KR100998861B1 - Cell-transducing SAG fusion protein - Google Patents

Abstract

The present invention relates to a SAG fusion protein, and more particularly, a method for preparing a fusion protein covalently bonded to one or both ends of the SAG protein and introducing into a lung cell or nucleus, and a pharmaceutical composition using the fusion protein, It relates to an external preparation composition for skin, cosmetics and health functional food.

Protein transport domain, fusion protein, SAG

Description

Cell-transducing SAG fusion protein

The present invention relates to a cell-permeable SAG fusion protein, and relates to a fusion protein in which an HIV Tat protein transport domain is covalently bound to at least one end of the SAG protein.

Reactive oxygen species are inevitably produced as by-products of various intracellular metabolism in all living organisms that use oxygen for energy. These reactive oxygen species damage biopolymers such as proteins, nucleic acids and fats in cells. It is deeply involved in the progress of various diseases in the human body. In particular, it is involved in carcinogenesis, stroke, arthritis, arteriosclerosis, radiation damage, and inflammatory reactions.

Parkinson's diseases are degenerative neurological diseases in which dopaminergic neurons, which are present in the substantia nigra of the cerebral vasculature, are lost, resulting in impaired motor function due to progressive dopamine deficiency. ) Symptoms such as rigidity, rigidity, postuer and bradykinesia. Parkinson's disease is one of the leading degenerative neurodegenerative diseases in addition to dementia (Alzheimer's disease) and Amyotrophic lateral sclerosis as the population ages.

Sensitive to apoptosis gene (SAG) protein is a new zinc ring finger protein that protects cells from apoptosis caused by oxidative stress, removes harmful oxygen, and acts as a chelator of metal ions such as copper and zinc to prevent cell death. Prevent it. SAG is a small protein present in the cytoplasm and nucleus, and much of the mechanism and properties of antioxidant activity are still unknown.

In recent years, gene therapy has attracted attention instead of chemical synthesis agents that are commonly used for the purpose of preventing or treating diseases. However, gene therapy is not easy to transport genes, low expression of genes in target cells, short duration of protein expression in cells, and very difficult to artificially control the amount of protein expressed in target cells. There are several problems.

Thus, interest in protein therapy instead of gene therapy is increasing. In order to move drugs or proteins for treating diseases into cells, a method of directly delivering a target protein through a cell membrane may be considered. However, proteins are very difficult to cross cell membranes due to their size and various biochemical properties. In general, it is known that substances having a molecular weight of 600 daltons or more are almost impossible to pass through cell membranes.

Tat (transactivator of transcription) protein, a type of Human Immunodeficiency Virus type-1 protein, has been found to efficiently migrate through the cell membrane and into the cytoplasm. This function is due to the nature of the protein transduction domain ("PTD"), which is the intermediate region of the Tat protein, and its exact mechanism is still unknown. However, it is understood that no specific receptors or carriers are involved in the transmembrane passage of Tat protein and that the protein transduction site occurs by directly interacting with the lipid bilayer of the membrane.

However, not all proteins fused with the protein transduction site of HIV-Tat are smoothly introduced into the cell to show protein activity. In other words, there are reports of Tat fusion proteins introduced into cells but not active (Sengoku, T. et al., Experimental) . Neurology 188 (2004) 161-170; Falnes PO et al. Biochemistry 2001 Apr 10; 40 (14): 4349-4358).

It is an object of the present invention to provide a method for efficiently introducing SAG proteins into cells which are useful for the treatment and prevention of immune and inflammatory diseases such as asthma and atopy.

In addition, the object of the present invention is skin inflammation, itching, immune and inflammatory diseases such as atopy, Parkinson's, dementia. To provide a SAG fusion protein that can be used as a prophylactic or therapeutic agent for brain diseases such as cerebral ischemia, cardiovascular disease, heavy metal poisoning.

It is also an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of immune and inflammatory diseases, such as asthma, atopy, including the cell-permeable SAG fusion protein as a main component.

In addition, an object of the present invention is to provide a health functional food composition comprising the cell-permeable SAG fusion protein as an active ingredient.

Therefore, the present inventors prepared a Tat-SAG fusion protein in which the protein transduction site of the Tat protein was fused to a human SAG protein that is an external protein, overexpressed the prepared fusion protein in E. coli, and subjected to metal chelating affinity chromatography. Purification was easy and convenient. It was confirmed in studies using neurons and animal models whether purified fusion proteins effectively penetrated into cells to increase expression of SAG protein. In other words, by increasing the SAG fusion protein was found to inhibit neuronal cell death caused by oxidative stress.

The present invention discloses for the first time that SAG fusion protein is transported into the cell at the protein level and can be applied as a protein therapeutic agent.

Hazardous oxygen damages cellular biopolymers and has been reported to be deeply involved in the progress of more than 100 human diseases. Therefore, SAG can be effectively used for the protection and treatment of damage caused by harmful oxygen.

Based on the present invention, Tat-SAG can be delivered directly into cells to protect cells and tissues from harmful oxygen harmful to the human body, thereby preventing diseases such as degenerative refractory diseases such as Lou Gehrig's disease, diabetes, cerebral ischemia and Parkinson's disease and atopic dermatitis. And / or a medicament or functional cosmetic to treat.

The inventors have first identified Tat-SAG, which overexpresses and easily purifies the Tat-SAG fusion protein. Expression vectors were developed. This expression vector is a continuous linkage of human SAG cDNA, Tat peptide and six histidines. By the over-expression of the Tat-SAG fusion protein using the expression vector in E. coli Ni ^{2 +} in the natural state were purified using an affinity chromatography column. Tat-SAG overexpression was high and the amount of purified protein was also high. It was confirmed that Tat-SAG fusion protein purified on neurons cultured by Western blot was delivered to cells in a concentration and time dependent manner, and Tat-SAG permeated into cells was maintained for 48 hours in cells and Effectively inhibited apoptosis. In addition, it was found that Tat-SAG effectively penetrates tissues of animal models of cerebral ischemia and effectively inhibits neuronal cell death by oxidative stress.

These results indicate that Tat-SAG is well permeable into cells and shows good SAG function in cells. Therefore, this Tat-SAG fusion protein suggests a variety of applications in the field of brain disease and various diseases in relation to harmful oxygen.

The present invention consists of 9 to 15 amino acid residues and a transport domain comprising at least 3/4 of arginine or lysine residues is covalently linked to at least one end of the SAG protein to provide a cell permeable SAG fusion protein.

The present invention provides a cell-permeable SAG fusion protein, wherein said transport domain is at least one of HIV Tat 49-57 residues, oligolysine, oligoarginine or oligo (lysine, arginine).

The present invention also provides a cell permeable SAG fusion protein, characterized in that the amino acid sequence of the fusion protein as SEQ ID NO: 7.

The present invention provides a recombinant polynucleotide encoding the cell permeable SAG fusion protein by binding the transport domain coding oligonucleotide sequence to at least one end of the SAG coding cDNA.

The present invention provides a recombinant polynucleotide encoding a SAG fusion protein, characterized in that the base sequence of the recombinant polynucleotide is the same as SEQ ID NO: 6.

In another aspect, the present invention provides a SAG fusion protein expression vector comprising the recombinant polynucleotide, for example, as shown in Figure 1 to express the fusion protein.

In addition, the present invention comprises the SAG fusion protein as an active ingredient and includes a pharmaceutically acceptable carrier, in Lou Gehrig's disease, diabetes, cerebral ischemia, Parkinson's disease, dementia, heavy metal poisoning, cardiovascular disease, skin inflammation, itching and atopic dermatitis Provided is a pharmaceutical composition for use as a prophylactic and therapeutic agent for one or more diseases.

In addition, the present invention is the SAG fusion protein as an active ingredient, preventing and improving the effects of one or more of Lou Gehrig's disease, diabetes, cerebral ischemia, Parkinson's disease, dementia, heavy metal poisoning, cardiovascular disease, skin inflammation, itching and atopic dermatitis. It provides a health functional food composition.

In addition, the present invention uses the SAG fusion protein as an active ingredient, and provides an external composition for skin, which has an effect of preventing and improving atopic dermatitis, skin diseases and itching.

Pharmaceutical compositions containing a transport domain fusion protein as an active ingredient, including a Tat-SAG fusion protein, can be formulated in oral or injectable form by conventional methods in combination with a carrier that is commonly acceptable in the pharmaceutical art. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, may contain diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants (e.g. silica, talc) , Stearic acid and its magnesium or calcium salts and / or polyethylene glycols, the tablets also contain binders (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone). ), And optionally a disintegrant (eg starch, agar, alginic acid or its sodium salt) or boiling mixture and / or absorbent, colorant, flavor and sweetener. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries such as preservatives, stabilizers, wetting or emulsifier solution promoters, salts or buffers for controlling osmotic pressure. They may also contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally as desired, or parenterally, i.e., intravenously, subcutaneously, intraperitoneally, or topically, and when applied to asthma in the art to which this invention pertains. As is well known to those skilled in the art, it may be formulated into inhaled or sprayed formulations. The dose may be administered by dividing the daily dose of 0.001-100 mg / kg in one to several times. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

The present invention also provides a health functional food composition comprising the SAG fusion protein as an active ingredient. SAG fusion protein of the present invention is a health functional food for the effect of preventing or treating brain diseases such as Lou Gehrig's disease, cerebral ischemia, Parkinson's disease, dementia, diabetes, heavy metal poisoning, cardiovascular disease, skin inflammation, itching and atopic dermatitis Can be used. Foods to which the SAG fusion protein according to the present invention may be added include various foods, drinks, teas, etc. in the form of powders, granules, tablets, and capsules. At this time, the amount of the extract in the food or beverage, that is, the general health functional food composition of the present invention can be added to 0.01 to 20% by weight of the total food weight. The beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract as an essential ingredient in the ratios indicated, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates are conventional sugars such as glucose, fructose, maltose, sucrose, dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural and synthetic flavoring agents can be added. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 10 g per 100 ml of the food composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids. Thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not particularly limited, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the food composition of the present invention.

The present invention also provides a topical skin composition including a cosmetic composition comprising the SAG fusion protein as an active ingredient. The coating agent which uses the fusion protein of this invention as an active ingredient can be easily manufactured in any form according to the conventional manufacturing method. For example, in preparing a cream coating agent, the SAG fusion protein of the present invention is contained in a creamy oil-in-water (O / W) or water-in-oil (W / O) cream base, and includes perfume, chelating agent, pigment, antioxidant, While preservatives and the like are used as necessary, synthetic or natural materials such as proteins, minerals and vitamins can be used in combination for the purpose of improving the properties.

In addition, the fusion protein of the present invention can be used as a cosmetic, by adding a pH adjuster, fragrance, emulsifier, preservatives, etc. as necessary, the usual cosmetic preparation method, lotion, cream, gel, shampoo, rinse, hair conditioner, makeup Soap, cleanser, water soluble powder, fat soluble powder, water soluble liquid, cream or essence and the like.

The present invention also provides a method for efficiently delivering SAG proteins into cells. Intracellular delivery of SAG protein molecules according to the present invention is carried out by constructing a fusion protein in the form of a covalently bonded HIV Tat protein transport domain. An example of the transport domain of the present invention is an HIV Tat peptide consisting of nine amino acids of aa 49-57 and consisting of an amino acid sequence such as SEQ ID NO: 1. However, the protein transport domain of the present invention is not limited to the Tat peptide of SEQ ID NO: 1, and the production of a peptide having a function similar to the Tat peptide by partial substitution, addition or lack of the amino acid sequence of Tat in the field of the present invention belongs. Since it is easy for a person skilled in the art, a protein transport domain composed of 7 to 15 amino acids, including four or more lysine or arginine, and a protein transport function that performs the same or similar protein transport function by partially replacing amino acids therefrom. It will be apparent that the fusion protein using the domain belongs to the scope of the present invention.

Specifically, the present invention relates to a SAG fusion protein, a recombinant nucleotide and a vector for producing the fusion protein, a therapeutic composition comprising the fusion protein, a pharmaceutical composition for the purpose of prevention, a health functional food, and the like.

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

"SAG fusion protein" refers to a covalent complex formed by genetic fusion or chemical bonding of a protein transport domain and a SAG, wherein the transport domain and the target protein (ie, SAG in the present invention). In this specification, it is mixed with "Tat-SAG" or "Tat-SAG fusion protein".

A “target protein” is a molecule that is not originally a transport domain or fragment thereof that cannot enter the target cell or is not able to enter the target cell at its original useful rate, and is a molecule of the transport domain or of the transport domain-target protein complex prior to fusion with the transport domain. Refers to the target protein portion. The target protein includes a polypeptide, a protein, a peptide, and means a SAG protein in the present invention.

"Fused protein" means a complex comprising a transport domain and one or more target protein moieties and formed by genetic fusion or chemical bonding of the transport domain and the target protein.

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

In addition, "target cell" means a cell to which a target protein is delivered by a transport domain, and a target cell refers to a cell in or outside the body. That is, a target cell is meant to include cells in the body, that is, cells constituting organs or tissues of a living animal or human, or microorganisms found in a living animal or human. In addition, target cells are meant to include extracellular cells, ie cultured animal cells, human cells or microorganisms.

In the present invention, the "transport domain" may be covalently bonded to a polymer organic compound such as oligonucleotide, peptide, protein, oligosaccharide or polysaccharide to introduce the organic compound into cells without requiring a separate receptor, carrier, or energy. Say what it is.

In addition, the specification is used interchangeably with the expressions "transport", "penetration", "transport", "delivery", "transmission", "pass" for "introducing" a protein, peptide, organic compound into a cell.

The present invention provides a SAG fusion protein having 9 to 15 amino acid residues and a transport domain comprising 3/4 or more of arginine or lysine residues covalently bonded to at least one end of the SAG, thereby improving cell penetration efficiency.

In the present invention, the transport domain is characterized in that at least one of HIV Tat 49-57 residue, oligolysine, oligoarginine or oligo (lysine, arginine).

In addition, the present invention is characterized in that the amino acid sequence of the fusion protein is the same as SEQ ID NO: 7.

The present invention also provides a recombinant polynucleotide encoding the SAG fusion protein by binding the transport domain coding oligonucleotide sequence to at least one end of the SAG coding cDNA.

In addition, the recombinant polynucleotide is characterized in that the nucleotide sequence is the same as SEQ ID NO: 6.

The present invention also provides a SAG fusion protein expression vector comprising a recombinant polynucleotide for expressing the fusion protein.

The present invention also provides a pharmaceutical composition comprising the SAG fusion protein as an active ingredient and a pharmaceutically acceptable carrier, and used as a prophylactic and therapeutic agent for immune diseases or inflammatory diseases such as asthma, dermatitis and atopic dermatitis.

Furthermore, the present invention provides a health functional food composition having the SAG fusion protein as an active ingredient and preventing and improving an immune disease or inflammatory disease such as asthma, dermatitis and atopic dermatitis.

The present invention relates to a cell-transducing SAG fusion protein consisting of 9-15 amino acids, wherein a protein transport domain comprising at least 4 lysine or arginine is covalently attached to at least one end of the SAG protein. .

In addition, silent changes may result in one or more amino acids in the sequence being substituted with other amino acid (s) of similar polarity that function functionally equivalently. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs.

For example, hydrophobic amino acid classifications include 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. Negative charged acidic amino acids include aspartic acid and glutamic acid. Also included within the scope of the invention are fragments or derivatives thereof having the same similar biological activity within a range of homology between the fusion protein and amino acid sequence of the invention, such as 85-100%.

In addition, the present invention relates to a recombinant polynucleotide comprising SEQ ID NO: 6, which binds a protein transport domain peptide coding DNA sequence to SAG cDNA and encodes the cell-inducible fusion protein. The scope of the present invention extends not only to recombinant polynucleotides of SEQ ID NO: 6 but also to nucleic acid molecules having sequences due to codon degeneracy of the genetic code.

Hereinafter, the configuration of the present invention will be described in more detail with reference to the following examples. However, it is apparent to those skilled in the art that the scope of the present invention is not limited only to the description of the following examples.

In particular, the examples below describe examples of the Tat-SAG fusion protein, which is a protein in which the Tat transport domain is fused to the N-terminus of the SAG protein. In addition, the present inventors have fused the SAG protein with a transport domain consisting of nine arginines. One type of SAG fusion protein was prepared and the same experiment as in the following example was performed, and the result was close to 90% of the Tat-SAG fusion protein in cell permeability and activity (data not shown). From this it is clear that the scope of the present invention is not limited to the Tat-SAG fusion protein of SEQ ID NO: 7.

material

Restriction enzyme and T4 DNA ligase were purchased from Promega (USA) and Pfu polymerase was purchased from stratagene (USA). Tat oligonucleotides were synthesized in Gibco BRL custom primer (USA). IPTG was purchased from Duchefa (Netherland). pET-15b and BL21 (DE3) plasmids were purchased from Novagen (USA) and Ni-nitrilotriacetic acid Sepharose superflow was purchased from Qiagen (Germany). Human SAG cDNA was isolated from human liver cDNA library by PCR method. All other reagents were used as express products.

Example  1: recombination Tat - SAG  Fusion protein of  Expression vector preparation and transformation

To produce a Tat-SAG fusion protein, first, a pET-Tat expression vector containing a basic domain (amino acids 49-57) of HIV-1 Tat was made. Two kinds of oligonucleotides corresponding to the Tat basic domain (upper chain, 5'-TAGGAAGAAGCGGAGA CAGCGACGAAGAC-3 '; lower chain, 5'-TCGAGTCTTCGTCGCTGTCT CCGCTTCTTCC-3') were inserted into pET15b cut with NdeI- XhoI restriction enzyme. . Next, two kinds of oligonucleotides were synthesized based on the sequence of human SAG cDNA. The forward primer has a Xho I restriction site at 5'-CTCGAGATGGCCGACGTGGAAGACGGAGAG-3 'and the sequence of the reverse primer has a Bam HI restriction site at 5'-GGATCC TCATTTGCCGATTCTTTGGACCAC-3'.

Polymerase chain reaction (PCR) was performed in a thermocycler (Perkin-Elmer, model 9600), and the reaction mixture was placed in a 50 μl silicon tube and heated at 94 ° C. for 5 minutes. PCR reactions were extended by 30 cycles of 40 seconds at 94 ° C, denaturation at 54 ° C for 1 minute, annealing at 70 ° C for 3 minutes, and final extension at 20 ° C for 10 minutes, and 20 ° C for 5 minutes. (final extension) was induced. After PCR, the reactants were separated by agarose gel electrophoresis and linked to a TA cloning vector (Invitrogen, Sandiego, USA). The vectors were transformed into competent cells and plasmids were isolated from the transformed bacteria by alkaline lysis. . The TA vector containing the human SAG cDNA and Xho Ⅰ After cutting with Bam HI, it was inserted into HIV-1 Tat expression vector. E. coli BL21 (DE3) transformed with Tat-SAG was selected, and colonies were inoculated in 100 ml LB medium and IPTG (0.5 mM) was added to the medium to induce overexpression of recombinant Tat-SAG. Overexpressed Tat-SAG was confirmed by SDS-PAGE and Western blot analysis.

Example  2: recombination Tat - SAG  Expression and Purification of Fusion Proteins

E. coli BL21 (DE3) cells containing human SAG cDNA prepared by the present inventors were placed in LB medium containing ampicillin and cultured at 37 ° C. at 200 rpm. When the bacterial concentration in the culture medium was OD ₆₀₀ = 0.5 to 1.0, IPTG was added to the medium to bring the final concentration to 0.5 mM, followed by further incubation at 30 ° C for 12 hours. The cultured cells were collected by centrifugation, and 5 ml of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) was added and pulverized by an ultrasonic grinder. Centrifugation and the supernatant is immediately Ni ^{2 +} - a nitrile tree ethyl Sepharose Super flow (Ni ^{2 +} -nitrilotriacetic acid superflow sepharose) load to the column, washed with buffer, binding buffer and 6 times the volume of 10-fold volume (60mM imidazole , 0.5M NaCl, 20mM Tris-HCl, pH 7.9) and then fusion protein was eluted with elution buffer (1M imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9). Fractions containing the fusion protein were collected to remove salts contained in the fractions using a PD-10 column. Purified protein concentration was determined by Bradford method using bovine serum albumin as standard.

Example  3: cell culture and SAG  Intracellular Permeation of Fusion Proteins

C6 astrocytes, a type of neuron, supply 95% air and 5% CO ₂ at 37 ° C, and 10% FBS (fetal bovine serum) and antibiotics (100 µg / ml streptomycin, 100 U / ml penicillin) Cultured in the included Dulbecco's Modified Eagle's Medium (DMEM).

To observe intracellular penetration of SAG fusion proteins, neurons were grown in 6-well plates for 4-6 hours, replaced with fresh DMEM culture containing 10% FBS, and recombinant Tat-SAG (1-3 μM) was cultured. Treated within. After 1 hour the cells were treated with trypsin-EDTA (Gibco BRL) and washed thoroughly with phosphate buffered saline ("PBS"). After crushing the cells, the amount of SAG permeated into the cells was measured by Western blot analysis.

Example  4: Weston Blot  analysis

Western blot methods were performed to confirm intracellular penetration of SAG fusion proteins. One hour after the cells were treated with the fusion protein of Tat-SAG (1-3 μM), only cells were collected and western blots were performed. Proteins in the cell mill were separated with a 12% SDS polyacrylamide gel and then the proteins in the gel were electrophoresed to a nitrocellulose membrane (Amersham, UK). Protein shifted nitrocellulose membrane was blocked with PBS containing 5% non-dry milk. The membrane was then treated with a rabbit anti-histidine polyclonal antibody (Santacruze, USA, 1: 1,000) for 1 hour. After washing, the cells were reacted with horseradish peroxidase-bound mouse anti-rabbit IgG antibody (1: 10,000 dilution) for 1 hour. Finally, protein bands were identified using an ECL kit (ECL; Amersham).

Example  5: MTT  analysis

Cultured neurons were treated with Tat-SAG (1-3 μM) and washed, and then cultured with 1.2 mM H ₂ O ₂ for 12 hours. Cell survival rate due to oxidative stress was measured using MTT [3- (4,5, -dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide].

Example  6: immunohistostaining ( Immunohistochemistry )

Tissue immunostaining was performed to confirm that the Tat-SAG fusion protein penetrates into the brain tissue to protect neurons. Experimental animals used male gerbil mice. The experimental animals were anesthetized with 3% isoflurane in a nitrogen and oxygen mixed gas, and brain tissue was made into frozen tissue sections.

Prior to immunohistochemical staining, the frozen tissue sections were dried at room temperature for 30 minutes and then completely dried at 37 ° C. for at least 2 hours before starting the experiment. Tissue sections were buffered with 0.01 M PBS (Phosphate Buffered Saline) for 10 minutes and washed again with distilled water for 10 minutes. After washing with PBS twice for 7 minutes, and then reacted for 20 minutes in a PBS solution containing 0.5% hydrogen peroxide to remove endogenous peroxidase (peroxidase) in the tissue. After washing twice with PBS for 7 minutes each, the tissues were reacted with 5% normal goat serum in PBS for 30 minutes to prevent non-specific reactions. Subsequently, the immunohistochemical reaction using conventional streptavidin-biotin peroxidase was performed.

The primary antibody was a rabbit anti-mouse histidine antibody (Santa cruz, USA). All antibodies used for immunohistochemistry were diluted in 0.1M PBS (pH 7.4) containing 2% normal goat serum and 0.1% triton X-100. The skin tissue was diluted 1: 500 primary antibody and reacted at 4 ° C. for 48 hours. Subsequently, the biotin-conjugated rabbit anti-mouse IgG (Vector, USA) with the secondary antibody was diluted 1: 200 and reacted at room temperature for 2 hours, followed by the peroxidase-bound streptavidin (Vector, USA) diluted 1: 200. 2 hours at room temperature. After each step, the reaction was washed 4-5 times with PBS solution.

Antigen-reacted tissues were developed in Tris buffer (pH 7.4) containing 0.03% hydrogen peroxide and 0.05% DAB (3,3'-diaminobenzidine tetrachloride; Sigma, USA) for 1-5 minutes. After completion of the color reaction, the tissue was encapsulated using a crystal mount (Biomeda, USA), which is a water-soluble encapsulant, and then photographed after a microscope observation (Axioscope microscope; Carl Zeiss, Germany).

The results obtained by the above method are as follows.

Result 1: recombination SAG  Overexpression and Purification of Fusion Proteins

To overexpress the Tat-SAG fusion protein, a Tat-SAG expression vector was developed that contains SAG, a transduction site of Tat (Tat49-57) and cDNAs for six histidines in succession (FIG. 1).

E. coli cells, which induced overexpression of the fusion protein with IPTG, were disrupted by an ultrasonic grinder at 4 ° C. and centrifuged to separate supernatant proteins by 12% SDS-PAGE. FIG. 2 shows the results of Tat-SAG overexpression and protein blotting stained with Coomassie Brilliant Blue (FIG. 2A) and Western blotting (FIG. 2B). 2A shows well overexpression of the fusion protein by treatment with 0.5 mM IPTG.

Since the Tat-SAG fusion protein contains six histidines at the N-terminus, immobilized metal-chelate affinity chromatography provides a single step for the fusion protein to be pure in nature (purity> 95%). Purification was possible (FIG. 2A). Refined Tat-SAG Fusion proteins were identified by staining with Coomassie Brilliant Blue.

Overexpressed and purified Tat-SAG fusion protein was once again confirmed by western blotting. As shown in FIG. 2B, the Tat-SAG band was observed at the same position as the protein band of FIG. 2A.

Result 2: SAG  Neuronal Permeation of Fusion Proteins

It was observed how the intracellular penetration of purified Tat-SAG fusion protein changes with concentration. As shown in FIG. 3, it was confirmed by Western blotting that Tat-SAG was penetrated into nerve cells in a concentration-dependent manner. 3A shows the results when 0.5-5 μM Tat-SAG was treated in cell culture for 1 hour. When the degree of intracellular permeation of Tat-SAG was observed for each concentration, it was confirmed that the amount of Tat-SAG permeated into cells increased in a concentration-dependent manner. These results show that Tat-SAG fusion protein permeates into cells in a concentration-dependent manner. In addition, when observed by time, the permeated fusion protein could be confirmed within 10 minutes of administration, and it was confirmed that the amount of Tat-SAG permeated into cells increased in proportion to the treatment time (FIG. 3B). In other words, it was found that the Tat-SAG fusion protein permeated into cells in a time and concentration-dependent manner.

Tat-SAG penetrated into neurons must remain stable in cells for a significant period of time before they can be effectively applied to protein therapy. As shown in FIG. 3C, Tat-SAG penetrated into the cell was degraded with time, and the amount of protein gradually decreased, but it was found to exist in the cell for at least 48 hours. Therefore, Tat-SAG penetrated into the cell is stable for at least 48 hours, which may be useful for protein treatment.

Result 3: SAG Biological Functions of Fusion Proteins

Fusion proteins permeated into neurons must maintain their inherent activity before they can be applied to protein therapy. Therefore, the degree of biological activity of the fusion protein penetrated into the cell is a very important problem. Therefore, in order to examine the biological function of the purified Tat-SAG proteins, the viability of cells induced by oxidative stress by H ₂ O ₂ was confirmed by MTT assay (FIG. 4). By treatment with purified Tat-SAG H ₂ O ₂ (1.2mM) and Tat-SAG (0.5-5μM) concentration of the fusion protein in the nerve cells after the cell viability was measured fusion protein concentration by the H ₂ O ₂ It was confirmed to inhibit cell death. Increasing the survival rate of cells infiltrated with SAG fusion protein indicates that the fusion protein effectively inhibits apoptosis by H ₂ O ₂ .

Result 4: In animal testing SAG  Cell Protection Effect of Fusion Proteins

To confirm the cell death inhibitory effect of recombinant fusion protein in animal models of cerebral ischemia, 300 μg of SAG fusion protein was intraperitoneally injected 30 minutes before cerebral ischemia induction and confirmed by tissue immunostaining 4 days later. As shown in FIG. 5, the Tat-SAG fusion protein almost completely protected neurons. These results indicate that SAG fusion proteins effectively penetrate into cells and inhibit neuronal cell death.

1 shows the expression vector of the Tat-SAG fusion protein.

2 shows purification of Tat-SAG fusion protein. Expressed and purified fusion proteins were analyzed by 12% SDS-PAGE (A) and western blotted with antirabbit polyhistidine antibodies (B).

3 shows that the Tat-SAG fusion protein is introduced into cultured astrocytes. 0.5-5 μM Tat-SAG fusion protein was added to the culture medium for 60 minutes (A). 5 μM of Tat-SAG fusion protein was added to the culture medium for 10-60 minutes (B). Cells into which 5 μM of Tat-SAG fusion protein were introduced were incubated for 1-60 hours (C). Analysis was by Western blotting.

4 shows the effect of the transduced Tat-SAG fusion protein on the viability of astrocytes. H ₂ O ₂ (1.2 mM) was added to astrocytes infiltrated with 0.5-5 μM Tat-SAG fusion protein for 1 hour, and cell viability was measured using MTT.

5 shows the effect of Tat-SAG fusion protein on neuronal survival after ischemic insult. (A) Crystal violet staining of hippocampus hippocampus 4 days after ischemic attack. Negative controls (A and B , normal), positive controls (C and D, carrier injection group), gerbils (E and F) injected with 300 μg Tat-SAG fusion protein after seizure. Scale bars: a, b and c, 400 μm, d, e and f, 50 μm. (B) neuronal density. Bars are mean ± standard deviation of seven gerbils.

<110> Industry Academic Foundation Corporation, Hallym University <120> CELL-TRANSDUCING SAG FUSION PROTEIN <130> hallymU-tatSAG <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 29 <212> DNA <213> Human immunodeficiency virus type 1 <400> 1 taggaagaag cggagacagc gacgaagac 29 <210> 2 <211> 31 <212> DNA <213> Human immunodeficiency virus type 1 <400> 2 tcgagtcttc gtcgctgtct ccgcttcttc c 31 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ctcgagatgg ccgacgtgga agacggagag 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggatcctcat ttgccgattc tttggaccac 30 <210> 5 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 5 Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 <210> 6 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> coding sequence which consists of HIV Tat coding sequence and          human SAG cDNA sequence <220> <221> CDS (222) (1) .. (372) <400> 6 agg aag aag cgg aga cag cga cga aga ctc gag atg gcc gac gtg gaa 48 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp Val Glu   1 5 10 15 gac gga gag gaa acc tgc gcc ctg gcc tct cac tcc ggg agc tca ggc 96 Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His Ser Gly Ser Ser Gly              20 25 30 tcc aag tcg gga ggc gac aag atg ttc tcc ctc aag aag tgg aac gcg 144 Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu Lys Lys Trp Asn Ala          35 40 45 gtg gcc atg tgg agc tgg gac gtg gag tgc gat acg tgc gcc atc tgc 192 Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp Thr Cys Ala Ile Cys      50 55 60 agg gtc cag gtg atg gat gcc tgt ctt aga tgt caa gct gaa aac aaa 240 Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys Gln Ala Glu Asn Lys  65 70 75 80 caa gag gac tgt gtt gtg gtc tgg gga gaa tgt aat cat tcc ttc cac 288 Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys Asn His Ser Phe His                  85 90 95 aac tgc tgc atg tcc ctg tgg gtg aaa cag aac aat cgc tgc cct ctc 336 Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn Asn Arg Cys Pro Leu             100 105 110 tgc cag cag gac tgg gtg gtc caa aga atc ggc aaa tgaggatc 380 Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly Lys         115 120 c 381 <210> 7 <211> 124 <212> PRT <213> Artificial Sequence <400> 7 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp Val Glu   1 5 10 15 Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His Ser Gly Ser Ser Gly              20 25 30 Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu Lys Lys Trp Asn Ala          35 40 45 Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp Thr Cys Ala Ile Cys      50 55 60 Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys Gln Ala Glu Asn Lys  65 70 75 80 Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys Asn His Ser Phe His                  85 90 95 Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn Asn Arg Cys Pro Leu             100 105 110 Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly Lys         115 120  

Claims (9)

A cell permeable SAG fusion protein comprising a amino acid sequence of SEQ ID NO: 7 consisting of 9 to 15 amino acid residues, wherein the transport domain comprising 3/4 or more of arginine or lysine residues is covalently attached to one end of the SAG protein. delete delete delete A base sequence of SEQ ID NO: 6, consisting of 9 to 15 amino acid residues at one end of the SAG encoding cDNA, to which an oligonucleotide sequence encoding a transport domain comprising at least 3/4 of an arginine or lysine residue is bound; A recombinant polynucleotide encoding the cell permeable SAG fusion protein of claim 1. SAG fusion protein expression vector comprising the recombinant polynucleotide of claim 5. A pharmaceutical composition used as an agent for preventing and treating cerebral ischemia comprising the cell permeable SAG fusion protein of claim 1 as an active ingredient and a pharmaceutically acceptable carrier. delete delete

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