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WO1998038213A1 - Inhibiteur de la transmission de signaux intracellulaires - Google Patents

Inhibiteur de la transmission de signaux intracellulaires Download PDF

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Publication number
WO1998038213A1
WO1998038213A1 PCT/JP1998/000836 JP9800836W WO9838213A1 WO 1998038213 A1 WO1998038213 A1 WO 1998038213A1 JP 9800836 W JP9800836 W JP 9800836W WO 9838213 A1 WO9838213 A1 WO 9838213A1
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Prior art keywords
dna
gly
protein
arg
seq
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English (en)
Japanese (ja)
Inventor
Hideaki Yoshida
Toshiaki Ohtsuka
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Institute of Cytosignal Research Inc
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Institute of Cytosignal Research Inc
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Priority to AU61184/98A priority Critical patent/AU6118498A/en
Publication of WO1998038213A1 publication Critical patent/WO1998038213A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • 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)).
  • 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)).
  • IL : 8 Since the relationship between IL : 8 and various diseases has been elucidated in this way, pharmaceuticals targeting IL-8 are also being developed.
  • 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). ).
  • 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.
  • IL-8 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.
  • 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)).
  • 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 ⁇ ⁇ ⁇ It is considered to be a strong candidate for a gene encoding quality.
  • the present inventors attempted to isolate a gene whose expression is induced by dexamethasone treatment.
  • 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.
  • 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.
  • the present invention relates to a factor that suppresses activation of the IL-8 promoter, and more specifically,
  • 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,
  • 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 ,
  • 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.
  • IL-1 Crin. Immunol. 2718-28 (1995)
  • IL-8 Clin. Immunol. 2780-85 (1995)
  • TNF Molecular Medicine 33 1010-1020 ( 1996)
  • IL-8 production is induced by stimulation of IL-1 and TNF (Mukaida, N. et al. Icrobiol. Immunol., 36773 (1977). 992)).
  • IL-8 gene is induced through an intracellular signal transduction process based on the binding of IL-1 and TNF to cell membrane receptors.
  • 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.
  • 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).
  • 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.
  • amino acid sequence of the amino acid sequence of the protein described in SEQ ID NO: 1 has an amino acid sequence in which one or several amino acids have been substituted, deleted, or added.
  • SEQ ID NO: 1 or SEQ ID NO: 3
  • amino acid sequence in which one or several amino acids have been substituted, deleted, or added has been substituted, deleted, or added.
  • 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. _
  • 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.
  • 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.
  • ⁇ -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)
  • 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.
  • 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.
  • 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.
  • a host for example, Escherichia coli, yeast, Bacillus subtilis and the like are mainly used.
  • 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
  • an enzyme such as a probe
  • RT-PCR Yamamoto, ES and Wang, AM, PCR Technology (Erlich, HA ed.) Stockton Press, 89-97 (1989), etc.
  • cDNA can be prepared by expression screening using Escherichia coli or the like as a host.
  • 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.
  • DNA 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.
  • E. coli for example, r p Bluescipt II "(STRATAGENE Co., Ltd.),” p CR T “II” (Invitrogen), and the like.
  • yeast for example, “pSR403j (Sikorski RS and Hieter, P., Genetics, 122, 19-27 (1989))”, etc.
  • pCDM8 Seed, B., Nature, 329, 840-842 (1987)
  • pSV2-neo Southern and Berg, J. Mol. App 1. Genet., 1, 327-341, (1982)
  • 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.
  • pKC30 Shimatake, H, and Rosenberg, M., Nature, 292, 128-132, (1981)
  • pTrc 99A Align, E. et al., Gene, 69, 30 1-315, (1988)
  • pCAGGS Niwa et al., Gene, 108, 193-200, (1991)
  • pcDL-SR 293 Takebe et al. Mo 1. Cell. Biol., 8, 466-472, (1988)
  • pAdexlw a transfer vector for adenovirus production (Kanegae et al., Experimental Medicine, 12, 316). -322, (1994)).
  • 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 logistic (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
  • suitable Escherichia coli strains include "X-Blue”, “SURE”, “DH5" (all available from RIKEN Genebank).
  • prokaryotic eg, Escherichia coli
  • eukaryotic eg, yeast, mammal, insect cells and the like can be used as host cells.
  • animal cells examples 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).
  • yeast examples include baker's yeast (Saccharomyces cerevisae) and ethanol-assimilating yeast (Pichia pastoris).
  • 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.
  • 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.
  • 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.
  • 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.
  • an appropriate partial peptide thereof for example, the peptides described in SEQ ID NO: 25 to SEQ ID NO: 30) can be used.
  • Antisera can be obtained from these animals with increased antibody titers, or cells can be collected to obtain polyclonal antibodies.
  • an animal having an increased antibody titer against the antigen is used as a source of antibody-producing cells.
  • 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.
  • 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.
  • MA Radioimnoassay
  • EIA Errawa et al., Enzymimnoadisei (New Chemistry Laboratory Course 12), 88-99, Tokyo Kagaku Dojin (1992)
  • 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
  • a pharmaceutical composition comprising the protein of the present invention as an active ingredient.
  • the protein of the present invention has an activity of suppressing activation of the IL-8 promoter.
  • 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.
  • the protein of the present invention can be directly administered, or can be used after being formulated by a known pharmaceutical production method.
  • 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.
  • 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.
  • 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.
  • Dex lipopolysaccharide Sa steward Lai de
  • Dex anti-inflammatory
  • 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 ⁇ g 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.
  • ZAP-cDNA SYNTHESIS KIT manufactured by STRATAGENE, Toyobo.
  • coli PLK-F (STRATAGENE, Toyobo), and about 50,000 pieces of 20-cm 15 cm plates
  • 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 3 ⁇ 4 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”.
  • 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
  • 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).
  • 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.
  • 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 ⁇ g of the above marauder was digested with restriction enzymes Bgll I (10u 1) 10/1 and PvuI I (10u / ⁇ l) 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).
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g of single-stranded DNA was obtained.
  • a solution containing NRK-52E cell Dex (-) phage library add an equal volume of a solution (203 ⁇ 4 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.
  • DE DIETHYLAMINOETHL CELUL0SE
  • 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.
  • RNase-free DNasel (8 ⁇ 1) was added for the purpose of removing type I DNA and incubated at 37 ° C for 15 minutes.
  • 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. .
  • VECTRE-AVIDIN VECTOR LABORATORIES, manufactured by Funakoshi
  • "Potinated copy RNA” and “hybridized product of biotinylated copy MA and single-stranded circular DNA” were removed.
  • add 250m of “VECTREX-AVID ⁇ _ ⁇ ” (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.
  • the composition of the reaction mixture 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)).
  • dNTPs dATP, dCTP, dGTP, dTTP
  • primer DNA synthetic 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 ⁇ g / 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.
  • L medium trypton 10 g, yeast extract 5 g, NaCl 10 g, pH 7.5
  • ampicillin 50 ⁇ g / ml
  • 1L plasmid automatic separation device
  • First strand cDNA synthesis reaction was performed using reverse transcriptase “Super Script TM II” (manufactured by GIBCO BRL) with 5 ⁇ g 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.
  • 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 ⁇ g / ml of salmon sperm DNA.
  • 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) polyviny
  • 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.).
  • 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.
  • 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.
  • Lanes 2, 4, and 6 show the mRNA of cells treated with dexamethasone
  • lanes 1, 3, and 5 show the mRNA of cells not treated with dexamethasone
  • Lanes 1, 3, and 5 show the lysis of dexamethasone.
  • 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).
  • the sequencing reaction was performed using “Taq DyeDeoxy TM 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.
  • 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 ⁇ .
  • 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.
  • the phage was adsorbed from the plate to a nylon filter “GeneScreen TM 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.
  • 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.
  • the plasmid of a clone having an insert cMA of less than 1.2 kb was named “pBlue-r # 5 1.2”.
  • the DNA base sequence of the insert cDNA of “ ⁇ 1 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
  • 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.
  • 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 1 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.
  • This gene had a region encoding a new protein consisting of 467 amino acid residues according to MOlbp's 0RF (open reading frame).
  • ESTs short cDNA fragments of unknown function
  • the gene for the renal tubular interstitial nephritis antigen tubu lointerstitical nepharitis antigen mRNA (LOCUS name: Ozono 270)
  • LCUS name Ozono 270
  • 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 ( ⁇ sagi version). Rather than the family or super family.
  • 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”.
  • 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
  • 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” 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.
  • the gene fusion method using PCR 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.
  • the first step mix the two PCR products in equal volumes (40 ⁇ g 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.
  • 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].
  • 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 ⁇ .
  • dNTPs dATP, dCTP, dGTP, TTP
  • primer-DNA 1 zM
  • 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”.
  • 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.
  • lung-derived cells “MRC-5 SV1 TGI cells” (Riken Cell Bank) were cultured with “RITC 80-7 medium” supplemented with 10% FCS.
  • 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.
  • GISP transcriptional repressive activity
  • 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 ⁇ l of ⁇ GISP '' expression vector ( ⁇ pEF-h # 5 '' or ⁇ pEF-r # 5 '').
  • “PRL-EF” was 0.05 / g.
  • dexamethasone final concentration 1 M
  • IL-15 final concentration 100 U / ml
  • Cell extracts were prepared using "Passive LysisBuffer” (Promega).
  • 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
  • Figure 5A shows the vector with the human luciferase gene downstream of the human IL-8 promoter.
  • 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.
  • GISP is not a significant factor due to the elongation factor—1 ⁇ -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.
  • 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
  • peptides having a cysteine residue added to the N-terminus were used.
  • TBS Tris-HC1, 0.5 M NaCl (pH 7.5)”
  • TTBS 0.1% Tween 20
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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))
  • TNF-H Shakhov, AN et.al. J. Exp.Med. 171, 35-47 (1990)
  • IL-6 Kerat, T. et.al. Proc. Nat. Acad. Sci. US 90, 10193-7 (1993)
  • NOS nitric oxide synthase
  • Gly Arg lie Tyr Pro Val Leu Gly Thr Tyr Trp Asp Asn Cys Asn Arg
  • Val Ala Ser Asp Arg Val Ser lie His Ser Leu Gly His Met Thr Pro
  • Asn Asp Lys Glu lie Met Lys Glu Leu Met Glu Asn Gly Pro Val Gin
  • Sequence type nucleic acid
  • Val Ser lie His Ser Leu Gly His Met Thr Pro lie Leu Ser Pro Gin
  • Sequence type nucleic acid
  • 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
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • CTCGCTCGCC CAATGTGGCG ATGTCCACTG GGGCTATGC 39 SEQ ID NO: 15
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthesis MA Array.
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic MA
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid Number of chains: single strand
  • 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

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Abstract

L'invention concerne une protéine présentant l'activité d'inhiber l'activation du promoteur d'IL-8 en réponse à une stimulation extracellulaire spécifique; un ADN codant cette protéine; un vecteur contenant cet ADN; un transformant portant ce vecteur; un anticorps contre la protéine et des compositions médicinales contenant la protéine en tant que principe actif. Du fait de l'activité d'inhibition de l'activation du promoteur d'IL-8, on pense que la protéine précitée est applicable à des médicaments de traitement de maladies liées à l'expression de l'IL-8, par exemple, des agents anti-inflammatoires, des agents anti-asthme bronchique, des agents antiallergiques et des agents anti-rhumatismaux.
PCT/JP1998/000836 1997-02-28 1998-02-27 Inhibiteur de la transmission de signaux intracellulaires Ceased WO1998038213A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999043831A1 (fr) * 1998-02-25 1999-09-02 Smithkline Beecham Plc Cprot03, cysteine protease humaine
WO1999014234A3 (fr) * 1997-09-17 1999-09-02 Genentech Inc Stimulation ou inhibition de l'angiogenese et de la cardiovascularisation
WO2000015792A3 (fr) * 1998-09-14 2000-09-21 Genentech Inc Promotion ou inhibition d'angiogenese et de cardiovascularisation

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Title
BRETON J. J., CHABOT-FLETCHER M. C.: "THE NATURAL PRODUCT HYMENIALDISINE INHIBITS INTERLEUKIN-8 PRODUCTION IN U937 CELLS BY INHIBITION OF NUCLEAR FACTOR-KAPPA B.", JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, US, vol. 282., no. 01., 1 January 1997 (1997-01-01), US, pages 459 - 466., XP002910635, ISSN: 0022-3565 *
HARANT H., ET AL.: "1ALPHA,25-DIHYDROXYVITAMIN D3 AND A VARIETY OF ITS NATURAL METABOLITES TRANSCRIPTIONALLY REPRESS NUCLEAR-FACTOR-KB-MEDIATED INTERLEUKIN-8 GENE EXPRESSION.", EUROPEAN JOURNAL OF BIOCHEMISTRY, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 282., no. 01., 1 November 1997 (1997-11-01), GB, pages 63 - 71., XP002915093, ISSN: 0014-2956, DOI: 10.1111/j.1432-1033.1997.00063.x *
MUKAIDA N., ET AL.: "NOVEL MECHANISM OF GLUCOCORTICOID-MEDIATED GENE REPRESSION. NUCLEAR FACTOR -KAPPA B IS TARGET FOR GLUCOCORTICOID-MEDIATED INTERLEUKIN 8 GENE REPRESSION.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 269., no. 18., 6 May 1994 (1994-05-06), US, pages 13289 - 13295., XP002910637, ISSN: 0021-9258 *
SIMEONOVA P. P., LUSTER M. I.: "ASBESTOS INDUCTION OF NUCLEAR TRANSCRIPTION FACTORS AND INTERLEUKIN8 GENE REGULATION.", AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY., AMERICAN LUNG ASSOCIATION, NEW YORK, NY, US, vol. 15., no. 06., 1 January 1996 (1996-01-01), NEW YORK, NY, US, pages 787 - 795., XP002910636, ISSN: 1044-1549 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999014234A3 (fr) * 1997-09-17 1999-09-02 Genentech Inc Stimulation ou inhibition de l'angiogenese et de la cardiovascularisation
WO1999043831A1 (fr) * 1998-02-25 1999-09-02 Smithkline Beecham Plc Cprot03, cysteine protease humaine
WO2000015792A3 (fr) * 1998-09-14 2000-09-21 Genentech Inc Promotion ou inhibition d'angiogenese et de cardiovascularisation

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