WO2020213913A1 - Gstt1 단백질 또는 이를 암호화하는 유전자를 유효성분으로 포함하는 조성물 - Google Patents

Gstt1 단백질 또는 이를 암호화하는 유전자를 유효성분으로 포함하는 조성물 Download PDF

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WO2020213913A1
WO2020213913A1 PCT/KR2020/005011 KR2020005011W WO2020213913A1 WO 2020213913 A1 WO2020213913 A1 WO 2020213913A1 KR 2020005011 W KR2020005011 W KR 2020005011W WO 2020213913 A1 WO2020213913 A1 WO 2020213913A1
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Prior art keywords
protein
disease
present
gstt1
composition
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English (en)
French (fr)
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천재희
김승원
김재현
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Industry Academic Cooperation Foundation of Yonsei University
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Industry Academic Cooperation Foundation of Yonsei University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01018—Glutathione transferase (2.5.1.18)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/9116—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • G01N2333/91165—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1)
    • G01N2333/91171—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5) general (2.5.1) with definite EC number (2.5.1.-)
    • G01N2333/91177—Glutathione transferases (2.5.1.18)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention uses GSTT 1 (glutathione S-transferases theta 1) protein or
  • compositions containing the encoding gene as an active ingredient It relates to compositions containing the encoding gene as an active ingredient.
  • IBD Inflammatory bowel disease
  • ulcerative colitis As a disease that induces inflammation, its symptoms include abdominal pain, fever, diarrhea, and bleeding.
  • UC ulcerative colitis
  • CD Crohn's disease
  • ulcerative colitis is a diffuse non-specificity of unknown cause of the large intestine that forms patinas or ulcers.
  • IEC intestinal epithelial cells
  • Infliximab was used to treat patients with Hookron's disease, which was approved by the US FDA as a treatment for Crohn's disease in 1998, but it has reduced pan blood cell count, drug-induced lupus,
  • An object of the present invention is to provide a composition comprising a GSTT 1 (glutathione S-transferases theta 1) protein or a gene encoding it as an active ingredient.
  • a GSTT 1 glutthione S-transferases theta 1
  • Another object of the present invention is to provide a method for screening a therapeutic agent for inflammatory growth disease, comprising measuring the expression level of the GSTT1 protein or a gene encoding it.
  • Another object of the present invention is to identify the GSTT1 protein or the gene encoding it.
  • Another object of the present invention is to determine the level at which the dimer of GSTT1 protein exists.
  • Another object of the present invention is to identify the GSTT1 protein or the gene that encodes it.
  • composition for preventing, improving or treating inflammatory growth disease is provided.
  • composition of the present invention can be used as a pharmaceutical composition or a food composition.
  • composition of the present invention contains as an active ingredient a GSTT 1 (glutathione S-transferases theta 1) protein or a gene encoding it.
  • GSTT 1 glutthione S-transferases theta 1
  • GSTT1 protein of the present invention is among glutathione S-transferase (GST)
  • the level at which the GSTT1 protein exists preferably the level at which the GSTT1 protein exists as a dimer
  • the level at which the GSTT1 protein exists is significantly increased, thereby causing phosphorylation of STAT3 and P38/JNK.
  • composition of the present invention further contains the 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.
  • the IL-22 protein of the present invention increases the expression of the GSTT1 protein, thereby increasing the expression of the GSTT1 protein. Since it induces an immune response, for the purposes of the present invention, when the IL-22 protein or a gene encoding it is further included, administration of the composition can induce a synergistic effect on the expected immune response of the mucosal barrier.
  • the GSTT1 protein of the present invention is an amino acid sequence represented by SEQ ID NO: 1.
  • the gene encoding the GSTT1 protein may be composed of, but is not limited to, the base sequence represented by SEQ ID NO: 2.
  • the IL-22 protein of the present invention may consist of an amino acid sequence represented by SEQ ID NO: 3, and the gene encoding IL-22 protein may consist of a base sequence represented by SEQ ID NO: 4, but It is not limited.
  • the GSTT1 protein or IL-22 protein of the present invention can be prepared by a known protein synthesis method or by culturing transformed host cells.
  • the protein of the present invention is encoded.
  • the transformant After preparing a transformant by introducing a recombination expression vector containing a base sequence to a host cell, the transformant can be cultured by appropriately using any method known in the art. Proteins from transformants can be obtained by separation and purification through methods known in the art such as centrifugation, chromatography, and electrophoresis.
  • the amino acid sequence of the present invention can be easily modified by one or more amino acid substitutions, deletions, insertions, or combinations thereof. Therefore, a protein or polypeptide having high homology with the amino acid sequence represented by SEQ ID NO: 1, for example, even if the homology is 80% or more, 90% or more, or 95% or more, all of the above proteins of the present invention are included as long as they maintain the function of overcoming antibacterial resistance.
  • the above-described 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 above the above-described sequence homology with the amino acid sequence of the present invention.
  • This homology can also be determined by visually comparing two sequences, but it can be determined using a bioinformatic algorithm that analyzes the degree of homology by arranging the sequences to be compared side by side.
  • a useful automated algorithm is 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 include Needleman & Wunsch, Pearson & Lipman, Smith & Waterman sequence alignment algorithms. Other useful alignment algorithms and homology determinations are FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL. It can be automated in software containing W.
  • the recombinant expression vector of the present invention is a recombinant expression vector capable of expressing a target protein or peptide in a target host cell, and refers to a gene construct comprising essential regulatory elements that are operatively linked to express a gene insert.
  • the expression vector includes expression control elements such as start codon, stop codon, promoter, operator, etc., and the start codon and stop codon are generally considered to be part of the nucleotide sequence encoding the polypeptide, and when the gene construct is administered, the individual must It should show an action and should be in frame (/n/rame) with the coding sequence.
  • the promoter of the vector may be a constitutive or inducible promoter.
  • the above recombination expression vector of the present invention is the promoter and the above GSTT1
  • the base sequence of the gene i.e., the polynucleotide may be operably linked.
  • the ⁇ operably linked'' of the present invention refers to a protein or RNA that is aimed at a nucleic acid expression regulatory sequence to perform a general function. It refers to the state in which the nucleic acid sequence encoding is functionally linked.
  • Polynucleotides are operably linked and may affect the expression of the coding sequence.
  • Operationally linked with a recombinant expression vector can be produced 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.), derived from E. coli
  • Plasmid (pYG601BR322, pGEX-4T-l, pET, pBR325, pUC118, pUC119, etc.), Bacillus subtilis derived plasmid (pUB110, pTP5, etc.), yeast-derived
  • Plasmid (YEpl3, YEp24, YCp50, etc.), Phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, XgtlO, AAAAAXgtl l, IZAP, etc.), Animal Virus
  • It may be a vector (retrovirus, adenovirus, vaccinia virus, etc.), a heavy virus vector (baculovirus, etc.), but is not limited thereto.
  • the inflammatory growth 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 pouchitis, but is limited thereto. Becomes 2020/213913 1»(:1 ⁇ 1 ⁇ 2020/005011 No.
  • 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 by targeting humans.
  • the pharmaceutical composition of the present invention is not limited to these, but is formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injectable solutions according to the usual methods.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is a binding agent, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, and a stabilizer for oral administration. Agents, suspending agents, pigments, flavors, etc. can be used. In the case of injections, buffers, preservatives, painless agents, solubilizing agents, stabilizing agents, etc.
  • the formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier for example, for oral administration, tablets, troches, and preservatives can be used. It can be manufactured in the form of capsules, elixir (manureuria), suspension, syrup, wafers, etc. In the case of injections, it can be manufactured in the form of unit dose ampoules or multiple doses. Others, solutions, suspensions, tablets, capsules, sustained release type It can be formulated as a formulation.
  • suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil can be used.
  • Anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may additionally be included.
  • the route of administration of the pharmaceutical composition of the present invention is not limited to these, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal tract , Topical, sublingual or rectal. Oral or parenteral delivery is preferred.
  • parenteral of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or injection techniques.
  • 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.
  • composition of the present invention is the activity of the specific compound used, age, weight,
  • the pharmaceutical composition can be formulated as a pill, sugar tablet, capsule, liquid, gel, syrup, slurry, and suspension.
  • the food composition of the present invention is a variety of foods, for example, beverages, gum, tea, vitamins
  • It can be manufactured in the form of complex drugs, powders, granules, tablets, capsules, sweets, rice cakes, and bread.
  • the amount can be added in a proportion of 0.1 to 50% of the total weight, but is not limited thereto.
  • natural carbohydrates monosaccharides such as glucose, disaccharides such as fructose, and conventional sugars such as sucrose and polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are included.
  • natural flavoring agents Trigger, Stevia
  • It may be an extract (for example, rebaudioside, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.)
  • the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavors, coloring agents, fectic acids and salts thereof, alginic acid and salts thereof, organic acids, ,Stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. may be further included.
  • the ingredients contained in the food composition of the present invention can be used independently or in combination.
  • the ratio of the additives does not correspond to the core element of the present invention, but 0.1 to 0.1 per 0 parts by weight of the food composition of the present invention It can be selected from a range of about 50 parts by weight, but is not limited thereto.
  • Another embodiment of the present invention provides a method for screening a therapeutic agent for inflammatory growth disease.
  • the screening method of the present invention is separated from patients with inflammatory growth disease.
  • the screening method of the present invention is performed after the treatment of the candidate material.
  • the candidate substance is selected. It may further include the step of selecting a therapeutic agent for inflammatory growth disease.
  • the biological sample of the present invention refers to any substance, biological fluid, tissue, or cell obtained from or derived from an inflammatory growth disease patient or an individual whose disease is suspected, for example, whole blood (whole blood).
  • blood whole blood
  • leukocytes peripheral blood mononuclear cells
  • leukocytosis (buffy coat)
  • blood including plasma and serum, sputum, tears, Mucus, nasal washes, nasal aspirate, bmati, 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, Nipple aspirate, bronchial aspirate, synovM fluid, joint aspirate, organ secretions, cells, cell extract or cerebrospinal fluid (cerebrospinal fluid) may contain, but is not limited to.
  • the above candidate material of the present invention has the activity to overcome inflammatory growth disease, that is, the level of expression of the GSTT1 protein or the gene encoding it, equal to or similar to the level of expression in normal persons without inflammatory growth disease. It refers to a drug for testing whether or not there is an activity that can be increased by, and includes arbitrary molecules such as proteins, oligopeptides, organic molecules, polysaccharides, polynucleotides, and a wide range of compounds. These candidates are not only natural substances but also No, it may contain all synthetic materials.
  • a preparation for confirming the expression level of a protein to be measured contained in a biological sample refers to a preparation for confirming the expression level of a protein to be measured contained in a biological sample.
  • a preparation for measuring the expression level of the protein may be an antibody or aptamer capable of specifically binding to the protein. .
  • the formulation is Western blot assay, ELISA (Enzyme linked immunosorbent assay), Bang! ⁇ Station ⁇ :Suk (RIA: Radioimmunoassay), room! Radioimmimodiffusion, Ouchterlony Immune Diffusion Method, Rocket Immunoelectrophoresis,
  • Antibodies or aptamers used in methods such as immunochromatography, fluorescenceactivated cell sorter analysis (FACS), and protein chip technology assays may include, but are not limited to.
  • the form of the antibody is not particularly limited, and is a polyclonal antibody, a monoclonal antibody, or an antigen-binding protein. If it is, 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 may contain two full-length light chains and two full-length antibodies. In addition to the complete form having a heavy chain, it includes functional fragments of antibody molecules. Functional fragments of antibody molecules refer to fragments that have at least an antigen-binding function, and Fab, F(ab'), F(ab') 2 , Fv, etc., but are not limited thereto.
  • the above "aptamer” of the present invention means a single stranded oligonucleotide
  • the aptamer can have various three-dimensional structures according to its base sequence, and can have high affinity for specific substances such as antigen-antibody reactions.
  • the aptamer can inhibit the activity of a given target molecule by binding to a given target molecule.
  • the aptamer may be RNA, DNA, modified nucleic acid or a mixture thereof, and the form may be straight or cyclic.
  • the antibody of the present invention is a protein (antigen) encoded by the gene by cloning the gene consisting of the base sequence of the GSTT1 gene, preferably the base sequence represented by SEQ ID NO: 2, into an expression vector according to a conventional method.
  • the aptamer can be manufactured by a conventional method, and the aptamer can be easily manufactured according to the method of public knowledge by a person with common knowledge in the relevant technical field by referring to each base sequence.
  • the formulation capable of measuring the expression level of the gene encoding the GSTT1 protein of the present invention is transcribed from the gene to be measured to confirm the expression level of the gene to be measured included in the biological sample. It refers to a formulation that can be used in a method of measuring the level of mRNA. Specifically, the formulation is RT-PCR, quantitative real time PCR, and competitive
  • RT-PCR Competitive RT-PCR
  • RT-PCR real time quantitative RT-PCR
  • RPA RNase protection assay
  • Northern blot assay DNA chip assay, etc. It may contain, but is not limited to, primer pairs or probes capable of specifically binding to the mRNA to be measured used.
  • the "primer" of the present invention is a short free 3-terminal hydroxyl group (free 3'hydroxyl).
  • the primer is an appropriate buffer solution and a base pair.
  • Reagents for polymerization reactions at temperature i.e. DNA polymerase or reverse transcriptase
  • 4 different temperatures i.e. DNA polymerase or reverse transcriptase
  • DNA synthesis can be initiated in the presence of nucleoside triphosphate.
  • 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 has the presence or absence of a specific mRNA, the level of expression ( Expression level)
  • the probe may be labeled as an oligonucleotide probe, a single strand DNA probe, or a double strand DNA probe.
  • RNA probe can be produced in the form of a DNA) probe or an RNA probe, but is not limited thereto.
  • the primer or probe of the present invention is the base sequence of the GSTT1 gene
  • composition is provided.
  • composition for promoting proliferation of the present invention may include a GSTT1 protein or a gene encoding it.
  • composition for promoting proliferation of the present invention is the IL-22 protein or
  • composition of the present invention protects the intestinal mucosa through phosphorylation of STAT3 and p38/JNK.
  • It can induce the immune response of the mucous membrane barrier by promoting the proliferation of the goblet cells with functions, and through this, it can be applied to the prevention or treatment of diseases that may be caused by the decrease of the goblet cells, for example, inflammatory growth disease. .
  • the term "proliferation" of the present invention means that the number of cells is increased by dividing while maintaining the same properties.
  • the purpose of the present invention is to increase the number of goblet cells present in the intestinal mucosa. have.
  • Another embodiment of the present invention provides a composition for diagnosis of inflammatory growth disease.
  • the above diagnostic composition of the present invention includes a formulation for measuring the level of the presence of a dimer of GSTT1 protein.
  • the agent for measuring the level of the presence of the dimer of the GSTT1 protein of the present invention may be an antibody or an aptamer that specifically binds to the GSTT1 protein present as a dimer.
  • the above diagnostic composition of the present invention is suitable for normal control in patients with inflammatory growth disease.
  • the dimer of the GSTT1 protein of the present invention is a form in which residues of amino acids present in two GSTT1 protein monomers are bonded, and in the presence of intracellular oxidation stimulation, the GSTT1 protein performs p38/MAPK signaling. Promoted 2020/213913 1»(:1/10 ⁇ 020/005011 It can relieve oxidative stress.
  • the GSTT1 protein dimer of the present invention is present at a high level in normal people who do not develop inflammatory growth disease. On the other hand, in patients with inflammatory growth disease, Gsrr/gene mutations, for example, deletion mutations, occur, and the GSTT1 protein does not form a dimer, which may induce or aggravate symptoms by not sufficiently alleviating the stimulation to oxidative stress. .
  • the description of the GSTT1 protein or the gene encoding it and the inflammatory growth disease is the same as described in the above pharmacy; and food composition, and is omitted in order to avoid excessive complexity in this specification.
  • the "antibody" of the present invention is specific to the GSTT1 protein present as a dimer.
  • a functional fragment of a molecule means a fragment that has 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 that has binding activity to the GSTT1 protein present as a dimer.
  • the aptamer can have various three-dimensional structures according to its base sequence. It can have high affinity for certain substances, such as antigen-antibody reactions. Aptamer can inhibit the activity of a given target molecule by binding to a given target molecule.
  • the aptamer is RNA, DNA, modified nucleic acid, or a mixture thereof.
  • kits for diagnosing inflammatory bowel disease comprising the above composition of the present invention is provided.
  • the kit of the present invention can be predicted as an inflammatory growth disease when the dimer of the GSTT1 protein is present at a lower level in the target object compared to the normal control without the inflammatory growth disease using the above composition of the present invention.
  • 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.
  • MRM multiple reaction monitoring
  • the substrate of the present invention may be a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin or polystyrene resin, and a slide glass made of glass, and the coloring enzyme is peroxidase, alkaline
  • Phosphatase Alkaline Phosphatase
  • FITC FITC
  • RITC RITC
  • the color developing substrate liquid is
  • 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (2,2'-azino-bis(3-ethylbenzothiazoli ne-6-sulphonic acid); ATBS), o-phenylenedimine ( o-Phenylenediamine (OPD), tetramethyl benzidine (TMB) may be used, but is not limited thereto.
  • the above method of the present invention is in a biological sample isolated from an object of interest
  • the target individual is an inflammatory growth disease.
  • the biological sample isolated from the object of the present invention is normal or
  • any substance, biological body fluid, tissue or cell obtained from or derived from an individual whose disease is suspected for example, whole blood, leukocytes, peripheral blood mononuclear cells. cells), leukocytosis (buffy coat), blood including plasma and serum, sputum, tears, mucus, nasal washes, nasal saws ( nasal aspirate), hosaw (breabi), urine (urine), semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meninges
  • the description of the GSTT1 protein or the gene encoding it, the inflammatory growth disease, the dimer of the GSTT1 protein, and the preparation for measuring the level of the present invention is the above pharmacy; and food It is the same as described in the composition and diagnostic composition, and is omitted to avoid excessive complexity in this specification.
  • the step of measuring the level of the protein of the present invention is to use the composition of the present invention to perform Western blot analysis, ELISA, radioimmunoassay, radioimmuno diffusion method, octeroni immunodiffusion method, rocket immunoelectrophoresis.
  • Immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, FACS, and protein chip assay can be performed through at least one selected from the group consisting of, preferably the above step is non-reducing It may be performed under conditions, but is not limited thereto.
  • the non-reduction condition of the present invention is an agent that breaks the disulfide bonds of the protein and facilitates analysis such as electrophoresis, for example (3-mercaptoethanol (p
  • -mercaptoethanol or DTT (dithiothreitol) is not included, and for the purposes of the present invention, it may be a suitable condition for detecting the oxidized AMPK protein.
  • the present invention is applied to a subject in need of administration.
  • GSTT1 glutthione S-transferases theta 1
  • the "administration" refers to a predefined copy to the subject in any appropriate way.
  • the "subject" requiring the administration may include both mammals and non-mammals.
  • the mammals include humans, non-human primates, such as chimpanzees, other apes or monkey species. ;Livestock animals, such as cattle, horses, sheep, goats, pigs; breeding animals, such as rabbits, dogs, or cats; laboratory animals, such as rodents, such as rats, mice, or guinea pigs, but are limited thereto. No.
  • examples of the non-mammals in the present invention may include, but are not limited to, birds or fish.
  • it may be administered as a solid formulation, a liquid formulation, or an aerosol formulation for inhalation, and may be administered as a solid formulation intended to be converted into a liquid formulation for oral or parenteral administration immediately before use,
  • it may be formulated and administered in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, but is not limited thereto.
  • a pharmaceutically acceptable carrier may be additionally administered together with the protein or gene formulation of the present invention upon the above administration.
  • Pharmaceutically acceptable carriers can use binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, and flavoring agents for oral administration.
  • buffers, preservatives, painless It can be used by mixing an agent, a solubilizing agent, a stabilizing agent, etc.
  • a base, an excipient, a lubricant, 2020/213913 1»(:1 ⁇ 1 ⁇ 2020/005011 Preservatives, etc. can be used.
  • the formulation of the protein or gene preparation of the present invention can be manufactured in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • oral administration it can be prepared in the form of tablets, troches, capsules, elixirs (611X11), suspension, syrup, wafers, etc.
  • elixirs (611X11)
  • suspension syrup
  • wafers etc.
  • Others can be formulated as solutions, suspensions, tablets, capsules, sustained-release preparations, etc.
  • suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil can be used. It can contain anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives at stock prices.
  • the route of administration of the protein or gene product according to the present invention is not limited to these, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, and intranasal This includes the minister, local, sublingual or workplace. Oral or parenteral delivery is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or injection techniques.
  • the pharmaceutical composition may also be administered in the form of suppositories for rectal administration.
  • a "effective amount” for therapeutic use is the amount of a protein or gene disclosed in the present invention that is desired to provide a clinically significant reduction in the disease.
  • the appropriate “effective” amount can be determined by one of skill in the art using routine experimentation.
  • the expression “effective amount” generally refers to the amount by which the active substance has a therapeutic effect.
  • the active substance is protein 08111 Or it is a gene that encodes it and is a prophylactic or therapeutic agent for inflammatory growth disease.
  • the protein or gene preparation of the present invention is the activity of the specific protein or gene used, age, weight, general health, sex, diet, administration time, route of administration, excretion rate, drug formulation and the severity of the specific disease to be prevented or treated. It may vary according to a number of factors, including, and the dosage of the protein or gene product depends on the patient's condition, weight, severity of disease, drug type, route and duration of administration, but can be appropriately selected by the person skilled in the art. From 0.0001 per day Or 0.001 to
  • Protein or genetic preparations according to the present invention Silver pill, party uijeong, It can be formulated as a capsule, liquid, gel, syrup, slurry, or suspension.
  • composition according to the present invention can be used to treat goblet cells through phosphorylation of STAT3 and p38/JNK.
  • Figure 1a is a result of performing immunohistochemical staining according to an embodiment of the present invention
  • Figure 1b and c is a quantitative real-time reverse transcriptase chain of the expression level of the GSTT1 gene according to an embodiment of the present invention It shows the results confirmed through the reaction (qRT-PCR).
  • Figure 2 shows the results of staining goblet cells in a colitis animal model injected with an antibody or IgG specific to GSTT1 protein according to an embodiment of the present invention.
  • FIG. 3A to 3D show changes in weight (a) of the GSTT1 gene according to an embodiment of the present invention through rectal injection, and a disease activity index (Disease activity index); DAI) (Fig. 3b), intestinal length change (Fig. 3c), and goblet cells (Fig. 3d) are shown.
  • DAI Disease activity index
  • Fig. 3b disease activity index
  • Fig. 3c intestinal length change
  • Fig. 3d goblet cells
  • Figures 4a to 4e show the weight change (Figure 4a), disease activity score (Figure 4b), survival rate in the mouse transformed by the GSTT1 gene according to an embodiment of the present invention through intraperitoneal injection. (Fig. 4c), intestinal length change (Fig. 4d) and goblet cells (Fig. 4e) are shown.
  • Figures 5a to 5c show the results of confirming the expression levels of the GSTT1 gene and the IL-22 gene according to an embodiment of the present invention, weight change (d of FIG. 5), disease activity score (d of FIG. 5) e) The result of confirming the change in intestinal length (fig. 5f) and goblet cells (fig. 5g) is shown.
  • 6A shows the expression level of the GSTT1 protein according to an embodiment of the present invention.
  • Fig. 6 shows the results of confirming the expression level of the GSTT1 gene through quantitative real-time reverse transcriptase chain reaction
  • Fig. 6 d is (Sr77 gene).
  • the result of confirming the expression level of GSTT1 protein through Western blot analysis, and e to h in FIG. 6 are in cells in which the expression of GSTT1 protein is inhibited. It shows the result of checking the expression level of the genes.
  • 7A and 7B show the expression level of the monomer and the dimer of the GSTT1 protein through Western blot analysis, and the expression level of the dimer GSTT1 protein according to the total GSTT1 as a ratio.
  • Figure 8a and b show the expression level of the GSTT1 protein monomer and the dimer through Western blot analysis, and show the expression level of the dimer GSTT1 protein according to the total GSTT1 as a ratio, and Figure 8c is This is the result of confirming the expression level of the cell signaling protein related to the GSTT1 protein through Western blot analysis.
  • FIG. 9 is a schematic diagram showing the mechanism of action of the GSTT1 protein according to an embodiment of the present invention.
  • An embodiment of the present invention relates to a composition for preventing, improving or treating inflammatory growth disease.
  • Another embodiment of the present invention relates to a method for screening a therapeutic agent for inflammatory growth disease.
  • composition for diagnosis of inflammatory growth disease [111] in another embodiment of the present invention, a composition for diagnosis of inflammatory growth disease
  • Another embodiment of the present invention relates to a kit for diagnosis of inflammatory bowel disease comprising the composition of the present invention.
  • the present invention is applied to a subject in need of administration.
  • the present invention relates to a method for preventing or treating inflammatory growth disease, comprising administering a pharmaceutically effective amount of GSTT1 (glutathione S-transferases theta 1) protein or a gene encoding it.
  • GSTT1 glutthione S-transferases theta 1
  • the HT-29 cell line (Korea Cell Line Bank, Korea) was cultured in a medium under conditions of 5% C0 2 and 37°C. Using Lipofectamine 2000 (Life Technologies, USA), the GSTT1 gene (SEQ ID NO: SiRNA or non-targeting control (non-targeting control; AccuTarget, Bioneer, Korea) that binds complementarily to number 2) was transformed.
  • SEQ ID NO: SiRNA or non-targeting control non-targeting control (non-targeting control; AccuTarget, Bioneer, Korea) that binds complementarily to number 2
  • Wild type C57BL/6 mice (OrientBio, Korea) were reared in a 12:12 dark cycle environment without pathogens. 2.5% (W ⁇ 0 DSS (Dextran Sulfate Sodium Salt, MW. Drinking water containing 36,00050,000; MP Biomedicals, USA) was supplied to 8-week-old mice for 7 days, followed by supplying only sterilized drinking water for 2 days to induce colitis in the mice, thereby constructing a colitis animal model. .
  • W ⁇ 0 DSS Extran Sulfate Sodium Salt, MW. Drinking water containing 36,00050,000; MP Biomedicals, USA
  • the vector described in Table 1 below was used twice (1 day and 5 days) in the rectum (100 Utg in PBS) or intraperitoneal (100/retention in 1 ml PBS) of the colitis animal model or wild-type mouse of the phase reference ratio 2.
  • the vector and the transformant PEI Polyetherimide
  • the vector and the transformant PEI were mixed in a ratio of l:l ((w: V, DNA: 1 PEI) to increase the efficiency of gene transfer.
  • Isofurane Ifrane liquid, MNS Korea
  • the mixed solution was used for the colitis animal model and wild-type mouse.
  • the colitis animal model and mouse were hung upside down for 20 seconds in order to be evenly distributed throughout the body of the body. Through this process, a mouse corresponding to a total of 8 cases was produced as shown in Table 1 below. .
  • Immunohistochemistry using quantitative real-time reverse transcriptase chain reaction (qRT-PCR), inflammatory growth disease patients (Crohn's disease (CD); intestinal Behcet's disease (intBD)) or The expression levels of the GSTT1 protein and the GSTT1 gene were confirmed in tissues isolated from the intestine of a normal person (CTL). In addition, the expression level of the GSTT1 gene was confirmed in the colitis animal model of the phase reference non-example 2 and the tissue isolated from the intestine of wild-type mice.
  • qRT-PCR quantitative real-time reverse transcriptase chain reaction
  • CD Crohn's disease
  • intBD intestinal Behcet's disease
  • the sections were cultured with biotin-conjugated IgG (1:500, Vector Laboratories, Burlingame, CA, USA), and then Vecta-Elite streptavidin-peroxidase kit (Vector Laboratories).
  • the sections were stained with diluted hematoxylin, and observed with an optical microscope (Olympus BX41, Olympus Optical, Tokyo, Japan)
  • Olympus BX41 Olympus Optical, Tokyo, Japan
  • the change in the length of the intestine and the goblet cells were checked, and shown in Figs. 3 (injection of 77 gene into the rectum) and in Fig. 4 (injection of 05777 gene into the abdominal cavity).
  • the weight change was measured by measurement, and the goblet cells were stained in the same manner as described in Example 2.
  • the three factors corresponding to weight loss, stool consistency, and bleeding were scored from 0 to 4 points.
  • the disease activity score was calculated through the process of summing.
  • the length of the animal model was excised and its length was measured.
  • 08111 Gene is injected through the abdominal cavity, as if injected into the rectum.
  • the reduction in body weight was recovered by gene injection (Fig. 4, the disease activity score increased (Fig. 4)), the recovery of the intestinal length reduction (Fig. 4 (1) and the number of goblet cells increased (Fig. 4 Ui-anaga, (as seen from 1 and 3 ⁇ 4-°1, 088(+)- ⁇ :08111(-) mouse in Fig. 4)
  • the survival rate was reduced to 20%, but In the case of mice, the survival rate was similar to that of mice whose colitis was not induced by 088.
  • the intestinal epithelial barrier is reduced by mucus secreted from the goblet cells Since it can be maintained, it can be seen that the GSTT1 protein expressed in the intestinal gland of the colon can significantly increase the proliferation of goblet cells, thereby enhancing the intestinal epithelial response as the first defense mechanism against pathogens.
  • Lymphocytes intestinal epkhelial lymphocytes; IEL
  • mucosal intrinsic lymphocytes lamina propria lymphocytes; LPL
  • the expression levels of the GSTT1 gene and IL-22 gene were checked in the same manner as in Example 1, The results are shown in a to c of FIG. 5.
  • Example + ( ⁇ 1 : 031 ⁇ 1(-) mouse (compared to that 13 ⁇ 4, 03 +) ( ⁇ 1:031 ' 1 ' 1(+ )
  • the expression levels of the 2 gene and the 11:22 gene were significantly increased in 0 1).
  • the culture medium of the HT-29 cell line or the transformed HT-29 cell line of Preparation Example 1 was 1 g/mL of LPS (Lipopolysaccaride), 500 nM of H 2 O 2 , 100 g/mL of recombinant human IL Replaced with RPMI medium containing -22, 40 ng/mL of TNF-a, flagenlin (fla) or Salmonella typhimurium (Sal), and cultured for 4 or 24 hours.
  • the control group In (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, 0.1 containing 5% or 1% BSA was fixed.
  • the cells were blocked using X-lOO (Triton X-lOO) and sufficiently cultured after adding a-GSTTl. After that, the GSTT1 protein present in the cell was further cultured with a secondary antibody conjugated with fluorescence. Visualized and observed through an optical microscope (Olympus BX41, Olympus Optical, Japan), and the results are shown in Fig. 6a.
  • X-lOO Triton X-lOO
  • a secondary antibody conjugated with fluorescence Visualized and observed through an optical microscope (Olympus BX41, Olympus Optical, Japan), and the results are shown in Fig. 6a.
  • DAPI was used to stain the cell nucleus.
  • the expression levels of GSTT1, CAMP, and MUC2 genes were checked in the cell line cultured for 4 hours in the same manner as in Example 1, and the results are shown in b to h of FIG. 6.
  • the human MMC2 The expression levels of the gene, CAMP gene, and TNF-a gene were measured using the primer sequence shown in Table 4 below.
  • the AW-a gene it is commercially produced and provided by Bioneer (Korea).
  • the expression level of the GSTT1 protein was significantly reduced after 2 hours.
  • the GSTT1 gene-specific siRNA was treated with 3 ⁇ 40 2 in the transformed HT-29 cell line.
  • the expression level of the TNF-a gene increased, whereas the expression level of the MUC2 gene decreased when the HT-29 cell line transfected with the GSTT1 gene-specific siRNA was treated with 3 ⁇ 40 2 or IL-22.
  • the GSTT1 protein and the gene encoding it are related to mucous or antibacterial peptides, and that pathogen infection and innate immune responses may be impaired by the defect of the GSrri gene.
  • Proteins were isolated using Pierce RIPA buffer (Cat # 89900, Thermo Fisher Scientific, USA) containing protein and phosphatase inhibitors. Controls in Table 5 below, IBD patients, Int BD TT patients and Int BD 11 patients The whole blood cells or the separated protein were heated at 58 O C for W minutes, centrifuged, and then 30-100 fig protein was added to the SDS-polyacrylamide gel and subjected to electrophoresis. Next, the gel was transferred to a PVDF membrane, reacted with a primary antibody (GSTT1 protein and an antibody specific to 3-actin protein), reacted with a secondary antibody, and then visualized and quantified, and the results are shown in a and It is shown in b.
  • a primary antibody GSTT1 protein and an antibody specific to 3-actin protein
  • the protein prevents the formation of dimers, and if the dimer of the protein is present at a lower level compared to the normal control, inflammatory growth disease is caused. You can see that it can be predicted.
  • the dimer of the GSTT1 protein was present at a lower level compared to the expression level of the total GSTT1 protein, and p38/MAPK and phosphorylated JNK (phosphor JNK), ERK (phosphor ERK) and STAT The expression level of the (phosphor STAT3) protein was also reduced.
  • the immune response of the mucosal barrier is not normally induced because the phosphorylation of STAT3 and p38/JNK does not occur at normal levels. Therefore, as shown in FIG. 9, it can be seen that the mucosal immune response is induced by p38 and STAT3 cell signaling when one or more of the IL-22 protein and GSTT1 protein are treated.
  • the present invention does not have side effects or is less effective in treating inflammatory growth diseases.
  • composition containing as an active ingredient a possible GSTT 1 (glutathione S-transferases theta 1) protein or a gene that encodes it.By inducing an immune response of the mucosal barrier through phosphorylation of STAT3 and p38/JNK, It can be used very effectively in prevention or treatment. Furthermore, by measuring whether a dimer of the GSTT1 protein, which regulates the cellular signaling, is present at a low level, inflammatory growth disease can be predicted very effectively.
  • GSTT 1 glutas theta 1
  • Sequence number 8 0 Ahsho 0( ⁇ 1:(3(:1 ' (:1 ' 1 ' (:1 ⁇ :

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Abstract

본 발명은 GSTT1(glutathione S-transferases theta 1) 단백질 또는 이를 암호화하는 유전자를 유효성분으로 포함하는 조성물에 관한 것으로서, STAT3와 p38/JNK의 인산화를 통해 점막 장벽의 면역반응을 유도함으로써, 염증성 장질환의 예방 또는 치료에 매우 효과적으로 사용될 수 있다. 나아가, 상기 세포신호전달을 조절하는 GSTT1 단백질의 이량체가 낮은 수준으로 존재하는지 여부를 측정함으로써 염증성 장질환을 매우 효과적으로 예측할 수 있다.

Description

2020/213913 1»（：1/10公020/005011 명세서
발명의명칭 : 08111단백질또는이를암호화하는유전자를 유효성분으로포함하는조성물
기술분야
[1] 본발명은 GSTT 1 (glutathione S-transferases theta 1)단백질또는이를
암호화하는유전자를유효성분으로포함하는조성물에관한것이다.
배경기술
[2] 염증성장질환 (Inflammatory bowel disease; IBD)은위장관내에만성적인
염증을유발하는질환으로서,그증상으로는복통,발열,설사,하혈등을 수반한다.대체로,궤양성대장염 (UC: ulcerative colitis)과크론병 (CD: Crohn's disease)의 2가지형태로염증성장질환이분류될수있다.궤양성대장염은 문드러짐이나궤양을형성하는대장의원인불명의확산성비특이성
염증 (diffuse nonspecific inflammation)의일종으로서,혈성설사를비롯하여 다양한전신증상을수반한다.또한,크론병은구강에서항문까지전소화관을 비연속성으로점막에서장관전층에궤양,섬유화,협착과병변이진전되는 원인불명의육아종성염증성병변으로,복통,만성설사,발열,영양장애등의 전신증상을수반한다.이와같은염증성장질환의발생율은종래에는 서양인에게높다고알려져 있었으나,최근식습관등생활습관의변화등으로 인하여우리나라를비롯한아시아권국가에서도환자수가급증하고있는 추세이다.
[3] 현재까지상기염증성장질환의발생원인이나병태생리에대해서는아직까지 명확하게알려진바없으나,유전적요인,장내세균또는음식물등의환경적인 요인,및면역학적요인등이복합적으로관여할것으로추측되고있다.이러한 발생원인중하나로서,장내에서발생되는산화스트레스와항산화방어기작 사이의불균형을들수있다.구체적으로,산화제 (Oxidant)및자유라디칼의 생산은염증유전자의발현을중재하는신호기작을활성화시킬뿐만아니라, 세포의분열,분화및사멸을조절하는유전자의발현을촉진시킬수있다. 그러나,이러한산화제및자유라디칼이과잉으로생산되는경우에는장 상피세포 (Intestinal epithelial cells; IEC)에서염증,세포손상및세포사멸이 유도될수있을뿐만아니라,점막장벽의기능장애까지유도됨으써위장관 내에만성적인염증이유발될수있을것으로추측하고있다.
[4] 이러한상황에도불구하고염증성장질환의경우에는근본적인치료법이 확립되어 있지않아완전한치료를목표로하는것이아닌증상의진행을지연 및완화시키는데불과한실정이다.나아가현재사용되고있는치료제인 설파살라진은구역질,구토,식욕부진,발진,두통,간장해,백혈구감소,이상 적혈구,단백뇨,설사등의부작용이존재하며,프레드니솔론의경우위궤양이나 장기사용에의한대퇴골두괴사등의부작용이존재하고,
인플리시맵 (Infliximab)는 1998년미국 FDA로부터크론병치료제로허가를받은 후크론병환자들을치료하기위해사용되었으나,범혈구감소,약물유발낭창,
B형간염/결핵 재활성등의부작용이존재한다.
[5] 이에,부작용이존재하지 않거나,적으면서 염증성장질환의치료가가능한 의약품의 개발에 대한연구가여전히 필요한상황이다.
발명의상세한설명
기술적과제
[6] 본발명의 일목적은 GSTT 1 (glutathione S-transferases theta 1)단백질또는이를 암호화하는유전자를유효성분으로포함하는조성물을제공하는것이다.
[7] 본발명의다른목적은 GSTT1단백질또는이를암호화하는유전자의 발현 수준을측정하는단계를포함하는염증성장질환의치료제를스크리닝하는 방법을제공하는것이다.
[8] 본발명의또다른목적은 GSTT1단백질또는이를암호화하는유전자를
포함하는술잔세포 (Goblet cell)의증식촉진용조성물을제공하는것이다.
[9] 본발명의또다른목적은 GSTT1단백질의 이량체가존재하는수준을
측정하는제제를포함하는염증성장질환의 진단용조성물과,이를포함하는 키트및이를사용하는진단에 관한정보를제공하는방법을제공하는것이다.
[1이 본발명의또다른목적은 GSTT1단백질또는이를암호화하는유전자를
사용하여 염증성장질환의 예방또는치료하는방법을제공하는것이다.
[11] 그러나본발명이 이루고자하는기술적과제는이상에서 언급한과제에
제한되지 않으며,언급되지 않은또다른과제들은아래의 기재로부터 당 업계에서통상의지식을가진자에게 명확하게 이해될수있을것이다.
과제해결수단
[12] 본발명의 일구현예에서는염증성장질환의 예방,개선또는치료용조성물을 제공한다.
[13] 본발명의상기조성물은약학조성물또는식품조성물로사용될수있다.
[14] 본발명의상기조성물은 GSTT 1 (glutathione S-transferases theta 1)단백질또는 이를암호화하는유전자를유효성분으로포함한다.
[15] 본발명의상기 GSTT1단백질은글루타티온 S-트랜스퍼라아제 (GST)중
하나로서, GSTT1단백질또는이를암호화하는유전자를투여하는경우에는 GSTT1단백질이존재하는수준,바람직하게는 GSTT1단백질이 이량체로 존재하는수준을현저하게증가시킴으로써그에 따라발생되는 STAT3와 P38/JNK의 인산화를통해장점막을보호하는기능을갖는술잔세포 (Goblet cell)의증식을촉진하여 점막장벽의 면역반응을유도함으로써 염증성장질환의 치료효과가발휘되도록할수있다.
[16] 본발명의상기조성물은 IL-22단백질또는이를암호화하는유전자를더 포함할수있다.
[17] 본발명의상기 IL-22단백질은술잔세포 (Goblet cell)의분화와창상의치유를 유도하는사이토카인이다.본발명의상기 IL-22단백질이 GSTT1단백질의 발현을증가시킴으로써점막장벽의면역반응을유도하기때문에,본발명의 목적상 IL-22단백질또는이를암호화하는유전자가더포함되는경우에는상기 조성물을투여함으로써예상되는점막장벽의면역반응에시너지효과를 유도할수있다.
[18] 본발명의상기 GSTT1단백질은서열번호 1로표시되는아미노산서열로
이루어진것일수있고, GSTT1단백질을암호화하는유전자는서열번호 2로 표시되는염기서열로이루어진것일수있으나,이에제한되는것은아니다.
[19] 본발명의상기 IL-22단백질은서열번호 3으로표시되는아미노산서열로 이루어진것일수있고, IL-22단백질을암호화하는유전자는서열번호 4로 표시되는염기서열로이루어진것일수있으나,이에제한되는것은아니다.
[2이 본발명의상기 GSTT1단백질또는 IL-22단백질은공지의단백질합성법또는 형질전환된숙주세포를배양함으로써제조될수있다.상기단백질을형질 전환된숙주세포를이용하는경우,본발명의단백질을암호화하는염기서열을 포함하는재조합발현벡터를숙주세포에도입하여형질전환체를제조한뒤, 당업계에공지된임의의방법을적절하게사용하여상기형질전환체를배양할 수있다.이렇게배양된상기형질전환체로부터단백질은당업계에서공지된 원심분리,크로마토그래피,전기영동등의방법을통해분리및정제함으로써 얻을수있다.
[21] 본발명의상기아미노산서열은하나이상의아미노산이치환,결실,삽입또는 이들조합에의해용이하게변형될수있다.따라서,서열번호 1로표시되는 아미노산서열과높은상동성을갖는단백질또는폴리펩티드,예를들면 상동성이 80%이상, 90%이상또는 95%이상인경우도항균제내성이극복될수 있는기능을유지하는한본발명의상기단백질에모두포함된다.
[22] 본발명의상기상동성이란,야생형 (Wild type)단백질의아미노산서열과의 유사한정도를나타내기위한것으로서，본발명의아미노산서열과상기와같은 서열상동성이상의동일한서열을가지는서열을포함한다.이러한상동성은두 서열을육안으로비교하여결정할수도있으나,비교대상이되는서열을나란히 배열하여상동성정도를분석해주는생물정보알고리즘 (bioinformatic algorithm)을사용하여결정할수있다.상기두개의아미노산서열사이의 상동성은백분율로표시될수있다.유용한자동화된알고리즘은 Wisconsin Genetics Software Package(Genetics Computer Group, Madison, W, USA)의 GAP, BESTFIT, FASTA와 TFASTA컴퓨터소프트웨어모듈에서이용가능하다.상기 모듈에서자동화된배열알고리즘은 Needleman & Wunsch와 Pearson & Lipman과 Smith & Waterman서열배열알고리즘을포함한다.다른유용한배열에대한 알고리즘과상동성결정은 FASTP, BLAST, BLAST2, PSIBLAST와 CLUSTAL W를포함하는소프트웨어에서자동화될수있다.
[23] 본발명의상기 GSTT1단백질을암호화하는유전자또는 IL-22단백질을
암호화하는유전자자체로서포함될수있고,바람직하게는재조합발현벡터에 포함되는형태로포함될수있으나,이에제한되는것은아니다.
[24] 본발명의상기재조합발현벡터는목적하는숙주세포에서목적하는단백질 또는펩타이드를발현할수있는재조합발현벡터로서,유전자삽입물이 발현되도록작동하게연결된필수적인조절요소를포함하는유전자제작물을 의미한다.상기발현벡터는개시코돈,종결코돈,프로모터 ,오퍼레이터등의 발현조절요소들을포함하는데,상기개시코돈및종결코돈은일반적으로 폴리펩티드를암호화하는뉴클레오티드서열의일부로간주되며,유전자 제작물이투여되었을때개체에서반드시작용을나타내야하며코딩서열과 인프레임 (/n/rame)에 있어야한다.벡터의프로모터는구성적또는유도성 프로모터일수있다.
[25] 본발명의상기재조합발현벡터는프로모터와본발명의상기 GSTT1
유전자를이루는염기서열즉,폴리뉴클레오티드가작동가능하게연결되어 있는것일수있다.본발명의상기’작동가능하게연결 (Operably linked)’이란, 일반적기능을수행하도록핵산발현조절서열과목적하는단백질또는 RNA를 코딩하는핵산서열이기능적으로연결 (Functional linkage)되어있는상태를 의미다.예를들어프로모터와단백질또는 RNA를코딩하는
폴리뉴클레오티드가작동가능하게연결되어코딩서열의발현에영향을미칠 수있다.재조합발현벡터와의작동가능하게연결되는것은당해기술분야에서 잘알려진유전자재조합기술을이용하여제조할수있으며,부위-특이적 DNA 절단및연결은당해기술분야에서일반적으로알려진효소등이사용될수 있다.
[26] 본발명의상기재조합발현벡터의백본 (Backbone)으로사용할수있는벡터는 본발명의상기단백질을생산할수있는한특별히제한되지는않으나,예를 들면,플라스미드 DNA,파아지 DNA,상업적으로개발된플라스미드 (pGEM® T 벡터 , pET22b, pUC18, pBAD, pIDTSAMRT-AMP등),대장균유래
플라스미드 (pYG601BR322, pGEX-4T-l, pET, pBR325, pUC118, pUC119등), 바실러스서브틸리스유래플라스미드 (pUB110, pTP5등),효모-유래
플라스미드 (YEpl3, YEp24, YCp50등),파아지 DNA(Charon4A, Charon21A, EMBL3, EMBL4, XgtlO, AAAAAXgtl l, IZAP등),동물바이러스
벡터 (레트로바이러스 (Retrovirus),아데노바이러스 (Adenovirus),백시니아 바이러스 (Vaccinia virus)등),곤중바이러스벡터 (배큘로바이러스 (Baculovirus) 등)일수있으나,이에제한되는것은아니다.
[27] 본발명의상기염증성장질환은궤양성대장염 ,크론병 (Crohn's disease), 장관형베체트병,출혈성직장궤양및회장낭염 (pouchitis)으로이루어진 군으로부터선택되는적어도하나인것일수있으나,이에제한되는것은 2020/213913 1»（：1^1{2020/005011 아니다.
[28] 본발명의상기 약학조성물은캡슐,정제,과립,주사제,연고제,분말또는음료 형태임을특징으로할수있으며 ,상기 약학조성물은인간을대상으로하는 것을특징으로할수있다.
[29] 본발명의상기 약학조성물은이들로한정되는것은아니지만,각각통상의 방법에 따라산제,과립제,캡슐,정제,수성 현탁액등의 경구형제형,외용제, 좌제 및멸균주사용액의 형태로제형화되어사용될수있다.본발명의 약학 조성물은약학적으로허용가능한담체를포함할수있다.약학적으로허용되는 담체는경구투여시에는결합제,활탁제,붕해제,부형제,가용화제,분산제, 안정화제,현탁화제,색소,향료등이사용될수있으며,주사제의 경우에는 완충제,보존제,무통화제,가용화제,등장제,안정화제등이혼합되어사용될수 있으며,국소투여용의 경우에는기제,부형제,윤활제,보존제등이사용될수 있다.본발명의 약학조성물의제형은상술한바와같은약제학적으로허용되는 담체와혼합하여다양하게제조될수있다.예를들어,경구투여시에는정제, 트로키,캡슐,엘릭서（만뇨뇨），서스펜션,시럽,웨이퍼등의 형태로제조할수 있으며,주사제의 경우에는단위투약엠플또는다수회투약형태로제조할수 있다.기타,용액,현탁액,정제,캡슐,서방형 제제등으로제형화할수있다.
[3이 한편,제제화에 적합한담체，부형제및희석제의 예로는,락토즈,덱스트로즈, 수크로즈,솔비톨,만니톨,자일리톨,에리스리톨,말디톨,전분,아카시아고무, 알지네이트,젤라틴,칼슘포스페이트,칼슘실리케이트,셀룰로즈,메틸 셀룰로즈,미정질셀룰로즈,폴리비닐피롤리돈,물,메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트,탈크,마그네슘스테아레이트또는광물유등이 사용될수있다.또한,중진제,항응집제,윤활제,습윤제,향료,유화제,방부제 등을추가로포함할수있다.
[31] 본발명의상기 약학조성물의투여 경로는이들로한정되는것은아니지만 구강,정맥내,근육내,동맥내,골수내,경막내,심장내,경피,피하,복강내, 비강내,장관,국소,설하또는직장이포함된다.경구또는비경구투하가 바람직하다.
[32] 본발명의상기 "비경구”란,피하,피내,정맥내,근육내,관절내,활액낭내, 흉골내,경막내,병소내 및두개골내주사또는주입기술을포함한다.
바람직하게는본발명의상기 약학조성물은또한직장투여를위한좌제의 형태로투여될수있으나,이에제한되는것은아니다.
[33] 본발명의상기 약학조성물은사용된특정화합물의 활성 ,연령 ,체중,
일반적인건강,성별,정식,투여시간,투여 경로,배출율,약물배합및 예방또는 치료될특정질환의중증을포함한여러요인에 따라다양하게 변할수있고, 상기 약학조성물의투여량은환자의상태,체중,질병의정도,약물형태,투여 경로및기간에 따라다르지만당업자에의해 적절하게선택될수있고, 1일 0.0001내지 5011¾/1¾또는 0.001내지 501¾/1¾으로투여할수있다.투여는 2020/213913 1»（：1^1{2020/005011 하루에 한번투여할수도있고,수회 나누어투여할수도있다.상기투여량은 어떠한면으로든본발명의범위를한정하는것은아니다.본발명에 따른의약 조성물은환제，당의정 ,캡슐,액제 ,겔,시럽 ,슬러리 ,현탁제로제형화될수있다.
[34] 본발명의상기식품조성물은각종식품류,예를들어,음료,껌,차,비타민
복합제,분말,과립,정제,캡슐,과자,떡,빵등의 형태로제조될수있다.
[35] 본발명의상기유효성분이식품조성물에포함될때그양은전체중량의 0.1 내지 50%의비율로첨가할수있으나,이에제한되는것은아니다.
[36] 본발명의상기식품조성물이음료형태로제조되는경우지시된비율로상기 식품조성물을포함하는것외에특별한제한점은없으며,통상의음료와같이 다양한향미제또는천연탄수화물등을추가성분으로서함유할수있다.
구체적으로,천연탄수화물로서포도당등의모노사카라이드,과당등의 디사카라이드,슈크로스등의 및폴리사카라이드,덱스트린,시클로덱스트린 등과같은통상적인당및자일리톨,소르비톨,에리트리톨등의 당알콜등을 포함할수있다.상기 향미제로서는천연향미제（타우마틴,스테비아
추출물（예를들어 레바우디오시드人글리시르히진등）및합성 향미제（사카린, 아스파르탐등）등일수있다.
[37] 본발명의상기식품조성물은여러 가지 영양제 ,비타민,광물（전해질）,합성 풍미제 및천연풍미제등의풍미제,착색제,펙트산및그의 염,알긴산및그의 염，유기산,
Figure imgf000008_0001
，안정화제，방부제，글리세린, 알콜,탄산음료에사용되는탄산화제등이 더포함될수있다.
[38] 본발명의상기식품조성물에포함되는성분들은독립적으로또는조합하여 사용될수있다.상기 첨가제의비율은본발명의 핵심적인요소에 해당하지 아니하지만,본발명의식품조성물 0중량부당 0.1내지 약 50중량부의 범위에서선택될수있으나,이에제한되는것은아니다.
[39]
[4이 본발명의다른구현예에서는염증성장질환의치료제를스크리닝하는방법을 제공한다.
[41] 본발명의상기스크리닝하는방법은염증성장질환환자로부터분리된
생물학적시료에목적하는후보물질을처리하는단계 ;및상기 생물학적 시료에서 08111단백질또는이를암호화하는유전자의 발현수준을측정하는 단계를포함한다.
[42] 본발명의상기스크리닝하는방법은상기후보물질의처리후에상기
생물학적시료에서측정된 08111단백질또는이를암호화하는유전자의 발현 수준이,상기 염증성장질환환자로부터분리된생물학적시료에서측정된 08111단백질또는이를암호화하는유전자의발현수준에비하여증가된경우, 상기후보물질을염증성장질환의치료제로선별하는단계를더포함할수있다.
[43] 본발명의상기스크리닝하는방법에서 , 08111단백질또는이를암호화하는 유전자및염증성장질환에 대한기재는상기 약학;및식품조성물에서 기재한 2020/213913 1»（：1/10公020/005011 바와동일하여,본명세서의과도한복잡성을피하기위하여생략한다.
[44] 본발명의상기생물학적시료는염증성장질환환자이거나,질환이의심되는 개체로부터얻어지거나개체로부터유래된임의의물질,생물학적체액,조직 또는세포를의미하는것으로,예를들면,전혈 (whole blood),백혈구 (leukocytes), 말초혈액단핵세포 (peripheral blood mononuclear cells),백혈구연증 (buffy coat), 혈장 (plasma)및혈청 (serum)을포함하는혈액,객담 (sputum),눈물 (tears), 점액 (mucus),세비액 (nasal washes),비강톱인물 (nasal aspirate),호톱 (bmati), 소변 (urine),정액 (semen),침 (saliva),복강세척액 (peritoneal washings),골반내 유체액 (pelvic fluids),낭종액 (cystic fluid),뇌척수막액 (meningeal fluid), 양수 (amniotic fluid),선액 (glandular fluid),췌장액 (pancreatic fluid),림프액 (lymph fluid),동수 (pleural fluid),유두톱인물 (nipple aspirate),기관지톱인물 (bronchial aspirate),활액 (synovM fluid),관절톱인물 (joint aspirate),기관분비물 (organ secretions),세포 (cell),세포주줄물 (cell extract)또는뇌척수액 (cerebrospinal fluid)을포함할수있으나,이에제한되는것은아니다.
[45] 본발명의상기후보물질은염증성장질환을극복하기위한활성,즉 GSTT1 단백질또는이를암호화하는유전자의발현수준을염증성장질환이존재하지 않는정상인에서의발현수준과동등,또는유사한정도의수준으로증가시킬수 있는활성이존재하는지여부에대해테스트하기위한약제를의미하며,단백질, 올리고펩타이드,유기분자,다당류,폴리뉴클레오티드및광범위한화합물 등의임의의분자를포함한다.이러한후보물질은천연물질뿐만아니라,합성 물질도모두포함하는것일수있다.
[46] 본발명의상기 GSTT1단백질의발현수준을측정할수있는제제는상기
생물학적시료에포함된측정의대상이되는단백질의발현수준을확인하기 위한제제를의미한다.구체적으로,상기단백질의발현수준을측정하는제제는 상기단백질에특이적으로결합할수있는항체또는앱타머일수있다.
구체적으로,상기제제는웨스턴블럿분석 (Western blot assay), ELISA(Enzyme linked immunosorbent assay),방 ! ^ 역 ^:석 (RIA: Radioimmunoassay),방 ! 면역확산법 (Radioimmimodiffusion),오우크테로니 (Ouchterlony)면역확산법 , 로케트면역전기영동 (Rocket Immunoelectrophoresis),
면역조직화학염색법 (Immunohistochemical staining),면역침전
분석법 (Immunoprecipitation Assay),보체고정분석법 (Complement Fixation
Assay),면역형광법 (Immunofluorescence),
면역크로마토그래피법 (Immunochromatography), FACS(Fluorescenceactivated cell sorter analysis)및단백질칩분석법 (protein chip technology assay)등의방법에 사용되는항체또는앱타머를포함할수있으나,이에제한되는것은아니다.
[47] 본발명의상기’’항체’，는단백질또는펩티드분자의항원성부위에
특이적으로결합할수있는단백질분자를의미한다.상기항체의형태는특별히 제한되지않으며폴리클로날항체,모노클로날항체또는항원결합성을갖는 것이라면,항체의일부인경우라도포함될수있고,모든종류의면역글로불린 항체가포함될수있다.또한,인간화항체등의특수항체가포함될수있고, 상기항체는 2개의전체길이의경쇄및 2개의전체길이의중쇄를가지는 완전한형태뿐만아니라항체분자의기능적인단편을포함한다.항체분자의 기능적인단편이란적어도항원결합기능을보유하고있는단편을의미하며 Fab, F(ab’), F(ab') 2, Fv등이될수있으나,이에제한되는것은아니다.
[48] 본발명의상기”앱타머”는단일가닥올리고뉴클레오티드를의미하는
것으로,소정의표적분자에대한결합활성을갖는핵산분자를말한다.상기 앱타머는그염기서열에따라다양한 3차원구조를가질수있으며,항원-항체 반응과같이특정물질에대하여높은친화력을가질수있다.앱타머는소정의 표적분자에결합함으로써소정의표적분자의활성을저해할수있다.상기 앱타머는 RNA, DNA,변형된 (modified)핵산또는이들의혼합물일수있으며,그 형태가직쇄상또는환상일수있다.
[49] 본발명의상기항체는 GSTT1유전자의염기서열,바람직하게서열번호 2로 표시되는염기서열로이루어진유전자를통상적인방법에따라발현벡터에 클로닝하여상기유전자에의해암호화되는단백질 (항원)을얻은뒤에통상적인 방법에의해제작될수있고,상기 앱타머는각각의염기서열을참조하여당해 기술분야에서통상의지식을가진자가공지의방법에따라쉽게제작될수 있다.
[5이 본발명의상기 GSTT1단백질을암호화하는유전자의발현수준을측정할수 있는제제는상기생물학적시료에포함된측정의대상이되는유전자의발현 수준을확인하기위하여,상기측정대상이되는유전자로부터전사된 mRNA의 수준을측정하는방법에사용될수있는제제를의미한다.구체적으로,상기 제제는 RT-PCR,정량실시간 PCR(quantified real time PCR),경쟁적
RT-PCR(Competitive RT-PCR),실시간 RT-PCR(real time quantitative RT-PCR), RNase보호분석법 (RPA; RNase protection assay),노던블럿분석 (Northern blot assay), DNA칩분석법등의방법에사용되는측정대상이되는 mRNA에 특이적으로결합할수있는프라이머쌍또는프로브를포함할수있으나,이에 제한되는것은아니다.
[51] 본발명의상기 "프라이머 "는짧은자유 3말단수산화기 (free 3'hydroxyl
group)를가지는염기서열로상보적인주형가닥 (template)와염기쌍 (base pair)을 형성할수있고,주형가닥복사를위한시작지점으로서기능을하는짧은염기 서열을의미한다.상기프라이머는적절한완충용액및온도에서중합반응 (즉, DNA폴리머레이즈또는역전사효소)을위한시약및상이한 4가지
뉴클레오사이드트리포스페이트의존재하에서 DNA합성이개시될수있다.
[52] 본발명의상기 "프로브”는상기 mRNA와특이적으로결합을이룰수있는 염기에해당하는 RNA또는 DNA등의핵산단편을의미하고,이와같은 프로브는특정 mRNA의존재유무,발현되는수준 (발현량)을확인할수있도록 라벨링되어 있을수있다.상기프로브는올리고뉴클레오타이드 (oligonucleotide) 프로브,단쇄 DNA(single strand DNA)프로브,이중쇄 DNA(double strand
DNA)프로브, RNA프로브등의 형태로제작될수있으나,이에제한되는것은 아니다.
[53] 본발명의상기프라이머또는프로브는 GSTT1유전자의 염기서열,
바람직하게는서열번호 2로표시되는염기서열을참조하여 당해기술분야에서 통상이지식을가진자가공지된방법에의해 쉽게제작될수있다.
[54]
[55] 본발명의또다른구현예에서는술잔세포 (Goblet cell)의증식촉진용
조성물을제공한다.
[56] 본발명의상기증식촉진용조성물은 GSTT1단백질또는이를암호화하는 유전자를포함하는것일수있다.
[57] 본발명의상기증식촉진용조성물은 IL-22단백질또는이를암호화하는
유전자를더포함할수있다.
[58] 본발명의상기조성물은 STAT3와 p38/JNK의 인산화를통해장점막보호
기능을갖는술잔세포의증식을촉진시킴으로써 점막장벽의 면역반응을 유도할수있고,이를통해궁극적으로술잔세포의감소로인해유발될수있는 질환,예를들면염증성장질환등의 예방또는치료에 적용할수있다.
[59] 본발명의상기 "증식”이란,세포가같은성질을유지하면서분열하여 세포의 개수가증가되는것을의미한다.본발명의목적상상기증식은장점막에 존재하는술잔세포의 개수가증가되는것일수있다.
[6이 본발명의상기증식촉진용조성물에서 GSTT1단백질또는이를암호화하는 유전자,염증성장질환, GSTT1단백질의 이량체,존재하는수준을측정하는 제제에 대한기재는상기 약학;및식품조성물및진단용조성물에서 기재한 바와동일하여,본명세서의과도한복잡성을피하기위하여 생략한다.
[61]
[62] 본발명의또다른구현예에서는염증성장질환의 진단용조성물을제공한다.
[63] 본발명의상기진단용조성물은 GSTT1단백질의 이량체가존재하는수준을 측정하는제제를포함한다.
[64] 본발명의상기 GSTT1단백질의 이량체가존재하는수준을측정하는제제는 이량체로존재하는 GSTT1단백질에특이적으로결합하는항체또는앱타머일 수있다.
[65] 본발명의상기진단용조성물은염증성장질환환자에서 정상대조군에
비하여상기 GSTT1단백질의 이량체가낮은수준으로존재하는지 여부를 확인함으로써,염증성장질환을진단할수있다.
[66] 본발명의상기 GSTT1단백질의 이량체란,두개의 GSTT1단백질단량체에 존재하는아미노산의잔기가결합되어 있는형태로서,세포내산화자극이 존재하는경우상기 GSTT1단백질은 p38/MAPK신호전달을촉진하여 2020/213913 1»（：1/10公020/005011 산화스트레스를완화시킬수있다.본발명의상기 GSTT1단백질이량체는 염증성장질환이발생되지 않는정상인에서는높은수준으로존재하여산화 스트레스에 대한자극을완화시키는반면,염증성장질환환자에서는 Gsrr/ 유전자돌연변이,예를들면결손돌연변이가발생되어 GSTT1단백질이 이량체를이루지못하여산화스트레스에 대한자극을충분히완화시키지 못함으로써증상을유발또는악화시키는것일수있다.
본발명의상기진단용조성물에서, GSTT1단백질또는이를암호화하는 유전자및염증성장질환에 대한기재는상기 약학;및식품조성물에서 기재한 바와동일하여,본명세서의과도한복잡성을피하기위하여 생략한다.
[68] 본발명의상기 "항체”는이량체로존재하는 GSTT1단백질에특이적으로
결합할수있는단백질분자를의미하며 ,상기항체의 형태는특별히 제한되지 않지만폴리클로날항체,모노클로날항체또는항원결합성을갖는것이라면, 항체의 일부인경우라도포함될수있고,모든종류의 면역글로불린항체가 포함될수있다.또한,인간화항체등의특수항체가포함될수있고,상기 항체는 2개의 전체길이의경쇄 및 2개의 전체길이의중쇄를가지는완전한 형태뿐만아니라항체분자의기능적인단편을포함한다.항체분자의기능적인 단편이란적어도항원결합기능을보유하고있는단편을의미하며 Fab, F(ab’), F(ab') 2, Fv등이 될수있으나,이에제한되는것은아니다.
본발명의상기“앱타머”는단일가닥올리고뉴클레오티드를의미하는 것으로,이량체로존재하는 GSTT1단백질에 대한결합활성을갖는핵산분자를 말한다.상기 앱타머는그염기서열에 따라다양한 3차원구조를가질수 있으며,항원-항체반응과같이특정물질에 대하여높은친화력을가질수있다. 앱타머는소정의표적분자에결합함으로써소정의표적분자의활성을저해할 스 수있다.상기 앱타머는 RNA, DNA,변형된 (modified)핵산또는이들의혼합물일
7 7 6 679211 수있으며，그형태가직쇄상또는환상일수있다.
[7이
[기] 본발명의또다른구현예에서는본발명의상기조성물을포함하는염증성 장질환의 진단용키트를제공한다.
본발명의상기키트는본발명의상기조성물을사용하여 염증성장질환이 존재하지 않는정상대조군에비하여목적하는개체에서 GSTT1단백질의 이량체가낮은수준으로존재하는경우염증성장질환으로예측할수있다.
[73] 본발명의상기진단용키트에서, GSTT1단백질또는이를암호화하는유전자, 염증성장질환, GSTT1단백질의 이량체,존재하는수준을측정하는제제에 대한 기재는상기 약학;및식품조성물및진단용조성물에서 기재한바와동일하여 , 본명세서의과도한복잡성을피하기 위하여 생략한다.
본발명의상기키트는 RT-PCR키트, DNA칩키트, ELISA키트,단백질칩 키트,래피드 (Rapid)키트또는 MRM (Multiple reaction monitoring)키트등일수 있으나,이에 제한되는것은아니다. P5] 본발명의상기키트가단백질의수준을측정하기위한경우에는항체의 면역학적검출을위하여기재,적당한완충용액,발색효소또는형광물질로 표지된 2차항체,발색기질등이포함될수있다.
6] 본발명의상기기재는니트로셀룰로오스막,폴리비닐수지또는폴리스티렌 수지로합성된 96웰플레이트및유리로된슬라이드글라스등이사용될수 있고,상기발색효소는퍼옥시다아제 (Peroxidase),알칼라인
포스파타아제 (Alkaline Phosphatase)가사용될수있으며 ,형광물질은 FITC, RITC 등이사용될수있고,발색기질액은
2,2’-아지노-비스 (3 -에틸벤조티아졸린- 6 -설폰산 (2,2’-azino-bis(3-ethylbenzothiazoli ne-6-sulphonic acid); ATBS), o-페닐렌디이민 (o-Phenylenediamine; OPD), 테트라메틸벤지딘 (tetramethyl benzidine; TMB)가사용될수있으나,이에 제한되는것은아니다.
7]
8] 본발명의또다른구현예에서는염증성장질환의진단을위한정보를
제공하는방법을제공한다.
9] 본발명의상기방법은목적하는개체로부터분리된생물학적시료에서,
GSTT1단백질의이량체가존재하는수준을측정하는단계를포함한다.
[8이 본발명의상기방법은목적하는개체로부터분리된생물학적시료에서측정된
GSTT1단백질의이량체가존재하는수준이정상대조군에비하여낮은경우, 상기목적하는개체가염증성장질환일것으로예측할수있다.
[81] 본발명의상기목적하는개체로부터분리된생물학적시료는정상또는
질환이의심되는개체로부터얻어지거나개체로부터유래된임의의물질, 생물학적체액,조직또는세포를의미하는것으로,예를들면,전혈 (whole blood), 백혈구 (leukocytes),말초혈액단핵세포 (peripheral blood mononuclear cells), 백혈구연증 (buffy coat),혈장 (plasma)및혈청 (serum)을포함하는혈액, 객담 (sputum),눈물 (tears),점액 (mucus),세비액 (nasal washes),비강톱인물 (nasal aspirate),호톱 (brea比 i),소변 (urine),정액 (semen),침 (saliva),복강세적액 (peritoneal washings),골반내유체액 (pelvic fluids),낭종액 (cystic fluid),뇌척수막
액 (meningeal fluid),양수 (amniotic fluid),선액 (glandular fluid),췌장액 (pancreatic fluid),림프액 (lymph fluid),몽수 (pleural fluid),유두톱인물 (nipple aspirate),기관지 톱인물 (bronchial aspirate),활액 (synovial fluid),관절톱인물 (joint aspirate),기관 분비물 (organ secretions),세포 (cell),세포주줄물 (cell extract)또는
뇌척수액 (cerebrospinal fluid)을포함할수있으나,이에제한되는것은아니다.
[82] 본발명의상기진단을위한정보를제공하는방법에서 , GSTT1단백질또는 이를암호화하는유전자,염증성장질환, GSTT1단백질의이량체,존재하는 수준을측정하는제제에대한기재는상기약학;및식품조성물및진단용 조성물에서기재한바와동일하여,본명세서의과도한복잡성을피하기위하여 생략한다. [83] 본발명의상기단백질이존재하는수준을측정하는단계는본발명의상기 조성물을이용하여웨스턴블럿분석, ELISA,방사선면역분석,방사면역 확산법,오우크테로니면역확산법,로케트면역전기영동,면역조직화학염색법, 면역침전분석법,보체고정분석법,면역형광법,면역크로마토그래피법, FACS 및단백질칩분석법으로구성된군으로부터선택되는적어도하나를통해 수행될수있고,바람직하게는상기단계는비-환원조건에서수행되는것일수 있으나,이에제한되는것은아니다.
[84] 본발명의상기비-환원조건은단백질의이황화결합등을끊어전기영동과 같은분석을용이하게해주는제제,예를들면 (3-메르캅토에탄올 (p
-mercaptoethanol)또는 DTT (dithiothreitol)가포함되어 있지않은조건으로서 ,본 발명의목적상산화된 AMPK단백질을검출하는데적합한조건일수있다.
[85]
[86] 본발명의또다른구현예에서는투여가필요한대상체에게본발명의
GSTT1 (glutathione S-transferases theta 1)단백질또는이를암호화하는유전자를 약학적으로유효한양으로투여하는단계를포함하는염증성장질환의예방 또는치료방법에관한것이다.
[87] 본발명에서상기 "투여”는임의의적절한방법으로대상체에소정의본
발명의화합물을제공하는것을의미한다.
[88] 본발명에서상기투여가필요한 "대상체”는포유동물및비-포유동물을모두 포함할수있다.여기서,상기포유동물의예로는인간,비-인간영장류,예컨대 침팬지,다른유인원또는원숭이종;축산동물,예컨대소,말,양,염소,돼지; 사육동물,예컨대토끼,개또는고양이;실험동물,예를들어설치류,예컨대 래트,마우스또는기니아피그등을포함할수있으나,이에제한되는것은 아니다.또한,본발명에서상기비-포유동물의예로는조류또는어류등을 포함할수있으나,이에제한되는것은아니다.
[89] 본발명에서상기와같이투여되는단백질또는유전자의제제는특별히
제한하지않으며,고체형태의제제,액체형태의제제또는흡인용에어로졸 제제로투여될수있으며 ,사용하기바로전에경구또는비경구투여용액체 형태제제로전환되도록의도되는고체형태제제로투여될수있고,예를들면, 산제,과립제,캡슐,정제,수성현탁액등의경구형제형,외용제,좌제및멸균 주사용액의형태로제형화하여투여될수있으나,이에제한되는것은아니다.
[9이 또한,본발명에서상기투여시본발명의단백질또는유전자의제제와함께 약학적으로허용가능한담체를추가로투여할수있다.여기서,상기
약학적으로허용되는담체는경구투여시에는결합제 ,활탁제 ,붕해제,부형제 , 가용화제,분산제,안정화제,현탁화제,색소,향료등을사용할수있으며 , 주사제의경우에는완충제 ,보존제 ,무통화제 ,가용화제 ,등장제 ,안정화제등을 혼합하여사용할수있으며,국소투여용의경우에는기제,부형제,윤활제, 2020/213913 1»（：1^1{2020/005011 보존제등을사용할수있다.본발명의단백질또는유전자제제의제형은 상술한바와같은약학적으로허용되는담체와혼합하여다양하게제조될수 있다.예를들어 ,경구투여시에는정제，트로키，캡슐,엘릭서 (611X11),서스펜션, 시럽,웨이퍼등의 형태로제조할수있으며,주사제의경우에는단위투약엠플 또는다수회투약형태로제조할수있다.기타,용액,현탁액,정제,캡슐,서방형 제제등으로제형할수있다.
[91] 한편,제제화에 적합한담체，부형제및희석제의 예로는,락토즈,덱스트로즈, 수크로즈,솔비톨,만니톨,자일리톨,에리스리톨,말디톨,전분,아카시아고무, 알지네이트,젤라틴,칼슘포스페이트,칼슘실리케이트,셀룰로즈,메틸 셀룰로즈,미정질셀룰로즈,폴리비닐피롤리돈,물,메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트,탈크,마그네슘스테아레이트또는광물유등이 사용될수있다.또한,중진제,항응집제,윤활제,습윤제,향료,유화제,방부제 등을주가로포함할수있다.
[92] 본발명에 따른단백질또는유전자제제의투여 경로는이들로한정되는것은 아니지만구강,정맥내,근육내,동맥내,골수내,경막내,심장내,경피,피하, 복강내,비강내,장관,국소,설하또는직장이포함된다.경구또는비경구 투하가바람직하다.
[93] 본발명에서，"비경구”는피하,피내,정맥내,근육내，관절내,활액낭내,흉골내 , 경막내,병소내 및두개골내주사또는주입기술을포함한다.본발명의 약학적 조성물은또한직장투여를위한좌제의 형태로투여될수있다.
[94] 본발명에서, "약학적으로유효한양”은바람직한생물학적 결과를제공하기 위한작용제의충분한양을지칭한다.상기 결과는질환의 징후,증상또는 원인의감소및/또는완화,또는생물계의 임의의다른바람직한변화일수있다. 예를들어,치료용도를위한 "유효량”은질환에서 임상적으로유의한감소를 제공하는데요구되는,본발명에 개시된단백질또는유전자의 양이다.임의의 개별적인경우에서 적절한”효과적인”양은일상적인실험을사용하여 당업자에 의해결정될수있다.따라서,표현”유효량”은일반적으로활성물질이치료 효과를갖는양을지칭한다.본발명의 경우에,활성물질은 08111단백질또는 이를암호화하는유전자이자,염증성장질환의 예방또는치료제이다.
[95] 본발명의 단백질또는유전자제제는사용된특정 단백질또는유전자의활성, 연령,체중,일반적인건강,성별,정식,투여시간,투여 경로,배출율,약물배합 및 예방또는치료될특정 질환의중증을포함한여러요인에따라다양하게 변할수있고,상기 단백질또는유전자제제의투여량은환자의상태,체중, 질병의 정도,약물형태,투여 경로및기간에따라다르지만당업자에 의해 적절하게선택될수있고, 1일 0.0001내지
Figure imgf000015_0001
또는 0.001내지
10011¾/1¾으로투여할수있다.투여는하루에 한번투여할수도있고,수회 나누어투여할수도있다.상기투여량은어떠한면으로든본발명의 범위를 한정하는것은아니다.본발명에따른단백질또는유전자제제는환제,당의정, 캡슐,액제 ,겔,시럽 ,슬러리 ,현탁제로제형될수있다.
[96] 본발명의단백질또는유전자제제는단독으로,또는수술,방사선치료,
호르몬치료,화학치료및생물학적반응조절제를사용하는방법들과병용하여 사용할수있다.
발명의효과
[97] 본발명에따른조성물은 STAT3와 p38/JNK의인산화를통해술잔세포의
증식을촉진하여장점막장벽의면역반응을유도함으로써,염증성장질환의 예방또는치료에매우효과적으로사용될수있다.
[98] 나아가,상기세포신호전달을조절하는 GSTT1단백질의이량체가낮은
수준으로존재하는지여부를측정함으로써염증성장질환을매우효과적으로 예측할수있다.
도면의간단한설명
[99] 도 1의 a는본발명의일실시예에따른면역조직화학염색을수행한결과이며, 도 1의 b및 c는본발명의실시예에따른 GSTT1유전자의발현수준을정량적 실시간역전사중합효소연쇄반응（qRT-PCR）을통해확인한결과를나타낸 것이다.
[10이 도 2는본발명의일실시예에따른 GSTT1단백질에특이적인항체또는 IgG가 주입된대장염동물모델에서술잔세포（goblet cell）를염색한결과를나타낸 것이다.
[101] 도 3의 a내지 d는본발명의일실시예에따른 GSTT1유전자가직장주입을 통해형질전환된마우스에서,체중변화（도 3의 a）,질병활성점수（Disease activity index; DAI）（도 3의 b）,장의길이변화（도 3의 c）및술잔세포（도 3의 d）를 확인한결과를나타낸것이다.
[102] 도 4의 a내지 e는본발명의일실시예에따른 GSTT1유전자가복강주입을 통해형질전환된마우스에서,체중변화（도 4의 a）,질병활성점수（도 4의 b）, 생존율（도 4의 c）,장의길이변화（도 4의 d）및술잔세포（도 4의 e）를확인한 결과를나타낸것이다.
[103] 도 5의 a내지 c는본발명의일실시예에따른 GSTT1유전자및 IL-22유전자의 발현수준을확인한결과를나타낸것이고,체중변화（도 5의 d）,질병활성 점수（도 5의 e）,장의길이변화（도 5의 f）및술잔세포（도 5의 g）를확인한결과를 나타낸것이다.
[104] 도 6의 a는본발명의일실시예에따른 GSTT1단백질의발현수준을
면역조직화학（immunohistochemistry）을통해확인한결과를나타낸것이고,도 6의 b및 c는 GSTT1유전자의발현수준을정량적실시간역전사중합효소 연쇄반응을통해확인한결과를나타낸것이며,도 6의 d는（ Sr77유전자에 특이적인 siRNA처리후,웨스턴블롯분석을통해 GSTT1단백질의발현수준을 확인한결과이고,도 6의 e내지 h는 GSTT1단백질의발현이억제된세포에서 유전자들의 발현수준을확인한결과를나타낸것이다.
[105] 도 7의 a및 b는 GSTT1단백질의단량체 및이량체의 발현수준을웨스턴블롯 분석을통해확인하고,전체 GSTT1에따른이량체 GSTT1단백질의발현수준을 비율로나타낸것이다.
[106] 도 8의 a및 b는 GSTT1단백질단량체 및이량체의 발현수준을웨스턴블롯 분석을통해확인하고,전체 GSTT1에따른이량체 GSTT1단백질의발현수준을 비율로나타낸것이며,도 8의 c는 GSTT1단백질과관련된세포신호전달 단백질의 발현수준을웨스턴블롯분석을통해확인한결과를나타낸것이다.
[107] 도 9는본발명의 일실시예에 따른 GSTT1단백질의작용기작을모식도로 나타낸것이다.
발명의실시를위한최선의형태
[108] 본발명의 일구현예에서는염증성장질환의 예방,개선또는치료용조성물에 관한것이다.
[109] 본발명의다른구현예에서는염증성장질환의치료제를스크리닝하는방법에 관한것이다.
[110] 본발명의또다른구현예에서는술잔세포 (Goblet cell)의증식촉진용
조성물에 관한것이다.
[111] 본발명의또다른구현예에서는염증성장질환의 진단용조성물에 관한
것이다.
[112] 본발명의또다른구현예에서는본발명의상기조성물을포함하는염증성 장질환의 진단용키트에 관한것이다.
[113] 본발명의또다른구현예에서는염증성장질환의 진단을위한정보를
제공하는방법에관한것이다.
[114] 본발명의또다른구현예에서는투여가필요한대상체에게본발명의
GSTT1 (glutathione S-transferases theta 1)단백질또는이를암호화하는유전자를 약학적으로유효한양으로투여하는단계를포함하는염증성장질환의 예방 또는치료방법에 관한것이다.
발명의실시를위한형태
[115] 이하,실시예를통하여본발명을더욱상세히 설명하고자한다.이들실시예는 오로지본발명을보다구체적으로설명하기위한것으로서，본발명의요지에 따라본발명의 범위가이들실시예에의해제한되지 않는다는것은당업계에서 통상의지식을가진자에게 있어서자명할것이다.
[116]
[117] 실시예
[118]
[119] 『주비예 11세포주배양밀형짐 저환
[12이 열을이용하여불활성화된 의우태아혈청 및 1%의 항생제가포함된 RPMI 배지에서 HT-29세포주 (Korea Cell Line Bank,한국)를 5%의 C02, 37°C의 조건에서배양하였다.리포펙타민 2000(Life Technologies,미국)을이용하여상기 세포주에 GSTT1유전자 (서열번호 2)에상보적으로결합하는 siRNA또는 비-표적화대조군 (non-targeting control; AccuTarget, Bioneer,한국)을형질 전환하였다.
[121]
[122] [준비예 2]마우스사육조거및대장염동물모델제작
[123] 병원체가없는 12:12암주기의환경에서야생형 C57BL/6마우스 (OrientBio, 한국)를사육하였다.필터를이용하여멸균된 2.5%(W八 0의 DSS(Dextran Sulfate Sodium Salt, MW. 36,00050,000; MP Biomedicals,미국)가포함된음용수를 8주된 마우스에 7일동안공급한뒤,이어서 2일동안멸균된음용수만을공급함으로써 상기마우스에대장염을유도하여대장염동물모델을제작하였다.
[124]
[125] [준비예 3]마우스에유저자저담
[126] 하기표 1에기재된벡터를상기준비예 2의대장염동물모델또는야생형 마우스의직장 (100 의 PBS중 30 Utg)또는복강 (lml의 PBS중 100/保)에 2회 (1일 및 5일)주입하였다.이때,상기벡터와형질전환물질 PEI(Polyetherimide)를 l:l((w : V,收 DNA : ¹ PEI)의비율로혼합된혼합용액을사용하여,유전자 전달의효율을높였다.또한,상기혼합용액의직장또는복강주입시에 이소퓨렌 (Isofurane; Ifrane liquid, MNS한국)을이용하여상기대장염동물모델 및야생형마우스를마취하였다.이때,상기혼합용액이대장염동물모델및 야생형마우스의몸에골고루분포될수있도록하기위하여, 20초동안상기 대장염동물모델및마우스를거꾸로매달았다.이러한과정을통해최종적으로 하기표 1에서와같이총 8가지의경우의수에해당하는마우스를제작하였다.
[127] [표 1]
Figure imgf000019_0001
Figure imgf000019_0002
Figure imgf000019_0003
[128]
[129] [실시예 1] GSTT1다백짐및유저자의밤혀수주확이
[130] 면역조직화학 (Immunohistochemistry)염색과정량적실시간역전사중합효소 연쇄반응 (qRT-PCR)을이용하여 ,염증성장질환환자 (크론병 (CD);장관형 베체트병 (intestinal Behcet's disease; intBD))또는정상인 (CTL)의장으로부터 분리된조직에서 GSTT1단백질및 GSTT1유전자의발현수준을확인하였다. 또한,상기준비예 2의대장염동물모델및야생형마우스의장으로부터분리된 조직에서 GSTT1유전자의발현수준을확인하였다.
[131] 면역조직화학염색을위해포르말린으로고정된파라핀포매조직절편을
탈파라핀화한뒤,증류수로세척하고 10 mM, pH 6.0의구연산 (Citrate)완충액에 넣고 W분동안가열하여항원이노출될수있도록하였다.그런다음,상기 항원이노출된절편을증류수로세척하고, 3%의과산화수소에넣어 5분동안 반응시켰다.이후, 0.1%트윈- 20(TBS-T)가포함된 Tris완충식염수로세척하고 GSTT1단백질에특이적으로결합하는항체 (Santa Cruz,미국)와밤새도록 반응시켰다.세척후,절편을바이오틴 -접합된 IgG (1 : 500, Vector Laboratories, Burlingame, CA, USA)와함께배양한뒤 ,발색을위한벤지딘기질을갖는 Vecta-Elite streptavidin-peroxidase kit (Vector Laboratories)의시약을처리하였다. 대조를위하여희석된헤마톡실린으로상기절편을염색하고,광학현미경 (Olympus BX41, Olympus Optical, Tokyo, Japan)으로관찰하였다.단백질의 발현을정량화하기위해,무작위로선택된필드를 W0배율로각샘플에대해 조사하고 0내지 3의점수를매겨,그결과를도 1의 a에나타내었다. [132] qRT-PCR을위해 , cDNA합성키트（High Capacity cDNA Reverse Transcription Kit, Applied Biosystems,미국）를이용하여 ,각조직으로부터 Ribospinä（GeneAll, 한국）을통해추출된전체 RNA의 1 ~ 4/ 를제조사가제공하는방법에따라 cDNA를합성하였다.제조된 cDNA는 SYBR그린마스터믹스（SYBR Green master mix, Applied Biosystems）및하기표 2의프라이머쌍（각프라이머 200 nmol,최종농도）과 3중으로혼합한뒤 , tepOne Plus실시간 PCR시스템（Applied Biosystems）에서 45-60사이클（95 °C 30초, 56-61 °C 30초, 72 °C 40초）동안 증폭시켰다.그런다음,각프라이머에따른증폭된값을 GAPDH또는 p-actin의 발현양에따른상대적발현수준으로정량화하여,도 1의 b및 c에나타내었다.
[133] [표 2]
Figure imgf000020_0004
[134] 도 1의 &에서보는바와같이,정상인（01）에비하여크론병및베체트병에 해당하는염증성장질환환자의장조직에서 08111단백질의발현이현저하게 감소되어 있었다.
[135] 도 1의 15및 에서보는바와같이 ,정상인（01）에비하여염증성장질환환자의 장조직에서 0 777유전자의발현이 2배이상감소되었으며,
Figure imgf000020_0001
에의해 대장염이유도된동물모델의장조직에서역시 05777유전자의발현이정상에 비하여감소되어있었다.
[136] 상기결과를통해,염증성장질환에서 08111단백질또는이를암호화하는 유전자의발현수준이감소되어있음을알수있다.
[137]
[138] [실시예 2]숨자세포（이 라 0태）확이
[139] 상기준비예 2의대장염동물모델에 2회（7일째또는 11일째） 08111단백질에 특이적인항체 ）또는 ¾（3를복강내주사한
Figure imgf000020_0003
사용하여상기대장염동물모델로부터
Figure imgf000020_0002
장샘 마）에존재하는술잔세포를염색한뒤,현미경으로관찰하여그결과를도 2020/213913 1»（：1^1{2020/005011
2에나타내었다.
[14이 도 2에서보는바와같이, 투여된경우와비교하여,대장염동물모델에
Figure imgf000021_0001
투여되었을때에술잔세포가감소되는것을확인하였다.
[141] 상기결과를통해,대장염이발생되었을때 08111단백질이존재하는수준이 감소되는경우술잔세포가감소되는것을알수있다.
[142]
[143] [실시예 3] (75777유저자저담음통한대장염와화효과확이
[144] 상기준비예 3의마우스들에서체중변화,질병활성점수
Figure imgf000021_0002
0시),장의길이변화및술잔세포를확인하여,도 3(직장에 ( 77유전자주입) 및도 4(복강에 05777유전자주입)에나타내었다.여기서,상기동물모델의 체중을 9일동안매일측정하여체중변화를측정하였고,상기실시예 2에 기재된방식과동일하게술잔세포를염색하였다.또한,체중감소,대변의일관성 및출혈여부에해당하는 3가지의요인에대하여 0내지 4점으로점수화하고 합산하는과정을통해상기질병활성점수를계산하였다.또한,상기동물 모델의장을적출하고,그길이를측정하였다.
[145] 보는바와같이, 03 +)- (太11必 1'1(-)마우스 (도 2의山
Figure imgf000021_0003
체중이약 40%감소되었다.그러나, 088(+)-16^1:08111(+) 마우스마우스 (
Figure imgf000021_0004
경우에는 8일또는 7일째되는날부터 체중의감소가회복되는양상을나타내었다.
[146] 도 3의 15에서보는바와같이, 03 +)- ( 11쑈31'11(-)마우스 (도 3의江
예3+!)(：11)의경우,질병활성점수가시간이지남에따라계속적으로증가되어 , 8일째 12점을나타내었다.반면,
Figure imgf000021_0005
마우스 (도 3의江
088+08111)£]경우에는약 5점또는약 7점으로질병활성점수가감소되었다.
[147] 도 3의。에서보는바와같이, 03 +)-^( 11:031'1'1(-)마우스의장길이는
088(-)-16^1^08111(-)마우스에비하여 2 «11감소되었으나, 08111단백질이 형질전환된예3(+)- 1必 1'1(+)마우스에서장길이감소가회복되었다.
[148] 도 3의 (1에서보는바와
Figure imgf000021_0006
088(-)-16^1:08111(-)마우스
\¥없 +!)(：11)와비교하여, 033(-) (血1不0 1'1(+)마우스 (도
Figure imgf000021_0007
Water+pGsttl)에서술잔세포의수가현저하게증가되었다.
[149]
Figure imgf000021_0008
08111유전자가복강을통해주입된경우, 직장에주입된경우와마찬가지로
Figure imgf000021_0009
유전자주입에의해체중의감소가 회복되고 (도 4의幻，질병활성점수가증가되었으며 (도 4의 )，장의길이감소의 회복 (도 4의 (1)및술잔세포의수가증가되었다 (도 4의아나아가,도 4의 (1에서 보는바와 ¾-°1 , 088(+)-^:08111(-)마우스
Figure imgf000021_0010
생존율이 20%로감소하였으나,
Figure imgf000021_0011
마우스 의 경우에는그생존율이 088에의해대장염이유도되지않은마우스와유사한 정도의생존율을보였다.
[15이 상기결과를통해 ,술잔세포에서분비되는점액에의하여장상피장벽이 유지될수있기 때문에,대장의장샘에서 발현되는 GSTT1단백질은술잔세포의 증식을현저하게증가시켜,병원체에 대한첫번째방어기작으로장상피 반응을 향상시킬수있음을알수있다.
[151]
[152] [실시예 4] GSTT1다백짐과 IL-22와의과계확이
[153] 상기준비예 3의직장으로유전자를주입한마우스들에서분리된장상피
림프구 (intestinal epkhelial lymphocyte; IEL)및점막고유증림프구 (lamina propria lymphocyte; LPL);또는조직으로부터분리된 mRNA에서상기실시예 1과 동일한방법으로 GSTT1유전자및 IL-22유전자의 발현수준을확인하여,그 결과를도 5의 a내지 c에 나타내었다.
[154] 또한,준비예 3의 DSS(+)-rectal:GSTTl(+)마우스및 DSS(+)-rectal:GSTTl(-) 마우스에상기실시예 2와동일한방법으로 IL-22단백질에특이적인
항체 (a-IL-22)를복강내로주사한뒤,상기실시예 3에서와동일한방법으로체중 변화,질병 활성 점수,장길이 변화및술잔세포를확인하여,그결과를도 5의 d 내지 g에나타내었다.여기서 IL-22유전자및 Muc2유전자의발현수준은하기 표 3의프라이머서열을이용하여측정하였다.
[155] [표 3]
Figure imgf000022_0001
[156] 스여(：11)와
비교하여,예 -:片 산必 江)마우스여0 1)으로부터분리된장상피 림프구 및 점막고유층림프구知)와장조직으로부터분리된 mRNA(b)에서 (^777 유전자및 11:22유전자의 발현수준이 현저하게증가되었다.
[157] 또한,도 5의。에서보는바와같이,예 +) (油1:031ᅵ1(-)마우스여(그1¾와 비교하여, 03 +) (油1:031’1’1(+)마우스여0 1)에서 2유전자및 11:22 유전자의 발현수준이 현저하게증가되었다.
[158] 도 5의 (1내지은에서보는바와같이,예 +)- (油1쑈 11(+)마우스에
01-1ᄂ22가투여되는경우 pCMV-GSTTl-GFP벡터가형질전환됨으로써 발생되는체중감소억제,질병활성 점수감소및술잔세포수가증가되는 효과가발휘되지 않았다.
[159] 상기결과를통해 , 08111단백질에 의한염증성장질환의 억제효과는 1 22 단백질과관련된세포신호전달에 의해유도되는것임을알수있다.
[16이 [161] 『심시예 51 GSTT1다백짐과장상피며역바유가의과계확이
[162] 상기준비예 1의 HT-29세포주또는형질전환된 HT-29세포주의배양배지를 1 g/mL의 LPS(Lipopolysaccaride), 500 nM의 H202, 100 g/mL의재조합인간 IL-22, 40 ng/mL의 TNF-a,플라젤린 (flagenlin; fla)또는살모넬라트리뮤리움 ( Salmonella typhimurium; Sal)이포함된 RPMI배지로교체하고 4시간또는 24시간 동안배양하였다.여기서,대조군 (Vehicle)에는상기세포주에아무것도 처리하지않았다. pH 7.4, 4%의파라포름알데히드 (paraformaldehyde)용액을 이용하여 24시간동안배양된상기세포를고정시키고, PBS로상기고정된 세포를세척하였다.그런다음, 5%또는 1% BSA가포함된 0.1%트리톤
X-lOO(Triton X-lOO)을이용하여상기세포를차단하고, a-GSTTl를넣은뒤에 충분히배양하였다.이후형광이결합된 2차항체와함께추가배양하는과정을 통해세포내존재하는 GSTT1단백질을시각화하여광학현미경 (Olympus BX41, Olympus Optical,일본)을통해관찰하고,그결과를도 6의 a에
나타내었다.이때, DAPI를이용하여세포핵을염색하였다.
[163] 또한,상기실시예 1과동일한방법으로상기 4시간동안배양된세포주에서 GSTT1, CAMP,및 MUC2유전자의발현수준을확인하여그결과를도 6의 b 내지 h에나타내었다.상기인간 MMC2유전자, CAMP유전자및 TNF-a유전자의 발현수준은아래표 4의프라이머서열을이용하여측정하였다.여기서, AW-a 유전자의경우바이오니아 (한국)에서상업적으로제작되어제공되는
프라이머를이용하여측정하였다.이때,형질전환된 HT-29세포주의형질전환 확인을위하여통상의방법에의해웨스턴블롯분석을수행하여,그결과를도 6의 d에나타내었다.
[164] [표 4]
Figure imgf000023_0001
[165] 도 6의 a에서보는바와같이, LPS가처리되지않은경우와비교하여, LPS가 처리된 HT-29세포주에서 GSTT1단백질의발현수준이현저하게감소되었다.
[166] 도 6의 b및세서보는바와같이,대조군 (Veh)에비하여, HT-29세포주에 LPS, 플라젤린 (Fla)및살모넬라트리뮤리움 (Sal)을처리한경우 GSTTJ및 CAMP 유전자의발현수준이감소된반면, H202, IL-22및 TNF-a를처리하였을때에는 GSTT1및 CAMP유전자의발현수준이증가되었다.
[167] 도 6의 d에서보는바와같이,형질전환된 HT-29세포주의경우 2시간이후부터 GSTT1단백질의발현수준이현저하게감소되었다. [168] 도 6의 e내지 h에서보는바와같이,비-표적화대조군이형질전환된 경우 (SCR)와비교하여, GSTT1유전자에특이적인 siRNA가형질전환된 HT-29 세포주에 ¾02가처리되었을때 , TNF-a유전자의발현수준은증가된반면, GSTT1유전자에특이적인 siRNA가형질전환된 HT-29세포주에 ¾02또는 IL-22가처리되었을때오히려 MUC2유전자의발현수준이감소되었다.
[169] 상기결과를통해, GSTT1단백질및이를암호화하는유전자는점액또는항균 펩타이드와관련성이있으며, GSrri유전자의결함에의해병원균감염및 선천성면역반응이손상될수있음을알수있다.
[17이
[171] [실시예 6]염증성장짐환환자에서 GSTT1다백짐확이
[172] 아래표 5의대조군; IBD환자; Int BDTT;및 Int BD 환자의조직으로부터
단백질및인산화분해효소억제제가포함되어있는 Pierce RIPA완충액 (Cat # 89900, Thermo Fisher Scientific,미국)을이용하여단백질을분리하였다.아래표 5의대조군, IBD환자, Int BDTT환자및 Int BD 11환자의전체혈액세포를 채취하였다.상기전체혈액세포또는분리된단백질을 58 OC에서 W분동안 가열한뒤원심분리하고, SDS-폴리아크릴아미드겔에 30 - 100 fig단백질을넣고 전기영동하였다.그런다음,상기겔을 PVDF막으로옮기고 1차항체 (GSTT1 단백질및 (3-actin단백질에특이적인항체)와반응시킨뒤 2차항체와반응시킨 뒤시각화및정량화하여 ,그결과를도 7의 a및 b에나타내었다.여기서 ,상기 단백질의비환원조건을유지하기위하여 (3-머캅토에탄올 (p-mercaptoethanol;
Sigma- Aldrich, MO,미국)을모든준비용액에서제외하였다.
[173] [표 5]
Figure imgf000024_0002
[174] 도 7의 &에서보는바와같이,대조군에비하여염증성장질환 (161))환자에서 08111단백질의이량체가낮은수준으로존재하였다.
[175] 도 7의 &및 15에서보는바와같이 ,조직및전체혈액세포모두에서 ,염증성 장질환환자중에서 05X11유전자결손돌연변이가없는경우여유)에비하여, 05777유전자의결손돌연변이가존재하는경우 。 ·111)에서 08111단백질의 이량체가낮은수준으로존재하였다.
[176] 상기결과를통해 (^ 77유전자돌연변이,특히결손돌연변이는
Figure imgf000024_0001
단백질이이량체가형성되지못하도록하는것을알수있으며 , 단백질의 이량체가정상대조군에비하여낮은수준으로존재하는경우염증성장질환을 예측할수있음을알수있다.
[177]
[178] [실시예 7] GSTT1다백짐과과려되세포스 I호저담확이
[179] 상기준비예 1의 HT-29세포주및형질전환된 HT-29세포주에 IL-22단백질을 상기실시예 5와동일한방법으로처리한뒤,단백질의발현수준을상기실시예 6과동일한방법으로확인하여,그결과를도 8의 a내지 c에나타내었다.
[180] 도 8의 a및 b에서보는바와같이, HT-29세포주 (CTL)에비하여 GSTT1
단백질의발현이억제되어 있는경우 (MT),전체 GSTT1단백질의발현수준에 비하여 GSTT1단백질의이량체가낮은수준으로존재하였고, p38/MAPK와 인산화된 JNK(phosphor JNK), ERK(phosphor ERK)및 STAT (phosphor STAT3) 단백질의발현수준도감소되었다.
[181] 상기결과를통해염증성장질환의경우, GSTT1단백질의이량체가낮은
수준으로존재하여, STAT3와 p38/JNK의인산화가정상적인수준으로일어나지 않음으로써점막장벽의면역반응이정상적으로유도되지않음을알수있다. 따라서,도 9에서보는바와같이, IL-22단백질및 GSTT1단백질중어느하나 이상을처리하는경우 p38및 STAT3세포신호전달에의해점막면역반응이 유도되는것을알수있다.
[182] 이상으로본발명의특정한부분을상세히기술하였는바,당업계의통상의 지식을가진자에게있어서이러한구체적인기술은단지바람직한구현예일 뿐이며,이에본발명의범위가제한되는것이아닌점은명백하다.따라서,본 발명의실질적인범위는첨부된청구항과그의등가물에의하여정의된다고할 것이다.
산업상이용가능성
[183] 본발명은부작용이존재하지않거나,적으면서염증성장질환의치료가
가능한 GSTT 1 (glutathione S-transferases theta 1)단백질또는이를암호화하는 유전자를유효성분으로포함하는조성물에관한것으로서, STAT3와 p38/JNK의 인산화를통해점막장벽의면역반응을유도함으로써,염증성장질환의 예방 또는치료에매우효과적으로사용될수있다.나아가,상기세포신호전달을 조절하는 GSTT1단백질의이량체가낮은수준으로존재하는지여부를 측정함으로써염증성장질환을매우효과적으로예측할수있다.
서열목록 Free Text
[184] 서열번호 1:
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[185] 서열번호 2:
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[186] 서열번호 3:
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[188]
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[189] 서열번호
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[191] 서열번호
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[193] 서열번호 10:
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[195] 서열번호 12:
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[196] 서열번호
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[197] 서열번호 14:
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[203] 서열번호 20:
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Claims

2020/213913 1»（：1/10公020/005011 청구범위
[청구항 1] GSTTl (glutathione S-transferases theta 1)단백질또는이를암호화하는 유전자를유효성분으로포함하는염증성장질환의 예방또는치료용 약학조성물.
[청구항 2] 제 1항에있어서,
상기약학조성물은 IL-22단백질또는이를암호화하는유전자를더 포함하는것인,약학조성물.
[청구항 3] 제 1항에있어서,
상기염증성장질환은궤양성대장염,크론병 (Crohn’s disease),장관형 베체트병,출혈성직장궤양및회장낭염 (pouchitis)으로이루어진 군으로부터선택되는적어도하나인것인,약학조성물.
[청구항 4] 제 1항에있어서,
상기 GSTT1단백질은서열번호 1로표시되는아미노산서열로이루어진 것인,약학조성물.
[청구항 5] GSTT1단백질또는이를암호화하는유전자를유효성분으로포함하는 염증성장질환의예방또는개선용식품조성물.
[청구항 6] 제 5항에있어서,
상기식품조성물은 IL-22단백질또는이를암호화하는유전자를더 포함하는것인,식품조성물.
[청구항 7] 염증성장질환환자로부터분리된생물학적시료에목적하는후보물질을 처리하는단계 ;및
상기생물학적시료에서 GSTT1단백질또는이를암호화하는유전자의 발현수준을측정하는단계를포함하는,염증성장질환의치료제를 스크리닝하는방법 .
[청구항 8] 제 7항에있어서,
상기후보물질의처리후에상기생물학적시료에서측정된 GSTT1 단백질또는이를암호화하는유전자의발현수준이,상기염증성장질환 환자로부터분리된생물학적시료에서측정된 GSTT1단백질또는이를 암호화하는유전자의발현수준에비하여증가된경우,상기후보물질을 염증성장질환의치료제로선별하는단계를더포함하는,염증성 장질환의치료제를스크리닝하는방법 .
[청구항 9] GSTTl (glutathione S-transferases theta 1)단백질또는이를암호화하는 유전자를유효성분으로포함하는술잔세포 (Goblet cell)의증식또는분화 촉진용조성물.
[청구항 ] GSTTl단백질의이량체가존재하는수준을측정하는제제를포함하는, 염증성장질환의진단용조성물.
[청구항 11] 제 W항에있어서, 상기단백질의이량체가존재하는수준을측정하는제제는상기
단백질에특이적으로결합하는항체또는앱타머인것인,염증성 장질환의진단용조성물.
[청구항 12] 제 W항에있어서,
상기염증성장질환은궤양성대장염,크론병,장관형베체트병,출혈성 직장궤양및회장낭염으로이루어진군으로부터선택되는적어도 하나인것인,염증성장질환의진단용조성물.
[청구항 13] 제 W항내지제 12항중어느한항의조성물을포함하는,염증성
장질환의 예후예측용키트.
[청구항 14] 목적하는개체로부터분리된생물학적시료에서,
GSTT1단백질의이량체가존재하는수준을측정하는단계를포함하는, 염증성장질환을진단하기위한정보를제공하는방법.
[청구항 15] 제 U항에있어서,
상기목적하는개체로부터분리된생물학적시료에서측정된 GSTT1 단백질의이량체가존재하는수준이정상대조군에비하여낮은경우, 상기목적하는개체가염증성장질환일것으로예측하는것인,염증성 장질환을진단하기위한정보를제공하는방법.
[청구항 16] 제 U항에있어서,
상기단백질이존재하는수준을측정하는단계는단백질의비-환원 조건에서수행되는것인,염증성장질환을진단하기위한정보를 제공하는방법 .
[청구항 17] 투여가필요한대상체에게 GSTH (glutathione S-transferases theta 1)
단백질또는이를암호화하는유전자를약학적으로유효한양으로 투여하는단계를포함하는염증성장질환의 예방또는치료방법 .
PCT/KR2020/005011 2019-04-16 2020-04-14 Gstt1 단백질 또는 이를 암호화하는 유전자를 유효성분으로 포함하는 조성물 Ceased WO2020213913A1 (ko)

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