KR101950035B1 - R12 -PIAS3 Fusion peptide having cell penetration for preventing or treating immune disease and pharmaceutical composition comprising thereof - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a cell permeable R12-PIAS3 fusion peptide and a composition for preventing or treating an immunological disease containing the same as an active ingredient. More particularly, the present invention relates to a composition comprising 12 consecutive arginine (R) A recombinant peptide fused with PIAS3, a vector containing a base sequence encoding the peptide, and a composition for preventing or treating an immunological disease comprising the peptide. The R12-PIAS3 fusion peptide according to the present invention is excellent in permeability into target cells, and is superior in the effect of inhibiting the differentiation of cytotoxic Th17 cells that produce and secretes inflammatory cytokines, compared with the use of PIAS3 alone, Immunoregulatory T cells (Tregs), which have the property of inhibiting the function of immune cells and controlling the inflammatory response, are highly effective in activating the immune cells, and thus can be used for the treatment of autoimmune diseases, inflammatory diseases and graft rejection diseases And can be usefully used as a pharmaceutical composition or immunosuppressive agent capable of preventing or treating immune diseases.

Description

(R12-PIAS3 Fusion Peptide Having Cell Penetration for Preventing or Treating Immune Disease and Pharmaceutical Composition Containing the R12-PIAS3 Fusion Peptide Having Cell Permeability and an Active Ingredient)

TECHNICAL FIELD The present invention relates to a fusion peptide of R12 and PIAS3 having cell permeability capable of improving the therapeutic effect of an immunological disease, and a composition for preventing or treating immunological diseases containing the peptide as an active ingredient.

T cells are one of the cell groups that plays a central role in the immune system as a bio-defense system for various pathogens. T cells are produced in the thymus of the human body, and undergo a series of differentiation processes, resulting in differentiation into T cells with inherent characteristics. The differentiated T cells are largely classified into type 1 (Th1) Auxiliary cells (Th2). Among these, Th1 cells are mainly involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations maintain the balance of the immune system through mutual checks to prevent mutual activation with each other.

Therefore, most of the immune diseases are caused by the imbalance between these two immune cells. For example, when the activity of Th1 cells abnormally increases, an autoimmune disease may occur and the activity of Th2 cells abnormally increases It is known that immune diseases are caused by hypersensitivity reactions.

On the other hand, recent studies on the differentiation of Th1 cells have revealed the existence of a new group of immunoregulatory T cells (Tregs) capable of regulating the activity of Th1 cells, , Treg cells have the property of suppressing the function of abnormally activated immune cells and controlling the inflammatory reaction, and there have been reported many experiments for treating immune diseases through the action of increasing the activity of Treg cells.

In addition, Th17 cells are known to be formed through a process similar to the differentiation of Treg cells in the differentiation process of undifferentiated T cells. That is, the differentiation of Treg cells and Th17 cells is commonly performed in the presence of TGF-β, whereas IL-6 is not required for Treg cells, while IL-6 is present along with TGF-β for Th17 cells Differentiate in situations. In addition, differentiated Th17 cells are characterized by secretion of IL-17.

Th17 cells, unlike Treg cells, are involved in the forefront of the inflammatory response seen in immune diseases, thereby maximizing the signal of the inflammatory response and accelerating the progression of the disease. Therefore, in the case of autoimmune diseases that are not controlled by Treg cells among autoimmune diseases, the development of therapeutic agents for autoimmune diseases targeting the inhibition of Th17 cell activation has been greatly emphasized.

Immunosuppressants that block the signal transduction pathway in T cells are the most commonly used therapeutic agents for immune diseases. Such immunosuppressants are toxic, infectious, lymphoid, diabetes, tremor, headache, diarrhea, hypertension, nausea , And a disorder of renal function.

In addition to the method of treating immune diseases through the method of inhibiting the activation of T cells, a method of regulating the amount of cytokine secreted from the immune cells and a method of using the antibody targeting the cytokine secreted from the immune cells have been developed . However, this method has a problem in that it takes much time to actually apply to patients through clinical experiments, and that the method using antibody is too expensive in the process of antibody production.

Therefore, there is a need to develop a novel therapeutic agent for immune diseases which has no side effects and is inexpensive and has a therapeutic effect.

Accordingly, an object of the present invention is to provide a recombinant peptide in which 12 consecutive arginine (R) fused with PIAS3 has cell permeability capable of effectively delivering a therapeutic agent for the treatment of immune diseases caused by abnormality of immune response .

Another object of the present invention is to provide a vector comprising a nucleotide sequence encoding a peptide according to the present invention.

It is another object of the present invention to provide a composition for preventing or treating an immunological disease comprising a peptide according to the present invention.

In order to achieve the above object, the present invention provides a recombinant peptide in which 12 consecutive arginines (R) having cell permeability are fused with PIAS3.

In one embodiment of the present invention, the recombinant peptide may have the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a vector comprising the nucleotide sequence encoding the peptide according to the present invention.

In one embodiment of the present invention, the nucleotide sequence may be the nucleotide sequence of SEQ ID NO: 2.

The present invention also provides a composition for preventing or treating an immunological disease comprising the peptide according to the present invention.

In one embodiment of the present invention, the peptide inhibits the production of IL-17, an inflammatory cytokine, promotes the activity of immunoregulatory T cells (Tregs), inhibits the invasion and proliferation of inflammatory cells Lt; / RTI >

In one embodiment of the invention, the immune disease is autoimmune disease; Inflammatory disease; And transplantation rejection diseases of cells, tissues or organs.

In one embodiment of the invention, the autoimmune or inflammatory disease is selected from the group consisting of autoimmune hepatitis, chronic rheumatoid arthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, scleroderma, , Multiple myositis / dermatomyositis, Hashimoto's disease, autoimmune cytopenias, Sjogren's syndrome, vasculitis syndrome and systemic lupus erythematosus.

The R12-PIAS3 fusion peptide according to the present invention is excellent in permeability into target cells, and is superior in the effect of inhibiting the differentiation of cytotoxic Th17 cells that produce and secretes inflammatory cytokines, compared with the use of PIAS3 alone, Immunoregulatory T cells (Tregs), which have the property of inhibiting the function of immune cells and controlling the inflammatory response, are highly effective in activating the immune cells, and thus can be used for the treatment of autoimmune diseases, inflammatory diseases and graft rejection diseases And can be usefully used as a pharmaceutical composition or immunosuppressive agent capable of preventing or treating immune diseases.

FIG. 1 is a schematic diagram of a pcDNA3.1 + PIAS3 vector and a pcDNA3.1 + R12 + PIAS3 vector prepared in an embodiment of the present invention.
2 is a schematic diagram of the pET15b / 6xHis + PIAS3 vector and pET15b / 6xHis + R12 + PIAS3 vector prepared in one embodiment of the present invention.

In order to enhance the therapeutic effect of PIAS3 protein which can prevent or treat immunological diseases through immunomodulating action, 12 arginines having excellent cell permeability and 12 consecutive arginines (R) having cell permeability fused with PIAS3 and PIAS3 And is characterized by providing a fused recombinant peptide.

The present inventors have focused on the PIAS3 gene to develop a novel immunomodulator for the treatment of immune diseases. The protein inhibitor of activated STAT3 (PIAS3) regulates the activity of the activated STAT (signal transducer and activator of transcription protein) It is known to regulate many transcription factors such as STAT, NF-kB, SMAD, and p53. PIAS has been shown to interfere with 1) the binding of transcription factors to DNA, 2) to inhibit transcriptional activity and to interact with facilitating cofactors, or 3) to promote protein sumoylation And transcription ability changes when the transcription factor is thermogenized). Recent studies have shown that PIAS plays an important role in the regulation of immune response.

STAT (signal transducers and activators of transcription) is a signal transduction and transcriptional regulatory protein that is activated by extracellular stimuli such as cytokines, hormones and growth factors to phosphorylate tyrosine residues, (dimer) is formed to enter the nucleus and bind to a specific promoter. This STAT protein signaling system can be inhibited by dephosphorylation and protein degradation.

When initial STAT3-related factors were discussed, PIAS3 and SOCS3 (suppressor of cytokine signaling 3) were suggested as STAT3 inhibitory feedback. SOCS protein inhibits JAKs and inhibits ligand-induced responses, PIAS protein is known to have an activity of inhibiting phosphorylation of STAT3 (Starr R, Hilton DJ. Negative regulation of the JAK / STAT pathway. Bioessays 1999; 21: 4752).

Recently, STAT3, STAT3, and STAT5 have been found to be active forms in various carcinomas. STAT3 is not only a blood cancer such as leukemia, but also breast cancer, head and neck cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer It is activated in a variety of solid tumors and has become an important cancer target (Hua Yu and Richard Jove, Nature Review Cancer ., 2004, 8, 945).

In addition, the activity of STAT3 has been known to inhibit apoptosis, induce angiogenesis, and induce immune evasion (Wang T. et al., Nature Medicine ., 2004, 10, 48). Therefore, inhibition of STAT3 activity has an effect of controlling the tumor by a complex anticancer mechanism. STAT3 protein is involved not only in tumor but also various cellular functions, so that its inhibitor can be developed as an immunosuppressant.

In general, the immune system controls a specific immune response to an autoantigen in a normal state, and also suppresses an immune response to an external antigen. For example, a response to a fetus of a pregnant woman and immunization against a microorganism in a chronic infection state Reaction. These phenomena are known to be induced by clonal deletion, clonal anergy, and active control by immunoregulatory T cells (Treg) as a mechanism by which antigen-specific immune tolerance can be induced. Some of the patients who have been successfully obtained immunostimulants for the transplantation antigen or those who have experimentally induced immune tolerance have been confirmed to be involved in the transplantation immunity tolerance in all three mechanisms. In particular, recently, immunocontrol T lymphocytes Has been attracting attention as an important cell involved in controlling virtually all immune responses in living bodies such as autoimmune, tumor immunity, and infectious immune response as well as transplantation immune response.

In particular, immunoregulatory T lymphocytes (Tregs), which have recently been discovered, can be divided into natural Treg and adaptive Treg cells, and CD4 + CD25 + T cells, which are natural Tregs, Is immunosuppressed from the time when it is newly produced in the thymus, and is present in a frequency of 5 to 10% of the peripheral CD4 + T lymphocytes of normal individuals. Although the immunosuppressive mechanism of this cell has not yet been elucidated yet, it has recently been shown that the expression control gene of Foxp3 plays an important role in the differentiation and activity of this cell. In addition, peripheral T cells can be differentiated into cells exhibiting an immunosuppressive effect upon being stimulated by a self or external antigen under a specific environment, which is called an adaptive or inducible Treg and secretes IL-10 Tr1 that secretes TGF-beta, Th3 and CD8 Ts that secrete TGF-beta, and the like.

In addition, as described in the prior art, T cells are differentiated into Th17 cells through differentiation of Treg cells. Th17 cells are commonly found in the presence of TGF-β in the presence of Treg cells, but in the case of Treg cells, IL -6, whereas Th17 cells are differentiated in the presence of TGF-beta and IL-6, and secrete IL-17.

In addition, Th17 cells have cytotoxic properties that maximize signal of inflammatory response and accelerate disease progression. Therefore, inhibition of differentiation or activity into Th17 cells is one of the methods for treating immune diseases.

Accordingly, the present inventors have confirmed that PIAS3 is effective in treating immune diseases by inhibiting the differentiation of T cells into Th17 cells, and that expression of Foxp3 is increased in regulatory T cells (Treg) , Demonstrating that PIAS3 can be used to activate or amplify regulatory T cells (Tregs) to prevent or treat immune disorders by confirming that secretion of IL-10, a cytokine secreted by Treg, And has been identified through application number 10-2010-0045058.

In the meantime, the present invention devised a method for further enhancing the therapeutic effect of PIAS3 on the immune diseases, that is, an optimal intracellular delivery system of PIAS3, that is, PIAS3 is connected to 12 consecutive arginine (Arg; R) peptides In the case of the fusion peptide, the arginine peptide is effectively transferred into the target cell as compared to the PIAS3 to which the arginine peptide is not ligated, and the effect of treating PIAS3 with the immunological disease is improved.

 That is, according to an embodiment of the present invention, a peptide fused with R12 and PIAS3 is prepared, a recombinant vector containing a gene sequence encoding a fusion peptide is prepared, and then R12-PIAS3 Inhibition of IL-17 cytokine production, activation of Treg cells, treatment of arthritis, and inflammatory response, the results showed that the R12-PIAS3-treated group had better efficacy than PIAS3 alone, which was not fused with R12 Respectively.

Therefore, the present invention can provide a recombinant peptide in which 12 consecutive arginines (R) having cell permeability are fused with PIAS3.

In the present invention, the recombinant peptide may consist of the amino acid sequence shown in SEQ ID NO: 1.

The present invention also relates to a vector comprising a nucleotide sequence (polynucleotide) encoding a recombinant peptide in which PIAS3 is fused with 12 consecutive arginines (R) having cell permeability according to the present invention, and a vector transformed with said vector The host cell may be a host cell transformed with the nucleotide sequence of SEQ ID NO: 2.

In the present invention, the polynucleotide encoding PIAS3 may be isolated from nature or artificially synthetically modified. The nucleotide sequence encoding the PIAS3 protein may be modified by substitution, deletion or insertion of one or more nucleotide bases, The protein expressed by the modification should not contain a significant change in its biological functionality. Such modifications include modifications to heterologous homologous genes.

Therefore, the polynucleotide encoding the PIAS3 protein is preferably characterized by being represented by the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto. The polynucleotide, that is, the polynucleotide having 70% or more, preferably 80% or more , More preferably 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 2.

In addition, the polynucleotide includes a codon degeneracy sequence that encodes an equivalent nucleic acid sequence, i. E., A DNA sequence that is different but encodes the same peptide of the invention. The compositions of various forms of the invention may comprise a polynucleotide or equivalent nucleic acid sequence consisting of a DNA sequence encoding the peptide, i. E. A vector comprising a codon degeneracy sequence encoding a peptide of the same sequence but different in sequence And the gene delivery method of the nucleotides is also included in the technical scope of the present invention. The nucleotides may be delivered intracellularly in a known form, for example, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, , A viral vector comprising a liposome, a polylysine, a polyethylenimine (PEI), a protamine, a histone, a polyester amine, and a cation thereof Non-viral vectors such as micelles, emulsions, and nanoparticles can be used as well as vaginal macromolecules. In addition to the vector system, known intracellular mass transfer peptides and the like can be used to efficiently transfer into cells.

The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. The vector of the present invention may be a conventional cloning vector or an expression vector, and the expression vector may contain an expression regulatory sequence such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer Signal sequence or leader sequence, and can be produced variously according to the purpose. The vector also includes a selection marker for selecting a host cell containing the vector and, if the vector is a replicable vector, a replication origin.

The transformant can be transformed by the vector. Preferably, the transformant is produced by transforming a vector comprising a polynucleotide encoding the peptide of the present invention with a method known in the art such as, but not limited to, transient transfection, microinjection, transfection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextranmediated transfection, polybrene-mediated transfection, ), An electroporation method, a gene gun, and other known methods for introducing a nucleic acid into a cell. The transformant may be Escherichia coli, , Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Star Lokomotiv kusu (Staphylococcus), may be an Agrobacterium tumefaciens Tome Pacific Enschede (Agrobacterium tumefaciens), but is not limited thereto.

The present invention also provides a composition for the prevention or treatment of immune diseases comprising the peptide according to the present invention and a composition for preventing or treating immunological diseases comprising the vector according to the present invention as an active ingredient Can be provided.

In the present invention, the immunological disease refers to a disease in which components of the mammalian immune system cause, mediate, or contribute to a pathological condition of a mammal. In addition, the stimulation or discontinuation of the immune response may include all diseases that have a compensatory effect on the progress of the disease, and the present invention may include diseases caused by an irritable immune response. Examples of such immune disorders include, but are not limited to, autoimmune diseases; Inflammatory disease; And transplantation rejection diseases of cells, tissues or organs.

In addition, one of the most important properties of all normal individuals is that they do not react negatively to the antigenic material that constitutes the self, while non-self antigens are recognized and reacted to remove I have the ability. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

However, when problems arise in inducing or maintaining such self-tolerance, an immune response to self-antigen occurs, thereby attacking the tissue of self. The disease caused by this process is called autoimmune disease .

In addition, inflammatory diseases include TNF-α, IL-1 (interleukin-1), IL-1 (interleukin-1 and IL-2) secreted from immune cells such as macrophages by overexpression of the human immune system due to harmful stimuli, Inflammatory substances such as inflammatory cytokines such as IL-1, IL-6, prostagladin, luecotriene or nitric oxide (NO).

In the present invention, the autoimmune diseases or inflammatory diseases include but are not limited to autoimmune hepatitis, chronic rheumatoid arthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, Scleroderma, severe muscular atrophy, multiple myelitis / dermatomyositis, Hashimoto's disease, autoimmune cytopenias, Sjogren's syndrome, vasculitis syndrome and systemic lupus erythematosus.

In the present invention, the term " treatment ", as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term " treatment ", as used herein, refers to an act of treating when " treating " is defined as described above. Thus, " treatment " or " treatment regimen " of an immune disorder in a mammal may include one or more of the following:

(1) inhibiting the growth of the immune system, i.e., inhibiting its development,

(2) preventing the spread of immune diseases, i.e., preventing metastasis,

(3) alleviation of immune diseases.

(4) preventing the recurrence of immune disorders, and

(5) palliating symptoms of immune disorders

The composition for the prevention or treatment of an immunological disease according to the present invention may comprise a pharmaceutically effective amount of a peptide according to the present invention or a vector containing a polynucleotide encoding the peptide, or may contain one or more pharmaceutically acceptable carriers, An excipient or diluent. A pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat symptoms of an immune disease.

The pharmacologically effective amount of the peptide according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmacologically effective amount may be appropriately changed depending on the severity of the immune disease symptoms, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term " pharmaceutically acceptable " as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition for preventing or treating immunological diseases according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular, and the dose of the active ingredient may be appropriately determined depending on the route of administration, The composition of the present invention can be appropriately selected according to various factors such as body weight and the severity of the patient and the composition for preventing or treating immune diseases according to the present invention can be used in combination with known compounds having the effect of preventing, Lt; / RTI >

Accordingly, the present invention can provide a pharmaceutical agent for the prevention or treatment of an immune disease, which comprises a composition comprising an expression vector comprising the peptide according to the present invention or a polynucleotide encoding the peptide as an active ingredient, The composition for immunosuppression comprising the active ingredient can be provided.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

R12 - PIAS3  Production of over-expression vectors and proteins

A novel fusion peptide, R12-PIAS3, which is highly permeable to the intracellular environment, was prepared to enhance the therapeutic effect of PIAS3 in diseases caused by immune responses such as autoimmune diseases. More specific experimental contents are as follows.

<1-1> PIAS3 and R12 - PIAS3 Production

For this, a PIAS3 cDNA clone purchased from 21C Human Gene Bank was digested with restriction enzymes of Kpn1 and Xho1, and pcDNA3.1 + (promega ) To construct pcDNA3.1 + PIAS3 vector. Then, for the production of R12-PIAS3, pcDNA3.1 + vector containing 12 arginine sequences was digested with restriction enzymes of Kpn1 and Xhol, and PIAS3 was inserted to construct a pcDNA3.1 + R12-PIAS3 vector (see FIG. Thereafter, E. coli was transformed with a recombinant vector, and plasmids were isolated. The recombinant plasmids were analyzed, and the recombinant plasmids having the correct nucleotide sequence were selected. Then, NIH3T3 mouse fibroblasts were transfected to express R12-PIAS3 Highly efficient recombinant vectors expressing were selected. At this time, pcDNA3.1 + PIAS3 in which R12 was not inserted was used as a control group.

<1-2> PIAS3 and R12 - PIAS3  Production of protein expression vector containing gene

The pET15b (novagen) vector containing 6 × His was digested with restriction enzymes NdeI and XhoI (pET15b / 6 × His PIAS3) and pET15b (novagen) containing 12 × 6 His and arginine sequences for the production of R12- The vector was digested with restriction enzymes Nde1 and Xho1 to prepare pET15b / 6xHis-R12-PIAS3 vector (see FIG. 2). At this time, pET15b / 6xHis-PIAS3 vector without R12 was used as a control group.

<1-3> R12 - PIAS3  Purification of proteins

pET15b / 6xHis-PIAS3 and pET15b / 6xHis-R12-PIAS3 were transformed into E. coli BL-21 competent cells, and then plated on LB agar plates supplemented with ampicillin and incubated at 37 ° C for 12 hours After culturing, each independent single colony was cultured in LB broth medium supplemented with ampicillin at 15 ° C for 12 hours.

Then, the E. coli was centrifuged, the precipitate was harvested, resuspended in phosphate buffered saline (PBS), 1% Triton-X 100 was added to the supernatant, and the mixture was allowed to stand at room temperature for 30 minutes. Thereafter, the mixture was centrifuged at 12,000 rpm at 4 DEG C for 20 minutes to obtain a supernatant. The proteins were purified according to the guidelines of Immobilized metal ion adsorption chromatography (IMAC) (GE healthcare Life sciences, USA) in order to separate His-tagged proteins from the supernatant. Purified protein was quantitated by protein assay, separated by electrophoresis on 12% SDS-polyacrylamide gel, and stained with coommasie blue staining reagent to analyze the degree of purification of R12-PIAS3 protein.

< Example  2>

Th17  In a cell R12 - PIAS3 of IL -17 generation Inhibition  analysis

In order to confirm whether the R12-PIAS3 protein purified in Example 1 can effectively treat immune diseases such as autoimmune diseases, the secretion of IL-17 cytokine by R12-PIAS3 in Th17 cells associated with autoimmune diseases Were measured. For this, T cell line LBRM was used and 1 ug of the recombinant vector containing R12-PIAS3 was diluted in serum-free RPMI medium, diluted in polymag (Chemicell) solution, and the mixture was left at room temperature for 30 minutes , And reacted on a magnet plate for 5 minutes, and then transfected into CD4 + T cells. At this time, a recombinant vector containing PIAS3 and a mock vector were used as a control. After one day, Th17 differentiation conditions were determined, namely, 0.5 ug / ml of anti-CD3 antibody, 1 ug / mL of anti-CD28 antibody and 20 ng / mL of IL-6, 2 ng / ml of TGF- IL-17 mRNA expression was measured by real-time PCR using IL-17 (2 ug / ml anti-IFNr antibody and 2 ug / ml anti-IL-2 antibody) Was confirmed by ELISA. Primer sequences for PCR were as follows.

Primer sequence primer Primer sequence IL-17 sense  5'-CCTCAAAGCTCAGCGTGTCC -3 ' IL-17 antisense  5'-GAGCTCACTTTTGCGCCAAG -3 ' β-actin sense  5'-GAAATCGTGCGTGACATCAAAG -3 ' β-actin antisense  5'-TGTAGTTTCATGGATGCCACAG-3 '

As a result, IL-17 mRNA expression was inhibited when STAT3 inhibitor PIAS3 was overexpressed, thus inhibiting the secretion of IL-17, an inflammatory cytokine. In contrast, when R12-PIAS3 was overexpressed in which intracellular transport was more smoothly expressed by R12, IL-17 secretion inhibition was superior to PIAS3 overexpression.

< Example  3>

Treg In a cell R12 - PIAS3 of Treg Generation  analysis

Regulatory T lymphocyte cells are known to play a key role in causing immune tolerance (Nat. Rev. Immunol. 2009, 7: 305), requiring the expression of Foxp3, It is known that it plays a role of suppressing the production of cain. Thus, the present inventors investigated the expression level of Foxp3 at the level of mRNA in cells subjected to R12-PIAS3-treated Th17 differentiation.

For this, PIAS3, R12-PIAS3 and mock vector were transfected into T cell line as in Example 2, and after one day, 0.5 ug / ml of anti-CD3 antibody, 1 ug / mL of anti-CD28 antibody and IL- Treg differentiation was performed for 3 days by treatment with 10ng / ml of TGF-beta, 5ng / ml of anti-IL-4 antibody, 2ug / ml of anti-IFNr antibody and 2ug / ml of anti-IL-6 antibody. RNA was then obtained from the cells and real time PCR was performed using the primer sequences described below.

Primer sequence primer Primer sequence Foxp3 Sense  5'-GGCCCTTCTCCAGGACAGA -3 ' Foxp3 antisense  5'-GCTGATCATGGCTGGGTTGT -3 '

As a result, in the cells overexpressing R12-PIAS3, Foxp3 expression was more increased than that of PIAS3-overexpressed cells. From these results, we found that R12 enhances the function of Treg cells by PIAS3 I was able to.

< Example  4>

In an arthritis animal model R12 - PIAS3 Of Arthritis Treatment

To confirm whether R12-PIAS3 can effectively treat immune diseases, arthritis-induced mice were treated with PIAS3 or R12-PIAS3, and the degree of symptom improvement of arthritis was confirmed. First, arthritis was induced by intra-dermal injection of type II collagen into DBA1 / J mice for the preparation of arthritic animal models. After induction of arthritis, PIAS3 and R12-PIAS3 overexpression vectors were injected into mice via Hydrodynamic injection method. Hydrodynamic injection Was injected into the tail vein via an over-expression vector diluted in physiological saline. The mice were injected with the MOCK vector as a control. For each group of mice, arthritic index, degree of tissue destruction and degree of inflammation were evaluated after arthritis induction.

 As a result, when R12-PIAS3 was injected to mice in which arthritis was induced, arthritis was effectively inhibited over time as compared with the mock vector and PIAS3 vector, . In addition, as a result of the tissue staining, it was found that the degree of destruction of the joint tissue was significantly reduced by R12-PIAS3 as compared with that of the inflammatory cells in the Mock vector.

Therefore, the above-mentioned results confirmed that R12-PIAS3 according to the present invention can actually treat immune diseases.

< Example  5>

In an arthritis animal model R12 - PIAS3  Analysis of inhibitory effect of inflammatory antibodies on treatment

In order to determine whether the production of inflammatory autoantibodies was inhibited by R12-PIAS3 in the serum of the arthritic living body model, blood was collected from each mouse group 3 weeks after arthritis induction of the arthritis-induced mouse of Example 4 described above and centrifuged The amount of Cll specific IgG2a expressed in the serum was measured using a mouse IgG2a ELISA kit supplied by Bethyl, after coating the 96 well plate with type II collagen.

As a result, the expression of Cll-IgG2a in the serum was markedly inhibited by R12-PIAS3, and the degree of inhibition was superior to that of PIAS3.

Therefore, it was found from the above results that R12-PIAS3 according to the present invention can selectively inhibit the Cll antigen-specific inflammatory autoantibodies in vivo in an animal model of arthritis.

< Example  6>

Suppression of antigen-specific immune cell proliferation

In order to investigate the inhibition of antigen-specific immune cell proliferation by R12-PIAS3 in an arthritic mouse model, cells obtained from spleen cells of the arthritis-induced mouse of Example 4 were treated with antigen (dysmorphic collagen) Were analyzed by FACs analysis with CFSE staining. Add 2 μl of CFSE to 1 μl of 0.1% BSA / PBS per 2 × 10 6 cells of each group. After addition, the cells are allowed to react for 15 minutes at 37 ° C. After the reaction, wash with cold 1% FBS / RPMI and plated CFSE-stained cells in 48 wells with 5x10 5 cells. Each cell was incubated for 3 days with 50 ug / ml anti CD3 and type II collagen. After incubation, the cells were analyzed by FL1-FITC channel using FACs caliber, and the cells that were transferred to the left were analyzed.

As a result, the proliferation of the PIAS3-injected mice was reduced compared to that of the control vector-injected mice, and the proliferation was significantly decreased in the R12-PIAS3-injected mice compared to the PIAS3 mice. Therefore, R12-PIAS3 inhibits the proliferation of immune cells by the antigen, and thus plays an important role in autoimmune diseases.

< Example  7>

In an arthritis animal model R12 - PIAS3  Analysis of inhibitory effects of inflammatory cytokines, neovascularization, and joint destruction factors on joints

Through the above examples, the inventors of the present invention found that R12-PIAS3 inhibits the expression of IL-17, which is an arthritic inflammatory cytokine, in vivo in an animal model of arthritis, increases Foxp3 expression and assists Treg differentiation, Inhibits the production of antibodies and inhibits joint destruction and infiltration of inflammatory cells. Therefore, in order to investigate whether R12-PIAS3 affects the expression of inflammatory cells in joint tissues, arthritic tissues were stained and observed. As described in Example 4, arthritis-induced mice were treated with PIAS3 and R12-PIAS3 IL-1b, IL-6, IL-17, TNF-a, and RANK were used as the factors to be stained for each stain after collecting the joints of mice from the injected mice and the mice injected with the mock vector. Antibodies to VEGF were purchased (purchased from Sigma) and each factor was stained.

As a result, the inflammatory cytokines L-1b, IL-6, IL-17 and TNF-a were strongly expressed in the cells in the arthritis-induced control group, And VEGF-expressing cells associated with neovascularization were also strongly invaded. On the other hand, in the arthritic mouse group treated with PIAS3 and R12-PIAS3, the expression of inflammatory cytokines, joint destruction factors and VEGF was markedly suppressed compared to the control group, and R12-PIAS3 was more suppressed than PIAS3 .

Therefore, the inventors of the present invention have found that R12-PIAS3 can effectively inhibit infiltration and proliferation of inflammatory cells in joints of arthritic animal models.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Catholic University <120> R12-PIAS3 Fusion peptide having cell penetration for preventing          or treating immune disease and pharmaceutical composition          The <130> np12-1047 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 641 <212> PRT <213> R12-PIAS3 peptide sequence <400> 1 Met Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Met Ala Glu   1 5 10 15 Leu Gly Glu Leu Lys His Met Val Met Ser Phe Arg Val Ser Glu Leu              20 25 30 Gln Val Leu Leu Gly Phe Ala Gly Arg Asn Lys Ser Gly Arg Lys His          35 40 45 Glu Leu Leu Ala Lys Ala Leu His Leu Leu Lys Ser Ser Cys Ala Pro      50 55 60 Ser Val Gln Met Lys Ile Lys Glu Leu Tyr Arg Arg Arg Phe Pro Arg  65 70 75 80 Lys Thr Leu Gly Pro Ser Asp Leu Ser Leu Leu Ser Leu Pro Pro Gly                  85 90 95 Thr Ser Pro Val Gly Ser Pro Gly Pro Leu Ala Pro Ile Pro Pro Thr             100 105 110 Leu Leu Thr Pro Gly Thr Leu Leu Gly Pro Lys Arg Glu Val Asp Met         115 120 125 His Pro Pro Leu Pro Gln Pro Val His Pro Asp Val Thr Met Lys Pro     130 135 140 Leu Pro Phe Tyr Glu Val Tyr Gly Glu Leu Ile Arg Pro Thr Thr Leu 145 150 155 160 Ala Ser Thr Ser Ser Gln Arg Phe Glu Glu Ala His Phe Thr Phe Ala                 165 170 175 Leu Thr Pro Gln Gln Leu Gln Gln Ile Leu Thr Ser Arg Glu Val Leu             180 185 190 Pro Gly Ala Lys Cys Asp Tyr Thr Ile Gln Val Gln Leu Arg Phe Cys         195 200 205 Leu Cys Glu Thr Ser Cys Pro Gln Glu Asp Tyr Phe Pro Pro Asn Leu     210 215 220 Phe Val Lys Val Asn Gly Lys Leu Cys Pro Leu Pro Gly Tyr Leu Pro 225 230 235 240 Pro Thr Lys Asn Gly Ala Glu Pro Lys Arg Pro Ser Arg Pro Ile Asn                 245 250 255 Ile Thr Pro Leu Ala Arg Leu Ser Ala Thr Val Pro Asn Thr Ile Val             260 265 270 Val Asn Trp Ser Ser Glu Phe Gly Arg Asn Tyr Ser Leu Ser Val Tyr         275 280 285 Leu Val Arg Gln Leu Thr Ala Gly Thr Leu Leu Gln Lys Leu Arg Ala     290 295 300 Lys Gly Ile Arg Asn Pro Asp His Ser Arg Ala Leu Ile Lys Glu Lys 305 310 315 320 Leu Thr Ala Asp Pro Asp Ser Glu Val Ala Thr Thr Ser Leu Arg Val                 325 330 335 Ser Leu Met Cys Pro Leu Gly Lys Met Arg Leu Thr Val Pro Cys Arg             340 345 350 Ala Leu Thr Cys Ala His Leu Gln Ser Phe Asp Ala Ala Leu Tyr Leu         355 360 365 Gln Met Asn Glu Lys Lys Pro Thr Trp Thr Cys Pro Val Cys Asp Lys     370 375 380 Lys Ala Pro Tyr Glu Ser Leu Ile Ile Asp Gly Leu Phe Met Glu Ile 385 390 395 400 Leu Asn Ser Cys Ser Asp Cys Asp Glu Ile Gln Phe Met Glu Asp Gly                 405 410 415 Ser Trp Cys Pro Met Lys Pro Lys Lys Glu Ala Ser Glu Val Cys Pro             420 425 430 Pro Pro Gly Tyr Gly Leu Asp Gly Leu Gln Tyr Ser Ala Val Gln Glu         435 440 445 Gly Ile Gln Pro Glu Ser Lys Lys Arg Val Glu Val Ile Asp Leu Thr     450 455 460 Ile Glu Ser Ser Ser Asp Glu Glu Asp Leu Pro Pro Thr Lys Lys His 465 470 475 480 Cys Pro Val Thr Ser Ala Ala Ile Pro Ala Leu Pro Gly Ser Lys Gly                 485 490 495 Ala Leu Thr Ser Gly His Gln Pro Ser Ser Val Leu Arg Ser Ser Ala             500 505 510 Met Gly Thr Leu Gly Ser Asp Phe Leu Ser Ser Leu Pro Leu His Glu         515 520 525 Tyr Pro Pro Ala Phe Pro Leu Gly Ala Asp Ile Gln Gly Leu Asp Leu     530 535 540 Phe Ser Phe Leu Gln Thr Glu Ser Gln His Tyr Gly Pro Ser Val Ile 545 550 555 560 Thr Ser Leu Asp Glu Gln Asp Thr Leu Gly His Phe Phe Gln Tyr Arg                 565 570 575 Gly Thr Pro Ser His Phe Leu Gly Pro Leu Ala Pro Thr Leu Gly Ser             580 585 590 Ser His Arg Ser Ser Thr Pro Ala Pro Pro Gly Arg Val Ser Ser         595 600 605 Ile Val Ala Pro Gly Ser Ser Leu Arg Glu Gly His Gly Gly Pro Leu     610 615 620 Pro Ser Gly Pro Ser Leu Thr Gly Cys Arg Ser Asp Val Ile Ser Leu 625 630 635 640 Asp     <210> 2 <211> 1893 <212> DNA <213> R12-PIAS3 DNA sequence <400> 2 atgcgacgga gacgacgtag acgcaggcgg cgacgccgta tgagtttccg agtgtctgag 60 ctccaggtgc tcctcggctt cgctggcagg aacaagagtg ggcggaaaca cgagctgctg 120 gccaaggccc tgcacctcct caagtctagc tgcgccccca gcgtccagat gaagatcaaa 180 gaactttatc gcaggcgctt tccccggaag accctggggc cttctgatct ctccttgctt 240 tctttgcccc ctggcacctc tcctgtaggc tcccccggcc cccttgctcc catccctccc 300 acccttctga cccctggcac cttgctaggc cctaagcgtg aggtggacat gcatcctcct 360 ctgccgcagc ctgtgcaccc cgatgtcacc atgaagccac tgcccttcta tgaagtctat 420 ggggagctca tccgacccac cacccttgcg tccacctcca gccagaggtt cgaggaagcc 480 cacttcacct tcgcgctcac tccccagcag ctgcagcaga ttctcacgtc cagggaggtt 540 ctgccaggag ccaagtgtga ttacaccata caagtgcagc tcagattctg tctctgtgag 600 accagctgcc ctcaggagga ctatttcccc cctaacctct ttgttaaggt taatgggaaa 660 ctctgccccc tgccgggtta cctccctcca accaagaatg gagctgagcc caagaggccc 720 agccgtccga tcaacatcac acccttggct cgactctcag ccactgtccc caacaccatc 780 gtagttaatt ggtcatctga gtttggacgg aattactcct tgtccgtgta cctggtgagg 840 caattgactg cagggaccct tctacaaaaa ctcagagcca aggggatccg gaatccagac 900 cattcccggg cactgatcaa ggagaaactg accgctgacc ccgacagtga agtggctact 960 acaagtctcc gggtgtcact catgtgcccg ctagggaaga tgcgcctgac tgtcccgtgt 1020 cgtgccctca cctgtgccca tctgcagagt ttcgatgctg ccctttatct acagatgaat 1080 gagaagaagc cgacatggac gtgtcctgtg tgtgacaaga aggctcccta tgagtcgctg 1140 attattgatg gtttattcat ggaaattctt aattcctgtt cggattgtga tgagatccag 1200 ttcatggaag atggatcctg gtgtccgatg aaacccaaga aggaggcatc agaggtttgc 1260 cccccgccag ggtatgggct ggatggtctc cagtacagcg cagtccagga gggaattcag 1320 ccagagagta agaagagggt cgaagtcatt gacttgacca tcgaaagctc atcagatgag 1380 gaggatttgc cccccaccaa gaagcactgc cctgtcacct cagcggccat tccagccctt 1440 cctggaagca aaggagccct gacctctggt caccagccat cctcggtgct gcggagccct 1500 gcaatgggca cactgggcag tgacttcctg tctagtctcc cgctacatga gtacccacct 1560 gccttcccac tgggggctga catccaaggt ttagatttat tttctttcct tcagactgag 1620 agtcagcact acggcccttc agttatcact tcgctagatg aacaggacac ccttggccac 1680 ttcttccagt accggggaac cccttcccac ttcctgggcc cactggcccc cacactgggg 1740 agctctcacc gcagctccac tccagcgccc cctcctggtc gtgtcagcag cattgtggct 1800 cctgggagct ccttgaggga agggcatgga ggacccctgc cttcaggtcc ctctttgact 1860 ggctgtcggt cagacgtcat ttccttggac tga 1893 <210> 3 <211> 1980 <212> DNA <213> PIAS3 DNA sequence <400> 3 cagcagactg ccggagctgc gcgagccctt tctgcaccgg ggctcacccc cgcaactctg 60 cggcacatgg tgatgagttt ccgagtgtct gagctccagg tgctcctcgg cttcgctggc 120 aggaacaaga gtgggcggaa acacgagctg ctggccaagg ccctgcacct cctcaagtct 180 agctgcgccc ccagcgtcca gatgaagatc aaagaacttt atcgcaggcg ctttccccgg 240 aagaccctgg ggccttctga tctctccttg ctttctttgc cccctggcac ctctcctgta 300 ggctcccccg gcccccttgc tcccatccct cccacccttc tgacccctgg caccttgcta 360 ggccctaagc gtgaggtgga catgcatcct cctctgccgc agcctgtgca ccccgatgtc 420 accatgaagc cactgccctt ctatgaagtc tatggggagc tcatccgacc caccaccctt 480 gcgtccacct ccagccagag gttcgaggaa gcccacttca ccttcgcgct cactccccag 540 cagctgcagc agattctcac gtccagggag gttctgccag gagccaagtg tgattacacc 600 atacaagtgc agctcagatt ctgtctctgt gagaccagct gccctcagga ggactatttc 660 ccccctaacc tctttgttaa ggttaatggg aaactctgcc ccctgccggg ttacctccct 720 ccaaccaaga atggagctga gcccaagagg cccagccgtc cgatcaacat cacacccttg 780 gctcgactct cagccactgt ccccaacacc atcgtagtta attggtcatc tgagtttgga 840 cggaattact ccttgtccgt gtacctggtg aggcaattga ctgcagggac ccttctacaa 900 aaactcagag ccaaggggat ccggaatcca gaccattccc gggcactgat caaggagaaa 960 ctgaccgctg accccgacag tgaagtggct actacaagtc tccgggtgtc actcatgtgc 1020 ccgctaggga agatgcgcct gactgtcccg tgtcgtgccc tcacctgtgc ccatctgcag 1080 agtttcgatg ctgcccttta tctacagatg aatgagaaga agccgacatg gacgtgtcct 1140 gtgtgtgaca agaaggctcc ctatgagtcg ctgattattg atggtttatt catggaaatt 1200 cttaattcct gttcggattg tgatgagatc cagttcatgg aagatggatc ctggtgtccg 1260 atgaaaccca agaaggaggc atcagaggtt tgccccccgc cagggtatgg gctggatggt 1320 ctccagtaca gcgcagtcca ggagggaatt cagccagaga gtaagaagag ggtcgaagtc 1380 attgacttga ccatcgaaag ctcatcagat gaggaggatt tgccccccac caagaagcac 1440 tgccctgtca cctcagcggc cattccagcc cttcctggaa gcaaaggagc cctgacctct 1500 ggtcaccagc catcctcggt gctgcggagc cctgcaatgg gcacactggg cagtgacttc 1560 ctgtctagtc tcccgctaca tgagtaccca cctgccttcc cactgggggc tgacatccaa 1620 ggtttagatt tattttcttt ccttcagact gagagtcagc actacggccc ttcagttatc 1680 acttcgctag atgaacagga cacccttggc cacttcttcc agtaccgggg aaccccttcc 1740 cacttcctgg gcccactggc ccccacactg gggagctctc accgcagctc cactccagcg 1800 ccccctcctg gtcgtgtcag cagcattgtg gctcctggga gctccttgag ggaagggcat 1860 ggaggacccc tgccttcagg tccctctttg actggctgtc ggtcagacgt catttccttg 1920 gactgagctt tttggattat gaaatcaatc tccattggcc ccagcactga gcagatcacg 1980                                                                         1980 <210> 4 <211> 628 <212> PRT <213> PIAS3 peptide sequence <400> 4 Met Ala Glu Leu Gly Glu Leu Lys His Met Val Met Ser Phe Arg Val   1 5 10 15 Ser Glu Leu Gln Val Leu Leu Gly Phe Ala Gly Arg Asn Lys Ser Gly              20 25 30 Arg Lys His Glu Leu Leu Ala Lys Ala Leu His Leu Leu Lys Ser Ser          35 40 45 Cys Ala Pro Ser Val Gln Met Lys Ile Lys Glu Leu Tyr Arg Arg Arg      50 55 60 Phe Pro Arg Lys Thr Leu Gly Pro Ser Asp Leu Ser Leu Leu Ser Leu  65 70 75 80 Pro Pro Gly Thr Ser Pro Val Gly Ser Pro Gly Pro Leu Ala Pro Ile                  85 90 95 Pro Pro Thr Leu Leu Thr Pro Gly Thr Leu Leu Gly Pro Lys Arg Glu             100 105 110 Val Asp Met His Pro Pro Leu Pro Gln Pro Val His Pro Asp Val Thr         115 120 125 Met Lys Pro Leu Pro Phe Tyr Glu Val Tyr Gly Glu Leu Ile Arg Pro     130 135 140 Thr Thr Leu Ala Ser Thr Ser Ser Gln Arg Phe Glu Glu Ala His Phe 145 150 155 160 Thr Phe Ala Leu Thr Pro Gln Gln Leu Gln Gln Ile Leu Thr Ser Arg                 165 170 175 Glu Val Leu Pro Gly Ala Lys Cys Asp Tyr Thr Ile Gln Val Gln Leu             180 185 190 Arg Phe Cys Leu Cys Glu Thr Ser Cys Pro Gln Glu Asp Tyr Phe Pro         195 200 205 Pro Asn Leu Phe Val Lys Val Asn Gly Lys Leu Cys Pro Leu Pro Gly     210 215 220 Tyr Leu Pro Pro Thr Lys Asn Gly Ala Glu Pro Lys Arg Pro Ser Arg 225 230 235 240 Pro Ile Asn Ile Thr Pro Leu Ala Arg Leu Ser Ala Thr Val Pro Asn                 245 250 255 Thr Ile Val Val Asn Trp Ser Ser Glu Phe Gly Arg Asn Ser Ser Leu             260 265 270 Ser Val Tyr Leu Val Arg Gln Leu Thr Ala Gly Thr Leu Leu Gln Lys         275 280 285 Leu Arg Ala Lys Gly Ile Arg Asn Pro Asp His Ser Arg Ala Leu Ile     290 295 300 Lys Glu Lys Leu Thr Ala Asp Pro Asp Ser Glu Val Ala Thr Thr Ser 305 310 315 320 Leu Arg Val Ser Leu Met Cys Pro Leu Gly Lys Met Arg Leu Thr Val                 325 330 335 Pro Cys Arg Ala Leu Thr Cys Ala His Leu Gln Ser Phe Asp Ala Ala             340 345 350 Leu Tyr Leu Gln Met Asn Glu Lys Lys Pro Thr Trp Thr Cys Pro Val         355 360 365 Cys Asp Lys Lys Ala Pro Tyr Glu Ser Leu Ile Ile Asp Gly Leu Phe     370 375 380 Met Glu Ile Leu Asn Ser Cys Ser Asp Cys Asp Glu Ile Gln Phe Met 385 390 395 400 Glu Asp Gly Ser Trp Cys Pro Met Lys Pro Lys Lys Glu Ala Ser Glu                 405 410 415 Val Cys Pro Pro Pro Gly Tyr Gly Leu Asp Gly Leu Gln Tyr Ser Ala             420 425 430 Val Gln Glu Gly Ile Gln Pro Glu Ser Lys Lys Arg Val Glu Val Ile         435 440 445 Asp Leu Thr Ile Glu Ser Ser Ser Asp Glu Glu Asp Leu Pro Pro Thr     450 455 460 Lys Lys His Cys Pro Val Thr Ser Ala Ala Ile Pro Ala Leu Pro Gly 465 470 475 480 Ser Lys Gly Ala Leu Thr Ser Gly His Gln Pro Ser Ser Val Leu Arg                 485 490 495 Ser Pro Ala Met Gly Thr Leu Gly Ser Asp Phe Leu Ser Ser Leu Pro             500 505 510 Leu His Glu Tyr Pro Pro Ala Phe Pro Leu Gly Ala Asp Ile Gln Gly         515 520 525 Leu Asp Leu Phe Ser Phe Leu Gln Thr Glu Ser Gln His Tyr Gly Pro     530 535 540 Ser Val Ile Thr Ser Leu Asp Glu Gln Asp Thr Leu Gly His Phe Phe 545 550 555 560 Gln Tyr Arg Gly Thr Pro Ser His Phe Leu Gly Pro Leu Ala Pro Thr                 565 570 575 Leu Gly Ser Ser His Arg Ser Ser Thr Pro Ala Pro Pro Gly Arg             580 585 590 Val Ser Ser Ile Val Ala Pro Gly Ser Ser Leu Arg Glu Gly His Gly         595 600 605 Gly Pro Leu Pro Ser Gly Pro Ser Leu Thr Gly Cys Arg Ser Serp Val     610 615 620 Ile Ser Leu Asp 625

Claims (8)

delete delete delete delete A composition for the prevention or treatment of an immunological disease comprising a recombinant peptide in which 12 consecutive arginines (R) having cell permeability are fused with PIAS3,
The recombinant peptide comprises the amino acid sequence of SEQ ID NO: 1,
Wherein said recombinant peptide has the activity of inhibiting the production of IL-17, an inflammatory cytokine, promoting the activity of immunoregulatory T cells (Treg), and inhibiting the infiltration and proliferation of inflammatory cells Or &lt; / RTI &gt; delete 6. The method of claim 5,
The immunological disease is autoimmune disease; Inflammatory disease; And transplantation rejection diseases of cells, tissues or organs. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; 8. The method of claim 7,
Wherein said autoimmune or inflammatory disease is selected from the group consisting of autoimmune hepatitis, chronic rheumatoid arthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, scleroderma, severe muscular atrophy, multiple myelitis / , Autoimmune cytopenia, Sjogren's syndrome, vasculitis syndrome and systemic lupus erythematosus.

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