KR20200121954A - A composition comprising GSTT1 protein or encoding gene thereof - Google Patents

Abstract

The present invention relates to a composition comprising a glutathione S-transferases theta 1 (GSTT1) protein or a gene coding same as an active component. By inducing immune responses of a mucus barrier by means of phosphorylation of STAT3 and p38/JNK, the present invention can be effectively used for preventing or treating inflammatory bowel diseases. Moreover, by measuring whether a dimer of the GSTT1 protein controlling the cell signal transmission is present in a low level, it is possible to effectively predict inflammatory bowel diseases.

Description

Composition comprising GSTT1 protein or a gene encoding it as an active ingredient {A composition comprising GSTT1 protein or encoding gene thereof}

The present invention relates to a composition comprising a GSTT1 (glutathione S-transferases theta 1) protein or a gene encoding it as an active ingredient.

Inflammatory bowel disease (IBD) is a disease that causes chronic inflammation in the gastrointestinal tract, and its symptoms include abdominal pain, fever, diarrhea, and bleeding. In general, inflammatory bowel disease can be classified into two types: ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is a type of diffuse nonspecific inflammation of an unknown cause of the large intestine that forms ulcers or rubbing, and is accompanied by various systemic symptoms including bloody diarrhea. In addition, Crohn's disease is a granulomatous inflammatory lesion of unknown cause in which ulcers, fibrosis, stenosis and lesions progress from the mucous membrane to the entire intestinal tract from the entire digestive tract from the oral cavity to the anus discontinuously. Is accompanied by systemic symptoms of. The incidence rate of such inflammatory bowel disease was conventionally known to be high in Westerners, but the number of patients in Asian countries including Korea is increasing rapidly due to changes in lifestyle habits such as eating habits.

Until now, the cause or pathophysiology of the inflammatory bowel disease has not yet been clearly known, but it is estimated that genetic factors, environmental factors such as intestinal bacteria or food, and immunological factors will be involved in a complex manner. . One of the causes of this is the imbalance between oxidative stress and antioxidant defense mechanisms that occur in the intestine. Specifically, the production of oxidizing agents and free radicals not only activates a signaling mechanism that mediates the expression of inflammatory genes, but can also promote expression of genes that regulate cell division, differentiation, and death. However, when these oxidizing agents and free radicals are excessively produced, inflammation, cell damage, and apoptosis can be induced in intestinal epithelial cells (IEC), as well as dysfunction of the mucous membrane barrier. It is speculated that chronic inflammation may be induced in the inside.

In spite of this situation, in the case of inflammatory bowel disease, a fundamental treatment has not been established, so it is not aimed at complete treatment, but only delays and alleviates the progression of symptoms. Furthermore, sulfasalazine, the currently used treatment, has side effects such as nausea, vomiting, loss of appetite, rash, headache, liver injury, leukocyte reduction, abnormal red blood cells, proteinuria, and diarrhea. There are side effects such as, and Infliximab was used to treat Crohn's disease patients after it was approved by the US FDA as a treatment for Crohn's disease in 1998, but it was reduced pan blood cells, drug-induced lupus, hepatitis B/tuberculosis. There are side effects such as reactivation.

Accordingly, research on the development of drugs capable of treating inflammatory bowel disease with no or little side effects is still needed.

An object of the present invention is to provide a composition comprising a GSTT1 (glutathione S-transferases theta 1) protein or a gene encoding it as an active ingredient.

Another object of the present invention is to provide a method for screening a therapeutic agent for inflammatory bowel disease comprising measuring the expression level of the GSTT1 protein or a gene encoding it.

Another object of the present invention is to provide a composition for promoting proliferation of goblet cells comprising GSTT1 protein or a gene encoding the same.

Another object of the present invention is to provide a composition for diagnosing inflammatory bowel disease comprising an agent for measuring the level of the presence of a dimer of GSTT1 protein, a kit comprising the same, and a method of providing information on diagnosis using the same .

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned may be clearly understood by those of ordinary skill in the art from the following description.

One embodiment of the present invention provides a composition for preventing, improving or treating inflammatory bowel disease.

The composition of the present invention can be used as a pharmaceutical composition or a food composition.

The composition of the present invention includes a GSTT1 (glutathione S-transferases theta 1) protein or a gene encoding it as an active ingredient.

The GSTT1 protein of the present invention is one of glutathione S-transferase (GST), and when the GSTT1 protein or a gene encoding it is administered, the level at which the GSTT1 protein exists, preferably the level at which the GSTT1 protein exists as a dimer. By remarkably increasing STAT3 and p38/JNK, which is generated accordingly, by promoting the proliferation of goblet cells that protect the intestinal mucosa and inducing an immune response of the mucous barrier, the treatment of inflammatory bowel disease You can make it work.

The composition of the present invention may further include an IL-22 protein or a gene encoding it.

The IL-22 protein of the present invention is a cytokine that induces differentiation of goblet cells and healing of wounds. Since the IL-22 protein of the present invention induces an immune response of the mucosal barrier by increasing the expression of the GSTT1 protein, for the purposes of the present invention, when the IL-22 protein or a gene encoding it is further included, the composition is administered. This can induce a synergistic effect on the expected immune response of the mucosal barrier.

The GSTT1 protein of the present invention may consist of the amino acid sequence represented by SEQ ID NO: 1, and the gene encoding the GSTT1 protein may consist of the nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.

The IL-22 protein of the present invention may be composed of the amino acid sequence represented by SEQ ID NO: 3, and the gene encoding the IL-22 protein may be composed of the nucleotide sequence represented by SEQ ID NO: 4, but is limited thereto. no.

The GSTT1 protein or IL-22 protein of the present invention can be prepared by known protein synthesis methods or by culturing transformed host cells. In the case of using a host cell transformed with the protein, a recombinant expression vector containing a nucleotide sequence encoding the protein of the present invention is introduced into the host cell to prepare a transformant, and then any method known in the art is used. The transformant can be cultured by appropriate use. Proteins from the transformants cultured in this way can be obtained by separation and purification through methods such as centrifugation, chromatography, and electrophoresis known in the art.

The amino acid sequence of the present invention may be easily modified by substitution, deletion, insertion or a combination of one or more amino acids. Therefore, a protein or polypeptide having high homology with the amino acid sequence represented by SEQ ID NO: 1, for example, if the homology is 80% or more, 90% or more, or 95% or more, it maintains a function that can overcome antimicrobial resistance. It is included in all the proteins of the present invention.

The homology of the present invention is intended to indicate the degree of similarity with the amino acid sequence of a wild-type protein, and includes a sequence having the same sequence with the amino acid sequence of the present invention and more than the above-described sequence homology. Such homology can also be determined by visually comparing two sequences, but can be determined using a bioinformatic algorithm that analyzes the degree of homology by arranging the sequences to be compared side by side. Homology between the two amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA). Alignment algorithms automated in the module include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Algorithms and homology determinations for other useful arrangements can be automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.

It may be included as the gene encoding the GSTT1 protein or the gene encoding the IL-22 protein of the present invention, preferably included in a recombinant expression vector, but is not limited thereto.

The recombinant expression vector of the present invention is a recombinant expression vector capable of expressing a protein or peptide of interest in a host cell of interest, and refers to a gene construct comprising essential regulatory elements operably linked to express a gene insert. The expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator, and the start and stop codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and when the gene construct is administered, in an individual It must show an action and must be in frame with the coding sequence. The promoter of the vector may be a constitutive or inducible promoter.

The recombinant expression vector of the present invention is a promoter and the GSTT1 of the present invention The nucleotide sequence constituting the gene, that is, a polynucleotide may be operably linked. The'operably linked' of the present invention means a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function. . For example, a promoter and a polynucleotide encoding a protein or RNA can be operably linked to affect the expression of the coding sequence. The operative linkage with the recombinant expression vector may be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage may be performed using enzymes generally known in the art. .

The vector that can be used as the backbone of the recombinant expression vector of the present invention is not particularly limited as long as it can produce the protein of the present invention, but, for example, plasmid DNA, phage DNA, commercially developed plasmid ( pGEM ^® T vector, pET22b, pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pGEX-4T-1, pET, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc. ), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, AAAAAλgt11, λZAP, etc.), animal virus vectors (Retrovirus, Adenovirus, etc.), Vaccinia virus (Vaccinia virus, etc.), insect virus vector (Baculovirus (Baculovirus), etc.) may be, but is not limited thereto.

The inflammatory bowel disease of the present invention may be at least one selected from the group consisting of ulcerative colitis, Crohn's disease, enteric Behcet's disease, hemorrhagic rectal ulcer and ileal cystitis, but is not limited thereto. .

The pharmaceutical composition of the present invention may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and the pharmaceutical composition may be characterized for human.

The pharmaceutical composition of the present invention is not limited thereto, but is formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. I can. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffers, preservatives, painlessness, etc. for injections. An agent, a solubilizing agent, an isotonic agent, a stabilizer, and the like may be mixed and used, and in the case of topical administration, a base agent, an excipient, a lubricant, a preservative, and the like may be used. The formulation of the pharmaceutical composition of the present invention can be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.In the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. have. Others, solutions, suspensions, tablets, capsules, can be formulated as sustained-release preparations.

On the other hand, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may additionally be included.

The route of administration of the pharmaceutical composition of the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Includes sublingual or rectal. Oral or parenteral administration is preferred.

The "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. Preferably, the pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration, but is not limited thereto.

The pharmaceutical composition of the present invention depends on a number of factors including the activity of the specific compound used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug formulation and the severity of the specific disease to be prevented or treated. It may vary in various ways, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, route and duration of administration, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/kg per day Alternatively, it may be administered at 0.001 to 50 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention. The pharmaceutical composition according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.

The food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, and bread.

When the active ingredient of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 50% of the total weight, but is not limited thereto.

When the food composition of the present invention is prepared in the form of a beverage, there is no particular limitation other than including the food composition in the indicated ratio, and may contain various flavoring agents or natural carbohydrates, etc. as an additional component like a normal beverage. . Specifically, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and common sugars such as sucrose and polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are used. Can include. The flavoring agent may be a natural flavoring agent (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and a synthetic flavoring agent (saccharin, aspartame, etc.).

The food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, A pH adjuster, a stabilizer, a preservative, a glycerin, an alcohol, a carbonation agent used in carbonated beverages, and the like may be further included.

The ingredients included in the food composition of the present invention may be used independently or in combination. Although the ratio of the additive does not correspond to the core element of the present invention, it may be selected from 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

Another embodiment of the present invention provides a method for screening a therapeutic agent for inflammatory bowel disease.

The screening method of the present invention comprises the steps of treating a desired candidate substance in a biological sample isolated from a patient with inflammatory bowel disease; And measuring the expression level of the GSTT1 protein or a gene encoding it in the biological sample.

The screening method of the present invention includes the expression level of the GSTT1 protein measured in the biological sample or the gene encoding the same after treatment with the candidate substance, the GSTT1 protein measured in the biological sample isolated from the patient with inflammatory bowel disease or encoding it. When the expression level of the gene is increased, the step of selecting the candidate material as a therapeutic agent for inflammatory bowel disease may be further included.

In the screening method of the present invention, the GSTT1 protein or a gene encoding it and a description of inflammatory bowel disease include the pharmaceutical; And the same as described in the food composition, omitted in order to avoid excessive complexity of the present specification.

The biological sample of the present invention refers to any substance, biological body fluid, tissue, or cell obtained from or derived from an individual who is a patient with inflammatory bowel disease or suspected of a disease, for example, whole blood, Blood, sputum, tears, mucus, including leukocytes, peripheral blood mononuclear cells, leukocytes soft coat, plasma and serum ), nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluid fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate (nipple aspirate), bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extract, or cerebrospinal fluid ) May be included, but is not limited thereto.

The candidate substance of the present invention increases the activity to overcome inflammatory bowel disease, that is, the expression level of the GSTT1 protein or the gene encoding it, to a level equal to or similar to the expression level in a normal person without inflammatory bowel disease. It refers to a drug for testing whether or not there is an activity capable of causing it, and includes any molecule such as proteins, oligopeptides, organic molecules, polysaccharides, polynucleotides and a wide range of compounds. These candidate substances may include not only natural substances but also synthetic substances.

The formulation capable of measuring the expression level of the GSTT1 protein of the present invention means a formulation for checking the expression level of the protein to be measured contained in the biological sample. Specifically, the agent for measuring the expression level of the protein may be an antibody or aptamer capable of specifically binding to the protein. Specifically, the formulation is Western blot assay, ELISA (Enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, rocket Rocket immunoelectrophoresis, Immunohistochemical staining, Immunoprecipitation Assay, Complement Fixation Assay, Immunofluorescence, Immunochromatography, FACS Antibodies or aptamers used in methods such as (Fluorescenceactivated cell sorter analysis) and protein chip technology assay may be included, but are not limited thereto.

The "antibody" of the present invention refers to a protein molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. The form of the antibody is not particularly limited, and as long as it has a polyclonal antibody, a monoclonal antibody, or an antigen-binding property, it may be included even if it is a part of the antibody, and all kinds of immunoglobulin antibodies may be included. In addition, special antibodies such as humanized antibodies may be included, and the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules. The functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') 2, Fv, etc., but is not limited thereto.

The "aptamer" of the present invention refers to a single-stranded oligonucleotide, and refers to a nucleic acid molecule having binding activity to a predetermined target molecule. The aptamer may have various three-dimensional structures according to its base sequence, and may have high affinity for a specific substance such as an antigen-antibody reaction. The aptamer can inhibit the activity of a given target molecule by binding to a given target molecule. The aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and the form may be linear or cyclic.

The antibody of the present invention is GSTT1 The gene consisting of the nucleotide sequence of the gene, preferably the nucleotide sequence represented by SEQ ID NO: 2, is cloned into an expression vector according to a conventional method to obtain a protein (antigen) encoded by the gene, and then can be produced by a conventional method. In addition, the aptamer can be easily prepared according to a known method by a person skilled in the art by referring to each nucleotide sequence.

The agent capable of measuring the expression level of the gene encoding the GSTT1 protein of the present invention is mRNA transcribed from the gene to be measured in order to confirm the expression level of the gene to be measured included in the biological sample. It means a formulation that can be used in a method of measuring the level of. Specifically, the formulation is RT-PCR, quantitative real time PCR (quantified real time PCR), competitive RT-PCR (Competitive RT-PCR), real time quantitative RT-PCR (real time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blot assay, DNA chip analysis, and the like may include primer pairs or probes capable of specifically binding to the target mRNA to be measured, but are limited thereto. no.

The "primer" of the present invention is a base sequence having a short free 3'hydroxyl group and can form a complementary template strand and a base pair, and start for template strand copying It refers to a short nucleotide sequence that functions as a point. The primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.

The "probe" of the present invention refers to a nucleic acid fragment such as RNA or DNA that corresponds to a base capable of specifically binding to the mRNA, and such a probe includes the presence or absence of a specific mRNA and the level of expression (expression amount ) May be labeled. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, etc., but is not limited thereto.

The primer or probe of the present invention is GSTT1 With reference to the nucleotide sequence of the gene, preferably the nucleotide sequence represented by SEQ ID NO: 2, it can be easily produced by a method known to those of ordinary skill in the art.

In another embodiment of the present invention, there is provided a composition for promoting proliferation of goblet cells.

The composition for promoting proliferation of the present invention may include a GSTT1 protein or a gene encoding it.

The composition for promoting proliferation of the present invention may further include an IL-22 protein or a gene encoding it.

The composition of the present invention can induce an immune response of the mucosal barrier by promoting the proliferation of goblet cells having a protective function of the intestinal mucosa through phosphorylation of STAT3 and p38/JNK, and ultimately caused by a decrease in goblet cells. It can be applied to the prevention or treatment of possible diseases, for example, inflammatory bowel disease.

The "proliferation" of the present invention means that the number of cells is increased by dividing while maintaining the same properties. For the purposes of the present invention, the proliferation may be an increase in the number of goblet cells present in the intestinal mucosa.

In the composition for promoting proliferation of the present invention, the GSTT1 protein or a gene encoding it, inflammatory bowel disease, a dimer of the GSTT1 protein, and a formulation for measuring the level present include the pharmaceutical; And it is the same as described in the food composition and diagnostic composition, omitted in order to avoid excessive complexity of the present specification.

Another embodiment of the present invention provides a composition for diagnosing inflammatory bowel disease.

The diagnostic composition of the present invention includes an agent for measuring the level at which a dimer of GSTT1 protein is present.

The agent for measuring the level of the dimer of the GSTT1 protein of the present invention may be an antibody or aptamer that specifically binds to the GSTT1 protein present as a dimer.

The diagnostic composition of the present invention can diagnose inflammatory bowel disease by checking whether a dimer of the GSTT1 protein is present at a lower level compared to a normal control in a patient with inflammatory bowel disease.

The dimer of the GSTT1 protein of the present invention is a form in which amino acid residues present in two GSTT1 protein monomers are bound, and when there is an oxidation stimulus in the cell, the GSTT1 protein is oxidized by promoting p38/MAPK signaling. It can relieve stress. The GSTT1 protein dimer of the present invention is present at a high level in normal people who do not develop inflammatory bowel disease to relieve stimulation of oxidative stress, whereas in inflammatory bowel disease patients, GSTT1 gene mutations, for example, deletion mutations, are generated. The GSTT1 protein does not form a dimer and thus does not sufficiently alleviate the stimulation to oxidative stress, which may cause or worsen symptoms.

In the diagnostic composition of the present invention, the GSTT1 protein or a gene encoding the same and a substrate for inflammatory bowel disease include the pharmaceutical; And the same as described in the food composition, omitted in order to avoid excessive complexity of the present specification.

The "antibody" of the present invention refers to a protein molecule capable of specifically binding to the GSTT1 protein present as a dimer, and the form of the antibody is not particularly limited, but polyclonal antibody, monoclonal antibody or antigen binding If it has, it may be included even if it is part of the antibody, and all kinds of immunoglobulin antibodies may be included. In addition, special antibodies such as humanized antibodies may be included, and the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules. The functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') 2, Fv, etc., but is not limited thereto.

The "aptamer" of the present invention refers to a single-stranded oligonucleotide, and refers to a nucleic acid molecule having binding activity to the GSTT1 protein present as a dimer. The aptamer may have various three-dimensional structures according to its base sequence, and may have high affinity for a specific substance such as an antigen-antibody reaction. The aptamer can inhibit the activity of a given target molecule by binding to a given target molecule. The aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and the form may be linear or cyclic.

Another embodiment of the present invention provides a kit for diagnosing inflammatory bowel disease comprising the composition of the present invention.

The kit of the present invention can be predicted as inflammatory bowel disease when the dimer of GSTT1 protein is present in a target individual at a low level compared to a normal control in which inflammatory bowel disease does not exist using the composition of the present invention.

In the diagnostic kit of the present invention, a description of the GSTT1 protein or a gene encoding it, inflammatory bowel disease, a dimer of GSTT1 protein, and an agent for measuring the present level may include the pharmaceutical; And it is the same as described in the food composition and diagnostic composition, omitted in order to avoid excessive complexity of the present specification.

The kit of the present invention may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit, but is not limited thereto.

When the kit of the present invention is for measuring the level of a protein, a substrate, an appropriate buffer solution, a secondary antibody labeled with a color developing enzyme or a fluorescent substance, a color developing substrate, etc. may be included for the immunological detection of the antibody.

The substrate of the present invention may be a nitrocellulose membrane, a 96-well plate synthesized from a polyvinyl resin or a polystyrene resin, and a slide glass made of glass, and the coloring enzyme is peroxidase, alkaline phosphatase (Alkaline). Phosphatase) can be used, fluorescent materials can be used, such as FITC, RITC, etc., and the color developing substrate solution is 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (2,2'-azino -bis (3-ethylbenzothiazoline-6-sulphonic acid); ATBS), o-Phenylenediamine (OPD), tetramethyl benzidine (TMB) may be used, but is not limited thereto.

Another embodiment of the present invention provides a method of providing information for diagnosis of inflammatory bowel disease.

The method of the present invention includes measuring the level of the presence of a dimer of GSTT1 protein in a biological sample isolated from a subject of interest.

In the method of the present invention, when the level of the presence of the dimer of GSTT1 protein measured in the biological sample isolated from the target individual is lower than that of the normal control, the target individual can be predicted to be inflammatory bowel disease.

The biological sample isolated from the object of interest of the present invention refers to any substance, biological fluid, tissue or cell obtained from or derived from a normal or suspected disease, for example, whole blood ), leukocytes, peripheral blood mononuclear cells, leukocytes soft coat, blood including plasma and serum, sputum, tears, mucus (mucus), nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, fluid in the pelvis (pelvic fluids), cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipples Nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extract, or cerebrospinal fluid ( cerebrospinal fluid), but is not limited thereto.

In the method of providing information for the diagnosis of the present invention, the description of the GSTT1 protein or a gene encoding it, inflammatory bowel disease, a dimer of the GSTT1 protein, and an agent for measuring the level present may include the pharmaceutical; And it is the same as described in the food composition and diagnostic composition, omitted in order to avoid excessive complexity of the present specification.

The step of measuring the level of the protein present of the present invention is Western blot analysis, ELISA, radioimmunoassay, radioactive immunity diffusion method, Ouchteroni immune diffusion method, rocket immunoelectrophoresis, immune tissue using the composition of the present invention. It can be carried out through at least one selected from the group consisting of chemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, FACS, and protein chip assay, preferably the step is non-reducing conditions It may be performed in, but is not limited thereto.

The non-reducing conditions of the present invention are agents that facilitate analysis such as electrophoresis by breaking the disulfide bonds of proteins, for example, β-mercaptoethanol or DTT (dithiothreitol). As a condition, it may be a condition suitable for detecting oxidized AMPK protein for the purposes of the present invention.

[Sequence List]

SEQ ID NO: 1: GSTT1 amino acid sequence

MVLELYLDLLSQPCRAIYIFAKKNNIPFQMHTVELRKGEHLSDAFARVNPMKRVPAMMDGGFTLCESVAILLYLAHKYKVPDHWYPQDLQARARVDEYLAWQHTGLRRSCLRALWHKVMFPVFLGEQIPPDLGPDKFLQDLPADLKDKFLQDLKDLKFLQDL

SEQ ID NO: 2: GSTT1 gene sequence

ATGGTTCTGGAGCTGTACCTGGATCTGCTGTCGCAGCCCTGTCGCGCCATTTATATCTTCGCCAAGAAGAACAATATCCCGTTCCAGATGCACACGGTGGAGCTGCGCAAGGGTGAGCACCTCAGCGATGCGTTTGCCCGGGTGAACCCCATGAAGAGGGTACCAGCCATGATGGATGGTGGCTTCACCCTGTGTGAGAGTGTGGCTATCTTGCTCTACCTGGCACACAAGTATAAGGTTCCTGACCACTGGTACCCCCAAGACCTGCAGGCTCGTGCTCGTGTAGACGAGTACCTGGCATGGCAGCATACGGGCCTTCGGAGAAGCTGCCTCAGGGCCCTGTGGCATAAGGTGATGTTCCCTGTTTTCCTTGGTGAGCAAATACCTCCTGAAACACTGGCAGCCACGTTGGCAGAACTGGATGTTAACCTACAGGTGCTTGAAGACAAGTTCCTCCAGGACAAAGACTTCCTTGTTGGGCCCCACATCTCCCTGGCCGACTTGGTGGCCATCACAGAGCTGATGCATCCTGTAGGTGGTGGCTGCCCAGTCTTTGAAGGGCATCCCAGGCTGGCTGCATGGTACCAGCGAGTGGAGGCAGCTGTGGGGAAGGACCTCTTCCGGGAAGCCCATGAAGTCATCCTGAAGGTGAAGGACTGTCCCCCTGCTGACCTCATCATAAAGCAGAAGCTGATGCCCAGAGTGCTGGCAATGATCCAG

SEQ ID NO: 3: IL-22 amino acid sequence

1 maalqksvss flmgtlatsc llllallvqg gaaapisshc rldksnfqqp yitnrtfmla

61 keasladnnt dvrligeklf hgvsmsercy lmkqvlnftl eevlfpqsdr fqpymqevvp

121 flarlsnrls tchiegddlh iqrnvqklkd tvkklgesge ikaigeldll fmslrnaci

SEQ ID NO: 4: IL-22 gene sequence

1 acaagcagaa tcttcagaac aggttctcct tccccagtca ccagttgctc gagttagaat

61 tgtctgcaat ggccgccctg cagaaatctg tgagctcttt ccttatgggg accctggcca

121 ccagctgcct ccttctcttg gccctcttgg tacagggagg agcagctgcg cccatcagct

181 cccactgcag gcttgacaag tccaacttcc agcagcccta tatcaccaac cgcaccttca

241 tgctggctaa ggaggctagc ttggctgata acaacacaga cgttcgtctc attggggaga

301 aactgttcca cggagtcagt atgagtgagc gctgctatct gatgaagcag gtgctgaact

361 tcacccttga agaagtgctg ttccctcaat ctgataggtt ccagccttat atgcaggagg

421 tggtgccctt cctggccagg ctcagcaaca ggctaagcac atgtcatatt gaaggtgatg

481 acctgcatat ccagaggaat gtgcaaaagc tgaaggacac agtgaaaaag cttggagaga

541 gtggagagat caaagcaatt ggagaactgg atttgctgtt tatgtctctg agaaatgcct

601 gcatttgacc agagcaaagc tgaaaaatga ataactaacc ccctttccct gctagaaata

661 acaattagat gccccaaagc gatttttttt aaccaaaagg aagatgggaa gccaaactcc

721 atcatgatgg gtggattcca aatgaacccc tgcgttagtt acaaaggaaa ccaatgccac

781 ttttgtttat aagaccagaa ggtagacttt ctaagcatag atatttattg ataacatttc

841 attgtaactg gtgttctata cacagaaaac aatttatttt ttaaataatt gtctttttcc

901 ataaaaaaga ttactttcca ttcctttagg ggaaaaaacc cctaaatagc ttcatgtttc

961 cataatcagt actttatatt tataaatgta tttattatta ttataagact gcattttatt

1021 tatatcattt tattaatatg gatttattta tagaaacatc attcgatatt gctacttgag

1081 tgtaaggcta atattgatat ttatgacaat aattatagag ctataacatg tttatttgac

1141 ctcaataaac acttggatat cctaa

SEQ ID NO: 5: GSTT1 forward primer (human)

TCTTTTGCATAGAGACCATGACCAG

SEQ ID NO: 6: GSTT1 reverse primer (human)

CTCCCTACTCCAGTAACTCCCGACT

SEQ ID NO: 7: β-actin forward primer (human)

CTCTTCCAGCCTTCCTTCCTG

SEQ ID NO: 8: β-actin reverse primer (human)

CAGCACTGTGTTGGCGTACAG

SEQ ID NO: 9: GSTT1 forward primer (mouse)

GTAGGTTAACATCCAGTTCTGC

SEQ ID NO: 10: GSTT1 reverse primer (mouse)

GGCACATGGCAGCATACGG

SEQ ID NO: 11: β-actin forward primer (mouse)

AGTGTGACGTTGACATCCGT

SEQ ID NO: 12: β-actin reverse primer (mouse)

TGCTAGGAGCCAGAGCAGTA

SEQ ID NO: 13: IL-22 forward primer (mouse)

GGCCAGCCTTGCAGATAACA

SEQ ID NO: 14: IL-22 reverse primer (mouse)

GCTGATGTGACAGGAGCTGA

SEQ ID NO: 15: Muc2 forward primer (mouse)

GGTCCAGGGTCTGGATCACA

SEQ ID NO: 16: Muc2 reverse primer (mouse)

GCTCAGCTCACTGCCATCTG

SEQ ID NO: 17: Muc2 forward primer(human)

AGGATGACACCATCTACCTCACC

SEQ ID NO: 18: Muc2 reverse primer (human)

GGTGTAGGCATCGCTCTTCTC

SEQ ID NO: 19: CAMP forward primer (human)

GCACGCTGACACCACTACC

SEQ ID NO: 20: CAMP reverse primer (human)

CGGGCTATTCCCTGTCCAC

The composition according to the present invention can be used very effectively in the prevention or treatment of inflammatory bowel disease by promoting the proliferation of goblet cells through phosphorylation of STAT3 and p38/JNK to induce an immune response of the intestinal mucosa.

Furthermore, it is possible to very effectively predict inflammatory bowel disease by measuring whether or not a dimer of the GSTT1 protein that regulates cell signaling is present at a low level.

1A is a result of immunohistochemical staining according to an embodiment of the present invention, and FIGS. 1B and C are quantitative real-time reverse transcription polymerase chain reactions for the expression level of the GSTT1 gene according to an embodiment of the present invention. It shows the results confirmed through (qRT-PCR).
2 shows the result of staining goblet cells in an animal model of colitis injected with an antibody or IgG specific to GSTT1 protein according to an embodiment of the present invention.
3A to 3D show changes in body weight (FIG. 3A), disease activity index (DAI) (FIG. 3) in mice in which the GSTT1 gene according to an embodiment of the present invention was transformed through rectal injection. B), the change in the length of the intestine (FIG. 3C) and the goblet cells (FIG. 3D) are shown.
4A to 4E show changes in body weight (FIG. 4A), disease activity score (FIG. 4B), and survival rate (FIG. 4A), in mice transformed by intraperitoneal injection of GSTT1 gene according to an embodiment of the present invention. 4C), the change in length of the intestine (FIG. 4D) and the results of confirming the goblet cells (FIG. 4E) are shown.
5A to 5C show the results of confirming the expression levels of the GSTT1 gene and IL-22 gene according to an embodiment of the present invention, and change in body weight (D in FIG. 5), and disease activity score (E in FIG. 5). , It shows the result of confirming the change in the length of the intestine (F in Fig. 5) and the goblet cells (G in Fig. 5).
6A shows the result of confirming the expression level of GSTT1 protein according to an embodiment of the present invention through immunohistochemistry, and FIG. 6B and C are quantitative real-time reverse transcription polymerization for the expression level of the GSTT1 gene. It shows the results confirmed through the enzyme chain reaction, Figure 6D is the result of confirming the expression level of the GSTT1 protein through western blot analysis after siRNA treatment specific to the GSTT1 gene, and E to H of Fig. 6 are the results of the GSTT1 protein. It shows the results of confirming the expression levels of genes in cells whose expression is suppressed.
7A and B show the expression levels of the monomers and dimers of the GSTT1 protein through Western blot analysis, and the expression levels of the dimer GSTT1 protein according to the total GSTT1 as a ratio.
8A and B show the expression level of the GSTT1 protein monomer and dimer through Western blot analysis, and show the expression level of the dimer GSTT1 protein according to the total GSTT1 as a ratio, and FIG. 8C shows the GSTT1 protein and It shows the result of confirming the expression level of the related cell signaling protein through Western blot analysis.
9 is a schematic diagram showing the mechanism of action of the GSTT1 protein according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

[Preparation Example 1] Cell line culture and transformation

The HT-29 cell line (Korea Cell Line Bank, Korea) was cultured in RPMI medium containing 10% fetal bovine serum and 1% antibiotic inactivated using heat under conditions of 5% CO ₂ and 37°C. . Transformation of siRNA or non-targeting control (non-targeting control; AccuTarget, Bioneer, Korea) complementarily binding to GSTT1 gene (SEQ ID NO: 2) to the cell line using Lipofectamine 2000 (Life Technologies, USA) I did.

[Preparation Example 2] Mouse breeding conditions and colitis animal model construction

Wild-type C57BL/6 mice (OrientBio, Korea) were reared in a pathogen-free 12:12 dark cycle environment. Drinking water containing 2.5% (W/V) of DSS (Dextran Sulfate Sodium Salt, MW. 36,00050,000; MP Biomedicals, USA) sterilized using a filter was supplied to 8-week-old mice for 7 days, Subsequently, colitis was induced in the mouse by supplying only sterilized drinking water for 2 days to prepare an animal model of colitis.

[Preparation Example 3] Gene delivery to mice

The vector shown in Table 1 was injected twice (day 1 and 5) into the rectal (30 µg in 100 µl PBS) or intraperitoneal (100 µg in 1 ml PBS) of the colitis animal model or wild-type mouse of Preparation Example 2 I did. At this time, the efficiency of gene transfer was improved by using a mixed solution in which the vector and the transformant PEI (Polyetherimide) were mixed in a ratio of 1:1 ((w:v, μg DNA:μl PEI). At the time of rectal or intraperitoneal injection of the solution, the colitis animal model and wild-type mouse were anesthetized using Isofurane (Ifrane liquid, MNS Korea) In this case, the mixed solution was evenly distributed in the body of the colitis animal model and wild-type mouse. In order to be able to do so, the colitis animal model and the mice were hung upside down for 20 seconds, and finally, as shown in Table 1 below, mice corresponding to the number of total 8 cases were produced.

Injection method vector DSS processing Display rectal pCMV-GSTT1-GFP - DSS(-)-rectal:pGSTT1(+) mouse + DSS(+)-rectal:pGSTT1(+) mouse pEGFP-N1 - DSS(-)-rectal:pGSTT1(-) mouse + DSS(+)-rectal:pGSTT1(-) mouse Abdominal cavity pCMV-GSTT1-GFP - DSS(-)-ip:pGSTT1(+) mouse + DSS(+)-ip:pGSTT1(+) mouse pEGFP-N1 - DSS(-)-ip:pGSTT1(-) mouse + DSS(+)-ip:pGSTT1(-) mouse pCMV-GSTT1-GFP: CMV vector containing GFP-tagged mouse GSTT1 gene (OriGene, USA)
pEGFP-N1: pCMV-(AC)-EGFP-N1, CMV vector containing EGFP (AddGene, USA)

[ Example One] GSTT1 Confirmation of protein and gene expression levels

Using Immunohistochemistry staining and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), patients with inflammatory bowel disease (Crohn's disease (CD); intestinal Behηet's disease (intBD)) or normal subjects (CTL) ) In the tissues isolated from the intestine, the expression levels of the GSTT1 protein and the GSTT1 gene were confirmed. In addition, the expression level of the GSTT1 gene in the tissue isolated from the intestine of the colitis animal model and wild-type mouse of Preparation Example 2 was confirmed.

Paraffin-embedded tissue sections fixed with formalin for immunohistochemical staining were deparaffinized, washed with distilled water, placed in a 10 mM, pH 6.0 citric acid buffer and heated for 10 minutes to expose the antigen. . Then, the antigen-exposed fragment was washed with distilled water, added to 3% hydrogen peroxide, and reacted for 5 minutes. Thereafter, it was washed with Tris buffered saline containing 0.1% Tween-20 (TBS-T) and reacted with an antibody that specifically binds to GSTT1 protein (Santa Cruz, USA) overnight. After washing, the sections were incubated with biotin-conjugated IgG (1:500, Vector Laboratories, Burlingame, CA, USA), and then the reagent of Vecta-Elite streptavidin-peroxidase kit (Vector Laboratories) with benzidine substrate for color development Was processed. For control, the sections were stained with diluted hematoxylin, and observed with an optical microscope (Olympus BX41, Olympus Optical, Tokyo, Japan). In order to quantify the expression of the protein, a randomly selected field was examined for each sample at 100 magnification and a score of 0 to 3 was scored, and the results are shown in FIG. 1A.

For qRT-PCR, using a cDNA synthesis kit (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, USA), 1 ~ 4㎍ of total RNA extracted from each tissue through Ribospin ^TM (GeneAll, Korea) CDNA was synthesized according to the method provided. The prepared cDNA was mixed in triplicate with SYBR Green master mix (Applied Biosystems) and primer pairs (each primer 200 nmol, final concentration) in Table 2 below, and then in tepOne Plus real-time PCR system (Applied Biosystems). It was amplified for 45-60 cycles (95° C. 30 seconds, 56-61° C. 30 seconds, 72° C. 40 seconds). Then, the amplified value according to each primer was quantified as a relative expression level according to the expression level of GAPDH or β-actin, and is shown in B and C of FIG. 1.

Bell gene Sequence number division Sequence (5'-3') human GSTT1 5 F TCTTTTGCATAGAGACCATGACCAG 6 R CTCCCTACTCCAGTAACTCCCGACT β-actin 7 F CTCTTCCAGCCTTCCTTCCTG 8 R CAGCACTGTGTTGGCGTACAG mouse GSTT1 9 F GTAGGTTAACATCCAGTTCTGC 10 R GGCACATGGCAGCATACGG β-actin 11 F AGTGTGACGTTGACATCCGT 12 R TGCTAGGAGCCAGAGCAGTA

As shown in FIG. 1A, the expression of GSTT1 protein was significantly reduced in the intestinal tissues of inflammatory bowel disease patients corresponding to Crohn's disease and Behcet's disease compared to normal people (CTL).

As shown in B and C of FIG. 1, the expression of the GSTT1 gene in the intestinal tissues of inflammatory bowel disease patients was reduced by more than 2 times compared to the normal human (CTL), and also in the intestinal tissues of the animal model in which colitis was induced by DSS. The expression of the GSTT1 gene was decreased compared to normal.

Through the above results, it can be seen that the expression level of the GSTT1 protein or the gene encoding it is decreased in inflammatory bowel disease.

[ Example 2] Goblet cell (goblet cell) check

After intraperitoneal injection of GSTT1 protein-specific antibody (α-GSTT1) or IgG to the colitis animal model of Preparation Example 2 twice (day 7 or 11), the colitis using PAS (periodic acid-Schiff) After staining the goblet cells present in the intestinal gland (cypt) of the colon tissue isolated from the animal model, it was observed under a microscope and the results are shown in FIG. 2.

As shown in FIG. 2, it was confirmed that when α-GSTT1 was administered to a colitis animal model, goblet cells were reduced compared to the case where IgG was administered.

From the above results, it can be seen that when the level of the GSTT1 protein is decreased when colitis occurs, goblet cells are reduced.

[Example 3] GSTT1 Confirmation of colitis relief effect through gene transfer

In the mice of Preparation Example 3, weight change, disease activity index (DAI), intestinal length change, and goblet cells were checked, and FIGS. 3 ( GSTT1 gene injection into the rectum) and FIG. 4 ( GSTT1 gene injection into the abdominal cavity) ). Here, the body weight of the animal model was measured daily for 9 days to measure the body weight change, and the goblet cells were stained in the same manner as described in Example 2. In addition, the disease activity score was calculated through a process of scoring and summing three factors corresponding to weight loss, stool consistency, and bleeding from 0 to 4 points. In addition, the intestine of the animal model was excised and its length was measured.

As shown in Fig. 3A, the body weight of DSS(+)-rectal:GSTT1(-) mice (Fig. 2A, DSS+pCTL) was reduced by about 40%. However, in the case of DSS(+)-rectal:GSTT1(+) mice (Fig. 3A, DSS+GSTT1), the weight loss was recovered from the 8th or 7th day.

As shown in FIG. 3B, in the case of DSS(+)-rectal:GSTT1(-) mice (FIG. 3B, DSS+pCTL), the disease activity score continued to increase over time, day 8 It showed 12 points. On the other hand, in the case of DSS(+)-rectal:GSTT1(+) mice (B in FIG. 3, DSS+GSTT1), the disease activity score decreased to about 5 points or about 7 points.

As shown in C of Figure 3, the intestine length of DSS(+)-rectal:GSTT1(-) mice was reduced by 2 cm compared to DSS(-)-rectal:pGSTT1(-) mice, but the GSTT1 protein was transformed. In the DSS(+)-rectal:GSTT1(+) mice, the intestinal length reduction was recovered.

As shown in Fig. 3D, compared to DSS(-)-rectal:GSTT1(-) mice (D, water+pCTL in Fig. 3), DSS(-)-rectal:pGSTT1(+) mice (Fig. 3 In D, Water+pGstt1), the number of goblet cells was remarkably increased.

As shown in A, B and E of Figure 4, when the GSTT1 gene is injected through the abdominal cavity, the weight loss is recovered by the GSTT1 gene injection as in the case of the rectal injection (Fig. 4A), and disease activity The score was increased (FIG. 4B), recovery of intestinal length reduction (FIG. 4D) and the number of goblet cells increased (FIG. 4E). Furthermore, as shown in D of FIG. 4, in the case of DSS(+)-ip:GSTT1(-) mice (DSS+pCTL), the survival rate decreased to 20% from 7 days, but DSS(+)-ip:GSTT1 In the case of (+) mice (DSS+pGSTT1), the survival rate was similar to that of mice in which colitis was not induced by DSS.

Through the above results, since the intestinal epithelial barrier can be maintained by the mucus secreted from the goblet cells, the GSTT1 protein expressed in the intestinal gland of the large intestine significantly increases the proliferation of goblet cells, as a first defense mechanism against pathogens. It can be seen that it can improve the epithelial response.

[Example 4] Confirmation of relationship between GSTT1 protein and IL-22

Intestinal epithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) isolated from mice injected with the gene into the rectum of Preparation Example 3; Alternatively, the expression levels of the GSTT1 gene and the IL-22 gene were confirmed in the same manner as in Example 1 in the mRNA isolated from the tissue, and the results are shown in FIGS. 5A to 5C.

In addition, in the DSS(+)-rectal:GSTT1(+) mouse and DSS(+)-rectal:GSTT1(-) mouse of Preparation Example 3, an antibody specific for IL-22 protein (α -IL-22) was injected intraperitoneally, and weight change, disease activity score, intestinal length change, and goblet cells were checked in the same manner as in Example 3, and the results are shown in Fig. 5D to G. . Here, the expression levels of the IL-22 gene and the Muc2 gene were measured using the primer sequences shown in Table 3 below.

gene Sequence number division Sequence (5'-3') IL-22 13 F GGCCAGCCTTGCAGATAACA 14 R GCTGATGTGACAGGAGCTGA Muc2 15 F GGTCCAGGGTCTGGATCACA 16 R GCTCAGCTCACTGCCATCTG

5A and 5B, compared to DSS(-)-rectal:GSTT1(-) mouse (pCTL), intestinal epithelium isolated from DSS(-)-rectal:GSTT1(+) mouse (pGstt1) The expression levels of the GSTT1 gene and IL-22 gene were significantly increased in the lymphocytes and mucosal lamina propria lymphocytes (A) and mRNA (B) isolated from intestinal tissues.

In addition, as shown in C of Figure 5, compared to DSS(+)-rectal:GSTT1(-) mouse (pCTL), the Muc2 gene and IL in DSS(+)-rectal:GSTT1(+) mouse (pGstt1) The expression level of the -22 gene was significantly increased.

As shown in Fig. 5D to G, when α-IL-22 is administered to DSS(+)-rectal:GSTT1(+) mice, the pCMV-GSTT1-GFP vector is transformed, resulting in weight loss inhibition, disease The effect of decreasing the activity score and increasing the number of goblet cells was not exhibited.

From the above results, it can be seen that the inhibitory effect of inflammatory bowel disease by the GSTT1 protein is induced by cell signaling related to the IL-22 protein.

[Example 5] Confirmation of the relationship between GSTT1 protein and intestinal epithelial immune response

The HT-29 cell line of Preparation Example 1 or the culture medium of the transformed HT-29 cell line was 1 μg/mL of LPS (Lipopolysaccaride), 500 nM of H ₂ O ₂ , 100 g/mL of recombinant human IL-22, It was replaced with RPMI medium containing 40 ng/mL of TNF-α, flagellin (flagenlin; fla) or Salmonella typhimurium (Sal), and cultured for 4 or 24 hours. Here, the cell line was not treated with anything in the control (Vehicle). The cells cultured for 24 hours were fixed using a pH 7.4, 4% paraformaldehyde solution, and the fixed cells were washed with PBS. Then, the cells were blocked using 0.1% Triton X-100 containing 5% or 1% BSA, and sufficiently cultured after adding α-GSTT1. After that, the GSTT1 protein present in the cell was visualized through the process of further culturing with the secondary antibody bound with fluorescence, and observed through an optical microscope (Olympus BX41, Olympus Optical, Japan), and the results are shown in FIG. 6A. Done. At this time, the cell nucleus was stained using DAPI.

In addition, the expression levels of GSTT1 , CAMP , and MUC2 genes were checked in the cell lines cultured for 4 hours in the same manner as in Example 1, and the results are shown in B to H of FIG. 6. Expression levels of the human Muc2 gene, CAMP gene, and TNF- α gene were measured using the primer sequences shown in Table 4 below. Here, in the case of the TNF- α gene, it was measured using a commercially prepared and provided primer in Bioneer (Korea). At this time, Western blot analysis was performed by a conventional method to confirm the transformation of the transformed HT-29 cell line, and the results are shown in FIG. 6D.

gene Sequence number division Sequence (5'-3') Muc2 17 F AGGATGACACCATCTACCTCACC 18 R GGTGTAGGCATCGCTCTTCTC CAMP 19 F GCACGCTGACACCACTACC 20 R CGGGCTATTCCCTGTCCAC

As shown in FIG. 6A, the expression level of GSTT1 protein was significantly reduced in the HT-29 cell line treated with LPS compared to the case where LPS was not treated.

As shown in B and C of FIG. 6, compared to the control (Veh), when the HT-29 cell line was treated with LPS, flagellin (Fla) and Salmonella trimurium (Sal), the expression levels of GSTT1 and CAMP genes were On the other hand, when H ₂ O ₂ , IL-22 and TNF-α were treated, the expression levels of GSTT1 and CAMP genes were increased.

As shown in FIG. 6D, in the case of the transformed HT-29 cell line, the expression level of the GSTT1 protein was significantly reduced from 2 hours later.

As shown in E to H of Figure 6, compared to the case where the non-targeting control was transformed (SCR), when H ₂ O ₂ was treated in the HT-29 cell line transformed with siRNA specific to the GSTT1 gene , While the expression level of the TNF- α gene was increased, the GSTT1 gene-specific siRNA was transformed into H ₂ O ₂ in the HT-29 cell line. Or, when IL-22 was treated, the expression level of the MUC2 gene was rather reduced.

Through the above results, it can be seen that the GSTT1 protein and the gene encoding it are related to mucus or antibacterial peptides, and that pathogen infection and innate immune response may be impaired by the defect of the GSTT1 gene.

[Example 6] GSTT1 protein identification in patients with inflammatory bowel disease

The control group in Table 5 below; IBD patients; Int BD ^TT ; And Pierce RIPA buffer (Cat # 89900, Thermo Fisher Scientific, USA) containing proteins and phosphatase inhibitors from tissues of Int BD ^null patients to isolate proteins. Whole blood cells from the control group, IBD patients, Int BD ^TT patients, and Int BD ^null patients of Table 5 below were collected. The whole blood cells or the separated protein were heated at 58° C. for 10 minutes and then centrifuged, and 30 to 100 μg protein was added to an SDS-polyacrylamide gel, followed by electrophoresis. Then, the gel was transferred to a PVDF membrane, reacted with a primary antibody (antibodies specific to GSTT1 protein and β-actin protein), reacted with a secondary antibody, and then visualized and quantified, and the results are shown in Fig. 7A and It is shown in B. Here, β-mercaptoethanol (β-mercaptoethanol; Sigma-Aldrich, MO, USA) was excluded from all prepared solutions in order to maintain the non-reducing conditions of the protein.

division Inflammatory bowel disease Genetic variation Control none none IBD has exist none; And/or GSTT1 gene deletion mutation Int BD ^TT has exist none Int BD ^null has exist GSTT1 gene deletion mutation

As shown in A of FIG. 7, the dimer of GSTT1 protein was present in patients with inflammatory bowel disease (IBD) at a lower level than in the control group.

As is shown in A and B of 7, if in both the tissue and whole blood cells, a defect mutation in, GSTT1 gene compared to (BD ^TT) if there is no GSTT1 gene-deficient mutant in inflammatory bowel disease patients present (Int DB ^null ) at low levels of the dimer of the GSTT1 protein.

From the above results, it can be seen that the GSTT1 gene mutation, in particular the deletion mutation, prevents the formation of a dimer of the GSTT1 protein, and when the dimer of the GSTT1 protein is present at a lower level compared to the normal control, inflammatory bowel disease can be predicted. I can.

[Example 7] Confirmation of cell signaling related to GSTT1 protein

After treating the HT-29 cell line of Preparation Example 1 and the transformed HT-29 cell line with the IL-22 protein in the same manner as in Example 5, the expression level of the protein was confirmed in the same manner as in Example 6, , The results are shown in A to C of FIG. 8.

8A and 8B, when the expression of the GSTT1 protein is suppressed compared to the HT-29 cell line (CTL) (MT), the dimer of the GSTT1 protein is present at a low level compared to the expression level of the total GSTT1 protein. Also, the expression levels of p38/MAPK and phosphorylated JNK (phosphor JNK), ERK (phosphor ERK), and STAT (phosphor STAT3) proteins were also reduced.

From the above results, it can be seen that in the case of inflammatory bowel disease, the dimer of the GSTT1 protein is present at a low level, so that the phosphorylation of STAT3 and p38/JNK does not occur at a normal level, so that the immune response of the mucosal barrier is not normally induced. Therefore, as shown in FIG. 9, it can be seen that when any one or more of IL-22 protein and GSTT1 protein is treated, mucosal immune response is induced by p38 and STAT3 cell signaling.

As described above, a specific part of the present invention has been described in detail, and it is obvious that this specific technology is only a preferred embodiment for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A composition comprising GSTT1 protein or encoding gene thereof <130> PDPB192087 <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 240 <212> PRT <213> Homo sapiens <400> 1 Met Val Leu Glu Leu Tyr Leu Asp Leu Leu Ser Gln Pro Cys Arg Ala 1 5 10 15 Ile Tyr Ile Phe Ala Lys Lys Asn Asn Ile Pro Phe Gln Met His Thr 20 25 30 Val Glu Leu Arg Lys Gly Glu His Leu Ser Asp Ala Phe Ala Arg Val 35 40 45 Asn Pro Met Lys Arg Val Pro Ala Met Met Asp Gly Gly Phe Thr Leu 50 55 60 Cys Glu Ser Val Ala Ile Leu Leu Tyr Leu Ala His Lys Tyr Lys Val 65 70 75 80 Pro Asp His Trp Tyr Pro Gln Asp Leu Gln Ala Arg Ala Arg Val Asp 85 90 95 Glu Tyr Leu Ala Trp Gln His Thr Gly Leu Arg Arg Ser Cys Leu Arg 100 105 110 Ala Leu Trp His Lys Val Met Phe Pro Val Phe Leu Gly Glu Gln Ile 115 120 125 Pro Pro Glu Thr Leu Ala Ala Thr Leu Ala Glu Leu Asp Val Asn Leu 130 135 140 Gln Val Leu Glu Asp Lys Phe Leu Gln Asp Lys Asp Phe Leu Val Gly 145 150 155 160 Pro His Ile Ser Leu Ala Asp Leu Val Ala Ile Thr Glu Leu Met His 165 170 175 Pro Val Gly Gly Gly Cys Pro Val Phe Glu Gly His Pro Arg Leu Ala 180 185 190 Ala Trp Tyr Gln Arg Val Glu Ala Ala Val Gly Lys Asp Leu Phe Arg 195 200 205 Glu Ala His Glu Val Ile Leu Lys Val Lys Asp Cys Pro Pro Ala Asp 210 215 220 Leu Ile Ile Lys Gln Lys Leu Met Pro Arg Val Leu Ala Met Ile Gln 225 230 235 240 <210> 2 <211> 720 <212> DNA <213> Homo sapiens <400> 2 atggttctgg agctgtacct ggatctgctg tcgcagccct gtcgcgccat ttatatcttc 60 gccaagaaga acaatatccc gttccagatg cacacggtgg agctgcgcaa gggtgagcac 120 ctcagcgatg cgtttgcccg ggtgaacccc atgaagaggg taccagccat gatggatggt 180 ggcttcaccc tgtgtgagag tgtggctatc ttgctctacc tggcacacaa gtataaggtt 240 cctgaccact ggtaccccca agacctgcag gctcgtgctc gtgtagacga gtacctggca 300 tggcagcata cgggccttcg gagaagctgc ctcagggccc tgtggcataa ggtgatgttc 360 cctgttttcc ttggtgagca aatacctcct gaaacactgg cagccacgtt ggcagaactg 420 gatgttaacc tacaggtgct tgaagacaag ttcctccagg acaaagactt ccttgttggg 480 ccccacatct ccctggccga cttggtggcc atcacagagc tgatgcatcc tgtaggtggt 540 ggctgcccag tctttgaagg gcatcccagg ctggctgcat ggtaccagcg agtggaggca 600 gctgtgggga aggacctctt ccgggaagcc catgaagtca tcctgaaggt gaaggactgt 660 ccccctgctg acctcatcat aaagcagaag ctgatgccca gagtgctggc aatgatccag 720 720 <210> 3 <211> 179 <212> PRT <213> Homo sapiens <400> 3 Met Ala Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr Leu 1 5 10 15 Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Gly Gly Ala 20 25 30 Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn Phe Gln 35 40 45 Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser 50 55 60 Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe 65 70 75 80 His Gly Val Ser Met Ser Glu Arg Cys Tyr Leu Met Lys Gln Val Leu 85 90 95 Asn Phe Thr Leu Glu Glu Val Leu Phe Pro Gln Ser Asp Arg Phe Gln 100 105 110 Pro Tyr Met Gln Glu Val Val Pro Phe Leu Ala Arg Leu Ser Asn Arg 115 120 125 Leu Ser Thr Cys His Ile Glu Gly Asp Asp Leu His Ile Gln Arg Asn 130 135 140 Val Gln Lys Leu Lys Asp Thr Val Lys Lys Leu Gly Glu Ser Gly Glu 145 150 155 160 Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn 165 170 175 Ala Cys Ile <210> 4 <211> 1165 <212> DNA <213> Homo sapiens <400> 4 acaagcagaa tcttcagaac aggttctcct tccccagtca ccagttgctc gagttagaat 60 tgtctgcaat ggccgccctg cagaaatctg tgagctcttt ccttatgggg accctggcca 120 ccagctgcct ccttctcttg gccctcttgg tacagggagg agcagctgcg cccatcagct 180 cccactgcag gcttgacaag tccaacttcc agcagcccta tatcaccaac cgcaccttca 240 tgctggctaa ggaggctagc ttggctgata acaacacaga cgttcgtctc attggggaga 300 aactgttcca cggagtcagt atgagtgagc gctgctatct gatgaagcag gtgctgaact 360 tcacccttga agaagtgctg ttccctcaat ctgataggtt ccagccttat atgcaggagg 420 tggtgccctt cctggccagg ctcagcaaca ggctaagcac atgtcatatt gaaggtgatg 480 acctgcatat ccagaggaat gtgcaaaagc tgaaggacac agtgaaaaag cttggagaga 540 gtggagagat caaagcaatt ggagaactgg atttgctgtt tatgtctctg agaaatgcct 600 gcatttgacc agagcaaagc tgaaaaatga ataactaacc ccctttccct gctagaaata 660 acaattagat gccccaaagc gatttttttt aaccaaaagg aagatgggaa gccaaactcc 720 atcatgatgg gtggattcca aatgaacccc tgcgttagtt acaaaggaaa ccaatgccac 780 ttttgtttat aagaccagaa ggtagacttt ctaagcatag atatttattg ataacatttc 840 attgtaactg gtgttctata cacagaaaac aatttatttt ttaaataatt gtctttttcc 900 ataaaaaaga ttactttcca ttcctttagg ggaaaaaacc cctaaatagc ttcatgtttc 960 cataatcagt actttatatt tataaatgta tttattatta ttataagact gcattttatt 1020 tatatcattt tattaatatg gatttattta tagaaacatc attcgatatt gctacttgag 1080 tgtaaggcta atattgatat ttatgacaat aattatagag ctataacatg tttatttgac 1140 ctcaataaac acttggatat cctaa 1165 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GSTT1 forward primer (human) <400> 5 tcttttgcat agagaccatg accag 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GSTT1 reverse primer (human) <400> 6 ctccctactc cagtaactcc cgact 25 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> beta actin forward primer (human) <400> 7 ctcttccagc cttccttcct g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> beta actin reverse primer (human) <400> 8 cagcactgtg ttggcgtaca g 21 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GSTT1 forward primer (mouse) <400> 9 gtaggttaac atccagttct gc 22 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GSTT1 reverse primer (mouse) <400> 10 ggcacatggc agcatacgg 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta actin forward primer (mouse) <400> 11 agtgtgacgt tgacatccgt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta actin reverse primer (mouse) <400> 12 tgctaggagc cagagcagta 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-22 forward primer (mouse) <400> 13 ggccagcctt gcagataaca 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-22 reverse primer (mouse) <400> 14 gctgatgtga caggagctga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Muc2 forward primer (mouse) <400> 15 ggtccagggt ctggatcaca 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Muc2 reverse primer (mouse) <400> 16 gctcagctca ctgccatctg 20 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Muc2 forward primer (human) <400> 17 aggatgacac catctacctc acc 23 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Muc2 reverse primer (human) <400> 18 ggtgtaggca tcgctcttct c 21 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CAMP forward primer (human) <400> 19 gcacgctgac accactacc 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CAMP reverse primer (human) <400> 20 cgggctattc cctgtccac 19

Claims (16)

GSTT1 (glutathione S-transferases theta 1) protein or a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease comprising as an active ingredient a gene encoding it. The method of claim 1,
The pharmaceutical composition further comprises an IL-22 protein or a gene encoding the same. The method of claim 1,
The inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, Crohn's disease, enteric Behcet's disease, hemorrhagic rectal ulcer and ileal cystitis (pouchitis). The method of claim 1,
The GSTT1 protein is composed of the amino acid sequence represented by SEQ ID NO: 1, pharmaceutical composition. A food composition for preventing or improving inflammatory bowel disease, including GSTT1 protein or a gene encoding it as an active ingredient. The method of claim 5,
The food composition further comprises an IL-22 protein or a gene encoding the same. Treating a desired candidate substance in a biological sample isolated from a patient with inflammatory bowel disease; And
A method for screening a therapeutic agent for inflammatory bowel disease, comprising measuring the expression level of the GSTT1 protein or a gene encoding it in the biological sample. The method of claim 7,
After treatment of the candidate substance, the expression level of the GSTT1 protein or the gene encoding it measured in the biological sample is increased compared to the expression level of the GSTT1 protein or the gene encoding it measured in the biological sample isolated from the patient with inflammatory bowel disease. If so, a method for screening a therapeutic agent for inflammatory bowel disease, further comprising the step of selecting the candidate substance as a therapeutic agent for inflammatory bowel disease. GSTT1 (glutathione S-transferases theta 1) protein or a composition for promoting proliferation or differentiation of goblet cells comprising a gene encoding the same as an active ingredient. A composition for diagnosing inflammatory bowel disease, comprising an agent measuring the level of the presence of a dimer of GSTT1 protein. The method of claim 10,
The agent for measuring the level at which the dimer of the protein is present is an antibody or aptamer that specifically binds to the protein, a composition for diagnosis of inflammatory bowel disease. The method of claim 10,
The inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, Crohn's disease, enteric Behcet's disease, hemorrhagic rectal ulcer and ileal cystitis, a composition for diagnosis of inflammatory bowel disease. A kit for predicting prognosis of inflammatory bowel disease, comprising the composition of any one of claims 10 to 12. In a biological sample isolated from the subject of interest,
A method of providing information for diagnosing inflammatory bowel disease, comprising measuring the level at which a dimer of GSTT1 protein is present. The method of claim 14,
When the level of the presence of the dimer of the GSTT1 protein measured in the biological sample isolated from the object of interest is lower than that of the normal control, the object of interest is predicted to be inflammatory bowel disease, for diagnosing inflammatory bowel disease How to provide information. The method of claim 14,
The step of measuring the level of the protein present is performed under non-reducing conditions of the protein, a method of providing information for diagnosing inflammatory bowel disease.

Images