KR20130039061A - Maturation method for dendritic cell using mycobacterium tuberculosis rv2299c - Google Patents

Abstract

PURPOSE: A method for maturing dendritic cells is provided to promote differentiation of immature dendritic cells using Mycobacterium tuberculosis Rv2299c, to effectively mature the dendriric cells, and to effectively enhance immune response. CONSTITUTION: A composition for inducing maturation of dendritic cells contains 1-5 microgram/ml of Rv2299c protein derived from Mycobacterium tuberculosis as an active ingredient. A method for inducing maturation of immature dendritic cells comprises: a step of treating Rv2299c to the immature dendritic cells; and a step of culturing the treated dendritic cells for 12-36 hours. The maturation of the immature dendritic cells indicates enhancement of TNF-alpha, IL-12p70, IL-6, and IL-1beta.

Description

Maturation method for Dendritic cell using Mycobacterium tuberculosis Rv2299c}

The present invention relates to dendritic cell maturation inducing compositions and methods of maturing dendritic cells. More specifically, the present invention relates to a dendritic cell maturation inducing composition comprising an Rv2299c protein derived from Mycobacterium tuberculosis as an active ingredient and a method of differentiating immature dendritic cells into mature dendritic cells using the same.

Dendritic cells or dendritic cells (DCs) 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 to 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. However, macrophages and B lymphocytes can only activate memory T-cells, while dendritic cells are the most potent antigen-expressing cells because they can activate both memory and naive T-cells.

Mature dendritic cells can be derived from monocytes, white blood cells that circulate in the body, which can be converted 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 TNFα 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 if, for example, a cellular immune response is required. The Th-1 type response induces and polarizes cytotoxic T lymphocytes (CTLs), which are the mechanism of attack of the cellular immune system. These action factor attack measures are 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. Therefore, the use of dendritic cells producing IL-12 is theoretically optimally suitable for use in immunostimulation.

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-α, IL-1β) that regulate (activate) the differentiation of dendritic cells are known, but as a side effect of biotoxicity, there are many problems in direct application to the living body.

Mycobacterium genus includes not only species that cause serious diseases in humans and animals, such as tuberculosis, tuberculosis and leprosy, but also species called opportunistic bacteria, About 72 species are known to date, including saprophytic species found in the natural environment, of which 25 are known to be associated with human diseases.

The most common disease among mycobacterial infections is tuberculosis , which is classified as M. tuberculosis complex (TB complex) with strong pathogenicity ( M. tuberculosis , M. bovis , M. africanum , M. microti). Four of them are causative bacteria, and 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

The present inventors have completed the invention by discovering that Rv2299c derived from Mycobacterium tuberculosis effectively matures dendritic cells as a result of repeated studies to satisfy the above requirements.

The present invention is an immature dendritic cell maturation inducible composition comprising Rv2299c protein as an active ingredient, which can easily and effectively mature immature dendritic cells, and immature dendritic cells are treated with Rv2299c to mature into mature dendritic cells. It provides a method for inducing maturation of dendritic cells. In another aspect, the present invention provides a composition for enhancing immunity comprising the dendritic cell maturation inducing composition.

In order to solve the above problems, the present invention provides a dendritic cell maturation inducing composition comprising the Rv2299c protein as an active ingredient.

The present invention also provides a method of inducing maturation of immature dendritic cells characterized in that Rv2299c is treated to immature dendritic cells.

In another aspect, the present invention provides a composition for enhancing immunity comprising the dendritic cell maturation inducing composition.

According to the present invention, by promoting the differentiation of immature dendritic cells can effectively mature dendritic cells through which can effectively enhance the body's immune response.

1A shows recombinant protein Rv2299c isolated via cloning. (a: SDS-PAGE comparison of His-tagged recombinant Rv2299c with vector control (CON), b: SDS-PAGE after purification with NTA, c: western blot analysis).
1B is a graph showing the results of experiments measuring the survival rate of dendritic cells treated with Rv2299c.
Figure 2a is a graph analyzing the expression of specific protein surface factors CD80, CD86 and MHC class I, II in Rv2299c-treated dendritic cells
Figure 2b is a graph showing the amount of expression of CD80, CD86 and MHC class I, II specific protein surface factors in Rv2299c-treated dendritic cells.
3A and 3B are graphs showing the results of cytokine (TNF-α, IL-6, IL-1β, IL-12 p70, IL-10) secretion experiments of dendritic cells treated with Rv2299c.
Figure 4a shows the results of analyzing the effect of thermal degeneration on TNF-α, IL-6 and IL-1β release of dendritic cells induced by Rv2299c.
4b shows the results of analyzing the effect of proteinase K digestion on TNF-α, IL-6 and IL-1β release of dendritic cells induced by Rv2299c.
Figure 4c shows the results of the analysis of the effect of PMB on TNF-α, IL-6 and IL-1β release of dendritic cells induced by Rv2299c.
5 is a graph showing the results of dextran-FITC phagocytosis of the dendritic cells treated with Rv2299c.
6A and 6B are graphs confirmed through cytokine (TNF-α, IL-6, IL-1β) secretion and protein surface factors CD80 and CD86 through inhibitory factors of changes in the signal transduction system in dendritic cells treated with Rv2299c. to be.
Figure 7a shows the results of TLR experiments involved in maturation of dendritic cells treated with Rv2299c.
7B shows the relationship between the maturation of dendritic cells treated with MyD88, TRIF and Rv2299c.

The present invention relates to a dendritic cell maturation inducing composition comprising Rv2299c as an active ingredient.

The present invention relates to a dendritic cell maturation inducing composition comprising Rv2299c as an active ingredient.

Rv2299c (HtpG protein, HSP-90) is a 72.8 KDa cytoplasmic antigen consisting of 647 amino acid sequences. HSP90 is one of the most expressed proteins in cells and one of the heat shock proteins secreted by stress. HSP90 is found in bacteria and in all eukaryotes and is involved in cell growth. Mycobacterium tuberculosis H37Rv standard strain (NCBI GenBank ID: CAB00972.1) The unique ID number of the protein of Rv2299c assigned through analysis of the entire gene map is 1449321.

The present invention can provide a composition for inducing dendritic cell maturation by cloning a gene for M. tuberculosis by using an E. coli expression system.

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 promote differentiation into mature dendritic cells via a composition comprising Rv2299c. Rv2299c prepared by an effective recombination method may be included in the composition at a concentration of 1 μg / ml to 5 μg / ml, preferably 1 μg / ml to 2 μg / ml.

In another aspect, the present invention can provide a method of inducing maturation of immature dendritic cells, characterized in that Rv2299c is treated to immature dendritic cells.

The Rv2299c protein of the present invention may be treated at a concentration of 1 μg / ml to 5 μg / ml, preferably 1 μg / ml to 2 μg / ml. The present invention can incubate cells treated with Rv2299c to induce maturation by treating Rv2299c with immature dendritic cells. In order to induce differentiation into appropriate mature dendritic cells, the cells may be matured by, for example, not less than 12 hours but not more than 36 hours as a preferred example.

Mature dendritic cells promoted maturation through Rv2299c protein treatment in the present invention can express the general characteristics of mature dendritic cells. According to the present invention, by treating Rv2299c with an active ingredient, immature dendritic cells are differentiated into mature dendritic cells, more preferably, mature dendritic cells with increased production of IL-6, IL-1ß and TNF-α. can do. Mature dendritic cells formed through the composition of the present invention may preferably be differentiated into cells that do not affect the secretion of IL-10 and thus may be biased into Th1 type cells.

In addition, the present invention can provide a composition for enhancing immunity comprising a dendritic cell maturation inducing composition comprising Rv2299c as an active ingredient. According to the present invention, immature dendritic cells are mature, and the expression of surface protein markers may be increased by maturation of the dendritic cells. More specifically, according to the immune enhancing composition of the present invention, through the treatment of the active ingredient Rv2299c can mature dendritic cells and consequently induce the activity of T cells to increase the immune response.

Maturation of dendritic cells 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).

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1. Dendritic Cell Isolation and Recombination Rv2299c 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 collected in a 6-well plate 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 Rv2299c of Cloning

Rv2299c was isolated and cloned from Mycobacterium tuberculosis genomic DNA. The Rv2299c site was amplified using (5'-CGC CATATGAACGCCCATGTCGAGC-3 ') and reverse primer (5'-CCCAAGCTT CAAGGTACGCGCGAGACGTTC-3') of the forward primer. 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 E. coli BL21 transformed with pET-22b (+) vector with the inserted rv2299c gene at 37 ° C with an absorbance (OD) of 0.4 to 0.5 Galactopyranoside (IPTG) was added and incubated for 6 hours. The expressed protein was purified according to the manufacturer's method using nickel-nitrilotriacetic acid (Ni-NTA, Invitrogen, Carlsbad, Calif., USA) agarose. Finally, the purified recombinant protein was analyzed by SDS-PAGE and confirmed at the position of 72.8 kDa (Fig. 1a- (b), (c)).

Example  2. Rv2299c Cytotoxicity

In order to measure the cell viability of dendritic cells stimulated with Rv2299c, dendritic cells were treated with Rv2299c at a concentration of 2 μg / ml or as a control and 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. As shown in FIG. 1 b, Rv2299c at a concentration of 2 μg / ml showed no toxicity in dendritic cells.

Example  3. Rv2299c Surface Factor Expression Analysis of Dendritic Cells by

First, the cells were maintained in OptiMEM medium containing GM-CSF and IL-4 to uniformize the maturity of all cells, and then LPS at Rg2299c and 100 ng / ml at 1 and 2 μg / ml concentrations in dendritic cells. Were treated for 24 hours and then dendritic cells were recovered. 1 μg / ml of Fcγ I / III (BD bioscience) was treated at 4 ° C. for 20 minutes to inhibit nonspecific binding, followed by staining at 4 ° C. for 20 minutes with CD11c-FITC (BD bioscience) for dendritic cell analysis. It was. 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) (FIG. 2). As shown in Figure 2, Rv2299c dose-dependently increased the expression of CD80, CD86 and MHC class I, II protein surface factors of dendritic cells. This confirmed that maturation of dendritic cells was achieved by Rv2299c.

Example 4 . Effect of Rv2299c on Cytokine Secretion in Dendritic Cells

The type of cytokine secreted depends on the maturation of dendritic cells. To confirm this, the dendritic cells were treated with 1 μg / ml or 2 μg / ml of Rv2299c or a control group with 100ng / ml of LPS and then incubated for 24 hours. Supernatants were applied to ELISA kits for TNF-α, IL-6, IL-1β, IL-10 and IL-12 p70 to measure cytokine secretion (FIG. 3A). IL-12 p70 and IL-10 are the 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 of 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) (Fig. 3b). As shown in Figure 3a, IL-12 p70 increased and pro-inflammatory cytokines TNF-a, IL-6, IL-1β also increased, indicating that Rv2299c matures dendritic cells and changes the amount of cytokines. Able to know. On the other hand, there was no significant increase in the secretion of IL-10, confirming the possibility that Rv2299c could deflect dendritic cells into Th1-type cells.

Example 5 . LPS  And Rv2299c Of dendritic cells induced by TNF -α and IL For -6 emission Heat denaturation , Proteinase  K digestive and PMB Impact on

5.1 Thermophilic  Effect measurement

To analyze the effect of LPS and Rv2299c's thermodegeneration on TNF-α and IL-6 release of dendritic cells induced by LPS and Rv2299c, immature dendritic cells from C57BL / 6 mice were treated with 100 ng / ml LPS or 2 μg /. ml Rv2299c, or boiled LPS or Rv2299c, or not. After 24 hours, the amount of TNF-α, IL-6, IL-1β in the supernatant of the dendritic cells was analyzed by ELISA. As shown in FIG. 4A, since Rv2299c is a protein, it is decomposed when it is boiled, so that the secreted TNF-α, IL-6, and IL-1β are inhibited in the case of boiled Rv2299c. On the other hand, LPS did not show a difference in secretion of TNF-α, IL-6, IL-1β even when boiled (Fig. 4a).

5.2 Measuring Effects on Protease K Treatment

It was also treated with proteinase K at 100 ng / ml LPS or 2 μg / ml Rv2299c and then added to the culture of immature dendritic cells. The concentration of proteinase K was treated with 1/20 of LPS and Rv2299c concentration, and then activated at 50 ° C for 1 hour and inactivated at 100 ° C. The amount of TNF-α, IL-6, IL-1β in the supernatant of the dendritic cells was analyzed by ELISA. As shown in FIG. 4B, since Rv2299c is a protein, the protein is degraded by proteinase K, which is a protease. Thus, in the case of Rv2299c treated with proteinase K, secretion of TNF-α, IL-6, and IL-1β was inhibited. On the other hand, in the LPS treatment with proteinase K, there was no difference in the secretion of TNF-α, IL-6, IL-1β (Fig. 4b).

5.3 polymyxin  B ( PMB ) Measure effect after treatment

 To confirm that the effect of dendritic cell activation of Rv2299c is not due to LPS contamination, immature dendritic cells of C57BL / 6 mice were pretreated with LPS inhibitor polymyxin B (PMB) or without 100ng / ml LPS or 10 μg. Treatment with / ml Rv2299c for 24 hours. After 24 hours, the amount of TNF-α, IL-6, IL-1β in the supernatant of the dendritic cells was analyzed by ELISA.

As shown in FIG. 4C, the secretion of TNF-α, IL-6, and IL-1β by LPS is reduced by PMB in the case of LPS, whereas TNF-α, IL-6, IL- is treated even if PMB is treated in Rv2299c. There was no effect on the secretion of 1β. This proves that Rv2299c is not due to LPS contamination (Figure 4c).

Example  6. Rv2299c On by Dendritic  Antigen Capability Analysis

Immature dendritic cells are good at antigen uptake, while mature dendritic cells are known to have reduced antigen uptake. Therefore, an experiment was conducted to determine the effect of Rv2299c protein on the endothelial activity of dendritic cells. Immature dendritic cells were incubated for 24 hours after treatment with 1 and 2 μg / ml of Rv2299c or 100 ng / ml LPS. 1 g / ml of fluorescein conjugated dextran (molecular weight 40,000; Molecular Probes, Eugene, OR) was added to these 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 dextran-FITC (Fluorescein isothiocyanate) phagocytosis was measured using a flow cytometer FACs Canto (BD Biosciences) (FIG. 5). As shown in FIG. 5, the dextran (antigen) phagocytic ability was reduced for dendritic cells treated with Rv2299c than they did not. In this respect, it can be seen that Rv2299c matures dendritic cells in terms of function. In order to determine the nonspecific binding of dextran to dendritic cells, the result was also corrected by measuring at 4 ° C. Through this, Rv2299c treatment was able to confirm functionally increased maturity of dendritic cells.

Example  7. Rv2299c In the activity of dendritic cells MAPK  And NF -κ Of B  Impact Analysis

To determine if dendritic cells activated by Rv2299c were induced by MAPK and NF-κB signaling systems, U0126 (ERK1 / 2), SB203580 (p38), SP600125 (JNK), Bay 11-0782 (NF-κB And MAPK and NF-κB inhibitors such as). The dendritic cells were pretreated for 1 hour with MAPK and NF-κB inhibitors and then incubated for 24 hours after treatment with Rv2299c at 2 μg / ml concentration or 100 ng / ml of LPS. For analysis of the effect of surface factor expression on dendritic cells, the cells were stained with CD11c-FITC (BD bioscience) for 30 minutes at 4 ° C. After staining using cell surface factor antibodies such as anti-CD80-PE (BD bioscience), anti-CD86-PE (BD bioscience) and analyzed by flow cytometry FACs Canto (BD Biosciences) (Fig. 6a).

In addition, cytokine secretion was measured by applying an ELISA kit (BD PharMingen, USA) for TNF-α, IL-6, and IL-1β through the supernatant to analyze cytokines secreted according to maturation of dendritic cells. (Figure 6b).

As shown in FIG. 6A, MAPK and NF-κB inhibitors reduced the expression of CD80 and CD86, co-stimulatory factors of dendritic cells by Rv2299c, while not SP600125 (JNK). In addition, inflammatory cytokines TNF-α, IL-6, IL-1β were reduced as shown in FIG. 6B, while SP600125 (JNK) was not reduced. These results confirmed that the activity of dendritic cells by Rv2299c induced the production of pro-inflammatory cytokines by dependence on MAPKs (ERK1 / 2, p38) and NF-κB signaling.

Example  8. Rv2299c Dendritic cell maturation TLR Association analysis with

Dendritic cells were isolated from TLR2-/-, TLR4-/-and WT mice in the same manner as in Example 1 to see the association of TLRs with the maturation of dendritic cells by Rv2299c. Expression of CD86 and MHC II was measured for each of the isolated dendritic cells in the same manner as in Example 3, and the production of TNF-α, IL-6, and IL-1β was measured in the same manner as in Example 4. Rv2299c significantly increased the expression of CD86 and MHC II and the production of TNF-α, IL-6, and IL-1β in dendritic cells isolated and differentiated from bone marrow of WT or TLR2-/-knockout mice, but TLR4-/- In the dendritic cells isolated at, the expression of CD86 and MHC II and the production of TNF-α, IL-6, IL-1β were significantly reduced (FIG. 7A).

In addition, when TNF-α, IL-6, and IL-1β production was confirmed using knockout mice of MyD88 or TRIF, which are signals of TLR4-/-, cytokine production was increased in MyD88-/-and TRIF-/-. It was confirmed that the decrease compared to. These results support that Rv2299c activated dendritic cells via TLR4-MyD88 / TRIF.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Maturation method for Dendritic cell using Mycobacterium          tuberculosis Rv2299c <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 cgccatatga acgcccatgt cgagc 25 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 2 cccaagcttc aaggtacgcg cgagacgttc 30

Claims (9)

Dendritic cell maturation inducing composition comprising Rv2299c protein as an active ingredient.
The dendritic cell maturation inducing composition according to claim 1, wherein the Rv2299c protein is a protein derived from Mycobacterium tuberculosis .
The composition of claim 1 or 2, wherein the Rv2299c protein is included at a concentration of 1 μg / ml to 5 μg / ml.
A method of inducing maturation of immature dendritic cells, characterized by treating Rv2299c to immature dendritic cells.
The method of inducing maturation according to claim 4, wherein the cells are cultured for 12 hours or more and 36 hours or less after the treatment.
The method of claim 4, wherein the maturation of immature dendritic cells is an increase in TNF-α, IL-12p70, IL-6, IL-1ß production.
The method of claim 4, wherein the Rv2299c protein is treated at a concentration of 1 μg / ml to 5 μg / ml.
Immunity enhancing composition comprising the composition of claim 1 or claim 2.
The composition of claim 8, wherein the immune is a Th1 type immune response.

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