KR102256159B1 - C omposition for preventing and treating autoimmune diseases comprising Gastrokine 1 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating autoimmune diseases containing GKN 1 as an active ingredient, and more specifically, an expression vector containing GKN 1 or an autoimmune disease containing GKN 1 protein as an active ingredient. It relates to a composition for prevention or treatment. GKN 1 according to the present invention has been shown to improve the symptoms of autoimmune disease when the protein is overexpressed in an animal model with autoimmune disease. In particular, it can effectively inhibit osteoclast differentiation and thus osteoarthritis and rheumatism. It has an effect that can be usefully used in the development of therapeutic agents for autoimmune diseases including arthritis.

Description

Composition for preventing or treating autoimmune diseases comprising GKN1 as an active ingredient {C omposition for preventing and treating autoimmune diseases comprising Gastrokine 1}

The present invention relates to a pharmaceutical composition for preventing or treating autoimmune diseases containing GKN 1.

Immunity is one of the body's self-protection systems against all foreign polymers (antigens) invading or injected into living tissues. The main component of the immune system is lymphocytes, which are made in the bone marrow and circulate to lymphatic tissues or organs along the blood, mainly lymph nodes, spleen, tonsils, etc. They are white blood cells. As a cell involved in the immune response, B cells proliferate rapidly when stimulated by an appropriate antigen to form a clone that produces a special antibody (immunoglobulin) to neutralize the antigen, and the antibodies produced by B cells circulate in body fluids. While performing humoral immunity. In addition, T cells are produced in the thymus and move to lymphoid tissues, which are responsible for cell-mediated immunity that directly attack antigens.

On the other hand, one of the most important characteristics of all normal individuals is that they do not react harmfully to the antigenic substances constituting self, whereas many non-self antigens are recognized and reacted to remove them. It is that they have the ability to have. In this way, the non-response of a living body to an autoantigen is called immunologic unresponsiveness or tolerance.

If there is a problem in inducing or maintaining such self-tolerance, an immune response to the self-antigen occurs, and as a result, a phenomenon that attacks the own tissue occurs, multiple sclerosis, type 1 diabetes, rheumatoid arthritis, Autoimmune diseases such as Hashimoto's thyroiditis occur, and immune rejection reactions occur after procedures such as transplantation.

Specifically, autoimmune diseases are diseases that appear as adverse reactions to their own cells. Currently, immunosuppressants that block the signal transduction pathway in T cells are most often used as therapeutic agents, but these immunosuppressants are toxicity, infection, lymphoma, diabetes, There is a problem that side effects such as tremor, headache, diarrhea, high blood pressure, nausea, and renal dysfunction occur. Therefore, it is necessary to develop a new therapeutic agent that has no side effects and is inexpensive and has excellent therapeutic effects.

Republic of Korea Patent Publication No. 2012-0051258

In this respect, an object of the present invention is to provide a composition for preventing or treating autoimmune diseases comprising a GKN 1 (Gastrokine 1) protein or a polynucleotide encoding a GKN 1 protein.

Another object of the present invention is an autoimmune disease comprising the step of culturing a GKN 1 protein or a recombinant cell expressing the protein and a candidate material, and measuring the effect of the GKN 1 protein on increasing the activity or expression level in the cell It is to provide a method for screening a therapeutic agent.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases comprising a GKN 1 (Gastrokine 1) protein or a polynucleotide encoding a GKN 1 protein.

In one embodiment of the present invention, the GKN 1 protein may be composed of the amino acid sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the polynucleotide encoding the GKN 1 protein may be composed of the nucleotide sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the polynucleotide may be included in an expression vector.

In one embodiment of the present invention, the expression vector may be pCMV-HA-GKN 1.

In one embodiment of the present invention, the autoimmune disease is rheumatoid arthritis, osteoarthritis, Behcet's disease, polymyositis or cutaneous myositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatitis, Goodfighter syndrome, autoimmune meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes, dystrophic bullous epidermolysis, epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, sarcoidosis, scleroderma, spondyloarthrosis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, scleroderma, It may be selected from the group consisting of mitochondrial-related syndrome and inflammatory bowel disease.

In addition, the present invention comprises the steps of treating a candidate substance in cells or tissues containing the GKN 1 gene or the GKN 1 protein; And it provides a screening method for an autoimmune disease treatment comprising the step of measuring the expression amount of the GKN 1 gene or the amount of the GKN 1 protein or the activity of the GKN 1 protein.

In one embodiment of the present invention, when the expression amount of the GKN 1 gene, the amount of GKN 1 protein, or the GKN 1 protein activity is increased compared to the control group not treated with the candidate substance, the candidate substance is used to prevent autoimmune diseases. Alternatively, a step of determining as a therapeutic substance may be further added.

In one embodiment of the present invention, the amount of expression of the GKN 1 gene or the amount of GKN 1 protein or the activity of the GKN 1 protein is determined by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Immunohistochemistry, Western Blotting, and flow cytometry (FACS) can be performed by any one method selected from the group consisting of.

In one embodiment of the present invention, the autoimmune disease is rheumatoid arthritis, osteoarthritis, Behcet's disease, polymyositis or cutaneous myositis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatitis, Goodfighter syndrome, autoimmune meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes, dystrophic bullous epidermolysis, epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, sarcoidosis, scleroderma, spondyloarthrosis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, scleroderma, It may be selected from the group consisting of mitochondrial-related syndrome and inflammatory bowel disease.

GKN 1 according to the present invention has been shown to improve the symptoms of autoimmune disease when the protein is overexpressed in an animal model with autoimmune disease. In particular, it can effectively inhibit osteoclast differentiation and thus osteoarthritis and rheumatism. It has an effect that can be usefully used in the development of therapeutic agents for autoimmune diseases including arthritis.

1 shows a schematic diagram of a recombinant expression vector prepared by introducing the GKN1 gene.
2 is a result of confirming the therapeutic effect of rheumatoid arthritis according to overexpression of GKN1 in a rheumatoid arthritis-induced mouse model, FIG. 2a is a result of measuring rheumatoid arthritis index, 2b is a result of measuring the amount of IgG2a in serum, 2c is It shows the results of analyzing whether or not intracellular overexpression by the recombinant expression vector of the present invention.
3 is a result of observing the degree of inhibition of osteoclast differentiation by overexpression of GKN1 in bone marrow cells isolated from rheumatoid arthritis-induced mice, 3a is a photograph observed by staining Trap-positive cells, and 3b is a count of the number of Trap-positive cells. This is the result of analysis.
FIG. 4 shows the results of analyzing the effect of overexpression of GKN1 on the expression of osteoclast differentiation-related factors in bone marrow cells isolated from rheumatoid arthritis-induced mice.

The present invention is characterized by providing a composition for preventing or treating autoimmune diseases containing GKN 1 (Gastrokine 1) as an active ingredient, and more specifically, GKN 1 (Gastrokine 1) protein or GKN 1 (Gastrokine 1). It is characterized by providing a composition for preventing or treating autoimmune diseases comprising a polynucleotide encoding a protein.

While researching a new therapeutic agent capable of treating autoimmune diseases, the present inventors paid attention to GKN 1.

GKN 1 (Gastrokine 1), also known as AMP-18 (antrum mucosal protein-18) or CA11, is known to be expressed in the antrum, protects the vestibular mucosa, and facilitates recovery and proliferation after wounding. It has been known to have an activity promoting healing (Toback. FG et al ., Am J Physiol Gastrointest Liver Physiol, 285: G344-353, 2003). However, little is known about the association of GKN 1 with autoimmune diseases.

Accordingly, the present inventors first identified the use of GKN 1 as a therapeutic agent for autoimmune diseases by confirming that the symptoms of the disease are improved when GKN 1 is overexpressed in an animal model in which an autoimmune disease is induced.

Specifically, according to an embodiment of the present invention, as a result of injecting a recombinant vector containing GKN 1 into a rheumatoid arthritis mouse model, one of autoimmune diseases, the arthritis index decreased compared to the group not injected with GKN 1 It was found to be (see Fig. 2).

In addition, according to another embodiment, after obtaining bone marrow cells from rheumatoid arthritis mice overexpressing GKN 1 and rheumatoid arthritis mice not injected with GKN 1, after culturing the cells under conditions capable of differentiating into osteoclasts, As a result of analyzing the degree of differentiation into osteoclasts, it was found that when GKN 1 was overexpressed, differentiation into osteoclasts was inhibited compared to the group to which GKN 1 was not injected (see FIG. 3), and this cause was GKN 1 These osteoclast differentiation factors were found to remarkably inhibit the expression of TRAP, MMP-1, Itgb3, DC-Stamp, and carcitonin receptors (see FIG. 4).

Therefore, the present inventors found that GKN 1 has excellent activity to inhibit the differentiation of osteoclasts, and thus can be used as a therapeutic agent for autoimmune diseases related to osteoclast differentiation.

Therefore, the present invention is characterized by providing the use of GKN 1 as a therapeutic agent for autoimmune diseases, and specifically, prevention of autoimmune diseases including a polynucleotide encoding GKN 1 (Gastrokine 1) protein or GKN 1 (Gastrokine 1) protein. Or it can provide a pharmaceutical composition for treatment.

Preferably, the GKN 1 protein may have an amino acid sequence described in SEQ ID NO: 2, and the polynucleotide encoding the GKN 1 protein may have a nucleotide sequence described in SEQ ID NO: 1.

In addition, the GKN 1 protein according to the present invention may preferably be a functional equivalent to a polypeptide having the amino acid sequence of SEQ ID NO: 2. The'functional equivalent' is a result of addition, substitution or deletion of amino acids, having at least 60%, preferably 70%, more preferably 80% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 2 It refers to a polypeptide exhibiting substantially the same activity as GKN 1 of the present invention. Here, "substantially homogeneous activity" means the activity of GKN 1 described above. The functional equivalents may include, for example, amino acid sequence variants in which some of the amino acids in the amino acid sequence of GKN 1 according to the present invention are substituted, deleted or added. Substitution of amino acids may preferably be conservative substitutions, and examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid may preferably be located at a portion that is not directly involved in the activity of GKN 1 of the present invention. In addition, the functional equivalent range may include a polypeptide derivative in which some of the chemical structures of the polypeptide have been modified while maintaining the basic backbone of GKN 1 and its physiological activity. For example, while maintaining the structural change and physiological activity to change the stability, storage, volatility or solubility of the polypeptide of the present invention, it may include a fusion protein made by fusion with another 　 protein.

In addition, the polynucleotide encoding the GKN 1 (Gastrokine 1) protein is introduced into an expression vector such as a plasmid or a viral vector by a known method, and then the expression vector is transduced by various methods known in the art or It can be introduced into a target cell as an expression type by transfection.

The plasmid expression vector is an FDA approved gene delivery method that can be used in humans, and is a method of delivering plasmid DNA directly to human cells (Nabel, EG et al ., Science, 249:1285-1288, 1990), unlike viral vectors, plasmid DNA has the advantage of being able to be homogeneously purified. As a plasmid expression vector that can be used in the present invention, a mammalian expression plasmid known in the art can be used, and in an embodiment of the present invention, a cleavage map of Fig. 1 in which the GKN 1 gene is inserted using a pCMV-HA vector A recombinant expression vector having pCMV-HA-GKN 1 was prepared.

The plasmid expression vector containing the nucleic acid according to the present invention is a method known in the art, such as, but not limited to, transient transfection, microinjection, transduction, Cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection , Electroporation, gene gun, and other known methods for introducing DNA into cells can be introduced into target cells (Wu. et al. al ., J. Bio. Chem., 267:963-967, 1992; Wu. et al ., Bio. Chem., 263:14621-14624, 1988).

In addition, the vector capable of expressing GKN 1 may be administered to cells, tissues or the body by a known method, for example, topically, parenteral, oral, nasal, intravenous, intramuscular, subcutaneous or It can be administered by other suitable means. In particular, the vector can be directly injected in an effective amount for treating a target tissue or target cell.

Furthermore, the present invention can provide a method for screening a therapeutic agent for autoimmune diseases using GKN 1.

That is, the screening method for an autoimmune disease therapeutic agent according to the present invention comprises the steps of treating a candidate substance to cells or tissues containing the GKN 1 gene or the GKN 1 protein; And measuring the expression amount of the GKN 1 gene or the amount of the GKN 1 protein or the activity of the GKN 1 protein.

The method according to the present invention is a method for discovering a new therapeutic agent for autoimmune diseases using GKN 1, and the “candidate material” refers to the expression amount of GKN 1 gene, the amount of GKN 1 protein, or the activity of GKN 1 protein. It refers to an unknown substance that is used in screening to check whether it has an effect on. The sample may include a compound, protein, peptide, oligonucleotide, or natural product extract, but is not limited thereto.

The treatment of the candidate substance may be performed by culturing after treating the candidate substance to cells or tissues containing the GKN 1 gene or GKN 1 protein.

In addition, the meaning of the increase in the intracellular level of the GKN 1 protein in the above means that the expression of the GKN 1 gene is increased or the degradation of the GKN 1 protein is suppressed, thereby increasing the amount of the GKN 1 protein. The GKN 1 gene expression includes transcription of the GKN 1 gene and translation into a protein. Therefore, when the candidate substance increases the expression of the GKN 1 gene and the level of the protein in the cell, the candidate substance can be determined as a new therapeutic agent capable of treating autoimmune diseases.

Measurement of the expression level of the gene is preferably to measure the level of mRNA, and as a method of measuring the level of mRNA, reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern Blots and DNA chips, and the like, but are not limited thereto.

In addition, an antibody may be used to measure the amount of the protein or the activity of the protein. In this case, the marker protein in the biological sample and the antibody specific thereto form a conjugate, that is, an antigen-antibody complex, and an antigen-antibody complex The amount of formation of can be quantitatively measured through the magnitude of the signal of the detection label. The detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but is not limited thereto. Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radiation immunity diffusion method, Ouchteroni immunity diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement fixation assay, FACS, protein chip, etc.

As described above, the method for measuring the expression amount of the GKN 1 gene or the amount of GKN 1 protein or the activity of the GKN 1 protein may be performed using various methods known in the art, preferably reverse transcription polymerase chain Reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, coimmunoprecipitation, enzyme-linked immunosorbentassay, radioimmunoassay (RIA), immunohistochemistry, western blotting, and Flow cytometry (FACS) can be used.

In addition, the screening method targeting GKN 1 of the present invention can apply high throughput screening (HTS). HTS is a method of simultaneously or almost simultaneously screening for the biological activity of multiple candidates by testing multiple candidates in parallel. In a specific embodiment, the cell line may be cultured in a 96-well microtiter plate or a 192-well microtiter plate, treated with a plurality of candidate substances, and then the level of expression of GKN 1 may be measured by an immunochemical method. These wells typically require assay volumes ranging from 50 μl to 500 μl, and in addition to plates, a number of instruments, instruments, pipets, robots, and more to make the 96-well format suitable for a wide range of homogeneous and heterogeneous assays. Plate washers and plate readers are commercially available.

In addition, the composition according to the present invention may be used as a pharmaceutical composition capable of preventing and treating autoimmune diseases, and the pharmaceutical composition may further include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause an allergic reaction such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, a carrier for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like, and a carrier for parenteral administration such as water, suitable oil, saline, aqueous glucose and glycol. And the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral administration forms according to known methods, and as a representative formulation for parenteral administration, an isotonic aqueous solution or suspension is preferable as a dosage form for injection. Formulations for injection can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be formulated for injection by dissolving it in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules.

The pharmaceutical composition formulated by the above method may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular. In the above, "effective amount" refers to an amount that exhibits a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention can be appropriately selected according to the route of administration, the subject of administration, age, sex, weight, individual differences, and disease conditions. Preferably, the pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the degree of disease, but preferably 1 to 10000 ㎍ / weight kg / day, more preferably 10 to 1000 mg / body weight kg It can be administered repeatedly several times a day with an effective dose of /day. In addition, the composition of the present invention may be administered in parallel with a known compound having an effect of preventing, improving or treating autoimmune diseases.

In addition, autoimmune diseases that can be treated and prevented with the composition containing GKN1 of the present invention are not limited thereto, but rheumatoid arthritis, osteoarthritis, Behcet's disease, multiple myositis or skin myositis, autoimmune cytopenia, autoimmune myocarditis, Atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Goodfighter syndrome, autoimmune meningitis, Sjogren syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin Dependent diabetes, dystrophic bullous epidermolysis, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, sarcoidosis, skin sclerosis, spondyloarthrosis, thyroiditis, vasculitis , Vitiligo, myxedema, pernicious anemia, scleroderma, mitochondrial-related syndrome and inflammatory bowel disease.

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

< Example 1>

GKN1 Recombinant Vector Introduced

To prepare a vector capable of overexpressing GKN1, a recombinant vector was prepared as follows. First, the mouse GKN1 cDNA was amplified using a PCR method, and a pCMV-HA-mGKN 1 recombinant vector having a cleavage map of FIG. 1 was prepared using a restriction enzyme site in a pCMV-HA (Addgene ID 32530) vector. More specifically, pCMV-HA vector and GKNI DNA were treated with EcoRI and XhoI restriction enzymes, and then pCMV-HA and GKNI were ligated to introduce the GKNI gene into the vector. Subsequently, colonies formed on an agar plate by transformation into E. coli were taken, cultured to extract DNA, and then sequencing was performed to confirm whether the GKNI gene was correctly introduced.

< Example 2>

Rheumatism In animal models of arthritis GKN1 Analysis of treatment effect by overexpression of

In order to confirm whether GKN1 can treat autoimmune diseases, the present inventors prepared a mouse model in which rheumatoid arthritis was induced, and then analyzed the severity of the disease by overexpressing GKN1 in the disease model.

First, a mouse model induced rheumatoid arthritis was prepared for this purpose, and arthritis was induced by subcutaneous injection of type 2 collagen into DBA1/J mice (intra-dermal), and GKN1 overexpression vector (pCMV-HA) 8 days after arthritis induction -mGKN 1) was injected into the mouse through a hydrodynamic injection method. The hydrodynamic injection method is a method of introducing a gene through the cell membrane and into the cytoplasm using physical force. The pCMV-HA-mGKN 1 vector diluted in physiological saline is injected into the tail vein. In order to promote disease (Booster), type 2 collagen was mixed with an adjuvant (IFA) in a volume ratio of 1:1 and injected into the tail. Two days later, an overexpression vector was injected into the right thigh. One week later, the overexpression vector was again in the left thigh. Was injected. At this time, as a control group, a group injected with only the pCMV-HA vector that did not contain the mGKN 1 gene was used.

As a result of the analysis, as shown in FIG. 2, the group injected with the GKN1 overexpression vector to the group of rheumatoid arthritis-induced mice showed an effective decrease in the CIA arthritis index compared to the control group, and thus GKN1 has an activity capable of treating arthritis. It can be seen that there is (see Fig. 1a).

In addition, after separating the serum from the blood of the mice to be tested, the amount of IgG2a contained in the serum was measured using the ELISA method.As a result, the group in which GKN1 was overexpressed in the arthritis-induced mice was compared with the control group. It was found that the expression was reduced (see Fig. 1b). Further, for reference, it was confirmed whether the GKNI overexpression vector produced in the present invention was overexpressed when introduced into the actual cells.As a result, as shown in FIG.1C, it was found that the expression of GKNI was increased when the overexpression vector was introduced compared to the control group.

< Example 3>

GKN1 Analysis of osteoclast differentiation inhibitory effect of

The present inventors performed the following experiment to confirm whether GKN1 affects the differentiation of osteoclasts that are closely related to arthritis. After separating the bone marrow from the mouse experimental group and control group used in Example 2, the isolated cells were ^{plated as 2×10 5} cells, and treated with 10 ng/ml of a macrophage-colony stimulating factor (M-CSF). Thus, the cells in each well were cultured for 3 days, the supernatant was removed, washed with phosphate buffer, and then replaced with a new medium. Each well was treated with 10 ng/ml of M-CSF and 50 ng/ml of RANKL (receptor activator of NF-κB ligand), incubated for 2 days, and then stained with TRAP to observe the degree of differentiation of osteoclasts.

As a result of the analysis, as shown in FIG. 3, it was found that the number of osteoclasts expressing TRAP was significantly smaller in the cells overexpressing GKN1 compared to the group in which GKN1 was not overexpressed, whereby GKN1 could inhibit the differentiation of osteoclasts. Was confirmed.

< Example 4>

GKN1 Analysis of Influence on Factors Related to Inhibition of Osteoblast Differentiation

The present inventors were able to confirm that GKN1 has the activity of inhibiting the differentiation of osteoclasts through Example 3, and further, by controlling the expression level of factors related to osteoclast differentiation with an action capable of causing such an effect. The following experiment was performed to confirm whether it was.

That is, the expression levels of the osteoclast differentiation-related factors TRAP, MMP-9, ITGB3 (Integrin beta-3), DC-Stamp and Carcitonin receptor were analyzed for the cells performed in Example 3, and the differentiation of osteoclasts was analyzed. After confirming, RNA was extracted and cDNA was synthesized with a cDNA kit (transcriptor first strand cDNA synthesis kit). Real-time RCR used SYBR Green I (Roche Diagnostic) as an intercalator, and analyzed using a Light-Cycler instrument (Roche Diagnostic). Then, the correction value was calculated from the CT value of the gene and the expression level of the housekeeping gene, and then the difference in the expression level was compared with the control group to calculate a relative value.

Primer sequence used primer Primer sequence TRAP S CC TGG CTC AAA AAG CAG TT TRAP AS ACA TAG CCC ACA CCG TTC TC MMP-9S CTG TCC AGA CCA AGG GTA CAG CCT MMP-9 AS GAG GTA TAG TGG GAC ACA TAG TGG Itgb3 S CTG TGG GCT TTA AGG ACA GC Itgb3 AS GAG GGT CGG TAA TCC TCC TC DC-stamp S GCA GTG AAT GCG CTG AAC AT DC-stamp AS AGG CAT TCC GTC TGC TTT GA Carcitonin receptor S CGG ACT TTG ACA CAG CAG AA Carcitonin receptor AS AGC AGC AAT CGA CAA GGA GT

As a result of the analysis, as shown in FIG. 4, as a result of comparing the group with overexpression of GKN1 and the group without overexpression of the bone marrow cells of rheumatoid arthritis mice, the overexpression group suppresses the expression level of factors related to the differentiation of osteoclasts. Appeared to be. Therefore, through these results, it was found that GKN1 can suppress the expression of osteoclast differentiation factor at the mRNA level and ultimately improve and treat arthritis through inhibition of osteoclast differentiation.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Catholic University <120> C omposition for preventing and treating autoimmune diseases comprising Gastrokine 1 <130> PN1410-328 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 555 <212> DNA <213> GKN 1 cDNA sequence <400> 1 atgaagctca caatgttcgt cgtgggtctg cttggcctcc ttgcagctcc tggttttgct 60 tacacggtca acatcaatgg taatgatggc aatgtagacg gaagtggaca gcagtcggtg 120 agcatcaatg gtgtgcacaa cgtggccaat atcgacaaca ataacggctg ggactcctgg 180 aatagcctct gggactatga aaacagtttc gctgccacga gactcttctc caagaagtca 240 tgcattgtgc acagaatgaa caaggatgcc atgccctccc ttcaggacct cgatacaatg 300 gtcaaggaac agaagggtaa agggcctgga ggagctcctc ccaaggactt gatgtactcc 360 gtcaacccta ccagagtgga ggacctgaat acattcggac caaagattgc tggcatgtgc 420 aggggcatcc ctacctatgt ggccgaggag attccaggac caaaccagcc tttgtactca 480 aagaagtgct acacagctga catactctgg attctgcgga tgtccttctg tggaacatca 540 gtggagacat actag 555 <210> 2 <211> 184 <212> PRT <213> GKN 1 aminoacid sequence <400> 2 Met Lys Leu Thr Met Phe Val Val Gly Leu Leu Gly Leu Leu Ala Ala 1 5 10 15 Pro Gly Phe Ala Tyr Thr Val Asn Ile Asn Gly Asn Asp Gly Asn Val 20 25 30 Asp Gly Ser Gly Gln Gln Ser Val Ser Ile Asn Gly Val His Asn Val 35 40 45 Ala Asn Ile Asp Asn Asn Asn Gly Trp Asp Ser Trp Asn Ser Leu Trp 50 55 60 Asp Tyr Glu Asn Ser Phe Ala Ala Thr Arg Leu Phe Ser Lys Lys Ser 65 70 75 80 Cys Ile Val His Arg Met Asn Lys Asp Ala Met Pro Ser Leu Gln Asp 85 90 95 Leu Asp Thr Met Val Lys Glu Gln Lys Gly Lys Gly Pro Gly Gly Ala 100 105 110 Pro Pro Lys Asp Leu Met Tyr Ser Val Asn Pro Thr Arg Val Glu Asp 115 120 125 Leu Asn Thr Phe Gly Pro Lys Ile Ala Gly Met Cys Arg Gly Ile Pro 130 135 140 Thr Tyr Val Ala Glu Glu Ile Pro Gly Pro Asn Gln Pro Leu Tyr Ser 145 150 155 160 Lys Lys Cys Tyr Thr Ala Asp Ile Leu Trp Ile Leu Arg Met Ser Phe 165 170 175 Cys Gly Thr Ser Val Glu Thr Tyr 180

Claims (10)

GKN 1 (Gastrokine 1) protein or a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis comprising a polynucleotide encoding the GKN 1 protein. The method of claim 1,
The GKN 1 protein composition, characterized in that consisting of the amino acid sequence of SEQ ID NO: 2. The method of claim 1,
The composition, characterized in that the polynucleotide encoding the GKN 1 protein consists of the nucleotide sequence of SEQ ID NO: 1. The method of claim 1,
The composition, characterized in that the polynucleotide is contained in the expression vector. The method of claim 4,
The composition, characterized in that the expression vector is pCMV-HA-GKN 1. delete Treating a candidate material to cells or tissues containing the GKN 1 gene or the GKN 1 protein; And
A method for screening a therapeutic agent for rheumatoid arthritis, comprising measuring the amount of expression of the GKN 1 gene or the amount of the GKN 1 protein or the activity of the GKN 1 protein. The method of claim 7,
When the expression amount of the GKN 1 gene, the amount of GKN 1 protein, or the GKN 1 protein activity is increased compared to the control group not treated with the candidate substance, determining the candidate substance as a substance for preventing or treating rheumatoid arthritis. Screening method, characterized in that to. The method of claim 8,
The amount of expression of the GKN 1 gene or the amount of GKN 1 protein or the activity of the GKN 1 protein is determined by reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, immunoprecipitation, enzyme immunoassay (enzyme). -linked immunosorbentassay), radioimmunoassay (RIA), immunohistochemistry, Western blotting (Western Blotting) and flow cytometry (FACS), characterized in that performed by any one method selected from the group consisting of. delete

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