonucleotide used in the preparation of the recombinant vector oligonucleotide (5'-3 ') 감정제 Forward (SEQ ID NO: 5) CGCATATGCA GTCCATCAAG CGTAC Nde I Reverse (SEQ ID NO: 6) CGGGATCCGC AGCGTTACCG TTG Bam HI

In the case of serine protease, aspartic acid (D), histidine (H) and serine (S) residues are commonly present in the enzyme active site Aspartic acid (asp162) and serine (ser349), which are important for dual protease activity, are mutated by substitution of alanine (alanine) for histidine (his193) with arginine (R) A recombinant vector (pET-21a-mutant AP) into which an allele gene was inserted was prepared. pET-21a contains the His-Tag sequence so that the expressed protein can be easily purified.

 (PET-21a-mutant AP) and a recombinant vector (pET-21a-wild AP) in which the gene of the mutant protein allergen gene was inserted into Escherichia coil (BL21) The recombinant microorganism was prepared by transforming.

The recombinant microorganism was cultured to express the mutant protein allergen (SEQ ID NO: 4) and the protein allergen (SEQ ID NO: 2), and to isolate the expressed mutant protein allergen and the protease allergen, The cell pellet was harvested and the cells were disrupted and centrifuged at 4 ° C and 16,000 rpm for 40 minutes. After centrifugation, the supernatant was passed through a His-tag adsorption column (affinity column, Ni column) to purify the mutant protein allergen and the protease allergen produced from the recombinant microorganism (FIG. 7).

In order to measure the activity of purified mutant protein allergen and protease allergen, protease assay kit using fluorogenic substrate capable of measuring serine protease specific activity was used.

Mutation Protocols  Asthma inhibition effect by allergen

5-1: mutation Protocols  Allergen activity measurement

A protease assay kit (Product code: PF 0100, Sigma-Aldrich) using a fluorogenic substrate capable of measuring serine protease specific activity to measure the activity of purified mutant protein allergens and protease allergens in Example 4, Aldrich, USA) was used.

Sample Fluorescence intensity (at 535 nm) (protease activity) Control (Water) 3,412 Trypsin 54,723 Aspergillus Protein from oryzae (AP) 315,437 Heat-inactivated AP 5,786 Mutant protein allergen 3,747

As a result of the measurement of the protease activity, it was confirmed that the mutant protein allergen of the present invention showed a lower protease activity than the heat-inactivated AP and showed a protein activity similar to that of the control (water) (Table 2).

5-2: Mutation Protocols  Confirming the effect of allergen on asthma

In order to confirm the asthma-suppressing effect of the purified mutant protein allergen in Example 4, the asthma-suppressing effect was compared with the inactivated protease allergen prepared by heating in Example 1.

5 μl of mutant protein allergen and 5 μl of ovalbumin, 5 μl of inactive protein and 45 μl of ovalbumin was injected through the nose of the mouse 5 times at intervals of 4 days 0 day, 4 day, 8 day, 12 day, and 16 day). After that, 5 μl of allergen having protease activity and 45 μl of ovalbumin were mixed and injected through the nose of the mouse 5 times at intervals of 4 days to induce asthma. As the positive control group, when inducing immune tolerance, the mice were injected with physiological saline, which is not an inert protein allergen, to induce asthma by injecting allergen with protease activity after inducing immune tolerance. Negative control group was saline salin) did not induce both immune tolerance and asthma.

To measure airway hyper-responsiveness (AHR), the main symptom of asthma, mice were anesthetized with pentobarbital sodium (Hanlim Pharma Co., Seoul, Korea, 60 mg / Kg), and a 20-gauge tube was intubated through the airway. After intubation, mechanical ventilation was connected to the flexiVent system (SCIREQ Inc., Montreal, Canada) and methacholine (Sigma-Aldrich, St.) was injected via airway nebulizer (SCIREQ) Louis, MO) and the airway resistance was measured. After each concentration of methacholine, the airway resistance dropped to basal levels, followed by the administration of the following concentrations of methacholine and the airway resistance of the next step.

 As a result of the measurement of the degree of airway hyperresponsiveness, the mutant protein allergen-treated group showed lower airway hyperresponsiveness than the group treated with the inactive protein allergen, and showed a similar or low airway hyperresponsiveness to the negative control group (Fig. 8A).

The degree of lung / airway inflammation and mucus production and secretion were measured by an ELISA method. As a result, mice immunized with the mutant protein allergen and induced with immune tolerance were treated with an inactive protein allergen (Fig. 8B). In order to analyze the degree of inflammation, bronchoalveolar lavage (BAL) was used to analyze the degree of inflammation The number of total cell (Fig. 8C) and eosinophilia (eosinophilia; Fig. 8D) was measured. As a result, the group to which the mutant protein allergen was administered showed the inhibition of the inflammatory reaction to a degree similar to that of the group to which the inactive protein allergen Respectively. This demonstrates that the mutant protease allergen of the present invention, in which the protease activity is removed, is effective in inhibiting disease through induction of immune tolerance rather than causing disease caused by immunity induction.

Mutation Protocols  Reduction of T cell activation by allergen

In order to investigate the mechanism by which the mutant protein allergen reduced asthma symptoms, activation of T cells and the degree of Treg formation were observed in the lung tissue of each mouse.

5 μl of mutant protein allergen (mutant AP) and 5 μl of ovalbumin, 5 μl of heat-inactivated AP and 45 μl of ovalbumin, Immune tolerance was induced by injecting 5 times at 4-day intervals (0, 4, 8, 12 and 16 days) through the nose. As a positive control group, allergen with protease activity (wild type AP) was injected and negative control group was injected with saline. After that, 5 μl of allergen having protease activity and 45 μl of ovalbumin were mixed and injected through the nose of the mouse 5 times at intervals of 4 days to induce asthma.

(CD25 + FoxP3 +) in each group, and CD4 T was identified by gating CD4 after making a single cell suspension. Then, And effector T cells (CD25 + FoxP3-) were analyzed using a fluorescence activated cell sorter (FACS).

As a result, it was confirmed that Treg cells and effector T cells were significantly increased in a group injected with a positive control (wild-type AP) compared to a negative control (saline injection), and heat-inactivated AP ) And mutant protein allergen (mutant AP), Treg were increased, but effector T cells were significantly reduced compared to the positive control (Fig. 9). It has been confirmed that the mutant protein allergen of the present invention can be injected through the respiratory tract to induce immune tolerance and thus can be used as a composition for prevention and treatment of asthma.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Korea Advanced Institute of Science and Technology <120> Composition for prevention or treating asthma comprising Mutant          protease allergen <130> P13-B184 <150> KR2012-0076149 <151> 2012-07-12 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 1211 <212> DNA <213> Aspergillus oryzae protease <400> 1 atgcagtcca tcaagcgtac cttgctcctc ctcggagcta tccttcccgc ggtcctcggt 60 gcccctgtgc aggaaacccg ccgggccgct gagaagcttc ctggaaagta cattgtcaca 120 ttcaagcccg gcattgacga ggcaaagatt caggagcata ccacctgggc taccaacatt 180 caccagcgca gtctggagcg tcgtggcgcc actggcggtg atcttcctgt cggtattgag 240 cgcaactaca agatcaacaa gttcgccgcc tatgcaggct ctttcgacga tgctaccatt 300 gaggagattc gcaagaacga agatgttgcc tacgtcgagg aggaccagat ctactacctc 360 gatggcctga ctacccagaa gagtgccccc tggggtctgg gcagcatttc ccacaagggc 420 cagcagagca ccgactacat ctacgacact agtgccggcg agggcaccta tgcctacgtg 480 gtggatagcg gtgtcaatgt cgaccatgag gagttcgagg gccgcgccag caaggcctac 540 aacgctgccg gtggtcagca tgtggacagc attggccatg gcacccacgt ttccggcacc 600 attgctggca agacttatgg tatcgccaag aaggccagca tcctttcggt caaagttttc 660 cagggtgaat cgagcagcac ttccgtcatt cttgacggct tcaactgggc tgccaacgac 720 attgttagca agaagcgtac cagcaaggct gcaatcaaca tgagcttggg cggtggctac 780 tctaaggctt tcaacgatgc ggtcgagaac gcattcgagc agggtgttct ctcggttgtc 840 gctgccggta acgagaactc tgatgccggc caaaccagcc ctgcctctgc ccctgatgcc 900 atcactgttg ccgctatcca gaagagcaac aaccgcgcca gtttctccaa ctttggcaag 960 gtcgttgacg tcttcgctcc cggtcaagat atcctttctg cctggattgg ctcttcctct 1020 gccaccaaca ccatctctgg tacctccatg gctactcccc acattgtcgg cctgtccctc 1080 tacctcgctg cccttgagaa cctcgatggc cccgctgccg tgaccaagcg catcaaggag 1140 ttggccacca aggacgtcgt caaggatgtt aagggcagcc ctaacctgct tgcctacaac 1200 ggtaacgctg c 1211 <210> 2 <211> 403 <212> PRT <213> Aspergillus oryzae protease <400> 2 Met Gln Ser Ile Lys Arg Thr Leu Leu Leu Leu Gly Ala Ile Leu Pro   1 5 10 15 Ala Val Leu Gly Ala Pro Val Gln Glu Thr Arg Arg Ala Ala Glu Lys              20 25 30 Leu Pro Gly Lys Tyr Ile Val Thr Phe Lys Pro Gly Ile Asp Glu Ala          35 40 45 Lys Ile Gln Glu His Thr Thr Trp Ala Thr Asn Ile His Gln Arg Ser      50 55 60 Leu Glu Arg Arg Gly Ala Thr Gly Gly Asp Leu Pro Val Gly Ile Glu  65 70 75 80 Arg Asn Tyr Lys Ile Asn Lys Phe Ala Ala Tyr Ala Gly Ser Phe Asp                  85 90 95 Asp Ala Thr Ile Glu Glu Ile Arg Lys Asn Glu Asp Val Ala Tyr Val             100 105 110 Glu Glu Asp Gln Ile Tyr Tyr Leu Asp Gly Leu Thr Thr Gln Lys Ser         115 120 125 Ala Pro Trp Gly Leu Gly Ser Ile Ser His Lys Gly Gln Gln Ser Thr     130 135 140 Asp Tyr Ile Tyr Asp Thr Ser Ala Gly Glu Gly Thr Tyr Ala Tyr Val 145 150 155 160 Val Asp Ser Gly Val Asn Val Asp His Glu Glu Phe Glu Gly Arg Ala                 165 170 175 Ser Lys Ala Tyr Asn Ala Gly Gly Gly Gln His Val Asp Ser Ile Gly             180 185 190 His Gly Thr His Val Ser Gly Thr Ile Ala Gly Lys Thr Tyr Gly Ile         195 200 205 Ala Lys Lys Ala Ser Ile Leu Ser Val Lys Val Phe Gln Gly Glu Ser     210 215 220 Ser Ser Thr Ser Val Ile Leu Asp Gly Phe Asn Trp Ala Ala Asn Asp 225 230 235 240 Ile Val Ser Lys Lys Arg Thr Ser Lys Ala Ile Asn Met Ser Leu                 245 250 255 Gly Gly Gly Tyr Ser Lys Ala Phe Asn Asp Ala Val Glu Asn Ala Phe             260 265 270 Glu Gln Gly Val Leu Ser Val Val Ala Ala Gly Asn Glu Asn Ser Asp         275 280 285 Ala Gly Gln Thr Ser Pro Ala Ser Ala Pro Asp Ala Ile Thr Val Ala     290 295 300 Ala Ile Gln Lys Ser Asn Asn Arg Ala Ser Phe Ser Asn Phe Gly Lys 305 310 315 320 Val Val Asp Val Phe Ala Pro Gly Gln Asp Ile Leu Ser Ala Trp Ile                 325 330 335 Gly Ser Ser Ser Ala Thr Asn Thr Ser Ser Ser Ser Ser Ser Ala Thr             340 345 350 Pro His Ile Val Gly Leu Ser Leu Tyr Leu Ala Ala Leu Glu Asn Leu         355 360 365 Asp Gly Pro Ala Ala Val Thr Lys Arg Ile Lys Glu Leu Ala Thr Lys     370 375 380 Asp Val Val Lys Asp Val Lys Gly Ser Pro Asn Leu Leu Ala Tyr Asn 385 390 395 400 Gly Asn Ala             <210> 3 <211> 1232 <212> DNA <213> Aspergillus oryzae mutant protease <400> 3 atgcaaagca taaagcgcac actgctactg ctgggagcaa ttctcccggc ggtactgggt 60 gcaccggtcc aggagacccg tagagcagca gaaaaactgc caggtaaata catcgttaca 120 ttcaaacccg gaatcgacga ggcaaagata caggaacata ccacatgggc aacaaatatt 180 caccagcgaa gcttagaacg tcggggtgca accggtggcg atttacccgt aggaattgaa 240 cgcaattaca aaattaacaa atttgccgcc tacgcaggtt ccttcgatga tgccaccatc 300 gaagaaattc gcaagaatga agatgtggca tacgtggaag aggatcaaat ctattacctg 360 gacggcctga ctacccagaa atctgctcca tggggtctgg gtagtatatc acataagggt 420 cagcaaagca ccgactatat ttatgatact tccgcagggg aaggcaccta tgcgtatgtc 480 gtagcaagcg gagtcaacgt ggatcatgaa gaatttgagg gtcgtgcaag caaagcatat 540 aatgcagcag gaggtcagca cgttgactca attggtcgtg ggacgcatgt tagcggtacc 600 attgcaggca aaacctatgg tatcgcaaag aaagctagca ttctgtctgt taaagtgttt 660 cagggtgaga gctcgtcgac gagcgttatc ctggatggtt ttaattgggc agcaaatgat 720 attgttagca agaaacgtac atccaaagcc gcgattaaca tgtctctggg aggtggttat 780 tctaaagcct ttaatgacgc cgttgaaaat gcatttgagc aaggggttct gagcgttgtt 840 gcagcaggta atgagaactc agacgcgggg cagacatctc cggcaagcgc acctgatgca 900 ataaccgttg cagcaattca gaaatctaat aacagggcta gtttcagcaa ttttggtaaa 960 gttgttgatg tctttgcacc tggtcaagat attctgagcg catggatagg ttctagctca 1020 gcgaccaata ccatcagtgg tactgcaatg gcaactccac atatcgttgg cctgagtttg 1080 tatctcgcgg cattagaaaa cctggatgga ccggcagcag tgactaaacg gattaaggaa 1140 ttggcaacga aagacgttgt caaagatgtg aaaggtagtc ctaaccttct tgcatataac 1200 gggaacgcag agaatttgta tttccagagc cg 1232 <210> 4 <211> 410 <212> PRT <213> Aspergillus oryzae mutant protease <400> 4 Met Gln Ser Ile Lys Arg Thr Leu Leu Leu Leu Gly Ala Ile Leu Pro   1 5 10 15 Ala Val Leu Gly Ala Pro Val Gln Glu Thr Arg Arg Ala Ala Glu Lys              20 25 30 Leu Pro Gly Lys Tyr Ile Val Thr Phe Lys Pro Gly Ile Asp Glu Ala          35 40 45 Lys Ile Gln Glu His Thr Thr Trp Ala Thr Asn Ile His Gln Arg Ser      50 55 60 Leu Glu Arg Arg Gly Ala Thr Gly Gly Asp Leu Pro Val Gly Ile Glu  65 70 75 80 Arg Asn Tyr Lys Ile Asn Lys Phe Ala Ala Tyr Ala Gly Ser Phe Asp                  85 90 95 Asp Ala Thr Ile Glu Glu Ile Arg Lys Asn Glu Asp Val Ala Tyr Val             100 105 110 Glu Glu Asp Gln Ile Tyr Tyr Leu Asp Gly Leu Thr Thr Gln Lys Ser         115 120 125 Ala Pro Trp Gly Leu Gly Ser Ile Ser His Lys Gly Gln Gln Ser Thr     130 135 140 Asp Tyr Ile Tyr Asp Thr Ser Ala Gly Glu Gly Thr Tyr Ala Tyr Val 145 150 155 160 Val Ala Ser Gly Val Asn Val Asp His Glu Glu Phe Glu Gly Arg Ala                 165 170 175 Ser Lys Ala Tyr Asn Ala Gly Gly Gly Gln His Val Asp Ser Ile Gly             180 185 190 Arg Gly Thr His Val Ser Gly Thr Ile Ala Gly Lys Thr Tyr Gly Ile         195 200 205 Ala Lys Lys Ala Ser Ile Leu Ser Val Lys Val Phe Gln Gly Glu Ser     210 215 220 Ser Ser Thr Ser Val Ile Leu Asp Gly Phe Asn Trp Ala Ala Asn Asp 225 230 235 240 Ile Val Ser Lys Lys Arg Thr Ser Lys Ala Ile Asn Met Ser Leu                 245 250 255 Gly Gly Gly Tyr Ser Lys Ala Phe Asn Asp Ala Val Glu Asn Ala Phe             260 265 270 Glu Gln Gly Val Leu Ser Val Val Ala Ala Gly Asn Glu Asn Ser Asp         275 280 285 Ala Gly Gln Thr Ser Pro Ala Ser Ala Pro Asp Ala Ile Thr Val Ala     290 295 300 Ala Ile Gln Lys Ser Asn Asn Arg Ala Ser Phe Ser Asn Phe Gly Lys 305 310 315 320 Val Val Asp Val Phe Ala Pro Gly Gln Asp Ile Leu Ser Ala Trp Ile                 325 330 335 Gly Ser Ser Ser Ala Thr Asn Thr Ile Ser Gly Thr Ala Met Ala Thr             340 345 350 Pro His Ile Val Gly Leu Ser Leu Tyr Leu Ala Ala Leu Glu Asn Leu         355 360 365 Asp Gly Pro Ala Ala Val Thr Lys Arg Ile Lys Glu Leu Ala Thr Lys     370 375 380 Asp Val Val Lys Asp Val Lys Gly Ser Pro Asn Leu Leu Ala Tyr Asn 385 390 395 400 Gly Asn Ala Glu Asn Leu Tyr Phe Gln Ser                 405 410 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence <400> 5 cgcatatgca gtccatcaag cgtac 25 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer sequence <400> 6 cgggatccgc agcgttaccg ttg 23

Claims (12)

A mutant protein allergen represented by the amino acid sequence of SEQ ID NO: 4;
The method according to claim 1, wherein the Aspergillus oryzae. &lt; / RTI &gt;
The mutant protease allergen according to claim 1, wherein the protease activity is eliminated.
A gene encoding the mutant protease allergen of claim 1.
5. The gene according to claim 4, which is represented by the nucleotide sequence of SEQ ID NO: 3.
A recombinant vector comprising a gene encoding the mutant protein allergen of claim 4 or 5.
7. The recombinant vector according to claim 6, which is pET-21a-mutant AP.
A recombinant microorganism having mutagenic protease allergen-producing ability, wherein the gene encoding the mutant protein allergen of claim 4 or a recombinant vector containing the gene is introduced into the host cell.
9. The method of claim 8, wherein said host cell is Escherichia wherein the recombinant microorganism is E. coli .
A method for producing a mutant protein allergen comprising the steps of:
(a) culturing a recombinant microorganism having the ability to produce a mutant protein allergen of claim 8 to produce a mutant protein allergen; And
(b) recovering the resulting mutant protein allergen.
A composition for preventing or treating asthma comprising the mutant protein allergen according to any one of claims 1 to 3.
12. The composition for preventing or treating asthma according to claim 11, wherein the mutant protein allergen induces immune tolerance to allergens by inducing immunoregulatory T cells.

Images