KR20160087010A - Pharmaceutical composition for autosomal dominant polycystic kidney disease containing cell-transducible Atox1 fusion protein - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating autosomal dominant polycystic kidney diseases and, more specifically, relates to a pharmaceutical composition for treating diseases or preventing polycystic kidney diseases, comprising the fusion protein of Atox1 protein which can be penetrated to the inside of the polycystic kidney disease cells. Provided in the present invention is a protein therapeutic agent, which is effective by selecting proteins for inhibiting Erk and by using the protein penetrating technology.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for prevention and treatment of polycystic kidney disease containing cell-permeable Atox1 fusion protein,

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing and treating polycystic kidney disease, and more particularly, to a pharmaceutical composition for preventing and treating polycystic kidney disease containing an Atox1 fusion protein capable of tissue and intracellular permeability as an active ingredient.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease with an incidence rate of at least 1 per 500 to 1,000 people. Autosomal dominant danangsin is characterized by the formation of a large number of liquid cysts in both kidneys and a 4 to 8 fold increase in kidney compared to healthy individuals. Until now, there has been no effective clinical treatment for polycystic kidney disease, and most of the polycystic kidneys progress to end stage renal disease (Gabow, 1993; Grantham, 1996). Cyst formation in human polycystic kidney is accompanied by increased cell proliferation and magnetic cell death of cystic epidermal cells.

Although many anticancer therapies have been developed so far, disappointing clinical results have been reported due to negative feedback inhibition.

WT 9-7 cells are basically polycontogenic cells with activated mTOR signal. Therefore, WT 9-7 cells should die when using therapeutic agents. Rapamycin is the most widely known treatment for polycystic kidney disease. Rapamycin is known to be a therapeutic agent for polycystic kidney disease by replacing S6, which is a signal of mTOR, activated in WT 9-7 cells with p-S6, but in contrast, it exhibits side effects of activating the inflammatory factor Erk signal.

There is a growing interest in using in vivo polymers for therapeutic purposes. At present, gene therapy is the focus of attention. Gene therapy, however, is not easy to carry out, and it is difficult to artificially regulate the amount of protein expressed in target cells because of low gene expression, low expression in target cells, .

In order to transfer a therapeutic drug or protein into a cell, a method of directly delivering the target protein through the cell membrane may be considered. However, proteins are very difficult to pass through cell membranes because of their molecular size and various biochemical properties. In general, substances with a molecular weight of 600 Daltons or more are known to be almost impossible to pass through cell membranes.

Recently, it has been found that one of the methods of transporting proteins is to transport naturally-occurring heterologous proteins into cells using Pep-1 peptide. Pep-1 peptide consists of 21 amino acids (KETWWETWWTEW SQP KKKRKV) and has three domains (hydrophobic domain, spacer, hydrophilic domain). So far, studies using Pep-1 peptides have revealed that when the Pep-1 peptide and the external protein are administered simultaneously to the cells, the protein can be transported into the cell in a natural state. In addition, Pep-1 peptide has several advantages as a protein therapeutic agent compared to Tat protein, that is, it efficiently penetrates the protein into cells, shows stability in physiological buffer solution, lack of sensitivity to serum, and the like. However, PEP peptides must be administered at a constant rate when transporting an external protein, such as green fluorescent protein (GFP), β-galactosidase, or β-Gal, Protein was confirmed to be transported. In addition, it is not yet clear whether all proteins including therapeutic proteins can be delivered into cells by Pep-1.

It is an object of the present invention to provide an effective prophylactic and therapeutic agent for polycyclic acid.

The present invention aims at providing a more effective protein therapeutic by identifying proteins that inhibit Erk, which is a target of polycyclic therapy, and confirming mechanism and protective effect against polycystic kidney using protein permeation technique.

In order to achieve the above object, the present inventors have studied to develop a protein preparation which is an immunosuppressive drug as a penetration enhancing effect, and the penetration effect of a protein preparation, the signal of Erk and Akt when treated with Atox1 fusion protein in polycystic kidney cell line And the effect of the Atox1 fusion protein on the polycationic effect was confirmed, thereby completing the present invention.

The pharmaceutical composition containing the Atox1 fusion protein as an active ingredient can be formulated in an oral or injection form by a conventional method in combination with a carrier which is conventionally acceptable in the pharmaceutical field. Oral compositions include, for example, tablets and gelatin capsules, which may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, , Magnesium stearate, stearic acid and its magnesium or calcium salt and / or polyethylene glycol) and the tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone ), And may optionally contain a disintegrant (e.g., starch, agar, alginic acid or a sodium salt thereof) or a boiling mixture and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The injectable composition is preferably an isotonic aqueous solution or suspension, and the composition mentioned is sterilized and / or contains adjuvants such as preservatives, stabilizers, wetting or emulsifying solution accelerators, salts for controlling osmotic pressure and / or buffering agents. They may also contain other therapeutically valuable substances.

Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of autosomal dominant polycystic kidney, which comprises the above Atox1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutical preparations thus prepared may be administered orally or parenterally, that is, intravenously, subcutaneously, intraperitoneally, or topically, as desired. The dose may be administered in a single dose of 0.01 to 100 mg / kg per day. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, time of administration, method of administration, excretion rate, severity of disease, and the like.

The present invention also provides a health functional food composition containing the Atox1 fusion protein as an active ingredient and preventing or improving autosomal dominant polycystic kidney.

The intracellular delivery of the Atox1 protein molecule according to the present invention is carried out in the form of a hydrophobic domain consisting of 15 to 30 amino acids in Atox1 and containing 5 or more tryptophan, a hydrophilic domain containing 4 or more lysines domain, and a spacer that separates the two domains is covalently linked to the N-terminus of Atox1, the target protein, and / or the transmembrane domain, including Pep-1, Terminus and a covalently linked form of the fusion protein with the C-terminus. Examples of the transport domain of the present invention include Pep-1 peptide and HIV Tat 49 to 57 peptide. However, the protein transport domain of the present invention is not limited to the Pep-1 peptide, the HIV Tat 49-55 residue peptide, and the Pep-1 peptide or HIV Tat Since it is easy for a person skilled in the art to make a peptide having a function similar to that of a peptide, a hydrophobic domain consisting of 15 to 30 amino acids and containing at least 5 tryptophan, , A hydrophilic domain containing four or more lysines, and a spacer for separating the two domains, and a protein transport domain carrying the same or similar protein-transporting function with partial amino acid substitution therefrom Domain, or six to twelve amino acid residues, wherein the arginine or lysine residue An oligolysine transport domain consisting of 6 to 12 lysines, an oligosaccharide transport domain consisting of 6 to 12 arginines, or an oligos (lysine, lysine, or lysine) chain consisting of 6 to 12 lysine or arginine, Arginine) transport domains are also within the scope of the invention.

The definitions of the main terms used in the description of the present invention and the like are as follows.

"Atox1 fusion protein" means a covalent complex formed by genetic fusion or chemical bonding between a protein transport domain and Atox1, and a protein transport domain and a cargo molecule (i.e., Atox1 in the present invention). As a specific example in the present specification and drawings, "Tat-Atox1" refers to the binding of the Tat protein transport domain to the N-terminus of Atox1 among the Atox1 fusion proteins.

In addition, the term "genetic fusion" means a link consisting of a linear, covalent bond formed through genetic expression of a DNA sequence encoding a protein.

The term "target cell" refers to a cell to which a cargo molecule is delivered by a transport domain. That is, the target cell is a cell, that is, a living organism or a cell or organ It is meant to include microorganisms that are found. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism. Specifically, in the present specification, the target cell means a brain cell.

As used herein, the term "protein transport domain" refers to a peptide or protein that is covalently bonded to a cargo molecule (target protein) peptide or protein and can introduce the peptide or protein into cells without requiring additional receptor, carrier, energy, For example, it refers to HIV-1 Tat.

As used herein, the term "target protein" refers to a molecule that is covalently bound to Tat protein transport domain and introduced into cells to exhibit activity. It is synonymous with "cargo molecule".

Also, in the present specification, the term "introduction", "transport", "penetration", "transport", "transfer", "permeation", "pass" Respectively.

In the present invention, the protein transport domain is a protein transport domain composed of 15 to 30 amino acids and composed of a non-hydrophilic domain containing 5 or more tryptophan, a hydrophilic domain containing many lysines, and a spacer separating the two domains, A transport domain comprising 6 to 12 amino acid residues and comprising ¾ or more arginine or a lysine residue, an oligolysine transport domain consisting of 6 to 12 lysines, an oligoroginalin transport domain consisting of 6 to 12 arginines, Or an oligo (lysine, arginine) transport domain consisting of 12 lysine or arginine. In addition, the target protein (cargo molecule) is Atox1. The protein transport domain and the target protein may be replaced by other amino acid (s) of similar polarity in which one or more amino acids functionally equivalently function in the sequence according to the silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, the hydrophobic amino acid class includes alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positive basic amino acids include arginine, lysine and histidine. Acidic amino acids with negative charge include aspartic acid and glutamic acid. In addition, fragments or derivatives thereof having the same or similar biological activity within a certain range of homology, such as 85-100%, between the fusion protein of the present invention and the amino acid sequence are included in the scope of the present invention.

The Atox1 fusion protein according to the present invention infiltrated into polycystic kidney cells in a time-dependent and dose-dependent manner.

In addition, the Atox1 fusion protein according to the present invention penetrated into cells and stably maintained for 15 hours or more.

In addition, the Atox1 fusion protein according to the present invention decreased Erk signal and Akt signal in polycystic kidney cells.

Accordingly, the Atoxl fusion protein according to the present invention is expected to be useful as a pharmaceutical composition for the prevention or treatment of autosomal dominant polycystic kidney.

FIG. 1 shows the results of SDS-PAGE after purification of Atox1 protein and Tat-Atox1 fusion protein. Further, Western blot analysis of the Atox1 protein and the Tat-Atox1 fusion protein revealed that the Atox1 protein and the Tat-Atox1 fusion protein were purified.
FIG. 2 is a Western blot analysis of WT 9-7 cells treated with 3 μM of Tat-Atox1 fusion protein at different times for 0 to 40 minutes. As a control, Atox1 protein without a protein transduction domain was used.
FIG. 3 is a photograph of western blot analysis after treatment of WT 9-7 cells with 0.5-3 .mu.M of the Tat-Atox1 fusion protein at different concentrations for 1 hour. As a control, Atox1 protein without a protein transduction domain was used.
FIG. 4 is a photograph of western blot analysis of WT 9-7 cells treated with 3 μM of Tat-Atox1 fusion protein for 1 hour. The fusion protein was stably maintained until 15 hours.
FIG. 5 is a photograph of WT 9-7 cells treated with 3 μM Tat-Atox1 fusion protein for 1 hour and then photographed using a confocal microscope. "Control" is an untreated control group.
FIG. 6 shows Western blot analysis of WT 9-7 cells to examine the effect of the Tat-Atox1 fusion protein on the p38, ERK signaling mechanism.
FIG. 7 shows Western blot analysis of WT 9-7 cells to examine the effect of Tat-Atoxl fusion protein on the Akt signaling mechanism.
FIG. 8 shows Western blot analysis of WT 9-7 cells to examine the effect of the Tat-Atox1 fusion protein on the p65, IκBα signaling mechanism.
FIG. 9 shows Western blot analysis of WT 9-7 cells to examine the inhibitory effect of Tat-Atox1 fusion protein on COX2, iNOS, IL-6, IL-1β and TNF-α signaling mechanisms.
FIG. 10 shows the effect of RT-PCR on WT 9-7 cells to inhibit COX2, iNOS, IL-6, IL-1β and TNF-α signaling of Tat-Atox1 fusion protein
Figure 11 shows Western blot analysis of WT 9-7 cells to show the effect of the Tat-Atoxl fusion protein on the caspase 3, cleaved caspase 3 signaling mechanism.
FIG. 12 shows Western blot analysis of WT 9-7 cells to examine the effect of Tat-Atox1 fusion protein on the S6K, p-S6K signaling mechanism.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments. Particularly, as a result of each experimental example, data on Tat-Atox1 fusion protein among the various fusion proteins prepared in the specific examples are described, but other fusion proteins are also similar to those obtained by using Tat-Atox1 fusion protein as a sample And the results are shown.

material

Restriction endonuclease and T4 DNA ligase were purchased from Promega (Madison, Wis., USA). Plasmid pET-15b and Escherichia coli strain BL21 (DE3) were purchased from Novagen (Hilden, Germany). Oligonucleotides were synthesized in Bioneer (Dajeon, Korea). Ni ^{2 +} -nitrile trichlorosilicate Sepharose Superflow column was obtained from Qiagen (Valencia, CA, USA). Fetal serum and antibiotics were purchased from Gibco BRL (Grand Island, NY, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted and the best available analytical grade products were used.

Cell culture and Atox1 Fusion protein  Introduction

WT 9-7 cells were cultured in EMEM (Eagle's Minimum) containing 20 mM HEPES / NaOH (pH 7.4), 5 mM NaHCO3, 10% fetal calf serum (FBS) and antibiotics (100 ug / ml streptomycin, 100 U / ml penicillin) Essential Medium) medium at 37 ° C, 95% air and 5% CO ₂ .

For delivery of the Tat-Atox1 fusion protein, cells were treated with various concentrations of Tat-Atox1 (0.5 to 3 mu m) for 1 hour. The cells were then treated with trypsin-EDTA and washed with PBS. Cells were harvested to produce cell extracts and Western blot analysis.

Western Blat  analysis

Cell lysates were analyzed by 15% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis). The protein migrated to the nitrocellulose membrane by the voltage difference, and the membrane was immersed in PBS with 5% skim milk solution. The membrane was first identified by immunoblotting using the manufacturer's recommended primary antibody. Proteins were determined by chemical fluorescence as directed by the manufacturer (Amersham, Franklin Lakes, NJ, USA).

Confocal  Fluorescence microscope

WT 9-7 cells were placed on glass cover slips and incubated with Atox1 and Tat-Atox1 fusion protein (3 μM) for one hour at 37 ° C. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 3 minutes. Cells were fixed with PBS containing 3% bovine serum albumin, 0.1% Triton X-100 and washed with PBS-BT. The anti-histidine primary antibody was diluted 1: 2,000 and incubated with each sample for one and a half hours at room temperature. Alexa fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, Calif., USA) was diluted 1: 15,000 and incubated at room temperature for 1 hour under dark conditions. Nuclei were stained with 4'6-diamidino-2-phenylindole (Roche Applied Science, Basel, Switzerland) diluted 1: 3,000 with 1 μg / ml for 3 minutes. Fluorescence was analyzed with an Olympus FV-300 confocal fluorescence microscope (Olympus, Tokyo, Japan).

iNOS  And COX -2 expression level measurement

WT 7-9 cells were cultured in 6-well plates for 12 hours to 70% saturation. After incubation, the cells were pretreated with Tat-Atoxl fusion protein (0.5-2 μM) for 2 hours and the culture medium was harvested. Expression of RNA levels as well as iNOS and COX-2 protein expression was measured by Western blot analysis and RT-PCR.

RT - PCR  analysis

Total RNA was isolated from Raw 264.7 cells using the Easy blue kit according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif., USA). RNA (2 μg) was then reverse transcribed using reverse transcriptase and oligo- (dT) primers. PCR amplification for cDNA was performed using the following sense and antisense primers.

COX-2 sense primer, 5'-CAAAGGCCTCCATTGACCAGA-3 ';

COX-2 antisense primer, 5'-TGGACCAGGTTTTTCCACCG-3 ';

IL-1 [beta] sense primer, 5'-TGCAGAGTTCCCCAACTGGTACTC-3 ';

IL-1? Antisense primer, 5'-GTGCTGCCTAATGTCCCTTGAATC-3 ';

IL-6 sense primer, 5'-CAAGAAAGACAAAGCCAGAGTCCTT-3 ';

IL-6 antisense primer, 5'-TGGATGGTCTTGGTCCCTTAGCC-3 ';

TNF-α sense primer, 5'-AAGTTCCCAAATGGCCTCCC-3 ';

TNF-α antisense primer, 5'-TGGCACCACTAGTTGGTTGTCTTT-3 '; And

GADPH sense primer, 5'-TGAAGGTCGGTGAACGGATTTGG-3 ';

GAPDH antisense primer, 5'-CATGTAGGCCATGAGGTCCAC CAC-3 '.

PCR primer kit (Intron Biotechnology, Seoul, Korea) was used for PCR, and each primer was heated at 72 ° C for 5 minutes to terminate the reaction. The PCR products were separated on 1% agarose gel, stained with ethidium bromide and observed with UV.

Result 1: Taste - Atox1 Of the fusion protein  Expression and purification

In order to construct the control Atox1 protein and Tat-Atox1 fusion protein, the Atox1 gene was transformed into Escherichia coli cells and overexpressed. The resultant was purified by Sepharose affinity chromatography with Ni ^{2 +} -nitriloic acid and then subjected to SDS-PAGE The size and purity of the protein were confirmed. Also, the control Atox1 protein and the Tat-Atox1 fusion protein were identified by Western blot analysis using a histidine antibody. Thus, it was confirmed that the target protein was expressed and purified (Fig. 1).

Result 2: WT  In 9-7 cells Taste - Atox1 Fusion protein permeability  Confirm

WT 9-7 cells were treated with 3 μM of Tat-Atox1 fusion protein and analyzed by histidine antibody using western blot analysis after time course. As a result, Tat-Atox1 fusion protein was detected in WT 9-7 It was confirmed that it penetrated into the cell. In contrast, it was confirmed that Atox1 protein did not penetrate into cells (Fig. 2).

In addition, WT 9-7 cells were treated with Tat-Atoxl fusion protein at different concentrations, and the permeation time was fixed to 1 hour. Analysis of this by Western blot analysis using histidine antibody revealed that Tat-Atoxl fusion protein infiltrated WT 9-7 cells in a concentration-dependent manner. In contrast, it was confirmed that Atox1 protein did not penetrate into cells (Fig. 3).

Result 3: WT  In 9-7 cells Taste - Atox1 Fusion protein  Confirm safety after penetration

WT 9-7 cells were treated with 3 μM of Tat-Atox1 fusion protein and the permeation time was fixed at 1 hour. The Tat-Atox1 fusion protein was then removed and the Tat-Atox1 fusion protein infiltrated into the cells was confirmed by Western blot analysis using a histidine antibody to determine how long the fusion protein retained in the cells. As a result, it was confirmed that Tat-Atox1 fusion protein was present in WT 9-7 cells by about 15 hours after infiltration (FIG. 4).

Result 4: WT  In 9-7 cells Taste - Atox1 Fusion protein  Confirm infiltration type after infiltration

WT 9-7 cells were treated with 3 μM of Tat-Atox1 fusion protein and the permeation time was fixed at 1 hour. This was confirmed by confocal microscopy. As a result, it was confirmed that the Tat-Atox1 fusion protein successfully penetrated into WT 9-7 cells. In contrast, it was confirmed that Atox1 protein did not penetrate into cells (Fig. 5).

Result 5: WT  In 9-7 cells Taste - Atox1 Of the fusion protein  Mechanism Research

One) p38 , ERK  Signal mechanism

Western blot analysis was performed on WT 9-7 cells and the p38, ERK signaling mechanism was confirmed using each antibody. As a result, it was confirmed that Tat-Atox1 fusion protein inhibited p38 and ERK signaling in a concentration-dependent manner 6).

2) Akt  Signal mechanism

Western blot analysis was performed on WT 9-7 cells, and the Akt signaling mechanism was confirmed using the antibody. As a result, it was confirmed that the Tat-Atox1 fusion protein inhibited the Akt signal in a concentration-dependent manner (FIG.

3) p65  And IκBα signaling mechanism

Western blot analysis was performed on WT 9-7 cells and the p65, IκBα signaling mechanism was confirmed using each antibody. As a result, it was confirmed that the Tat-Atox1 fusion protein inhibited the p65 and IκBα signals in a concentration-dependent manner 8).

4) COX2 , iNOS , IL -6, IL -1β and TNF -α signal mechanism

Western blot analysis was performed on WT 9-7 cells and the signaling mechanism of COX2, iNOS, IL-6, IL-1β, and TNF-α was examined using each antibody. As a result, Tat- , iNOS, IL-6, IL-1 [beta] and TNF- [alpha] signals (Fig. 9).

5) COX2 , iNOS , IL -6, IL -1β and TNF -α signal mechanism

The expression of COX2, iNOS, IL-6, IL-1β, and TNF-α was examined by RT-PCR using WT 9-7 cells. , iNOS, IL-6, IL-1 [beta] and TNF- [alpha] (Fig. 10).

6) CAS PHASE  3, cut CAS PHASE  3 signaling mechanism

Western blot analysis was performed on WT 9-7 cells, and the results of caspase 3, cleaved caspase 3 signaling mechanism using each antibody were examined. As a result, it was found that Tat-Atoxl fusion protein was expressed in a concentration- It was confirmed that phase 3 was suppressed (FIG. 11).

7) S6K , p- S6K  Signal mechanism

Western blot analysis was performed on WT 9-7 cells to confirm the S6K and p-S6K signaling mechanism using the mTOR antibody. As a result, it was confirmed that the Tat-Atox1 fusion protein inhibited S6K and p-S6K in a concentration-dependent manner (Fig. 12).

From these results, it can be concluded that Atox1 fusion protein can penetrate intracellularly smoothly and show stability for more than 15 hours, and decrease Erk signal and Akt signal. Therefore, Atox1 fusion protein can be used for prevention and treatment of polycystic It is expected to be available as a composition.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for autosomal dominant polycystic          kidney disease containing cell-transducible Atox1 fusion protein <130> HallymU-syCHOI-Atox1-PCKD <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ctcgagatgc cgaagcacg 19 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggatccctac tcaaggccaa gg 22 <210> 3 <211> 270 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Tat-Atox1 fusion protein <400> 3 agcagcggcc tggtgccgcg cggcagccat aggaagaagc ggagacagcg acgaagactc 60 gagatgccga agcacgagtt ctctgtggac atgacctgtg gaggctgtgc tgaagctgtc 120 tctcgggtcc tcaataagct tggaggagtt aagtatgaca ttgacctgcc caacaagaag 180 gtctgcattg aatctgagca cagcatggac actctgcttg caaccctgaa gaaaacagga 240 aagactgttt cctaccttgg ccttgagtag 270 <210> 4 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> Tat-Atox1 fusion protein <400> 4 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Pro Lys His Glu   1 5 10 15 Phe Ser Val Asp Met Thr Cys Gly Gly Cys Ala Glu Ala Val Ser Arg              20 25 30 Val Leu Asn Lys Leu Gly Gly Val Lys Tyr Asp Ile Asp Leu Pro Asn          35 40 45 Lys Lys Val Cys Ile Glu Ser Glu His Ser Met Asp Thr Leu Leu Ala      50 55 60 Thr Leu Lys Lys Thr Gly Lys Thr Val Ser Tyr Leu Gly Leu Glu  65 70 75 <210> 5 <211> 278 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Atox1-Tat fusion protein <400> 5 agcagcggcc tggtgccggc ggcagccact cgagatgccg aagcacgagt tctctgtgga 60 catgacctgt ggaggctgtg ctgaagctgt ctctcgggtc ctcaataagc ttggaggagt 120 taagtatgac attgacctgc ccaacaagaa ggtctgcatt gaatctgagc acagcatgga 180 cactctgctt gcaaccctga agaaaacagg aaagactgtt tcctaccttg gccttgagta 240 gggatcctag gaagaagcgg agacagcgac gaagatag 278 <210> 6 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> Atox1-Tat fusion protein <400> 6 Met Pro Lys His Glu Phe Ser Val Asp Met Thr Cys Gly Gly Cys Ala   1 5 10 15 Glu Ala Val Ser Arg Val Leu Asn Lys Leu Gly Gly Val Lys Tyr Asp              20 25 30 Ile Asp Leu Pro Asn Lys Lys Val Cys Ile Glu Ser Glu His Ser Met          35 40 45 Asp Thr Leu Leu Ala Thr Leu Lys Lys Thr Gly Lys Thr Val Ser Tyr      50 55 60 Leu Gly Leu Glu Arg Lys Lys Arg Arg Gln Arg Arg Arg  65 70 75 <210> 7 <211> 277 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Tat-Atox1-Tat fusion protein <400> 7 aggaagaagc ggagacagcg acgaagactc gagatgccga agcacgagtt ctctgtggac 60 atgacctgtg gaggctgtgc tgaagctgtc tctcgggtcc tcaataagct tggaggagtt 120 aagtatgaca ttgacctgcc caacaagaag gtctgcattg aatctgagca cagcatggac 180 actctgcttg caaccctgaa gaaaacagga aagactgttt cctaccttgg ccttgagtag 240 ggatcctagg aagaagcgga gacagcgacg aagatag 277 <210> 8 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Tat-Atox1-Tat fusion protein <400> 8 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Pro Lys His Glu   1 5 10 15 Phe Ser Val Asp Met Thr Cys Gly Gly Cys Ala Glu Ala Val Ser Arg              20 25 30 Val Leu Asn Lys Leu Gly Gly Val Lys Tyr Asp Ile Asp Leu Pro Asn          35 40 45 Lys Lys Val Cys Ile Glu Ser Glu His Ser Met Asp Thr Leu Leu Ala      50 55 60 Thr Leu Lys Lys Thr Gly Lys Thr Val Ser Tyr Leu Gly Leu Glu Gly  65 70 75 80 Ser Arg Lys Lys Arg Arg Gln Arg Arg Arg                  85 90 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sense primer of COX-2 <400> 9 caaaggcctc cattgaccag a 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer of COX-2 <400> 10 tggaccaggt ttttccaccg 20 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sense primer of IL-1b <400> 11 tgcagagttc cccaactggt actc 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer of IL-1b <400> 12 gtgctgccta atgtcccttg aatc 24 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sense primer of IL-6 <400> 13 caagaaagac aaagccagag tcctt 25 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer of IL-6 <400> 14 tggatggtct tggtccctta gcc 23 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer of TNF-a <400> 15 aagttcccaa atggcctccc 20 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer of TNF-a <400> 16 tggcaccact agttggttgt cttt 24 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sense primer of GADPH <400> 17 tgaaggtcgg tgaacggatt tgg 23 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer of GADPH <400> 18 catgtaggcc atgaggtcca ccac 24

Claims (3)

A pharmaceutical composition for the prevention and treatment of polycysticosis comprising an Atox1 fusion protein in which a protein transport domain is covalently bound to one or more of the N-terminal and C-terminal of an Atox1 (Antioxidant 1) protein.
The method according to claim 1,
Wherein the protein transport domain is an HIV Tat peptide. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
The method according to claim 1,
Wherein the fusion protein is SEQ ID NOS: 4, 6, or 8.

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