KR20130118633A - Compositon for maturating dendritic cell comprising mycobacterium tuberculosis rv2769c - Google Patents

Abstract

PURPOSE: A composition for inducing maturation of dendritic cells is provided to stimulate immature dendritic cells by Rv2769c, to secrete proinflammatory cytokines, to promote maturation to mature dendritic cells with enhanced expression levels of surface factors, and to effectively increase immune response. CONSTITUTION: A composition for inducing maturation of dendritic cells contains Rv2769c proteins as active ingredients. The proteins are derived from Mycobacterium tuberculosis. The concentration of the proteins is 0.1-5 ug/ml. The proteins have an amino acid sequence of sequence number 2. The proteins are encoded by a base sequence of sequence number 1.

Description

Composition for inducing maturation of dendritic cells comprising Rb2776bc protein of Mycobacterium tuberculosis {Compositon for maturating dendritic cell comprising Mycobacterium tuberculosis Rv2769c}

The present invention relates to a composition for inducing dendritic cell maturation comprising Rv2769c protein from Mycobacterium tuberculosis as an active ingredient, and a method for differentiating immature dendritic cells into mature dendritic cells using the same.

Dendritic cells or dendritic cells (DC) are immune cells that form part of the mammalian immune system. These cells act as antigen-expressing cells that process the antigenic material and make it appear on the surface so that other cells in the immune system can recognize it. Dendritic cells are mainly present in small amounts in tissues that contact the external environment, such as the inner walls of the skin, nose, lungs, stomach, and intestine, and in particular, the cells in the skin are called Langerhans cells. Dendritic cells can be found immature in the blood and, when activated, migrate to lymphoid organs and interact with T and B cells to initiate an immune response. At certain stages of development they extend into processes called dendrites.

Dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially turn into immature dendritic cells and are characterized by high endocytosis and T-cell activity. Immature dendritic cells constantly feed on pathogens such as viruses and bacteria in their surroundings. This is possible through pattern recognition receptors (PRRs) such as toll-like receptors (TLRs). TLRs recognize certain chemical features found on a subset of pathogens, and immature dendritic cells devour cell membranes through a process called nibbling from living autologous cells. When they come into contact with existing antigens, they are activated into mature dendritic cells and migrate to lymph nodes. Immature dendritic cells devour pathogens and break down their proteins into small pieces that, when mature, appear on the cell surface using major histocompatibility complex (MHC) molecules. At the same time, it increases cell surface receptors that act as co-receptors in T-cell activation, such as Cluster of Differentiation (CD) 80, CD86, and CD40. They display antigens derived from pathogens with non-antigenic specific costimulatory signals to activate B cells as well as helper T-cells, killer T-cells.

All helper T-cells are specific for one particular antigen. Only specialized antigen expressing cells (macrophages, B lymphocytes and dendritic cells) activate the remaining helper T-cells when the correct antigen is present. Macrophages and B lymphocytes, however, can only activate memory T-cells, while dendritic cells can activate both memory and naive T-cells, making them the most potent antigen-expressing cells.

Mature dendritic cells can be derived from monocytes, white blood cells circulating in the body, which can also be transformed into dendritic cells or macrophages according to appropriate signals. Monocytes are derived from stem cells of bone marrow. Monocyte-derived dendritic cells can be produced from peripheral blood mononuclear cells (PBMC) in a laboratory. PBMCs can be planted in tissue culture flasks to allow monocytes to attach, which can be differentiated into immature dendritic cells by treatment with IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF). Subsequent treatment with TNF-a differentiates immature dendritic cells into mature dendritic cells.

Fully mature dendritic cells differ qualitatively and quantitatively from immature DCs. Fully mature DC expresses high levels of Major Histocompatibility Complex (MHC) type I and II antigens, and high levels of T cell costimulatory molecules, namely CD80 and CD86. These changes increase dendritic cells' ability to activate T cells, which not only increases the antigen density on the cell surface, but also increases the amount of T cell activation through counterparts of costimulatory molecules on T cells, such as, for example, CD28. Because it increases. In addition, mature DCs produce large amounts of cytokines, which stimulate and induce T cell responses. Two of these cytokines are interleukin 10 (IL-10) and interleukin 12 (IL-12). These cytokines have the opposite effect on the direction of the induced T cell response. The production of IL-10 induces a Th-2 type response, while the production of IL-12 induces a Th-1 type response. The latter response is particularly preferred when a cellular immune response is desired, for example in cancer immunotherapy. The Th-1 type response induces and polarizes cytotoxic T lymphocytes (CTLs), which are the mechanism of attack of the cellular immune system. This effector attack is most effective in inhibiting tumor growth. IL-12 also induces the growth of natural killer (NK) cells and has anti-angiogenic activity, all of which are effective anti-tumor attack means.

Because dendritic cells are rare and difficult to isolate, only the approximate formation and development of dendritic cells of different types and subsets and their interrelationship are known. Dendritic cells are in constant communication with other cells in the body. This communication can take the form of direct cell-to-cell contact based on the interaction of cell surface proteins.

The cytokines produced by dendritic cells depend on the type of cell. Lymphoblastic dendritic cells can produce large amounts of type 1 IFN, which collects more activated macrophages to enable phagocytosis. Lymphoblastic dendritic cells are involved in central and peripheral immune regulation, and myeloid dendritic cells are known to be involved in inducing immunity to foreign antigens or infections. Therefore, when the dendritic cells do not function normally, autoimmune diseases such as diabetes, rheumatoid arthritis, and allergic hypersensitivity reactions may occur, or normal immune responses may not occur to infectious diseases or cancers.

In cancer patients, the function of dendritic cells is reduced and it is difficult to induce normal anticancer immunity. Until now, many substances (LPS, TNF-a, IL-1β) that regulate (activate) the differentiation of dendritic cells are known, but there are many problems in direct application to living organisms as a side effect of biotoxicity.

On the other hand, Mycobacterium is not only a species that causes serious diseases in humans and animals such as tuberculosis, tuberculosis, and leprosy, but also the species called opportunistic bacteria and the object parasitics found in the natural environment. About 72 species, including saprophytic species, are known to date, and 25 of them are known to be related to human diseases.

Tuberculosis is the most common disease among mycobacterial infections. Four species of M. tuberculosis , M. bovis , M. africanum and M. microti are classified into M. tuberculosis complex (TB complex). M. tuberculosis is the most common and important causative agent. Tuberculosis is still an important disease in Korea, and it is reported that approximately 8 million new cases occur worldwide each year.

As described above, dendritic cells play an important role in enhancing the body's own immune function. Therefore, dendritic cells promote the differentiation and maturation of dendritic cells. Has become an important challenge for cellular immune therapy using dendritic cells

As such, dendritic cells have an advantage that they can be usefully used for the treatment using an immune response when they mature, so there is an urgent need for new methods for effectively maturing immature dendritic cells.

KR10-2010-0013088

The present inventors conducted a study to satisfy the above requirements, and confirmed that Rv2769c derived from Mycobacterium tuberculosis can effectively induce maturation of immature dendritic cells to promote Th1-biased mature dendritic cells and complete the present invention. It was.

Disclosure of Invention It is an object of the present invention to provide a composition for inducing dendritic cell maturation, comprising Rv2769c from Mycobacterium tuberculosis, which can induce immature dendritic cells to differentiate into Th1 type mature dendritic cells.

The present invention provides a composition for inducing maturation of dendritic cells comprising Rv2769c protein as an active ingredient.

The present invention also provides a method for inducing maturation of immature dendritic cells, characterized in that immature dendritic cells are treated with Rv2769c to differentiate into mature dendritic cells.

The present invention also provides an immune enhancing composition comprising the Rv2769c protein as an active ingredient.

Rv2769c according to the present invention stimulates immature dendritic cells, secretes pro-inflammatory cytokines TNF-α, IL-6, IL-1β, and expresses the protein surface factors CD80, CD86, MHC class I and II. It promotes maturation to differentiate into increased mature dendritic cells, thereby effectively enhancing the body's immune response.

1 is a diagram showing a recombinant protein Rv2769c (PE family protein) isolated through cloning (a: SDS-PAGE comparison of His-tagged recombinant Rv2769c and vector control, b: SDS-PAGE, c after purification with NTA) Western blot analysis).
2 is a graph showing the results of experiments measuring the survival rate of dendritic cells treated with Rv2769c.
Figure 3 is a graph analyzing the expression of the specific protein surface factors CD80, CD86 and MHC class I, II in Rv2769c-treated dendritic cells (a) and a graph showing the expression amount of the protein surface factor as a numerical value (b) to be.
Figure 4 is a graph showing the change in the secretion of cytokines (TNF-α, IL-6, IL-1β, IL-12 p70, IL-10) secreted from dendritic cells following Rv2769c treatment and IL in dendritic cells Figure (b) shows the result of analyzing the secretion amount of -12p70 and IL-10 by flow cytometry.
5 is a graph showing the results of measuring the dextran-FITC phagocytosis of Rv2769c-treated dendritic cells at 4 ° C. and 37 ° C., respectively.
Figure 6 shows the change in the signaling system in dendritic cells treated with Rv2769c by phosphorylation measurement of ERK, JNK, p38, IκB-α, NF-κB p65 and protein degradation using Western blot (a), NF-κB p65 Shows the result of confirming that (b) is transferred to the nucleus by the Rv2769c treatment.
Figure 7 shows the results of analysis by flow cytometry FACs Canto after staining with anti-CD80-PE, anti-CD86-PE cell surface factor antibody after treatment of MAPK and NF-κB inhibitors to dendritic cells (a) and (B) shows the result of confirming by using the ELISA kit that the secretion amount of TNF-α, IL-6, IL-1β in the dendritic cells treated with the inhibitor.

The present invention provides a composition for inducing maturation of dendritic cells comprising Rv2769c as an active ingredient.

Rv2769c according to the present invention can stimulate immature dendritic cells to differentiate into mature dendritic cells, immature dendritic cells stimulated with Rv2769c become mature dendritic cells to express higher levels of MHC type I and II antigens In addition to increasing the expression of protein surface factors, CD80 and CD86, the secretion of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β is markedly increased.

Rv2769c consists of 275 amino acid sequences and has a size of 26.2KDa. Rv2769c belongs to the PE family, a family of repeat genes that comprise a large proportion of 10% of the entire genome of Mycobacterium tuberculosis H37Rv standard strain, and the function of Rv2769c is not yet clear. Mycobacterium tuberculosis H37Rv standard strain (NCBI GenBank ID: CAB00972.1) The unique ID number of the protein of Rv2769c assigned through the analysis of the entire gene map is 57117023. The nucleotide sequence of the tuberculosis bacteria-derived Rv2769c protein is shown in SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 2, respectively.

The present invention can clone the Rv2769c gene of M. tuberculosis , and then isolated and purified using an E. coli expression system to induce dendritic cell maturation.

Immature dendritic cells mature to form mature dendritic cells. Mature dendritic cells lose the ability to exhibit up-regulated expression of various cytokines and cell surface molecules that capture and co-stimulate antigens. In particular, mature dendritic cells express higher levels of MHC type I and II antigens than immature dendritic cells and regulate CD (Cluster of Differentiation) 80+, CD83 +, CD86 + and CD14−. More MHC expression leads to an increase in antigen density on dendritic cell surfaces, while up-regulation of co-stimulatory molecules CD80 and CD86, T cell activity through co-stimulatory molecular counterparts such as CD28 on T cells Strengthen the signal

The present invention can facilitate the differentiation of immature dendritic cells into Th1 biased mature dendritic cells through a composition comprising Rv2769c. Rv2769c prepared by an effective recombinant method may be included in the composition at a concentration of 0.1 μg / ml to 5 μg / ml, preferably 0.5 μg / ml to 1 μg / ml.

In addition, the present invention can provide a method of inducing maturation of immature dendritic cells characterized in that the immature dendritic cells are treated with Rv2769c to differentiate into mature dendritic cells.

The Rv2769c protein may be treated in immature dendritic cells at a concentration of 0.1 μg / ml to 5 μg / ml, preferably 0.5 μg / ml to 1 μg / ml.

The present invention can culture cells stimulated with Rv2769c to induce maturation by stimulating immature dendritic cells with Rv2769c. In order to induce differentiation into appropriate mature dendritic cells, the cells may be matured by culturing for 12 to 36 hours as a preferred example, but are not limited thereto.

Dendritic cell maturation can be monitored by methods known in the art and cell surface markers can be detected by assays familiar to the art such as flow cytometry and immunohistochemistry. The cells can also be monitored via cytokine production (eg, ELISA, FACS and other immune assays).

Mature dendritic cells promoted maturation through Rv2769c protein treatment in the present invention can express the general characteristics of mature dendritic cells. That is, according to the present invention, by treating Rv2769c to immature dendritic cells, immature dendritic cells are differentiated into mature dendritic cells, and more preferably, immature dendritic cells are treated with TNF-α and IL- according to Rv2769c protein stimulation. The production of 12p70, IL-6, IL-1ß may be changed to increased mature dendritic cells, which are preferably mature dendritic cells biased to Th1 type cells.

In addition, the present invention provides an immune enhancing composition comprising Rv2769c as an active ingredient.

By treating Rv2769c according to the present invention to immature dendritic cells, immature dendritic cells are mature, and the maturation of dendritic cells can increase the expression of surface protein markers. More specifically, by treating Rv2769c to immature dendritic cells, the dendritic cells can be matured and consequently induce the activity of T cells to increase the immune response.

Therefore, Rv2769c of the present invention can be usefully used for medicines and health foods for immune boosting.

Immunostimulating pharmaceutical composition comprising the Rv2769c protein as an active ingredient, in addition to the Rv2769c protein preferably contains other ingredients that can give a synergistic effect to the main effect within the scope that does not impair the main effect of the present invention. can do.

In addition, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient and diluent in addition to the active ingredient described above for administration.

For example, carriers, excipients and diluents include lactose, textose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil.

The pharmaceutical composition of the present invention may be prepared in various parenteral or oral administration forms according to known methods. As representative of formulations for parenteral administration, isotonic aqueous solutions or suspensions are preferred for injectable formulations. The injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the active ingredient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The effective dosage of the pharmaceutical composition of the present invention may vary depending on the age and gender of the patient, but may be characterized as being administered at 0.01 to 1000 mg / kg, and preferably at 0.1 to 100 mg / kg. Can be.

Immunity-enhancing food compositions containing Rv2769c protein as an active ingredient may be added as it is or used with other food or food ingredients, and may be appropriately used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, gums, ice cream, soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The beverage composition of the present invention may include various flavors or natural carbohydrates, and the like as additional ingredients, as in general beverages. Such natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like . The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may include a pulp for the production of natural fruit juice, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples and Formulation Examples. However, the following Examples and Preparation Examples are provided to illustrate the present invention more easily, and the content of the present invention is not limited by Examples and Preparation Examples.

Example  1. Isolation and Recombination of Dendritic Cells Rv2769c of Cloning

1.1 Isolation and Induction of Dendritic Cells

Femoral bone marrow was harvested using a bone marrow harvesting injection from C57BL / 6 mice. After the collected bone marrow was washed, red blood cells were removed using ammonium chloride. The isolated cells were harvested in 6-well plates using RPMI 1640 (10% FBS (Fetal bovine serum, calf serum), 2 mM L-glutamine, 100 U / ml penicillin / streptomycin, 50 μM mercaptoethanol, 0.1 mM non-essential amino acid, 1 mM sodium pyruvate, 20 ng / ml GM-CSF, 20 ng / ml IL-4) was added and incubated for 8 days. GM-CSF and IL-4 were used to induce differentiation into dendritic cells.

1.2 Recombination Rv2769c of Cloning

Rv2769c was isolated and cloned from Mycobacterium tuberculosis genomic DNA. Rv2769c sites were amplified using forward primers (5'-CATATGTCATTCCTGACGACACAGCCT-3 ', SEQ ID NO: 3) and reverse primers (5'-AAGCTTTACCGCCGGCTTGGGCACGAC-3', SEQ ID NO: 4). The PCR product was digested with NdeI and HindIII enzymes and inserted into the expression vector pET-22b (+) vector (Novagen, Madison, Wis.). 1 mM isopropyl-D-thio after incubation of the E. coli BL21 transformed with the pET-22b (+) vector with the Rv2769c gene at 37 ° C. at an absorbance (OD) of 0.4 to 0.5 Galactopyranoside (IPTG) was added and incubated for 6 hours. The expressed protein was purified using nickel-nitrilotriacetic acid (Ni-NTA, Invitrogen, Carlsbad, Calif., USA) agarose according to the manufacturer's method. Finally, the purified recombinant protein was confirmed by analysis by SDS-PAGE and Western blot.

The results are shown in Fig.

As shown in FIG. 1, recombinant Rv2769c protein was found at the 26.2 kDa position.

Example 2. Confirmation of cytotoxicity of Rv2769c

In order to confirm the cytotoxicity of Rv2769c, cell viability of dendritic cells stimulated with Rv2769c was measured. Immature dendritic cells were treated with Rv2769c at a concentration of 0.5, 1 μg / ml, or lipopolysaccharide (LPS) at a concentration of 100 ng / ml for 24 hours. This was then double stained with propidium iodide (PI) and Annexin-V and analyzed via flow cytometry.

 The results are shown in Fig.

As shown in FIG. 2, Rv2769c at a concentration of 0.5 μg / ml as well as Rv2769c at 1 μg / ml did not show toxicity in dendritic cells.

Example 3 Increasing Surface Factor Expression of Dendritic Cells by Rv2769c Stimulation

First, the cells were maintained in OptiMEM medium containing GM-CSF and IL-4 in order to uniformize the maturity of all cells, and then RD2769c and 0.5 ng / ml LPS at concentrations of 0.5 and 1 μg / ml were applied to dendritic cells for 24 hours, respectively. Dendritic cells were recovered after treatment. In order to inhibit nonspecific binding, 1 μg / ml of Fc I / III (BD bioscience) was treated at 4 ° C. for 30 minutes, and then stained with CD11c-FITC (BD bioscience) at 30 ° C. for 30 minutes for dendritic cell analysis. . After staining with cell surface factor antibodies such as anti-CD80-PE (BD bioscience), anti-CD86-PE (BD bioscience), anti-MHC I and II-PE (BD bioscience), flow cytometry FACs Canto (BD Biosciences).

The results are shown in FIG.

As shown in FIG. 3, stimulation of Rv2769c dose-dependently increased the expression of CD80, CD86 and MHC class I and MHC class II, protein surface factors of dendritic cells. Through this, it was confirmed that the maturation of dendritic cells was effectively performed by Rv2769c.

Example 4 . Effect of Rv2769c Stimulation on Cytokine Secretion in Dendritic Cells

The type of cytokine secreted depends on the maturation of dendritic cells. Therefore, in order to confirm this, the immature dendritic cells were treated with Rv2769c at a concentration of 0.5, 1 μg / ml or 100 ng / ml of LPS with a control group, and then cultured for 24 hours. Supernatants were subjected to ELISA kits (ebioscience) for TNF-α, IL-6, IL-1β, IL-10 and IL-12 p70 to determine cytokine secretion. IL-12 p70 and IL-10 are major cytokines secreted by dendritic cells, and play an important role in the differentiation of T cells into Th1 and Th2, respectively. IL-12 is a representative Th1 cytokine, and IL-10 is known to interfere with the activity of cytokines (including IL-12 p70) produced by activated monocytes, NK cells and Th1 cells. Based on these results, staining anti-IL-12 p70-APC (BD bioscience) and anti-IL-10-APC to measure intracellular IL-12 p70 and IL-10 secretion flow cytometry FACScallibur (Beckson Dikinson) , USA).

The results are shown in Fig.

As shown in Figure 4, ELISA kit analysis showed that IL-12 p70 significantly increased in dose-dependent Rv2769c, and also pro-inflammatory cytokines TNF-α, IL-6, IL-1β also markedly increased (a). This shows that Rv2769c matures dendritic cells and changes the amount of cytokines. In particular, as a result of measuring the secretion of IL-12 p70 and IL-10 in the cell by flow cytometry, unlike IL-12 p70, IL-10 secretion did not increase significantly, so Rv2769c deflected dendritic cells to Th1-type cells. It was possible to confirm the possibility (b).

Example  5. Rv2769c Analysis of antigen acquisition ability of dendritic cells by

Immature dendritic cells are known to have good antigen uptake, while mature dendritic cells are known to have reduced antigen uptake capacity. Therefore, in order to confirm the effect of Rv2769c protein on the endocytic activity of dendritic cells, the following experiment was performed. Immature dendritic cells were incubated for 24 hours after treatment with 0.5, 1 μg / ml Rv2769c or 100 ng / ml LPS. 1 μg / ml of fluorescein-conjugated dextran (molecular weight 40,000; Molecular Probes, Eugene, OR) was added to the cultured dendritic cells for 1 hour. After incubation at 37 ° C. and 4 ° C. for 30 minutes, the reaction was stopped by the addition of cold staining buffer and the cells were washed. After staining with PE (Phycoerythrin) -binding CD11c (BD bioscience), the phagocytosis of dextran-FITC (Fluorescein isothiocyanate) was measured using flow cytometry FACs Canto (BD Biosciences).

The results are shown in Fig.

As shown in FIG. 5, dendritic cells treated with Rv2769c at 37 ° C. had a lower dextran (antigen) phagocytic capacity than dendritic cells not treated with Rv2769c. According to these results, it was found that Rv2769c matures dendritic cells in terms of function. In order to determine the nonspecific binding of dextran to dendritic cells, measurement was also performed at 4 to correct the results. Through this Rv2769c treatment it was functionally confirmed that the maturity of dendritic cells increased.

Example  6. Rv2769c Signaling System Activation of Dendritic Cells by

Dendritic cells are known to activate mitogenactivated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signaling systems such as ERK, JNK, and p38 during maturation. Thus, after immature dendritic cells were treated with Rv2769c 1 μg / ml for 0 minutes 5 minutes, 15 minutes, 30 minutes, and 60 minutes, the cells were disrupted to isolate all proteins, MAPK (mitogen-activated protein kinases) and NF-κB. (nuclear factor-kappa B) The phosphorylation and degradation of ERK, JNK, p38, IκB-α, and NF-κB p65, which are factors related to the signaling system, were confirmed. The amount of protein was measured by Western blot method using a specific antibody. In addition, it was confirmed by fluorescence staining whether NF-κB p65 was transcribed into the nucleus by Rv2769c treatment.

The results are shown in Fig.

As shown in Figure 6, it was confirmed that the amount of p-p38, p-ERK, p-JNK phosphorylated by Rv2769c treatment increased. In addition, it was confirmed that p-IκB-a is increased and IκB-a is degraded (a). NF-κB p65, the final signaling factor, was also transcribed into the nucleus by Rv2769c treatment (b). This confirmed that Rv2769c is involved in MAPKs and NF-κB signaling system of dendritic cells.

Example  7. Rv2769c In the activity of dendritic cells MAPK  And NF -κ Of B  Impact Analysis

To determine whether dendritic cells activated by Rv2769c were activated by the signaling systems of MAPK and NF-κB, U0126 (ERK1 / 2), SB203580 (p38), SP600125 (JNK), Bay 11-0782 (NF Analysis was performed using MAPK and NF-κB inhibitors such as -B). Specifically, dendritic cells were pretreated with MAPK and NF-κB inhibitors for 1 hour, followed by incubation for 24 hours after treatment with Rv2769c at 1 μg / ml concentration or 100 ng / ml LPS with the control group. For analysis of the effect of surface factor expression on dendritic cells, the cells were stained with CD11c-PE-cy7 (ebioscience) for 30 minutes at 4 ° C. Thereafter, the cells were stained using cell surface factor antibodies such as anti-CD80-PE (ebioscience) and anti-CD86-PE (ebioscience), and analyzed by flow cytometry FACs Canto (BD Biosciences). In addition, the cytokine secretion was measured by applying an ELISA kit (ebioscience) for TNF-α, IL-6, IL-1β to the culture supernatant to analyze the cytokine secreted according to the maturation of dendritic cells, MAPK and The amount of NF-κB p65 expression in the nucleus was measured using an NF-κB inhibitor.

The results are shown in Fig.

As shown in Figure 7, MAPK and NF-κB inhibitors were confirmed by flow cytometry FACs Canto analysis to reduce the costimulatory factors CD80 and CD86 expression of dendritic cells induced by Rv2769c stimulation (a), ELISA kit As a result, it was confirmed that the inflammatory cytokines TNF-α, IL-6, IL-1β are also reduced (b). It was confirmed that the activity of dendritic cells by Rv2769c is dependent on MAPKs (ERK1 / 2, p38, JNK) and NF-κB signaling system, which induces the production of pro-inflammatory cytokines.

Formulation example  1. Preparation of pharmaceutical compositions

1.1 Manufacture of Powder

Rv2769c 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1.2 Preparation of tablets

Rv2769c 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1.3 Preparation of capsules

Rv2769c 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1.4 Manufacture of Injection

Rv2769c 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na2HPO4 占 H2O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

1.5 Preparation of Liquids

Rv2769c 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  2. Preparation of food composition

2.1. Manufacture of health food

Rv2769c 100 mg

Vitamin mixture quantity

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2.2 Preparation of Beverages

Rv2769c 100 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered and sterilized in a sterilized 2 L container, ≪ / RTI >

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Compositon for maturating dendritic cell comprising Mycobacterium          tuberculosis Rv2769c <130> 1.2p <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 828 <212> DNA <213> Mycobacterium tuberculosis <400> 1 ctataccgcc ggcttgggca cgacgatggg tttggcgccg tagcgtggcg caccgaagcc 60 cgcgctgctg cgcgtcgccg aggccaaccc cggcatcccg ggaatgaccg tccccgcggc 120 accatgcggt gccgcggtgg tccagccagc gccctgcagt gtgctggtgc tcgataccag 180 gttggcctgt cccgcccagc tgggcggcac cgacaatgcg ccgattgacg acgcccgact 240 aaggccggcg gctagcggag ccgcacccag accggccgcg atcggcgcct cgccgacggc 300 cgcctccgcc gcccccagct ccgataggcc cgcgccctcc aagccctcct cgagggcggc 360 ttcctcggca gccggaagaa gaccaccgct ggccagccct agcaagtccg acgcggcgga 420 ggcccagttc ccagccccaa tgttgaagat attggcaata tctgaaatcc aggagggcac 480 cttcccgggc gtggaaccca agatgctcgc gatacccgac aacggcgaag cggccgcgga 540 tgagttggcg gcctcggtgg ccgcataggt gccagcgctg acccccaggg tcttcacaaa 600 caggtcgtat accgcagctg cttcagcact gacctgctgg tagagagtgc cgtacgcggt 660 gaacaacggc gcctgtagca ctgatatctc atcagcggcg gcgggaatca cgcccgtggt 720 ggtcggggcg gccgcggccg cgttctgggc gaccatcgcc gagccgatgg tctcgagctt 780 gccggccgca gccgccaact cttcaggctg tgtcgtcagg aatgacat 828 <210> 2 <211> 275 <212> PRT <213> Mycobacterium tuberculosis <400> 2 Met Ser Phe Leu Thr Thr Gln Pro Glu Glu Leu Ala Ala Ala Gly   1 5 10 15 Lys Leu Glu Thr Ile Gly Ser Ala Met Val Ala Gln Asn Ala Ala Ala              20 25 30 Ala Ala Pro Thr Thr Thr Gly Val Ile Pro Ala Ala Ala Asp Glu Ile          35 40 45 Ser Val Leu Gln Ala Pro Leu Phe Thr Ala Tyr Gly Thr Leu Tyr Gln      50 55 60 Gln Val Ser Ala Glu Ala Ala Ala Val Tyr Asp Leu Phe Val Lys Thr  65 70 75 80 Leu Gly Val Ser Ala Gly Thr Tyr Ala Ala Thr Glu Ala Ala Asn Ser                  85 90 95 Ser Ala Ala Ala Ser Pro Leu Ser Gly Ile Ala Ser Ile Leu Gly Ser             100 105 110 Thr Pro Gly Lys Val Pro Ser Trp Ile Ser Asp Ile Ala Asn Ile Phe         115 120 125 Asn Ile Gly Ala Gly Asn Trp Ala Ser Ala Ala Ser Asp Leu Leu Gly     130 135 140 Leu Ala Ser Gly Gly Leu Leu Pro Ala Ala Glu Glu Ala Ala Leu Glu 145 150 155 160 Glu Gly Leu Glu Gly Ala Gly Leu Ser Glu Leu Gly Ala Ala Glu Ala                 165 170 175 Ala Val Gly Glu Ala Pro Ile Ala Ala Gly Leu Gly Ala Ala Pro Leu             180 185 190 Ala Ala Gly Leu Ser Arg Ala Ser Ser Ile Gly Ala Leu Ser Val Pro         195 200 205 Pro Ser Trp Ala Gly Gln Ala Asn Leu Val Ser Ser Thr Ser Thr Leu     210 215 220 Gln Gly Ala Gly Trp Thr Thr Ala Ala Pro His Gly Ala Ala Gly Thr 225 230 235 240 Val Ile Pro Gly Met Pro Gly Leu Ala Ser Ala Thr Arg Ser Ser Ala                 245 250 255 Gly Phe Gly Ala Pro Arg Tyr Gly Ala Lys Pro Ile Val Val Pro Lys             260 265 270 Pro ala val         275 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Rv2769c forward primer <400> 3 catatgtcat tcctgacgac acagcct 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Rv2769c reverse primer <400> 4 aagctttacc gccggcttgg gcacgac 27

Claims (11)

A composition for inducing maturation of dendritic cells comprising Rv2769c protein as an active ingredient.
The method of claim 1, wherein the Rv2769c protein is Mycobacterium tuberculosis , and a composition for inducing maturation of dendritic cells.
The composition for inducing maturation of dendritic cells according to claim 1, wherein the Rv2769c protein is included at a concentration of 0.1 μg / ml to 5 μg / ml.
The composition for inducing maturation of dendritic cells according to claim 1, wherein the Rv2769c protein has an amino acid sequence of SEQ ID NO.
The composition for inducing maturation of dendritic cells according to claim 1, wherein the Rv2769c protein is encoded by the nucleotide sequence of SEQ ID NO: 1.
A method of inducing maturation of immature dendritic cells, characterized in that immature dendritic cells are treated with Rv2769c to differentiate into mature dendritic cells.
The method of claim 6, wherein the immature dendritic cells are incubated for 12 to 36 hours after Rv2769c treatment.
The method of claim 6, wherein the mature dendritic cells have increased secretion of TNF-α, IL-6, IL-1β.
The method of claim 6, wherein Rv2769c is treated at a concentration of 0.1 μg / ml to 5 μg / ml.
Immune enhancer pharmaceutical composition comprising the Rv2769c protein as an active ingredient.
Immune enhancing food composition comprising an Rv2769c protein as an active ingredient.

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