KR20210114579A - Pharmaceutical composition for treating cerebral ischemia containing endophilin-A1 fusion protein - Google Patents

Abstract

The present invention is directed to providing a pharmaceutical preparation for treating brain ischemia which has little or no side effects and shows an excellent effect. The present invention relates to a pharmaceutical composition for treating brain ischemia including cytopermeable endophilin-A1 fusion protein. According to the present invention, the endophilin-A1 fusion protein permeated into cells increases the cell survival rate against hydrogen peroxide toxicity, and reduces the level of intracellular active oxygen species and DNA fragmentation induced by hydrogen peroxide. In an animal model, the endophilin-A1 fusion protein shows a protective effect against the nerve cell apoptosis occurring when transient ischemia is induced at the site of forebrain hippocampus CA1. Such results suggest that the endophilin-A1 fusion protein has a protective effect against apoptosis and a therapeutic effect of protecting nerve cells from brain ischemia damages.

Description

A pharmaceutical composition for treating cerebral ischemia comprising a cell-permeable endophylline-A1 fusion protein {Pharmaceutical composition for treating cerebral ischemia containing endophilin-A1 fusion protein}

The present invention relates to a pharmaceutical composition for treating cerebral ischemia comprising a cell-permeable endophylline-A1 fusion protein.

"Free radicals" called reactive oxygen compounds are responsible for oxidative stress, and reactive oxygen species (ROS) are unavoidable by-products of various cellular processes, including interactions with oxygen. . If cells are exposed to relatively more active compounds, they are subjected to oxidative stress, resulting in oxidative damage to important elements such as proteins, DNA, and fats. Oxidative stress is the cause of many diseases, such as cancer and Alzheimer's, and these diseases are also associated with aging.

Excessive reactive oxygen species generated from abnormal cellular processes are known to cause various human diseases such as inflammation, ischemia, diabetes and Parkinson's disease. During the metabolic process of cells, reactive oxygen species have many effects such as damaging not only DNA in cells but also macromolecules such as proteins and lipids. Among them, cerebral ischemia is a disease in which brain cells do not produce enough energy when an artery in the brain is blocked, resulting in apoptosis. Hypoxia conditions such as cerebral ischemia induce the translocation of cytoplasmic Bax to the outer membrane mitochondria and then promote the release of anti-apoptotic factors from the mitochondria with a decrease in mitochondrial membrane potential. In contrast, Bcl-2 functions to block cytochrome c release from mitochondria and prevent apoptosis. Under hypoxia, the expression level of Bcl-2 is greatly reduced, which is a major cause of apoptosis. Therefore, it is expected that if a substance with the ability to regulate reactive oxygen species is found, it will be able to show a protective effect against cerebral ischemia damage.

Endophilin-A1 (endophilin-A1) is encoded by the SH3GL2 gene, and maintains homeostasis by mediating protein-protein interactions that are important for endocytosis of epidermal growth factor receptor (EGFR). Downregulation of endophylline-A1 is associated with the development of cancer. The SH3GL2 gene, encoding endophyllin-A1, is a tumor suppressor gene. Endophylline-A1 is mainly distributed in the central nervous system and is particularly concentrated in presynaptic ganglia. Endophylline-A1 plays an important biological role in cell proliferation, differentiation and other physiological functions, mainly through SH3 domain proteins and other downstream proteins.

However, the function or effect of endophyllin-A1 on neuronal apoptosis by reactive oxygen species has not yet been clearly elucidated.

An object of the present invention is to provide an effective therapeutic agent for treating cerebral ischemic injury and nerve cell damage and death caused by cerebral ischemic injury.

In order to solve the above problems, the present inventors recombined protein transport domains such as PEP-1 and HIV Tat peptide with endophyllin-A1 protein and confirmed that they penetrate into HT22 cells, and cell-penetrating endophyllin-A1 fusion against oxidative stress The protective effect of the protein was studied.

The present inventors tried to determine whether the endophyllin-A1 fusion protein bound to the protein transport domain has a protective effect on oxidative stress-induced neuronal cell death in vitro. To study the potential effect of the endophyllin-A1 fusion protein on ischemic neuronal apoptosis, the present inventors constructed an endophyllin-A1 fusion protein that can penetrate into cells. The endophyllin-A1 fusion protein effectively penetrated into neurons in a concentration-dependent and time-dependent manner. The endophyllin-A1 fusion protein permeated into the cell increased the cell viability against hydrogen peroxide toxicity and lowered the level of intracellular reactive oxygen species. These results suggest that the endophyllin-A1 fusion protein exhibits a protective effect against apoptosis in vitro and can be used as a therapeutic agent for oxidative stress-related brain ischemia damage.

To describe the configuration of the present invention in more detail, in an embodiment of the present invention, the present inventors first developed an endophylline-A1 fusion protein expression vector capable of overexpressing and easily purifying the endophylline-A1 fusion protein. This expression vector contains cDNA capable of expressing human endophyllin-A1 protein, at least one protein transport domain consisting of 7 to 21 amino acids, and 6 histidine residues at the N-terminus. As an example, the amino acid sequence of the endophyllin-A1 fusion protein is characterized as SEQ ID NO: 1 in the sequence listing.

Using this expression vector, the endophyllin-A1 fusion protein was overexpressed in E. coli and purified using Ni-affinity chromatography. It was confirmed by Western blot that the endophyllin-A1 fusion protein was transported to the cells in a time- and concentration-dependent manner in the cultured cells. The endophyllin-A1 fusion protein permeated into the cell was continuously maintained for up to 12 hours in the cell, and suppressed apoptosis caused by oxidative stress.

These results indicate that the endophyllin-A1 fusion protein is well permeated into the cell and that the function of the endophyllin-A1 protein is well expressed in the cell. Therefore, this endophylline-A1 fusion protein suggests the possibility of application to cranial nerve diseases, such as protecting nerve cells from reactive oxygen species-related symptoms or cerebral ischemia damage.

The pharmaceutical composition containing the cell-penetrating endophylline-A1 fusion protein as an active ingredient may be formulated in oral or injection form by a conventional method by mixing with a carrier commonly acceptable in the pharmaceutical field. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, contain diluents (eg lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), glidants (eg silica, talc). , stearic acid and its magnesium or calcium salts and/or polyethylene glycol), and tablets also contain binders (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone) ), preferably containing a disintegrant (eg, starch, agar, alginic acid or sodium salt thereof) or a boiling mixture and/or absorbent, colorant, flavoring agent and sweetening agent as the case may be. Compositions for injection are preferably isotonic aqueous solutions or suspensions, the compositions mentioned are sterile and/or contain adjuvants (eg preservatives, stabilizers, wetting or emulsifying agents, solution promoters, salts/or buffers for regulating the osmotic pressure). In addition, they may contain other therapeutically useful substances.

The pharmaceutical preparation prepared as described above may be administered orally, or parenterally, ie, intravenously, subcutaneously, intraperitoneally, or applied topically, as desired. The dose may be administered in one to several divided doses of 0.0001 to 100 mg/kg per day. The dosage level for a specific patient may vary depending on the patient's weight, age, sex, health status, administration time, administration method, excretion rate, severity of disease, and the like.

Furthermore, the present invention provides a pharmaceutical composition useful for preventing or treating cerebral ischemia, comprising the endophylline-A1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides a method for efficiently delivering the endophyllin-A1 protein into a cell. The intracellular delivery of the endophyllin-A1 protein molecule according to the present invention is performed by constructing a fusion protein in which a protein transport domain such as an HIV Tat peptide is covalently bound. An example of the protein transport domain of the present invention may be an HIV Tat peptide. However, the protein transport domain of the present invention is not limited only to the HIV Tat peptide, and it is in the field of the present invention to prepare a peptide having a function similar to that of the HIV Tat peptide by partial substitution, addition, or lack of amino acid sequence of the HIV Tat peptide. Since it is easy for those of ordinary skill in the art, it is a protein transport domain composed of 7 to 21 amino acids and comprising four or more lysine or arginine and a protein transport that performs the same/similar protein transport function by substituting some amino acids therefrom It will be apparent that fusion proteins using domains also fall within the scope of the present invention.

Specifically, the present invention relates to an endophylline-A1 fusion protein, an oligonucleotide encoding an endophylline-A1 fusion protein, a vector comprising an oligonucleotide encoding an endophylline-A1 fusion protein, a treatment for cerebral ischemia comprising the fusion protein, or It relates to a pharmaceutical composition for the purpose of prevention and a health functional food composition for preventing or improving cerebral ischemia comprising the fusion protein.

The polynucleotide encoding the endophylline-A1 fusion protein is specifically characterized in that it is SEQ ID NO: 1.

Foods to which the cell-permeable endophylline-A1 fusion protein of the present invention can be added include various foods, for example, beverages, gum, tea, vitamin complexes, health supplements, and the like, and include pills, powders, granules, It can be used in the form of pills, tablets, capsules or beverages. In this case, the amount of the endophylline-A1 fusion protein in the food or beverage is generally 0.01 to 15% by weight, preferably 0.2 to 10% by weight of the total food weight in the case of the health food composition of the present invention. In the case of a beverage composition, it may be added in a ratio of 0.1 to 30 g, preferably 0.2 to 5 g, based on 100 ml. The health drink composition of the present invention has no particular limitation on the liquid component except that it contains the endophylline-A1 fusion protein as an essential component in the indicated ratio, and various flavoring agents or natural carbohydrates, such as a conventional beverage, are added. It can contain as an ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides, for example, disaccharides such as glucose and fructose, for example, polysaccharides such as maltose and sucrose, for example, conventional sugars such as dextrin, cyclodextrin, etc., and xylitol , sorbitol, and sugar alcohols such as erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention. In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts. salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Definitions of key terms used in the detailed description of the present invention are as follows.

The term "endophyllin-A1 fusion protein" includes a protein transport domain and an endophyllin-A1 protein, and a genetic fusion or chemical bond between the protein transport domain and a target protein (that is, in the present invention, it refers to the endophyllin-A1 protein) means a covalent complex formed by In the present specification, since the HIV-Tat protein transport domain was used as a specific example, "Endophilin-A1 fusion protein" was used interchangeably with "Tat-SH3GL2", "Tat-SH3GL2 fusion protein", and the like.

"Target protein" refers to a molecule that is not a transport domain or fragment thereof that cannot originally enter a target cell or cannot enter a target cell at an intrinsically useful rate, the molecule itself or transport domain-target protein complex before fusion with the transport domain. refers to the target protein portion. The target protein includes a polypeptide, a protein, and a peptide, and in the present invention refers to endophyllin-A1.

The term "fusion protein" refers to a complex comprising a transport domain and one or more target protein portions, and formed by genetic fusion or chemical bonding of the transport domain and target protein.

In addition, the "genetic fusion" refers to a linear, covalent linkage formed through genetic expression of a DNA sequence encoding a protein.

In addition, "target cell" refers to a cell to which a target protein is delivered by a transport domain, and the target cell refers to a cell in or outside the body. That is, the target cell means a cell in the body, that is, a cell constituting an organ or tissue of a living animal or human, or a microorganism found in a living animal or human. In addition, the target cell is meant to include cells in vitro, that is, cultured animal cells, human cells or microorganisms.

"Protein transport domain" in the present invention is a high molecular weight organic compound, such as oligonucleotide, peptide, protein, oligosaccharide or polysaccharide, etc. covalently bound to introduce the organic compounds into the cell without the need for a separate receptor, carrier, or energy. say what you can

In addition, in this specification, the expressions "transport", "penetration", "transport", "transfer", "permeation", and "pass" are used interchangeably with respect to "introduction" of a protein, peptide, or organic compound into a cell.

The endophylline-A1 fusion protein of the present invention is composed of 7 to 21 amino acid residues, and a transport domain containing 3/4 or more of arginine or lysine residues is covalently bound to at least one end of endophylline-A1 to improve cell penetration efficiency Endophylline-A1 fusion protein. In addition, the transport domain refers to one or more of HIV Tat 49-57 residues, Pep-1 peptide, oligolysine, oligoarginine, or oligo (lysine, arginine).

In addition, the amino acid sequence of the endophyllin-A1 fusion protein of the present invention includes SEQ ID NO: 4, 6 or 8, and the like. In preparing the endophyllin-A1 fusion protein, fusion proteins of various sequences can be obtained according to the selection of restriction site sequences, etc., which is apparent to those of ordinary skill in the art to which the present invention pertains. It is apparent that the above amino acid sequence is merely exemplary, and the amino acid sequence of the endophyllin-A1 fusion protein is not limited to the above-listed sequences.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cerebral ischemia, comprising the endophylline-A1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

In addition, the present invention provides a health functional food composition comprising the endophylline-A1 fusion protein as an active ingredient, and for preventing or improving brain ischemia.

The present invention is composed of 7 to 21 amino acids and a cell-transducing endophylline-A1 fusion in which a protein transport domain comprising 4 or more lysine or arginine is covalently linked to at least one end of the endophylline-A1 protein It's about protein. In addition, in the endophylline-A1 fusion protein, one or more amino acids in the sequence may be substituted with other amino acid(s) of similar polarity that function equally and functionally according to silent change. An amino acid substitution in a 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. Negatively charged acidic amino acids 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 between the fusion protein of the present invention and the amino acid sequence, such as 85-100%, are also included in the scope of the present invention.

In the present invention, the endophyllin-A1 fusion protein permeated into the cell increased the cell viability against hydrogen peroxide toxicity. These results indicate that the endophyllin-A1 fusion protein has a protective effect against apoptosis caused by cerebral ischemia due to reactive oxygen species, and has a therapeutic effect to protect nerve cells from cerebral ischemia damage. This indicates that the protein is useful as a pharmaceutical composition for preventing and treating cerebral ischemia.

1 shows the construction of the Tat-endophyllin-A1 expression vector on the pET-15b vector. Synthetic Tat oligomers were cloned into the Nde I and Xho I sites, and human endophyllin-A1 cDNA was cloned into the Xho I and BamHI sites of pET-15b. (A) shows the construction of expression vectors of endophyllin-A1 and Tat-endophyllin-A1 fusion proteins. The Tat-endophyllin-A1 fusion protein contains six histidine residues. (B) was expressed by adding IPTG. The purified endophyllin-A1 and Tat-endophyllin-A1 fusion proteins were purified using 15% SDS-PAGE and western blotted with anti-rabbit polyhistidine antibody.
Figure 2 shows the permeation of Tat-endophyllin-A1 fusion protein into HT22 cells. (A) is Tat-endophyllin-A1 fusion protein (0.5-5 μM) and endophyllin-A1 protein (0.5-5 μM) are treated in culture medium for 1 hour, (B) is Tat-endophyllin-A1 fusion protein And endophyllin-A1 protein was treated in the culture medium for 15 to 60 minutes and analyzed by Western blot. (C) The intracellular distribution of the Tat-endophyllin-A1 fusion protein was visualized by confocal fluorescence microscopy. (D) The stability of the Tat-endophyllin-A1 fusion protein transduced into HT22 cells was evaluated. The Tat-endophyllin-A1 fusion protein (5 μM) transduced into the cells was cultured for 1 to 60 hours, followed by western blotting.
FIG. 3a shows that hydrogen peroxide (100 μM) was added to HT22 cells pretreated with Tat-endophyllin-A1 fusion protein and endophylline-A1 protein for 1 hour, and left for 1 hour. Cell viability was determined by colorimetric assay using MTT.
Figure 3b shows the intracellular reactive oxygen species level was measured by DCF-DA staining.
Figure 3c shows DNA fragmentation was measured by TUNEL staining.

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 description of the examples. In particular, in the embodiment of the present invention, HIV Tat peptide was used as the protein transport domain constituting the endophyllin-A1 fusion protein, but those of ordinary skill in the art to which the present invention pertains, the PEP-1 peptide is N-terminal. By applying the endophyllin-A1 fusion protein bound to the endophylline-A1, various protein transport domains such as PEP-1 peptide, oligoarginine, oligolysine and oligo (arginine + lysine) can be transferred to either the N-terminus or the C-terminus of endophyllin-A1. A fusion protein bound to one or more sites can be easily formed, and although there may be slight differences between such fusion proteins, it can be easily seen that they exhibit similar activities.

ingredient

Ni ²⁺ -nitrilostriacetic acid sepharose superflow was purchased from Qiagen, and PD-10 column chromatography (Amersham, Brauncschweig, Germany) was purchased. Mouse hippocampal nerve cells, HT22 cells, were provided by the Korea Cell Line Research Foundation (KCLF). 2',7'-DCF-DA (Dichlorofluorescein diacetate) and Bcl-2, Bax, β-actin, P-p53, p53, Casphase 3, Casphase 9, p-p38, p38, p-Erk, Erk, p-JNK and primary antibodies to JNK were purchased from Cell signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). For all other reagents, special grade products were used.

Expression and purification of Tat-endophyllin-A1 fusion protein

Expression vectors of the endophyllin-A1 protein and the Tat-endophyllin-A1 fusion protein were constructed. The SH3GL2 gene encoding the human endophyllin-A1 protein was amplified by polymerase chain reaction (PCR) using two primers together with cDNA. The sense primer consists of 5'-CTCGAGGGCAACGCGCAG-3', and a restriction enzyme site called Xho1 exists on the 5' side. And the antisense primer consists of 5'-GGATCCTCAGGAATCTTCGGACTC-3', and a restriction enzyme action site called BamH1 is present on the 5' side. The result obtained through PCR was ligated to a TA vector, cut using Xho1 and BamH1, and then linked to an expression vector to prepare a Tat-SH3GL2 fusion protein. Likewise, the control endophyllin-A1 protein was prepared using a vector lacking the Tat peptide. After transforming the recombinant Tat-endophyllin-A1 plasmid into E. coli BL21, it was induced with 0.5 mM IPTG (isopropyl-β-D-thiogalactoside) and cultured overnight at 18°C. The cultured cells were pulverized by ultrasonication to obtain a Tat-endophyllin-A1 fusion protein and purified using a ^{Ni 2+- Nitrilotriacetate Sepharose Superflow column.} Protein concentration was determined using the Bradford method using bovine serum albumin as a standard material.

Tat-endophyllin-A1 fusion protein introduced into HT22 cells

Mouse hippocampal neurons, HT22 cells, maintain conditions of 37°C, 95% air and 5% CO2, 10% fetal calf serum (FBS) and 5 mM NaHCO ₃ , antibiotics (100 mg/ml streptomycin, 100 U/ml penicillin) , and cultured under humid conditions using DMEM (Dulbecco's Modified Eagle's Medium) composed of 20 mM HEPES/NaOH (pH 7.4).

The time dependence and concentration dependence of the Tat-endophyllin-A1 fusion protein and the control endophyllin-A1 protein into cells were evaluated. Cells were treated in a 60 mm dish at different times (15 to 60 minutes) and at different doses (0.5 to 5 μM) of each protein. After treatment with trypsin-EDTA (Gibco) and washing with PBS, the fusion protein permeated into the cells was quantified by the Bradford method, and analyzed by Western blot.

Western blot analysis

For Western blot analysis, proteins in the cell suspension were separated using a 12% SDS polyacrylamide gel, and then the proteins in the gel were electrophoresed on a nitrocellulose membrane (Amersham, UK). The membrane was blocked with TBS-T buffer (25 mM Tris-HCl, 140 mM NaCl, 01% Tween 20, pH 7.5). Membranes were analyzed by western blot using the primary antibody recommended by the manufacturer. Bound antibody complexes were detected using an enhanced chemiluminescent agent according to the manufacturer's instructions (Amersham, Franklin Lakes, NJ, USA).

confocal microscopy

To detect Tat-endophyllin-A1 fusion protein and endophyllin-A1 protein in HT22 cells, cells were seeded on glass coverslips, and Tat-endophyllin-A1 fusion protein and endophyllin-A1 protein were added at a concentration of 5 μM for 1 hour. treated during Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 5 minutes at room temperature. HT22 cells were blocked with PBS (PBS-BT) containing 3% bovine serum albumin and 01% Triton X-100 for 40 minutes at room temperature, and washed with PBSBT. The primary antibody (His-probe, Santa Cruz Biotechnology) was diluted at a ratio of 1:10000, and the secondary antibody (Alexa fluor 488, Invitrogen) was diluted at a ratio of 1:15000 and incubated for 1 hour at room temperature in the dark. Cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole 1 mg/ml; Roche, Mannheim, Germnay) for 3 minutes. Stained HT22 cells were observed under an Fv-300 confocal fluorescence microscope (Olympus, Tokyo, Japan).

Cell viability assay

The viability of HT22 cells treated with hydrogen peroxide was confirmed by colorimetric analysis using MTT {3-(4,5-dimethylthiazol -2-yl)-2,5-dipheyltetrazolium bromide}. Cell death was induced by exposing the cells to 100 μM hydrogen peroxide for 1 hour, with or without pre-treating the cells with 0.5-5 μM of Tat-endophyllin-A1 fusion protein. Absorbance was measured at 570 nm with an ELISA microplate reader (Labsystems Multiskan MCC/340). Cell viability was expressed as a percentage relative to the untreated control.

Measurement of intracellular reactive oxygen species levels

Intracellular reactive oxygen species levels were measured using DCF-DA (2',7'-dichlorodihydrofluorescein diacetate). DCF-DA is converted to DCF in cells by reactive oxygen species and fluoresces. The level of reactive oxygen species when HT22 cells were treated with Tat-endophyllin-A1 fusion protein and when not treated was compared. Tat-endophyllin-A1 fusion protein was treated with 5 μM for 1 hour and then hydrogen peroxide was treated with 700 μM for 30 minutes. And washed twice with PBS and treated with DCF-DA at a concentration of 20 μM for 30 minutes. Fluorescence intensity was measured using a Fluoroskan ELISA plate reader (Labsystems, Helsinki, Finland) at 485 nm excitation wavelength and 538 nm emission wavelength.

TUNEL analysis

HT22 cells were treated with hydrogen peroxide (100 μM) by adding hydrogen peroxide (100 μM) to the culture medium of the group not treated with Tat-endophyllin-A1 fusion protein and the group treated at a concentration of 5 μM for 1 hour. To measure apoptosis, TUNEL (Terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling staining) technique was performed using an apoptosis detection kit (Roche Applied Science). The results were analyzed using a fluorescence microscope (Nikon eclipse 80i, Japan).

Result 1: Purification and cell permeation of Tat-endophyllin-A1 fusion protein

The present inventors recombined the SH3GL2 gene with the Tat-vector containing Tat, which is a type of protein transduction domain (PTD), to prepare a permeable Tat-endophyllin-A1 fusion protein (FIG. 1A). It was overexpressed in Escherichia coli and ^{purified using Ni 2+} -nitrilotriacetic acid sepharose affinity chromatography column and PD-10 column chromatography, and Tat-endophyllin-A1 fusion using SDS-PAGE and Wespun blot analysis. It was confirmed that the protein was purified (FIG. 1A, B).

Result 2: Permeation of Tat-endophyllin-A1 fusion protein into HT22 cells

The degree of permeation into HT22 cells was measured for each time and concentration of the Tat-endophyllin-A1 fusion protein. As a result, intracellular permeation occurred effectively in a time- and concentration-dependent manner, and permeation did not occur in the endophylline-A1 protein (FIG. 2A, B). In addition, cell nuclei were stained with DAPI using a confocal microscope, and the Tat-endophyllin-A1 fusion protein was stained with green fluorescence to confirm effective penetration into cells ( FIG. 2C ).

Result 3: Cell protective effect of Tat-endophyllin-A1 fusion protein on cell viability, reactive oxygen species generation and DNA fragmentation due to oxidative stress

Cells were pretreated with Tat-endophyllin-A1 fusion protein by concentration, and then oxidative stress was induced with hydrogen peroxide. MTT assay was performed to confirm cell viability. When HT22 cells were treated with 100 μM hydrogen peroxide, the number of viable cells decreased to about 40%, but when Tat-endophyllin-A1 was pretreated, the cell viability increased in a concentration-dependent manner ( FIG. 3A ).

In addition, in order to confirm that the Tat-endophyllin-A1 fusion protein effectively inhibits reactive oxygen species induced when hydrogen peroxide is treated, the degree of intracellular oxidation was analyzed using 8-OHdG fluorescent dye. When HT22 cells were exposed to 100 μM hydrogen peroxide for 1 hour, the 8-OHdG signal was significantly increased by hydrogen peroxide. However, the reactive oxygen species induced by hydrogen peroxide was decreased by the Tat-endophyllin-A1 fusion protein (FIG. 3B).

The protective effect of the fusion protein against DNA fragmentation caused by oxidative stress was investigated by TUNEL staining using a fluorescent dye that specifically attaches to the DNA fragmentation site. It was performed under the same conditions as the previous experiment, and when HT22 cells were exposed to 100 μM hydrogen peroxide for 1 hour, it was confirmed that the Tat-endophyllin-A1 fusion protein had a protective effect against DNA fragmentation (FIG. 3C) .

Therefore, the present invention suggests the possibility that the Tat-endophyllin-A1 fusion protein can be effectively applied as a therapeutic agent for various diseases and neurodegenerative diseases such as cerebral ischemia due to oxidative stress.

<110> Industry Academic Cooperation Foundation, Hallym University <120> A pharmaceutical composition containing cell-transducing endophilin-A1 fusion protein for preventing and treating neuronal disorder <130> HallymU-SYChoi-SH3GL2 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1092 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding Tat-endophilin-A1 fusion protein <400> 1 aggaagaagc ggagacagcg acgaagactc gagatgtcgg tggccggcct caagaagcag 60 ttccataaag ccactcagaa agtgagtgag aaggttggag gagctgaagg aaccaagcta 120 gatgatgact tcaaagagat ggaaaggaaa gtggatgtca ccagcagggc tgtgatggaa 180 ataatgacta aaacaattga ataccttcaa cccaatccag cttccagagc taagctcagc 240 atgatcaaca ccatgtcaaa aatccgtggc caggagaagg ggccaggcta tcctcaggca 300 gaggcgctgc tggcagaggc catgctcaaa tttggaagag agcttggaga tgattgcaac 360 tttggcccag cacttggtga ggtcggggag gccatgcggg aactgtcgga ggtcaaagac 420 tctttggaca tagaagtgaa gcagaacttc attgaccctc ttcagaatct tcatgacaaa 480 gatcttaggg aaattcaaca tcatctaaag aagttggagg gtcgacgcct ggattttgat 540 tataagaaga aacgacaagg caagattccg gatgaagagc ttcgtcaagc tctagagaaa 600 tttgatgagt ctaaggaaat tgctgagtca agcatgttca atctcttgga gatggatatt 660 gaacaagtga gccagctctc tgcacttgtg caagctcagc tggagtacca caagcaggca 720 gtccagatcc tgcagcaagt cacggtcaga ctggaagaaa gaataagaca ggcttcatct 780 cagcctagaa gggaatatca acctaaacca cgaatgagcc tggagtttcc aactggagac 840 agtactcagc ccaatggggg tctctcccac acaggcactc ccaaaccttc aggtgtccaa 900 atggatcagc cctgctgccg agctctgtac gactttgaac ctgaaaatga aggggagttg 960 ggatttaaag agggcgatat catcacactc actaaccaaa ttgatgagaa ctggtatgag 1020 gggatgctgc atggccattc aggcttcttc cccatcaatt atgtggaaat tctggttgcc 1080 ctgccccatt ag 1092 <210> 2 <211> 271 <212> PRT <213> Artificial Sequence <220> <223> Tat-endophilin-A1 fusion protein <400> 2 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ser Val Ala Gly 1 5 10 15 Leu Lys Lys Gln Phe His Lys Ala Thr Gln Lys Val Ser Glu Lys Val 20 25 30 Gly Gly Ala Glu Gly Thr Lys Leu Asp Asp Asp Phe Lys Glu Met Glu 35 40 45 Arg Lys Val Asp Val Thr Ser Arg Ala Val Met Glu Ile Met Thr Lys 50 55 60 Thr Ile Glu Tyr Leu Gln Pro Asn Pro Ala Ser Arg Ala Lys Leu Ser 65 70 75 80 Met Ile Asn Thr Met Ser Lys Ile Arg Gly Gln Glu Lys Gly Pro Gly 85 90 95 Tyr Pro Gln Ala Glu Ala Leu Leu Ala Glu Ala Met Leu Lys Phe Gly 100 105 110 Arg Glu Leu Gly Asp Asp Cys Asn Phe Gly Pro Ala Leu Gly Glu Val 115 120 125 Gly Glu Ala Met Arg Glu Leu Ser Glu Val Lys Asp Ser Leu Asp Ile 130 135 140 Glu Val Lys Gln Asn Phe Ile Asp Pro Leu Gln Asn Leu His Asp Lys 145 150 155 160 Asp Leu Arg Glu Ile Gln His His Leu Lys Lys Leu Glu Gly Arg Arg 165 170 175 Leu Asp Phe Asp Tyr Lys Lys Lys Arg Gln Gly Lys Ile Pro Asp Glu 180 185 190 Glu Leu Arg Gln Ala Leu Glu Lys Phe Asp Glu Ser Lys Glu Ile Ala 195 200 205 Glu Ser Ser Met Phe Asn Leu Leu Glu Met Asp Ile Glu Gln Val Ser 210 215 220 Gln Leu Ser Ala Leu Val Gln Ala Gln Leu Glu Tyr His Lys Gln Ala 225 230 235 240 Val Gln Ile Leu Gln Gln Val Thr Val Arg Leu Glu Glu Arg Ile Arg 245 250 255 Gln Ala Ser Ser Gln Pro Arg Arg Glu Tyr Gln Pro Lys Pro Arg 260 265 270

Claims (6)

A pharmaceutical composition for preventing or treating cerebral ischemia, comprising an endophylline-A1 fusion protein, wherein the HIV-Tat protein transport domain is covalently bound to at least one end of the endophylline-A1 protein to improve cell penetration efficiency.
The method according to claim 1,
The endophylline-A1 fusion protein is a pharmaceutical composition for preventing or treating cerebral ischemia containing an endophylline-A1 fusion protein, characterized in that the amino acid sequence is SEQ ID NO: 2.
A pharmaceutical composition for preventing or treating cerebral ischemia, comprising a recombinant polynucleotide encoding an endophylline-A1 fusion protein by binding an oligonucleotide sequence encoding an HIV-Tat protein transport domain to at least one end of cDNA encoding an endophylline-A1 protein.
4. The method according to claim 3,
The recombinant polynucleotide is a pharmaceutical composition for preventing or treating cerebral ischemia, characterized in that the base sequence is SEQ ID NO: 1.
Endophylline-A1 fusion protein expression vector containing a recombinant polynucleotide encoding an endophyllin-A1 fusion protein by binding an oligonucleotide sequence encoding an HIV-Tat protein transport domain to at least one end of the endophyllin-A1 coding cDNA. Prevention of cerebral ischemia or a therapeutic pharmaceutical composition.
A health functional food composition for preventing or improving cerebral ischemia, comprising the endophylline-A1 fusion protein of claim 1 or 2 as an active ingredient.

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