KR20170029671A - Pharmaceutical composition for treating Parkinson's disease containing cell-transducible ATOX1 fusion protein - Google Patents
Pharmaceutical composition for treating Parkinson's disease containing cell-transducible ATOX1 fusion protein Download PDFInfo
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- KR20170029671A KR20170029671A KR1020150126033A KR20150126033A KR20170029671A KR 20170029671 A KR20170029671 A KR 20170029671A KR 1020150126033 A KR1020150126033 A KR 1020150126033A KR 20150126033 A KR20150126033 A KR 20150126033A KR 20170029671 A KR20170029671 A KR 20170029671A
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- fusion protein
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- C—CHEMISTRY; METALLURGY
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Abstract
Description
The present invention relates to the protective action of dopamine neurons of ATOX1 fusion proteins, wherein the ATOX1 fusion protein covalently linked to the protein transport domain smoothly migrates into the brain tissue and significantly inhibits reactive oxygen species generation and DNA fragmentation, Indicating that it protects dopamine neurons in the substantia nigra. ATOXl fusion proteins are available for the prevention and treatment of Parkinson ' s disease.
Parkinson's disease is a representative neurodegenerative disorder with signs of stable tremor, stiffness, slow motion, and postural instability. Parkinson's disease is characterized by morphological features such as the specific death of dopaminergic brain cells in Substantia nigra . The causes of active cell tumors (ROS), oxidative stress, abnormal structure of proteins, and mitochondrial damage have been identified, but the exact cause has not yet been elucidated.
ATOX1 (
The present inventors used protein transduction domains (PTDs) to transport proteins into living cells. The present inventors efficiently transduced Tat-Catalase in various cells in the prior art. As a result of experiments using astrocytes, it was found that when Tat-catalase increased, radical scavenger activity was increased. In addition, it has been shown that various transduction fusion proteins function to protect against cell death on Invitro and Invivo.
The present inventors examined whether ATOX1 fusion protein effectively permeates cell membrane and cerebrovascular barrier to show neuroprotective effect in an animal model of Parkinson's disease and aims to provide a pharmaceutical composition for prevention and treatment of Parkinson's disease comprising ATOX1 fusion protein do.
The present inventors have now found that Tat-ATOX1 fusion protein induces dopaminergic apoptosis induced by MPP + and MPTP, and that the Tat-ATOX1 fusion protein induces apoptotic cell death in the Parkinson's disease model Protection efficacy was demonstrated. As a result, it was confirmed that the cell-permeable Tat-ATOX1 fusion protein has potential as an effective protein therapeutic agent in various apoptotic diseases including Parkinson's disease.
The present inventors have examined the protective effect of the Tat-ATOX1 fusion protein in animal models of SH-SY5Y cells and Parkinson's disease. Parkinson's disease is a neurodegenerative disease in which dopaminergic neurons gradually disappear in the substantia nigra of the brain. The inventors have found that Tat-ATOXl fusion proteins can protect dopamine neurons from oxidative stress in SH-SY5Y neuroblastoma cells and Parkinson's disease animal models. The Tat-ATOX1 fusion protein was confirmed to be well transferred into SH-SY5Y cells and into the substantia nigra of the brain by Western blot analysis. The Tat-ATOX1 fusion protein significantly inhibited the production of MPP + -induced reactive oxygen species and DNA fragmentation, resulting in the survival of SH-SY5Y cells. The neuroprotective effect is obtained by the Tat-ATOX1 fusion protein affecting the levels of pro-apoptotic mediator and anti-apoptotic mediator. Further, immunohistochemistry data using TH antibody and cresyl violet staining indicate that Tat-ATOXl fusion protein significantly protects dopamine cells in the substantia nigra against oxidative stress such as MPTP. Thus, the Tat-ATOXl fusion protein is available for the prophylactic and therapeutic use of Parkinson's disease.
The pharmaceutical composition containing the Tat-ATOX1 fusion protein as an active ingredient can be formulated together with a carrier that is conventionally accepted in the pharmaceutical field and can be formulated by oral methods or injections by a conventional method. 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.
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 definitions of the main terms used in the description of the present invention and the like are as follows.
"Tat-ATOX1 fusion protein" refers to a covalent complex formed by genetic fusion or chemical bonding between a protein transport domain and an ATOX1 protein and a protein transport domain and a cargo molecule (i. E., ATOX1 in the present invention) it means. As one specific example in the present specification and drawings, "Tat-ATOX1" refers to the HIV-Tat protein transport domain bound to the N-terminus of the ATOX1 protein among 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 or a nerve 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, HIV-Tat peptide.
As used herein, the term "target protein" means a molecule that is covalently bound to a 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.
The present invention relates to a pharmaceutical composition for the prevention and treatment of Parkinson's disease comprising an ATOX1 fusion protein in which a protein transport domain is covalently bonded to one or more of the N-terminal and C-terminal of a human ATOX1 protein.
In addition, the present invention relates to a pharmaceutical composition for preventing and treating Parkinson's disease containing the ATOX1 fusion protein, wherein the protein transport domain is an HIV-Tat peptide.
In addition, the present invention relates to a pharmaceutical composition for preventing and treating Parkinson's disease containing the ATOX1 fusion protein, wherein the fusion protein is SEQ ID NO: 4.
The ATOX1 fusion protein of the present invention was permeabilized intracellularly in a time-dependent, dose-dependent manner into SH-SY5Y cells, and further maintained intracellular ATOX1 fusion protein level for a considerable period of time.
In addition, the ATOXl fusion protein of the present invention significantly inhibited the production of reactive oxygen species, a response to MPP + .
The ATOXl fusion protein of the present invention can protect cells from oxidative stress induced by MPP + .
The ATOX1 fusion protein of the present invention can effectively cross the cerebral vascular barrier and protects dopamine neurons against MPTP toxicity in MPTP-induced Parkinson's disease mouse models. MPTP-induced dopaminergic neurons It is predicted that it will be possible to reduce motor dysfunction by inhibiting the death.
FIG. 1 shows the result of purification of the control ATOX1 and Tat-ATOX1 fusion protein by SDS-PAGE and Western blotting.
FIG. 2 shows permeation of Tat-ATOX1 fusion protein after treatment of cells with immunofluorescence staining.
FIG. 3 is a graph showing the results of measuring the cell viability by treating Tat-ATOX fusion protein with dopaminergic neurons.
FIG. 4 shows that Tat-ATOX1 fusion protein was treated with SH-SY5Y cells to inhibit production of reactive oxygen species by DCF-DA staining.
Fig. 5 shows the results of TUNEL analysis confirming the DNA fragmentation inhibitory effect of the Tat-ATOX1 fusion protein.
FIG. 6 shows the cytoprotective effect of the Tat-ATOX1 fusion protein by assaying the expression of caspase-3, Bax and Bcl2, which are apoptosis markers.
Figure 7 shows the penetration of Tat-ATOX1 fusion protein into tissue in an animal model of Parkinson's disease.
Figure 8 shows the tissue protective efficacy of the Tat-ATOXl fusion protein in an animal model of Parkinson's disease.
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 by the descriptions of the embodiments. Particularly, as a result of each experimental example, data of Tat-ATOX1 fusion protein among various fusion proteins prepared in the specific examples were described, but other fusion proteins were also similar to the result of using Tat-ATOX1 fusion protein as a sample And the results are shown.
material
Restriction enzymes and T4 DNA ligase were purchased from promega (USA) and Tat-oligonucleotides were synthesized from Gibco BRL (USA). IPTG was purchased from Duchefa (Netherland). The pET15b vector and the BL21 (DE3) plasmid were purchased from Novagen (USA), and the Ni 2 + -nitrile trichlorosaccharide sepharose superfluous column was purchased from Qiagen (Germary). Anti-Akt antibody, anti-ATOX1 antibody and pATOX1 were purchased from Santa Cruz (CA, USA), anti-pAkt primary antibody, Bax rabbit antibody and Bcl-2 rabbit antibody were purchased from Cell Signaling Technology (Denvers, MA, USA) Respectively. Other primary antibodies and secondary anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (USA). Tat-peptide synthesis was custom-made on PEPTRON (Deajeon, Korea). All of the other reagents were made using the express product. Other chemicals and reagents not specified were purchased at the highest level in Sigma-Aldrich (St. Louis, Mo., USA).
Experimental Method
Tat-ATOX1 Of the fusion protein Expression and purification
Tat-expression vectors were prepared as in the prior art of the present inventors. Briefly, human ATOX1 cDNA was amplified by PCR using the following sense and antisense primers. ATOX1 sense primer used: 5'-CTCGAGATGCCGAAGCACG-3 ', ATOX1 antisense primer: 5'-GGATCCCTACTCAAGGCCAAGG-3'. The result obtained by PCR was subcloned into TA vector and ligated to Tat expression vector to prepare Tat-ATOX1 expression vector. Likewise, ATOX1 protein without a Tat peptide was prepared as a control. The recombinant Tat-ATOX1 plasmid was transformed with Escherichia coli BL21 (DE3) and then induced with 0.5 mM IPTG (isopropyl-β-D-thio-galactoside) (Duchefa, Haarlem, Netherlands) and incubated overnight at 18 ° C. Using a nitro reels three seconds acid Sepharose affinity chromatography column and PD-10 column chromatography (Amersham, Pisctaway, NY, USA ) - by crushing the cultured cells by sonication to obtain a Tat-ATOX1 fusion protein Ni 2 + Lt; / RTI > The concentration of the purified protein was measured by the Bradford assay.
Tat-ATOX1 Of the fusion protein SH - SY5Y Neuroblastoma cell permeation
SH-SY5Y human neuroblastoma cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium containing heat-inactivated fetal calf serum (10% FBS) and antibiotics (100 μg / ml streptomycin, 100 U / ml penicillin) % Air and 5% CO 2 And cultured under a humid environment.
In order to observe intracellular permeation of Tat-ATOX1 fusion protein, cultured cells were grown in 6-well plate for 4 to 6 hours, replaced with 1 ml of fresh medium without FBS, and mixed with various concentrations of Tat-ATOX1 fusion Proteins were treated in media. After one hour, the cells were treated with trypsin-EDTA (Gibco Grand Island, NY, USA), washed thoroughly with PBS (phosphate-buffered saline), and the amount and activity of the fusion protein permeated into the cells were analyzed and measured by Western blot .
Fluorescence microscopy analysis
SH-SY5Y human neuroblastoma cells were cultured on cover slips and treated with 3 uM Tat-ATOX1 fusion protein. Then, the cells were incubated at 37 ° C for 2 hours, washed twice with PBS, and fixed with 4% paraformaldehyde for 5 minutes at room temperature. After making the cells permeable, they were blocked with 3% bovine serum albumin, PBS containing 0.1% Triton X-100 (PBS-BT) for 40 minutes, and washed with PBS-BT. The primary antibody (His-probe, Santa Cruze Biotechnology) was diluted 1: 2000 and over-night cultured at 4 ° C. The secondary antibody (Alexa flour 488, Invitrogen) was diluted 1: 15000 and incubated in the dark for one hour at room temperature. Nuclei were stained with 1 μg / ml DAPI (Roche) for 2 minutes. Fluorescence distribution was analyzed with a confocal laser scanning system (Bio-Rad MRC-1024ES).
MTT analysis
The biological activity of the Tat-ATOX1 fusion protein can be measured by determining the number of viable cells after treating the cells with hydrogen peroxide. SH-SY5Y human neuroblastoma cells were plated in 96-well plates at 80%, and the Tat-ATOX1 fusion protein was first treated at a concentration of 0.5 to 3 μM. And treated with 0.8 mM of hydrogen peroxide and cultured for 10 hours. Cell viability was evaluated by color development using MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-dipheyltetrazolium bromide}. Absorbance was measured at 570 nm using an ELISA microplate reader (Labsystems Multiskan MCC / 340) and the cell viability was expressed as a percentage of control cells that did not receive the fusion protein.
Intracellular Active oxygen species Measure
The level of reactive oxygen species in the cells was measured using DCF-DA (2 ', 7'-dichlorodihydrofluorescein diacetate). DCF-DA converts to DCF in cells by reactive oxygen species and emits fluorescence. We compared the levels of reactive oxygen species in the SH-SY5Y human neuroblastoma cells treated with and without the Tat-ATOX1 fusion protein. Tat-ATOX1 fusion protein was treated for 1 hour and then hydrogen peroxide was treated at 0.8 mM for 2 hours. Then, the cells were washed twice with PBS and DCF-DA was treated at a concentration of 20 μM for 1 hour. Fluorescence images were obtained using a fluorescence microscope (Nikon eclipse 80i, Japan).
Western Blat analysis
For Western blot analysis, the proteins in the cell lysate were separated by 12% SDS polyacrylamide gel, and the proteins in the gels were electrochromatically transferred to a nitrocellulose membrane (Amersham, UK). The membranes were blocked with 5% skim milk powder in TBS-T buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1
Confocal Microscopic observation
To detect Tat-ATOX1 fusion protein and ATOX1 protein in SH-SY5Y cells, cells were seeded on glass cover slip and treated with Tat-ATOX1 fusion protein and ATOX1 protein at 3 μM concentration for 3 hours, respectively. Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 5 minutes at room temperature, and incubated with anti-histidine primary antibody and Alexa Fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, Calif., USA). The nuclei were stained with 1 [mu] g / ml of 4'6-diamidino-2-phenylindole (Roche Applied Science, Basel, Switzerland) for 5 minutes. Fluorescence was analyzed with an Olympus FV-300 confocal fluorescence microscope (Olympus, Tokyo, Japan).
TUNEL (Terminal deoxynucleotidyl transferase -mediated dUTP nick-end-labeling) analysis
Tat-ATOX1 fusion protein and 2mM of ATOX1 protein were added to SH-SY5Y cells on glass cover slip, respectively, and incubated at 37 ° C for 3 hours and then exposed to MPP + (4.0 mM) for 14 hours and 30 minutes. TUNEL staining was performed using a cell death kits (Roche Applied Science, Basel, Switzerland). Fluorescence microscopy photographs were taken with an Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan).
Experimental animal
Male, 6 weeks old, C57BL / 6 mice were purchased from Hallym University Laboratory Animals Center. The mice were bred at a constant temperature (23 ° C) and at a constant relative humidity (60%) for 12 hours. Water and feed were freely accessible. All laboratory procedures and controls related to animals have been followed by the National Veterinary Research and Quarantine Service of Korea, which is guided by the Committee on Animal Protection and Utilization. Parkinson 's disease model was constructed as previously studied. Mice were injected with MPTP at a dose of 20 mg / kg every two hours. To confirm the protective effect of Tat-ATOX1 fusion protein on Parkinson's disease, 2.0 mg / kg protein was subcutaneously injected 12 hours before MPTP treatment. Mice were divided into five groups as follows: 1) untreated control group, 2) MPTP treated group, 3) MPTP + Tat-ATOX1 fusion protein treated group, 4) MPTP + PEP-1 Peptide treated group, and 5) MPTP + control ATOXl protein treated group. Mice were sacrificed one week after the last injection.
Immunohistochemistry
Immunostaining was performed as in the previous study. Tissue sections were blocked for 30 min at room temperature in PBS containing 3% bovine serum albumin, blocked to detect histidine antibodies or dopamine neurons to detect Tat-ATOX1 fusion protein and ATOX1 protein, and tyrosine hydroxylase TH) antibody. Biotin - conjugated goat anti - rabbit antibody was used as a secondary antibody. Cresyl violet staining was performed to stain Nissl bodies, which are granules found in living neurons and considered as rough endoplasmic reticulum. The sections were visualized with DAB (3,3'-diaminobenzidine) in 0.1 M Tris buffer and placed on gelatin-coated slides. The images were taken and analyzed with an Olympus DP72 digital camera and DP2-BSW microscope digital camera software. The drawing was made using Adobe Photoshop 7.0 (San Jose, CA, USA).
Statistical analysis
Data were expressed as mean ± standard deviation from each independent experiment. The differences between the mean values were analyzed using one - way ANOVA. Newman-Keuls post hoc analyzes were employed when differences were observed in the ANOVA test ( p <0.01).
Result 1: Tat-ATOX1 Of the fusion protein Purification and cell permeation
In order to prepare a cell-permeable Tat-ATOX1 fusion protein, Tat vector and ATOX1 gene containing the HIV-Tat peptide coding sequence, which is a type of protein transport domain, were recombinantly overexpressed in E. coli to produce Ni 2 + -nitriloacetic acid sepharose affinity column And PD-10 column chromatography, and confirmed by SDS-PAGE and Western blotting (FIG. 1).
In order to confirm whether Tat-ATOX1 fusion protein smoothly penetrates into cells, it was treated with SH-SY5Y human neuroblastoma cells having properties of dopaminergic neurons to stain green fluorescence with Tat-ATOX1 fusion protein specific antibody, And confirmed by observable confocal microscopy. As a result, the intracellular permeation of Tat-ATOX1 fusion protein occurred effectively, and the negative control and the control ATOX1 protein without Tat did not permeate (FIG. 2).
Result 2: MPP + Lt; RTI ID = 0.0 > Tat- ATOX1 Of the fusion protein Inhibitory effect
The inhibition of cell death by Tat-ATOX1 fusion protein was confirmed by MTT assay, which can specifically identify living cells, by inducing apoptosis by treating MPP + , a cell death-inducing factor, in a Parkinson's disease model. In the case of SH-SY5Y cells treated with MPP + alone, the number of viable cells decreased to about 55%, but when the Tat-ATOX1 fusion protein was treated, the cell viability was increased in a concentration-dependent manner. On the other hand, it was confirmed that there was no change in cell viability when ATOX1 protein having no cell penetration ability was treated (FIG. 3).
In addition, in order to confirm that Tat-ATOX1 fusion protein effectively suppresses the active oxygen species induced by MPP + treatment, H 2 DCFDA (2 ', 7'-dichlorofluorescin diacetate) (2 ', 7'-dichloro-fluorescein) by hydrolysis and hydrolysis of impermeable H 2 DCF (2', 7'-dichlorofluorescin) by reaction with DCF-DA staining method Respectively.
In the MPP + untreated cells, reactive oxygen species were generated to show green fluorescence, whereas in the pretreatment of Tat-ATOX1 fusion protein, production of reactive oxygen species in the cells was inhibited by fluorescence microscopy (FIG. 4). In order to confirm DNA fragmentation induced by active oxygen species, TUNEL staining was performed by attaching fluorescent pigment to the cut DNA fragment to confirm DNA fragmentation. As in the case of the DCF-DA staining method, it was confirmed that the increased DNA fragment obtained by single treatment of MPP + was very effectively inhibited when the Tat-ATOX1 fusion protein was treated (FIG. 5).
The expression of caspase-3 and Bax, Bcl2, which are markers of apoptosis induced by MPP + , was confirmed by treatment of Tat-ATOX1 fusion protein. It was found that the truncated caspase-3 and Bax induced by MPP + decreased in concentration-dependent manner as Tat-ATOX1 fusion protein was treated, confirming that Bcl2 increased. Conversely, there was no change in the treatment with the control ATOX1 protein without Tat (Fig. 6).
Result 3: Cell permeability Tat- ATOX1 Protective Effects of Fusion Proteins in Animal Model of Parkinson's Disease
Cell permeability to Parkinson's disease To investigate the protective effect of Tat-ATOX1 fusion protein, C57BL / 6 male mice were injected with 2 mg / kg of Tat-ATOX1 fusion protein and ATOX1 protein, respectively, and induced Parkinson's disease MPTP was injected intraperitoneally to produce an animal model of Parkinson 's disease.
It was confirmed that the fusion protein penetrated the blood-brain barrier (SN) region where dopaminergic neurons were present. As a result of tissue immunofluorescence staining, it was confirmed that the control ATOX1 protein did not infiltrate, but when Tat-ATOX1 fusion protein was treated, it penetrated into brain tissue effectively (FIG. 7). CV (Cresyl Violet) staining for staining only living cells and TH (Tyrosine Hydroxylase) staining for dopaminergic neurons were confirmed by immunohistochemical staining. In animals treated with Tat-ATOX1 fusion protein for 4 days after induction of Parkinson's disease, protective effects against dopaminergic neuron apoptosis were shown, whereas no protection against cell death was observed in animals treated with control ATOX1 protein (Fig. 8).
From these results, it was confirmed that Tat-ATOX1 fusion protein effectively penetrates into cells and tissues and protects against dopaminergic neuronal apoptosis and Parkinson's disease caused by MPP + and MPTP. Thus, the present inventors have found that Tat-ATOX1 fusion protein Suggesting the possibility of a therapeutic agent for Parkinson's disease.
<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for treating Parkinson's disease containing cell-transducible ATOX1 fusion protein <130> HallymU-syCHOI-Atox1-PD <160> 8 <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
Claims (3)
Wherein the protein transport domain is an HIV-Tat peptide. ≪ RTI ID = 0.0 > 11. < / RTI >
Wherein said fusion protein is SEQ ID NO: 4. 4. A pharmaceutical composition for preventing and treating Parkinson's disease comprising the ATOX1 fusion protein, wherein said fusion protein is SEQ ID NO:
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