KR20140102441A - Anti-inflammatory composition comprising 14-3-3 sigma gene and protein - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition comprising 14-3-3 sigma gene and/or protein as an active ingredient. The 14-3-3 sigma gene is preferred to be composed of a base sequence of SEQ ID NO.1; and provided in the present invent is an anti-inflammatory composition comprising a 14-3-3 sigma protein as an active ingredient. In an embodiment of the present invention, the 14-3-3 sigma protein is preferred to be composed of a base sequence of SEQ ID NO. 2, but is not limited to the same; and provided in the present invention is an expression vector controlling inflammatory cytokine comprising the 14-3-3 sigma gene composed of SEQ ID NO.1 as an active ingredient.

Description

An anti-inflammatory composition comprising a 14-3-3 sigma gene and / or a protein as an active ingredient.

The present invention relates to an anti-inflammatory composition comprising a 14-3-3 sigma gene and / or a protein as an active ingredient.

Congenital immunity and inflammation are the primary defense mechanisms against pathogens. Abnormal control of innate immune and inflammatory responses responsible for these defenses is known to be responsible for various immune related diseases such as sepsis, asthma, and autoimmune diseases. In addition, if the innate immune and inflammatory responses are not precisely resolved, persistent cell damage will occur and ultimately provide an important cause of chronic diseases such as cancer and diabetes.

Thus, innate immunity and inflammation control technology is a key technology for the treatment and prevention of chronic inflammatory diseases as well as infectious diseases and immune diseases of pathogens.

Recent research trends distinguish two types of inflammatory reactions. In other words, it is classified into therapeutic inflammation involving innate immunity and pathological inflammation caused by chronic inflammatory reaction. In other words, it has been studied as a pathological inflammation that induces cellular inflammation and cell damage by continuous and repeated inflammatory reaction, which is beneficial to the cell as a defense mechanism due to invasion of pathogens (Aggawal et al. Biochem Pharmacol., 2006).

In fact, this research direction has been accepted by researchers as having considerable relevance in terms of the pathogenesis of cell damage and disease. This concept suggests that strict regulation of the actual inflammatory response will play an important role in the prevention of disease, and therefore fundamental key proteins that are altered in response to the anti-inflammatory signal that resolves the inflammatory response or that are shifted from inflammatory to anti- Something is becoming an important issue.

Recently, in the March 2010 issue of Cell 2010, review papers on such inflammation and anti-inflammation were comprehensively presented, and one of them addressed the importance of developing anti-inflammatory medicines under the heading "Anti-inflammatory agents: Present and Future" All. These academic trends strongly suggest that the study of anti-inflammatory mechanisms is important for the prevention and treatment of inflammation-related diseases.

Inflammation reaction is the primary defense mechanism of the living body and has both sides that cause tissue damage. In general, resolution of the inflammatory response is mediated by several mechanisms, including short-half life of the inflammatory mediator, changes in reactive oxygen intermediates, or anti-inflammatory signals.

(Nathan and Ding., Cell 2010). In the absence of inflammatory response, nonresolving inflammation is a chronic inflammatory disease.

It has been reported that nonresolving inflammation can be caused by the continuous stimulation of inflammatory factors such as persistent infection of microorganisms, as well as by the deficiency of the anti-inflammatory mechanism itself.

The concept of this nonresolving inflammation and its link to disease have been addressed in Essays and Leading Edge Review on inflammation in 2010, Cell 140, Issue 6, Vol. 140, Issue 6, all of which address chronic inflammatory or nonresolving inflammation Suggesting that it is the core of the most important disease treatments.

Recent studies have shown that sustained inflammation is caused not only by microbial infection but also by stimulation of the inflammatory response receptor by the host product released by necrotic cell death or extracellular matrix damage in host cells, despite the absence of microbial infection . This concept is understood not only in external infections, but also in cell-damaging stimuli, which are implicated in individuals, induce intrinsic inflammatory responses and cause nonresolving inflammation, thereby providing the basis for disease development (Nathan and Ding. 140: 871-882).

Mediators that contribute to the resolution of nonresolving inflammation have been reported, and TGF-β, an anti-inflammatory cytokine, is an important factor in this resolution process.

TGF-β is known to be involved in cell growth inhibition, differentiation, and apoptosis in most cells (Roberts and Sporn, Growth Factor, 1993) -Out) The phenotype of mice shows that TGF- is a potent cytokine that mediates anti-inflammation. It is known that mice that have TGF- [beta] are prematurely killed by inducing inflammation as a whole, and that inflammatory responses occur in the mucosal surface of mice that have been infected with Smad3 gene (Geiser et al., PNAS, 1993; al., EMBO J., 1993). Recent studies have shown that TGF-β induces anti-inflammatory activity as well as the development and survival of the antigen presenting cell (APC), mast cell, natural killer (NK), CD4 +, CD8 + T cells, (Wahl, Curr. Opin. Immunol., 2007).

Although the role of TGF-β in the immune response is still required, it will be a very interesting subject to control the immune function in the body including innate immunity using the TGF-β signaling system.

In addition, the TGF-β signal transduction system has been reported to be a rationale for the control of immune function in the human body using inhibitory Smad proteins (Choi et al., 2006). , Nat. Immunol., 2007). The molecular mechanism by which TGF-β, one of the anti-inflammatory cytokines, inhibits innate immunity (IL-1R / TLR) and TNF-alpha signaling through inhibitory Smad proteins (Smad6 and Smad7) (Choi et al., Nature Immunol., 2006; Hong et al., Nature Immunol., 2007).

Related Prior Art European Patent 1 127 942 (Aug. 29, 2001) describes the immortalization of keratinocytes by down-regulation of 14-3-3 sigma.

The present invention has been made in view of the above needs, and an object of the present invention is to provide a novel anti-inflammatory composition.

Another object of the present invention is to provide a method for screening novel anti-inflammatory compounds.

In order to accomplish the above object, the present invention provides an anti-inflammatory composition comprising the 14-3-3 sigma gene as an active ingredient.

In one embodiment of the present invention, the 14-3-3 sigma gene is preferably composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The present invention also provides an anti-inflammatory composition comprising 14-3-3 sigma protein as an active ingredient.

In one embodiment of the present invention, the 14-3-3 sigma protein is preferably composed of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The present invention also provides an expression vector for inflammatory cytokine inhibition comprising the 14-3-3 sigma gene consisting of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient.

In one embodiment of the present invention, the expression vector has a cleavage map shown in FIG. 4, but is not limited thereto.

The invention is also directed to a recombinant vector containing one or more polynucleotides according to the invention.

A number of expression systems can be used, such as, for example, chromosomes, episomes and induced viruses. More particularly, the recombinant vector used may be a bacterial plasmid, a transposon, a yeast episome, an insertion factor, a yeast chromosome factor, a virus such as baculovirus, a papilloma virus such as SV40, a vaccine virus, an adenovirus, Rabies virus, retrovirus.

These recombinant vectors may also be cosmid or phagemid derivatives. The nucleotide sequence is known to those skilled in the art as described, for example, in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Lt; RTI ID = 0.0 > recombinant < / RTI > expression vector.

The recombinant vector may comprise a nucleotide sequence that controls the regulation of polynucleotide expression as well as a nucleotide sequence that enables the expression and transcription of the polynucleotides of the invention and the decoding of the polypeptides of the invention, .

Thus, for example, a suitable secretion signal can be incorporated into the recombinant vector, so that the polypeptide encoded by the polynucleotides of the invention is directed to the lumen of the cytoplasmic network, to the periplasmic space, to the cell membrane, or to the extracellular environment will be.

The introduction of the recombinant vector into the host cell may be accomplished by transfection with calcium phosphate, transfection with DEAE dextran, transfection with microinjection, Such as those described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., 1986, and MOLECULAR CLONING: A LABORATORY MANUAL, Same as Transfection by cationic lipid, electroporation, transduction or infection Can be carried out according to methods well known to those skilled in the art.

Host cells include, for example, streptococci, staphylococci, Bacterial cells such as E. coli or Bacillus subtilis, yeast cells and cells of fungi such as Aspergillus, Streptomyces cells, Drosophila ) S2 and Spodoptera Sf9, CHO, COS, HeLa, C127, BHK, HEK 293 cells and human cells of the subject to be treated.

Host cells can be used, for example, to express the polypeptides of the invention or as active products in pharmaceutical compositions.

The present invention also provides a method of co-transfection of a plasmid containing 14-3-3 sigma gene and NF-kB into an in vitro cell to reduce NF-kB activity in vitro.

The present invention also provides a method for reducing p65 / RelA protein expression in in vitro by treating 14-3-3 sigma gene in vitro cells.

The present invention also relates to a method for preparing a recombinant vector comprising the steps of transfecting a 14-3-3 sigma gene into a macrophage and then culturing the transformed cell, contacting the cell with a compound to be screened before, during, or after the transfection step, Measuring the expression of the 14-3-3 sigma gene; And a step of rejecting the compound which increases the expression of the 14-3-3 sigma gene by contacting with the compound in the compound to be screened, as a lead compound and does not increase the expression of the 14-3-3 sigma gene A method for screening a compound for identifying a compound useful for the treatment or prevention of anti-inflammatory activity.

The present invention is also directed to pharmaceutical compositions containing pharmaceutically acceptable excipients.

In these pharmaceutical compositions, the active agent is advantageously present in a physiologically effective dose.

These pharmaceutical compositions may be, for example, solid or liquid, and may be in the form of, for example, tablets, capsules, granules, caramels, suppositories, Lt; / RTI > These constraint forms can be prepared according to conventional methods.

The active agent (s) may be selected from the group consisting of talc, gum arabic, lactose, starch, dextrose, glycerol, ethanol, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, animal or vegetable fatty acid materials, Or an excipient commonly used in pharmaceutical compositions such as emulsifying and preservative agents.

The active agent (s) according to the present invention may be used alone or in combination with other compounds such as therapeutic compounds such as, for example, interleukins or other cytokines such as interferon.

The different formulations of the pharmaceutical composition are applied according to the mode of administration.

The pharmaceutical compositions may be administered by a different route of administration known to those skilled in the art.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by injection into the skin, oral, rectal, intravenous, intraperitoneal, muscle, subcutaneous, intra-uterine or intracerebroventricular.

The administration may contain one or more active ingredients which exhibit the same or similar functions. For administration, one or more additional pharmaceutically acceptable carriers may be included. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, Other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulations for injection, powders, tablets, capsules, rings, granules or injection solutions such as aqueous solutions, suspensions and emulsions. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.

The dosage may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the method of administration, and the dose may vary depending on the patient's body weight, age, sex, The range varies depending on time, method of administration, excretion rate, and severity of the disease. The daily dose of the composition of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, once or several times a day.

Hereinafter, the present invention will be described in detail.

Anti-inflammatory effect of TGF-beta1 in peritoneal macrophages

Peritoneal macrophages isolated from rat peritoneal macrophages were treated with LPS (0.1 mg / ml) for 24 hours in the absence of pretreatment or pretreatment of TGF-beta1 (5 ng / ml) for 30 min. (IL-6), IL-1beta and cyclooxygenase-2 (COX-2) mRNA were compared between the two groups. Expression of inflammatory cytokines induced by LPS was significantly reduced under conditions in which TGF-beta1 was pretreated (FIG. 1). These results demonstrate the anti-inflammatory effects of TGF-beta1 in peritoneal macrophages.

Increased expression of 14-3-3 sigma by TGF-beta1 in peritoneal macrophages

The effects of peritoneal macrophages isolated from mouse peritoneal cavity on 14-3-3 sigma expression in TGF-beta1 (5 ng / ml) were investigated. At the time of TGF-beta1 treatment, the expression level of 14-3-3 sigma mRNA was maximally increased at 1 hour and increased up to 12 hours (Fig. 2a). To investigate whether the increase of 14-3-3 sigma gene expression was specific to TGF-beta1, the effect of SB431542, an inhibitor of TGF-beta type 1 receptor, was investigated. The increase in the expression of 14-3-3 sigma decreased SB431542 concentration (Fig. 2b) under the condition that SB431542 was treated with 1, 5 and 10 uM of TGF-beta1 for 1 hour.

The transcription inhibitor actinomycin D (ActD) and the translation inhibitor cyclohexamide (CHX) were used to determine whether the increased expression of 14-3-3 sigma by TGF-beta1 occurs at transcriptional or post-transcriptional levels Respectively. The increased expression of 14-3-3 sigma mRNA by TGF-beta1 was completely inhibited by ActD irradiation, but not by CHX treatment (FIG. 2c). These results show that the expression of 14-3-3 sigma by TGF-beta1 is increased in the transcription stage.

The isoforms that make up the 14-3-3 family are known from mammals to the present day. In order to confirm that the expression of 14-3-3 sigma by TGF-beta1 is increased in comparison with other isoforms, the expression of mRNA was analyzed after treatment with time. As shown in FIG. 3, the increase in expression of 14-3-3 isoform by TGF-beta1 was confirmed only in 14-3-3 sigma, 14-3-3 epsilon expression was decreased, and the remaining isoforms were not significantly affected.

Inhibitory effect of 14-3-3 sigma on the expression of LPS-induced inflammatory cytokines in peritoneal macrophages

We analyzed the effect of 14-3-3 sigma on the expression of inflammatory cytokine genes induced by LPS in peritoneal macrophages isolated from mouse peritoneal cavity. To analyze the effect of 14-3-3 sigma, two kinds of lentiviral recombinant plasmids with 14-3-3 sigma gene sequences were constructed. The HIV-derived letivirus vector is a very efficient vector system for increasing or suppressing expression by introducing the gene of interest into the cell because it is highly infectious even in non-dividing cells. In order to induce overexpression of 14-3-3 sigma gene into peritoneal macrophages, a new recombinant plasmid was constructed by introducing mouse 14-3-3 sigma gene cDNA into the EcoRI and KpnI restriction sites of the lentivirus vector pCAG (Fig. 4 ). The 14-3-3 sigma overexpressing lentiviral recombinant plasmid was transfected into 293T cell line, a virus host cell, and viral soup was obtained from the medium and then infected into peritoneal macrophages. As shown in FIG. 5, it can be seen that the expression of 14-3-3 sigma (lane 7 & 8) is significantly increased than that of endogenous 14-3-3 sigma level (lane 1 to 4) in the cells. On the other hand, a small hairpin (sh) RNA lentiviral plasmid containing a part of the 14-3-3 sigma gene sequence was constructed to inhibit the expression of 14-3-3 sigma in the cell (FIG. 5). The pLKO1 lentiviral vector contains the U6 RNA promoter and produces small hairpin 14-3-3 sigma RNA. This shRNA 14-3-3 sigma is fragmented by the dicer system in the cell and is made into a single strand form, which binds to the complementary sequence of the original 14-3-3 sigma mRNA in the cell to produce partial double-stranded RNA . This type of RNA eventually degrades and results in a decrease in the expression of 14-3-3 sigma in the cells. In the case of peritoneal macrophages infected with pLKO1-14-3-3 sigma lentivirus, the basic expression level of intracellular 14-3-3 sigma is completely reduced (FIG. 6, lane 5 & 6). The expression of inflammatory cytokine genes induced by LPS treatment was analyzed in peritoneal macrophages infected with 14-3-3 sigma lentivirus. It was confirmed that the expression of IL-6, IL1-beta and COX-2 by LPS was almost suppressed under the condition of overexpressing 14-3-3 sigma (Fig. 6, lane 1 to 4 and lane 7 & 8 ). In contrast, the expression of these inflammatory cytokine genes by LPS was further increased under shRNA 14-3-3 sigma expression (lane 1 to 4 vs. lane 5 & 6). These results suggest that 14-3-3 sigma induced by TGF-beta1 has an anti-inflammatory effect that inhibits the production of inflammatory cytokines.

Inhibitory effect of 14-3-3 sigma on LPS-induced NF-kB activity in peritoneal macrophages

Peritoneal macrophages isolated from mouse abdominal cavity were cultured, co-transfected with NF-kB-Luc reporter plasmid and 14-3-3 sigma, and treated with LPS for 24 hours to analyze luciferase activity. NF-kB is known as a key transcriptional regulator that mediates LPS-induced inflammatory cytokine gene expression. The activity of NF-kB-Luc was increased 12-fold under LPS-treated condition, but the activity of NF-kB-Luc by LPS was significantly reduced to 5.4-fold under the condition of expressing 14-3-3 sigma together (FIG. In the peritoneal macrophages treated with TGF-beta as an important mechanism, the expression of p65 / RelA, a key member of the NF-kB family, was not changed at the mRNA level but decreased at the protein level (FIG. Lentivirus shRNA 14-3-3 sigma infected with 14-3-3 sigma inhibited the expression of p65 / RelA protein by TGF-beta (Fig. 7c). Furthermore, a decrease in p65 / RelA protein was observed under conditions in which only 14-3-3 sigma was expressed without TGF-beta treatment, and this effect was not observed in other 14-3-3 isotypes (FIG. 7d).

TGF-beta is one of intracellular cytokines with potent anti-inflammatory activity. Inflammation reaction is a biological defense system to remove injured or infected cells by invasion of virus or germs from the outside, but if it is not properly controlled and maintained continuously, it leads to chronic inflammation, which is a source of various diseases including cancer. Thus, identifying novel targets of TGF-beta and identifying the mechanism of action of the anti-inflammatory responses mediated by it is crucial in the search for and the discovery of therapeutic agents for various diseases caused by inflammation. In the results of the present invention, 14-3-3 sigma is a new target gene of TGF-beta that mediates anti-inflammatory activity. Even when expressed alone, it induces p65 / RelA protein degradation and inhibits inflammation suppressing the expression of inflammatory cytokine genes Function. Thus, the use of 14-3-3 sigma gene expression can be a very efficient system for exploring and discovering anti-inflammatory materials.

Figure 1 shows the inhibitory effect of TGF-beta1 on LPS-induced inflammatory cytokine expression;
Figure 2 shows the increase in 14-3-3 sigma gene expression by TGF-beta1;
Figure 3 shows the selective increase of 14-3-3 sigma gene expression by TGF-beta1;
Figure 4 shows the preparation of a lentiviral vector for mouse 14-3-3 sigma overexpression;
Figure 5 shows shRNA lentiviral vector preparation for mouse 14-3-3 sigma expression inhibition;
6 shows the inhibitory effect of 14-3-3 sigma on the expression of inflammatory cytokines by LPS;
FIG. 7 is a graph showing the inhibitory effect of 14-3-3 sigma on p65 / R elA activity and expression by LPS.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example 1: Isolation and culture of peritoneal macrophages from mice

Peritoneal macrophages were separated from mouse abdominal cavity using cervical dislocation. The epidermis of the mouse was cut and complete medium containing 8 ~ 10 ml of RPMI and 10% FBS in a 10 ml syringe was injected into the peritoneal cavity, avoiding the area of the intimal lining. In order to allow the cells attached to the wall to fall off well from the inflated abdominal cavity due to the media injection, the hands were massaged for 5 minutes and then the syringe was re-injected to collect the media containing the cells. The extracted cells (1 × 10 ⁷ ) were washed twice with media, seeded in 6-well culture plate using complete media, and cultured for 24 hours.

Example 2: Preparation of 14-3-3 sigma lentivirus expression vector

MRNA was purified from mouse peritoneal macrophages and 14-3-3 sigma cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR). For mRNA extraction, TRIzol reagent was added to the cells, and the mixture was allowed to stand at room temperature for 3 minutes, mixed with Chloroform for 15 seconds, and centrifuged at 12,000 g for 15 minutes. The supernatant was transferred to a new tube and Isopropyl alcohol was added to precipitate the RNA, followed by washing with 75% ethanol. RNA precipitates were dissolved in DEPC solution and stored frozen until use. RT-PCR was performed using 2 ug of extracted RNA and reverse transcription at a volume of 20 μl. (DT) 18 primer was added to each well and reacted at 70 ° C for 10 minutes. The oligonucleotide (dT) was ligated to the RNA and reacted with 2.5 mM dNTP, 50 mM KCl, 10 mM Tris-HCl, 5 mM MgCl 2, Reverse transcriptase was added to the reaction mixture at 42 ° C for 60 minutes and at 70 ° C for 15 minutes to synthesize 14-3-3 sigma cDNA. The primer sequences used in the synthesis of 14-3-3 sigma cDNA are as follows: Forward sequence 5'-ctt cat cgc a gt cat gga ga-3 ', Reward sequence 5'-cca cat ctg aag cag cgg aa-3 '

The 14-3-3 sigma cDNA fragment was purified from agarose gel and then double digested with EcoRI and KpnI restriction enzymes. This fragment was inserted into the EcoRI and KpnI cloning sites of the lentivirus vector pCAG to construct a new recombinant plasmid, pCAG-14-3-3 sigma. The 14-3-3 sigma cDNA contained in the recombinant plasmid was finally confirmed by homologous restriction enzyme cleavage and DNA sequencing.

Example 3: Preparation of shRNA 14-3-3 sigma lentivirus expression vector

A lentivirus vector capable of producing 14-3-3 sigma RNA (shRNA 14-3-3 sigma) in the form of a small hairpin was prepared as a system to suppress 14-3-3 sigma expression. The pLKO1 lentivirus vctor has a U6 RNA promoter capable of producing small nuclear transcripts by RNA polymerase III, making it suitable for producing shRNA products. We constructed shRNA 14-3-3 sigma oligonucleotides with Mouse 14-3-3 sigma (target 46 to 56 (red) from coding start site). sence 5'-ccgggccgaacggtatgaagacatgctcgagcatgtcttcataccgttcggctttttg-3 '

anti-sence 5'-aattcaaaaagccgaacggtatgaagacatgctcgagcatgtcttcataccgttcggc-3 '

Two types of oligonucleotides (10 mM) were added to the annealing buffer (5 ml each), reacted at 95 ° C for 4 minutes, and annealed at a temperature of 30 ° C or lower. Annealed oligonucleotides were ligated to pLKO1 lentivirus vector digested with EcoRI and AgeI restriction enzymes. The recombinant pLKO1-shRNA 14-3-3 sigma plasmid was finally identified by DNA sequencing.

Example 4: Lentivirus infection

To produce the 14-3-3 sigma lentivirus, HEK293T cell line, a virus production host cell, was used. PCAG-14-3-3 sigma was co-transfected with the virus packaging plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.VSVG in 2 × 10 ⁵ cells / 6-well plate by lipofectamin 2000 method. In the case of pLKO1-shRNA 14-3-3 sigma, the virus soup was collected from the cell culture medium at 72 hours after transfection with paPAX2 and pMD2.VSVG packaging plasmids and purified using a 0.45 mM filter. The purified virus was treated with 8 mg / ml polybrene in peritoneal macrophages (5 × 10 ⁵ cells / 6-well plate) three times at 8-hour intervals. Virus was cultured for 24 hours, and cell extracts were collected and analyzed for the effect of 14-3-3 sigma gene expression by RT-PCR.

Example 5: RT-PCR

RT-PCR was performed using the Access RT-PCR system (Promega, Madison, Wis.). Each primer sequence used was as follows:

mouse COX-2; forward 5'-caa tga gta ccg caa acg ct-3 ', reverse 5'-agg tgc tcg gct tcc agt at-3', mouse IL-1beta; forward 5'-gtt gac gga ccc caa aa at-3 ', reverse 5'-aag gtc cac ggg gac ac-3', mouse IL-6; forward 5'-ttg gga ctg atg ctg gtg ac-3 '; reverse 5'-tgc aag tgc atc atc gtt gt-3 ', mouse 14-3-3 sigma primers; forward 5'-CCT GCT GGA CTC GCA CCT CA-3 'and reverse, 5'-TGT CGG CTG TCC ACA GCG TC-3', mouse 14-3-3 beta primers; forward 5'-GAG CGC TAC GAC GAC ATG GCC-3 'and reverse, 5'-AAT TCC AGG ACC GTG GTG CAG ATG-3', mouse 14-3-3 ε primers; forward 5'-GAG CGA TAC GAC GAA ATG GTG-3 'and reverse 5'-CCT TGG ACT CGC CAG TGT TAG-3', mouse 14-3-3 η primers; forward 5'-CTG GCG GAG CAG GCG GAG CGC-3'and reverse 5'-CTG TCT CCA GCT CCT TTT CAA TCT TCT CCC-3 ', mouse gamma 14-3-3 primers; forward 5'-CCG GGA GAA GAT CGA GAA GGA GT-3'and revere 5'-CTG CAT GAT CAG AGT GGA GTC CTT G-3 ', mouse 14-3-3 τ primers; forward 5'-GCA GCT GAT CAA GGA CTA TCG GG-3 'and reverse 5'-GCC TCT TGG TAG GCT CCT TGG G-3', mouse 14-3-3 ζ primers; forward 5'-CCC ACT CCG GAC ACA GAA TAT CAG-3 'and reverse 5'-CTC TGT ATT CTC GAG CCA TCT GCT G-3' and mouse beta-actin were amplified as a control using the following primers ; forward, 5'-ACG TTG CTA TCC AGG CTG TG-3 ', and reverse, 5'-GCG ACG TAG CAC AGC TTC TC-3'. PCR amplification conditions are as follows; 25 cycles of 94 ° C for 30 s, 1 min at 55 ° C, and 1 min at 72 ° C.

Example 7: Reporter gene assay

The HEK293 cell line was seeded in a 5 x 10 ⁵ cells / 6-well plate and then treated with 0.5 mg / ml NF-kB-Luciferase reporter plasmid, 0.2 mg / ml CMV-b-galactosidase plasmid, 0.5 mg / ml pHMV6 or pHMV6-14 -3-3 sigma co-transfected. After 24 hours of transfection, the cells were treated with LPS for 24 hours and luciferase activity was analyzed. Reporter activity levels were measured by luminometer using Luciferin as substrate. CMV-beta-galactosidase was used to correct transfection efficiency.

Example 8: Western blotting

Was used to analyze the expression of the protein. 14-3-3 sigma, HA, p65 / ReA antibodies were purchased from Santa Cruz Biotechology, and beta-actin antibodies were purchased from Sigma.

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<110> KNU-Industry Cooperation Foundation <120> Anti-inflammatory composition comprising 14-3-3 sigma gene and          protein <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 747 <212> DNA <213> Mouse <400> 1 atggagagag ccagtctgat ccagaaggcc aagttggctg aacaggccga acggtatgaa 60 gacatggcag ctttcatgaa gagcgccgtg gaaaagggcg aggagctctc ctgcgaggag 120 cgaaacctgc tttccgtagc ttacaagaac gtggtgggcg gccagagagc ggcctggagg 180 gtcctgtcca gcatcgagca gaagagcaac gaggaggggt cagaagagaa gggccccgag 240 gtgaaagagt accgggagaa ggtagagacc gagctcagag gtgtgtgcga caccgtactc 300 ggcctgctgg actcgcacct catcaaaggg gctggagatg cagagagccg cgtcttctac 360 ctgaagatga agggtgacta ctaccgctac ctagccgagg tggccactgg cgatgacaag 420 aagcgcatca tcgattctgc ccggtcagcc taccaggagg ccatggacat cagcaagaag 480 gagatgccgc ctaccaaccc catccgcctg ggcctggccc tgaacttttc agtcttccac 540 tacgagatag ccaacagccc cgaggaggcc atctcgctgg ccaagaccac cttcgacgag 600 gccatggccg acctgcacac cctcagtgag gactcctaca aggacagcac cctcatcatg 660 cagctcctga gagacaacct gacgctgtgg acagccgaca gtgctgggga agagggtggt 720 gaggctccgg aggagcccca gagctga 747 <210> 2 <211> 248 <212> PRT <213> Mouse <400> 2 Met Glu Arg Ala Ser Leu Ile Gln Lys Ala Lys Leu Ala Glu Gln Ala   1 5 10 15 Glu Arg Tyr Glu Asp Met Ala Ala Phe Met Lys Ser Ala Val Glu Lys              20 25 30 Gly Glu Glu Leu Ser Cys Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr          35 40 45 Lys Asn Val Val Gly Gly Gln Arg Ala Ala Trp Arg Val Leu Ser Ser      50 55 60 Ile Glu Gln Lys Ser Asn Glu Glu Gly Ser Glu Glu Lys Gly Pro Glu  65 70 75 80 Val Lys Glu Tyr Arg Glu Lys Val Glu Thr Glu Leu Arg Gly Val Cys                  85 90 95 Asp Thr Val Leu Gly Leu Leu Asp Ser His Leu Ile Lys Gly Ala Gly             100 105 110 Asp Ala Glu Ser Arg Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr         115 120 125 Arg Tyr Leu Ala Glu Val Ala Thr Gly Asp Asp Lys Lys Arg Ile Ile     130 135 140 Asp Ser Ala Arg Ser Ala Tyr Gln Glu Ala Met Asp Ile Ser Lys Lys 145 150 155 160 Glu Met Pro Pro Thr Asn Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe                 165 170 175 Ser Val Phe His Tyr Glu Ile Ala Asn Ser Pro Glu Glu Ala Ile Ser             180 185 190 Leu Ala Lys Thr Thr Phe Asp Glu Ala Met Ala Asp Leu His Thr Leu         195 200 205 Ser Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg     210 215 220 Asp Asn Leu Thr Leu Trp Thr Ala Asp Ser Ala Gly Glu Glu Gly Gly 225 230 235 240 Glu Ala Pro Glu Glu Pro Gln Ser                 245

Claims (12)

A composition for anti-inflammation comprising 14-3-3 sigma gene as an active ingredient. 2. The anti-inflammatory composition according to claim 1, wherein the 14-3-3 sigma gene comprises the nucleotide sequence of SEQ ID NO: 1. 14. An antiinflammatory composition comprising 14-3-3 sigma protein as an active ingredient. 4. The anti-inflammatory composition according to claim 3, wherein the 14-3-3 sigma protein comprises the amino acid sequence of SEQ ID NO: 2. An expression vector for inflammatory cytokine inhibition comprising the 14-3-3 sigma gene consisting of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient. 6. The inflammatory cytokine inhibitory expression vector according to claim 5, wherein the expression vector has a cleavage map of Fig. Wherein the NF-kB activity is reduced in vitro by cotransfection of a plasmid comprising the 14-3-3 sigma gene and NF-kB into the in vitro cell. 8. The method according to claim 7, wherein the 14-3-3 sigma gene consists of the nucleotide sequence of SEQ ID NO: 1. A method of reducing p65 / RelA protein expression in vitro by treating 14-3-3 sigma genes in vitro. 10. The method according to claim 9, wherein the 14-3-3 sigma gene consists of the nucleotide sequence of SEQ ID NO: 1. Transforming the 14-3-3 sigma gene into macrophages and then culturing them;
Contacting the cell with a compound to be screened before, during, or after the transfection step;
Measuring the expression of the 14-3-3 sigma gene in the cell; And
Wherein the compound that increases the expression of the 14-3-3 sigma gene by contact with the compound in the compound to be screened is adopted as the lead compound and the compound that does not increase the expression of the 14-3-3 sigma gene is rejected A method for screening a compound for identifying a compound useful for the treatment or prevention of inflammation. 12. The method of claim 11,
Wherein the 14-3-3 sigma gene is composed of the nucleotide sequence of SEQ ID NO: 1. 2. A method for screening a compound for identifying a compound useful for the treatment or prevention of anti-inflammatory activity.

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