KR101229807B1 - Novel Drug Target against Hypersensitivity Immune or Inflammatory Disease - Google Patents

Abstract

The present invention relates to a method for screening a substance for preventing or treating an overactive immune disease or an inflammatory disease, a pharmaceutical composition for preventing or treating an overactive immune disease or an inflammatory disease, and a diagnostic kit for an overactive immune or inflammatory disease. According to the present invention, swiprosin-1 is over-expressed when activating a mast cell associated with inflammatory response control or in an inflammatory response animal model, and swiprocin in a mast cell. It was first identified that expression of -1 occurs through the protein kinase C βI / η pathway. Therefore, the screening method of the present invention uses PKCβI / η (protein kinase C βI / η) and swiprocin-1 as molecular targets on which substances for the prophylaxis or treatment of hypersensitivity immune diseases or inflammatory diseases act.

Sweeprocin-1, Inflammatory Disease, Allergy, Atopic Dermatitis

Description

Novel Drug Target against Hypersensitivity Immune or Inflammatory Disease

The present invention relates to a method for screening a substance for preventing or treating an overactive immune disease or an inflammatory disease.

Swiprosin-1 was first identified in human lymphocytes, prominent in CD8 ⁺ lymphocytes (1) and subsequently identified in immature, resting and active B cells, and nonlymphoid tissues, especially the brain ( 2). Swiprosin-1 has four myristylation sites, three binding sites for the SH3 domain containing proteins, two potential EF-hand domains, and one twisted-coil domain at the C-terminus. Collected databases have shown that they can inferred and act as small adapter proteins involved in calcium signaling (1). Consistent with this prediction, swiprosin-1 is associated with phosphotyrosine-based signaling events related to cell stimulation and actin rearrangement of early growth factor (EGF) (3).

However, the function of Swiprosin-1 is still largely unknown. The only report on function is that Swiprocin-1 is involved in lipid rafts in immature B-cell line WEHI231 and is involved in the contribution of B cell receptor (BCR) signal enhancement and BCR-induced apoptosis ( 2,4). Potential signaling pathways associated with Swiprocin-1 expression regulation and Swiprosin-1 expression have not yet been reported. With high expression in immature bone marrow B cells, it has been reported by the same group that Swiprocin-1 is expressed through differentiation of B-cells (2). However, when stimulated with immunoglobulin M (IgM) F (ab) ₂ / IL-4, lipopolysaccharide (LPS) or anti-CD40 / IL-4, the level of Swiprosin-1 changes during B cell activation. They demonstrated that they did not (2).

Mast cells are widely distributed in mammalian tissues and play an important role in various biological reactions (5-7). Typically, mast cells are considered in connection with immediate type hypersensitivity (5). However, several recent reports have demonstrated the possibility of involvement of mast cells in more persistent and even chronic inflammatory and immunological responses (8, 9). Notably, various cytokines are produced in mast cells, including IL-3, IL-4, IL-5, IL-6, IL-8, TNF-α, and IFN-γ (10-12), It plays a more important role in the immune process than IgE-mediated hypersensitivity. Because of the potential importance of mast cell-induced cytokines in physiological or pathological immune responses, it will be necessary to understand the signaling pathways or molecules involved in cytokine regulation in mast cells. However, until recently, the mechanism of mast cell degranulation mediated by high affinity IgE receptors (FcεR1) is relatively well established, while the number of reports on the regulatory mechanisms of cytokine expression in mast cells is only limited. (7).

HMC-1 is a cell line established from mast cell leukemia patients and exhibits various properties of immature mast cells (13). Although these cells do not express cell surface FcεRI and thus cannot be activated by antigens, they can still be activated by treatment of phorbol esters and calcium ionophore. (14, 15). These cells thus serve as a useful system for initiating investigation of mast cell expression of human cytokines or chemokines.

A number of patent documents are referred to throughout this specification and their citations are indicated. The disclosures of the cited patent documents are hereby incorporated by reference herein in their entirety to better illustrate the state of the art to which the invention pertains and the teachings of the present invention.

The present inventors have tried to develop a method for screening a substance that can effectively suppress an overactive immune disease or an inflammatory disease. As a result, the present inventors have found that the protein kinase C βI / η or the swiprosin-1 gene or protein may be a molecular target of a substance for the treatment or prevention of an overactive immune disease or an inflammatory disease. The present invention has been completed by discovering that there is.

Accordingly, an object of the present invention is to provide a method for screening a substance for preventing or treating an overactive immune disease or an inflammatory disease.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of irritable immune diseases or inflammatory diseases.

It is another object of the present invention to provide a diagnostic kit for irritable immune disease or inflammatory disease.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a method for screening a substance for preventing or treating an overactive immune disease or an inflammatory disease, comprising the following steps: (a) PKCβI / η (protein kinase C βI / η) Or contacting a sample with a swiprosin-1 gene or protein; And (b) measuring the expression level or activity of the PKCβI / η or swiprocin-1, when the expression level or activity is determined to be down-regulated. Or a substance for treating or preventing an inflammatory disease.

The present inventors have tried to develop a method for screening a substance that can effectively suppress an overactive immune disease or an inflammatory disease. As a result, the present inventors have found that the protein kinase C βI / η or Swiprosin-1 gene or protein may be a molecular target of a substance for the treatment or prevention of an overactive immune disease or an inflammatory disease. It was found.

The term "PKCβI / η" is used herein to generically express "PKCβI and / or PKC η".

According to the screening method of the present invention, a sample is first contacted with PKCβI / η (protein kinase C βI / η) or swiprosin-1 gene or protein. For example, the sample may be contacted with purified PKCβI / η or Swiprosin-1 protein.

According to a preferred embodiment of the present invention, the PKCβI / η or Swiprosin-1 gene or protein used in the present invention is included in the cell. More preferably, the cell comprising the PKCβI / η or swiprosin-1 gene or protein is a mast cell.

Preferably, the PKCβI / η gene used in the present invention is a nucleotide sequence described in SEQ ID NOs: 3 and 5, respectively, and the Swiprosin-1 gene is a nucleotide sequence described in SEQ ID NO: 1, and a PKCβI / η protein. Are amino acid sequences described in SEQ ID NO: 4 and 6, respectively, and the Swiprosin-1 protein is the amino acid sequence described in SEQ ID NO: 2 sequence.

As used to refer to the screening methods of the present invention, the term “sample” refers to an unknown substance used in screening to test whether it affects the expression level or activity of PKCβI / η or swiprocin-1. Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.

Then, the expression level or activity of PKCβI / η or swiprocin-1 in the cells treated with the sample is measured. As a result of the measurement, if the expression level or activity is measured to be down-regulated, the sample is determined to be a substance for treating or preventing an overactive immune disease or an inflammatory disease.

According to a preferred embodiment of the present invention, the method of the present invention is carried out by analyzing (i) a decrease in the activity of PKCβI / η or (ii) a decrease in the amount of expression of swiprosin-1. .

Determination of the change in the amount of Swiprosin-1 expression can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC press ), hybridization (Sambrook using a cDNA microarray such as, Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)) or in situ (in situ ) Hybridization reaction (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)).

When performing according to the RT-PCR protocol, first, total RNA is isolated from cells treated with a sample, and then a first-chain cDNA is prepared using oligo dT primers and reverse transcriptase. Subsequently, the first chain cDNA is used as a template, and a PCR reaction is performed using a swiprosin-1 gene-specific primer set. Then, PCR amplification products are electrophoresed, and the formed bands are analyzed to measure changes in the expression level of the Swiprosin-1 gene.

Changes in the amount of Swiprosin-1 protein can be carried out through various immunoassay methods known in the art. For example, changes in the amount of protein include, but are not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood analysis, and sandwich analysis. no. The method of immunoassay or immunostaining is Enzyme Immunassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme - linked immunosorbent assay ( ELISA ), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotopes (e.g., ¹⁴ C, ¹²⁵ I, ³² P And PKCβI / η or swiprocin-1 protein-specific antibodies labeled S ³⁵ ) can be used.

When the method of the present invention is carried out in an ELISA manner, certain embodiments of the present invention comprise the steps of: (i) coating an extract from the cells treated with the sample onto the surface of a solid substrate; (Ii) reacting said cell extract with a swiprosin-1 protein-specific antibody as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P ₄₅₀ . When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine) Substrates such as ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and o -phenylenediamine (OPD) can be used.

Measurement of the final enzyme activity or signal in the ELISA method can be carried out according to various methods known in the art. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

On the other hand, the decrease in the activity of PKCβ I / η can be carried out by analyzing the degree of phosphorylation of PKCβ ~ / η. For example, by performing the immunoassay method using an antibody that specifically binds to phosphorylated PKCβI / η, a decrease in activity of PKCβI / η can be analyzed.

According to a preferred embodiment of the present invention, the irritable immune disease is an allergic disease, more preferably allergic rhinitis, high fever or asthma.

According to a preferred embodiment of the present invention, the inflammatory disease is atopic dermatitis, encephilitis, inflammatory enteritis, chronic obstructive pulmonary disease, pulmonary pulmonary shock, pulmonary fibrosis, undifferentiated spinal arthrosis, undifferentiated arthrosis, arthritis, Chronic inflammatory diseases caused by inflammatory osteolysis, chronic viral or bacterial infections, colitis, inflammatory bowel disease, type 1 diabetes, type 2 diabetes, rheumatoid arthritis, reactive arthritis, osteoarthritis, psoriasis, scleroderma, porosity, Atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease (Lyme), Borreliasis, neurotic-Borrelia, tuberculosis, Sarcoidosis, lupus , Disc lupus, alumni lupus, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, irritable bowel syndrome, Croce's disease, shogran It includes syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immune dysfunction syndrome, myalgia encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, autism spectrum disorders, attention deficit disorder and attention deficit hyperactivity disorder.

As used herein, referring to the expression amount or activity of PKCβI / η or Swiprocin-1, the term “reduction-regulation” refers to PKCβI / η or Sweep in normal cells that are not immune stimulated (eg, normal mast cells). Relative expression compared to expression level or activity of cin-1. For example, the term “reduce-regulation” means a 1.5-20 fold reduction, preferably 2-10 fold reduction, compared to normal cells that have not been immunostimulated.

According to another aspect of the present invention, the present invention provides a hypersensitive immune disease or inflammation comprising an inhibitor or an activity inhibitor of PKCβ I / η (protein kinase C βI / η) or swiprosin-1 as an active ingredient. It provides a pharmaceutical composition for the prevention or treatment of diseases.

The pharmaceutical composition of the present invention may include chemicals, nucleotides, antisenses, siRNA oligonucleotides, and natural extracts as active ingredients.

According to a preferred embodiment of the present invention, the expression inhibitor included in the composition of the present invention is an antisense or siRNA (small interference RNA) oligonucleotide comprising a sequence complementary to the PKCβI / η or swiprocin-1 coding nucleotide sequence.

As used herein, the term "antisense oligonucleotide" means a DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to a complementary sequence in the mRNA to inhibit translation of the mRNA into a protein. An antisense sequence of the invention refers to a DNA or RNA sequence that is complementary to a PKCβI / η or swiprocin-1 coding nucleotide sequence and capable of binding to a PKCβI / η or swiprosin-1 coding nucleotide sequence and Antisense nucleic acids may inhibit the essential activity of the translation, translocation into the cytoplasm, maturation or any other overall biological function of the PKCβI / η or swiprosin-1 coding nucleotide sequence. To 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases.

The antisense nucleic acid can be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol . , 5 (3): 343-55 (1995)). The nucleic acid backbone can be modified with phosphorothioate, phosphoroester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic intersaccharide linkages and the like. In addition, antisense nucleic acids may include one or more substituted sugar moieties. Antisense nucleic acids can include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-me pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acids of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense nucleic acids. Cholesterol moieties, cholesteryl moieties, cholic acid, thioethers, thiocholesterols, fatty chains, phospholipids, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexylamino-carbonyl-oxy Fat-soluble moieties such as a cholesterol ester moiety, and the like. Oligonucleotides, including liposoluble moieties, and methods of preparation are well known in the art (U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid can increase stability to nucleases and increase the binding affinity of the antisense nucleic acid with the target mRNA.

Antisense oligonucleotides can be synthesized in vitro by conventional methods to be administered in vivo or to allow antisense oligonucleotides to be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin is in the opposite direction of the recognition site (MCS). Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.

As used herein, the term “siRNA” refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99 / 07409 and WO 00/44914) siRNAs are provided as an efficient gene knockdown method or as a gene therapy method because they can inhibit the expression of target genes siRNA was first discovered in plants, worms, fruit flies and parasites. siRNA was developed and used for mammalian cell research.

The siRNA molecules of the present invention are sequences complementary to the sense strand (corresponding sequence corresponding to the PKCβI / η or swiprocin-1 coding nucleotide sequence) and the antisense strand (PKCβI / η or swiprocin-1 coding nucleotide sequence). ) May be positioned opposite to each other to form a double chain. In addition, according to another embodiment, siRNA molecules of the present invention may have a single chain structure having self-complementary sense and antisense strands.

siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.

The siRNA terminal structure can be either blunt or cohesive, as long as it can inhibit the expression of the Swiprosin-1 gene by the RNAi effect. The cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures.

SiRNA molecules of the invention may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between self-complementary sense and antisense strands, in which case the expression of the nucleotide sequence The formed siRNA molecules form a hairpin structure by intramolecular hybridization and form a stem-and-loop structure as a whole. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

The activity inhibitor of PKCβI / η (protein kinase C βI / η) or swiprosin-1 used in the present invention may be a chemical or an antibody. Preferably, the activity inhibitor is an antibody that specifically binds to PKCβI / η or Swiprosin-1 protein and inhibits activity, and is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies to PKCβI / η or swiprosin-1 protein may be prepared by methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies are obtained by injecting a PKCβI / η or Swiprosin-1 protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using known affinity techniques. Can be.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be oral or parenteral (eg, intravenous, intraperitoneal, intramuscular, subcutaneous, or topical).

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-100 mg / kg body weight per day.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the invention, the invention relates to (i) a swiprosin-1 coding nucleotide sequence, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide or a protein encoded in the nucleotide sequence Antibodies or aptamers that specifically bind; And (ii) an antibody or aptamer specifically binding to a phosphorylated form of PKCβI / η (protein kinase C βI / η).

The sequence complementary to the nucleotide sequence of the Swiprosin-1 gene described in SEQ ID NO: 1 sequence used in the diagnostic kit of the present invention is used as a probe or primer. As used herein, the term “complementary” refers to having sufficient complementarity to selectively hybridize to the nucleotide sequence of the Swiprosin-1 gene described in SEQ ID NO: 1 under certain specific hybridization or annealing conditions. . Thus, the term “complementary” has a different meaning from the term perfectly complementary, and the primers or probes of the present invention may selectively hybridize to the nucleotide sequence of the Swiprosin-1 gene described in SEQ ID NO: 1. If possible, it may have one or more mismatch sequences.

As used herein, the term “probe” refers to a linear oligomer of natural or modified monomers or linkages, including deoxyribonucleotides and ribonucleotides, capable of specifically hybridizing to a target nucleotide sequence, and naturally occurring Or artificially synthesized. The probe of the present invention is preferably single chain. Preferably, the probe is an oligodioxyribonucleotide.

As used herein, the term “primer” refers to a single- which can act as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in suitable buffers at suitable temperatures. Refers to stranded oligonucleotides. The suitable length of the primer is typically 15-30 nucleotides, although it varies with various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form hybrid complexes that are sufficiently stable with the template.

The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence of the swiprosin-1 gene, which is a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence to act as a primer. Do.

According to a preferred embodiment of the present invention, the kit for diagnosing irritable immune disease or inflammatory disease comprising a probe is included in a microarray format, more preferably a DNA or cDNA microarray, most preferably cDNA micro It is an array.

In the microarray of the present invention, the probe is used as a hybridizable array element and immobilized on a substrate. Preferred gases include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. The hybridization array elements are arranged and immobilized on the substrate. This immobilization is carried out by chemical bonding methods or by covalent binding methods such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bound by UV at the polylysine coating surface. In addition, the hybridization array element can be coupled to the gas via a linker (eg, ethylene glycol oligomer and diamine).

On the other hand, the sample DNA applied to the microarray of the present invention is labeled and hybridized with the array element on the microarray. Hybridization conditions can vary. The detection and analysis of the hybridization degree can be variously carried out according to the labeling substance.

According to a preferred embodiment of the present invention, the primer is used for gene amplification reactions.

The term " amplification reaction " as used herein refers to a reaction to amplify a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including PCR (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Methods of ligase chain reaction (LCR) (17, 18), Gap-HI (WO 89/06700) and Davey, C. et al (EP 329,822) (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) 19 (WO 88/10315), self sustained sequence replication) 20 (WO 90/06995), selective amplification of target polynucleotide sequences (U.S. Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence-based amplification acid sequence based amplification (NASBA) (U.S. Patent Nos. 5,130,238, 5,4 09,818, 5,554,517, and 6,063,603), strand displacement amplification (21, 22) and loop-mediated isothermal amplification (LAMP) (23) It is not limited. Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and US Pat. No. 09 / 854,317.

In addition, the diagnostic kit for an overactive immune disease or inflammatory disease of the present invention is an antibody that specifically binds to Swiprosin-1 protein and an antibody that specifically binds to the phosphorylated form of PKCβI / η (protein kinase C βI / η). It may be produced to include.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. The antibody may be prepared by methods commonly practiced in the art, for example, by fusion methods (Kohler and Milstein, European). Journal of Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. The polyclonal antibody is injected into a suitable animal with an antigen in the phosphorylated form of swiprocin-1 protein or protein kinase C βI / η (PKCβI / η), the antiserum collected from the animal, and then known affinity. The antibody can be obtained by separating the antibody from the antiserum.

The kit of the present invention may further include other components in addition to the above components. For example, when the kit of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus). aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus thermally stable DNA polymerases obtained from literalis or Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

The diagnostic kit of the present invention can analyze the onset, development and alleviation of irritable immune disease or inflammatory disease. Therefore, the term "diagnosis" used in referring to the kit in the present specification has a meaning including not only determining the presence or absence of the disease, but also determining the onset, development and alleviation of the disease.

Preferably, the kit of the present invention is characterized by the overexpression of PKCβI / η and Swiprosin-1 in mast cells associated with overactive immune disease or inflammatory disease. It is especially useful for diagnosing them.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for screening a substance for preventing or treating an overactive immune disease or an inflammatory disease, a pharmaceutical composition for preventing or treating an overactive immune disease or an inflammatory disease, and a diagnostic kit for an overactive immune or inflammatory disease. .

(b) The present invention was the first to identify the relationship between the Swiprosin-1 overexpression in the mast cells involved in the regulation of inflammatory response, the overexpression of the Swiprosin-1 and PKCβ I / η (protein kinase C β / η) pathway .

(c) Therefore, the screening method of the present invention uses PKCβI / η (protein kinase C βI / η) and swiprocin-1 as molecular targets on which substances for the prophylaxis or treatment of hypersensitivity immune diseases or inflammatory diseases act.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art. Will be self explanatory.

Example

Materials and Methods

Antibodies and reagents

A goat polyclonal antibody against Swiprocin-1 (GeneBank Accession Number: NM_024329) was purchased from Imgenex (San Diego, CA) and used. Antibodies to protein kinase C (PKC) -α, PKC-βI (Genbank Accession Number: NM_002738), PKC-η (Genbank Accession Number: NM_006255), PKC-ζ, Actin, and I-κBα were tested by Santa Cruz Biotechnology. Purchased from Santa Cruz, CA). Antibodies against PKC-δ, PKC-θ, p-PI3 kinase, p-Akt and GFP were purchased from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated anti-goat, anti-rabbit and anti-mouse IgG was purchased from GE Healthcare (Chalfont St. Giles, UK). G, G, rottlerin, staurosporin, SB203580, PD98059, MG132, cyclosporine A, and PP2 were purchased from Calbiochem-Behring (La Jolla, Calif.). Total RNA isolation reagent was purchased from WelPrep ^™ Join Bio Innovation (Daegu, South Korea). Maxime RT Premix (oligo dT primer), Maxime PCR PreMix and plasmid purification kits were purchased from iNtRON Biotechnology (Daejon, South Korea). SYBR premix Ex Taq was purchased from Takara Bio Co., Ltd. (Shiga, Japan). A dual-luciferase assay system was purchased from Promega Corporation (Madison, WI) and used. An Enzyme-linked immunosorbent assay (ELISA) kit for hIL-8 was purchased from R & D Systems (Minneapolis, MN). Small interfering RNA targeting siRNAs were obtained as a pool of four or more siRNA duplexes from Dharmacon (Chicago, IL) targeting Swiprosin-1, PKC isotypes and scrambled siRNAs. All other reagents used in the examples of the present invention were purchased from Sigma Chemical (St. Louis, Mo.).

Cell culture

HMC-1 cells were cultured in IMDM medium. Jurkat T and Molt-4 T cells (T cells), Raji B cells (B cells), THP-1 cells (monocytes), and 293-T and CHO-K1 cells (epithelial cells) were maintained in RPMI 1640 medium. J774A.1 cells (macrophages), HT-29 cells (epithelial cells), COS-7 cells (fibroblasts) and RBL-2H3 cells (mast cells) were cultured in DMEM medium. Human umbilical vascular endothelial cells (HUVECs) were cultured in EBM medium. All culture media used in the examples of the present invention were supplemented with 10% heat inactivated FBS, 100 units / ml penicillin G, and 100 μg / ml streptomycin.

Experimental animal

Original stocks of male ICR mice and Balb / c J mice (6-8 weeks old) were purchased from the Experimental Animal Center (Daejeon, South Korea) and SLC (Hamamatsu, Japan), respectively. Experimental animals were bred 5-10 per cage in a laminar air flow (LAF) room (general conditions) and maintained at a relative humidity of 55 ± 5% and a temperature of 22 ± 2 ° C. throughout the examples of the present invention. Protection and treatment of mice was carried out in accordance with the guidelines established by the Public Health Service Policy on the Protection and Use of Laboratory Animals and was approved by GIST's Institutional Animal Care and Use Committee.

Manual skin Anaphylaxis ( passive cutaneous anaphylaxis , PCA ) reaction

IgE-dependent skin responses were investigated according to the methods previously described (16). In summary, 0.5 μg of anti-DNP IgE was injected into the dermis of mice. After 48 hours, 1 μg of DNP-HSA containing 4% evans blue (1: 4) was injected into each mouse through the tail vein at designated time intervals. Control mice were injected with 1XPBS. After a designated time, the mouse is sacrificed; Dorsal skin was removed and used to assess mRNA expression of Swiprosin-1.

Atopic eczema / dermatitis atopic eczema Of dermatitis syndrome , AEDS ) -Like skin disorder ( skin lesion Induction of)

Induction of AEDS-like skin disorders using a mite antigen was performed according to a previously established method (17) with several modifications. In summary, 30 minutes after tape-striping the surface of a sheep idol (Balb / c J mouse) three times, first the amount of 20 μl of 1% DNCB dissolved in acetone / olive oil solution (acetone: olive oil = 1: 3) Applied to the surface of the ear. Four days after applying DNCB, 20 μl of 10 mg / ml mite extract (Yonsei University, Seoul, South Korea) was reapplied on the surface of each tape-striped ear. The tape-striping, DNCB and tick antigen paintings were performed once a week for four weeks. Control mice received PBS solution instead of DNCB and tick antigen solution. On day 21, mice were sacrificed, ears were removed, and used for evaluation and pathologic observation of mRNA expression of Swiprosin-1.

Histopathology

For PCA, Evans blue dye images of the dorsal skin were taken using a simple camera. For AEDS-like skin disorders, ears were fixed in 10% formaldehyde, embedded in paraffin and made into thin sections. Skin sections were stained with hematoxylin and eosin and images were taken with a microscope (200X).

Recombination DNA Construct

To generate the Swiprosin-1 / pEGFP-C1 construct, a human Swiprosin-1 clone encoding the full-length open reading frame of Swiprosin-1 in the pOTB7 vector was constructed from the RZPD German Resource Center (Berlin, Germany). Was purchased from, used as a template, and PCR amplification was carried out using the following primers: Sense 5'-AAGAATTCTATGGCCACGGACGAGCTGGCCACC-3 '(SEQ ID NO: 7) containing an EcoRI restriction site and Bam HI restriction site Anti-sense 5'-TTTGGATCCCTACTTAAAGGTGGACTGCAGCTC-3 '(SEQ ID NO: 8). PCR products were subcloned into EcoRI / Bam HI fragments in the pEGFP-C1 vector (Clontech Laboratory, Inc.) of the neomycin resistance gene, resulting in in-plane fusion of Swiprosin-1 at the COOH terminus of GFP. The amino acid sequence of the linker polypeptide between EGFP and swiprosin-1 is SGLRSRAQAS (10 aa). To generate wild type swiprocin-1 in pcDNA3 vector or pHJ-1 vector (lenti-virus vector), the swiprosin-1 cDNA from pOTB7 vector to EcoD / XhoI to pcDNA3 vector or by Not I restriction digestion Transfer to pHJ-1 vector was confirmed by automated sequencing.

Establish stable cell lines

Establishing stable HMC-1 transformants using a Nucleofector device and corresponding kits (Amaxa, Cologne, Germany) by introducing pEGFP-C1 or swiprocin-1 / pEGFP-C1 cDNA, followed by 1 mg / ml Geneti Selected by gene (invitrogen). Cells were then injected into a fluorescence-activated cell sorter (FACS) and sorted to selectively obtain GFP-positive cells; Incubation in complete medium supplemented with the same concentration of antibiotics. Stable cells expressing EGFP or EGFP fused with Swiprosin-1 were designed as H-swip-1_GFP cells or H-GFP cells, respectively.

RNA  Separation and RT - PCR

Tissue sample or in Cells were collected from in vitro cultures and total RNA was isolated using the WelPrep ^™ JBI method (iNtRON Biotechnology, Daejon, Korea) according to the manufacturer's instructions. Reverse transcription of RNA was performed using dT primer Maxime RT-PCR PreMix (iNtRON Biotechnology, Daejeon, South Korea). Two micrograms of RNA were transferred to a dT primer mixture tube. The reaction volume was 20 μl. cDNA synthesis was performed at 45 ° C. for 60 minutes and then RT inactivated at 95 ° C. for 5 minutes. The RT-generating DNA was then diluted to 40 μl in distilled water. The diluted RT-producing DNA (2 μl) was amplified using Maxime PCR PreMix (iNtRON Biotechnology, Daejeon, South Korea). Primers used for cDNA amplification were as follows: hSwip-1 (p1), sense 5'-ATCTTCCGCAAGGCGGCGGCCGGGGAG-3 '(SEQ ID NO: 9) and antisense 5'-GACTGCAGCTCCTTGAAGGCCGCTTTC-3' (SEQ ID NO: 10) ; hSwip-1 (p2), sense 5'-CTCGAGGCCACAGTTATGCAA-3 '(SEQ ID NO: 11) and antisense 5'-ATCCGCTAAGGCAAACGCA-3' (SEQ ID NO: 12); rSwip-1 (p1), sense 5'-CGTTATGCAATGTTCCTCGTGTCACTG-3 '(SEQ ID NO: 13) and antisense 5'-GATTCTCACAGGTTCTAAGACCGAAAA-3' (SEQ ID NO: 14); rSwip-1 (p2), sense 5'-TACAATGCCTCAAGAGCCCCCGGGACC-3 '(SEQ ID NO: 15) and antisense 5'- CCAAAAGTAAAGGGAATTTATATTCCT-3' (SEQ ID NO: 16); mSwip-1, sense 5'-GAGGCTCCCGACGAGACTGCCCAGGCG-3 '(SEQ ID NO: 17) and antisense 5'-CCGGAAGCTGAGTTTGCTGTCGAAATC-3' (SEQ ID NO: 18); hIL-3, sense 5'-CTTTGCCTTTGCTGGACTTC-3 '(SEQ ID NO: 19) and antisense 5'-CGAGGCTCAAAGTCGTCTG-3' (SEQ ID NO: 20); hIL-8, sense 5'-GTGCAGTTTTGCCAAGGAGT-3 '(SEQ ID NO: 21) and antisense 5'-CTCTGCACCCAGTTTTCCTT-3' (SEQ ID NO: 22); hTNF-α, sense 5'-GGCTCCAGGCGGTGCTTGTTC-3 '(SEQ ID NO: 23) and antisense 5'-AGACGGCGATGCGGCTGATG-3' (SEQ ID NO: 24); hGAPDH, sense 5'-CGGAGTCAACGGATTTGGTCGTAT-3 '(SEQ ID NO: 25) and antisense 5'-AGCCTTCTCCATGGTGGTGAAGAC-3' (SEQ ID NO: 26); rβ-actin, sense 5'-ATTGAACACGGCATTGTCAC-3 '(SEQ ID NO: 27) and antisense 5'-GTCTCAAACATGATCTGGGTC-3' (SEQ ID NO: 28). Amplification conditions were performed by denaturation at 94 ° C. for 30 seconds for 30-35 cycles, annealing at 58-68 ° C. for 20 seconds, and extension at 94 ° C. for 40 seconds. PCR products were dissolved and visualized on 1 or 1.5% agarose gels stained with ethidium bromide.

real -Time quantification RT - PCR

In all experiments, the expression levels of the genes of interest were assessed using real-time RT-PCR, except as indicated. Continuous fluorescence detection system (MJ Research, Waltham) at a total volume of 20 μl with 2 μl cDNA / control and gene specific primers using the SYBR premix Ex Taq kit (Takara, Shiga, Japan) PCR amplification was performed in DNA Engine Opticon, MA). PCR reactions were carried out using the following conditions: 94 ° C. 30 sec, 58-68 ° C. 30 sec, 72 ° C. 30 sec, and 40 cycles of plate read (detection of fluorescent product) followed by 72 ° C. 7 minute extension in. Melting curve analysis was performed to identify dsDNA products with fluorescence data collected at 0.2 ° C. intervals by slowly increasing the temperature (0.2 ° C./s) from 65 ° C. to 95 ° C. The degree of expression normalized by GAPDH or β-actin (swiprocin-1, IL-3, IL-8 and TNF-α) was expressed as the maximum relative value% relative to the control. The maximum expression level of the results in each experiment was considered 100%.

Immunofluorescence staining and Confocal Imaging  analysis

Stable HMC-1 cells (H-GFP or H-swip-1_GFP; 1x10 ⁵ ) or COS-7 cells (transformed with GFP or swip-1_GFP) were not poly-L-lysine (PLL) -coated or uncoated. Seeds on 18-mm glass coverslips. Cells were then fixed for 10 minutes in 3.7% formaldehyde and washed twice with 1XPBS. F-actin was detected by staining with paloidine-TRITC (50 μg / ml). In addition, 293-T cells (sweeprocin-1 transformants) were grown on glass coverslips. Cells were immobilized as described above, reacted with a primary goat polyclonal Sweeprocin-1 antibody and then with a secondary FITC-conjugate anti-goat antibody. The localization of sweprocin-1 and actin was investigated with an FV1000 confocal laser scanning microscope (Olympus Corporation, Japan) equipped with 40X, 63X, and 100X objectives. In some cases, cells were not immobilized and live images could be obtained by confocal microscopy. For co-localization analysis, the Z section cutting area was selected through the apical surface. Overlapping degree of intensity (ODI) values were calculated using FLUOVIEW software ver.1.5. The co-position ratio was then derived by multiplying the ODI value by 100.

For live-cell time-lapse confocal imaging, H-swip-1_GFP cells were seeded with 2 × 10 ⁵ cell numbers on 18-mm glass coverslips mounted to the chamber device. Cells were stabilized at 37 ° C. for 10 minutes and then activated by adding PMA / A23187. During live-cell imaging, the chamber device was maintained in a 5% CO ₂ atmosphere using a live-cell instrumentation system (Chanlide Inc., Korea). Confocal series of fluorescence and differential interference contrast (DIC) images were simultaneously obtained at 20 second intervals using a 100 × oil immersion objective on an FV1000 confocal microscope (Olympus). The images were processed and assembled into video using Olympus FLUOVIEW software ver.1.5.

Cell extract preparation and Western Blot ( Western blot ) analysis

PKC isoforms including Swiprocin-1, GFP, p-PI3 kinase, p-Akt, PKC-α, PKC-βI, PKC-θ, PKC-δ, PKC-η and PKC-ζ, and I- For the analysis of κB, HMC-1 cells (parent or transformed cells) were rinsed twice with ice-cold PBS and then ice-cold lysis buffer (10 mM Tris-HCl pH 7.4, 50 mM NaCl, 1% Triton X-100 and Protease Inhibitor Cocktail Tablet). The cell lysate was centrifuged at 14,000 rpm for 20 minutes at 4 ° C. and the same amount of protein supernatant was mixed with 1/4 volume of 4 × SDS sample buffer, heated for 5 minutes and then 8 or 10% SDS-PAGE gels. Isolate through. After electrophoresis, proteins were transferred to nitrocellulose membranes using a trans-blot SD semi-dry transfer cell (Bio-Rad, Hercules, Calif.). The membrane was blocked in 5% skim milk (1 h), rinsed and reacted with the primary antibody in TBS containing 0.1% Tween 20 (TBS-T) and 3% skim milk overnight at 4 ° C. Excess primary Ab was then removed by washing the membrane three times in TBS-T and the membrane was reacted with 0.1 μg / ml hoseperoxidase-labeling secondary Ab (for goat, rabbit, or mouse) for 2 hours. After washing three times in TBS-T, the bands were visualized with ECL western blotting detection reagent and exposed to x-ray film.

IL -8 measurements and Histaamine  Release Assay

Stable HMC-1 cells (H-GFP or H-swip-1_GFP; 2 × 10 ⁶ ) were seeded in 12-well plates. Cells were further stimulated with PMA (200 nM) / A23187 (1 μM). At the indicated times, supernatants were collected and measured for IL-8 or histamine levels using ELISA (R & D Systems) according to the manufacturer's instructions.

NF -KB Luciferase  Activity analysis

With 100 μl of Amaxa's Nucleofector solution (Amaxa, Cologne, Germany) containing 1 μg of Swiprosin-1 / pEGFP-C1 or pEGFP-C1 and 1 μg of pRL-TK renilla and 1 μg of pGL3 / NF-κB HMC-1 cells (1.5 × 10 ⁶ ) were transformed and then immediately transferred to 2.0 ml of complete medium and incubated in 37 ° C. 6-well plates. 24 hours after transformation, the medium was transferred to IMDM medium containing 10% FBS and antibiotics. Cells were stimulated as described in the literature and reacted at 37 ° C. for 12 hours. Cell lysates were prepared and analyzed for luciferase activity using a dual luciferase assay system (Promega, Madison, Wis.) According to the manufacturer's instructions.

Statistical analysis

Average values were calculated from data taken from at least three (usually four or more) individual experiments conducted on different days. When running the significance test, an independent t test (student's; two populations) was used. P values below 0.05 were considered as indicators of statistical significance.

Experimental method on-line FIGS. 10-14

DNA Microarray  analysis

DUTP-labeled cDNA was amplified from total RNA samples (5 μg / sample) using RT-IVT labeling reagent (Applied Biosystems, MA). 15 micrograms of labeled cDNA were hybridized on microarray chips containing 33,096 human genes (Applied Biosystems) and further proceeded according to Applied Biosystems' protocol.

Microarray images were obtained with an AB 1700 microarray analyzer (Applied Biosystems). Signal quantification and data processing were performed using AVADIS (Strand Genomics, India). Each experiment was repeated three times to reduce the risk for false-positive or false-negative results. Significant differences in gene expression between data from two different groups (control, PMA / A23187) were identified using the One-Way ANOVA Kruskal-Wallis test with a significance threshold of p <0.05. The K-means clustering method was used to cluster the genes selected by the test. To evaluate fold induction, the ratio of treatment signal strength to no signal-signal strength was calculated. The log2 values of each ratio were determined to identify the magnitude of the deviation of the up-regulated and down-regulated genes, and differences in gene expression were graded based on absolute values. Of the 1467 genes filtered by the significance test ( p <0.05), 208 genes were expressed at least 3-fold differentially and treated as dendrograms for treatment with PMA / A23187 (FIG. 10A). Green blocks represent down-regulated genes, red blocks represent up-regulated genes, and assay blocks represent genes that are relatively unmodified relative to the control. Semi-quantitative RT-PCR was performed to confirm gene expression data from microarray analysis. Although not well identified, 11 genes highly expressed in mast cells by PMA / A23187 are shown in FIG. 10B. Amplification of GAPDH and hTNF-α were used as internal control and activated mast cell markers, respectively.

Lentivirus ( Lentiviral ) infection

Lentiviral vector (10 μg pHJ-1) with appropriate insult (1 μg pHDM-Hgpm2, 1 μg pRC / CMV-Rev1b, and 3 μg pHDM.G) using Lipofectaamine 2000 (Invitrogen) Or swiprosin-1 / pHJ-1) was transfected with 293T cells. Culture supernatants were collected 48 hours after transfection. All supernatants were collected, concentrated by ultracentrifugation, and stored at -80 ° C until use. For the lentivirus infection, it was mixed with 15-ml centrifuge tube viral particles HMC-1 cells ^{(2 x 10 5/500 μl} ) in the presence of 8 μg / ml polybrene (polybrene). After centrifugation at 2,000 rpm for 1 hour, cells were transferred to 60-mm culture dishes and infection efficiency was confirmed 48 hours after infection by Western blot.

Experiment result

Swiprosin -1 is mast  Expressed in cells in vitro  And in vivo  Up-regulation at all.

Gene chip technology is characterized by high communication, economics and miniaturization (18, 19), thus providing a technical platform for discovering new genes behind biological processes that are essential in various model systems. As a genome-level approach to understanding mast cell activation at the molecular level, we performed high density oligonucleotide microarrays with mRNA prepared from human mast cell line HMC-1 cells stimulated with PMA / A23187 (FIG. 10). From this approach, we have found some meaningful genes that have not been studied in mast cells involved in cytokine regulation. Of these gene candidates, the novel gene, Swiprosin-1, was of particular interest because it was not investigated in mast cells, and more interestingly, in contrast to previous reports on B-cell activation (2), It was overinduced in mast cells during the activation process (Figure 10).

We examined the expression of Swiprosin-1 in various cell types and swept in most immune cells, including T / B cells, monocytes, and mast cells, and in some non-immune cells such as epithelial and endothelial cells. It was found that Shin-1 was expressed, but there was some difference in the degree of expression (FIG. 1A). These results indicate that swiprosin-1 is not limited to T and B cells but is expressed in most cell types including mast cells. By using three different methods, including semi-quantitative RT-PCR, real-time quantitative PCR, and western blot, we found that sproprosin when stimulated with PMA (100 nM) / A23187 (1 nM) -1 was found to be significantly over-induced in HMC-1. Time-dependent experiments showed that maximal induction was observed 12-24 hours after stimulation in HMC-1 cells. Swiprocin-1 was also over-induced in rat mast cell line RBL-2H3 cells via cross-linking of FcεR1 by treatment of IgE and DNP-HSA, while the induction rate was faster than in HMC-1 cells. (2-6 hours after stimulation) (FIGS. 1E and 1F).

PCA reaction is a local allergic reaction in One of the best characterized in vivo models (20, 21). We further examined the expression of Swiprocin-1 in tissue samples prepared from dorsal skin after the PCA response. Blue represents Evans blue as an indicator of the PCA reaction (FIG. 2A). Interestingly, Swiprocin-1 was significantly induced in tissue samples of PCA responses (FIG. 2B). In addition, we applied to a mouse model of atopic dermatitis, a generally chronic type of eczema (inflammatory skin disease), and Swiprosin-1 is highly expressed in a sample of atopic dermatitis, but in control Balb / c mice (C and 2D) Taken together, these results also indicate that Swiprosin-1 expression is not the same as in acute dermal anaphylaxis and chronic atopic dermatitis. It is shown that it is regulated transcriptionally in vivo .

Swiprosin -1 expression is PKC It is induced through the -β1 / η-dependent pathway but down-regulated by calcium signals.

Calcium and PKC pathways are important and essential for the activation of various cells as well as for the progression of various cells (23-25). When Swiprocin-1 is over-induced in HMC-1 cells by simple co-treatment of PMA and A2387, we stand alone to further identify the corresponding signaling pathway that is responsible for Swiprosin-1 expression. The effect of the treatment was investigated. Surprisingly, Swiprosin-1 was induced only in HMC-1 cells treated with PMA (FIGS. 3A and 3B). Calcium ionophores A23187 or ionomycin alone did not affect the expression of Swiprocin-1; Rather, in part by inhibiting PMA-induced swiprocin-1 in HMC-1 cells (FIGS. 3A and 3B), it can be seen that the calcium signal has a negative effect on the expression of swiprocin-1 in mast cells.

Since PKC is the primary target of PMA, we next examined whether PKC-isoform could be responsible for the expression of Swiprocin-1 in HMC-1 cells. Initially, we used drugs that could modulate certain PKC isoforms. Because of this, HMC-1 cells were pretreated with various concentrations of various PKC inhibitors, and then the cells were stimulated with PMA. As can be seen in Figures 3c and 3d, G (inhibitors of PKC α and PKCβI) and rottlerin (inhibitors of PKCδ and PKCι) do not affect the expression of PMA-induced Swiprocin-1. G (inhibitors of PKCs α, βI, βII, δ and ζ) shows only minimal blocking effect. On the other hand, staurosporin, a non-specific PKC inhibitor, significantly blocks the expression of PMA-induced swiprocin (FIGS. 3C and 3D). These data show the need for PKC (s) in the expression of PMA-induced swiprosin-1, but it is not clear whether PKC (s) isoforms are involved in this process. Thus, we used siRNA to knock down the target PKC isoform. Mast cells express PKCα, βI, θ, δ, η, and ζ forms (FIG. 4A), and transfection of siRNA down-regulates each target PKC in a time-dependent manner, with maximum inhibition at 72 hours of transfection. Adjusted (FIG. 4B) can be seen by Western blot analysis. As can be seen in FIGS. 4C and 4D, while investigating six PKC isoforms, the knockdown of PKC-βI and PKC-η was targeted but significantly inhibited PMA-induced swiprocin-1 expression, which was PKC -βI and PKC-η are meant to play a major role in regulating swiprocin-1 expression. Consistent with these assumptions, it was found that knockdown of PKC-βI / η by siRNA significantly inhibited Swiprosin-1 expression, whereas knockdown of PKC-α / δ had no effect (FIGS. 4E and 4F). .

Sweeprosin -1 is Phorbol  Esters and calcium In ionophore  by mast  Improves cell activation.

Induction of Swiprosin-1 during activation of mast cells, linked to the fact that Swiprocin-1 has potential as a new signaling adapter protein (1-4), suggests that Swiprosin-1 is a mast adapter molecule as a signal adapter molecule. It is suggested that it may be involved in the process of cell activation. To clarify this point, we present HMC-1 cells that transiently or stably over-express GFP (H-GFP cells) or GFP (H-swip-1_GFP cells) fused to Swiprosin-1. Were prepared (FIGS. 5A and 5B). As can be seen in FIGS. 5C-5E, over-expression of Swiprocin-1 significantly increased PMA / A23187-induced cytokine expression. It also increased histaamine release by PMA / A23187 stimulation (FIG. 5F). In order to eliminate the potential effect of GFP on mast cell activation, we also used HMC- over-expressing wild type Swiprosin-1 without GFP by using a lentiviral vector comprising a Swiprosin-1 cDNA. 1 was prepared (FIG. 11A) and it was found that I-κBα degradation as well as cytokine expression (IL-3 and IL-8) was significantly increased in cells overexpressing Swiprosin-1 (FIGS. 11B and 11C). This shows that GFP has little effect on mast cell activation.

Next, we tested whether silencing of Swiprosin-1 can alter cytokine expression in HMC-1 cells. To this end, we transfected HMC-1 cells with siRNA targeting swiprocin-1 and then evaluated the cells for cytokine expression. As can be seen in Figures 6a and 6b, only the knockdown of Swiprosin-1 showed a minor effect on PMA / A23187-induced IL-3 and IL-8 mRNA expression in HMC-1 cells.

Actin Depolymerizing Agent , Cytocarcin  B and PI3K  Inhibitors, wortmannin silver

Sweeprosin Expressing -1 HMC Selectively inhibits cytokine expression in -1 cells.

The negligible effect of Swiprosin-1 knock-down on cytokine expression by PMA / A23187 is not related to the major pathway of cytokine expression, but rather by controlling other pathways when overexpressed It may mean that it has additional effects. Thus, we applied a variety of agents to systemically regulate the intercellular signaling pathways potentially involved in mast cell activation.

First, the NF-κB and NF-AT pathways are important for IgE-mediated cytokine production (26, 27). We investigated the effects of two agents (MG132 and cyclosporin A) on PMA / A23187-induced cytokine expression in H-GFP cells and H-swip-1_GFP cells. As can be seen in Figure 7a (left), each drug significantly inhibited cytokine expression to the same extent in both cells. Second, cleavage-active protein kinase (MAPK) signaling pathways are also involved in cytokine expression in mast cells (28-30). For this reason, the effects of two pharmaceutical inhibitors of extracellular signal-regulating kinase (ERK) and p38 kinase (eg PD98059 (10 μM) and SB203580 (10 μM)) were investigated, respectively. As can be seen in FIG. 7A (right), each of the two inhibitors significantly inhibited PMA / A23187-induced cytokine expression in both cells. Taken together, the results show that Swiprocin-1 is present at least upstream or at sites of two major signaling pathways in terms of cytokine expression in HMC-1 cells.

Third, Src family kinases and PI3 kinases play a central role in mast cell degranulation mediated by cross-linking of FcεR1 (7). However, it is not well understood in the cytokine expression of HMC-1 cells mediated by phorbol esters and calcium ionophores. We therefore tested whether increased cytokine expression in H-swip-1_GFP is also involved in the kinase. As can be seen in FIG. 7B (left), PP2, an Src kinase inhibitor, significantly inhibited cytokine expression in H-GFP and H-swip-1_GFP cells, and Src family kinase in HMC-1 cells. PMA / A23187-induced cytokine expression. Interestingly, however, wortmannin, a PI3 kinase inhibitor, inhibited only cytokine expression in H-swip-1_GFP cells (FIG. 7B, left). These results indicate that whiskeysin-1 may influence cytokine expression in HMC-1 cells through activation of the PI3 kinase pathway, whereas PI3 kinase is not primarily involved in PMA / A23187-induced cytokine expression. Shows. In particular, we found that treatment with PMA / A23187 of HMC-1 cells did not have a significant effect on PI3 kinase, whereas p-Akt, a downstream effector of PI3 kinase, slightly after 5-30 minutes of treatment. It was found to increase (FIG. 12). On the other hand, even at O time both p-PI3 kinase and p-Akt were observed in H-swip-1_GFP cells (FIG. 12).

Fourth, PI3 kinase also has important regulatory functions for receptor-mediated cytoskeletal rearrangement in mast cells (31, 32). We therefore assessed the potential involvement of the two most important components in the cytoskeleton (eg, actin and microtubules). The destruction of actin microfibers by cytocarcin B has been reported to improve the degranulation response in RBL-2H mast cells (33, 34). On the other hand, microtubule destruction has been known to inhibit the degranulation reaction through inhibition of calcium influx in the same cell (35). As can be seen in FIG. 7B (right), microtubule depolymerizer colchicine significantly inhibited cytokine expression in H-GFP and H-swip-1_GFP cells, which showed microtubule HMC-1 cells In PMA / A23187-induced cytokine expression. However, actin depolymerizer cytokaracin B inhibited the cytokine expression seen only in H-swip-1_GFP cells (FIG. 7B, right). In addition, the same results were obtained from lentiviral-transfected cells in the same experimental format (data not shown), showing that actin polymerization was not usually associated with cytokine expression of HMC-1 cells stimulated by PMA / A23187. However, these results strongly suggest that Swiprocin-1 can act via PI3 kinase and actin regulation.

Sweeprosin -1 is mast  Cell Actin -rich( rich ) Fine process ( microvilli )-Is located in a similar area.

In many cases, the location of the protein at the site of differentiation of the cell reflects the specific function of the protein. In addition, reduced cytokine expression in H-swip-1_GFP cells by actin depolymerizer cytokaracin B suggests a particular role of swiprocin-1 in relation to actin cytoskeleton (FIG. 7B, right). Thus, we used confocal microscopy to examine the location of Swiprosin-1 in HMC-1 cells and other cell types. As can be seen in Figure 8a, swip-1_GFP is located in the microprojection-like protrusions of HMC-1 cells, while GFP was distributed throughout the cytosol and nucleus. To better understand, wild type Swiprosin-1 (without GFP) is also transfected into 293T cells, stained with an antibody against Swiprosin-1 followed by a FITC-conjugated secondary antibody, followed by high resolution confocal microscopy. It was evaluated using. Interestingly, it accumulates high in the apical ridge area of the membrane of 293T cells (FIG. 8B). This observation is consistent with the report that Sweeprocin-1 is a lipid raft protein in B cells (4). Since it is known that F-actin is abundant in the microprojection-like region (36), we tested whether Sweeprocin-1 is present with F-actin in both HMC-1 and COS-7 cells. As can be seen in FIGS. 8C and 8D, Swiprocin-1 is located in a significant amount with F-actin in both HMC-1 and COS-7 cells, and COS-7 cells when the cells are stimulated with PMA It can be seen that it is repositioned with the movement of F-actin at (Fig. 8d). Taken together, with the fact that the actin degrading agent cytocarcin B inhibits cytokines only in H-swip-1_GFP cells, swiprocin-1 in the microprojection-like region (FIG. 8A) or the membrane peak region (FIG. 8B) The position of and association with F-actin (FIGS. 8C and 8D) suggest that Swiprocin-1 acts through actin regulation and reorganization. Interestingly, as determined by staining with paloidine-TRITC, we observed that the ectopic expression of Swiprosin-1 slightly reduced the F-actin content (FIG. 13B). We also observed that the content of F-actin gradually decreased while Swiprocin-1 was over-induced by PMA treatment in mast cells (FIGS. 13A and 13B). These observations support the above conclusion that Swiprosin-1 regulates actin polymerization or reorganization in mast cells, but the present invention does not reveal the mechanism.

Discuss Experiment Results

Gene chip technology is one of the powerful ways to discover new genes behind biological processes that are essential in various model systems (18, 19). Because of this approach, the inventors have discovered that a novel gene, Swiprosin-1, is expressed in human mast cells and is over-induced during mast cell activation. In addition, the expression of Swiprosin-1 is expressed by cross-linking of phorbol ester or FcεR1 in Mast Cells Cultured in vitro , and PCA and Atopic Dermatitis in vivo Up-regulation in model tissue. SiRNA approaches targeting the PKC isotype showed that PKC-βI / η was the primary target for phorbol ester-mediated induction in HMC-1 cells of swiprocin-1. In addition, the present inventors have Ca ^{2 +} - it was found that the negative effect for the new -1 expressed as a signaling sweep.

Molecules generated from mast cells can also activate mast cells through self-secreting methods. For example, TNF-α is one of the major cytokines that activate HMC-1 cells in a self-secreting manner (37). In a similar manner, ectopic expression of swiprocin-1 increased cytokine expression as well as PMA / A23187-induced NF-κB promoter activity. Since the degradation of actin or inhibition of PI3-kinase by cytocarcin B blocks only cytokines in swiprosin-1-overexpressing cells, the inventors have found that Sproprocin in microprotrusion-like membrane protrusions with F-actin. It was confirmed in the present invention that the position of −1 may be important for acting by activating mast cells.

Previous studies have shown that by Northern blot analysis, Swiprocin-1 was detected in the spleen, lung, and liver while being most abundantly detected in the brain (2). According to these data, we showed that Swiprosin-1 is expressed during B-cell differentiation, with high expression in immature bone marrow B cells (2). In addition, recent reports have shown that swiprocin-1 is associated with tau protein, a known cause of human degenerative neuropathy tauopathy (38). Or pathologically important action. However, the expression of Swiprosin-1 may affect the expression of physiological / pathological conditions in in vitro or in Whether it is regulated in vivo models is currently unknown. Indeed, there have been conventional reports that Swiprocin-1 expression does not change during B cell activation by IgM / IL-4, LPS, or anti-CD40 / IL-4 (2). However, in contrast to previous studies, we found that swiprocin-1 expression was in phorbol ester-induced activation model in vitro and in It was clearly shown that all of the in vivo pathological models regulate transcription. These experimental results strongly indicate that Swiprosin-1 may be importantly involved in physiological and / or pathological processes. Moreover, increased expression of Swiprocin-1 in the tissue and atopic dermatitis models of PCA suggests a strong role of Swiprosin-1 in the inflammatory response.

PKC families are expressed in a variety of cell types, which are known to regulate a variety of cellular processes that affect cell growth and differentiation, cytoskeletal remodeling and gene expression in response to various stimuli (39). In particular, PKC-regulated signaling pathways play an important role in various aspects of the immune response, from lymphocyte development, differentiation, activation and survival to macrophage activation (39). It is also clear that PKC activation is required for the early and delayed response of mast cell-mediated inflammatory processes (40). In this respect, the induction of Swiprocin-1 by the PKC-βI / η pathway also means that Swiprocin-1 is involved in the inflammatory response of mast cells. However, the unpublished experimental results of the present inventors showed that Swiprosin-1 by PKC is not only limited to mast cells but also in other immune cells including human T cells. It is not currently clear whether swiprosin-1 requires specific PKC isoforms in various cell types, but these results indicate that swiprosin-1 is a PKC-responsible gene in various cell types. Can be.

Calcium signals are important for initiating signaling cascades leading to degranulation of mast cells and release of inflammatory mediators (41, 42). Crossing of FcεR1 by IgE-Ag-inflammatory cytokine gene-binding continuously extracellular Ca ^{2 +} and influx and consequently results in the activation of various transcription factors, different before (pre) or precursor (pro) through the plasma membrane Induced (41, 43-45). However, it is surprising that the increase of calcium by A23187 or ionomycin in the present invention induces down-regulation of Swiprocin-1 rather than induction. Thus, analysis of the promoter region in the Swiprosin-1 gene will be important in understanding why and how calcium signals inhibit Swiprosin-1 expression in HMC-cells. It may also be interesting to investigate whether this phenomenon is observed in other immune cells, such as lymphocytes, because of their important role in lymphocyte activation (46, 47). There is one prediction that swiprocin-1 has two EF-hand motifs that effectively bind calcium (38). It is hypothesized that these substantial properties of Swiprosin-1 can modulate intracellular calcium signals or levels, so that calcium signals must regulate Swiprocin-1 levels in cells, which is not only external to intracellular calcium homeostasis It is important to maintain calcium signaling induced by stimulation.

As experimental results show that Swiprosin-1 lies on a signaling pathway that regulates the activity of HMC-1 cells, the PMA / A23187-induced HMC- of Swiprosin-1 in terms of cytokine expression. 1 The effect on the activation of cells is of interest. In this regard, it is important to investigate whether Swiprocin-1 is also involved in high affinity IgE-mediated degranulation and cytokine expression. Since HMC-1 cells do not express cell surface FcεRI and thus cannot be activated by antigen (48), further studies will be needed in other model systems such as bone marrow-derived mast cells (BMMC) and RBL-2H3 cells. Nevertheless, the HMC-1 model adopted in the present invention is of sufficient value because the cells have various properties of immature human mast cells (13), in particular, as with normal or transformed FcεR ⁺ mast cell lines ( 15), because immature human mast cells can still be activated by treatment with phorbol esters and calcium ionophores.

The location of proteins at the site of differentiation of cells may reflect their specific function (49-51). For example, SLP-76, an important adapter protein for FcεR1-induced calcium flux, degranulation, and cytokine secretion in mast cells, is found in the cytosol of unstimulated cells. As soon as FcεR1 cross-links, SLP-76 translocates to the cell membrane and clusters with FcεR1, tyrosine kinase Syk, adapter LAT, and phosphotyrosine. Breakdown of this position has been reported to inhibit FccR1-mediated signaling in mast cells (50). Along with this trend, the experimental results of the present invention showing the position of the highest sweeprocin-1 in the microprojection-like membrane constructs indicate that the protein forms a signaling complex in the plasma membrane or binds to actin or actin regulatory molecules to function It can mean. In addition, the reduction of actin content by over-expression of Swiprosin-1 also supports a strong role in actin reorganization. Indeed, the proline-rich element in the predicted secondary structure of Swiprosin-1 is the best-class member of the growing family of protein-interaction modules that include signaling in the dynamic actin network ( 52, 53), known as potential binding sites for the SH3 domain (2). Consistent with the predicted secondary structure, the results of our unpublished experiments show that the deletion of the proline-rich element in Swiprosin-1 is significantly reduced with respect to F-actin in COS-7 cells. (Data not shown).

Based on evidence for the physical or functional association of actin and swiprocin-1 in the present invention, it is not surprising that disruption of actin polymerization by cytocarcin B inhibits cytokine expression in HMC-1 cells. Induction of p-PI3 kinase and p-Akt and inhibition of cytokine expression by wortmannin also mean that swiprocin-1 affects the PI3 kinase pathway linked to the regulation of actin dynamics. However, the fact that the knockdown of the desired Swiprosin-1 shows a negligible effect on cytokine expression is not related to the major pathway of cytokine expression, but in the mast cells when it is over-expressed. It may mean that it has an additional effect of activating the actin reorganization. An interesting result in the present invention is that Swiprosin-1 is rapidly (<5 min) redistributed to secretory granules of HMC-1 cells during activation by PMA / A23187 (FIG. 14). Results from this time-lapse video microscope indicate that Swiprosin-1 can also be associated with the mast cell degranulation process by binding to actin dynamics.

Mast cells are central to allergies and asthma. Activation of these cells induces inflammatory mediators and de novo synthesis performed on differentiated granules and secretion of cytokines, chemokines, and eicosanoids (7). Upon stimulation, the response of mast cells can be regulated by the equilibrium of events of positive and negative intercellular molecules (7). In the present invention, we propose that Swiprocin-1 is a novel regulator for positive feedback activation of mast cells. Induction of swiprocin-1 is limited to the pathway of PKC-βI / η. Once induced, the protein is significantly located in the actin-rich microprotuberant-like membrane overhang in mast cells. In pathological situations, the protein can modulate local allergic or inflammatory responses through amplifying pre-required signals for mast cell activation.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

References

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1A-1F show the results of expression analysis of swiprosin-1 mRNA and protein by RT-PCR, real time RT-PCR, and Western blot. 1A shows the expression of Swiprosin-1 mRNA in various cell types. RNA is Jurkat T and Molt-4 T (T cells), Raji B (B cells), THP-1 (monocytes), 293T, CHO-K1, HT-29 (epithelial cells), and rat RBL-2H3 (mast cells) ), And mRNA levels of swiprocin-1 were determined using RT-PCR. Note : Expression of Swiprocin-1 in RBL-2H3 was determined using rat swiprocin-1-specific primers. 1B-1D show the induction of Swiprocin-1 by PMA / A23187 in HMC-1 cells. HMC-1 cells were treated with PMA (200 nM) and A23187 (1 μM). At the indicated time, expression of Swiprocin-1 was determined using RT-PCR (FIG. 1B), quantitative RT-PCR (FIG. 1C), and Western blot (FIG. 1D). 1E and 1F show the induction of Swiprosin-1 by cross-linking FcεR1 in RBL-2H3 cells. RBL-2H3 cells were sensitized to antigen with anti-DNP-IgE (1 μg / ml) for 18 hours and then the cells were subjected to anti-DNP-HSA (1 μg / ml). At the indicated time, expression of Swiprosin-1 was determined using RT-PCR (FIG. 1E) and quantitative RT-PCR (FIG. 1F). Amplification of GAPDH and hTNF-α was used as internal control and activated mast cell markers, respectively. β-actin was used as loading control for Western blot. All data represent at least three individual experiments. Data shown in bar graphs represent mean ± SD of three independent experiments.

2A-2D show that Swiprocin-1 is up-regulated in skin tissue of PCA and AEDS-like models. FIG. 2A is a photograph showing the dorsal skin of the mouse before (0 h) and after (0.5 h) PCA reaction. FIG. 2B shows total RNA was extracted at the designated time (0-6 h) after PCA reaction, and the swiprocin-1 level was RT-PCR ( top ) and quantitative RT-PCR ( bottom ), as described in the Examples. Determined using. 2C is a photograph showing the histological status of control or AEDS-like mouse ear skin. 2D was determined for Sweeprocin-1 levels using RT-PCR ( top ) and quantitative RT-PCR ( bottom ). β-actin was used as internal control. Gel data represents two separate experiments. Data shown in bar graphs represent mean ± SD values of three experiments.

3A-3D show the results of down-regulation of swiprocin-1 with phorbol ester but with calcium activator. HMC-1 cells were prepared with calcium modulators (FIG. 3A and 3B) or G (100 nM), G (100 nM), including BAPTA-AM (50 μM), A23187 (1 μM), and ionomycin (1 μM). , pre-treated with PKC inhibitors (FIGS. 3C and 3D), including rottlerin (10 μM) and staurosporin (500 nM), or without the modulator or inhibitor for 30 minutes, and then additionally the cells with PMA (200 nM) or Stimulated without. Expression of Swiprocin-1 was determined using RT-PCR (FIGS. 3A and 3C) and quantitative RT-PCR (FIGS. 3B and 3D). Amplification of GAPDH was used as internal control. The data represents three separate experiments. Data shown as bar graphs represent mean ± SD values of three experiments.

4A-4F show that Swiprosin-1 expression is dependent on the PKC-βI / η pathway in mast cells. 4A shows the results of PKC isoform detection by Western blot in HMC-1 cells. 4B shows knock-down of PKC isoforms by specific siRNAs. HMC-1 cells were transfected with siRNAs targeting specific PKC isoforms. At the indicated time, expression of PKC isoforms was analyzed using Western blot. In addition, blots were probed with antibodies against β-actin to confirm the same loading. 4C-4F show that knock-down of PKC-β1 / η inhibits phorbol ester-induced swiprocin-1 expression in HMC-1 cells. After 72 hours of siRNA transfection for the designated PKC isoform, cells were treated for 6 hours with or without PMA (200 nM) and RT-PCR (FIGS. 4C and 4E) and quantitative RT-PCR (FIG. 4D). And 4f) were used to analyze the expression of Swiprocin-1. Amplification of GAPDH was used as internal control. The data represents three separate experiments.

5A-5F show that ectopic expression of swiprosin-1 improves mast cell activation by phorbol esters and calcium ionophores. 5A and 5B show the establishment of stable cell lines expressing GFP or swip-1_GFP. After establishing H-GFP or H-swip-1_GFP cells as described in the Examples, the expression of Sweeprocin-1 was determined using flow cytometry (FIG. 5A) and Western blot (FIG. 5B). It was. Note: At least 95% of GFP-positive cells were used in the examples of the present invention. 5C-5F show that over-expression of Swiprosin-1 improves PMA / A23187-induced cytokine expression and histamine release. H-GFP or H-swip-1_GFP cells were treated with PMA (200 nM) for various times and then cytokine expression was determined using RT-PCR (FIG. 5C) and quantitative RT-PCR (FIG. 5D). . Supernatants were also used to determine IL-8 secretion (FIG. 5E) and histamine release (FIG. 5F). Amplification of GAPDH was used as internal control. The data represents three separate experiments. Data shown as bar graphs represent mean ± SD values of three experiments.

Figure 6 shows the negligible effect of cytokine expression in knock-down activated mast cells of Swiprocin-1. 6A shows targeting knock-down of swiprocin-1 by siRNA. HMC-1 cells were transformed with siRNAs targeting swiprocin-1. At the indicated time, the expression of Swiprosin-1 was analyzed using Western blot. FIG. 6B shows that after 72 hours of siRNA transformation, cells were treated with PMA / A23187 for various times (0-4 h) and cytokine expression (IL-) using RT-PCR (left) and quantitative RT-PCR (right). 3 and IL-8) were analyzed. GAPDH amplification was used as internal control. The data represents three separate experiments. Data shown in bar graphs represent mean ± SD values of four individual experiments.

7A-7B show inhibition of cytokine expression using various agents in HMC-1 cells expressing GFP or swip-1_GFP. 7A and 7B show MG132 (1 μM), cyclosporin A (CSA, 1 μg / ml), PD98059 (PD, 10 μM), SB203580 (SB, 10 μM), wortmannin (WOT, 100 nM), PP2 (10 μM) Pretreatment with H-GFP or H-swip-1_GFP cells for 30 minutes without the various pharmaceutical inhibitors, including colchicine (Col, 20 μM), and cytocarcin B (CTB, 1 μg / ml) or the inhibitor Shows. Expression of cytokines was determined using RT-PCR (top) and quantitative RT-PCR (bottom). Amplification of GAPDH was used as internal control. The data represents three separate experiments. Data shown in bar graphs represent mean ± SD values of three experiments.

8A-8D show that Sweeprocin-1 is located in the actin-rich microprotrusion-like membrane overhang in mast cells. 8A shows that H-GFP or H-swip-1_GFP cells are attached to glass coverslips and then imaged using confocal microscopy with serial z-sections. The images are shown as single series images and reconstructed images of the z-axis. FIG. 8B shows 293T cells were transformed with Swiprosin-1 / pcDNA3, stained with anti-Swiprosin-1 antibody and reacted with FITC-conjugate secondary antibody. The cells were then imaged using confocal microscopy. 8C and 8D fix HMC-1 cells expressing GFP or swip-1_GFP (FIG. 8C), or COS-7 cells expressing swip-1_GFP and stained with palodine-TRITC. The cells were then imaged by confocal microscopy. Co-localization analysis was performed as described in the examples. Bars represent 10 μm.

9A shows the results of analysis of the expression of I-κBα using Western blot at the indicated time. 9B shows the results of assaying luciferase activity using a dual luciferase assay system (Promega, Madison, Wis.).

10A-10B show the dendrogram of gene expression profile and the results of semi-quantitative RT-PCR. 10A stimulated HMC-1 cells (1 × 10 ⁷ ) for 4 hours with or without PMA (200 nM) / A23187 (1 μM). Microarrays and data were performed as described in the Examples. CON, mean value of three experiments; # 1-3 P / A, three PMA / A23187-treated cells. Green blocks represent down-regulated genes and red blocks represent up-regulated genes. FIG. 10B shows stimulation of HMC-1 cells (1 × 10 ⁶ ) with PMA (200 nM) / A23187 (1 μM) for various times, followed by RT-PCR analysis of mRNA levels of designated genes. Amplification of GAPDH was used as internal control. The data represents two separate experiments.

11A-11C show that ectopic expression of Swiprosin-1 improves mast cell activation by phorbol esters and calcium ionophores. 11A shows that Sweeprocin-1 is over-expressed by lentiviral infection in HMC-1 cells. Expression of Swiprocin-1 was determined using Western blot. 11B shows that over-expression of Swiprosin-1 in HMC-1 cells improves PMA / A23187-induced cytokine expression. HMC-1 cells infected with lentiviral vectors containing pHJ-1 or swiprocin-1 / pHJ-1 were treated with PMA (200 nM) / A23187 (1 μM) for 4 hours, followed by RT-PCR ( left ) And quantitative RT-PCR ( right ) were used to determine cytokine expression. Amplification of GAPDH was used as internal control. The data shown in the bar graph represents one of two experiments. FIG. 11C shows the result of reacting the cells from FIG. 11B for various times (0-90 min). At the indicated time, the cells were lysed and I-κBα degradation was determined using Western blot.

Figure 12 shows that the increase in phosphorylation (phosphorylation) of the above scan procedure ectopic expression of new -1 (ectopic expression) The PI3-kinase in HMC-1 cells, and Akt. H-GFP or H-swip-1_GFP cells were treated with PMA (200 nM) / A23187 (1 μM). At the indicated time, the cells were lysed and p-PI3 kinase and p-Akt were determined using Western blot.

13A-13B show that over-expression of Swiprosin-1 reduces the content of F-actin in HMC-1 cells. FIG. 13A shows that H-GFP or H-swip-1_GFP cells were treated with PMA (200 nM) for a specified time (26 h), and then the level of GFP signal was measured using RT-PCR ( top ) and flow cytometer ( bottom ). Show the decision result. Note: We found that the pCMV promoter of the pEGFP-C1 vector was activated in response to PMA and then improved protein expression as can be seen in the figure. FIG. 13B stained cells from FIG. 13A with Palodine-TRITC and then examined using a flow cytometer. The data represents two separate experiments.

14 shows the dynamic migration of Swiprosin-1 clusters during activation of HMC-1 cells. H-swip-1_GFP cells were treated with PMA / A23187 and fluorescence and DIC images were obtained at 20 second intervals using a 100 × oil immersion objective confocal microscope. Note, Swiprosin-1 was rapidly and significantly redistributed to secreting granules of HMC-1 cells.

<110> Gwangju Institute of Science and Technology <120> Novel Drug Target against Hypersensitivity Immune or Inflammatory          Disease <160> 28 <170> Kopatentin 1.71 <210> 1 <211> 2424 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (78) .. (797) <223> swiprosin-1 <400> 1 aggaagagga agagcgcggc cggcggcgct gcgctgagag caggggcccg gccaaggcga 60 gtgccgcgcg ggccacc atg gcc acg gac gag ctg gcc acc aag ctg agc 110                       Met Ala Thr Asp Glu Leu Ala Thr Lys Leu Ser                         1 5 10 cgg cgg ctg cag atg gag ggc gag ggc ggc ggc gag acc ccg gag cag 158 Arg Arg Leu Gln Met Glu Gly Glu Gly Gly Gly Glu Thr Pro Glu Gln              15 20 25 ccc ggg ctg aac ggg gca gcg gcg gcg gcg gcg ggg gca ccc gac gag 206 Pro Gly Leu Asn Gly Ala Ala Ala Ala Ala Ala Gly Ala Pro Asp Glu          30 35 40 gcg gcc gag gcg ctg ggc agc gcg gac tgc gag ctg agc gcc aag ctg 254 Ala Ala Glu Ala Leu Gly Ser Ala Asp Cys Glu Leu Ser Ala Lys Leu      45 50 55 ctg cgg cgc gca gac ctc aac cag ggc atc ggc gag ccc cag tcg ccc 302 Leu Arg Arg Ala Asp Leu Asn Gln Gly Ile Gly Glu Pro Gln Ser Pro  60 65 70 75 agc cgc cgc gtc ttc aac ccc tac acc gag ttc aag gag ttc tcc agg 350 Ser Arg Arg Val Phe Asn Pro Tyr Thr Glu Phe Lys Glu Phe Ser Arg                  80 85 90 aag cag atc aag gac atg gag aag atg ttc aag cag tat gat gcc ggg 398 Lys Gln Ile Lys Asp Met Glu Lys Met Phe Lys Gln Tyr Asp Ala Gly              95 100 105 cgg gac ggc ttc atc gac ctg atg gag cta aaa ctc atg atg gag aaa 446 Arg Asp Gly Phe Ile Asp Leu Met Glu Leu Lys Leu Met Met Glu Lys         110 115 120 ctt ggg gcc cct cag acc cac ctg ggc ctg aaa aac atg atc aag gag 494 Leu Gly Ala Pro Gln Thr His Leu Gly Leu Lys Asn Met Ile Lys Glu     125 130 135 gtg gat gag gac ttt gac agc aag ctg agc ttc cgg gag ttc ctc ctg 542 Val Asp Glu Asp Phe Asp Ser Lys Leu Ser Phe Arg Glu Phe Leu Leu 140 145 150 155 atc ttc cgc aag gcg gcg gcc ggg gag ctt cag gag gac agc ggg ctg 590 Ile Phe Arg Lys Ala Ala Ala Gly Glu Leu Gln Glu Asp Ser Gly Leu                 160 165 170 tgc gtg ctg gcc cgc ctc tct gag atc gac gtc tcc agt gag ggt gtc 638 Cys Val Leu Ala Arg Leu Ser Glu Ile Asp Val Ser Ser Glu Gly Val             175 180 185 aag ggg gcc aag agc ttc ttt gag gcc aag gtc cag gcc atc aac gtg 686 Lys Gly Ala Lys Ser Phe Phe Glu Ala Lys Val Gln Ala Ile Asn Val         190 195 200 tcc agc cgc ttc gag gag gag atc aag gca gag cag gag gaa agg aag 734 Ser Ser Arg Phe Glu Glu Glu Ile Lys Ala Glu Gln Glu Glu Arg Lys     205 210 215 aag cag gcg gag gag atg aag cag cgg aaa gcg gcc ttc aag gag ctg 782 Lys Gln Ala Glu Glu Met Lys Gln Arg Lys Ala Ala Phe Lys Glu Leu 220 225 230 235 cag tcc acc ttt aag tag cgggggctgc agccgaccgc cctgctccgg 830 Gln Ser Thr Phe Lys                 240 ccccagtgtg gtgggcgagg gtggcgcatg ggaggccgag cctgaatcct tgcctgtgtc 890 tgacgggacc actactaaaa acctaaaaat atctgtgaat ggagcaagtt caggggtctt 950 atggaggtgg cccggcccct ccccgctccc ttccactctg cacgaggccg ccacaccggc 1010 gctggctccc tgcccggccc ggccctccct ggcaatccct gggctctctt gcacccctaa 1070 ctgccccctg cctgctccgg cactgcccca ggcccagctc ctggccctag gtccctccca 1130 gccccatgtg cctgccgcct gccctccaca catccctgtc cccccaaccc gggaacccct 1190 gccctcctcc agcaggccgc accgcccctg gggccccctg ccagcccctt cccaggctgg 1250 gagacggcag aagagataga atcagggctg cccccacaga gtgggaccca aggggctaat 1310 tggaggcacg aggggacccc tccccagggc cttttcctcc tctgcgtctt ccatctactg 1370 aaatgggaga gggggtgggg agcttctgtt ctggtgaagg gacccgggca ggcccccagc 1430 accccatgct gacttggaga accccagatc tctggggccc agccaggcag ggtgtggggg 1490 cagctgtgcc aatctacctc acaggcccac cccctgccgg gcatgccgtg ggatcatggg 1550 cagggaaggc tctgggggtc ggagacaccg ctgcttagca cccccagcca gaacaccctg 1610 agggtctcgg ggctctggag agagtggggc gggaggaaga attggcacct tcctagggaa 1670 ggagacgagc gcttcgcctt gattctccga gaagcctccg agaagtgctt taagtgtgtt 1730 tgcatgcgcc aggcggtggg cagcgggggc ctgtccagcc ctctcccgcc atccttcccc 1790 aagtgacgtc cactgccttg tcaccagcga cctgcctgtc atgcccaccc cctgaggaag 1850 catggggacc ctaacaccct ggtgccctgc accagacagg ccgtggtcag gcccaggcca 1910 ccggccgggt tctgccacag cttcccacgt gcttgctgac atgcgtgtgc ctgtgtgtgg 1970 tgtctgttgc tgtgtcgtga aactgtgacc atcactcagt ccaaacaagt gagtggccct 2030 cgaggccaca gttatgcaac tttcagtgtg tgtcataacg acgtcactgc tttttaaact 2090 cgataactct ttattttagt aaaatgccca ggagtcctgg aagctacgcg gacttgcaga 2150 ggttttattt tttggcctta gaatctgcag aaattaggag gcaccgagcc cagcgcagca 2210 gcctcggacc cggattgcgt ttgccttagc ggatatgttt atacagatga atataaaatg 2270 tttttttctt tgggcttttt gcttcttttt tccccccctt ctcaccttcc cttctccccg 2330 accccacccc ccaaaaaagc tacttcttca ttccgtggta cgattatttt ttttaactaa 2390 aggaagataa aattctatat tcttatgtga aaaa 2424 <210> 2 <211> 240 <212> PRT <213> Homo sapiens <400> 2 Met Ala Thr Asp Glu Leu Ala Thr Lys Leu Ser Arg Arg Leu Gln Met   1 5 10 15 Glu Gly Glu Gly Gly Gly Glu Thr Pro Glu Gln Pro Gly Leu Asn Gly              20 25 30 Ala Ala Ala Ala Ala Ala Gly Ala Pro Asp Glu Ala Ala Glu Ala Leu          35 40 45 Gly Ser Ala Asp Cys Glu Leu Ser Ala Lys Leu Leu Arg Arg Ala Asp      50 55 60 Leu Asn Gln Gly Ile Gly Glu Pro Gln Ser Pro Ser Arg Arg Val Phe  65 70 75 80 Asn Pro Tyr Thr Glu Phe Lys Glu Phe Ser Arg Lys Gln Ile Lys Asp                  85 90 95 Met Glu Lys Met Phe Lys Gln Tyr Asp Ala Gly Arg Asp Gly Phe Ile             100 105 110 Asp Leu Met Glu Leu Lys Leu Met Met Glu Lys Leu Gly Ala Pro Gln         115 120 125 Thr His Leu Gly Leu Lys Asn Met Ile Lys Glu Val Asp Glu Asp Phe     130 135 140 Asp Ser Lys Leu Ser Phe Arg Glu Phe Leu Leu Ile Phe Arg Lys Ala 145 150 155 160 Ala Ala Gly Glu Leu Gln Glu Asp Ser Gly Leu Cys Val Leu Ala Arg                 165 170 175 Leu Ser Glu Ile Asp Val Ser Ser Glu Gly Val Lys Gly Ala Lys Ser             180 185 190 Phe Phe Glu Ala Lys Val Gln Ala Ile Asn Val Ser Ser Arg Phe Glu         195 200 205 Glu Glu Ile Lys Ala Glu Gln Glu Glu Arg Lys Lys Gln Ala Glu Glu     210 215 220 Met Lys Gln Arg Lys Ala Ala Phe Lys Glu Leu Gln Ser Thr Phe Lys 225 230 235 240 <210> 3 <211> 2711 <212> DNA <213> Homo sapiens <220> <221> CDS (198) .. (2210) <223> PKC beta I <400> 3 agctggacga gcggcagcag ctgggcgagt gacagccccg gctccgcgcg ccgcggccgc 60 cagagccggc gcaggggaag cgcccgcggc cccgggtgca gcagcggccg ccgcctcccg 120 cgcctccccg gcccgcagcc cgcggtcccg cggccccggg gccggcacct ctcgggctcc 180 ggctccccgc gcgcaag atg gct gac ccg gct gcg ggg ccg ccg ccg agc 230                       Met Ala Asp Pro Ala Ala Gly Pro Pro Pro Ser                         1 5 10 gag ggc gag gag agc acc gtg cgc ttc gcc cgc aaa ggc gcc ctc cgg 278 Glu Gly Glu Glu Ser Thr Val Arg Phe Ala Arg Lys Gly Ala Leu Arg              15 20 25 cag aag aac gtg cat gag gtc aag aac cac aaa ttc acc gcc cgc ttc 326 Gln Lys Asn Val His Glu Val Lys Asn His Lys Phe Thr Ala Arg Phe          30 35 40 ttc aag cag ccc acc ttc tgc agc cac tgc acc gac ttc atc tgg ggc 374 Phe Lys Gln Pro Thr Phe Cys Ser His Cys Thr Asp Phe Ile Trp Gly      45 50 55 ttc ggg aag cag gga ttc cag tgc caa gtt tgc tgc ttt gtg gtg cac 422 Phe Gly Lys Gln Gly Phe Gln Cys Gln Val Cys Cys Phe Val Val His  60 65 70 75 aag cgg tgc cat gaa ttt gtc aca ttc tcc tgc cct ggc gct gac aag 470 Lys Arg Cys His Glu Phe Val Thr Phe Ser Cys Pro Gly Ala Asp Lys                  80 85 90 ggt cca gcc tcc gat gac ccc cgc agc aaa cac aag ttt aag atc cac 518 Gly Pro Ala Ser Asp Asp Pro Arg Ser Lys His Lys Phe Lys Ile His              95 100 105 acg tac tcc agc ccc acg ttt tgt gac cac tgt ggg tca ctg ctg tat 566 Thr Tyr Ser Ser Pro Thr Phe Cys Asp His Cys Gly Ser Leu Leu Tyr         110 115 120 gga ctc atc cac cag ggg atg aaa tgt gac acc tgc atg atg aat gtg 614 Gly Leu Ile His Gln Gly Met Lys Cys Asp Thr Cys Met Met Asn Val     125 130 135 cac aag cgc tgc gtg atg aat gtt ccc agc ctg tgt ggc acg gac cac 662 His Lys Arg Cys Val Met Asn Val Pro Ser Leu Cys Gly Thr Asp His 140 145 150 155 acg gag cgc cgc ggc cgc atc tac atc cag gcc cac atc gac agg gac 710 Thr Glu Arg Arg Gly Arg Ile Tyr Ile Gln Ala His Ile Asp Arg Asp                 160 165 170 gtc ctc att gtc ctc gta aga gat gct aaa aac ctt gta cct atg gac 758 Val Leu Ile Val Leu Val Arg Asp Ala Lys Asn Leu Val Pro Met Asp             175 180 185 ccc aat ggc ctg tca gat ccc tac gta aaa ctg aaa ctg att ccc gat 806 Pro Asn Gly Leu Ser Asp Pro Tyr Val Lys Leu Lys Leu Ile Pro Asp         190 195 200 ccc aaa agt gag agc aaa cag aag acc aaa acc atc aaa tgc tcc ctc 854 Pro Lys Ser Glu Ser Lys Gln Lys Thr Lys Thr Ile Lys Cys Ser Leu     205 210 215 aac cct gag tgg aat gag aca ttt aga ttt cag ctg aaa gaa tcg gac 902 Asn Pro Glu Trp Asn Glu Thr Phe Arg Phe Gln Leu Lys Glu Ser Asp 220 225 230 235 aaa gac aga aga ctg tca gta gag att tgg gat tgg gat ttg acc agc 950 Lys Asp Arg Arg Leu Ser Val Glu Ile Trp Asp Trp Asp Leu Thr Ser                 240 245 250 agg aat gac ttc atg gga tct ttg tcc ttt ggg att tct gaa ctt cag 998 Arg Asn Asp Phe Met Gly Ser Leu Ser Phe Gly Ile Ser Glu Leu Gln             255 260 265 aaa gcc agt gtt gat ggc tgg ttt aag tta ctg agc cag gag gaa ggc 1046 Lys Ala Ser Val Asp Gly Trp Phe Lys Leu Leu Ser Gln Glu Glu Gly         270 275 280 gag tac ttc aat gtg cct gtg cca cca gaa gga agt gag gcc aat gaa 1094 Glu Tyr Phe Asn Val Pro Val Pro Pro Glu Gly Ser Glu Ala Asn Glu     285 290 295 gaa ctg cgg cag aaa ttt gag agg gcc aag atc agt cag gga acc aag 1142 Glu Leu Arg Gln Lys Phe Glu Arg Ala Lys Ile Ser Gln Gly Thr Lys 300 305 310 315 gtc ccg gaa gaa aag acg acc aac act gtc tcc aaa ttt gac aac aat 1190 Val Pro Glu Glu Lys Thr Thr Asn Thr Val Ser Lys Phe Asp Asn Asn                 320 325 330 ggc aac aga gac cgg atg aaa ctg acc gat ttt aac ttc cta atg gtg 1238 Gly Asn Arg Asp Arg Met Lys Leu Thr Asp Phe Asn Phe Leu Met Val             335 340 345 ctg ggg aaa ggc agc ttt ggc aag gtc atg ctt tca gaa cga aaa ggc 1286 Leu Gly Lys Gly Ser Phe Gly Lys Val Met Leu Ser Glu Arg Lys Gly         350 355 360 aca gat gag ctc tat gct gtg aag atc ctg aag aag gac gtt gtg atc 1334 Thr Asp Glu Leu Tyr Ala Val Lys Ile Leu Lys Lys Asp Val Val Ile     365 370 375 caa gat gat gac gtg gag tgc act atg gtg gag aag cgg gtg ttg gcc 1382 Gln Asp Asp Asp Val Glu Cys Thr Met Val Glu Lys Arg Val Leu Ala 380 385 390 395 ctg cct ggg aag ccg ccc ttc ctg acc cag ctc cac tcc tgc ttc cag 1430 Leu Pro Gly Lys Pro Pro Phe Leu Thr Gln Leu His Ser Cys Phe Gln                 400 405 410 acc atg gac cgc ctg tac ttt gtg atg gag tac gtg aat ggg ggc gac 1478 Thr Met Asp Arg Leu Tyr Phe Val Met Glu Tyr Val Asn Gly Gly Asp             415 420 425 ctc atg tat cac atc cag caa gtc ggc cgg ttc aag gag ccc cat gct 1526 Leu Met Tyr His Ile Gln Gln Val Gly Arg Phe Lys Glu Pro His Ala         430 435 440 gta ttt tac gct gca gaa att gcc atc ggt ctg ttc ttc tta cag agt 1574 Val Phe Tyr Ala Ala Glu Ile Ala Ile Gly Leu Phe Phe Leu Gln Ser     445 450 455 aag ggc atc att tac cgt gac cta aaa ctt gac aac gtg atg ctc gat 1622 Lys Gly Ile Ile Tyr Arg Asp Leu Lys Leu Asp Asn Val Met Leu Asp 460 465 470 475 tct gag gga cac atc aag att gcc gat ttt ggc atg tgt aag gaa aac 1670 Ser Glu Gly His Ile Lys Ile Ala Asp Phe Gly Met Cys Lys Glu Asn                 480 485 490 atc tgg gat ggg gtg aca acc aag aca ttc tgt ggc act cca gac tac 1718 Ile Trp Asp Gly Val Thr Thr Lys Thr Phe Cys Gly Thr Pro Asp Tyr             495 500 505 atc gcc ccc gag ata att gct tat cag ccc tat ggg aag tcc gtg gat 1766 Ile Ala Pro Glu Ile Ile Ala Tyr Gln Pro Tyr Gly Lys Ser Val Asp         510 515 520 tgg tgg gca ttt gga gtc ctg ctg tat gaa atg ttg gct ggg cag gca 1814 Trp Trp Ala Phe Gly Val Leu Leu Tyr Glu Met Leu Ala Gly Gln Ala     525 530 535 ccc ttt gaa ggg gag gat gaa gat gaa ctc ttc caa tcc atc atg gaa 1862 Pro Phe Glu Gly Glu Asp Glu Asp Glu Leu Phe Gln Ser Ile Met Glu 540 545 550 555 cac aac gta gcc tat ccc aag tct atg tcc aag gaa gct gtg gcc atc 1910 His Asn Val Ala Tyr Pro Lys Ser Met Ser Lys Glu Ala Val Ala Ile                 560 565 570 tgc aaa ggg ctg atg acc aaa cac cca ggc aaa cgt ctg ggt tgt gga 1958 Cys Lys Gly Leu Met Thr Lys His Pro Gly Lys Arg Leu Gly Cys Gly             575 580 585 cct gaa ggc gaa cgt gat atc aaa gag cat gca ttt ttc cgg tat att 2006 Pro Glu Gly Glu Arg Asp Ile Lys Glu His Ala Phe Phe Arg Tyr Ile         590 595 600 gat tgg gag aaa ctt gaa cgc aaa gag atc cag ccc cct tat aag cca 2054 Asp Trp Glu Lys Leu Glu Arg Lys Glu Ile Gln Pro Pro Tyr Lys Pro     605 610 615 aaa gct aga gac aag aga gac acc tcc aac ttc gac aaa gag ttc acc 2102 Lys Ala Arg Asp Lys Arg Asp Thr Ser Asn Phe Asp Lys Glu Phe Thr 620 625 630 635 aga cag cct gtg gaa ctg acc ccc act gat aaa ctc ttc atc atg aac 2 150 Arg Gln Pro Val Glu Leu Thr Pro Thr Asp Lys Leu Phe Ile Met Asn                 640 645 650 ttg gac caa aat gaa ttt gct ggc ttc tct tat act aac cca gag ttt 2198 Leu Asp Gln Asn Glu Phe Ala Gly Phe Ser Tyr Thr Asn Pro Glu Phe             655 660 665 gtc att aat gtg taggtgaatg caaactccat cgttgagcct ggggtgtaag 2250 Val Ile Asn Val         670 acttcaagcc aagcgtatgt atcaattcta gtcttccagg attcacggtg cacatgctgg 2310 cattcaacat gtggaaagct tgtcttagag ggcttttctt tgtatgtgta gcttgctagt 2370 ttgttttcta catttgaaaa tgtttagttt agaataagcg cattatccaa ttatagaggt 2430 acaattttcc aaacttccag aaactcatca aatgaacaga caatgtcaaa actactgtgt 2490 ctgataccaa aatgcttcag tatttgtaat ttttcaagtc agaagctgat gttcctggta 2550 aaagttttta cagttattct ataatatctt ctttgaatgc taagcatgag cgatattttt 2610 aaaaattgtg agtaagcttt gcagttactg tgaactattg tctcttggag gaagtttttt 2670 gtttaagaat tgatatgatt aaactgaatt aatatatgca a 2711 <210> 4 <211> 671 <212> PRT <213> Homo sapiens <400> 4 Met Ala Asp Pro Ala Ala Gly Pro Pro Pro Glu Gly Glu Glu Ser   1 5 10 15 Thr Val Arg Phe Ala Arg Lys Gly Ala Leu Arg Gln Lys Asn Val His              20 25 30 Glu Val Lys Asn His Lys Phe Thr Ala Arg Phe Phe Lys Gln Pro Thr          35 40 45 Phe Cys Ser His Cys Thr Asp Phe Ile Trp Gly Phe Gly Lys Gln Gly      50 55 60 Phe Gln Cys Gln Val Cys Cys Phe Val Val His Lys Arg Cys His Glu  65 70 75 80 Phe Val Thr Phe Ser Cys Pro Gly Ala Asp Lys Gly Pro Ala Ser Asp                  85 90 95 Asp Pro Arg Ser Lys His Lys Phe Lys Ile His Thr Tyr Ser Ser Pro             100 105 110 Thr Phe Cys Asp His Cys Gly Ser Leu Leu Tyr Gly Leu Ile His Gln         115 120 125 Gly Met Lys Cys Asp Thr Cys Met Met Asn Val His Lys Arg Cys Val     130 135 140 Met Asn Val Pro Ser Leu Cys Gly Thr Asp His Thr Glu Arg Arg Gly 145 150 155 160 Arg Ile Tyr Ile Gln Ala His Ile Asp Arg Asp Val Leu Ile Val Leu                 165 170 175 Val Arg Asp Ala Lys Asn Leu Val Pro Met Asp Pro Asn Gly Leu Ser             180 185 190 Asp Pro Tyr Val Lys Leu Lys Leu Ile Pro Asp Pro Lys Ser Glu Ser         195 200 205 Lys Gln Lys Thr Lys Thr Ile Lys Cys Ser Leu Asn Pro Glu Trp Asn     210 215 220 Glu Thr Phe Arg Phe Gln Leu Lys Glu Ser Asp Lys Asp Arg Arg Leu 225 230 235 240 Ser Val Glu Ile Trp Asp Trp Asp Leu Thr Ser Arg Asn Asp Phe Met                 245 250 255 Gly Ser Leu Ser Phe Gly Ile Ser Glu Leu Gln Lys Ala Ser Val Asp             260 265 270 Gly Trp Phe Lys Leu Leu Ser Gln Glu Glu Gly Glu Tyr Phe Asn Val         275 280 285 Pro Val Pro Pro Glu Gly Ser Glu Ala Asn Glu Glu Leu Arg Gln Lys     290 295 300 Phe Glu Arg Ala Lys Ile Ser Gln Gly Thr Lys Val Pro Glu Glu Lys 305 310 315 320 Thr Thr Asn Thr Val Ser Lys Phe Asp Asn Asn Gly Asn Arg Asp Arg                 325 330 335 Met Lys Leu Thr Asp Phe Asn Phe Leu Met Val Leu Gly Lys Gly Ser             340 345 350 Phe Gly Lys Val Met Leu Ser Glu Arg Lys Gly Thr Asp Glu Leu Tyr         355 360 365 Ala Val Lys Ile Leu Lys Lys Asp Val Val Ile Gln Asp Asp Asp Val     370 375 380 Glu Cys Thr Met Val Glu Lys Arg Val Leu Ala Leu Pro Gly Lys Pro 385 390 395 400 Pro Phe Leu Thr Gln Leu His Ser Cys Phe Gln Thr Met Asp Arg Leu                 405 410 415 Tyr Phe Val Met Glu Tyr Val Asn Gly Gly Asp Leu Met Tyr His Ile             420 425 430 Gln Gln Val Gly Arg Phe Lys Glu Pro His Ala Val Phe Tyr Ala Ala         435 440 445 Glu Ile Ala Ile Gly Leu Phe Phe Leu Gln Ser Lys Gly Ile Ile Tyr     450 455 460 Arg Asp Leu Lys Leu Asp Asn Val Met Leu Asp Ser Glu Gly His Ile 465 470 475 480 Lys Ile Ala Asp Phe Gly Met Cys Lys Glu Asn Ile Trp Asp Gly Val                 485 490 495 Thr Thr Lys Thr Phe Cys Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile             500 505 510 Ile Ala Tyr Gln Pro Tyr Gly Lys Ser Val Asp Trp Trp Ala Phe Gly         515 520 525 Val Leu Leu Tyr Glu Met Leu Ala Gly Gln Ala Pro Phe Glu Gly Glu     530 535 540 Asp Glu Asp Glu Leu Phe Gln Ser Ile Met Glu His Asn Val Ala Tyr 545 550 555 560 Pro Lys Ser Met Ser Lys Glu Ala Val Ala Ile Cys Lys Gly Leu Met                 565 570 575 Thr Lys His Pro Gly Lys Arg Leu Gly Cys Gly Pro Glu Gly Glu Arg             580 585 590 Asp Ile Lys Glu His Ala Phe Phe Arg Tyr Ile Asp Trp Glu Lys Leu         595 600 605 Glu Arg Lys Glu Ile Gln Pro Pro Tyr Lys Pro Lys Ala Arg Asp Lys     610 615 620 Arg Asp Thr Ser Asn Phe Asp Lys Glu Phe Thr Arg Gln Pro Val Glu 625 630 635 640 Leu Thr Pro Thr Asp Lys Leu Phe Ile Met Asn Leu Asp Gln Asn Glu                 645 650 655 Phe Ala Gly Phe Ser Tyr Thr Asn Pro Glu Phe Val Ile Asn Val             660 665 670 <210> 5 <211> 3522 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (306) .. (2354) <223> PKC eta <400> 5 aggggcgagt cctgcgcgag tccccgggag gcgccgcgcg cttggaaggg acggtcgggc 60 ttccccggcc cgctgagggc tcggcggcgg gctcccctcc tttccacctc gggagggagg 120 gaaggagggg agggaaaagt cccacggagg aggcagaatg gccagtcgag gggcgcttag 180 gcgctgcctt tccccagggc tgcctcgact cctgcacctg tcccgagggc tggcctgaga 240 cgggactccc ggttctcccg ctgcgaagca gcgcggcccc ccggggccgg ggcagcggcg 300 ccggc atg tcg tct ggc acc atg aag ttc aat ggc tat ttg agg gtc 347            Met Ser Ser Gly Thr Met Lys Phe Asn Gly Tyr Leu Arg Val              1 5 10 cgc atc ggt gag gca gtg ggg ctg cag ccc acc cgc tgg tcc ctg cgc 395 Arg Ile Gly Glu Ala Val Gly Leu Gln Pro Thr Arg Trp Ser Leu Arg  15 20 25 30 cac tcg ctc ttc aag aag ggc cac cag ctg ctg gac ccc tat ctg acg 443 His Ser Leu Phe Lys Lys Gly His Gln Leu Leu Asp Pro Tyr Leu Thr                  35 40 45 gtg agc gtg gac cag gtg cgc gtg ggc cag acc agc acc aag cag aag 491 Val Ser Val Asp Gln Val Arg Val Gly Gln Thr Ser Thr Lys Gln Lys              50 55 60 acc aac aaa ccc acg tac aac gag gag ttt tgc gct aac gtc acc gac 539 Thr Asn Lys Pro Thr Tyr Asn Glu Glu Phe Cys Ala Asn Val Thr Asp          65 70 75 ggc ggc cac ctc gag ttg gcc gtc ttc cac gag acg ccc ctg ggc tac 587 Gly Gly His Leu Glu Leu Ala Val Phe His Glu Thr Pro Leu Gly Tyr      80 85 90 gac cac ttc gtg gcc aac tgc acc ctg cag ttc cag gag ctg ctg cgc 635 Asp His Phe Val Ala Asn Cys Thr Leu Gln Phe Gln Glu Leu Leu Arg  95 100 105 110 acg acc ggc gcc tcg gac acc ttc gag ggt tgg gtg gat ctc gag cca 683 Thr Thr Gly Ala Ser Asp Thr Phe Glu Gly Trp Val Asp Leu Glu Pro                 115 120 125 gag ggg aaa gta ttt gtg gta ata acc ctt acc ggg agt ttc act gaa 731 Glu Gly Lys Val Phe Val Val Ile Thr Leu Thr Gly Ser Phe Thr Glu             130 135 140 gct act ctc cag aga gac cgg atc ttc aaa cat ttt acc agg aag cgc 779 Ala Thr Leu Gln Arg Asp Arg Ile Phe Lys His Phe Thr Arg Lys Arg         145 150 155 caa agg gct atg cga agg cga gtc cac cag atg aat gga cac aag ttc 827 Gln Arg Ala Met Arg Arg Arg Val His Gln Ile Asn Gly His Lys Phe     160 165 170 atg gcc acg tat ctg agg cag ccc acc tac tgc tct cac tgc agg gag 875 Met Ala Thr Tyr Leu Arg Gln Pro Thr Tyr Cys Ser His Cys Arg Glu 175 180 185 190 ttt atc tgg gga gtg ttt ggg aaa cag ggt tat cag tgc caa gtg tgc 923 Phe Ile Trp Gly Val Phe Gly Lys Gln Gly Tyr Gln Cys Gln Val Cys                 195 200 205 acc tgt gtc gtc cat aaa cgc tgc cat cat cta att gtt aca gcc tgt 971 Thr Cys Val Val His Lys Arg Cys His His Leu Ile Val Thr Ala Cys             210 215 220 act tgc caa aac aat att aac aaa gtg gat tca aag att gca gaa cag 1019 Thr Cys Gln Asn Asn Ile Asn Lys Val Asp Ser Lys Ile Ala Glu Gln         225 230 235 agg ttc ggg atc aac atc cca cac aag ttc agc atc cac aac tac aaa 1067 Arg Phe Gly Ile Asn Ile Pro His Lys Phe Ser Ile His Asn Tyr Lys     240 245 250 gtg cca aca ttc tgc gat cac tgt ggc tca ctg ctc tgg gga ata atg 1115 Val Pro Thr Phe Cys Asp His Cys Gly Ser Leu Leu Trp Gly Ile Met 255 260 265 270 cga caa gga ctt cag tgt aaa ata tgt aaa atg aat gtg cat att cga 1163 Arg Gln Gly Leu Gln Cys Lys Ile Cys Lys Met Asn Val His Ile Arg                 275 280 285 tgt caa gcg aac gtg gcc cct aac tgt ggg gta aat gcg gtg gaa ctt 1211 Cys Gln Ala Asn Val Ala Pro Asn Cys Gly Val Asn Ala Val Glu Leu             290 295 300 gcc aag acc ctg gca ggg atg ggt ctc caa ccc gga aat att tct cca 1259 Ala Lys Thr Leu Ala Gly Met Gly Leu Gln Pro Gly Asn Ile Ser Pro         305 310 315 acc tcg aaa ctc gtt tcc aga tcg acc cta aga cga cag gga aag gag 1307 Thr Ser Lys Leu Val Ser Arg Ser Thr Leu Arg Arg Gln Gly Lys Glu     320 325 330 agc agc aaa gaa gga aat ggg att ggg gtt aat tct tcc aac cga ctt 1355 Ser Ser Lys Glu Gly Asn Gly Ile Gly Val Asn Ser Ser Asn Arg Leu 335 340 345 350 ggt atc gac aac ttt gag ttc atc cga gtg ttg ggg aag ggg agt ttt 1403 Gly Ile Asp Asn Phe Glu Phe Ile Arg Val Leu Gly Lys Gly Ser Phe                 355 360 365 ggg aag gtg atg ctt gca aga gta aaa gaa aca gga gac ctc tat gct 1451 Gly Lys Val Met Leu Ala Arg Val Lys Glu Thr Gly Asp Leu Tyr Ala             370 375 380 gtg aag gtg ctg aag aag gac gtg att ctg cag gat gat gat gtg gaa 1499 Val Lys Val Leu Lys Lys Asp Val Ile Leu Gln Asp Asp Asp Val Glu         385 390 395 tgc acc atg acc gag aaa agg atc ctg tct ctg gcc cgc aat cac ccc 1547 Cys Thr Met Thr Glu Lys Arg Ile Leu Ser Leu Ala Arg Asn His Pro     400 405 410 ttc ctc act cag ttg ttc tgc tgc ttt cag acc ccc gat cgt ctg ttt 1595 Phe Leu Thr Gln Leu Phe Cys Cys Phe Gln Thr Pro Asp Arg Leu Phe 415 420 425 430 ttt gtg atg gag ttt gtg aat ggg ggt gac ttg atg ttc cac att cag 1643 Phe Val Met Glu Phe Val Asn Gly Gly Asp Leu Met Phe His Ile Gln                 435 440 445 aag tct cgt cgt ttt gat gaa gca cga gct cgc ttc tat gct gca gaa 1691 Lys Ser Arg Arg Phe Asp Glu Ala Arg Ala Arg Phe Tyr Ala Ala Glu             450 455 460 atc att tcg gct ctc atg ttc ctc cat gat aaa gga atc atc tat aga 1739 Ile Ile Ser Ala Leu Met Phe Leu His Asp Lys Gly Ile Ile Tyr Arg         465 470 475 gat ctg aaa ctg gac aat gtc ctg ttg gac cac gag ggt cac tgt aaa 1787 Asp Leu Lys Leu Asp Asn Val Leu Leu Asp His Glu Gly His Cys Lys     480 485 490 ctg gca gac ttc gga atg tgc aag gag ggg att tgc aat ggt gtc acc 1835 Leu Ala Asp Phe Gly Met Cys Lys Glu Gly Ile Cys Asn Gly Val Thr 495 500 505 510 acg gcc aca ttc tgt ggc acg cca gac tat atc gct cca gag atc ctc 1883 Thr Ala Thr Phe Cys Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile Leu                 515 520 525 cag gaa atg ctg tac ggg cct gca gta gac tgg tgg gca atg ggc gtg 1931 Gln Glu Met Leu Tyr Gly Pro Ala Val Asp Trp Trp Ala Met Gly Val             530 535 540 ttg ctc tat gag atg ctc tgt ggt cac gcg cct ttt gag gca gag aac 1979 Leu Leu Tyr Glu Met Leu Cys Gly His Ala Pro Phe Glu Ala Glu Asn         545 550 555 gaa gat gac ctc ttt gag gcc ata ctg aat gat gag gtg gtc tac cct 2027 Glu Asp Asp Leu Phe Glu Ala Ile Leu Asn Asp Glu Val Val Tyr Pro     560 565 570 acc tgg ctc cat gaa gat gcc aca ggg atc cta aaa tct ttc atg acc 2075 Thr Trp Leu His Glu Asp Ala Thr Gly Ile Leu Lys Ser Phe Met Thr 575 580 585 590 aag aac ccc acc atg cgc ttg ggc agc ctg act cag gga ggc gag cac 2123 Lys Asn Pro Thr Met Arg Leu Gly Ser Leu Thr Gln Gly Gly Glu His                 595 600 605 gcc atc ttg aga cat cct ttt ttt aag gaa atc gac tgg gcc cag ctg 2171 Ala Ile Leu Arg His Pro Phe Phe Lys Glu Ile Asp Trp Ala Gln Leu             610 615 620 aac cat cgc caa ata gaa ccg cct ttc aga ccc aga atc aaa tcc cga 2219 Asn His Arg Gln Ile Glu Pro Pro Phe Arg Pro Arg Ile Lys Ser Arg         625 630 635 gaa gat gtc agt aat ttt gac cct gac ttc ata aag gaa gag cca gtt 2267 Glu Asp Val Ser Asn Phe Asp Pro Asp Phe Ile Lys Glu Glu Pro Val     640 645 650 tta act cca att gat gag gga cat ctt cca atg att aac cag gat gag 2315 Leu Thr Pro Ile Asp Glu Gly His Leu Pro Met Ile Asn Gln Asp Glu 655 660 665 670 ttt aga aac ttt tcc tat gtg tct cca gaa ttg caa cca tagcct 2360 Phe Arg Asn Phe Ser Tyr Val Ser Pro Glu Leu Gln Pro                 675 680 tatggggagt gagagagagg gcacgagaac ccaaagggaa tagagattct ccaggaattt 2420 cctctatggg accttcccag catcagcctt agaacaagaa ccttaccttc aaggagcaag 2480 tgaagaactc tgtgaaggat ggaactttca gatatcaact atttagagtc cagagggagc 2540 catggcacta gaaatagttg ataatgaaat gagattttat gaagtatacc gctccaccta 2600 tgagcgtctg tctctgtggg cttgggatgt taacaggagc caaaaggagg gaaagtgtga 2660 agaataaagt agatctgaga aattctgagc caatcaggct tcttaattca agagacaaac 2720 caagacgttc tgtcaactgt gctgtgctct tctttaagcc aatgaacccc aattcctggc 2780 agtctacaag aagtctctta atgctaatga agaatttaaa ggtcttttta aggaaatgaa 2840 gggctttcca aatagaatga tttactctga agaaacaaac aatggtatct ctgaaactca 2900 caacctaaag cccaatcttg aaaatatgtt gtgcaccaag acgactgctt cagcttcttc 2960 tcttatcctt actttcttta atagatattt attaaactgt ccagtgaaaa ggtgccacaa 3020 tgcccagtat tgtaaacaac aggtttgcat tcatgaagct ttcattcatt ctggagtcta 3080 ctaatttacc tgaatggtgt ttgcattctg tgaaatgcct ctccacgttg catatgtcac 3140 acttttgtct gcacataact cttttttcac aagaagggtc actgccacaa cagcacagtc 3200 agcgggtgaa ttacaggtgc ctgctgcctg cctacctggg taatctgatc ttgtctgtat 3260 cgccgtgtgc tcatcactga agaattgcag gccactcatg tcagtgacca gatttgtggc 3320 ttataaacat tagcagttta tttatgtttt aagatgcaaa gatgtgtgtt tgatattcac 3380 tttaataatt agaaatggat cttgtaaaca gggcatatat caaagatgac cttataatat 3440 gtacccgaat atacagttca agaattttgt ctgactggaa ataaatgcat tttgtagcaa 3500 aaggaaaaaa aaaaaaaaaa aa 3522 <210> 6 <211> 683 <212> PRT <213> Homo sapiens <400> 6 Met Ser Ser Gly Thr Met Lys Phe Asn Gly Tyr Leu Arg Val Arg Ile   1 5 10 15 Gly Glu Ala Val Gly Leu Gln Pro Thr Arg Trp Ser Leu Arg His Ser              20 25 30 Leu Phe Lys Lys Gly His Gln Leu Leu Asp Pro Tyr Leu Thr Val Ser          35 40 45 Val Asp Gln Val Arg Val Gly Gln Thr Ser Thr Lys Gln Lys Thr Asn      50 55 60 Lys Pro Thr Tyr Asn Glu Glu Phe Cys Ala Asn Val Thr Asp Gly Gly  65 70 75 80 His Leu Glu Leu Ala Val Phe His Glu Thr Pro Leu Gly Tyr Asp His                  85 90 95 Phe Val Ala Asn Cys Thr Leu Gln Phe Gln Glu Leu Leu Arg Thr Thr             100 105 110 Gly Ala Ser Asp Thr Phe Glu Gly Trp Val Asp Leu Glu Pro Glu Gly         115 120 125 Lys Val Phe Val Val Ile Thr Leu Thr Gly Ser Phe Thr Glu Ala Thr     130 135 140 Leu Gln Arg Asp Arg Ile Phe Lys His Phe Thr Arg Lys Arg Gln Arg 145 150 155 160 Ala Met Arg Arg Arg Val His Gln Ile Asn Gly His Lys Phe Met Ala                 165 170 175 Thr Tyr Leu Arg Gln Pro Thr Tyr Cys Ser His Cys Arg Glu Phe Ile             180 185 190 Trp Gly Val Phe Gly Lys Gln Gly Tyr Gln Cys Gln Val Cys Thr Cys         195 200 205 Val Val His Lys Arg Cys His His Leu Ile Val Thr Ala Cys Thr Cys     210 215 220 Gln Asn Asn Ile Asn Lys Val Asp Ser Lys Ile Ala Glu Gln Arg Phe 225 230 235 240 Gly Ile Asn Ile Pro His Lys Phe Ser Ile His Asn Tyr Lys Val Pro                 245 250 255 Thr Phe Cys Asp His Cys Gly Ser Leu Leu Trp Gly Ile Met Arg Gln             260 265 270 Gly Leu Gln Cys Lys Ile Cys Lys Met Asn Val His Ile Arg Cys Gln         275 280 285 Ala Asn Val Ala Pro Asn Cys Gly Val Asn Ala Val Glu Leu Ala Lys     290 295 300 Thr Leu Ala Gly Met Gly Leu Gln Pro Gly Asn Ile Ser Pro Thr Ser 305 310 315 320 Lys Leu Val Ser Arg Ser Thr Leu Arg Arg Gln Gly Lys Glu Ser Ser                 325 330 335 Lys Glu Gly Asn Gly Ile Gly Val Asn Ser Ser Asn Arg Leu Gly Ile             340 345 350 Asp Asn Phe Glu Phe Ile Arg Val Leu Gly Lys Gly Ser Phe Gly Lys         355 360 365 Val Met Leu Ala Arg Val Lys Glu Thr Gly Asp Leu Tyr Ala Val Lys     370 375 380 Val Leu Lys Lys Asp Val Ile Leu Gln Asp Asp Asp Val Glu Cys Thr 385 390 395 400 Met Thr Glu Lys Arg Ile Leu Ser Leu Ala Arg Asn His Pro Phe Leu                 405 410 415 Thr Gln Leu Phe Cys Cys Phe Gln Thr Pro Asp Arg Leu Phe Phe Val             420 425 430 Met Glu Phe Val Asn Gly Gly Asp Leu Met Phe His Ile Gln Lys Ser         435 440 445 Arg Arg Phe Asp Glu Ala Arg Ala Arg Phe Tyr Ala Ala Glu Ile Ile     450 455 460 Ser Ala Leu Met Phe Leu His Asp Lys Gly Ile Ile Tyr Arg Asp Leu 465 470 475 480 Lys Leu Asp Asn Val Leu Leu Asp His Glu Gly His Cys Lys Leu Ala                 485 490 495 Asp Phe Gly Met Cys Lys Glu Gly Ile Cys Asn Gly Val Thr Thr Ala             500 505 510 Thr Phe Cys Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile Leu Gln Glu         515 520 525 Met Leu Tyr Gly Pro Ala Val Asp Trp Trp Ala Met Gly Val Leu Leu     530 535 540 Tyr Glu Met Leu Cys Gly His Ala Pro Phe Glu Ala Glu Asn Glu Asp 545 550 555 560 Asp Leu Phe Glu Ala Ile Leu Asn Asp Glu Val Val Tyr Pro Thr Trp                 565 570 575 Leu His Glu Asp Ala Thr Gly Ile Leu Lys Ser Phe Met Thr Lys Asn             580 585 590 Pro Thr Met Arg Leu Gly Ser Leu Thr Gln Gly Gly Glu His Ala Ile         595 600 605 Leu Arg His Pro Phe Phe Lys Glu Ile Asp Trp Ala Gln Leu Asn His     610 615 620 Arg Gln Ile Glu Pro Pro Phe Arg Pro Arg Ile Lys Ser Arg Glu Asp 625 630 635 640 Val Ser Asn Phe Asp Pro Asp Phe Ile Lys Glu Glu Pro Val Leu Thr                 645 650 655 Pro Ile Asp Glu Gly His Leu Pro Met Ile Asn Gln Asp Glu Phe Arg             660 665 670 Asn Phe Ser Tyr Val Ser Pro Glu Leu Gln Pro         675 680 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> sense primer comprising restriction site of EcoRI <400> 7 aagaattcta tggccacgga cgagctggcc acc 33 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> antisense primer comprising restriction site of Bam HI <400> 8 tttggatccc tacttaaagg tggactgcag ctc 33 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hSwip-1 (p1) sense primer <400> 9 atcttccgca aggcggcggc cggggag 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hSwip-1 (p1) antisense primer <400> 10 gactgcagct ccttgaaggc cgctttc 27 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hSwip-1 (p2) sense primer <400> 11 ctcgaggcca cagttatgca a 21 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> hSwip-1 (p2) antisense primer <400> 12 atccgctaag gcaaacgca 19 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> rSwip-1 (p1) sense primer <400> 13 cgttatgcaa tgttcctcgt gtcactg 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> rSwip-1 (p1) antisense primer <400> 14 gattctcaca ggttctaaga ccgaaaa 27 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> rSwip-1 (p2) sense primer <400> 15 tacaatgcct caagagcccc cgggacc 27 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> rSwip-1 (p2) antisense primer <400> 16 ccaaaagtaa agggaattta tattcct 27 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> mSwip-1 sense primer <400> 17 gaggctcccg acgagactgc ccaggcg 27 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> mSwip-1 antisense primer <400> 18 ccggaagctg agtttgctgt cgaaatc 27 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hIL-3 sense primer <400> 19 ctttgccttt gctggacttc 20 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> hIL-3 antisense primer <400> 20 cgaggctcaa agtcgtctg 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hIL-8 sense primer <400> 21 gtgcagtttt gccaaggagt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hIL-8 antisense primer <400> 22 ctctgcaccc agttttcctt 20 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hTNF-alpha sense primer <400> 23 ggctccaggc ggtgcttgtt c 21 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hTNF-alpha antisense primer <400> 24 agacggcgat gcggctgatg 20 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hGAPDH sense primer <400> 25 cggagtcaac ggatttggtc gtat 24 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hGAPDH antisense primer <400> 26 agccttctcc atggtggtga agac 24 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rbeta-actin sense primer <400> 27 attgaacacg gcattgtcac 20 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rbeta-actin antisense primer <400> 28 gtctcaaaca tgatctgggt c 21  

Claims (8)

A method for screening a substance for preventing or treating allergic disease or atopic dermatitis comprising the following steps: (a) contacting a sample with a swiprosin-1 gene or protein; And (b) measuring the expression level or activity of Swiprocin-1, wherein the sample is determined to be down-regulated, wherein the sample is used for the treatment of allergic disease or atopic dermatitis or It is determined as a prophylactic substance. The method of claim 1, wherein the Swiprosin-1 gene or protein is contained in a cell. 3. The method of claim 2, wherein said cell is a mast cell. delete delete delete (i) an antibody or aptamer that specifically binds to a swiprosin-1 coding nucleotide sequence, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded in the nucleotide sequence; And (ii) an antibody or aptamer specifically binding to a phosphorylated form of protein kinase C βI / η (PKCβI / η). delete

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