WO1998038213A1

WO1998038213A1 - Intracellular signal transmission inhibitor

Info

Publication number: WO1998038213A1
Application number: PCT/JP1998/000836
Authority: WO
Inventors: Hideaki Yoshida; Toshiaki Ohtsuka
Original assignee: Institute Of Cytosignal Research, Inc.
Priority date: 1997-02-28
Filing date: 1998-02-27
Publication date: 1998-09-03
Also published as: AU6118498A

Abstract

A protein having the activity of inhibiting the activation IL-8 promoter in response to specific extracellular stimulation; a DNA encoding this protein; a vector containing this DNA; a transformant carrying this vector; an antibody against the protein; and medicinal compositions containing the protein as the active ingredient. Because of the activity of inhibiting the activation of IL-8 promoter, the above protein is expected to be applicable to drugs for treating diseases connected with the expression of IL-8, for example, anti-inflammatory agents, anti-bronchial asthma agents, antiallergic agents and antirheumatic agents.

Description

Specification

-. Inhibitors of intracellular signaling

The present invention relates to a protein having an activity of suppressing the activation of IL-8 promoter in response to a specific extracellular stimulus, a DNA encoding the protein, a vector containing the DNA, a host holding the vector, The present invention relates to an antibody against the protein, and a pharmaceutical composition containing the protein as an active ingredient. Background art

Interleukin-8 (hereinafter referred to as “IL-8”) is a type of site-active protein that is a proteinaceous chemical that is responsible for signal transmission between cells. IL-8 is known as a site force-in with effects such as neutrophil migration, lymphocyte migration, neutrophil activation, and angiogenesis (Matsushima, K. et. Al. J. Exp. Med.167, 1883-93 (1988), Larsen, G.et.al.Science 243,1464-6 (1989), Daniels, RHet.al.Immunol.75, 157-63 (1992), Koch, AE et. al. Science 258, 1798-804 (1992), Harada, A. et al. Mol. Med. Today 2, 482-489 (1996)).

On the other hand, for IL-8, in addition to these normal functions in living organisms, inflammation (Watanabe, K. et al Infection & Immunity 60, 1268 (1992)) and reperfusion tissue damage in myocardial infarction (reperfusion injury) (Sekido, N. et al. Nature 365, 654-657 (1993)).

In particular, there have been many reports on the relationship between inflammation and IL-8, and various inflammatory diseases such as rheumatoid arthritis, gouty arthritis, psoriasis, contact dermatitis, sepsis, and spontaneous pulmonary fibrosis , Adult respiratory distress syndrome, inflammatory bowel disease, immune vasculitis, glomerulonephritis, urinary tract infection, myocardial infarction, respiratory tract infection, bronchial asthma, perinatal infection, transplant organ rejection, etc. Has been investigated (Matsushima, K. Chem Immunol 51, 236-265 (1992), Matsushima Tsunaharu Immunopharmacology 12, 15-21, 1994, Harada, A. et al. Mo 1. Med. Today 2, 482-489 (1996)).

Since the relationship between IL ^: 8 and various diseases has been elucidated in this way, pharmaceuticals targeting IL-8 are also being developed. For example, a drug discovery example based on the anti-inflammatory activity of anti-IL-8 antibody (Sekid 0, N. et. Al. Nature 365, 654-657 (1993)) is described (Nikkei Bio Yearbook 97, 321, 1996). ).

However, such drug development has problems such as loss of efficacy due to the production of neutralizing antibodies against antibodies and the production of allergy-induced antibodies. Disclosure of the invention

Therefore, an object of the present invention is to provide a factor having an anti-inflammatory effect that does not have such a problem, particularly a factor having an activity of suppressing activation of an IL-8 promoter.

As a finding related to IL-8, expression of IL-8 is observed in many inflammations (Watanabe, K., et al. Infection & Immunity 60, 1268 (1992), Matsushima, K. et al. Chem Immunol 51, 236-265 (1992)) and that inflammation is suppressed when IL-8 function is inhibited by IL-8 antibody (Harada, A. et al. Int. Immunol. 5 681-). 690 (1993) and Sekido, N. et al. Nature 365 654-657 (1993)). Therefore, it is considered that the expression of IL-8 is closely related to inflammation.

By the way, it has been reported that dexamethasone, which is known as an anti-inflammatory steroid, has an inhibitory effect on the activation of the IL-8 promoter overnight (Mukaida N. et al. J. Immunol. 146 1212- 1215 (1991), Mukaida, NJ Biol. Chem. 269 13289 (1994)).

Therefore, if a gene that is expressed in response to stimulation by dexamethasone can be isolated, the gene becomes a promising candidate for a gene that encodes a protein that has an inhibitory effect on the activation of IL-8 promoter, and as a result, anti-inflammatory Protein with action になる It is considered to be a strong candidate for a gene encoding quality.

Thus, the present inventors attempted to isolate a gene whose expression is induced by dexamethasone treatment. As a result, screening by the subtraction method and the differential hybridization method using a cDNA library extracted from cells treated with dexamethasone and a cDNA library extracted from cells not treated with dexamethasone, We succeeded in isolating a gene whose expression was induced in the treated cells.

Furthermore, the present inventors examined whether the isolated gene suppressed the activation of the IL-8 promoter. As a result, it was found that the isolated gene actually suppressed the activation of the IL-8 promoter by IL-1 stimulation.

The present inventors determined the nucleotide sequence of the isolated gene and performed a homology search on a DNA database basis, and found that the gene was a novel gene that had not been isolated before. Was found to be a gene.

That is, the present invention relates to a factor that suppresses activation of the IL-8 promoter, and more specifically,

(1) The protein according to SEQ ID NO: 1, or an amino acid sequence in the protein, which has an amino acid sequence in which one or several amino acids are substituted, deleted, or added, and responds to a specific extracellular stimulus A protein having an activity of suppressing the activation of the IL-8 promoter,

(2) a protein encoded by a DNA that hybridizes to the DNA of SEQ ID NO: 2, which has an activity of suppressing activation of an IL-8 promoter in response to a specific extracellular stimulus;

(3) The protein of SEQ ID NO: 3, or an amino acid sequence in the protein having one or several amino acids substituted, deleted, or added, and responded to a specific extracellular stimulus A protein having an activity of suppressing the activation of the IL-8 promoter, (4) a protein encoded by a DNA that hybridizes to the DNA of SEQ ID NO: 4, which has an activity of suppressing activation of an IL-8 promoter in response to a specific extracellular stimulus ,

(5) the protein according to any one of (1) to (4), wherein the specific extracellular stimulus is stimulation by an interleukin;

(6) a DNA encoding the protein of (1) or (3),

(7) A protein that hybridizes with the DNA of SEQ ID NO: 2 and / or the DNA of SEQ ID NO: 4 and has an activity of suppressing activation of the IL-8 promoter in response to a specific extracellular stimulus DNA to code,

(8) the DNA according to (7), wherein the specific extracellular stimulus is stimulation by interleukin;

(9) A vector containing the DNA according to any one of (6) to (8),

(10) a host carrying the vector according to (9),

(11) an antibody that reacts with the protein according to any one of (1) to (5),

(12) a pharmaceutical composition comprising the protein according to any one of (1) to (5) as an active ingredient;

(13) The pharmaceutical composition according to (12), which is an anti-inflammatory agent.

About.

The present invention relates to a protein having an activity of suppressing activation of an IL-8 promoter in response to a specific extracellular stimulus.

In various diseases including inflammation, IL-1 (Clin. Immunol. 2718-28 (1995)), IL-8 (Clin. Immunol. 2780-85 (1995)), TNF (Molecular Medicine 33 1010-1020 ( 1996)) and that IL-8 production is induced by stimulation of IL-1 and TNF (Mukaida, N. et al. Icrobiol. Immunol., 36773 (1977). 992)). It has also been suggested that the expression of this IL-8 gene is induced through an intracellular signal transduction process based on the binding of IL-1 and TNF to cell membrane receptors. on the other hand, The protein of the present invention has a property that its expression is induced by treatment with dexamethasone, an anti-inflammatory agent, and a property that it inhibits the activity of IL-8 promoter by inhibiting the above-mentioned intracellular signal transmission process. Have. From these facts, it is considered that the protein of the present invention has an anti-inflammatory effect.

The protein of the present invention may be of natural origin or a recombinant protein produced using genetic engineering techniques. A natural protein can be prepared, for example, using an affinity column to which the antibody of the present invention described later is bound. Further, the recombinant protein can be prepared, for example, using the transformant of the present invention described below.

The protein of the present invention also includes a variant in which an amino acid has been substituted, deleted, or added to a native form (for example, the protein described in SEQ ID NO: 1 or SEQ ID NO: 3). . Such a variant may occur naturally, but those skilled in the art, for example, synthesize a double-stranded DNA using the single-stranded DNA of the target gene as type III and the mutant oligonucleotide as a primer, Various methods based on the principle of eliminating DNA derived from normal type II genes and selecting plasmids derived from mutant DNA strands using genetic techniques (Lesley, SA and Bohnsack, RN, Promega Notes Magazine, 46, 6-10 (1994), Kunkel, T. et al. Methods Enzymol., 154, 367-382 (1987), Sayers, J. et al. Bioteches, 13, 592-596 (1992)). Is possible. In addition, a method for obtaining amplified DNA derived from a mutated primer by a combination of PCR (Cormack, B., Current Protocols in Molecular Biology (Ausubel, FM et al eds.) 8.5.1-8.5.9. (1987), It can be prepared using well-known techniques such as Erlich, H Press, (ed.) PCR Technology, Stocken Press, New York (1989)).

As described above, the amino acid sequence of the amino acid sequence of the protein described in SEQ ID NO: 1 (or SEQ ID NO: 3) has an amino acid sequence in which one or several amino acids have been substituted, deleted, or added. Of IL-8 promoter in response to extracellular stimuli in mice A protein having an activity of suppressing the formation of an enzyme is included in the scope of the present invention. _

For those skilled in the art, well-known techniques of hybridization (Sambrook, J., Fritsch, EF and Maniatis, T. (ed.), Molecular Cloning 2nd ed. Cold Spring Habor Laboratory Press, New York ( 1989)), and based on the DNA sequence (or a part thereof) of SEQ ID NO: 2 (or SEQ ID NO: 4), a DNA highly homologous thereto is isolated, and the DNA of the present invention is isolated from the DNA. Obtaining a functional equivalent of this protein is also routine. Thus, a protein encoded by a DNA that hybridizes with the DNA consisting of the DNA sequence of SEQ ID NO: 2 (or SEQ ID NO: 4), and the activity of the IL-8 promoter in response to a specific extracellular stimulus Proteins having the activity of inhibiting the formation of proteins are also included in the scope of the present invention. The protein obtained by the hybridization technique preferably has a homology of 60% or more, more preferably 80% or more, with the protein of the present invention described in SEQ ID NO: 1 or 3 in the amino acid sequence. More preferably, the homology is more preferably 90% or more. The activity of suppressing the activation of the IL-8 promoter in response to a specific extracellular stimulus can be measured, for example, by detecting a reporter gene as a marker. That is, a vector having a repo overnight gene downstream of the IL-8 promoter region is introduced into a host cell, a specific extracellular stimulus is applied to the cell, and the activity of the reporter gene product is detected. can do. Examples of reporter genes include, but are not limited to, white luciferase (de Wet, JR et al. Mol. Cell. Biol. 7, 725-737 (1987)), and mushroom luciferase (Sherf, BA et al. Prome ga Note 57, 2-9 (1996)), CAT (chloramphenico-l-acetyltransferase) (Gorman, CM et al. Mol. Cell. Biol. 2, 1044-1051 (1982)), β-galactosidase (Jain, V. et al. Anal.Biochem. 199, 119-124 (1991)), glucuronidase (Gallagher, SR GUS Protocol: Using the GUS Gene as a Reporter of Gene Expression, Acasemic Press 47-59 (1992) ), Azore force phosphatase (Cullen, B. et al. Methods in Enzymology 216, 362-368 (19 92)) can be used. In the present invention, “specific extracellular stimulation” refers to stimulation of cells, particularly cell membrane receptors of cells, by cytokines. Examples of site power-in include — for example, IL-1, TNF, etc., but are not particularly limited as long as they cause activation of IL-8.

The present invention also relates to a DNA encoding the protein of the present invention. The form of the MA encoding the protein of the present invention is not particularly limited. For example, in addition to cDNA genomic DNA which can be prepared from mRNA isolated from cells capable of producing the protein of the present invention, chemically synthesized DNA is also included in the DNA of the present invention.

The DNA of the present invention can be prepared, for example, by the following method. If it is a cDNA, first, a cDNA library is prepared to obtain the cDNA. A cDNA library is obtained by ligating a cDNA synthesized based on mRNA purified from cells producing the protein of the present invention to an appropriate vector having a microbial replicon, and then introducing it into a suitable host. It is made. As a method for synthesizing cDNA from the purified mRNA, a method using an oligo dT primer or a random primer, a method using a synthetic primer having a specific base sequence, and the like are usually used. As a vector used for library production, for example, a plasmid vector, a phage vector, a cosmid vector, a phagemid vector, a YAC vector, and the like are mainly used. As the host, for example, Escherichia coli, yeast, Bacillus subtilis and the like are mainly used. From a cDNA library one prepared, as a method for selecting clones having cDNA of the present invention, for example, using those labeled with the Origonukureo tides comprising the nucleotide sequence of the DNA of the present invention ^{3 2} P, an enzyme such as a probe In addition to the screening method, there is a method using a DNA fragment obtained by PCR (Polymerase Chain Reaction) using a primer comprising a sequence specific to the DNA of the present invention as a probe. In addition, by using RT-PCR (Kawasaki, ES and Wang, AM, PCR Technology (Erlich, HA ed.) Stockton Press, 89-97 (1989), etc.), a process for screening a cDNA library to be introduced into a host can be performed. The present invention directly without going through CDNA can also be prepared. When an antibody that reacts with the protein of the present invention can be used, cDNA can be prepared by expression screening using Escherichia coli or the like as a host. '

In the case of genomic DNA, the cells are lysed using the appropriate cells of the target organism as a material, and the proteins bound to genomic MA are removed using a protein denaturant and protease. Can be prepared by a method for purifying the same (Muramatsu Edition, Labomanual Genetic Engineering, Supplementary Edition, Maruzen, 61-62, (1990)).

In the case of chemically synthesized DNA, it is produced by nucleic acid chemical synthesis according to a conventional method such as the phosphite triester method (Hunka piller, M. et al. Nature, 3, 10. 105-111 (1984)). can do. The codon for the desired amino acid is known per se and may be selected arbitrarily. For example, it can be determined according to a conventional method in consideration of the codon usage of the host to be used (Grantham, R. et al., Nucleic Acids Res., 9, p43-p74 (1981)).

The nucleotide sequence of the DNA of the present invention thus cloned can be analyzed by a dideoxy method using a radiolabel or a fluorescent label, the Maxam-Gilbert method, or the like.

The present invention also relates to a vector into which the DNA of the present invention has been inserted. The vector of the present invention is not particularly limited as long as it can be propagated and replicated in a host cell (for example, Escherichia coli, yeast, animal cells, etc.) and has an appropriate selection marker gene. As for E. coli, for example, r _p Bluescipt II "(STRATAGENE Co., Ltd.)," p CR ^T "II" (Invitrogen), and the like. For yeast, for example, “pSR403j (Sikorski RS and Hieter, P., Genetics, 122, 19-27 (1989))”, etc. For animal cells, for example, “pCDM8” ( Seed, B., Nature, 329, 840-842 (1987)), "pSV2-neo" (Southern and Berg, J. Mol. App 1. Genet., 1, 327-341, (1982)), etc. No. When a vector is used for the purpose of producing the protein of the present invention, an expression vector is particularly useful. There are no particular restrictions on the expression vector as long as it is generally one to which a promoter 'and a control sequence compatible with the host cell are added.

For example, for Escherichia coli, “pKC30” (Shimatake, H, and Rosenberg, M., Nature, 292, 128-132, (1981)), “pTrc 99A” (Amann, E. et al., Gene, 69, 30 1-315, (1988)), pCAGGS (Niwa et al., Gene, 108, 193-200, (1991)) or pcDL-SR 293 (Takebe et al. Mo 1. Cell. Biol., 8, 466-472, (1988)), as a viral vector, pAdexlw, a transfer vector for adenovirus production (Kanegae et al., Experimental Medicine, 12, 316). -322, (1994)). Examples of the insect cells include a transfer vector for recombinant virus production, pAc373 (Luckow et al., Bio / Technology, 6, 47-55, (1988)).

Insertion of the DNA of the present invention into a vector can be performed by a conventional method (Molecular Cloning. A Laboratory anual (Maniatis, T., et al (eds), Cold Spring Habor Laboratory Press, New York)) or a laboratory manual genetic engineering (Muramatsu edition). , Maruzen))).

The present invention also relates to a host cell into which the vector of the present invention has been introduced. The host cells into which the vector of the present invention is introduced include prokaryotes (eg, Escherichia coli) and eukaryotes

Cells (eg, yeast, mammals, insects) can be used, and there is no particular limitation. For example, suitable Escherichia coli strains include "X-Blue", "SURE", "DH5" (all available from RIKEN Genebank). For the purpose of producing the protein of the present invention, prokaryotic (eg, Escherichia coli), eukaryotic (eg, yeast, mammal, insect) cells and the like can be used as host cells. Examples of animal cells include “C0S-1 cell” (RIKEN Cell Development Bank, RCB0143), human embryonic kidney line (293) (Dainippon Pharmaceutical Co., Ltd.), Baby-Hamus Yuichi kidney cell (BHK, ATCC) CCL10), Chinese hamster ovary cells (CH0-Kl, RIKEN Cell Development Bank, RCB0285), Monkey kidney cells (CV 1, ATCC CCL70). Examples of yeast include baker's yeast (Saccharomyces cerevisae) and ethanol-assimilating yeast (Pichia pastoris). Examples of insect cells include silkworm cultured cells.

The introduction of the vector into the host cell can be carried out using a calcium phosphate method or an electroporation method, which is common to those skilled in the art.

In order to produce the protein of the present invention as a recombinant protein, a host cell into which the above-described vector has been introduced, that is, a transformant is cultured, and the recombinant protein expressed in the transformant is recovered. It is possible to manufacture. The transformant can be cultured according to a conventional method, and the protein of the present invention is produced intracellularly or extracellularly by the culture. The medium used for the culture can be appropriately selected from those commonly used depending on the host cell used. For example, in the case of the above-mentioned COS cells, the “RPMI-1640” medium or the Dulbecco's modified Eagle's minimum essential medium ( A medium such as DMEM) to which serum components such as fetal bovine serum (FBS) are added as necessary can be used. The recombinant protein expressed in the cultured transformant can be separated and purified by various known separation procedures utilizing the physical and chemical properties of the protein. Separation and purification methods include, for example, treatment with ordinary protein precipitants, ultrafiltration, molecular sieve chromatography (gel filtration), various types of liquid chromatography such as adsorption chromatography, high-performance chromatography (HPLC), and dialysis. And combinations thereof.

The present invention also relates to an antibody that reacts with the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a monoclonal antibody as well as a polyclonal antibody. Also included are humanized antibodies and all antibody classes.

The antibody of the present invention can be prepared by a conventional method known to those skilled in the art (New Cell Engineering Experimental Protocol, edited by Cancer Inhibition Research Division, Institute of Medical Science, The University of Tokyo, 202-217 (1993), etc.). It is. Examples of the antigen include the whole protein of the present invention (for example, the protein of SEQ ID NO: 1 or SEQ ID NO: 3) produced using recombinant DNA technology. Or an appropriate partial peptide thereof (for example, the peptides described in SEQ ID NO: 25 to SEQ ID NO: 30) can be used. This is administered together with adjuvants to mammals, birds, etc., which are generally used for the production of antibodies, such as egrets, mice, rats, goats, higgies, birds, hams, gulls, and guinea pigs. Antisera can be obtained from these animals with increased antibody titers, or cells can be collected to obtain polyclonal antibodies. In addition, for a monoclonal antibody, an animal having an increased antibody titer against the antigen is used as a source of antibody-producing cells. For example, spleen cells prepared from immunized mouse spleen and mouse-derived myeloma By screening a hybridoma obtained by fusing cells, a monoclonal antibody specific to the protein of the present invention can be obtained. Furthermore, it is also possible to clone an antibody gene or a part thereof from a cell expressing the antibody of the present invention, and to express this by genetic engineering to obtain an antibody or a part thereof. It is also possible to obtain humanized antibodies. The antibody of the present invention can be used as an excellent means for detecting, quantifying, and further purifying the protein of the present invention.

As a detection method using the antibody of the present invention, a conventional method, for example, Western blot method (Towbin, H. et al., Proc. Natl. Acad. Sci. USA, 76, 4350-4354 (1979)) and the like, The quantitative method includes the Radioimnoassay (MA) method (Kitakawa et al., Radioimnoassay (New Chemistry Laboratory 12), 79-88, Tokyo Kagaku Doujin (1992)) (EIA) method (Ishikawa et al., Enzymimnoadisei (New Chemistry Laboratory Course 12), 88-99, Tokyo Kagaku Dojin (1992)) and the like.

Use as a purification method is, for example, an affinity chromatography method in which the antibody of the present invention is bound to an insoluble support (Nishimichi et al., Affinity Chromatography Using Immobilized Antibodies (New Biochemistry). Chemistry Experiment Course 1), 403-406, Tokyo Kagaku Dojin (1990)).

The present invention also relates to a pharmaceutical composition comprising the protein of the present invention as an active ingredient. As described above, the protein of the present invention has an activity of suppressing activation of the IL-8 promoter. Further, the protein of the present invention also has a property induced by dexamethasone which is an anti-inflammatory agent. Therefore, the protein of the present invention is useful as an agent for treating various diseases associated with the expression of IL-8, particularly as an anti-inflammatory agent. Diseases to which the protein of the present invention can be applied include, for example, rheumatoid arthritis, gouty arthritis, psoriasis, contact dermatitis, sepsis, idiopathic pulmonary fibrosis, adult respiratory distress syndrome, inflammatory bowel disease , Immune vasculitis, glomerulonephritis, urinary tract infection, myocardial infarction, respiratory tract infection, bronchial asthma, perinatal infection, transplant organ rejection and the like. When the protein of the present invention is used for the treatment of the above-mentioned diseases, the protein can be directly administered, or can be used after being formulated by a known pharmaceutical production method. For example, they can be used in appropriate combination with a pharmacologically acceptable carrier or medium. Those skilled in the art can appropriately select the administration method and dosage of the pharmaceutical composition containing the protein of the present invention as an active ingredient according to the patient's condition, age, body weight, and the like. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is an electrophoresis image of Northern blot hybridization detecting expression of rat "GISP" mRNA by treatment with the anti-inflammatory drug dexamethasone (Dex). FIG. 2 is an electrophoresis image of rat “GISP” mRNA expression in rat and human tissues detected by Northern blot hybridization.

FIG. 3 shows a comparison of amino acid sequences between human and rat “GISP”. The upper row is the amino acid sequence of rat “GISP”, and the lower row is the amino acid sequence of human “GISP”.

Figure 4 shows the effect of the expression of the “GISP” gene on the light CINC Promoter activity (A) and the elongation factor 1-1 activity (B) by the reporter gene method. It is a figure showing a detection result. Figure 5 shows the effect of the "GISP" gene on the human IL-8 promoter activity (A) and the elongation factor 1-1-hyperpromotor activity (B) detected by the repo-Ichiichii gene method. It is a figure showing a result. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Acquisition of rat "GISP" (Glucocorticoid-Inducible Suppressor Protein) gene

Genetic manipulation techniques that are not specifically described include “Molecular Cloning. A Laboratory Ma Thigh 1” (Maniatis, T., et al (eds), Cold Spring Habor Laboratory Press, New York) and laboratory manual genetic engineering (Muramatsu And Maruzen Co., Ltd.). General reagents used in genetic engineering experiments, such as restriction enzymes, were purchased and used from Boehringer Mannheim, Takara Shuzo.

(1) Preparation of mRNA (poly (A) ⁺ RNA) from NRK52E cells

Anti-inflammatory steroid-treated or untreated NRK52E cells (cultured cells derived from rat kidney) (Dainippon Pharmaceutical) and poly (A) ⁺ RNA were prepared. Steroid treatment cells are lipopolysaccharide Sa steward Lai de (LPS) 10 zg / ml RPMI1640 containing medium (LIFE TECHNO LOGIES, Inc., Inc.) was cultured overnight NRK52E cells, about 2.5Xl0 ⁸ cells anti-inflammatory (hereinafter abbreviated as Dex) scan Teroi de of which is one type dexamethasone (final concentration 1x10 one ⁶ M, dissolved in ethanol) were further cultured for 5 hours with the addition of the. On the other hand, cells not treated with steroids were similarly cultured overnight in RPMI1640 medium containing LPS, and then cultured for 5 hours by adding ethanol as a solvent. Thereafter, each NRK52E cell was recovered by centrifugation. From each cell, an ISOGEN reagent (Nitsubon Gene Co., Ltd.) based on the "Acid Guanidium Thiocyanate Phenol Chloroform (A GPC) method" (Chomczynski, P., and Sacchi, N., (1987) Anal. Biochem., 162, 156-159) Made by steroids About 5.0 mg of RNA was extracted from physiological cells, and about 6.0 mg of RNA was extracted from untreated cells. Of these, using Oligotex-dT30 (manufactured by Takara Shuzo Co., Ltd.) using l.Omg of MA as a material, approximately 12 μg of mRNA derived from steroid-treated cells (hereinafter abbreviated as “Dex (+) mRNA”), About 10 μg of mRNA derived from steroid-untreated cells (hereinafter abbreviated as “Dex (−) mRNA”) was purified as poly (A) ⁺ RNA.

(2) Preparation of cDNA library

The integration of the cDNA into the phage vector was performed using a ZAP-cDNA synthesis kit (ZAP-cDNA SYNTHESIS KIT) (manufactured by STRATAGENE, Toyobo). The process is described below. The operation was performed according to the instruction manual of the ZAP-cDNA synthesis kit. That is, a first-strand cDNA synthesis reaction using reverse transcriptase (M-MuLV Reverse Transcriptase) was performed using 5 μg each of Dex (+) mRNA and Dex (−) mMA as templates. Subsequently, a second strand synthesis reaction using E. coli RNase H and E. coli DNA polymerase was performed. Further, after an end blunting reaction with T4 DNA polymerase, an EcoRI adapter was added. The end of the adapter was phosphorylated with T4 Polynucleotide Kinase, cut with the restriction enzyme XhoI, and low-molecular-weight DNA was removed with a Sephacryl S-400 column. ) About 0.6 μg of double-stranded cDNA was obtained for each of the mRNA and De x (−) mRNA. The lOOng of cDNA was ligated to the arm of ZAPI I (Eco RI / Xho I digested, CIAP treated) using T4 DNA ligase. Next, "; DNA in vitro 'Packaging. Kit Gigapak I I Gold" (in vitro Packaginng kit Gigapak I I Gold)

(STRATAGENE, Toyobo) to perform in vitro packaging to prepare a cDNA library. Recombinant phages of about lxlO ⁶ were obtained from both prepared packaging extracts 500-1. Each of the packaging extracts (containing about 1 million recombinant phages) thus obtained was infected with host E. coli PLK-F, (STRATAGENE, Toyobo), and about 50,000 pieces of 20-cm 15 cm plates After plating on the plate and forming plaques on the plate, add SM buffer (NaCl 5.8 g, M _{_{gS0 4 - 7H 2 02.0g, lM}} Tris -HCl (pH7.5) 50ml, and allowed to stand overnight by adding ¾ gelatin (gelatin) _50ml / l physician 1 0 ml. A phage solution of two plates (containing about 107 recombinant phages) was stored as two sub-libraries, and a mixture of equal amounts of 10 sub-libraries was stored as an entire library. . The entire library was designated as "NRK-52E cell Dex (+) phage library 1" and "NRK-52E cell Dex (-) phage library".

(3) Preparation of deduction library

Genes derived from steroid-treated cells The genes that are also present in steroid-untreated cells were removed from the cDNA library (NRK-52E cell Dex (+) phage library) to enrich steroid-induced genes. The creation of the "deduction ripplery" was performed with reference to the method of A. Swaroop et al. (Nucleic Acid Res., 19, 8, 1954, (1991)).

(3-1) Preparation of single-stranded circular MA (Single-Stranded circular DNA) from NRK-52E cell Dex (+) phage library

A) Conversion from a phage vector to a plasmid (pBluescript SK-) library

Escherichia coli cultured to "0D _6O . = 5.0" in 5 ml of XLlBlue MRF'j (STRATAGENE, Toyobo) ("Helper phage ExAssistj (l.Ox10 ¹⁰ pfu / ml) (STRATAGENE, Toyobo) lml, and NRK-52E cells Dex (+) phage library (7.0xl0 ⁹ pfu / ml) 700〃 1 were mixed and incubated for 15 minutes at 37 ° C. then tetracycline (

Was added to 251111, and incubated at 37 ° C (2.5 hours). Heat treatment was performed at 70 ° C for 15 minutes, and the supernatant was centrifuged at 4,000 g for 15 minutes. were collected. then, the collected supernatant 100 ^ 1 E. coli strain (XL0LR) (ST MTAGENE Co., Toyobo) (OD ₆ .. = 1.0) was added to 20 ml, 37 ° C for 15 min Incubate base one After addition, the mixture was added to 30 ml of LB medium containing tetracycline (12.5 mg / ml) and cultured at 37 ° C for 45 minutes, followed by tetracycline (12.5 mg / 1111) and ampicillin (50/1111). ) Was added to 1 L of LB medium and cultured at 37 ° C overnight.

B) Preparation of single-stranded DM (Single-Stranded maraud) from plasmid DNA (pBluescript SK-) ''

After adding 10 ml of the above culture solution to 100 ml of 2 X YT medium and culturing at 37 ° C for 2 hours, add “M08Helper phage j (STRATAGENE, Toyobo) (7.5 × 10 ¹ ° pfu / ml) 140〃1 The cells were cultured for 8 hours at 37 ° C to produce single-stranded phage, and the supernatant was recovered by centrifugation at 12,000 g for 10 minutes (4 ° C). One-quarter volume of a solution containing 3.5 M ammonium acetate and 20% PEG was added and allowed to stand at room temperature for 15 minutes to precipitate single-stranded phage particles. The single-stranded phage particles were collected as a precipitate by the centrifugation procedure described in step 1. The resulting precipitate was suspended in 10 ml of TE buffer, then extracted with phenol (TE saturated), and phenol: chloroform (1: 1). Extraction and chloroform extraction were performed once each, and 1/10 volume of 3M sodium acetate (pH 5.2) and 2 volumes of ethanol were added. After keeping the temperature at-20 ° C, centrifugation at 12,000 g was performed for 10 minutes at 4 ° C to recover the DNA.The recovered DNA was rinsed with 70% ethanol, lightly dried, and then TE buffer 300 Dissolved in # 1 About 300 mg of DM was finally obtained.

C) Removal of double-stranded DNA

Since the double-stranded DNA remaining in the above DNA becomes the background in the subsequent subtraction step, the enzyme is digested with restriction enzymes and the phenol is extracted in the presence of magnesium ions (Nucleic Acid Res. 18.4833-4842 (1990). )). Specifically, 100 μg of the above marauder was digested with restriction enzymes Bgll I (10u 1) 10/1 and PvuI I (10u / Δl) 10 // 1. To the reaction mixture (300/1), add 10% SDS 3 il (final concentration 0.1%) and 0.5 M EDTA 7.5〃1 (final concentration 12.5 mM), mix, and then mix phenol (Tris-HC1 buffer (PH7.5) (Saturated). After centrifugation, the upper layer (aqueous layer) was recovered. The buffer layer was re-extracted by adding 300-1 Buffer-1 to the lower layer (phenol layer), and the upper layer solution after centrifugation was combined with the previous upper layer (aqueous layer) to proceed to the next step. After adding 1/10 volume of 1M MgCl ₂ to them, add an equal volume of phenol. And stirred vigorously. Centrifugation was performed at 16,000 g for 10 minutes to remove the aqueous layer containing the double-stranded DNA. The remaining phenol layer contains single-stranded DNA.Add a lOOmM MgCl ₂ solution in 300/1 TE buffer, mix vigorously, and centrifuge at 16,000 g for 10 minutes. The step of removing the aqueous layer was performed twice to remove double-stranded DNA. Next, after adding 100 mM 50 mM EDTA and stirring, the single-stranded DNA was transferred to the aqueous layer by centrifugation at 16,000 g for 10 minutes, and the aqueous layer was recovered. To the remaining ethanol layer, 100 zl of 50 mM EDTA was added again, and the aqueous layer was recovered by the same operation and combined with the previous aqueous layer. 1/10 volume of sodium acetate was added, and 2.5 times volume of ethanol was further added, and DNA was recovered by an ethanol precipitation method. Finally, about 30 μg of single-stranded DNA was obtained.

(3-2) Preparation of biotinylated copy A

A) Preparation of Dex (-) phage DNA from Dex (-) phage library

To a solution containing NRK-52E cell Dex (-) phage library, add an equal volume of a solution (20¾ PEG 2M NaCl dissolved in SM buffer), incubate on ice for 1 hour, and run at 3,000 rpm The phage particles were collected by centrifugation for 20 minutes. The precipitate of the phage particles is suspended in 7.5 ml of LB medium, an equal amount of “DE (DIETHYLAMINOETHL CELUL0SE) 52” (manufactured by Whatman) (suspended in LB medium) is added, mixed, and then mixed at 15,000 rpm for 5 minutes. Unnecessary substances other than the phage particles were adsorbed and removed on DE52 by performing the centrifugation operation of and collecting the centrifuged supernatant. After adding proteinase K (final concentration 200 zg / ml) and 10% SDS (final concentration 0.1%) to the collected supernatant and incubating at room temperature for 5 minutes, 3M potassium acetate (final concentration 0.5M) was added. The mixture was heated at 88 ° C for 20 minutes. After standing on ice for 10 minutes, the mixture was centrifuged at 15,000 rpm for 10 minutes, an equal volume of isopropanol was added to the supernatant, and DNA was precipitated at -20 ° C and collected by centrifugation. The obtained phage DNA was dissolved in a TE (pH 8.0) buffer.

B) Synthesis of copy MA

Corresponds to the insert cMA portion introduced into the vector with the phage DNA as type II A biotinylated cRNA was prepared. Details are shown below.

Preparation of Kupo type DNA>

After 50 zg of phage DNA was digested with the restriction enzyme Xhol, proteinase K (final concentration of 100 g / ml) and SDS (final concentration of 0.55) were added and the mixture was treated at 37 ° C for 30 minutes. The phenol / chloroform (1: 1) The mixture was added, extraction was performed, and DNA was recovered by ethanol precipitation.

RNA synthesis reaction>

Using “EGAscript ^™ ” (Ambion, Funakoshi) according to the protocol, the reaction solution of 160〃1 (IxTranscription Buffer, ATP, CTP, GTP ヽ 3.25 mM UTP, final concentration 7.5 mM, 8 µg of the above type I DNA (Xhol digested) and enzyme mixture (20 U / 〃1 pateriophage T3A polymerase, 46 U / j placental ribonuclease inhibitor (0.5 mM Biotin-2 UTP)) Placental Ribonuc lease Inhibitor)) 16〃1 was added and reacted at 37 ° C for 6 hours. Next, RNase-free DNasel (8〃1) was added for the purpose of removing type I DNA and incubated at 37 ° C for 15 minutes. In addition, add 240〃1 of RNase-free sterile water and 2001 of lithium chloride solution (attached to MEGAscript ^T ), leave at -20 ° C for 30 minutes, and centrifuge at 15,000 rpm for 15 minutes The RNA was recovered as a precipitate, rinsed with 70% ethanol, dissolved in 100〃1 TE (pH 8.0) buffer, and the yield was measured, yielding about 500〃g of biotinylated RNA. .

(3-3) Deduction Hybridization

After ethanol precipitation of the single-stranded circular DNA 2 / g from the NRK-52E cell Dex (+) phage library obtained in (3-1) and the above-mentioned (3-2) biotinylated copy RNA 50〃g RNase free sterile water was suspended in 20〃1. 5x hybridase buffer (2.0 M NaCls 250 mM HEPES (pH 7.6), lOmM EDTA) 20/1 and formamide 20〃1 and keep at 70 ° C for 2 minutes, then 52 ° C, 45 hours Was conducted. Next, using “VECTRE-AVIDIN” (VECTOR LABORATORIES, manufactured by Funakoshi), "Potinated copy RNA" and "hybridized product of biotinylated copy MA and single-stranded circular DNA" were removed. Specifically, add 250m of “VECTREX-AVID ΙΝ_Τ” (equilibrated in advance with lOOmM Tris-HCl (pH 7.5) and OmM NaCl buffer) per 25mg of the biotinylated copy RNA for 2 hours at room temperature. After keeping the temperature, centrifugation was performed at 3,000 rpm for 30 seconds to collect the supernatant, and the supernatant was repeatedly treated with a new and similar VECTREX-AVIDIN, and the collected supernatant was precipitated with ethanol and hybridized. The single-stranded circular DNA that escaped the lysis was recovered, and the resulting single-stranded circular DNA was synthesized with a synthetic DNA (BLU-T3, “Synthetic DNA” complementary to the vector (pBluescriptSK-). DNA (SEQ ID NO: 5) ") was converted to double-stranded circular DNA by a reaction using Taq DNA polymerase as a primer. The composition of the reaction mixture (the final concentration) was lxTaq DNA Polymerase-zebuffer (10 mM Tris-HCl (pH 8.3), 50 mM KCK 1.5 mM MgCL, 0.001% gelatin), and 0.2 mM each. The final concentrations were dNTPs (dATP, dCTP, dGTP, dTTP), and primer DNA (synthetic DNA (SEQ ID NO: 5)). Using "DNA Thermal Cycler Model PJ2 000" (Takara Shuzo, PERKIN ELMER CETUS) as an apparatus, temperature conditions are "95 ° C, 1 minute", "55 ° C, 30 minutes", "72 ° C, 30 minutes". After stopping the reaction with 0.1 SDS and 12.5 mM EDTA pH 8.0j (both final concentrations), phenol- / chloroform treatment was performed, and the DNA was recovered by ethanol precipitation. Escherichia coli strain “XL1-Blue MRF,” was transformed in 20〃1 TE buffer and transformed. A total of about 1000 transformants were obtained. Proceeded to the next step by using these

(4) Selection of genes whose expression is induced by steroids by differential hybridization method

A) Preparation of membrane with spots of each plasmid DNA

Escherichia coli transformants were transformed into L medium (trypton 10 g, yeast extract 5 g, NaCl 10 g, pH 7.5) containing 2 ml of ampicillin (50 μg / ml). 1L) overnight, and the plasmid DNA was purified using a plasmid automatic separation device (Clavo, PI-100). An equivalent amount of an alkaline solution (3M NaCl, 1N NaOH) 'was added to the TE buffer in which those DNAs were dissolved, and the mixture was allowed to stand at room temperature for 5 minutes. Then, an equivalent amount (2 / 1) Two spots were created, and two identical fills were created.

B) Screening by differential hybridization

First strand cDNA synthesis reaction was performed using reverse transcriptase “Super Script ^™ II” (manufactured by GIBCO BRL) with 5 μg each of Dex (+) mRNA and Dex (−) mRNA as a template. Subsequently, a second strand synthesis reaction using E. coli RNase H and E. coli DNA polymerase (STMTAGE NE, Toyobo) was performed. Thus obtained et the two cDNA it it to hexa nucleotides, Klenow fragment, Non - ³² P] probe was ³² P labeled with multi-prime DNA labeling system (manufactured by full Alma shear Biotech) using dCTP like Produced. Professional one Bed specific activity of about lxl0 ⁹ cpm per lg of each cDNA could be created. A probe synthesized from Dex (+) mRNA was designated as "probe A", and a probe derived from Dex (-) mRNA was designated as "probe B". Next, for two duplicates shown in (4) -A, hybridization was performed separately for these two types of probes. The hybridization between Phil and Yoichi was carried out under the following conditions. The filter was placed in a plastic bag, filled with a prehybridization solution, and incubated at 65 ° C for 4 hours with shaking. The composition of the pre hybrida I See Chillon solution (final concentration), 5xSSPE (20xSSPE: 3.6M NaCl, 0.2 M sodium phosphate _{(pH7.7), 0.02M Na 2 EDTA} ), 5x Denhardt's solution (100x Denhardt's solution: 2% (W / V) BSA (bovine serum albumin), 2% (W / V) Ficoll 400, 2% (W / V) polyvinylpyrrolidone (PVP)), 0.5% SDS and heat-denatured 20 μg / ml of salmon sperm DNA. Subsequently, hybridization was performed in a solution obtained by adding a ³² P-labeled probe to a new prehybridization solution, and further kept at 65 ° C. for 18 hours. Filtration is performed using a 2xSSPE solution containing 0.1% SDS at 65 ° C for 30 minutes. Washing was performed once with an O.lxSSPE solution containing 0.1% SDS at 65 ° C for 30 minutes. After washing, the filter was affixed to filter paper (Whatman 3MM), exposed to an imaging plate (manufactured by Fuji Film Co., Ltd.), and analyzed using a “Bio Image Analyzer Bas 2000” (manufactured by Fuji Film Co., Ltd.). By comparing the intensities of the hybridization signals of the two fils that form a pair, 57 clones showing a stronger signal with “Probe A” were picked up. In the following examples, one of these clones (this clone was named “r # 5 (rat # 5)”) was a gene encoding “GISP” (Glucocortic oid—Inducible Suppressor Protein). The following shows the analysis.

(5) Detection of mRNA by Northern blot hybridization method

MRNA (poly (A) ⁺ RNA) was prepared from N52E cells subjected to various steroid treatment conditions according to the method described in (1) of [Example 1]. Subsequently, Northern plot analysis was performed according to a conventional method. That is, poly (A) ⁺ RNA of each sample was electrophoresed on a 0.8% agarose gel containing formaldehyde. After the electrophoresis, the RNA was transferred from the gel to a nylon filter (Biodyne A, manufactured by Nippon Pol Co., Ltd.) using capillary action. The DNA was immobilized on the membrane by UV treatment of the filter. On the other hand, a probe used for hybridization was prepared as follows. In other words, Escherichia coli clones of plasmid DNA (hereinafter referred to as “pBlue-r # 5”) containing the rat “GISP” gene are cultured and the nucleic acid separation / purification chip “QIAGEN-tip 500” (QIAGENinc. Funakoshi Co., Ltd.) Was used to prepare plasmid DNA. 10 〃g of this plasmid DNA is treated with restriction enzymes XhoI and EcoRI, electrophoresed on a 0.8% low-melting-point agarose gel, and a DNA fragment inserted into a vector of about 0.3 kb is cut out. PrepA- gene was purified by Matrix j (manufactured by Nippon Bio-Rad Laboratories Ichizu Co.) thus prepared DNA (approximately 30 ng) and the hexa nucleotides, click Renou fragment, [shed - ^{3 2} P]. multi-prime using dCTP like the ^{3 2} P-labeled probe was prepared by labeling method.

The hybridization between Phil Yuichi and the above probe is Northern Plot High. This was performed according to the standard method of bridging. The filter was placed in a plastic bag, filled with a prehybridization solution for Northern plot, and incubated at 65 ° C for 4 hours with shaking. The composition of the prehybridization solution was `` 50% (W / V) formamide, 5xSSPE, 5x Denhardt's solution, 2% (W / V) Ficoll 400, 2% (W / V) polyvinylpyrrolidone (PVP)), 0.5% SDS and heat denatured 20 g / ml salmon sperm DNA. Subsequently, a new pre hybrida I peptidase one Chillon solution ^{3 2} plus P-labeled probe solution was further subjected to Haiburidi zero one Chillon by incubating 18 hours at 42 ° C. Thereafter, the filter was washed three times in a 2xSSPE solution containing 0.1% SDS at 65 ° C for 30 minutes, followed by a 30 minute wash at 42 ° C in an O.lxSSPE solution containing 0.1% SDS. . After light air drying, autoradiography was performed. Figure 1 shows the results. Dexamethasone treatment was found to induce the expression of rat “GISP”. Lanes 2, 4, and 6 show the mRNA of cells treated with dexamethasone, and lanes 1, 3, and 5 show the mRNA of cells not treated with dexamethasone. Lanes 1, 3, and 5 show the lysis of dexamethasone. In the control containing only the ethanol used in the experiment, the cells treated with dexamethasone in lanes 2, 4, and 6 and the same time (lanes 1 and 2: 2 hours, lanes 3 and 4: 6 hours, Lanes 5 and 6: 24 hours).

(6) DNA base sequence determination

The DNA base sequence of the insert cDNA of “pBlue-r # 5”, which was confirmed to be induced by steroid stimulation, was determined. The sequencing reaction was performed using “Taq DyeDeoxy ^™ Termination Cycle Sequncing Kit” (manufactured by Applied Biosystems) according to the protocol. After completion of the reaction, the reaction product purified by a spin column (Bio-Spin 30 manufactured by Bio-Rad) was analyzed by a DNA sequencer (ABI 373A DNA Sequencing System). Using the homology search system “GeneBright” (manufactured by Hitachi Software) for the obtained DNA base sequence, a homology search was performed for GeneBank Release 97, which is a DNA database. It turned out to be a gene. (7) Acquisition of rat "GISP" gene from rat NRK cell-derived cDNA library As shown in Fig. 1, in the results of Northern blot analysis, this gene was a much longer mRNA than that obtained from the subtraction library. It was suggested that the insert cDNA of “pBlue-r # 5” was part of the original gene. Therefore, in order to obtain a more complete gene, rescreening was performed from a rat NM cell-derived cDNA library. Of the 10 NRK-52E cell Dex (+) sublibraries shown in (2) of [Example 1], clones having a DNA fragment longer than the insert cDNA of "pBlue-r # 5" were included. Was examined by PCR. Specifically, a synthetic MA primer (“synthetic DNA (SEQ ID NO: 6)” and “synthetic DNA (SEQ ID NO: 7)”) that binds to the DNA portion of the phage vector and the primer of “pBlue-r # 5” DNA primers (“synthetic DNA (SEQ ID NOs: 8-11)”) that bind to the cDNA are prepared, and the phage DNA contained in each sub-library is subjected to PCR to perform PCR, and “pBlue-r # 5” It was examined whether there was a supply library in which a longer PCR product was amplified than in the case of 錡.

The reaction composition of PCR is the same as in the above (3-3), except that the temperature conditions are “94 ° (for 1 minute)”, “60 ° C for 1 minute”, “72 ° C for 2 minutes”. As a result, bands of several sizes were observed. Furthermore, in order to detect those having the insert cDNA sequence of pBlue-r # 5j in the PCR reaction products, The PCR reaction product was electrophoresed on a 0.8% agarose gel and transcribed to Nylon Fil Yuichi according to a conventional method.The screening probe was as described in (5) of [Example 1] above. pBlue-r # 5 "to about 0.3 kb cDNA fragments was a ^{3 2} P-labeled by use of a. filter and Haiburidize one Chillon probes were carried out under the same conditions as (4) of example 1 After hybridization, filter the 2xSSPE solution containing 0.1% SDS, 6 The plate was washed four times for 30 minutes at 5 ° C. After light air drying, autoradiography was performed, and as a result, clones having longer DNA fragments existed in three of the sublibraries. Therefore, these sub-libraries were infected with the host E. coli XL1-Blue, and about 50,000 of them were inserted into 15 cm diameter The plates were plated to form plaques. The phage was adsorbed from the plate to a nylon filter “GeneScreen ™ Plus Hybridization Transfer Membrane j” (manufactured by Daiichi Pure Chemicals Co., Ltd.) to prepare screening filters for screening. For 5 minutes, and then neutralized by treatment with 1 M Tris-HCl (pH 7.5) for 5 minutes, followed by UV treatment to immobilize the DNA. It was the use of a fragment of about 0.3kb cMA of "pBlue- r # 5" ^{3 2} P-labeled by. Positive plaques were picked from the results of the plaque hybridization and transferred to 1 ml of SM buffer. In addition, plaques were again formed on the plate, and the steps of making a filter and hybridization with a probe were repeated to isolate a single clone. The obtained phage clone was converted from a ZAP phage vector into a plasmid (pBluescript SK-) by the method described in (3-1)-(A) of (3) in [Example 1], and the inserts thereof were inserted. The DNA base sequence of the cDNA was determined. Among them, the plasmid of a clone having an insert cMA of less than 1.2 kb was named “pBlue-r # 5 1.2”. When the DNA base sequence of the insert cDNA of “ρΒ1 ue-r # 5 1.2” was determined, it contained the entire sequence of the insert cDNA of “pBlue-r # 5”. It turned out to be.

[Example 2] Confirmation of expression of “GISP” gene in rat and human tissues To examine the expression of “GISP” gene in rat and human tissues, mRNAs from various tissues were transcribed. Northern plot analysis was carried out using Yuichi Nichiru (CL0NTECH, Toyobo). After purification of the DNA fragment of about 1.2kb obtained by cutting the "pBlue-r # 5 1.2" DNA restriction enzymes EcoRI and Xhol as probes, using ^{3 2} those P-labeled. Other experimental conditions are as shown in [Example 1] (5). Figure 2 shows the results. In the rat, a band of about 2 kb was detected in the various cells examined, but high expression was observed particularly in the heart, skeletal muscle, and kidney. In humans, no signal is seen in the brain and peripheral blood leukocytes However, in most other tissues, a slightly larger (about 2.4 kb) hybridizing band was detected than in rats (Fig. 2). A in the figure indicates poly (A) + RNA in each tissue of the rat (heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis) B shows poly (A) + RNA detected in human tissues (in order from lane 1 to 8, heart, brain, placenta, lung, liver, skeletal muscle, kidney, and kidney). C is poly poly (A) + RNA detected in human tissues (in order from lane 1 to 8, spleen, thymus, prostate, testis, ovary, small intestine, large intestine, and peripheral blood leukocytes). This figure shows the detection of poly (A) + RNA from human fetal tissues (brain, lung, liver, kidney, in order from lanes 1 to 4).

[Example 3] Acquisition of human "GISP" gene

From the results of [Example 2], it was confirmed that a similar gene (homolog) of the rat "GISP" gene was expressed in human kidney and the like. Therefore, a human kidney cDNA library was prepared and the human "GISP" gene was cloned.

(1) Preparation of human kidney cDNA library

Starting from 5 μg of human kidney mMA (poly (A) + RNA) (CL0NTECH, Toyobo) using the ZAP-cDNA Synthesis Kit (STRATAGENE, Toyobo) Fabrication was performed. A library consisting of about 1 × 10 6 recombinant phages was prepared by the same steps as in (2) of [Example 1].

(2) Cloning of human "GISP" gene

After purification excised cDNA moiety from containing the gene of the rat "pBlue- r # 5 1.2", used as a probe to ^{3 2} P-labeled by the multi-prime labeling method, in Example 1 (7 Hybridization was carried out as indicated in section). About 6Xl0 ⁵ or phage plaques showing a positive signal were screened recombinant Ekarada phage were peak Dzukuappu. Then, for each of them, the process of making a filter and hybridization with a probe was repeated to isolate 28 single phage clones. Of these 12 phage clones, (3-) of (3) of [Example 1] 1) The human ZAP phage was converted from plasmid Yuichi to plasmid (pBluescriptSK-) by the method described in -A. Plasmid DNA was prepared and analyzed for restriction enzyme cleavage patterns and partial DNA base sequence. Based on the results, “pBlue-h # 5-22” (“h # 5”) was used as a clone containing the human gene corresponding to the rat gene. "Means" human # 5 "), and its entire DNA base sequence and its encoded amino acid sequence were determined. The determined amino acid sequence and base sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. This gene had a region encoding a new protein consisting of 467 amino acid residues according to MOlbp's 0RF (open reading frame). Among the ESTs (short cDNA fragments of unknown function) currently published on the database, there are those whose DNA base sequence matches a part of this gene. As high as possible, the gene for the renal tubular interstitial nephritis antigen (tubu lointerstitical nepharitis antigen mRNA (LOCUS name: Ozono 270)) in the egret in the GeneBank database (Nelson, TR, J. Biol. Chem. 270 (27), 16265-) 16270 (1995)). The protein encoded by this gene and the human “GISP” gene have about 47% similarity at the amino acid level. This similarity is estimated to be due to the coding of the rat “GISP” gene and the human “GISP” gene. The similarity between the proteins (89% for rat (upper) and human (lower), see Fig. 3) is clearly lower, and “0CU24270” is called the homologue of the gene in this specification (ゥ sagi version). Rather than the family or super family.

[Example 4] Construction of expression vector for human "GISP" gene

The method for constructing the expression vector for the human "GISP" gene is described below. "PEF-18S" (0 hashi, H., Proc. Natl. Acad. Sci. USA 91 158-162 (1994)) having a promoter of the elongation factor was used as an expression vector. "PEF-l8S" DNA was digested with restriction enzymes EcoRI and Notl, and then purified with a DNA purification reagent "prepA-gene Matrix" (Nippon Bio-Rad Laboratories). On the other hand, in order to introduce this, the gene was transformed into “pBlue-h # 5-22” DNA, Using NAj (SEQ ID NO: 12) and “synthetic DNA” (SEQ ID NO: 13) as primers, PCR was performed using “LA taq” (Takara Shuzo) to perform the protein coding region of the hidden “GISP” gene, about 1400 bp Was prepared. The reaction mixture was composed of lxLAtaq buffer (Takara Shuzo), dNTPs (dATP, dCTP, dGTP, TTP) at a concentration of 0.2 mM (all final concentrations), primer DNA (1 M) and type I The total reaction volume is 50/1, and the reaction is kept at 94 ° C for 5 minutes, followed by 20 cycles of 98 ° C for 10 seconds and 68 ° C for 2 minutes. Furthermore, the test was performed under the conditions of three steps of keeping the temperature at 72 ° C for 10 minutes. After the PCR was completed, digestion was performed with restriction enzymes EcoRI and Notl, and the PCR product was further purified using DNA purification reagent “prepA-gene Matrix”. These were ligated with a “DNA Ligation Kit” (manufactured by Takara Shuzo Co., Ltd.) using T4 MA ligase, and then introduced into E. coli K-12 strain X-Blue competent cells to obtain ambicillin-resistant colonies. Plasmid was recovered from cultured cells of several colonies, and plasmid “pEF-h # 5” into which human “GISPj gene DNA was inserted” was obtained as expected from the restriction enzyme cleavage pattern. With respect to the obtained plasmid DNA, it was confirmed by analyzing the DNA base sequence that there was no error in the entire base sequence of the human “GISP” gene portion inserted into the vector.

[Example 5] Preparation of expression vector for rat "GISP" gene

(1) Cloning of the 5 'end of the rat "GISP" gene

As a result of the analysis of the human “GISP” gene shown in the above [Example 4], the plasmid “pBlue-r # 5 1.2” containing the rat “GISP” gene has five or more ends of the human “GISP” mRNA. It turned out that the part corresponding to the side was missing. Therefore, we applied PCR to clone the 5 'end of the rat “GISP” gene. The 5 'primer "synthetic DM (SEQ ID NO: 14)" refers to the human gene (cDNA) sequence, and the 3' primer "synthetic DNA (SEQ ID NO: 15)" refers to the rat gene (SEQ ID NO: 15). The cDNA corresponding to the sequence was designed and used. PCR using “LA Taq” (Takara Shuzo) was performed using cDNA synthesized from NRK52E Dex (+) mRNA as type I DNA as type I. The composition of the reaction solution was the same as in [Example 4], and the reaction conditions were "96 ° C for 20 seconds" and "68 ° C for 1 minute". Was performed in 25 cycles. Approximately 700 bp amplified by PCR was run on a 0.8% low melting point agarose gel, the target DNA fragment was excised, and purified using the DNA preparation reagent prepA-gene Matrix. The purified DNA fragment was purified from the “PCR ligation independent cloning (LIC) method” (Nucleic Acid Res. 18: 6069-6074 (1990)) by PCR Direct cloning systemj (CLONTECH, Toyobo Co., Ltd.). ) Was used to perform subcloning into “pDIRECT vector” (manufactured by CL0NTECH, Toyobo). The obtained plasmid DNA was designated as “pDirect700j”.

(2) Connection of the 5 'end and 3' end of the rat "GISP" gene

The gene fusion method using PCR (Va) was used to ligate the 5 and end portions of the rat “GISP” gene of PDirect700j to “pBlue-r # 5 1.2” described in (7) of [Example 1]. et al, Nucleic Acid Res. 17 723-733 (1989)). First, two types of DNA fragments containing the 5 'end and the 3' end were prepared by PCR. The 5 'end uses pD irect700 DNA as a type II, "synthetic DNA (SEQ ID NO: 14)" and "synthetic DNA (SEQ ID NO: 16)", and the 3' end uses "pBlue-r The first step PCR was performed using “Synthetic DNA (SEQ ID NO: 17)” and “Synthetic DNA (SEQ ID NO: 18)” with # 5 1.2 DNA as type II. The first-stage PCR product has a primer sequence that has an overlapping portion (30-40 bp) to ligate DNA fragments. The obtained two DNA fragments were purified and further subjected to the second-stage PCR. The second step PCR consists of a two-step reaction.In the first step, mix the two PCR products in equal volumes (40 μg each) and add 30 minutes at 96 ° C without adding the primers. For 5 seconds, the reaction was performed for 2 seconds and at 72 ° C for 2 minutes. As a second step, add primers (“Synthetic DNA (SEQ ID NO: 14)” and “Synthetic DNA (SEQ ID NO: 18)”) (0.2〃M each) to the reaction solution, and add “96 ° The reaction was performed for 25 cycles of “20 seconds at C”, “4 minutes at 65 ° C.”, and 6 minutes at 72 ° C. As a result, an expected DNA fragment of about 1.8 kb was obtained. The resulting fragment was purified and then subcloned by the PCR Direct Cloning system using the LIC method. Plasmid DNA was extracted from several clones and analyzed, and the desired plasmid was analyzed. DNA CpDirect r # 5-1.8kj was obtained. After preparing plasmid DNA according to a conventional method, the DNA base sequence was determined. It was confirmed that there was no change from the respective sequences before ligation. However, of the determined base sequence, the 24 bases from the 5 'end are sequences of a primer derived from a human sequence. [Example 1] Therefore, a primer was used that is directed 5 'upstream from the portion determined as the rat cDNA sequence, and a primer that is directed from the vector side toward the 5' end of the insert cDNA was used [Example 1]. Using the NM52E cell Dex (+) sublibrary shown in (2) above as type III, three-step PCR was performed. The phage solution 5〃1 was heat-treated at 98 ° C for 10 minutes, and then subjected to PCR using LAtaq (Takara Shuzo). The composition of the reaction mixture was IX LA taq buffer (Takara Shuzo Co., Ltd.) and dNTPs (dATP, dCTP, dGTP, TTP) at a concentration of 0.2 mM (all final concentrations), primer-DNA (1 zM) and 錡. A total of 50 ju1 volume is added with the template DNA, and the reaction is performed at (96 ° C, 30 seconds) (58 ° C, 30 seconds) (72 ° C, 2 minutes) for 25 cycles, and (° C, 10 minutes). In a total of three PCRs, synthetic DNA (SEQ ID NO: 6) was used as the 5 'primer on the upstream side of the cDNA (hybridizes to one vector sequence), and three primers (synthetic DNA ( By using SEQ ID NOs: 22, 23, and 24 in sequence, a PCR product of the desired size could be obtained.The PCR product was excised from the gel, purified, and the DNA base sequence was determined. The nucleotide sequence and amino acid sequence of the rat cDNA determined

: 3 and SEQ ID NO: 4.

(3) Preparation of expression vector for rat "GISP" gene

An expression vector for the rat “GISP” gene was constructed by the same steps as in [Example 4]. The DNA fragment to be inserted into the vector was prepared using LADirect and the sequences shown in “Synthetic DNA (SEQ ID NO: 12)” and “Synthetic DNA (SEQ ID NO: 19)” using “pDirect r # 5-1.8kj”. Using this primer, about 1400 bp of the protein coding region of the PCI trophy “GISP” gene was amplified. Reaction conditions and the like are as shown in [Example 4]. The PCR reaction product was purified by digestion with the restriction enzymes Ecoill and Notl. After ligating to pEF-18S DNA digested with EcoRI and Notl using T4 DNA ligase, E. coli K-12 strain was used. It was transfected into a competent cell of a certain XL Blue to obtain an ampicillin-resistant D21. Plasmid was recovered from cultured cells of several colonies, and plasmid DNA into which rat "GISP" gene DNA had been inserted was obtained as expected from restriction enzyme digestion proteins. The resulting plasmid DNA “pEF-r # 5” was sequenced with the rat “GISP” gene to confirm that the nucleotide sequence was correct.

[Example 6] Effect of "GISP" gene on human IL-8 and rat CINC (rat IL-8) promoter activities

(1) Cell culture

As human cells, lung-derived cells “MRC-5 SV1 TGI cells” (Riken Cell Bank) were cultured with “RITC 80-7 medium” supplemented with 10% FCS. As rat cells, "NRK-52E cells" (Flow Laboratories Inc. Tokyo, Japan), a cell line derived from renal epithelium, were cultured with "DMEM medium" supplemented with 10% FCS. Dexamethasone was dissolved in 100% ethanol, diluted with the medium, and added. In the control group, the solvent was added at the same concentration as the solvent in the dexamethasone-treated group.

(2) Construction of luciferase expression vector

1) Preparation of “pGL3-hIL8-185”

As a PCR primer, "5, -ATGTCTCGAGAATTCAGTAACCCAGGCATTATTTTATC-3 '(SEQ ID NO: 20)" and "5'-TTGTCCTAGAAGCTTGTGTGCTCTGCTGTC-3' (SEQ ID NO: 21)" were synthesized and human "VA-13 cells" (Riken Cell Bank) genomic DNA was subjected to PCR, and the promoter region of human IL-8 (neutrophil chemotactic cytokine) (-1481 to +44 bp) (K. Matsushima et. Al.: J Immunol., 143, 1366-1371, 1989). This was cut with restriction enzymes Dra I and Hind III, and a DNA fragment of the IL-8 promoter region of -185 to +44 bp was recovered and purified. On the other hand, a plasmid "pGL3-Promoter Vectorj (Promega)" was cut with Sma I and Hind III to prepare the SV40 initial promoter, and a fragment from -185 to +44 bp was prepared. Insert Then, a luciferase expression vector “pGL3-hIL8-185” having a human IL-8 promoter was prepared.

2) Preparation of "pGL3-CINC'-164"

"Preparing a plasmid cloned with the rat CINC (rat IL-8) promoter region (-164 to +7 bp) between the Kpn I and Hind III cleavage sites of pGL2-Basic Vector j (Promega) (T. Ohtsuka et. Al .: J. Biol. Chem., 271, 1651-1659, 1996.) This plasmid was digested with the restriction enzymes Sma I and Hind I II and the fragment was cut from -164 to +7 bp. The plasmid "pGL3-Promoter Vectorj (Promega)" was cleaved with Sma I and Hind 111 to remove the SV40 early promoter. Then, a +7 bp fragment from -164 described above was inserted into it to prepare a plasmid luciferase expression vector "pGL3-CINC-164" having a rat CINC promoter.

3) Preparation of "pRL-EF"

Factory pEF-18S ”The vector was cut with HindII and EcoRI to recover and purify a DNA fragment of about 1.2 kbp containing the promoter region of E. On the other hand, a plasmid was prepared by cutting the plasmid “pRL-null Vectorj (Promega)” with Hind I II and Eco RI. In addition, the above-mentioned elongation factor 1-1 and 1 The DNA fragment was inserted to prepare a "PRL-EF" expression vector for Mycobacterium luciferase, which had a promotion factor 1-1.

(3) Evaluation of "GISP" by repo-one-night genetic method

To analyze the transcriptional repressive activity of “GISP”, we performed an assay using the reporter gene method. That is, together with the “GISP” expression vector (“pEF-h # 5” and “pEF-r # 5”), the plasmid for expression of luciferase (pGL3-hIL8-185 or pGL3-CINC-164) ) And Plasmid luciferase expression plasmid ("pRL-EF"), a total of three types of plasmid DNA were cotransfected into cultured cells, and changes in promoter activity were examined. The method is as follows: On the first day of the experiment, seed the “O-5 SV1 TG1 cell” or “NRK-52E cell” on a 6-wel 1 plate (manufactured by Corning) at 3 to ⁵ x 10 ⁵ / well, and on the second day of the experiment. Using the DEAE-dextran method in the eyes: The plasmid DNA was cotransfected into cells. In addition, the amount of DNA used was 0.75 ^ g + in the case of `` MRC-5 SV1 TGI cells, '' per 100 μl of `` GISP '' expression vector (`` pEF-h # 5 '' or `` pEF-r # 5 ''). 0.25 g of “pGL3-hIL8-185” + 0.025 μg of pRL-EFj pRL-EFj, and in the case of “NRK-52E cells”, “GISP” expression vector per 1 μl (“pEF-h # 5” or “pEF -r # 5 ") l.O ^ g +" pGL3-CINC-164 "0.5 / g M +

“PRL-EF” was 0.05 / g. On the third day of the experiment, add dexamethasone (final concentration 1 M), culture at 37 ° C for 2 hours, add IL-15 (final concentration 100 U / ml), and 5 hours later Cell extracts were prepared using "Passive LysisBuffer" (Promega). Then, using a Dua Luciferase Repoter Assay Systemj (manufactured by Promega), the activity of the hydrogen and P. luciferase in this cell extract was measured using a Lumat model LB953j (Berthold, Germany). (Figures 4 and 5) As a control for “pEF-h # 5” and “pEF-r # 5”, the “pEF-18S” vector into which “# 5 cDNA” was not inserted was used. Figure 4A shows the vector with the human luciferase gene ligated downstream of the rat IL-8 (CINC) promoter, and Figure 5A shows the vector with the human luciferase gene downstream of the human IL-8 promoter. One of the ligated vectors, FIG. 4B and FIG. 5B, was detected in cells into which the vector linked to the MYC luciferase gene downstream of the EF motor was introduced.

As a result, in cells into which the “pEF-18S” vector into which the “# 5 cDNA” was not inserted, when IL-1 was added, human IL-8 and rat CINC promoter were activated, Strong firefly luciferase activity was induced, but suppression was observed by adding dexamethasone treatment (Figs. 4A and 5A). With the introduction of the “GISP” expression vector (“pEF-h # 5” or “pEF-r # 5”), suppression of the expression of luciferase at the same level or higher than that of dexamethasone treatment was observed. (Figures 4A and 5A). However, “GISP” is not a significant factor due to the elongation factor—1 α-promo It did not suppress the expression of C. luciferase (FIGS. 4B and 5B). Therefore, the specificity of the inhibitory effect of GI SPj on human IL-8 and rat CINC promoter was demonstrated.

[Example 7]

Six amino acid regions (shown in Table 1) were selected from the amino acid sequence of the rod “GISP” shown in SEQ ID NO: 3, and a peptide containing this sequence was synthesized. Immunization was performed by preparing an antigen solution to which thyroglobulin was bound as a carrier protein and an emulsion of oily adjuvant (manufactured by Immune Biology Laboratories, Inc.), and exercising the heron eight times in 100 〃g doses. Three small inoculations were made. Table 1 Amino acid sequence of rat “GISP” contained in synthetic peptide antigen Peptide 5-1 (SEQ ID NO: 25) MTPILSPQNLLSCD

Beptide 5-2 (SEQ ID NO: 26) CRGGRLDGAWWFLRRRGVVSD

Peptide 5- 3 (SEQ ID NO: 27) SGREQNDEASPTPRC

Peptide 5-4 (SEQ ID NO: 28) HSRAMGRGKRQATSRC

Veptide 5-5 (SEQ ID NO: 29) VSQGRPEQYRRHGTHSVKI

Peptide 5-6 (SEQ ID NO: 30) ETFVLGVWGRVGMED GHH

As for “peptide 5-5” and “peptide 5-6”, peptides having a cysteine residue added to the N-terminus were used.

ところ When a test sample of the heron was collected and its antibody level was examined using an enzyme immunoassay, an increase in the antibody level against the synthetic peptide was confirmed in any of the sera. These were used as antisera. In order to evaluate the obtained antisera, the following tests were performed. A protein fraction prepared from C0S1 cells (RIKEN Cell Development Bank, RCB0143), into which the gene encoding the amino acid sequence of rat “GISP” shown in SEQ ID NO: 3 was introduced and expressed, was subjected to SDS-polyacrylamide electrolysis. Electrophoresis (SDS-PAGE) followed by PVDF membrane

(Fluorotrans, manufactured by Nippon Genetics) using a BioRad semi-driving device (manufactured by Nippon Bio-Rad Laboratories). Transfer the membrane

After washing with “20 mM Tris-HC1, 0.5 M NaCl (pH 7.5)” (hereinafter referred to as “TBS”) for 5 minutes, washing with TBS containing 0.1% Tween 20 (hereinafter referred to as “TTBS”). After washing twice for 2 minutes, the plate was treated at room temperature for 60 minutes with a blocking agent composed of TTBS containing 2% of a blocking reagent, Smileite (manufactured by Sumitomo Metal Industries, Ltd.). A stamp lot analysis was performed using each antiserum containing an antibody against each peptide diluted 1000-fold with a TTBS solution containing 2% smileite as the primary antibody. That is, the plate was treated with a TTBS solution containing an anti-GISP peptide antibody and 2% smileite at room temperature for 60 minutes, and then washed twice with TTBS for 5 minutes. Next, alkaline phosphatase-labeled anti-Egret IgG (manufactured by Nippon Bio-Rad Laboratories) was diluted 1000-fold with a TTBS solution containing 2% Smartlight, treated as a secondary antibody at room temperature for 60 minutes, and treated with TTBS for 5 minutes. Two washes were performed for 2 minutes. Further, after washing twice with TBS for 5 minutes, the color was developed using an alkaline phosphatase color development kit (AP color development kit) (manufactured by Nippon Bio-Rad Laboratories). As a result, it was confirmed that the “GISP” protein was recognized and detected in the antisera of “peptide 5-3” and “peptide 5-5”. Industrial applicability

According to the present invention, a protein having an activity of suppressing activation of an IL-8 promoter in response to a specific extracellular stimulus, a DNA encoding the protein, a vector containing the DNA, and a transformation retaining the vector A body, an antibody that reacts with the protein, and a pharmaceutical composition containing the protein as an active ingredient are provided. Since the protein of the present invention has an activity of suppressing the activation of the IL-8 promoter, it is used as a drug for treating a disease associated with IL-8 expression, for example, an anti-inflammatory drug, an anti-bronchial asthma drug It is expected to be used for antiallergic drugs, antirheumatic drugs, etc. In addition, the protein of the present invention has no risk of producing neutralizing antibodies and the like in the human body when it is of the human type. In addition, both human and rat types are proinflammatory proteins other than IL-8, which are activated and expressed by the same pathway as that of IL-8 promoter overnight, such as TNF-H (Shakhov, AN et.al. J. Exp.Med. 171, 35-47 (1990)) and IL-6 (Kishimoto, T. et.al. Proc. Nat. Acad. Sci. US 90, 10193-7 (1993)) It is also possible to suppress the production of nitric oxide synthase (NOS) (Xie. QW et.al. J. Biol. Chem. 269, 4705-8 (1994)), and a broad anti-inflammatory effect can be expected. .

Sequence listing ―

(1) Name of applicant: Site Signal Research Institute, Inc.

(2) Title of invention ': Intracellular signaling inhibitor

(3) Reference number: S 1—9 0 1 P C T

(4) Application number:

(5) Application:

(6) Name and application number of the application that filed the priority application: Japan, Japanese Patent Application No. 9-62008

(7) Priority date: February 28, 1997

(8) Number of sequences: 30 Sequence number: 1

Sequence length: 467

Sequence type: amino acid

Topology: linear

Sequence type: Protein

Rooster

Met Trp Arg Cys Pro Leu Gly Leu Leu Leu Leu Leu

1 5 10

Pro Leu Ala Gly His Leu Ala Leu Gly Ala Gin Gin Gly Arg Gly Arg

15 20 25

Arg Glu Leu Ala Pro Gly Leu His Leu Arg Gly lie Arg Asp Ala Gly

30 35 40

Gly Arg Tyr Cys Gin Glu Gin Asp Leu Cys Cys Arg Gly Arg Ala Asp

45 50 55 60

Asp Cys Ala Leu Pro Tyr Leu Gly Ala lie Cys Tyr Cys Asp Leu Phe 65 70 75

Cys Asn Arg Thr Val Ser Asp Cys Cys Pro Asp Phe Trp Asp Phe Cys

80 85 90

Leu Gly Val Pro Pro Pro Phe Pro Pro lie Gin Gly Cys Met His Gly

95 100 105

Gly Arg lie Tyr Pro Val Leu Gly Thr Tyr Trp Asp Asn Cys Asn Arg

110 115 120

Cys Thr Cys Gin Glu Asn Arg Gin Trp Gin Cys Asp Gin Glu Pro Cys 125 130 135 140

Leu Val Asp Pro Asp Met lie Lys Ala lie Asn Gin Gly Asn Tyr Gly

145 150 155

Trp Gin Ala Gly Asn His Ser Ala Phe Trp Gly Met Thr Leu Asp Glu

160 165 170

Gly lie Arg Tyr Arg Leu Gly Thr lie Arg Pro Ser Ser Ser Val Met

175 180 185

Asn Met His Glu lie Tyr Thr Val Leu Asn Pro Gly Glu Val Leu Pro

190 195 200

Thr Ala Phe Glu Ala Ser Glu Lys Trp Pro Asn Leu lie His Glu Pro 205 210 215 220

Leu Asp Gin Gly Asn Cys Ala Gly Ser Trp Ala Phe Ser Thr Ala Ala

225 230 235

Val Ala Ser Asp Arg Val Ser lie His Ser Leu Gly His Met Thr Pro

240 245 250

Val Leu Ser Pro Gin Asn Leu Leu Ser Cys Asp Thr His Gin Gin Gin

255 260 Z65

Gly Cys Arg Gly Gly Arg Leu Asp Gly Ala Trp Trp Phe Leu Arg Arg 270 275 280

Arg Gly Val Val Ser Asp His Cys Tyr Pro Phe Ser Gly Arg Glu Arg 285 '290 295 300

Asp Glu Ala Gly Pro Ala Pro Pro Cys Met Met His Ser Arg Ala Met

305 310 315

Gly Arg Gly Lys Arg Gin Ala Thr Ala His Cys Pro Asn Ser Tyr Val

320 325 330

Asn Asn Asn Asp lie Tyr Gin Val Thr Pro Val Tyr Arg Leu Gly Ser

335 340 345

Asn Asp Lys Glu lie Met Lys Glu Leu Met Glu Asn Gly Pro Val Gin

350 355 360

Ala Leu Met Glu Val His Glu Asp Phe Phe Leu Tyr Lys Gly Gly lie 365 370 375 380

Tyr Ser His Thr Pro Val Ser Leu Gly Arg Pro Glu Arg Tyr Arg Arg

385 390 395

His Gly Thr His Ser Val Lys lie Thr Gly Trp Gly Glu Glu Glu Thr Leu

400 405 410

Pro Asp Gly Arg Thr Leu Lys Tyr Trp Thr Ala Ala Asn Ser Trp Gly

415 420 425

Pro Ala Trp Gly Glu Arg Gly His Phe Arg lie Val Arg Gly Val Asn

430 435 440

Glu Cys Asp lie Glu Ser Phe Val Leu Gly Val Trp Gly Arg Val Gly 445 450 455 460

Met Glu Asp Met Gly His His

465 SEQ ID NO: 2

Array length: 2187-

Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: cDNA to mRNA

Array features

Characteristic symbol: CDS

Location: 77.. 1477

How the feature was determined: E

Arrangement

CGCAGAGCCA GGAGGCGGAG GCGCGCGGGC CAGCCTGGGC CCCAGCCCAC ACCTTCACCA 60 GGGCCCAGGA GCCACC ATG TGG CGA TGT CCA CTG GGG CTA CTG CTG TTG CTG 112

Met Trp Arg Cys Pro Leu Gly Leu Leu Leu Leu Leu

1 5 10

CCG CTG GCT GGC CAC TTG GCT CTG GGT GCC CAG CAG GGT CGT GGG CGC 160 Pro Leu Ala Gly His Leu Ala Leu Gly Ala Gin Gin Gly Arg Gly Arg

15 20 25

CGG GAG CTA GCA CCG GGT CTG CAC CTG CGG GGC ATC CGG GAC GCG GGA 208 Arg Glu Leu Ala Pro Gly Leu His Leu Arg Gly He Arg Asp Ala Gly

30 35 40

GGC CGG TAC TGC CAG GAG CAG GAC CTG TGC TGC CGC GGC CGT GCC GAC 256 Gly Arg Tyr Cys Gin Glu Gin Asp Leu Cys Cys Arg Gly Arg Ala Asp

45 50 55 60

GAC TGT GCC CTG CCC TAC CTG GGC GCC ATC TGT TAC TGT GAC CTC TTC 304 Asp Cys Ala Leu Pro Tyr Leu Gly Ala lie Cys Tyr Cys Asp Leu Phe 65 70 75

TGC AAC CGC ACG GTC TCC GAC TGC TGC CCT GAC TTC TGG GAC TTC TGC 352 Cys Asn Arg Thr Val Ser Asp Cys Cys Pro Asp Phe Trp Asp Phe Cys

80 85 90

CTC GGC GTG CCA CCC CCT TTT CCC CCG ATC CAA GGA TGT ATG CAT GGA 400 Leu Gly Val Pro Pro Pro Phe Pro Pro lie Gin Gly Cys Met His Gly

95 100 105

GGT CGT ATC TAT CCA GTC TTG GGA ACG TAC TGG GAC AAC TGT AAC CGT 448 Gly Arg lie Tyr Pro Val Leu Gly Thr Tyr Trp Asp Asn Cys Asn Arg

110 115 120

TGC ACC TGC CAG GAG AAC AGG CAG TGG CAG TGT GAC CAA GAA CCA TGC 496 Cys Thr Cys Gin Glu Asn Arg Gin Trp Gin Cys Asp Gin Glu Pro Cys

125 130 135 140

CTG GTG GAT CCA GAC ATG ATC AAA GCC ATC AAC CAG GGC AAC TAT GGC 544 Leu Val Asp Pro Asp Met lie Lys Ala lie Asn Gin Gly Asn Tyr Gly

145 150 155

TGG CAG GCT GGG AAC CAC AGC GCC TTC TGG GGC ATG ACC CTG GAT GAG 592 Trp Gin Ala Gly Asn His Ser Ala Phe Trp Gly Met Thr Leu Asp Glu

160 165 170

GGC ATT CGC TAC CGC CTG GGC ACC ATC CGC CCA TCT TCC TCG GTC ATG 640 Gly lie Arg Tyr Arg Leu Gly Thr lie Arg Pro Ser Ser Ser Val Met

175 180 185

AAC ATG CAT GAA ATT TAT ACA GTG CTG AAC CCA GGG GAG GTG CTT CCC 688 Asn Met His Glu lie Tyr Thr Val Leu Asn Pro Gly Glu Val Leu Pro

190 195 200

ACA GCC TTC GAG GCC TCT GAG AAG TGG CCC AAC CTG ATT CAT GAG CCT 736 Thr Ala Phe Glu Ala Ser Glu Lys Trp Pro Asn Leu lie His Glu Pro

205 210 215 220

CTT GAC CM GGC "AAC TGT GCA GGC TCC TGG GCC TTC TCC ACA GCA GCT 784

Leu Asp Gin Gly Asn Cys Ala Gly Ser Trp Ala Phe Ser Thr Ala Ala

225 230 235

GTG GCA TCC GAT CGT GTC TCA ATC CAT TCT CTG GGA CAC ATG ACG CCT 832 Val Ala Ser Asp Arg Val Ser lie His Ser Leu Gly His Met Thr Pro

240 245 250

GTC CTG TCG CCC CAG AAC CTG CTG TCT TGT GAC ACC CAC CAG CAG CAG 880 Val Leu Ser Pro Gin Asn Leu Leu Ser Cys Asp Thr His Gin Gin Gin

255 260 265

GGC TGC CGC GGT GGG CGT CTC GAT GGT GCC TGG TGG TTC CTG CGT CGC 928 Gly Cys Arg Gly Gly Arg Leu Asp Gly Ala Trp Trp Phe Leu Arg Arg

270 275 280

CGA GGG GTG GTG TCT GAC CAC TGC TAC CCC TTC TCG GGC CGT GAA CGA 976 Arg Gly Val Val Ser Asp His Cys Tyr Pro Phe Ser Gly Arg Glu Arg

285 290 295 300

GAC GAG GCT GGC CCT GCG CCC CCC TGT ATG ATG CAC AGC CGA GCC ATG 1024 Asp Glu Ala Gly Pro Ala Pro Pro Cys Met Met His Ser Arg Ala Met

305 310 315

GGT CGG GGC AAG CGC CAG GCC ACT GCC CAC TGC CCC AAC AGC TAT GTT 1072 Gly Arg Gly Lys Arg Gin Ala Thr Ala His Cys Pro Asn Ser Tyr Val

320 325 330

AAT AAC AAT GAC ATC TAC CAG GTC ACT CCT GTC TAC CGC CTC GGC TCC 1120 Asn Asn Asn Asp lie Tyr Gin Val Thr Pro Val Tyr Arg Leu Gly Ser

335 340 345 AAC GAC AAG GAG ATC ATG AAG GAG CTG ATG GAG AAT GGC CCT GTC CAA 1168 Asn Asp Lys Glu He Met Lys Glu Leu Met Glu Asn Gly Pro Val Gin

350 355 360

GCC CTC ATG GAG GTG CAT GAG GAC TTC TTC CTA TAC AAG GGA GGC ATC 1216 Ala Leu Met Glu Val His Glu Asp Phe Phe Leu Tyr Lys Gly Gly lie

365 370 375 380

TAC AGC CAC ACG CCA GTG AGC CTT GGG AGG CCA GAG AGA TAC CGC CGG 1264 Tyr Ser His Thr Pro Val Ser Leu Gly Arg Pro Glu Arg Tyr Arg Arg

385 390 395

CAT GGG ACC CAC TCA GTC AAG ATC ACA GGA TGG GGA GAG GAG ACG CTG 1312 His Gly Thr His Ser Val Lys lie Thr Gly Trp Gly Glu Glu Thr Leu

400 405 410

CCA GAT GGA AGG ACG CTC AAA TAC TGG ACT GCG GCC AAC TCC TGG GGC 1360 Pro Asp Gly Arg Thr Leu Lys Tyr Trp Thr Ala Ala Asn Ser Trp Gly

415 420 425

CCA GCC TGG GGC GAG AGG GGC CAC TTC CGC ATC GTG CGC GGC GTC AAT 1408 Pro Ala Trp Gly Glu Arg Gly His Phe Arg lie Val Arg Gly Val Asn

430 435 440

GAG TGC GAC ATC GAG AGC TTC GTG CTG GGC GTC TGG GGC CGC GTG GGC 1456 Glu Cys Asp lie Glu Ser Phe Val Leu Gly Val Trp Gly Arg Val Gly

445 450 455 460

ATG GAG GAC ATG GGT CAT CAC TGAGGCTGCG GGCACCACGC GGGGTCCGGC 1507 Met Glu Asp Met Gly His His

465

CTGGGATCCA GGCTAAGGGC CGGCGGAAGA GGCCCCAATG GGGCGGTGAC CCCAGCCTCG 1567 CCCGACAGAG CCCGGGGCGC AGGCGGGCGC CAGGGCGCTA ATCCCGGCGC GGGTTCCGCT 1627 /. d ει〇

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65 70 75 80

Ser Asp Cys Cys Pro Asp Phe Trp Asp Phe Cys Leu Gly lie Pro Pro

"85 90 95

Pro Phe Pro Pro Val Gin Gly Cys Met His Ala Gly Arg He Tyr Pro

100 105 110 l ie Phe Gly Thr Tyr Trp Glu Asn Cys Asn Arg Cys Thr Cys His Glu

115 120 125

Lys Gly Gin Trp Glu Cys Asp Gin Glu Pro Cys Leu Val Asp Pro Ala

130 135 140

Met lie Lys Ala lie Asn Arg Gly Asn Tyr Gly Trp Gin Ala Gly Asn 145 150 155 160

His Ser Ala Phe Trp Gly Met Thr Leu Asp Glu Gly lie Arg Tyr Arg

165 170 175

Leu Gly Thr lie Arg Pro Ser Ser Ser Val Met Asn Met Asn Glu He

180 185 190

Tyr Thr Val Leu Gly Gin Gly Glu Val Leu Pro Thr Ala Phe Glu Ala

195 200 205

Ser Glu Lys Trp Pro Asn Leu lie His Glu Pro Leu Asp Gin Gly Asn

210 215 220

Cys Ala Gly Ser Trp Ala Phe Ser Thr Ala Ala Val Ala Ser Asp Arg 225 230 235 240

Val Ser lie His Ser Leu Gly His Met Thr Pro lie Leu Ser Pro Gin

245 250 255

Asn Leu Leu Ser Cys Asp Thr His His Gin Lys Gly Cys Arg Gly Gly

260 265 270

Arg Leu Asp Gly Ala Trp Trp Phe Leu Arg Arg Arg Gly Val Val Ser 275 280 285

Asp Asn Cys Tyr Pro Phe Ser Gly Arg Glu Gin Asn Asp Glu Ala Ser

290 '295 300

Pro Thr Pro Arg Cys Met Met His Ser Arg Ala Met Gly Arg Gly Lys 305 310 315 320

Arg Gin Ala Thr Ser Arg Cys Pro Asn Ser Gin Val Asp Ser Asn Asp

325 330 335

He Tyr Gin Val Thr Pro Val Tyr Arg Leu Ala Ser Asp Glu Lys Glu

340 345 350 l ie Met Lys Glu Leu Met Glu Asn Gly Pro Val Gin Ala Leu Met Glu

355 360 365

Val His Glu Asp Phe Phe Leu Tyr Gin Arg Gly lie Tyr Ser His Thr

370 375 380

Pro Val Ser Gin Gly Arg Pro Glu Gin Tyr Arg Arg His Gly Thr His 385 390 395 400

Ser Val Lys l ie Thr Gly Trp Gly Glu Glu Thr Leu Pro Asp Gly Arg

405 410 415

Thr lie Lys Tyr Trp Thr Ala Ala Asn Ser Trp Gly Pro Trp Trp Gly

420 425 430

Glu Arg Gly His Phe Arg lie Val Arg Gly lie Asn Glu Cys Asp lie

435 440 445

Glu Thr Phe Val Leu Gly Val Trp Gly Arg Val Gly Met Glu Asp Met

450 455 460

Gly His His SEQ ID NO: 4

Sequence length: 1930-

Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: cDNA to mRNA

Array features

Characteristic symbol: CDS

Location: 67.. 1467

How the feature was determined: E

Rooster

CGAGCTTGGA GGCGGAGGCG CGCAGGGCAG CCTGGGTCCA GCCCACACCC 50 CTCACCAGCA GGCACC ATG TGG GGA TGT CCG CTA GGG CTG CTA TTG 96

Met Trp Gly Cys Pro Leu Gly Leu Leu Leu

1 5 10

CTG CTG CTG GCT GGC CAG GCT GCC CTG GAG GCC CGG CGG AGT CGT TGG 144 Leu Leu Leu Ala Gly Gin Ala Ala Leu Glu Ala Arg Arg Ser Arg Trp

15 20 25

CGC AGG GAG CTG GCG CCA GGG CTG CAC CTG CGG GGC ATC CGG GAC GCC 192 Arg Arg Glu Leu Ala Pro Gly Leu His Leu Arg Gly lie Arg Asp Ala

30 35 40

GGT GGC AGA TAC TGC CM GAG CAG GAC ATG TGC TGC CGC GGC CGT GCT 240 Gly Gly Arg Tyr Cys Gin Glu Gin Asp Met Cys Cys Arg Gly Arg Ala

45 50 55

GAC GAG TGT GCT TTG CCC TAC CTG GGA GCC ACC TGT TAC TGT GAC CTC 288 Asp Glu Cys Ala Leu Pro Tyr Leu Gly Ala Thr Cys Tyr Cys Asp Leu 60 65 70

TTC TGC AAC CGC ACC GTC TCT GAC TGC TGC CCT GAC TTT TGG GAC TTC 336 Phe Cys Asn Arg `` Thr Val Ser Asp Cys Cys Pro Asp Phe Trp Asp Phe

75 80 85 90

TGC CTC GGG ATT CCA CCC CCC TTC CCT CCT GTC CAA GGT TGC ATG CAT 384 Cys Leu Gly lie Pro Pro Pro Phe Pro Pro Val Gin Gly Cys Met His

95 100 105

GCG GGC CGG ATC TAC CCA ATC TTC GGA ACC TAT TGG GAA AAC TGC AAT 432 Ala Gly Arg lie Tyr Pro He Phe Gly Thr Tyr Trp Glu Asn Cys Asn

110 115 120

CGG TGC ACC TGC CAT GAG AAA GGG CAG TGG GAA TGT GAC CAG GAG CCA 480 Arg Cys Thr Cys His Glu Lys Gly Gin Trp Glu Cys Asp Gin Glu Pro

125 130 135

TGT CTA GTG GAC CCA GCC ATG ATT AAA GCC ATC AAC CGG GGC AAC TAC 528 Cys Leu Val Asp Pro Ala Met He Lys Ala lie Asn Arg Gly Asn Tyr

140 145 150

GGG TGG CAG GCT GGG AAC CAC AGT GCC TTC TGG GGC ATG ACC CTG GAT 576 Gly Trp Gin Ala Gly Asn His Ser Ala Phe Trp Gly Met Thr Leu Asp

155 160 165 170

GAG GGC ATT CGA TAC CGG CTG GGC ACA ATC CGC CCA TCT TCC TCT GTC 624 Glu Gly lie Arg Tyr Arg Leu Gly Thr He Arg Pro Ser Ser Ser Val

175 180 185

ATG AAT ATG AAT GAA ATT TAT ACA GTG CTG GGA CAA GGA GAA GTG CTA 672 Met Asn Met Asn Glu lie Tyr Thr Val Leu Gly Gin Gly Glu Val Leu

190 195 200

CCC ACT GCC TTT GAG GCT TCC GAG AAG TGG CCC AAC CTG ATC CAT GAG 720 nsi SJV J QJd J¾ Ι¾Λ U] 9 J 9Π dsy usy s dsy ΐ¾ ^ ΐθ Οίΐ SID 003 DV1 010 100 33V 319 9V0 OVX OIV 3V9 IW 031 IVO 1X9 3V0

J8S usy OJJ S (Q Say J8S J¾ ¾IV u [9 Sjy Say ¾iv 990Ϊ I3V XW DD3 001 393 131 IDV 330 DVD D03 SW DOS 093 D3D 91V 000

OTC 90C 00S

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800 ΐ 093 ODV OVO 3XV OIV 331 V03 133 I3V 333 09V 039 9V9 DV9 OW OVD

2 062 98Z

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096 9V3 103 3D9 331 Oil V3D 3VX 101 OW XV3 101 9X9 D19 ΰΰΰ V93 ODD

08Z SZZ O Z

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Z 993 913 3X1 991 931 139 199 IV9 313 193 999 X99 V93 391 330 9W

92 09Z 992

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9 £ 800 / 86df / I3d € IZ8 £ / 86 OAV 335 340 345

GCC TCC GAT GAG AAG GAG ATC ATG AAG GAG CTA ATG GAA AAT GGC CCC 1152 Ala Ser Asp Glu "Lys Glu He Met Lys Glu Leu Met Glu Asn Gly Pro

350 355 360

GTT CAA GCG CTT ATG GAA GTA CAC GAA GAC TTC TTC TTG TAC CAG CGA 1200 Val Gin Ala Leu Met Glu Val His Glu Asp Phe Phe Leu Tyr Gin Arg

365 370 375

GGC ATC TAC AGC CAC ACA CCT GTA AGC CAG GGG AGG CCA GAG CAG TAC 1248 Gly lie Tyr Ser His Thr Pro Val Ser Gin Gly Arg Pro Glu Gin Tyr

380 385 390

CGC CGA CAC GGG ACT CAC TCT GTC AAG ATC ACA GGG TGG GGA GAA GAG 1296 Arg Arg His Gly Thr His Ser Val Lys lie Thr Gly Trp Gly Glu Glu

395 400 405 410

ACA CTG CCA GAC GGA AGG ACG ATC AAG TAC TGG ACT GCT GCC AAC TCG 1344 Thr Leu Pro Asp Gly Arg Thr lie Lys Tyr Trp Thr Ala Ala Asn Ser

415 420 425

TGG GGC CCA TGG TGG GGT GAG AGG GGC CAC TTC CGA ATC GTG CGT GGC 1392 Trp Gly Pro Trp Trp Gly Glu Arg Gly His Phe Arg He Val Arg Gly

430 435 440

ATC AAC GAG TGT GAC ATC GAG ACC TTC GTG CTG GGC GTC TGG GGC CGC 1440 l ie Asn Glu Cys Asp lie Glu Thr Phe Val Leu Gly Val Trp Gly Arg

445 450 455

GTA GGA ATG GAG GAC ATG GGG CAC CAC TGAGTCTCGG CCACTAAGCT 1487 Val Gly Met Glu Asp Met Gly His His

460 465

AGGTGGGATC CACAGCCACG GAAGAGGCCT TGGGGGCCAC GCCGATGAGG CCTTGGGACC 1547 AGGTGCTAAT CCCCTCAGAC TCAGATCCAC ACAAACGCAG AACCCCACCT GGGAGCTAAT 1607

GTCCCTCAGG GAGCAACAGT GAGGCTGGAG GGAACCCTCA GACATCACAG CCGGAACTGG 1667

AAAGGGCCCG GCTTGGAAAC TGCAGGGAGT AAAATTCCCA GGCCCCTGGT CAGCCAGCCC 1727

AAGACCATGG GAGCTAAGAC ACCCCAACCT CTTTATCCTC CTACCCAACC TCATTCTTAT 1787

TTTTTCATTC TCTTTGGTGG ATTCTGCCCA TCCCCCTGGC CTCCGTTCTG GCTGGACCTT 1847

TCCATCCTCA ACACTGCTTT CTTACTCTTT AAAAATATTT ATTTTTCTTT TCATTAAAAT 1907

AAAACCAAAG TATTGATAAT TGC 1930 SEQ ID NO: 5

Array length: 29

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

ATTAACCCTC ACTAAAGGGA ACAAAAGCT 29 SEQ ID NO: 6

Array length: 21

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

TCTAGAACTA GTGGATCCCC C Zl SEQ ID NO: 7

Array length: 21

Sequence type: nucleic acid ''

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

GCCCCCCCTC GAGTTTTTTT T 21

SEQ ID NO: 8

Array length: 27

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

GGCCGCGGAA TTATCAATAC TTTGGTT 27

SEQ ID NO: 9

Array length: 24

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

GAGTAAGAAA GCAGTGTTGA GGAT 24 SEQ ID NO: 10 ——

Array length: 21 '

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthesis MA

Array

CAGAACGGAG GCCAGGGGGA T 21 SEQ ID NO: 11

Array length: 23

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

ACTGCAGGGA GTAAAATTCC CAG 23 SEQ ID NO: 12

Array length: 33

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array ATGAATTCCA CCATGTGGCG ATGTCCACTG GGG 33 SEQ ID NO: 13 "-Sequence length: 33

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

ATGCGGCCGC TTATCAGTGA TGACCCATGT CCT 33 SEQ ID NO: 14

Array length: 39

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthesis MA

Array

CTCGCTCGCC CAATGTGGCG ATGTCCACTG GGGCTATGC 39 SEQ ID NO: 15

Array length: 35

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthesis MA Array.

CTGGTTCGGC CCAGATGGAC ACTCGGTCAG ATGCG 35 SEQ ID NO: 16

Array length: 22

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

GATGGACACT CGGTCAGATG CG 22 SEQ ID NO: 17

Array length: 23

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthesis MA

Array

CAGCTGTCGC ATCTGACCGA GTG 23 SEQ ID NO: 18

Array length: 34

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear Sequence type: other nucleic acid synthetic DNA

Array

CTGGTTCGGC CCAGCAATTA TCAATACTTT GGTT 34 SEQ ID NO: 19

Array length: 33

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

ATGCGGCCGC TTATCAGTGG TGCCCCATGT CCT 33 SEQ ID NO: 20

Array length: 38

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

ATGTCTCGAG AATTCAGTAA CCCAGGCATT ATTTTATC 38 SEQ ID NO: 21

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand Topology: linear

Sequence type: other nucleic acid synthetic MA

Array

TTGTCCTAGA AGCTTGTGTG CTCTGCTGTC 30 SEQ ID NO: 22

Array length: 25

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthesis MA

Array

AACAGTTATC AGACACCACC CCTCG 25 SEQ ID NO: 23

Array length: 21

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthetic DNA

Array

GTCATGCCCC AGAAGGCACT G 23 SEQ ID NO: 24

Array length: 24

Sequence type: nucleic acid Number of chains: single strand

Topology: linear

Sequence type: other nucleic acid synthesis MA

Array

TCCCGAGGCA GAAGTCCCAA AAGT 24 SEQ ID NO: 25

Array length: 14

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

Met Thr Pro lie Leu Ser Pro Gin Asn Leu Leu Ser Cys Asp

1 5 10 SEQ ID NO: 26

Array length: 21

Sequence type: amino acid

Topology: linear

Sequence type: Peptide

Array

Cys Arg Gly Gly Arg Leu Asp Gly Ala Trp Trp Phe Leu Arg Arg Arg Gly Val 1 5 10 15

Val Ser Asp

20 SEQ ID NO: 27

Array length: 15 '

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

Ser Gly Arg Glu Gin Asn Asp Glu Ala Ser Pro Thr Pro Arg Cys

1 5 10 15 SEQ ID NO: 28

Array length: 16

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

His Ser Arg Ala Met Gly Arg Gly Lys Arg Gin Ala Thr Ser Arg Cys

1 5 10 15 SEQ ID NO: 29

Array length: 19

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

Val Ser Gin Gly Arg Pro Glu Gin Tyr Arg Arg His Gly Thr His Ser Val Lys 1 5 10 15 lie SEQ ID NO: 30'- Sequence length: 19

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

Glu Thr Phe Val Leu Gly Val Trp Gly Arg Val Gly Met Glu Asp Met Gly His 1 5 10 15

His

Claims

The scope of the claims . -

I. The protein according to SEQ ID NO: 1, or an IL having an amino acid sequence in which one or several amino acids have been substituted, deleted, or added in the amino acid sequence thereof, and which responded to a specific extracellular stimulus -A protein that has the activity to suppress the activation of 8 promoter.

2. A protein encoded by a DNA that hybridizes with the DNA of SEQ ID NO: 2, which has an activity of suppressing activation of the IL-8 promoter in response to a specific extracellular stimulus.

3. The protein according to SEQ ID NO: 3, or an IL having an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of the protein, and which responds to a specific extracellular stimulus -8 Promote A protein that has the activity of inhibiting the activation of the protein.

4. A protein encoded by a DNA that hybridizes with the DNA of SEQ ID NO: 4, which has an activity of inhibiting the activation of IL-8 promoter overnight in response to a specific extracellular stimulus.

5. The protein according to any one of claims 1 to 4, wherein the specific extracellular stimulus is a stimulus by interleukin-1 / 3.

6. DNA encoding the protein according to claim 1 or 3.

7. The activity of hybridizing with the DNA of SEQ ID NO: 2 and / or the DNA of SEQ ID NO: 4 and inhibiting the activation of IL-8 promoter overnight in response to a specific extracellular stimulus DNA encoding a protein having

8. The DM of claim 7, wherein the particular extracellular stimulus is a stimulus by interleukin-1 / 5.

9. A vector comprising the DNA according to any one of claims 6 to 8.

10. A host carrying the vector according to claim 9.

II. An antibody that reacts with the protein according to any one of claims 1 to 5.

12. A pharmaceutical composition comprising the protein according to any one of claims 1 to 5 as an active ingredient.

13. The pharmaceutical composition according to claim 12, which is an anti-inflammatory agent.