KR20170018225A - Pharmaceutical composotion for treating ischemia containing cell-transducible Heat Shock Protein 22 fusion protein - Google Patents
Pharmaceutical composotion for treating ischemia containing cell-transducible Heat Shock Protein 22 fusion protein Download PDFInfo
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- KR20170018225A KR20170018225A KR1020150111561A KR20150111561A KR20170018225A KR 20170018225 A KR20170018225 A KR 20170018225A KR 1020150111561 A KR1020150111561 A KR 1020150111561A KR 20150111561 A KR20150111561 A KR 20150111561A KR 20170018225 A KR20170018225 A KR 20170018225A
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- fusion protein
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- hsp22
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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
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Abstract
Description
The present invention relates to a pharmaceutical composition for treating cerebral ischemia comprising a cell permeable heat shock protein 22 fusion protein, wherein the heat shock protein 22 fusion protein permeated into the cell has an increased cell survival rate against hydrogen peroxide toxicity, Induced DNA fragmentation. In the animal model, the heat shock protein 22 fusion protein showed a protective effect against the neuronal apoptosis induced by inducing transient ischemia in the CA1 part of the whole hippocampus. These results indicate that the heat shock protein 22 fusion protein has a protective effect against apoptosis and has a therapeutic effect for protecting nerve cells from damage of cerebral ischemia.
Due to a variety of causes, the blood vessels to the brain block, blocking glucose and oxygen, which are essential for brain function. Continuous energy supply and oxygen deficiency lead to oxidative stress leading to stroke, total cerebral ischemia or severe brain damage. When ischemic reperfusion proceeds, reactive oxygen species are produced in the mitochondria of brain cells. The resulting reactive oxygen species will lead to neurodegenerative brain diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease.
In order to transfer a therapeutic drug or protein into a cell, a method of directly delivering the target protein through the cell membrane may be considered. However, proteins are very difficult to pass through cell membranes because of their size and various biochemical properties. In general, substances with a molecular weight of 600 Daltons or more are known to be almost impossible to pass through cell membranes.
It has been found that the Tat (Transactivator of transcription) peptide, a type of human immunodeficiency virus type-1 protein, efficiently migrates into the cytoplasm through the cell membrane. This function is due to the characteristics of the protein transduction domain, which is the middle part of the Tat peptide, and the precise mechanism is still unknown. It is also known that the PEP-1 protein transport domain also covalently binds to other proteins and smoothly transports the other proteins into the cells. Recent studies have shown that other proteins are directly transported into cells and tissues when fused with HIV-1 Tat peptide, PEP-1 peptide, oligo lysine, oligoarginine, oligo (lysine + arginine)
However, not all of the proteins can be permeated into the cell via the protein transport domain and the fusion protein. In 2001, 2004 and 2007, it was reported that the proteins bound to Tat transduction sites were introduced into cells but did not show activity (Sengoku, T. et al. Experimental Neurology 188 (2004) 161-170, Falnes PO et al. Biochemistry 2001 Apr 10; 40 (14): 4349-4358, Daniele Peroni et al., Neuroscience letters 421 (2007) 110-114, etc.). That is, when the protein transport domain is fused to a protein which is not easily introduced into cells through the above-mentioned references, it can be seen that all the fused proteins are not activated smoothly after being introduced into the cells.
It is an object of the present invention to provide a composition capable of effectively preventing and treating brain ischemic injury.
Heat Shock protein 22 (HSP22) is an antioxidant enzyme that plays an important role in various diseases. However, HSP22 has been known to play an important role in cytoprotection, neurodegeneration and apoptosis of reactive oxygen species in mitochondria in some studies, but mechanisms and functions for the prevention or treatment of cerebral ischemia are unknown. Thus, we have studied the protective effect of the Tat-HSP22 fusion protein on oxidative stress in neuronal and brain ischemia animal models by recombining HIV-Tat peptide, one of the protein transport domains, with HSP22 protein. As a result, we confirmed that Tat-HSP22 fusion protein has a protective effect against apoptosis induced by oxidative stress and explored the possibility of being useful for the treatment of oxidative stress related diseases such as brain diseases.
The present invention relates to a pharmaceutical composition for preventing or treating cerebral ischemia comprising a heat shock protein 22 fusion protein in which a Tat peptide is covalently bonded to the N-terminus of a heat shock protein 22.
In addition, the present invention relates to a pharmaceutical composition for preventing or treating cerebral ischemia comprising the heat shock protein 22 fusion protein, wherein the heat shock protein 22 fusion protein is composed of the amino acid sequence of SEQ ID NO: 2.
In one embodiment of the present invention, the present inventors first developed an HSP22 expression vector capable of overexpressing and easily purifying an HSP22 fusion protein. This expression vector contains a human HSP22 protein, a Tat protein transport domain, and a cDNA capable of expressing six histidine residues at the amino terminal end.
Using this expression vector, the HSP22 fusion protein was overexpressed in E. coli and purified using Ni-affinity chromatography. Western blotting confirmed that the HSP22 fusion protein was delivered to the cells in a time- and concentration-dependent manner in the cultured cells. The HSP22 fusion protein permeated into the cells was maintained in the cells for up to 24 hours and inhibited cell death by oxidative stress.
These results indicate that the HSP22 fusion protein is well permeated into the cell and is well expressed in the function of the HSP22 protein in the cell. Therefore, this HSP22 fusion protein provides a possibility to apply to brain diseases such as diseases associated with reactive oxygen species or protection of nerve cells from damage of cerebral ischemia.
The pharmaceutical composition containing the cell-permeable HSP22 fusion protein as an active ingredient can be formulated together with a carrier that is conventionally acceptable in the pharmaceutical field, and can be formulated by an ordinary method, such as oral or injection form. 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 preparation thus prepared may be administered orally, parenterally, that is, intravenously, subcutaneously, intraperitoneally, or topically, depending on the purpose, and in the case of application to asthma, As known to those of ordinary skill in the art, it can be formulated into a formulation by inhalation or spray. The dose may be administered in a single daily dose of 0.0001 to 100 mg / kg dividedly in several doses. 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.
Further, the present invention provides a pharmaceutical composition useful for the prevention and treatment of cerebral ischemia, which comprises the HSP22 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.
The present invention also provides a method for efficiently delivering HSP22 protein into a cell. Intracellular delivery of the HSP22 protein molecule according to the present invention is carried out by constructing a fusion protein in which the protein transport domain including the Tat peptide is covalently bonded. Examples of the transport domain of the present invention include PEP-1 peptide, Tat peptide, oligo lysine, and the like. However, the protein transport domain of the present invention is not limited to a Tat peptide, and it may be desirable to prepare a peptide having a function similar to that of a Tat peptide due to partial substitution, addition or deletion of the amino acid sequence of Tat, It is obvious to those skilled in the art that the Tat protein transport domain and the fusion protein using the peptide transport domain and the protein transduction domain that carry out the same or similar protein transport function as a partial amino acid substitution therefrom also fall within the scope of the present invention will be.
Specifically, the present invention relates to an HSP22 fusion protein, a pharmaceutical composition for treating or preventing cerebral ischemia comprising the fusion protein.
The definitions of the main terms used in the description of the present invention and the like are as follows.
"HSP22 fusion protein" means a covalent binding complex formed by genetic fusion or chemical bonding between a protein transport domain and an HSP22 protein, and a transport domain and a target protein (i.e., HSP22 protein in the present invention). In this specification, "Tat-HSP22 "," Tat-HSP22 fusion protein "
"Target protein" is a molecule which is not originally able to enter the target cell, or which is not a transport domain or a fragment thereof that can not enter the target cell at an inherently useful speed, as a molecule itself before being fused with the transport domain, Means the target protein portion. The target protein includes a polypeptide, a protein, and a peptide, and the term " HSP22 protein "
"Fusion protein" means a complex comprising a transport domain and one or more target protein fragments, formed by genetic fusion or chemical bonding of the transport domain and the target protein.
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 target protein is delivered by a transport domain, and the target cell refers to a cell in the body or in vitro. That is, the target cell is meant to include a body cell, that is, a living animal, or a cell or living organism of a human organ or tissue, or a microorganism found in a human being. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism.
The term "protein transport domain" in the present invention refers to a protein transport domain that is covalently bonded to a polymer organic compound such as an oligonucleotide, peptide, protein, oligosaccharide or polysaccharide to introduce the organic compound into cells without requiring additional receptor, It can be said.
Also, in the present specification, the terms "transport", "penetration", "transport", "delivery", "permeation" and "passage" are used interchangeably with respect to "introducing" proteins, peptides and organic compounds into a cell.
The HSP22 fusion protein of the present invention refers to an HSP22 fusion protein in which the protein transduction domain is covalently bound to at least one terminal of HSP22 to improve cell penetration efficiency. Also, the transport domain refers to at least one of HIV Tat 49-57 residue, PEP-1 peptide, oligo lysine, oligoarginine or oligo (lysine, arginine).
In addition, the HSP22 fusion protein amino acid sequence of the present invention comprises SEQ ID NO: 4. The fusion protein of various sequences can be obtained according to the selection of the restriction site sequence in the production of the HSP22 fusion protein, and this is obvious to a person having ordinary skill in the art. It is obvious that the above amino acid sequence is only exemplary and the amino acid sequence of HSP22 fusion protein is not limited to the above sequence.
The present invention also provides a pharmaceutical composition comprising the HSP22 fusion protein as an active ingredient and a pharmaceutically acceptable carrier, for the prevention and treatment of cerebral ischemia.
The present invention also provides a health functional food composition comprising the HSP22 fusion protein as an active ingredient and having an effect of preventing and ameliorating cerebral ischemia.
The present invention relates to a cell-transducing HSP22 fusion protein in which a protein transport domain is covalently bonded to at least one end of an HSP22 protein. Also, depending on the silent change, one or more amino acids within the sequence may be replaced with other amino acid (s) of similar polarity functionally equivalent. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs.
For example, the hydrophobic amino acid class includes alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positive basic amino acids include arginine, lysine and histidine. Acidic amino acids with negative charge include aspartic acid and glutamic acid. Also included within the scope of the invention are fragments or derivatives thereof having homologous homology, for example within the range of 85-100%, between the fusion protein of the present invention and the amino acid sequence, having the same similar biological activity.
The nucleic acid sequence and the amino acid sequence of Table 1 below illustrate one embodiment of the nucleic acid sequence encoding the Tat-HSP22 fusion protein and the amino acid sequence of the Tat-HSP22 fusion protein, respectively, but the nucleic acid sequence encoding the Tat- It will be apparent to those skilled in the art that the amino acid sequence of the HSP22 fusion protein is not limited to the sequence shown in Table 1. [
GGATCC
In the present invention, the Tat-HSP22 fusion protein permeated into cells increased the cell survival rate to hydrogen peroxide toxicity. In addition, the Tat-HSP22 fusion protein of the present invention reduced intracellular reactive oxygen species levels and hydrogen peroxide-induced DNA fragmentation. In addition, the Tat-HSP22 fusion protein of the present invention showed a protective effect against the neuronal apoptosis that occurs when transient ischemia was induced in the CA1 region of the hippocampus in the animal model. From these results, it can be seen that the cell-permeable Tat-HSP22 fusion protein of the present invention can be used as a pharmaceutical composition for preventing and treating cerebral ischemia.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing a construction diagram and purification of a Tat-HSP22 fusion protein. FIG. 1A is a schematic diagram of a constructed Tat-HSP22 fusion protein. The Tat-HSP22 fusion protein contains a His tag consisting of six histidine residues. The purified fusion protein was analyzed by 12% SDS-PAGE (left of FIG. 1b) and Western blot analyzed with anti-rabbit polyhistidine antibody (right of FIG. 1b).
Figure 2 shows the results of an experiment in which the Tat-HSP22 fusion protein is permeated into HT22 cells. The cells were cultured in a 60 mm culture dish and treated with Tat-HSP22 fusion protein at a concentration (0.5 to 5 μM) and added to the culture medium for 2 hours. As a control, HSP22 protein without a protein transduction domain was used (FIG. 2A). In addition, a 5 占 농도 concentration of Tat-HSP22 fusion protein was added to the culture medium for 10 to 120 minutes (Fig. 2b). This was analyzed by Western blotting. The stability of the Tat-HSP22 fusion protein permeated into the cells was evaluated by Western blotting (Fig. 2c) after treatment of the 5 占 Tat Tat-HSP22 fusion protein for 2 hours and then with different times (1-60 h). The intracellular distribution of the Tat-HSP22 fusion protein permeated into the cells was observed with a confocal microscope (Fig. 2d). Size bar = 20 mm.
FIG. 3 shows the cytotoxic effect of Tat-HSP22 fusion protein on cell viability, DCF-DA, DNA fragmentation after addition of H 2 O 2 . H 2 O 2 (1 mM 12 h) was added to HT22 cells pretreated with Tat-HSP22 (5 mM) for two hours. Cell viability was evaluated by colorimetric assay using MTT (Fig. 3A). ** P and 0.01 are values compared with cells treated with H 2 O 2 . The effect of the Tat-HSP22 fusion protein on cells that induced the production of reactive oxygen species with H 2 O 2 was tested. For two hours, cells were treated with Tat-HSP22 fusion protein and stimulated with 1 mM H 2 O 2 for 5 minutes. Intracellular reactive oxygen species levels were measured after staining with DCF-DA and fluorescence intensity was measured with an ELISA plate reader (Fig. 3B). Size bar = 25 mm. * P and 0.01 are values compared with cells treated with H 2 O 2 alone. Cells were treated with Tat-HSP22 fusion protein (5 mM) for two hours and then exposed to H 2 O 2 (1 mM) for four hours. DNA fragmentation was detected by TUNEL staining. Size bar = 50 mm.
Figure 4 shows the effect of permeable Tat-HSP22 fusion protein on H 2 O 2 -induced mitochondrial membrane potential activation. Mitochondrial membrane potential was detected using a mitochondrial membrane potential assay kit (Fig. 4C). Size bar = 50 mm. Cells were treated with Tat-HSP22 fusion protein (5-0.3 mM) for two hours and then exposed to H 2 O 2 (1 mM). Bax and Bcl-2 were detected by Western blotting and the band intensity was measured by density meter (FIG. 4b). * P and 0.01 are values compared with cells treated with H 2 O 2 alone.
Figure 5 shows the results of NeuN immunohistochemistry analysis of neuronal cell viability. Immunohistochemistry using histidine antibody showed that the Tat-HSP22 fusion protein permeated across the blood-brain barrier. Sham group, carrier group, Tat peptide treated group, HSP22 protein treated group and Tat-HSP22 fusion protein treated group were analyzed by immunohistochemistry after 7 days of cerebral ischemia / reperfusion. SP: stratum pyramidale, SO: stratum oriens, SR: stratum radiatum. Relative analysis of the number of immunoreactive neurons is a significant difference from the carrier group. * P <0.01 compared with the carrier group. Size bar = 50 μm.
FIG. 6 is a result of immunohistochemical analysis of the neuroprotective effect of Tat-HSP22 fusion protein permeated into cells. Immunohistochemistry for CV, GFAP, Iba-1 and FJB in the CA1 region of the Tat-HSP22 fusion protein-treated group was performed 7 days after cerebral ischemia / reperfusion Respectively. SP: stratum pyramidale, SO: stratum oriens, SR: stratum radiatum. Relative analysis of the number of immunoreactive neurons is a significant difference from the carrier group. * P <0.01 compared with the carrier group. Size bar = 50 μm.
Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the scope of the embodiments.
1) Purification of Tat-HSP22 fusion protein and cell permeation
In order to prepare the permeable Tat-HSP22 fusion protein, Tat vector containing Tat, which is a kind of protein transduction domain (PTD), and HSP22 gene were recombined (Fig. 1A) and overexpressed in E. coli to produce Ni 2 + Purification was carried out using a leuracetate sepharose affinity chromatography column and PD-10 column chromatography, and confirmed by SDS-PAGE and Western blotting (Fig. 1B).
To determine whether the Tat-HSP22 fusion protein infiltrated into cells, HT-22 cells were treated with hippocampal neuronal cells at different times and concentrations. As a result, intracellular permeation was effectively effected in a time- and concentration-dependent manner, and HSP22 protein not bound with negative control (Control) and protein transduction domain Tat did not permeate (Figs. 2a and 2b). The nucleus was stained with DAPI using a confocal microscope, and the Tat-HSP22 fusion protein was stained with green fluorescence to be effectively penetrated into the cells (FIG. 2d). The permeabilized fusion protein was stably maintained in the cells for 24 hours (Fig. 2C).
2) Protection effect of Tat-HSP22 fusion protein on cell viability, reactive oxygen species level and DNA fragmentation due to oxidative stress
The cells were pretreated with Tat-HSP22 fusion protein at different concentrations and then subjected to oxidative stress with H 2 O 2 . MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide} analysis was performed to confirm cell viability. Single-treatment of hippocampal neuron HT-22 cells with 1 mM H 2 O 2 reduced the number of viable cells to 56%, but when the Tat-HSP22 fusion protein was treated, the cell viability was increased in a concentration-dependent manner ).
In addition, in order to confirm that Tat-HSP22 fusion protein effectively suppresses the increase of reactive oxygen species due to H 2 O 2 , active oxygen species generated in the cell and 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 . In the H 2 O 2 -treated cells, the green fluorescence was strongly colored by lipopolysaccharide (LPS) compared to the control HSP22 protein, whereas the permeable Tat-HSP22 fusion protein reduced the fluorescence signal (FIG.
The protection effect against DNA fragmentation caused by oxidative stress was examined by TUNEL staining method using fluorescent pigment which specifically attaches to the fragmented part of DNA. The experiment was carried out in the same condition as the previous experiment, and it was confirmed that the Tat-HSP22 fusion protein had a protective effect against DNA fragmentation (FIG. 3C).
3) Protective effect of Tat-HSP22 fusion protein on mitochondrial damage
The activity of mitochondria was confirmed by JC-1 staining. JC-1 staining is a technique to measure mitochondrial membrane permeabilization (MMP) of mitochondria damaged by oxidative stress. When mitochondrial membrane permeation is damaged, JC-1 can not penetrate and is present as a monomer. , Whereas when the membrane permeation proceeds normally, the JC-1 staining material aggregates to form a j-aggregate, resulting in red fluorescence. H 2 O 2 treated with the Tat-HSP22 fusion protein protected the damage to the oxidative stress, whereas the membrane permeation damage was reduced, resulting in red fluorescence (Fig. 4A).
The expression level of Bcl-2 and Bax during cell death in mitochondria was examined. Treatment of H 2 O 2 resulted in a decrease in Bcl-2 expression and a marked increase in Bax expression. In the case of treatment with Tat-HSP22 fusion protein, the expression of Bcl-2 was increased in a concentration-dependent manner similar to the negative control without any treatment , And the expression of Bax was markedly decreased (Fig. 4B).
4) Confirmation of penetration of Tat-HSP22 fusion protein in an animal model inducing cerebral ischemia
In order to confirm the protective effect of Tat-HSP22 fusion protein on neurons, immunohistochemical staining was performed. In the experimental group without the Tat-HSP22 fusion protein, the number of cells stained with NeuN (neuronal nuclei) antibody decreased significantly in the CA1 region of Zeville while the number of cells stained with Tat-HSP22 fusion protein was decreased The inhibition of CA1 local neuronal apoptosis was confirmed by NeuN antibody staining. In addition, the penetration of Tat-HSP22 fusion protein into the tissue was confirmed by anti-histidine antibody, and nuclear staining was confirmed by DAPI (FIG. 5).
5) Cytoprotective effect of Tat-HSP22 fusion protein on ischemic brain injury
In order to examine the protective effect of Tat-HSP22 fusion protein against ischemic brain injury, Hybil rats were used to reduce cerebral blood flow continuously to induce brain hypoxia and cerebral ischemia, Animal models were constructed using surgical procedures to induce neuronal death through acute ischemic injury. Tissue fluorescence using F-JB (Fluoro-Jade B) staining of damaged brain cells with CV (Cresyl Violet) staining, which stains live cells only to see if the infiltrated Tat-HSP22 fusion protein protects against cerebral ischemia (histofluorescent) staining. Among animal models inducing cerebral ischemia, Tat-HSP22 fusion protein-treated animals showed protection against apoptosis, whereas animals treated with the control HSP22 protein showed no protective effect against apoptosis. In addition, Iba-1 (ionized calcium-binding adapter molecule 1) is a new Ca ++ -binding protein, specifically expressed in brain activated microglia. Thus, Iba-1 is used as a marker for micrographs in these studies, confirming activation of microglial cells by neuronal death. Also, GFAP (Glial fibrillary acidic protein), which is a component of intermediate fibers (intermediate filament) and a marker of astrocytes, was also used. GFAP is a protein that is expressed when activated to compensate for damage to astrocyte cells, especially when damaged, and it can be observed that astrocytes are activated and aggregated when brain damage occurs. The effect of Tat-HSP22 fusion protein on microglial and astrocytic activity and aggregation in the cerebral ischemia model was confirmed. As a result, it was confirmed that when the ischemia induced, the activity of the stained microglial cells and astrocytes increased and coagulated with each other, whereas when the Tat-HSP22 fusion protein was treated, the activation and aggregation phenomena decreased 6).
Therefore, the present invention suggests that Tat-HSP22 fusion protein can be effectively applied as a therapeutic agent for neurodegenerative diseases such as cerebral ischemia due to oxidative stress and various diseases.
<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for treating ischemia containing cell-transducible Heat Shock Protein 22 fusion protein <130> HallymU-SYCHOI-HSP22-ischemia <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding Tat-HSP22 fusion protein <400> 1 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggctgac ggtcagatgc ccttctcctg ccactaccca 120 agccgcctgc gccgagaccc cttccgggac tctcccctct cctctcgcct gctggatgat 180 ggctttggca tggacccctt cccagacgac ttgacagcct cttggcccga ctgggctctg 240 cctcgtctct cctccgcctg gccaggcacc ctaaggtcgg gcatggtgcc ccggggcccc 300 actgccaccg ccaggtttgg ggtgcctgcc gagggcagga cccccccacc cttccctggg 360 gagccctgga aagtgtgtgt gaatgtgcac agcttcaagc cagaggagtt gatggtgaag 420 accaaagatg gatacgtgga ggtgtctggc aaacatgaag agaaacagca agaaggtggc 480 attgtttcta agaacttcac aaagaaaatc cagcttcctg cagaggtgga tcctgtgaca 540 gtatttgcct cactttcccc agagggtctg ctgatcatcg aagctcccca ggtccctcct 600 tactcaacat ttggagagag cagtttcaac aacgagcttc cccaggacag ccaggaagtc 660 acctgtacct gaggatcc 678 <210> 2 <211> 207 <212> PRT <213> Artificial Sequence <220> <223> Tat-HSP22 fusion protein <400> 2 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Asp Gly Gln 1 5 10 15 Met Pro Phe Ser Cys His Tyr Pro Ser Arg Leu Arg Arg Asp Pro Phe 20 25 30 Arg Asp Ser Pro Leu Ser Ser Arg Leu Leu Asp Asp Gly Phe Gly Met 35 40 45 Asp Pro Phe Pro Asp Asp Leu Thr Ala Ser Trp Pro Asp Trp Ala Leu 50 55 60 Pro Arg Leu Ser Ser Ala Trp Pro Gly Thr Leu Arg Ser Gly Met Val 65 70 75 80 Pro Arg Gly Pro Thr Ala Thr Ala Arg Phe Gly Val Pro Ala Glu Gly 85 90 95 Arg Thr Pro Pro Phe Pro Gly Glu Pro Trp Lys Val Cys Val Asn 100 105 110 Val His Ser Phe Lys Pro Glu Glu Leu Met Val Lys Thr Lys Asp Gly 115 120 125 Tyr Val Glu Val Ser Gly Lys His Glu Glu Lys Gln Gln Glu Gly Gly 130 135 140 Ile Val Ser Lys Asn Phe Thr Lys Lys Ile Gln Leu Pro Ala Glu Val 145 150 155 160 Asp Pro Val Thr Val Phe Ala Ser Leu Ser Pro Glu Gly Leu Leu Ile 165 170 175 Ile Glu Ala Pro Gln Val Pro Pro Tyr Ser Thr Phe Gly Glu Ser Ser 180 185 190 Phe Asn Asn Glu Leu Pro Gln Asp Ser Gln Glu Val Thr Cys Thr 195 200 205
Claims (2)
Wherein the heat shock protein 22 fusion protein is SEQ ID NO: 2. 22. A pharmaceutical composition for preventing or treating cerebral ischemia comprising a heat shock protein 22 fusion protein, wherein the heat shock protein 22 fusion protein is SEQ ID NO:
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