KR20180037399A - Process for preparing exosome loading Peroxiredoxin I or II protein, and pharmaceutical composition for use in antioxidant containing the same as an active ingredient - Google Patents

Abstract

The present invention relates to a method for mass producing an exosome (Prx I/II:EXPLOR) comprising Peroxiredoxin (Prx) I or II protein, a vector for producing an exosome, which can be used to produce the exosome, an exosome comprising Peroxiredoxin (Prx) I or II protein, which is produced by the method; and an antioxidant pharmaceutical composition comprising the same as an effective component. In particular, by using the method provided in the present invention for producing an exosome comprising Peroxiredoxin (Prx) I or II protein, it is possible to produce an exosome comprising Peroxiredoxin (Prx) I or II protein in a high yield. Further, the produced exosome may be widely used in an antioxidant composition; or a pharmaceutical composition for preventing or treating any one disease caused by active oygen, selected from the group consisting of cancer, arteriosclerosis, respiratory diseases, osteoporosis, obesity, and degenerative dementia; or a cosmetic composition for antioxidation or controlling skin aging.

Description

TECHNICAL FIELD The present invention relates to a process for preparing an exosome comprising a peroxiredoxin I or II protein and an antioxidant pharmaceutical composition containing an exosome prepared by the process as an active ingredient, pharmaceutical composition for use in antioxidant containing the same as an active ingredient}

The present invention relates to a method for producing an exosome containing peroxiredoxin (Prx) I or II protein using a photo-specific binding protein, and an antioxidant containing an exosome prepared by the above- A pharmaceutical composition, or a cosmetic composition.

The human body is composed of about 200 kinds of 100 trillion cells, and its bioactivity is regulated by the action of various proteins in the cell to maintain its life.

Cells are surrounded by a bilayer membranous membrane composed of phospholipids, which blocks the entry of foreign substances into cells. Most of the protein therapeutics developed so far also pass through the cell membrane and can not enter the cytoplasm. They have physiological effects by acting outside the cell or by acting on a receptor on the cell membrane to transmit signals into the cell.

There are numerous proteins in the cytoplasm, which interact with each other to regulate their physiological activity. If the protein therapeutic can be put directly into the cell, that is, into the cytoplasm, the cell activity will be controlled more effectively.

Recently, studies on the direct introduction of the target protein into the cell through the cell membrane have been actively conducted. Protein Transduction Domains (PTDs), which are peptides that pass through the cell membrane, and recombinant proteins of the target protein can be prepared and introduced into the cytoplasm through the cell membrane (FIG. 1). The PTDs include HIV-1 TAT, HSV VP22, Antp, dfTAT, and Hph-1. Fusion proteins that bind these proteins with target proteins need to be produced and separated in the form of recombinant proteins. There is a problem in that refolding of the protein is not performed properly, the activity is decreased, the protein is nonspecifically transmitted, the risk of causing an immune reaction in vivo is large, the cost is high, and the yield is low.

The target protein bound to various nanoparticles can enter the cytoplasm through the cell membrane by endocytosis (Fig. 2). The nanoparticles include Gold NP, Liposome NP, Magnetic NP, and Polymeric NP. Most of the degradation products of these proteins and proteins bind to lysosomes in the cell, , It is difficult for the target protein and the nanoparticles to separate from each other in the cytoplasm, and the toxicity of the nanoparticles may become a problem.

Exosome refers to a small vesicle of membrane structure with a size of 50 - 200 nm that is secreted out of the cell with protein, DNA, RNA, etc., for intercellular signaling.

Exosomes were originally found in the process of leaving hemoglobin in the red blood cells by eliminating and exiting the intracellular proteins at the last stage when the red blood cells were matured. These exosomes were found in the electron microscopic study, But rather from a specific compartment within the cell called Multivesicular bodies (MVBs), which is released and released outside the cell. That is, when fusions between the polycation and the plasma membrane occur, such follicles are released into the extracellular environment, which is called exosomes (FIG. 3).

Although it is unclear how these exosomes are produced by any molecular mechanism, it is possible to identify a variety of immune cells and stem cells, including B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, Tumor cells are also known to produce and secrete exosomes in a living state.

Exosomes contain various proteins, DNA, and RNA in the cell. The substances contained in the exosome and secreted out of the cell may be re-introduced into other cells by fusion or endocytosis with the cell membrane and serve as intercellular communication partners. In addition, the substances contained in the exosome and secreted out of the cell can be analyzed and used for diagnosis of a specific disease.

Several types of microRNAs are contained in exosomes, and a method for diagnosing disease by detecting the presence or abundance thereof is known (see KR 10-2010-0127768A). In particular, WO2009-015357A discloses a method for detecting exosomes present in a cancer-derived sample (blood, saliva, tears, etc.) and measuring the amount of microRNA change. When the increase or decrease is compared with the control, And a method for diagnosing the abnormality is disclosed. Particularly, exosomes obtained from patients having a specific disease (lung disease) are analyzed to specifically describe the relationship between a specific microRNA and lung disease. Also, in addition to pulmonary disease, methods for diagnosing renal disease using protein contained in exosome have been studied.

Exosomes also contain antigens. On the other hand, in antigen presenting cells (APCs), antigen peptides are loaded on MHC (major histocompatibility complex) class II molecules in intracellular compartments containing membranes, including polycations. Therefore, Peptide-MHC class II complex. Thus, exosomes can present antigen peptides to CD4 + T lymphocytes as transporters of immunogens, thereby inducing an immune response such as proliferation of T lymphocytes. In addition, since exosomes are enriched with molecules capable of stimulating an immune response, such as MHC class I, heat-shock proteins (HSPs), they are immune to autoimmune disease or tumor treatment May be used for the purpose of enhancing or reducing.

In addition, the exosome in which the target protein is contained can be used for the treatment of various diseases in vivo. For example, a protein exhibiting an anti-cancer effect as a target protein, or an exosome containing siRNA can be prepared, and then treated with cancer cells to treat cancer (FIG. 4).

Thus, exosomes containing the desired protein can be used for the treatment of disease. For this purpose, it is necessary to be able to efficiently produce exosome containing the target protein. Korean Patent Publication No. 2004-0015508 describes a method for producing an exosome comprising a specific antigen. There is disclosed a method in which a gene for a specific antigen is introduced into a cell line and the protein of the introduced gene is stably expressed in the cell line and is released to the outside of the cell through the exosome and a method of using such exosome as a vaccine .

However, since the exosome is formed naturally in the cell, even if a gene encoding the target protein is introduced into the cell that produces the exosome, the exosome in which the expressed protein is contained can be produced Is very low.

Accordingly, the present inventors have made efforts to develop a method for more effectively producing an exosome containing a target protein. As a result, they have found that a fusion protein obtained by fusing an exosome-specific marker and a target protein is expressed in a cell producing a large amount of exosome , It was confirmed that the exosome containing the target protein can be efficiently produced (Fig. 5).

In addition, in the above method, the target protein is attached to the membrane of the exosome, and a fusion protein obtained by fusing each of the light-specific binding protein pairs to each of the exosome-specific marker and the target protein is called a cell that produces a large amount of exosome When these fusion proteins are introduced into the exosome by the exosome-specific marker and the irradiation of the light is stopped after the introduction of the exosome-specific marker, the exosome- It was confirmed that the fusion protein of the red binding protein was isolated and the exosome containing the target protein in the free state could be effectively produced (FIG. 6).

Peroxiredoxin (Prx) is a representative antioxidant enzyme present in the cytoplasm, accounting for 0.1-0.8% of the water-soluble proteins in mammalian cells. Prx plays a role in reducing the hydroperoxide (ROOH) to water and ROH by receiving two electrons (2e-) in the cells. In addition, it is involved in the formation and disappearance of hydrogen peroxide (H ₂ O ₂ ) at nmol concentration, which is involved in cell signaling such as cell proliferation, differentiation and cell death. Prx is divided into 1-Cys Prx and 2-Cys Prx depending on the number of cycteine amino acids directly involved in the catalytic action of the redox reaction, depending on the cysteine amino acid that is sensitive to redox reaction in the enzyme. do. In particular, 2-Cys Prx is subdivided into 'typical' and 'atypical' 2-Cys Prx depending on the structural and mechanism differences. In all three Prx groups, active cycteine (Cys-SH) reacts with the ROOH group to form Cys-SOH, but there is a difference in the redox reaction from the second step. Prx I to Prx IV corresponds to typical 2-Cys Prx, PrxV to atypical 2-Cys Prx, and PrxVI to 1-Cys Prx. In case of 2-Cys Prx, up to 10 units may form aggregates to form oligomers.

Prx I and II are involved in the activation of receptor-related signaling pathways by regulating the concentration of intracellular hydrogen peroxide produced by growth factors and tumor necrosis factor (TNF-α). In particular, PrxII protects cells from stimulation of cell death inducers such as serum starvation, ceramide, or etoposide.

In normal cells, Prx I inhibits the oxidation of PTEN phosphatase and plays a role in maintaining its activity well. However, when the oxidative stress is increased, Prx is irreversibly oxidized and separated from PTEN, so that the activity of PTEN is inhibited by hydrogen peroxide. As a result, the cell proliferation signal such as intracellular Akt is continuously activated and cancer is induced.

Intracellular quantitative changes in Prx are closely related to disease. The quantitative changes of reactive oxygen species are closely related to the development and progression of carcinogenesis, atherosclerosis, the production of respiratory inflammation, osteoporosis, obesity and degenerative dementia.

Accordingly, the present inventors prepared an exosome containing peroxiredoxin (Prx) I or II protein and, when treating the exosome, detected that peroxiredoxin (Prx) I or II protein And confirming that exosome carrying the Prx I or II protein of the present invention can be used as an antioxidant composition. Thus, the present invention has been completed.

It is an object of the present invention to provide a method for mass production of exosome comprising a fusion protein of an exosome-specific marker and a peroxiredoxin (Prx) I or II protein.

Another object of the present invention is to provide a method for mass production of exosome containing peroxiredoxin (Prx) I or II protein isolated from exosomal membrane using a photo-specific binding protein pair.

Another object of the present invention is to provide a vector for producing an exosome which can be used for the production of the exosome.

Another object of the present invention is to provide a pharmaceutical composition for antioxidant containing the exosome as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for prevention or treatment of diseases caused by at least one active oxygen selected from the group consisting of cancer, arteriosclerosis, respiratory disease, osteoporosis, obesity and degenerative dementia containing the exosome as an active ingredient To provide a composition.

According to an aspect of the present invention,

a) obtaining a transformed cell by introducing into the exosome-producing cell a polynucleotide encoding a fusion protein in which a exoxom-specific marker and a peroxiredoxin (Prx) I or II protein are bound; And b) culturing the transformed cells. The present invention provides a method for mass-producing exosome having peroxiredoxin (Prx) I or II protein attached to an exosome membrane .

In addition,

a) a polynucleotide encoding a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the exosome-producing cell, and a polynucleotide that binds to the first photo- Introducing a polynucleotide encoding a fusion protein (second fusion protein) in the form of a combination of a second photo-specific binding protein and a peroxiredoxin (Prx) I or II protein; b) irradiating the exosome-producing cell with light capable of inducing binding of the first photo-specific binding protein and the second photo-specific binding protein; And c) stopping the irradiation of the exosome after the exosome is produced in the exosome-producing cell, followed by stopping the irradiation of the exosome. .

In addition,

(a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in which a first light-specific binding protein and an exosome-specific marker are bound; And (b) a polynucleotide capable of introducing a polynucleotide encoding a Peroxiredoxin (Prx) I or II protein and a second photo-specific binding capable of binding to the first photo- And a second expression vector comprising a polynucleotide encoding a protein.

The present invention also relates to an exosome comprising an inner part of a peroxiredoxin (Prx) I or II protein produced using the above method, and a cytoplasmic peroxiredoxin Peroxiredoxin (Prx) I or II protein.

The present invention also relates to a pharmaceutical composition for antioxidant comprising the exosome as an active ingredient or a composition for prevention of diseases caused by at least one active oxygen selected from the group consisting of cancer, atherosclerosis, respiratory diseases, osteoporosis, obesity and degenerative dementia Or a pharmaceutical composition for therapeutic use.

In addition, the present invention provides an antioxidant or skin anti-aging cosmetic composition containing the exosome as an active ingredient.

Using the method of producing exosome containing peroxiredoxin (Prx) I or II protein provided in the present invention, exosome containing peroxiredoxin (Prx) I or II protein is expressed in high Since the peroxiredoxin (Prx) I or II protein exists separately from the exosmomic membrane, the exosome can be used for the composition for antioxidation or the composition for cancer, atherosclerosis, respiratory diseases, Osteoporosis, obesity and degenerative dementia, or a composition for preventing or treating diseases caused by at least one active oxygen selected from the group consisting of obesity and degenerative dementia, or a cosmetic composition for antioxidation or skin aging prevention.

FIG. 1 is a diagram showing a cytoplasmic delivery method of a target protein through a cell membrane transduction domain (PTD) and a recombinant protein of a target protein (Steven R. et al., Protein transduction: unrestricted delivery into all cells, Trends in Cell Biology, .
FIG. 2 is a graph showing the intracellular delivery of a target protein through endocytosis to a conjugate comprising nanoparticles and a target protein (Munish Chanana et al., Physicochemical properties of protein-coated gold nanoparticles in biological fluids and cells before and after proteolytic digestion, Angew, Chem., Int.
FIG. 3 is a view showing the process in which exosomes are released from the cells and released from the cells of the multi-vesicular bodies (MVBs) (Grace Raposo and Willem Stoorvogel, Extracellular vesicles: Exosomes, microvesicles, and friends. (4), 373-383, 2013).
FIG. 4 is a diagram showing a process of delivering siRNA in vivo through a targeted exosome to treat cancer (Alvarez-Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature biotechnology 29, 341-345, 2011).
FIG. 5 is a schematic diagram showing a process for producing protein-carrying exosomes (EXPLORs) designed as optogenetically-designed proteins according to the present invention.
FIG. 6 is a diagram showing a process of separating the fusion protein of the target protein and the light-specific binding protein from the inside of the exosome when the light irradiation of EXPLORs according to the present invention is stopped.
FIG. 7 is a fluorescence photograph showing the change in the intracellular location of mCherry protein in a transformant into which CIBN-EGFP-CD9 gene and mCherry-CRY2 gene are introduced into HEK293T cells according to irradiation of blue light.
8 is a diagram showing an experimental procedure for obtaining EXPLORs according to the present invention.
FIG. 9 is an immunoblot analysis image showing the result of measurement of a change in the content of a target protein (mCherry protein) captured inside the exosome according to the intensity of blue light.
FIG. 10 is an electron micrograph showing the result of treating target cells (HT1080 cells) treated with exosome containing a target protein (mCherry) and confirming the introduction of a target protein into the target cells. And the right side represents the target cell treated with exosome.
FIG. 11 is a graph showing the fluorescence image (a) showing the result of examining whether the target protein is introduced into the target cell (HT1080 cell) after treating the exosome containing the target protein (mCherry) (B) showing the results of comparing the ratios of the apoptotic cells induced by the apoptotic cells.
FIG. 12 is a fluorescence image showing the change in intracellular location of mCherry protein according to whether blue light was irradiated in a transformant into which GIGANTEA-EGFP-CD9 gene and mCherry-FKF1LOV were introduced into HEK293T cells.
13 shows the degree of expression (a) of luciferase-mCherry fusion protein using fluorescence imaging and the number of luciferase activity and the number of molecules in the produced cells (b):
Control: HEK293T cells without any treatment;
OVER: HEK293T cells transfected with Luciferase-mCherry-CRY2 alone;
XP: HEK293T cells transfected with XPACK-Luciferase-mCherry using XPACK (Systems Biosciences), a commercial vector designed for exosome loading technology;
EXPLOR: HEK293T cells transfected with Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the method of the present invention.
Fig. 14 is a diagram showing the measurement of the luciferase activity (a) and the number of molecules (b) in the produced exosome:
NEG: Exosomes produced in HEK293T cells that were not treated with anything;
OVER: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2;
XP: Exosomes produced in HEK293T cells using XPACK (Luciferase-mCherry XPACK) using XPACK (Systems Biosciences), a commercial vector designed for exosome loading technology;
EXPLOR: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the present invention;
ON: Exosomes produced by culturing in 200 μW of blue light for 72 hours,
OFF: Exosome produced by culturing for 72 hours in the absence of light.
FIG. 15 is a diagram showing the loading efficiency of a target protein in the produced exosome. FIG.
FIG. 16 shows the efficiency of target protein transfer to target cells (HeLa) using exosomes:
Control: Exosomes produced in HEK293T cells without any treatment;
OVER: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2;
XP: Exosomes produced in HEK293T cells using XPACK (Luciferase-mCherry XPACK) using XPACK (Systems Biosciences), a commercial vector designed for exosome loading technology;
EXPLOR: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the present invention;
ON: Exosomes produced by culturing in 200 μW of blue light for 72 hours,
OFF: Exosome produced by culturing for 72 hours in the absence of light.
FIG. 17 shows that Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 are expressed in the same position in HEK293T cells.
FIG. 18 shows that Prx I / II-mCherry-CRY2 and CIBN-EGFP-CD9 are expressed in the same position in HEK293T cells.
FIG. 19 is a graph showing inhibition of cytotoxicity by oxidative stress induced by hydrogen peroxide (H ₂ O ₂ ) when PrL I / II: EXPLOR according to the present invention was pre-treated in HeLa cells (Scale bars, 20 μm) :
None: Control group treated with hydrogen peroxide;
Cre: EXPLOR:
Prx I: EXPLOR: exosome with peroxiredoxin I; And
Prx II: EXPLOR: Exosome with Peroxiredoxin II.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for efficiently and mass-producing an exosome containing peroxiredoxin (Prx) I or II protein.

The present inventors focused on exosome-specific markers (CD9, CD63, CD81 or CD82) while carrying out various studies in order to develop a method for more effectively producing exosomes containing the target protein therein. Since the exosome-specific marker has a common property that it is a membrane protein of 4-fold penetration type belonging to the tetraspanin family, when the desired protein is bound to the membrane protein of the exosome, it can be relatively easily contained in the exosome Respectively.

The exosome-specific marker that is specifically present in the exosome membrane, which is specifically present in the exosome membrane, and the exosome-specific marker and the fusion protein of the target protein are expressed in a cell that mass-produces the exosome, a large amount of the exosome containing the target protein can be produced .

Specifically, a method for producing an exosome comprising a peroxiredoxin (Prx) I or II protein, which is a target protein of the present invention, is a method for producing exosome-specific markers and peroxiredoxin (Prx) Characterized by introducing a polynucleotide encoding a fusion protein in which the I or II protein is bound and expressing the polynucleotide.

The resulting exosome is fused with exosome-specific markers in which peroxiredoxin (Prx) I or II protein is embedded in the exosome membrane.

The target protein is bound to the membrane protein of the exosome and is not separated even after reaching the target cell. In order to solve these problems, various studies have been conducted. As a result, it has been found that, by using a photo-specific binding protein, a technology for temporarily producing an exosome containing the target protein by binding the target protein with the marker protein Respectively. For example, CIBN and CRY2, which are light specific binding proteins, can be used. Specifically, CIBN is expressed in a form fused with CD9, which is one of the marker proteins, and CRY2 is fused with a target protein A gene encoding these fusion proteins was introduced into the exosome-producing cells. The CIBN-CD9 fusion protein expressed in the exosome-producing cell is included in the exosome due to CD9. When the cell is irradiated with blue LED light, the target protein-CRY2 fusion protein expressed in the exosome- Is bound to the CIBN domain fused to CD9, a reversibly derived fusion protein of the target protein -CRY2-CIBN-CD9 form is formed, and such fusion protein is contained in the exosome due to CD9. When the blue light LED illumination is no longer irradiated after the exosome containing the target protein is produced, the CIBN-CRY2 binding is released and the target protein is not bound to the cell membrane of the exosome, , So that an exosome containing the target protein can be produced as a result (FIGS. 5 to 10).

The exosome thus produced may exhibit a completely different effect from the exosome including the conventional target substance. Since the conventional exosome has been expressed in the form of being fused to the exosome-specific marker in order to trap the target protein inside the exosome, the target protein is trapped in the exosome, but is still attached to the exosome membrane The target protein can be transferred to the target cell only when the exosome is fused to the cell membrane of the target cell. Thus, even when the target protein is fused to the target cell, the target protein can be bound to the exosome fused to the exosome membrane , There is a limitation that the target protein has a very low probability of exhibiting its effect in the target cell. However, since the exosome provided in the present invention is trapped in the exosome without the target protein bound to the membrane, when the exosome is endocytosed by the target cell and enters the cytoplasm, the exosome is attached to the exosome membrane Therefore, when the exosome is degraded, the target protein can be transferred to the cytoplasm of the target cell, and the target protein can freely move within the cytoplasm of the target cell, so that the target protein has an advantage that it can sufficiently exhibit the physiological activity in the target cytoplasm ( 11).

In addition, since the binding level of the target protein to be bound to the marker protein is changed according to the intensity of the light irradiated upon light irradiation, the content of the target protein captured in the exosome can be controlled when the light intensity is controlled .

Therefore, a method for producing an exosome containing a target protein using a photo-specific binding protein has not been known at all and has been developed for the first time by the present inventors.

Accordingly, the present invention provides a method for producing an exosome comprising a Peroxiredoxin (Prx) I or II protein comprising the following steps:

(a) a polynucleotide encoding a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the exosome-producing cell, and a polynucleotide encoding the first specific binding protein Introducing a polynucleotide encoding a fusion protein (second fusion protein) in a form in which a second photo-specific binding protein capable of binding to a peroxiredoxin (Prx) I or II protein is bound;

(b) irradiating the exosome-producing cell with light capable of inducing binding of the first photo-specific binding protein and the second photo-specific binding protein; And

(c) after the exosome is produced in the exosome-producing cell, stopping the irradiation of the light.

The term "exosome " of the present invention means a small vesicle of a plasma membrane structure originating from a specific compartment within a cell called multivesicular bodies (MVBs) and released or secreted out of the cell.

In the present invention, the exosome may serve as a carrier for transporting a target protein to a target cell or tissue of a target protein by including a target protein therein. The target protein carried by the exosome may be a target cell Or may be used to treat certain diseases or to diagnose certain diseases by acting on the tissues.

The term "exosome-producing cell" of the present invention means a cell capable of producing the exosome.

In the present invention, the exosome-producing cell may be, but not limited to, a B-lymphocyte, a T-lymphocyte, a dendritic cell, a megakaryocyte, a macrophage, a stem cell, . For example, in the example of the present invention, HEK293T cells, which are a kind of immortalized cell line, were used as the exosome-producing cells.

The term "exosome-specific marker " of the present invention means a protein abundantly present in the membrane of the exosome.

In the present invention, the exosome-specific marker may be, for example, CD9, CD63, CD81, CD82, and the like. For example, in the examples of the present invention, CD9 was used as an exosome-specific marker. CD9, CD63, CD81, and CD82 are 4-fold penetration type membrane proteins. When the desired protein is bound to the exosome membrane protein, the desired protein is easily present in the exosome.

The term "photo-specific binding protein" of the present invention is also referred to as a light-oil-type heterodimer forming protein or a photoinduced homodimer forming protein. When light of a specific wavelength is irradiated, it may bind to a different protein to form a heterodimer Refers to a protein capable of binding to a protein or another protein of the same kind to form a homodimer.

In the present invention, the photo-specific binding protein may be, for example, a light oil-releasing duplex forming protein, but not limited thereto. As another example, a cryptochrome-interacting basic-helix-loop-helix protein, CIBN, phytochrome B, phytochrome interacting factor, FKF1, GIGANTEA, CRY (chryptochrome), PHR (phytolyase homolgous) region), and the like.

In particular, when forming a heterodimer, two photo-specific binding proteins (a first photo-specific binding protein and a second photo-specific binding protein) may be used, wherein the first photo-specific binding protein is CIB or CIBN , The second photo-specific binding protein may be CRY or PHR, and when the first photo-specific binding protein is PhyB, the second photo-specific binding protein may be PIF, When the first photo-specific binding protein is GIGANTEA, the second photo-specific binding protein may be FKF1.

For example, in an embodiment of the present invention, CIBN is used as the first photo-specific binding protein, CRY2 is used as the second photo-specific binding protein, and the wavelength of the used light is set to 460 to 490 nm And the intensity of the light was set to 20 to 50 μW.

Meanwhile, in order to confirm the expression of the first fusion protein in which the exosome-specific marker and the first photo-specific binding protein are bound and the position in the cell, marker proteins may be fused together. For example, in an embodiment of the present invention, expression of the first fusion protein in which CIBN and CD9, or GIGANTEA and CD9 are combined, in a form in which the fluorescent protein EGFP is inserted, And to confirm the intracellular location.

The term "target protein" of the present invention means a protein expressed in a form fused with the second photo-specific binding protein to be contained in the exosome.

In the present invention, the target protein is peroxiredoxin (Prx) I or II protein, and the protein has an effect of inhibiting cytotoxicity due to oxidative stress.

The peroxiredoxin I protein of the present invention is preferably composed of the amino acid sequence shown in SEQ ID NO: 1, but not limited thereto, and the peroxiredoxin II protein is preferably composed of the amino acid sequence shown in SEQ ID NO: 2 But is not limited thereto.

The term "cultivation" of the present invention means a method of growing a cell or a microorganism under an appropriately artificially controlled environmental condition.

In the present invention, the transformant is cultivated for 1 to 3 days, then replaced with a medium not containing fetal bovine serum, and further cultured for 2 to 5 days.

In the present invention, the method for culturing the transformant may be carried out by a method well known in the art.

The medium means a known medium used for animal cell culture and may be selected from a group such as commercially available serum free media, protein free media and chemically defined media .

Preferably, the serum-free medium is SFM4CHO (HyClone), EX-Cell (JHR Bioscience) or the like, and the insulin-like growth factor I -I), ethanolamine, ferric chloride, and phosphatidyl choline may be added, but the present invention is not limited thereto.

The animal cell culture medium in which the protein derived from animal, in particular, the protein having a molecular weight of 10 kDa or more is removed in the serum-free medium from which the bovine serum component has been removed, is selected as Prostate (Lonza) and PF-CHO (HyClone) But is not limited thereto.

The chemically defined medium is an animal cell culture medium having no known animal-derived components and all constituent components of which are known chemical structures. These media include CDM4CHO (HyClone), PowerCHO2CD (Lonza), and CD-optiCHO ) May be selected, but the present invention is not limited thereto.

The term "first fusion protein" of the present invention means a fusion protein in which the exosome-specific marker and the first photo-specific binding protein are bound.

In the present invention, the sequence of the exosome-specific marker and the first photo-specific binding protein contained in the first fusion protein is such that the first fusion protein binds to the first light in the exosome- As long as it can be expressed so that the specific binding protein can be expressed so as to be positioned so that the N-terminal of the first photo-specific binding protein is bound to the C-terminus of the exosome-specific marker .

The exosome-specific marker constituting the first fusion protein and the first photo-specific binding protein may be directly linked to each other, or may be linked through a linker. The linker is not particularly limited as long as the first fusion protein can be expressed so that the first photo-specific binding protein is located in the exosome-producing cell in the inside of the exosome-producing cell, but a peptide linker composed of an amino acid can be used And more preferably a flexible peptide linker can be used. The peptide linker may be operably linked to an expression vector by linking the nucleic acid encoding the linker in-frame between the nucleic acids encoding each domain.

The term "second fusion protein" of the present invention means a fusion protein in which the second photo-specific binding protein and the target protein are combined.

In the present invention, the order of arrangement of the second photo-specific binding protein and the target protein contained in the second fusion protein is such that the second fusion protein is the first photo-specific of the first fusion protein in the exosome- As long as it can bind to the binding protein site and be located within the exosome, it is not particularly limited. For example, the N-terminal of the target protein is bound to the C-terminus of the second photo- specific binding protein .

In addition, the second photo-specific binding protein and the target protein constructing the second fusion protein may be directly coupled to each other, or may be connected to each other through a linker. The linker is not particularly limited as long as the second fusion protein can be located in the exosome by binding to the first photo-specific binding protein region of the first fusion protein in the exosome-producing cell, Linkers can be used, and more preferably, flexible peptide linkers can be used. The peptide linker may be operably linked to an expression vector by linking the nucleic acid encoding the linker in-frame between the nucleic acids encoding each domain.

In addition, each of the fusion proteins may include a polypeptide having a sequence that differs from a wild-type amino acid sequence of each domain contained therein by one or more amino acid residues. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, such as heat, pH and the like.

Finally, the polypeptide of each domain constituting the fusion protein or the fusion protein may be prepared by a chemical peptide synthesis method known in the art, or may be prepared by amplifying a gene encoding the domain by PCR (polymerase chain reaction) Followed by expression and cloning into an expression vector.

On the other hand, each fusion protein can be expressed in the exosome-producing cell by introducing a polynucleotide encoding the fusion protein into the exosome-producing cell. As the method for introducing the polynucleotide into the exosome-producing cell, Method can be used. For example, a method of introducing using an expression vector can be used.

The term "expression vector" of the present invention refers to a recombinant vector capable of expressing a target peptide in a desired host cell, and a gene product containing an essential regulatory element operatively linked to the expression of the gene insert. The expression vector includes expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc. The initiation codon and termination codon are generally regarded as part of the nucleotide sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

The term "operably linked" of the present invention means a state in which a nucleic acid sequence encoding a desired protein or RNA is functionally linked to a nucleic acid expression control sequence so as to perform a general function . For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with an expression vector can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.

In addition, the expression vector may include a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture medium. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers.

According to a preferred embodiment of the present invention, the expression vector can be expressed by binding a tag to the target protein to confirm whether the target protein is inserted into the exosome. Since the tag is for confirming the presence or absence of the target protein, the target protein can be bound to the opposite site to which the second photo-specific binding protein is bound. For example, fluorescence of a red fluorescent protein, The protein can be used as a tag to bind to the C-terminal region of the target protein.

It is possible to confirm whether or not the exosome contains the target protein by expressing the designed protein in the exosome-producing cell and confirming whether the fluorescent protein tag is detected in the exosome after the exosome is produced .

The term "light" of the present invention means light irradiated to temporarily bind the first light-specific binding protein and the second light-specific binding protein expressed in the exosome-producing cell.

As described above, in the exosome-producing cell, the first photo-specific binding protein is expressed in the form of the first fusion protein together with the exosome-specific marker, and the second photo-specific binding protein is expressed in the form of the second fusion protein Specific binding protein is irradiated with light necessary for binding of the first specific binding protein and the second specific binding protein to the exosome-producing cell, the first specific binding protein and the second specific Binding protein is bound and consequently a fusion protein complex having the shape of the exosome-specific marker-first light-specific binding protein-second light-specific binding protein-target protein is temporarily formed. The exosome-producing cell , The target protein may be linked to the exosome due to the exosome-specific marker. In this case, the target protein is present inside the exosome, and when irradiation of light is stopped after the exosome is produced, the first photo-specific binding protein and the second photo-specific binding protein are separated, The target protein present in the inside is released to the outside in the form contained in the exosome upon the release of the exosome. It is also preferable that the light is intermittently irradiated rather than continuously irradiated so that the target protein can be more efficiently introduced into the exosome. That is, when light is intermittently irradiated, the binding and separation of the first photo-specific binding protein and the second photo-specific binding protein are repeated, so that the probability that the target protein is introduced into the exosome can be improved .

Meanwhile, since the wavelength of the light that induces the binding of the first photo-specific binding protein and the second photo-specific binding protein depends on the kind of the first photo-specific binding protein and the second photo-specific binding protein, The wavelength of the light can be selected. That is, when CRY2 and CIBN are combined, light having a wavelength of 460 to 490 nm is irradiated, and when the light is not irradiated for 10 minutes or more, CRY2 and CIBN are dissociated from each other; When PhyB and PIF are combined, light having a wavelength of 650 nm is irradiated for 10 minutes, and light having a wavelength of 750 nm is irradiated for 5 minutes, PhyB and PIF dissociate from each other; When combining FKF1 and GIGANTEA, light with a wavelength of 460 nm can be irradiated for 30 minutes. In the embodiment of the present invention, light having a wavelength of 460 to 490 nm was irradiated to induce the binding of CIBN and CRY2.

According to the embodiment of the present invention, the expression of the fusion protein of CRY2 and mCherry and the fusion protein of CIB and CD9 was expressed in HEK293T cell, an immortal cell line producing a large amount of exosome. As a result, the distribution of mCherry protein uniformly distributed in cytoplasm When irradiated with blue light, it was observed that it is in a membrane such as a cell membrane and an endosome-like structure (Fig. 7). Similar results were also observed when FKF1 and mCherry fusion proteins and GIGANTEA and CD9 fusion proteins were expressed in HEK293T cells (Fig. 12). Furthermore, when the fusion proteins of CRY2 and mCherry and the fusion proteins of CIBN and CD9 were expressed in HEK293T and the intensity of blue light was controlled, it was found that mCherry protein captured in the exosomes (FIG. 9). As a result of treatment of HT1080 cells, which are isolated from exocytes produced from the cells, at a concentration of about 250 μg / ml, they did not show any cytotoxicity against HT1080 cells, MCherry protein was transferred to the cytoplasm of HT1080 cells (Fig. 10).

In order to compare the introduction efficiency of the target protein in the exosome of the present invention and the exosome transfer efficiency to the target cell with the conventional method, XPACK vector was used as the conventional method, and the CRY2 and mCherry proteins of the present invention Type fusion protein with CIBN and CD9 protein was introduced into HEK293T, and the amount of target protein produced in the exosome was compared. As a result, when the method of the present invention was used, (Fig. 15). In addition, when the exosome isolated from the exosome-producing cell is treated with target cells (HeLa) and the degree of expression of the target protein is compared, when the exosome isolated by the method of the present invention is used, And the highest expression was confirmed (Fig. 16).

The present invention provides, in another aspect, (a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in which a first photo-specific binding protein and an exosome-specific marker are bound; And (b) a polynucleotide encoding a multicloning site into which a polynucleotide encoding a target protein can be introduced and a second photo-specific binding protein capable of binding to the first photo-specific binding protein, And a second expression vector comprising the exogenous expression vector.

In the vector for producing an exosome provided by the present invention, the exosome-specific marker, the first photo-specific binding protein, the exosome-producing cell and the second photo-specific binding protein are the same as described above.

The term "transforming cell for producing exosome" of the present invention refers to a cell that encodes a fusion protein (first fusion protein) in which exosome-specific markers and a first photo-specific binding protein are bound to the exosome- Refers to an exosome-producing cell into which a nucleotide has been introduced to express the first fusion protein.

In the present invention, the second expression vector may include a polynucleotide encoding the second photo-specific binding protein, and may be constructed to include a polyclonal region adjacent to the polynucleotide. The second expression vector When a polynucleotide encoding a target protein is introduced into the polyclonal region, the second light specific binding protein and the target protein can be expressed as a fusion form (second fusion protein).

The vector for producing exosome provided by the present invention can be used not only for transfecting cells and expression vectors for production of exosome but also for introducing the expression vector, culturing transgenic cells for production of exosome, producing transgenic cells for producing exosome One or more other component compositions, solutions or devices suitable for isolating and purifying exosomes may also be included. For example, it may further comprise an appropriate buffer necessary for introduction of the expression vector, a medium and a container necessary for culturing the transformed cell for producing the exosome, and the like.

In another aspect, the present invention provides an exosome produced by the above method, wherein the peroxiredoxin (Prx) I or II protein is contained therein.

The exosome produced by the above-mentioned method contains a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the plasma membrane constituting the exosome, A second photo-specific binding protein capable of binding to the first photo-specific binding protein and a fusion protein (second fusion protein) in which a peroxiredoxin (Prx) I or II protein is bound Therefore, when the exosome is treated to cells in the target tissue, the second fusion protein contained in the exosome can be transferred to the cytoplasm in the target tissue through fusion of the plasma membrane.

The present invention also provides a pharmaceutical composition for antioxidant containing an exosome carrying peroxiredoxin (Prx) I or II protein prepared by the above method as an active ingredient.

The present invention also relates to a pharmaceutical composition comprising an exosome containing peroxiredoxin (Prx) I or II protein prepared by the above method as an active ingredient, cancer, arteriosclerosis, respiratory disease, osteoporosis, obesity and degenerative dementia The present invention provides a pharmaceutical composition for preventing or treating diseases caused by at least one active oxygen selected from the group consisting of

The present invention also provides an antioxidant cosmetic composition containing, as an active ingredient, an exosome on which a peroxiredoxin (Prx) I or II protein prepared by the above method is mounted.

In addition, the present invention provides a cosmetic composition for preventing skin aging comprising, as an active ingredient, an exosome carrying peroxiredoxin (Prx) I or II protein prepared by the above method.

In a specific embodiment of the present invention, the present inventors have found that an exosome (Prx I / II :: EXPLOR) equipped with peroxiredoxin (Prx) I or II protein prepared by the above- To confirm the cytotoxic inhibitory effect due to oxidative stress, it was confirmed that cytotoxicity due to oxidative stress was statistically significantly inhibited by pretreating Prx I / II :: EXPLOR with HeLa cells (see FIG. 19).

Accordingly, the exoprotein (Prx I / II :: EXPLOR) carrying the peroxiredoxin (Prx) I or II protein of the present invention is a pharmaceutical composition for preventing or treating diseases caused by antioxidant or active oxygen, It can be used as a cosmetic composition for antioxidant or skin aging prevention.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Exosome  Produce

<1-1> Confirmation of binding of CIBN and CRY2 for the production of exosome

The pcDNA3.1 (+) vector containing the CIBN-EGFP-CD9 gene and the pcDNA3.1 (+) vector containing the mCherry-CRY2 gene were introduced into the exosome-producing HEK293T cells under light-free conditions, , Then replaced with medium without fetal bovine serum, and further cultured for 48 hours. After completion of the cultivation, blue light having a wavelength of 460 to 490 nm was irradiated, and the positions of red fluorescence appearing in the mCherry before and after irradiation of the blue light were confirmed through a confocal microscope (Fig. 7).

FIG. 7 is a fluorescence photograph showing the change in the intracellular location of mCherry protein in a transformant into which CIBN-EGFP-CD9 gene and mCherry-CRY2 gene are introduced into HEK293T cells according to irradiation of blue light. As shown in FIG. 7, mCherry protein is uniformly distributed in the cytoplasm before blue light that induces the binding of CIBN and CRY2, which is a photo-specific binding protein, is observed. However, after the blue light is irradiated, Of the total population. This clustering of mCherry protein was analyzed to be caused by the combination of CIBN and CRY2, a light specific binding protein.

<1-2> Confirmation of binding of GIGANTEA and FKF1 for the production of exosome

Using pcDNA3.1 (+) vector containing GIGANTEA-EGFP-CD9 gene and pcDNA3.1 (+) vector containing mCherry-FKF1LOV gene and using the same method as in Example <1-1> Exosomes in the cells were confirmed. (In the above FKF1LOV, LOV is an abbreviation of light-oxygen-voltage domain. It indicates a domain in which FKF1 protein actually binds to another protein by light. Thus, FKF1 and FKF1LOV are used in the same meaning.)

As shown in FIG. 12, the mCherry protein is uniformly distributed in the cytoplasm before the blue light that induces the binding of GIGANTEA and FKF1, which is a photo-specific binding protein, is irradiated. However, The mCherry protein was dense in the membrane (Fig. 12). Thus, similar to the results of <1-1>, it was confirmed that the dense mCherry protein can be induced by the binding of GIGANTEA and FKF1 of the photo-specific binding protein.

Exosome  Production and Exosome  Effect of light intensity on production

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene, respectively, was irradiated with light of a wavelength of 460 nm at an intensity of 0, 5, 20, 50 or 200 μW, , HEK293T cells, cultured for 24 hours, then replaced with medium without fetal bovine serum, and further cultured for 48 hours. After completion of the culture, the culture broth was separated and centrifuged (3000 × g, 15 minutes) to obtain a supernatant from which cellular debris was removed. The obtained supernatant was mixed with ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, California, USA) of 5 times the volume of the supernatant and centrifuged (1500 x g, 30 minutes) Cotton was obtained, and the resulting exosome was suspended in PBS to give an exosome suspension. The exosome suspension was filtered through a 0.2 탆 filter using a syringe equipped with a 27-G needle to obtain a single-sized exosome (Fig. 8). Then, Exosome Lysate was prepared using Lysis buffer, and the amount of mCherry protein contained in exosome was compared through immunoblot analysis (FIG. 9).

FIG. 9 is an immunoblot analysis image showing the result of measurement of a change in the content of a target protein (mCherry protein) captured inside the exosome according to the intensity of blue light. As shown in FIG. 9, when the blue light was irradiated at an intensity of 20 to 50 μW, it was confirmed that the content of the target protein (mCherry protein) captured in the exosome exhibited the maximum value. From the above results, it was found that the content of the target protein captured in the exosome can be controlled by controlling the intensity of the light irradiated upon binding of the photo-specific binding protein.

Exosome  Treatment effect

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene was introduced into HEK293T cells, which are exosome-producing cells, under the LEDs that emit light with a wavelength of 460 nm at an intensity of 50 μW Exosome was extracted by the method of Example 2. Then, the extracted exosome was treated with HT1080 cells at a concentration of 250 μg / ml for 24 hours. Then, the HT1080 cells were fixed with 0.1 M phosphate buffer (pH 7.4) containing 4% PFA and 0.01% GA and fixed on 10% gelatin gel. The cells adhered onto the gelatin gel were cooled with liquid nitrogen for one day and a slice cut to a thickness of 45 nm was obtained at -120 캜 using a cryoultramicrotome. Next, the flakes were immunostained with anti-mCherry antibody and protein A-gold, and mCherry protein was observed through Tecnai G2 Spirit Twin TEM (FIG. 10).

FIG. 10 is an electron micrograph showing the result of treating target cells (HT1080 cells) treated with an exosome containing a target protein (mCherry) and confirming the introduction of a target protein into the target cell. And the right side represents the target cell treated with exosome. As shown in FIG. 10, when the exosome of the present invention was treated on the target cell, it was confirmed that the target protein was transferred into the target cell.

Containing the desired protein Exosome  analysis

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene was introduced into HEK293T cells, which are exosome-producing cells, under the LEDs that emit light with a wavelength of 460 nm at an intensity of 50 μW Exosome was extracted by the method of Example 2. Then, the extracted exosome was treated with HT1080 cells at a concentration of 250 μg / ml for 24 hours. Then, the red fluorescence of mCherry protein was confirmed by fluorescence microscopy, and the ratio of dead cells of cells treated with exosome and untreated cells was compared through LDH cell death assay (FIG. 11).

FIG. 11 is a graph showing fluorescence (a) showing the result of examining whether the target protein is introduced into the target cell (HT1080 cell) after treating the exosome containing the target protein (mCherry) to the target cell (B) showing the results obtained by comparing the ratios of the apoptotic cells induced by the apoptotic cells. As shown in Fig. 11, it was confirmed that apoptosis did not induce apoptosis.

Exosome  Produced and produced Exosome  Comparison of introduction efficiency of target protein

<5-1> Confirmation of exosome production efficiency

In order to confirm the expression of the target protein in the exosome-producing cells in order to compare the exosome production of the present invention and the degree of introduction of the target protein in the produced exosome, the luciferase activity measurement experiment was performed .

In the conventional method, XPACK-Luciferase-mCherry was introduced into HEK293T cells (XP) using XPACK (Systems Biosciences), which is a commercial vector designed for exosome mounting technology, and Luciferase-mCherry-CRY2 And CIBN-EGFP-CD9 were introduced into HEK293T cells (EXPLOR), and then the intracellular luciferase activity was measured to compare the efficiency of the two methods. The luciferase activity was measured according to the manufacturer's instructions (Luciferase Assay Reagent, Promega), and the standard curve of the result was plotted, and then the number of exocytes in the cell was quantitatively calculated.

As shown in FIG. 14, it was confirmed that the method using the photo-specific binding proteins CIBN and CRY2 of the present invention was significantly higher in the introduction efficiency into exosomes than the conventional method XP (FIG. 14).

<5-2> Expression of the target protein in the produced exosome

The cells of the above Example <5-1> were cultured for 72 hours, and the exosomes were separated (Exoquick-TC, Systems biosciences) As a result of indirectly comparing the amount of the target protein with the activity of luciferase activity, as shown in FIG. 15, the method of the present invention can produce an exosome containing a remarkably large amount of the target protein (Fig. 15).

<5-3> Comparison of introduction efficiency of target protein

Using the measured luciferase activity values in the above Examples <5-1> and <5-2>, the introduction efficiency (E) of the target protein was calculated by the following equation (1).

[Equation 1]

As shown in FIG. 15, it was confirmed that the exosomes produced using the CRY2 and CIBN binding of the present invention were 4 to 120 times more efficient than the other comparative groups (FIG. 15).

Target cell Exosome  Comparison of transfer efficiency

In order to compare the efficiency of treatment of the exosome into which the target protein was introduced to the target cells, 5 × 10 ⁹ exosomes were treated with HeLa cells for 24 hours and the fluorescence intensity expressed in the cells was measured. As a result, As shown, it was confirmed that the fluorescence intensity of EXPLOR of the present invention was remarkably high (FIG. 16).

Therefore, it can be seen that the method of using the exosome of the present invention can more effectively deliver the target protein to the target cell than the conventional method.

< Experimental Example  1> Prx  I or Prx  II protein-loaded Of exosome (Prx I / II: EXPLOR)  Produce

<1-1> Exosome  Internal Prx  Identification of I / II proteins

To confirm that Prx I (SEQ ID NO: 1) or Prx II (SEQ ID NO: 2) protein is loaded on the exosome, the present inventors found that the CIBN-EGFP-CD9 and Prx I / II-mCherry- , The presence of CIBN and CRY2 was confirmed.

Specifically, a pcDNA3.1 (+) vector containing the CIBN-EGFP-CD9 gene and a pcDNA3.1 (+) vector containing the Prx I-mCherry-CRY2 or Prx II-mCherry-CRY2 gene under light- The cells were transfected into HEK293T cells, an exosome-producing cell, cultured for 24 hours, then replaced with a medium containing no fetal bovine serum, and further cultured for 48 hours. After completion of the cultivation, blue light having a wavelength of 488 nm was irradiated, and the positions of red fluorescence appearing in the mCherry before and after irradiation of the blue light were confirmed through a confocal microscope. The experiment was repeated 5 or more times.

As a result, as shown in FIG. 18, Prx I-mCherry-CRY2 or Prx II-mCherry-CRY2 (red) was characterized by aggregation by blue light stimulation (FIG. 18). Therefore, Prx I or Prx-II protein is loaded inside the exosome by confirming that Prx I-mCherry-CRY2 or Prx II-mCherry-CRY2 (red) and CIBN-EGFP-CD9 .

<2-2> Prx  I or Prx  II protein-loaded Of exosome (Prx I / II: EXPLOR)  production

The present inventors conducted the following experiment to obtain an exosome carrying a Prx I or Prx II protein.

Specifically, each of the expression vectors containing the CIBN-EGFP-CD9 gene and the Prx I-mCherry-CRY2 or Prx II-mCherry-CRY2 gene under an LED that emits light with a wavelength of 488 nm at an intensity of 50 μW The cells were transfected into HEK293T cells, an exosome-producing cell, cultured for 24 hours, then replaced with a medium containing no fetal bovine serum, and further cultured for 48 hours. After completion of the culture, the culture broth was separated and centrifuged (3000 × g, 15 minutes) to obtain a supernatant from which cellular debris was removed. The obtained supernatant was mixed with ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, California, USA) of 5 times the volume of the supernatant and centrifuged (1500 x g, 30 minutes) Cotton was obtained, and the resulting exosome was suspended in PBS to give an exosome suspension. The exosome suspension was filtered through a 0.2 탆 filter using a syringe equipped with a 27-G needle to obtain a single-sized exosome (Fig. 8).

< Experimental Example  2> Prx  I or Prx  II-loaded In exosome (Prx I / II: EXPLOR)  Of cytotoxicity by stress

The present inventors conducted the following experiments to confirm the cytotoxic inhibitory effect of oxidative stress using Prx I / II: EXPLOR prepared in the above <Experimental Example 1>.

Specifically, HeLa cells were replaced with medium containing no fetal bovine serum, and then treated with 100 μg / mL of Prx I: EXPLORs or Prx II: EXPLORs and cultured for 18 hours. H ₂ O ₂ (0, 0.5, 1 mM) was then added and incubated for an additional 8 h. Cell viability was analyzed using WST assy.

As a result, it was confirmed that cytotoxicity due to oxidative stress was statistically significantly inhibited by pretreatment of Prx I: EXPLORs or Prx II: EXPLORs as shown in Fig. 19 (Fig. 19).

<110> Korea Advanced Institute of Science and Technology          Cellex Life Sciences Inc. <120> Process for preparing exosome loading Peroxiredoxin I or II          protein, and pharmaceutical composition for use in antioxidant          containing the same as an active ingredient <130> 2016P-06-001 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> Peroxiredoxin I <400> 1 Met Ser Ser Gly Asn Ala Lys Ile Gly His Pro Ala Pro Asn Phe Lys   1 5 10 15 Ala Thr Ala Val Met Pro Asp Gly Gln Phe Lys Asp Ile Ser Leu Ser              20 25 30 Asp Tyr Lys Gly Lys Tyr Val Val Phe Phe Phe Tyr Pro Leu Asp Phe          35 40 45 Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Ala Glu      50 55 60 Glu Phe Lys Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val Asp Ser  65 70 75 80 His Phe Cys His Leu Ala Trp Val Asn Thr Pro Lys Lys Gln Gly Gly                  85 90 95 Leu Gly Pro Met Asn Ile Pro Leu Val Ser Asp Pro Lys Arg Thr Ile             100 105 110 Ala Gln Asp Tyr Gly Val Leu Lys Ala Asp Glu Gly Ile Ser Phe Arg         115 120 125 Gly Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu Arg Gln Ile Thr Val     130 135 140 Asn Asp Leu Pro Val Gly Arg Ser Val Asp Glu Thr Leu Arg Leu Val 145 150 155 160 Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu Val Cys Pro Ala Gly                 165 170 175 Trp Lys Pro Gly Ser Asp Thr Ile Lys Pro Asp Val Gln Lys Ser Lys             180 185 190 Glu Tyr Phe Ser Lys Gln Lys         195 <210> 2 <211> 198 <212> PRT <213> Artificial Sequence <220> <223> Peroxiredoxin II <400> 2 Met Ala Ser Gly Asn Ala Arg Ile Gly Lys Pro Ala Pro Asp Phe Lys   1 5 10 15 Ala Thr Ala Val Val Asp Gly Ala Phe Lys Glu Val Lys Leu Ser Asp              20 25 30 Tyr Lys Gly Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp Phe Thr          35 40 45 Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asn Arg Ala Glu Asp      50 55 60 Phe Arg Lys Leu Gly Cys Glu Val Leu Gly Val Ser Val Asp Ser Gln  65 70 75 80 Phe Thr His Leu Ala Trp Ile Asn Thr Pro Arg Lys Glu Gly Gly Leu                  85 90 95 Gly Pro Leu Asn Ile Pro Leu Leu Ala Asp Val Thr Arg Arg Leu Ser             100 105 110 Glu Asp Tyr Gly Val Leu Lys Thr Asp Glu Gly Ile Ala Tyr Arg Gly         115 120 125 Leu Phe Ile Ile Asp Gly Lys Gly Val Leu Arg Gln Ile Thr Val Asn     130 135 140 Asp Leu Pro Val Gly Arg Ser Val Asp Glu Ala Leu Arg Leu Val Gln 145 150 155 160 Ala Phe Gln Tyr Thr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp                 165 170 175 Lys Pro Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu             180 185 190 Tyr Phe Ser Lys His Asn         195

Claims (18)

a) obtaining a transformed cell by introducing into the exosome-producing cell a polynucleotide encoding a fusion protein in which a exoxom-specific marker and a peroxiredoxin (Prx) I or II protein are bound; And
and b) culturing the transformed cells. The method for producing a large amount of exosome in which the peroxiredoxin (Prx) I or II protein is present in the exosome.
The method according to claim 1, wherein, when the peroxiredoxin (Prx) I or II protein is attached to the exosomal membrane, the peroxiredoxin (Prx) Further comprising the step of isolating the exosome from the membrane. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
3. The method according to claim 1 or 2, wherein the peroxiredoxin I comprises a protein represented by the amino acid sequence of SEQ ID NO: 1, and the peroxiredoxin II comprises a protein represented by the amino acid sequence of SEQ ID NO: 2 Wherein the exosome is produced in a large amount.
The method of claim 1 or 2, wherein the exosome-producing cell is selected from the group consisting of B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells, Of the total amount of the exosome.
The method according to claim 1, wherein the exosome-specific marker is CD9, CD63, CD81 or CD82.
a) a polynucleotide encoding a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the exosome-producing cell, and a polynucleotide that binds to the first photo- Introducing a polynucleotide encoding a fusion protein (second fusion protein) in the form of a combination of a second photo-specific binding protein and a peroxiredoxin (Prx) I or II protein;
b) irradiating the exosome-producing cell with light capable of inducing binding of the first photo-specific binding protein and the second photo-specific binding protein; And
c) producing a large amount of exosome comprising peroxiredoxin (Prx) I or II protein, comprising the step of producing exosome in said exosome-producing cell and then ceasing irradiation of said light.
7. The method of claim 6, wherein the first photo-specific binding protein is selected from the group consisting of cryptochrome-interacting basichelix-loop-helix protein (CIBN), N-terminal domain of CIB, phytochrome B, phytochrome interacting factor (PIF) , FKF1 (Flavinbinding, Kelch repeat, Fbox 1), GIGANTEA, CRY (chryptochrome) and PHR (phytolyase homolgous region).
7. The method of claim 6, wherein when the first photo-specific binding protein is CIB or CIBN, the second photo-specific binding protein is CRY or PHR, and the second photo- Wherein the binding of the protein is carried out by irradiating light having a wavelength of 460 to 490 nm.
7. The method of claim 6, wherein when the first photo-specific binding protein is PhyB, the second photo-specific binding protein is PIF, and the binding of the first photo- specific binding protein and the second photo- And irradiating light having a wavelength of 600 to 650 nm. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
7. The method of claim 6, wherein when the first photo-specific binding protein is GIGANTEA, the second photo-specific binding protein is FKF1, and the binding of the first photo- specific binding protein and the second photo- Characterized in that the method is carried out by irradiating light having a wavelength of 460 to 490 nm.
An exosome comprising a peroxiredoxin (Prx) I or II protein incorporated therein, which is produced using the method of any one of claims 1, 2 or 6.
A method for delivering peroxiredoxin (Prx) I or II protein to a cytoplasm, characterized by using the exosome of claim 11.
A pharmaceutical composition for antioxidation comprising the exosome of claim 11 as an active ingredient.
A pharmaceutical composition for preventing or treating diseases caused by at least one active oxygen selected from the group consisting of cancer, arteriosclerosis, respiratory disease, osteoporosis, obesity and degenerative dementia comprising the exosome of claim 11 as an active ingredient.
11. An antioxidant cosmetic composition comprising the exosome of claim 11 as an active ingredient.
11. A cosmetic composition for preventing skin aging comprising the exosome of claim 11 as an active ingredient.
(a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in which a first light-specific binding protein and an exosome-specific marker are bound; And
(b) a polynucleotide capable of introducing a polynucleotide encoding a peroxiredoxin (Prx) I or II protein and a second photo-specific binding protein capable of binding to the first photo- And a second expression vector comprising a polynucleotide encoding said exonome.
[18] The method according to claim 17, wherein the second expression vector comprises a fusion protein (second fusion protein) in which the second photo-specific binding protein and the Peroxiredoxin (Prx) I or II protein are fused to the exosome Lt; RTI ID = 0.0 &gt; production cell. &Lt; / RTI &gt;

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