WO2002033071A1 - Polypeptides analogues a la survivine et leurs adn - Google Patents

Polypeptides analogues a la survivine et leurs adn Download PDF

Info

Publication number
WO2002033071A1
WO2002033071A1 PCT/JP2001/009071 JP0109071W WO0233071A1 WO 2002033071 A1 WO2002033071 A1 WO 2002033071A1 JP 0109071 W JP0109071 W JP 0109071W WO 0233071 A1 WO0233071 A1 WO 0233071A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
salt
dna
activity
present
Prior art date
Application number
PCT/JP2001/009071
Other languages
English (en)
Japanese (ja)
Inventor
Hiroshi Tanaka
Isao Kaieda
Original Assignee
Takeda Chemical Industries, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Chemical Industries, Ltd. filed Critical Takeda Chemical Industries, Ltd.
Priority to AU2001294267A priority Critical patent/AU2001294267A1/en
Publication of WO2002033071A1 publication Critical patent/WO2002033071A1/fr

Links

Classifications

    • 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
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a survivin-like polypeptide having a BIR (baculovirus IAP repeat) domain or a BIR domain and a RING--nger domain and its DNA, and a screening method for an anticancer agent or the like, characterized by using the polypeptide or the like.
  • BIR Baculovirus IAP repeat
  • Apoptosis is, for example, cell shrinkage, chromatin condensation, nucleus condensation, cell surface microvilli loss, blebbing of large and small processes, apoptotic body formation, gaps with peripheral cells accompanying cell shrinkage, neighboring cells (Japanese clinical practice, Vol. 54, No. 7 (1996)).
  • Apoptosis or programmed cell death plays an important role in ontogeny, maintenance of homeostasis, and so on. It is becoming increasingly clear that abnormalities in apoptosis can cause diseases such as cancer, autoimmune diseases, and neurological diseases.
  • molecules involved in apoptosis regulation have been isolated one after another, and the regulation mechanism has been gradually revealed.
  • the caspase family is a group of molecules involved in the execution of apoptosis.
  • the caspase family plays a central role in the signal transduction process leading to apoptosis.
  • the Inhibitor of apoptosis protein (hereinafter also referred to as “IAPJ”) family. (The latest medicine, vol. 54, p. 861, (1999), clinical immunity, vol. 32, p. 1 (1999)).
  • the structure of the IAP family proteins is very well conserved, and there are two domains called BIR (baculovirus IAP repeat) and RING-finger.
  • BIR baculovirus IAP repeat
  • the BIR domain is found in all IAP-family proteins, and is mainly repeated three times at the N-terminal side. This sequence consists of about 70 amino acids, cysteine and conserved sequences of the histidine residues (RX 7 PX n ⁇ U) GXGX, DX; i CX, characterized in CX B ff3 ⁇ 4DX 5 HX B C) .
  • RING-finger domain is present in the C-terminal side, also cysteine and histidine residues are well conserved (C3 ⁇ 4CX (11 _ 2 CX HX M C3 ⁇ 4CX t5 _ 17] CX 2 C), this Domein all IAP family
  • This RING-finger domain is a sequence found in the consensus sequence of ubiquitin ligase and has been reported to ubiquitinate and degrade the target protein or itself [Science ), 288, 874 (2000)].
  • Survivin also inhibits caspases 3, 7 and exerts an apoptosis-suppressing action [Cancer Research, 58, 5315 (1998)]. However, unlike other IAP family proteins, Survivin is rarely expressed in normal tissues and is very frequently expressed in cancer tissues [Nature Medicine, 3, 917 P. (1997)]. In addition, it has been reported that the survival rate of colorectal cancer patients who express Survivin is lower than that of patients who do not, indicating that Survivin is involved not only in the development of cancer but also in malignant transformation It is suggested that this is the case [Oncogene, vol. 17, p. 3247 (1998)].
  • the Survivin-like polypeptide which belongs to the IAP family of proteins due to the characteristics of Survivin described above, is an excellent therapeutic agent for apoptosis disorder and can be used for screening excellent anticancer agents, etc. The development of such DNA is expected. Disclosure of the invention
  • SLIPJ survivin-like protein
  • Splicing varia nt (SLIP-long gene (consisting of the amino acid sequence represented by SEQ ID NO: 5)
  • S It was expressed as a LIP-short gene (consisting of the amino acid sequence represented by SEQ ID NO: 7)
  • SLIP-ring gene (consisting of the amino acid sequence represented by SEQ ID NO: 9).
  • SLIP-long has a BIR domain that is conserved between IAP families, and was found to be involved in apoptosis suppression via caspase inhibition. Also, since SLIP-long does not have a RING-finger domain like Survivin, its protein is not easily degraded. In addition, SLIP-long is thought to be involved in the process of canceration.
  • the SLIP-short gene which is a single nucleotide polymorphism (SNPs) of the SLIP-long gene, has a BIR domain conserved between IAP families, but does not have an inhibitory effect on apoptosis via caspase inhibition Became.
  • SNPs single nucleotide polymorphism
  • SLIP-ring has a RING-finger domain in addition to the BIR domain and promotes apoptosis induced by etoposide. Furthermore, SLIP-long, SLIP-short, and SLIP-ring are useful as cancer antigens because their expression is specifically observed in certain types of cancer tissues. Therefore, it promotes apoptosis in cancer cells by (1) reducing the amount of SUP-long protein in vivo or (2) inhibiting the binding of SLIP-long to caspases. Can be expected. Furthermore, since the isolated SLIP-long has lower expression in normal tissues than Survivin, it is considered that suppressing its expression and activity from the viewpoint of side effects may be an effective cancer treatment.
  • the present invention provides the following inventions.
  • polypeptide of the above-mentioned [1] which comprises the amino acid sequence represented by SEQ ID NO: 5, its amide, its ester, or a salt thereof.
  • a polynucleotide comprising a polynucleotide encoding the polypeptide of [1].
  • the polypeptide according to [1] which comprises culturing the transformant according to [7], producing and accumulating the polypeptide according to [1], and collecting the polypeptide.
  • a medicament comprising the polypeptide of the above-mentioned [1], an amide or an ester thereof, or a salt thereof.
  • a medical comprising the polynucleotide according to [3].
  • the antibody of the above-mentioned [14] which is a neutralizing antibody that inactivates the activity of the polypeptide of the above-mentioned [1], its amide or its ester, or a salt thereof.
  • a diagnostic agent comprising the antibody of the above [14].
  • a diagnostic agent comprising the polynucleotide of [3].
  • a pharmaceutical comprising the antisense DNA according to the above [22].
  • [26] Function is caspase inhibitory activity, apoptosis inhibitory activity, Tumor Necrosis Receptor Associated Factor (TRAF) binding activity, Receptor Interacting Protein (RIP) binding activity or Drosophila apoptosis
  • TNF Tumor Necrosis Receptor Associated Factor
  • RIP Receptor Interacting Protein
  • the ratio of the amount of the labeled polypeptide bound to the antibody when the polypeptide is competitively reacted with the polypeptide, and (ii) cells having the ability to express the gene of the polypeptide are compared with the test compound.
  • the antibody to the polypeptide is reacted with the cell culture solution (test solution) and the labeled polypeptide in a competitive manner, the antibody bound to the polypeptide is labeled with the antibody.
  • polypeptide or amide or ester thereof or polypeptide or amide thereof of [1] which can be obtained using the screening method of [25] or the screening kit of [32].
  • polypeptide or amide or ester thereof or the amide or ester thereof of the above-mentioned (1) which can be obtained by using the screening method of the above-mentioned [25] or the screening kit of the above-mentioned [32]. Promotes or inhibits the function of salt Comprising a compound having an active activity or a salt thereof.
  • a prophylactic or therapeutic agent for cancer comprising a compound having an activity of inhibiting the function of a salt or a salt thereof.
  • polypeptide or amide or ester thereof or polypeptide thereof according to [1] which can be obtained by using the screening method according to [25] or the screening kit according to [32].
  • a prophylactic / therapeutic agent for an apoptosis disorder comprising a compound having an activity of promoting the function of a salt or a salt thereof.
  • a method for preventing or treating apoptosis disorder which comprises administering to a mammal an effective amount of the polypeptide according to [1], the amide thereof, the ester thereof, or a salt thereof.
  • a method for preventing and treating abnormal apoptosis which comprises administering to a mammal an effective amount of the polynucleotide according to [3].
  • a method for preventing or treating cancer which comprises administering an effective amount of the antibody according to [14] to a mammal.
  • a method for preventing and treating cancer which comprises administering to a mammal an effective amount of the antisense DNA described in [22].
  • polypeptide of the above-mentioned [1] or an amide or an ester thereof which can be obtained from a mammal using the screening method of the above-mentioned [25] or the screening kit of the above-mentioned [32].
  • a method for preventing and treating cancer which comprises administering an effective amount of a compound having an activity of inhibiting the function of a salt thereof or a salt thereof.
  • polypeptide of the above-mentioned [49] which comprises the amino acid sequence represented by SEQ ID NO: 9 and has an apoptosis-promoting effect, or an amide or ester thereof, or a salt thereof;
  • An apoptosis-promoting agent comprising the polypeptide of the above-mentioned [49], an amide thereof, an ester thereof or a salt thereof.
  • a prophylactic / therapeutic agent for cancer comprising the polypeptide described in [49] or an amide or ester thereof or a salt thereof.
  • (53) contains a polynucleotide encoding the polypeptide of (49) above An apoptosis promoting agent comprising a polynucleotide.
  • a prophylactic / therapeutic agent for cancer comprising a polynucleotide comprising a polynucleotide encoding the polypeptide of [49].
  • a diagnostic agent for cancer comprising an antibody against the polypeptide according to [49] or an amide or ester thereof, or a salt thereof.
  • a diagnostic agent for cancer comprising a polynucleotide comprising a polynucleotide encoding the polypeptide of [49].
  • [58] has a nucleotide sequence complementary to or substantially complementary to the DNA encoding the polypeptide of [49] or a part thereof, and has an effect of suppressing expression of the DNA; Prevention of abnormal apocytic diseases containing antisense DNA.
  • a cell capable of expressing the polypeptide gene according to [49] is cultured, and an antibody against the polypeptide and a cell culture solution (test solution) and labeled The ratio of the amount of the labeled polypeptide bound to the antibody when the polypeptide is competitively reacted with the polypeptide, and (ii) a cell capable of expressing the polypeptide gene Is cultured in the presence of the test compound, and the antibody against the relevant peptide and the cell culture solution (test solution) and the labeled
  • a cell capable of expressing the polypeptide gene described in [49] above is cultured, and a cell culture solution (test solution) and an antibody against the polypeptide insolubilized on a carrier are obtained. And the activity of the labeling agent on the insolubilized carrier when simultaneously or sequentially reacted with another labeled antibody against the polypeptide, and (ii) the ability of the polypeptide to express the gene.
  • the cells are cultured in the presence of a test compound, and a cell culture solution (test solution) is simultaneously or continuously treated with an antibody of the polypeptide insolubilized on a carrier and an antibody against another labeled polypeptide of interest.
  • the screening method according to the above [59] wherein the activity is compared with the activity of the labeling agent on the insolubilized carrier when the reaction is carried out.
  • the polypeptide or amide or amide thereof of [49] which can be obtained using the screening method of [59] or the screening kit of [66].
  • polypeptide according to [49], its amide or ester thereof which can be obtained by using the screening method according to [59] or the screening kit according to [66].
  • a medicament comprising a compound having an activity of promoting or inhibiting the apoptosis promoting activity of those salts or a salt thereof.
  • polypeptide or amide or ester thereof or the amide or ester thereof of the above [49] which can be obtained using the screening method of the above [59] or the screening kit of the above [66].
  • a prophylactic / therapeutic agent for cancer comprising a compound having an activity of promoting the apoptosis promoting activity of those salts or a salt thereof.
  • the polypeptide or amide or ester thereof or the amide or ester thereof of the above [49] which can be obtained by using the screening method of the above [59] or the screening kit of the above [66].
  • a prophylactic / therapeutic agent for an apoptotic disorder comprising a compound having an activity of inhibiting the apoptosis-promoting activity of those salts or a salt thereof.
  • a method for preventing and treating cancer which comprises administering to a mammal an effective amount of the polypeptide according to [49], the amide thereof, the ester thereof, or a salt thereof.
  • a method for preventing and treating cancer which comprises administering to a mammal an effective amount of a polynucleotide containing a polynucleotide encoding the polypeptide of [49].
  • prevention of apoptosis dysfunction which comprises administering to a mammal an effective amount of an antibody against the polypeptide of the above [49], an amide thereof, an ester thereof, or a salt thereof; Method of treatment.
  • a mammal having a base sequence complementary to or substantially complementary to a DNA encoding the polypeptide of [49] or a portion thereof with respect to a mammal, and suppressing the expression of the DNA;
  • a method for preventing and treating apoptosis-disorder disease which comprises administering an effective amount of an antisense DNA having an effect of obtaining the same.
  • a polynucleotide comprising a polynucleotide encoding the polypeptide of [81].
  • the above-mentioned [81] which comprises culturing the transformant of the above-mentioned [87], producing and accumulating the polypeptide of the above-mentioned [81], and collecting this.
  • a diagnostic agent comprising the antibody of the above [89].
  • a diagnostic agent comprising the polynucleotide of [83].
  • [96] A method for diagnosing cancer, comprising using the antibody according to [89] and the antibody according to [14].
  • FIG. 1 shows the results of expression distribution of SLIP in human tissues.
  • FIG. 2 shows the results of expression distribution of Survivin and SLIP genes.
  • FIG. 3 shows the amount of DNA fragmentation when etoposide was added to the cell line.
  • FIG. 4 shows the amount of DNA fragmentation when the cell line was irradiated with ultraviolet light.
  • FIG. 5 shows the results of fluorescence microscopy of cDNA3.1 myc-SLIP-long, pcDNA3.1 myc-SLIP-short and pcDNA3.1 myc-SLIP-ring-introduced cells.
  • FIG. 6 shows the expression level of the SLIP gene in cancer tissues and normal tissues.
  • FIG. 7 shows the results of Western plotting using a polyclonal antibody of the egret.
  • FIG. 8 shows the results of Western blotting using a peptide antibody.
  • SLIP-long a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5 of the present invention
  • SLIP-shorU a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 7 of the present invention
  • the polypeptide of the present invention having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 9 is a warm animal (eg, , Human, guinea pig, rat, mouse, nit Cells (eg, hepatocytes, spleen cells, nerve cells, glial cells, knee 3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, etc.) Epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils , Monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, breast cells, hepatocytes or stromal cells, or precursor
  • SLIPs such as SLIP-ling, SLIP-short and SLIP-ring, or their amides or their esters, or their salts may be collectively referred to as “Po of the present invention.
  • the polypeptide containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 includes, for example, an activity substantially the same as the polypeptide containing the amino acid sequence represented by SEQ ID NO: 5 (eg, Caspase (especially lower caspases such as caspase 3 and caspase 7) inhibitory activity, apoptosis inhibitory activity, TRAF (Tumor Necrosis Receptor Associated Factor) binding activity, RIP (Receptor interacting Protein) binding activity, RPR (apotos is— inducing protein of (Drosophia, Reaper) binding activity).
  • Caspase especially lower caspases such as caspase 3 and caspase 7
  • TRAF Tumor Necrosis Receptor Associated Factor
  • the caspase inhibitory activity described in the present specification means an activity of inhibiting caspase activity by a mechanism such as inhibition of activation of a protease, a kind of protease, and more specifically, caspase. Means an activity of inhibiting caspase activation by binding to caspase.
  • the caspase inhibitory activity can be measured according to a known method.
  • the caspase inhibitory activity can be measured according to a screening method described later.
  • polypeptide having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 7 of the present invention include, for example, the same as the polypeptide containing the amino acid sequence represented by SEQ ID NO: 7, Caspase (especially lower caspases such as caspase 3 and caspase 7) inhibitory activity, apoptosis inhibitory activity, TRAF (Tumor Necrosis Receptor Associated Factor) binding activity, RIP (Receptor Interacting Protein) thread p-combining activity, RPR (apotos is- (Inducing protein of Drosophia, Reaper) Polypeptides having no activity such as binding activity are preferred.
  • polypeptide having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 9 of the present invention include, for example, a polypeptide substantially containing the amino acid sequence represented by SEQ ID NO: 9 Polypeptides having the same activity (for example, activity of promoting apoptosis by etoposide) are preferable.
  • polypeptide of the present invention examples include: (1) one or two or more (preferably about 1 to 10) in the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9; More preferably, an amino acid sequence in which a number (1 to 5), particularly preferably 1, 2 or 3 amino acids have been deleted, (2) the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 An amino acid sequence obtained by adding one or two or more (preferably about 1 to 10, more preferably 1 to 5 and particularly preferably 1, 2 or 3) amino acids to the amino acid sequence; 5, 1 or 2 or more (preferably about 1 to 10, more preferably 1 to 5), particularly preferably 1, 2, or 3 in the amino acid sequence represented by SEQ ID NO: 7 or SEQ ID NO: 9 Amino acid with 3) amino acids inserted Sequence, 4 SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 1 or 2 or more (preferably in the amino acid sequence represented by 9 Is an amino acid sequence in which about 1 to 10,
  • polypeptide of the present invention is a polypeptide of the present invention.
  • polypeptide in the present specification is an N-terminus (amino terminus) at the left end and a C-terminus (carboxyl terminus) at the right end according to the convention of peptide labeling. It is.
  • the polypeptides of the present invention including polypeptides containing the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, generally have a carboxyl group at the C-terminus (one COOH), It may be a carboxylate (one C ⁇ —), an amide (one CONH 2 ) or an ester (one C ⁇ R).
  • R in the ester e.g., methyl, Echiru, n- propyl, C alkyl group such as isopropyl or ⁇ - butyl, Shikurobe pentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, C 6 one 12 Ariru groups such as ⁇ one naphthyl, for example, benzyl, full of such phenethyl Eniru - ( ⁇ _ 2 alkyl or ⁇ - naphthylmethyl etc. ⁇ - Nafuchiru C 1 over such 2 Arukiru group (7 In addition to ⁇ 4 aralkyl groups, pivaloyloxymethyl groups commonly used as oral esters are used.
  • the polypeptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the polypeptide of the present invention.
  • the ester of this bain for example, the above-mentioned C-terminal ester and the like are used.
  • the amino group of the amino acid residue at the ⁇ -terminal (eg, methionine residue) is protected by a protecting group (for example, a C-acyl group such as a formyl group or an acetyl group, etc.).
  • a protecting group for example, a C-acyl group such as a formyl group or an acetyl group, etc.
  • N-terminal glutamine residue formed by pyroglutamine oxidation substituent on the side chain of amino acid in the molecule (eg, 1 OH, —SH, amino group, imidazole group, indole group, guanidino group) a suitable protecting group (e.g., formyl group, a C i-s Ashiru groups such as C Bok 6 Arukanoiru group such as ⁇ Se ethyl group) in shall be protected, a sugar chain such as glycopeptides bound polypeptide And other composite polypeptides.
  • a suitable protecting group e.g., formyl group, a C i-s Ashiru groups such as C Bok 6 Arukanoiru group such as ⁇ Se ethyl group
  • a salt of the polypeptide of the present invention a salt with a physiologically acceptable acid (eg, an inorganic acid, an organic acid) or a base (eg, an alkali metal salt) is used, and particularly, a physiologically acceptable salt is used. Acid addition salts are preferred.
  • a physiologically acceptable acid eg, an inorganic acid, an organic acid
  • a base eg, an alkali metal salt
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid
  • polypeptide of the present invention can also be produced from the cells or tissues of warm-blooded
  • the tissues or cells of the warm-blooded animal are homogenized, extracted with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Purification and isolation can be performed by combining them.
  • resins for polypeptide synthesis can be usually used.
  • resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin , 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenylhydroxymethyl ) Phenoxy resin, and 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target polypeptide according to various known condensation methods.
  • the polypeptide is cleaved from the resin and, at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target polypeptide or an amide thereof.
  • the protected amino acids described above various activating reagents that can be used for polypeptide synthesis can be used, and carbodiimides are particularly preferable.
  • the carbodiimides DCC, ⁇ , ⁇ ′-diisopropyl carbodiimide, ⁇ -ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used.
  • the protected amino acid may be added directly to the resin along with the racemization inhibitor additive (eg, HOBt, HOOBt) or may be pre-protected as a symmetrical acid anhydride or HOBt ester or HOOB ester. Can be added to the resin after activation.
  • the solvent used for activating the protected amino acid or for condensing with the resin can be appropriately selected from solvents known to be usable for the polypeptide condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, chloroform, alcohols such as trifluoroethanol , Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof.
  • the reaction temperature is appropriately selected from the range known to be usable for the reaction for forming a polypeptide bond, and is usually selected from the range of about ⁇ 20 ° C. to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the starting amino group include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, UZ, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, fluoryl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the carboxyl group may be, for example, an alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) linear, branched or cyclic alkyl.
  • Esterification aralkyl esterification (eg, benzyl ester, 4-ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide, etc. Can be protected.
  • an alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclo
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • Examples of a group suitable for etherification include a benzyl group, a tetrahydrovinyl group, a t-butyl group, and the like.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz l, CI 2 -Bz K 2- nitrobenzyl, Br @ - Z, such as t- butyl are used.
  • imidazole protecting group for histidine for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, BoTrt, Fmoc and the like are used.
  • Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2, 4-dinito ⁇ -phenol, cyanomethyl alcohol , Paranitrophenol, H0NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt).
  • active ester for example, pentachlorophenol, 2,4,5-trichlorophenol, 2, 4-dinito ⁇ -phenol, cyanomethyl alcohol , Paranitrophenol, H0NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt.
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or the like.
  • Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia Also used.
  • the elimination reaction by the above acid treatment is generally performed at a temperature of about ⁇ 20 ° C. to 40 ° C.
  • an amide form of the polypeptide for example, first, after amidating and protecting the ⁇ -carboxyl group of the carboxy terminal amino acid, a peptide (polypeptide) chain is added to the amino group side to a desired length. After the elongation, a polypeptide was prepared by removing only the protecting group of the ⁇ -terminal monoamino group of the peptide chain, and a polypeptide was obtained by removing only the protecting group of the carboxyl group at the C-terminus. In such a mixed solvent. Details of the condensation reaction are the same as described above.
  • ester of the polypeptide for example, after condensing the a-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the ester of the desired polypeptide can be obtained in the same manner as the amide of the polypeptide. You can get your body.
  • the polypeptide of the present invention can also be produced according to a known peptide synthesis method, or by cleaving a precursor containing the polypeptide of the present invention with an appropriate peptidase.
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, a partial peptide or amino acid capable of constituting the polypeptide of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the desired peptide. be able to .
  • Examples of the known condensation method and elimination of the protecting group include the methods described in the following 1 to 5.
  • the polypeptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
  • the polypeptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto. It can be converted to a free form or another salt by an analogous method.
  • a part of the nucleotide sequence of the DNA encoding the polypeptide ffl Is a polynucleotide containing a part of a nucleotide sequence complementary to the DNA, and is used to mean not only DNA encoding the polypeptide of the present invention but also RNA.
  • an antisense polynucleotide capable of inhibiting the replication or expression of the polypeptide gene of the present invention is cloned or determined to be a polypeptide of the present invention. It can be designed and synthesized based on the base sequence information of the DNA to be coded.
  • a polynucleotide can hybridize with the RNA of the polypeptide gene of the present invention, inhibit the synthesis or function of the RNA, or interact with the polypeptide-related RNA of the present invention.
  • the expression of the polypeptide gene of the present invention can be regulated and controlled.
  • Polynucleotides complementary to the selected sequence of the polypeptide-related RNA of the present invention and polynucleotides capable of specifically hybridizing with the polypeptide-related RNA of the present invention can be used in vivo and in vitro. It is useful for regulating and controlling the expression of a polypeptide gene, and for treating or diagnosing diseases.
  • the term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of a peptide (protein) specified by a sequence derived from the nucleotide (nucleic acid) sequence or its complement.
  • the untranslated region, the 3'-end palindrome region, and the 3'-end hairpin loop may be selected as preferred regions of interest, but any region within the polypeptide of the present invention may be selected.
  • the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region can be said to be “antisense J.
  • Polynucleotides are polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, N-nucleotides of purine or pyrimidine bases.
  • polynucleotides that are lycosides, or other polymers with non-nucleotide skeletons (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing specialized linkages, except The polymer contains a nucleotide having a configuration permitting base pairing and base attachment as found in DNA and RNA). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can further comprise unmodified polynucleotides (or unmodified oligonucleotides).
  • Nucleotides as well as those with known modifications, e.g., those labeled in the art, capped, methylated, one or more natural nucleotides replaced with analogs , Modified with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate) ), Such as proteins (nucleases, nucleases and inhibitors, Compounds having side chain groups such as amino acids, antibodies, signal peptides, poly-L-lysine, etc., sugars (eg, monosaccharides), and interactive compounds (eg, acridine, psoralen).
  • an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, oleb
  • nucleic acid Having a chelating compound (eg, metal, radioactive metal, boron, oxidizing metal, etc.), containing an alkylating agent, or having a modified bond (eg, ⁇ -anomer) Type nucleic acid).
  • a chelating compound eg, metal, radioactive metal, boron, oxidizing metal, etc.
  • alkylating agent e.g., boron, oxidizing metal, etc.
  • modified bond eg, ⁇ -anomer
  • nucleic acid may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
  • Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. May be converted to
  • the antisense 'polynucleotide (hereinafter, also referred to as antisense nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acids include, but are not limited to, sulfur derivatives of nucleic acids, thiophospho derivatives, and polynucleoside amides, which are resistant to degradation of oligonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, it makes the antisense nucleic acid more stable in the cell, enhances the cell permeability of the antisense nucleic acid, increases the affinity for the target sense strand, and is toxic. If so, make the toxicity of the antisense nucleic acid smaller.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional form.
  • Antisense nucleic acids used in such an additional form include polycations such as polylysine, which acts to neutralize the charge of the phosphate backbone, and lipids, which enhance the interaction with cell membranes and increase the uptake of nucleic acids. (Eg, phospholipid, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • nucleic acid can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond.
  • Other groups are cap groups specifically arranged at the 3 'end or 5' end of nucleic acids to prevent degradation by nucleases such as exonuclease and RNase. Is mentioned.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the antisense nucleic acid inhibitory activity can be determined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the peptide of the present invention. You can find out.
  • the nucleic acid can be applied to cells by various known methods.
  • the polynucleotide encoding the polypeptide of the present invention may be any polynucleotide that encodes the above-described polypeptide of the present invention.
  • DNA is preferred as such a polynucleotide.
  • Such DNA may be any of genomic DNA, genomic DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • amplification can be performed directly by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a preparation of total RNA or mRNA fraction from the above-mentioned cells and tissues.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • Examples of the DNA encoding the SUP-long of the present invention include: (1) DNA containing the base sequence represented by SEQ ID NO: 6, or DNA containing the base sequence represented by SEQ ID NO: 6 and high stringency Activity that is substantially the same as that of SLIP-long containing DNA that hybridizes under natural conditions and containing the amino acid sequence represented by SEQ ID NO: 5 (for example, caspases (particularly lower caspases such as caspase 3 and caspase 7).
  • Inhibitory activity apoptosis inhibitory activity, TRAF (Tumor Necrosis Re Geptor Associated Factor) binding activity, RIP (Receptor Interacting Protein) binding activity, RPR (apotosis-inducing protein of DrosopKia, Reaper) binding activity, etc.
  • TRAF Tumor Necrosis Re Geptor Associated Factor
  • RIP Receptor Interacting Protein binding activity
  • RPR apotosis-inducing protein of DrosopKia, Reaper binding activity, etc.
  • a DNA encoding a polypeptide or the like is used.
  • Examples of the DNA encoding the SLIP-short of the present invention include: (1) DNA containing the base sequence represented by SEQ ID NO: 8, or DNA containing the base sequence represented by SEQ ID NO: 8 and high stringency As with SLIP-short containing DNA that hybridizes under simple conditions and containing the amino acid sequence represented by SEQ ID NO: 7, caspase (especially lower caspases such as caspase 3 and caspase 7) inhibitory activity, Abortion not having apoptosis inhibitory activity, TRAF (Tumor Necrosis Receptor Associated Fa dor) binding activity, RIP (Receptor Interacting Protein) binding activity, RPR (apotosis-inducing protein of Drosophia, Reaper) binding activity
  • a DNA code is used.
  • DNA having the nucleotide sequence represented by SEQ ID NO: 10 or DNA having the nucleotide sequence represented by SEQ ID NO: 10 is highly stringent. It has a DNA that hybridizes under the conditions and has substantially the same activity as the SUP-ring containing the amino acid sequence represented by SEQ ID NO: 9 (for example, an activity to promote apoptosis by etoposide). Any DNA may be used as long as it encodes a polypeptide to be expressed.
  • DNAs that hybridize under high stringent conditions to DNA having the nucleotide sequence represented by SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 1.0 include, for example, SEQ ID NO: 6, SEQ ID NO: Has a homology of about 95% or more, preferably about 98% or more, more preferably about 99% or more, particularly preferably about 99.6% or more with the base sequence represented by 8 or SEQ ID NO: 10 DNA having a base sequence is used.
  • Hybridization is carried out according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Samrook et al., Cold Spring Harbor Lab. Press, 1989). be able to.
  • the procedure can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under highly stringent conditions.
  • High stringency conditions refer to, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C (preferably about 60 to 65 ° C). In particular, it is most preferable when the sodium concentration is about 19 mM and the temperature is about 65 ° C.
  • the DN encoding SUP-Iong containing the amino acid sequence represented by SEQ ID NO: 5 includes a DNA containing the base sequence represented by SEQ ID NO: 6, and the like.
  • Examples of the DNA encoding the SUP short having the amino acid sequence represented by SEQ ID NO: 8 include a DNA having the base sequence represented by SEQ ID NO: 8, and a SUP-ring having the amino acid sequence represented by SEQ ID NO: 9.
  • DNA containing the base sequence represented by SEQ ID NO: 10 is used as the DNA to be transferred. You can.
  • the nucleotide sequence represented by SEQ ID NO: 2 is represented by SEQ ID NO: 3 above.
  • the DNA sequence corresponding to the amino acid sequence corresponding to the RING-Finger domain includes, for example, the base sequence represented by SEQ ID NO: 4.
  • the DNA incorporated in the evening may be selected by hybridization with a DNA fragment coding for a part or the entire region of the polypeptide of the present invention or labeled with a synthetic DNA.
  • the hybridization method can be carried out, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When using a commercially available library, the method can be performed according to the method described in the attached instruction manual.
  • the DNA base sequence can be converted by PCR or a known kit such as Mutan TM -Super Express Km (Takara Shuzo) or Mutan TM -K (Takara Shuzo) using the 0DA-LA PCR method or the like. It can be carried out according to a known method such as the Gaed duplex method or the Kunkel method, or a method analogous thereto.
  • the DNA encoding the cloned polypeptide can be used as it is depending on the purpose, or can be used by digesting with a restriction enzyme or adding a linker, if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and may have TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.
  • the expression vector for the polypeptide of the present invention can be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the polypeptide of the present invention, and (mouth) appropriately expressing the DNA fragment. It can be manufactured by connecting to the downstream of the promoter in the vector.
  • the vector examples include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast (eg, pSH19, pSH15), bacteriophages such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., ⁇ 1-11, ⁇ 1, ⁇ Rc CMV, pRcZRSV, pc DNA I / Neo > pc DNA3.1 (+) is used.
  • Escherichia coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis eg, pUB110, pTP5, pC194
  • yeast eg, pSH19, pSH15
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SR promoter when animal cells are used as host, SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc.
  • CMV (cytomegalovirus) promoter It is preferable to use a promoter such as SRo; When the host is a genus Escherichia, the tr ⁇ promoter, lac promoter, recA promoter, ⁇ PL promoter, lpp promoter, T7 promoter, etc., and when the host is a Bacillus genus, When the host is yeast, such as SP-1 promoter, SP02 promoter, penP promoter, etc., PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV400 ri), if desired. Anything can be used.
  • the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) ⁇ Ko [meso Bok Rekise one Bok (MTX) resistance], ampicillin phosphorus resistance Yadenko (hereinafter, abbreviated as Amp r there is a case to be), neomycin resistance gene leading frame (hereinafter sometimes referred to as Ne o r, G418 resistance) and the like can be ⁇ up.
  • dhfr gene as a selection marker using Chinese hamster cells deficient in dhfr gene,! ⁇ Target genes in thymidine-free medium You can also select one.
  • a signal sequence suitable for the host is added to the N-terminal side of the polypeptide of the present invention.
  • a PhoA ′ signal sequence, a 0-A / signal sequence, etc. is used.
  • an ⁇ -amylase / signal sequence, a subtilisin / signal sequence, etc. are included in the host.
  • the host is an animal cell, such as MFa signal sequence and SUC2 signal sequence. If the host is an animal cell, the insulin signal sequence, ⁇ - ⁇ interferon signal sequence, and antibody molecule. 'Signal sequences can be used.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia examples include, for example, Escherichia coli Escheric hia coli K12 and DH1 [Procedures' ob 'the' National 'academy'ob' Sciences'ob 'the' USA (Pro Natl. Acad. Sc. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, Vol. 9, 309 (1 981)] , JA 22 1 [Journal of Molecular Biology], 120 volumes, 5 17 (1 978)], HB 10 1 [Journal of Molecular Biology, 41 volumes] , 459 (1969)], C600 [Genetics, Vol. 39, 440 (1954)], JMl09, etc. are used.
  • Bacillus ⁇ bacteria examples include, for example, Bacillus subtilis MI114 (Gene, 24, 255 (1983)), 207-21 (Journal oi Biochemistry), 95, 87 (1 1984)].
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, ⁇ 22R ⁇ , ⁇ 87-11A, DD-5D, 20B-12, Schizosaccharomyces pombe NC YC 1913, NCYC 2036, Pichia pastoris KM71 and the like are used.
  • the bizoid cells include, for example, when the virus is AcNPV, a cell line derived from a larva of night roth moth (Spodoptera irugiperda cell; Sf cell), an MGl cell derived from the midgut of Trichoplusia ni, and an egg of Trichoplusia ni High Five TM cells, cells derived from Mamestra b rassicae or cells derived from Eslgmena acrea are used.
  • TM cells derived from Mamestra b rassicae or cells derived from Eslgmena acrea are used.
  • the virus is BmNPV
  • a silkworm-derived cell line Boombyx mori N cell; BmN cell
  • S ⁇ cells include Sf9 cells (iVTCC CRL1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like. Used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey cells COS-7, Vero, Chinese Hams Yuichi cell CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (abbreviated as CHO (dhfr-) cells).
  • CHO cells monkey cells COS-7, Vero, Chinese Hams Yuichi cell CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (abbreviated as CHO (dhfr-) cells).
  • CHO cells monkey cells COS-7, Vero, Chinese Hams Yuichi cell CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (abbreviated as CHO (dhfr-) cells).
  • Mouse L cells mouse AtT-20, mouse myeloma cells, rat GH3, and human FL cells.
  • Proc. Natl. Acad. Sci. USA, 69 can be used to transform a microorganism belonging to the genus Escherichia. Vol. 2110 (1972) and Gene (17), 107 (1982), etc., can be used to transform Bacillus ⁇ bacteria by, for example, molecular 'and' dieneral- Genetics (Molecular & General Genetics), 168, 111 (1979).
  • Transformation of bizoid cells or insects can be carried out according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as the medium used for the culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salt, nitrate, cone chip liquor, peptone, casein, meat extract, soybean meal, and potato extract.
  • Inorganic or organic substances such as liquids, and inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • ⁇ of the medium is preferably about 5 to 8.
  • Examples of a medium for culturing Escherichia bacteria include, for example, ⁇ 9 medium containing glucose and casamino acid (Miller, Journal 'Ob' Experimentin ',' Journal of Experiments in Molecular Genetics' ), 431-433, Cold Spring Harbor Laboratory, New York 1972).
  • a drug such as 33-indolylacrylic acid can be added to make the promoter work efficiently if necessary.
  • the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
  • culturing is usually performed at about 30 to 40 ° C. for about 6 to 24 hours. If necessary, aeration and stirring can be added.
  • the culture medium When culturing a transformant in which the host is an insect cell or an insect, the culture medium is 10% immobilized in Grace> s Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). A solution to which an additive such as a serum is appropriately added is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • the medium When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science '122, 501 (1952)], DMEM Medium [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association, 1999, 519, 519] (1967)], 199 medium (Proceeding ⁇ the Society for the Biological Medicine, Vol. 73, 1 (1950)) Can be Preferably, the pH is about 6-8. Cultivation is usually carried out at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • polypeptide of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • polypeptide of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells are cultured, and then the cells are rested or cells in a known manner. After disrupting the cells or cells by waves, lysozyme and / or freeze-thawing, a method of obtaining a crude polypeptide extract by centrifugation or filtration is used as appropriate.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-1000 TM .
  • Purification of the polypeptide contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods.
  • These known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method using a difference in hydrophobicity such as isoelectric point electrophoresis and a method using a difference in isoelectric point are used.
  • the polypeptide thus obtained when obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto. It can be converted to a free form or other salts by a method analogous thereto.
  • the polypeptide produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, tribcine, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the presence or activity of the thus produced polypeptide of the present invention can be measured by enzyme immunoassay using a specific antibody or the like.
  • the antibody against the polypeptide of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the polypeptide of the present invention, but a monoclonal antibody is more preferable.
  • An antibody against the polypeptide of the present invention can be produced by using the polypeptide of the present invention as an antigen according to a known antibody or antiserum production method.
  • the polypeptide of the present invention is administered to a warm-blooded animal itself or together with a carrier or diluent at a site capable of producing an antibody upon administration.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration is usually performed once every 2 to 6 weeks, preferably about 2 to 10 times in total.
  • a power mouse and a rat including a monkey, a rabbit, a dog, a guinea pig, a mouse, a rat, a sheep, a goat, and a chicken are preferably used.
  • a warm-blooded animal immunized with an antigen for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them.
  • an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled polypeptide described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975) 3].
  • the fusion promoter include polyethylene glycol (PJEG) and Sendai virus, but PEG is preferably used.
  • PEG polyethylene glycol
  • myeloma cells include bone marrow femoral cells of warm-blooded animals such as NS-1, P3U1, SP 2/0, and AP-1, but P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is used at a concentration of about 10 to 80%.
  • Cell fusion can be carried out efficiently by adding the mixture and incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes.
  • Screening of monoclonal antibody-producing hybridomas can be carried out by a faded method.
  • polypeptide antigens are adsorbed on or in contact with a carrier.
  • Anti-globulin antibody is added to a solid phase (eg, microplate), and then labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody if the cells used for cell fusion are mice). Or a method to detect monoclonal antibodies bound to the solid phase by adding protein A, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, and adding radioactive substances, enzymes, etc. A method of adding a polypeptide labeled with, and detecting a monoclonal antibody bound to a solid phase.
  • the selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow eight hybridomas.
  • RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.)
  • a serum-free medium for hybridoma cultivation SFM-101, Nissui Pharmaceutical Co., Ltd.
  • the culture temperature is usually from 20 to 40 ° C, preferably about 37 ° C.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)).
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody.
  • the polyclonal antibody of the present invention is produced according to a known method or a method analogous thereto. can do. For example, an immunizing antigen (the polypeptide antigen of the present invention) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting an antibody-containing substance against the polypeptide of the present invention and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier. Any material may be cross-linked at any ratio if it can be efficiently used.For example, serum albumin, thyroglobulin, hemocyanin, etc., in a weight ratio of about 0.1 to 2 per hapten per hapten. A method of coupling at a rate of 0, preferably about 1 to 5 is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration is usually made once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.
  • an antibody against the polypeptide of the present invention examples include, for example, partial sequences of SLIP-long and SUP short: Glu-Pro-G 1 yA 1 -G 1 An antibody that recognizes yProG1yProProG1y (SEQ ID NO: 31) is used.
  • the use of the polypeptide of the present invention, the DNA encoding the polypeptide of the present invention (hereinafter, also referred to as “DNAJ of the present invention”), and the use of the antibody of the present invention will be described below.
  • the SLIP-long of the present invention or its amide, ester or salt thereof includes, for example, caspase (especially lower caspases such as caspase 3 and caspase 7) inhibitory activity, apoptosis inhibitory activity, TRAF (Tumor Necrosis Receptor) DNAs encoding SLIP-long of the present invention because they have physiological activities such as Associated Factor) binding activity, RIP (Receptor Interacting Protein) binding activity, and RPR (apotos is-inducing protein of Drosophia, Reaper) binding activity.
  • caspase especially lower caspases such as caspase 3 and caspase 7
  • TRAF Tumor Necrosis Receptor
  • RIP Receptor Interacting Protein binding activity
  • RPR apotos is-inducing protein of Drosophia, Reaper
  • the SLIP-long of the present invention and the DNA encoding the SLIP-long of the present invention can be used as, for example, a therapeutic / prophylactic agent for various diseases causing abnormalities in apoptosis (eg, Alzheimer's disease, autoimmune disease, etc.). It can be used as a medicine such as.
  • the DNA encoding the SUP-long of the present invention is inserted into (oral) cells, After expressing the SUP-long of the present invention, transplanting the cells into a patient, or (8) administering the SLIP-Iong of the present invention to the patient, or the like, to thereby obtain the SL of the present invention in the patient.
  • the role of IP-1 ong can be fully or normally exhibited.
  • the SUP-ring or its amide or its ester or a salt thereof of the present invention has, for example, an activity of promoting apoptosis by etoposide stimulation (more specifically, an activity of promoting etoposide stimulation on HEK293 cells).
  • DNA that encodes the SUP ring of the present invention because it has a physiological activity such as the activity of promoting apoptosis). Expression When the amount is reduced, diseases such as cancer develop.
  • the SLIP-ring of the present invention and the DNA encoding the SLIP-ring of the present invention include, for example, various cancers (eg, endometrial cancer, endometrial tumor, breast cancer, colorectal cancer, rectal cancer, prostate gland) It can be used as a drug for the treatment and prevention of cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.).
  • various cancers eg, endometrial cancer, endometrial tumor, breast cancer, colorectal cancer, rectal cancer, prostate gland
  • the function of SLIP-ring in cells may not be sufficiently or normally exhibited in some patients.
  • the DNA encoding the SLIP-ring of the present invention is administered to the patient, and the SLIP-ring of the present invention is expressed in vivo, whereby the (mouth) cell encodes the SUP-ring of the present invention.
  • transplanting the cells into a patient, or (8) administering the SLIP-ring of the present invention to the patient The role of SL IP-dng of the present invention in the patient can be fully or normally exerted.
  • the DNA of the present invention particularly the DNA encoding SLIP-long or the DNA encoding SLIP-ring is used as the above-mentioned therapeutic or prophylactic agent
  • the DNA may be used alone or as a retroviral vector. 1.
  • an appropriate vector such as an adenovirus vector or an adenovirus associated virus vector
  • it can be administered to a human or a warm-blooded animal according to a conventional method.
  • the DNA of the present invention can be administered as it is or in the form of a formulation with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and can be administered by a gene gun or a catheter such as a hide mouth gel catheter.
  • polypeptide of the present invention When used as the above-mentioned therapeutic / prophylactic agent, it is purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. It is preferred to use
  • polypeptide of the present invention particularly SLIP-long or SLIP-ring, or an amide or ester thereof, or a salt thereof can be used, for example, as a sugar-coated tablet, capsule, elixir, microcapsule, etc. It can be used orally or parenterally in the form of injections, such as sterile solutions with water or other pharmaceutically acceptable liquids, or suspensions.
  • injections such as sterile solutions with water or other pharmaceutically acceptable liquids, or suspensions.
  • Manufactured by mixing in physically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in the unit dosage form required for generally accepted drug practice. be able to.
  • the amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
  • a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • Various solubilizing agents for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 TM, HC 0-5 0, etc.).
  • the oily liquid include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, proforce hydrochloride, etc.
  • stabilizers eg, human blood albumin, polyethylene glycol, etc.
  • a preservative eg, benzyl alcohol, phenol, etc.
  • an antioxidant e.g, an antioxidant and the like.
  • the vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used non-periodically.
  • the preparations obtained in this way are safe and have low toxicity, for example warm-blooded animals (Eg, humans, rats, mice, guinea pigs, egrets, birds, higgs, bushes, pacific animals, pomas, cats, dogs, monkeys, chimpanzees, etc.).
  • warm-blooded animals Eg, humans, rats, mice, guinea pigs, egrets, birds, higgs, bushes, pacific animals, pomas, cats, dogs, monkeys, chimpanzees, etc.
  • the dose of the polypeptide of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like.
  • the SLIP-Iong of the present invention when orally administered for the purpose of treating a disorder of apoptosis, general In general, for adults (assuming 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of SLIP-long is administered daily.
  • the single dose of SLIP-Iong varies depending on the administration target, target disease, etc.
  • the SLIP-long of the present invention may be administered as an injection for the treatment of apoptotic disorder.
  • SLIP-long When administered to adults (assuming a body weight of 60 kg) in the form of SLIP-long, about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 mg of SLIP-long per day It is convenient to administer by injecting about 1010 mg into the affected area. In the case of other animals, the amount converted per 60 kg can be administered.
  • the SLIP-ring of the present invention When the SLIP-ring of the present invention is orally administered for the purpose of treating cancer, generally, in adults (as 60 kg), about 0.1 mg to 100 mg of SUP-ring per day, preferably About 1.0 to 5 Omg, more preferably about 1.0 to 20 mg is administered.
  • the single dose of the SLIP-ring varies depending on the administration target, target disease, etc.
  • the SLIP-ring of the present invention may be administered in the form of an injection (in the form of an injection) to treat cancer.
  • the amount of SLIP-ring should be about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day. It is convenient to administer by injection into the affected area. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the SUP-long of the present invention has caspase (especially lower caspases such as caspase 3 and caspase 7) inhibitory activity, apoptosis inhibitory activity, TRAF (Tumor Necrosis Receptor Associated Factor) binding activity, RIP (Receptor Interacting Protein) binding activity, RPR (apotosis-inducing protein of Drosophia, Reaper) binding activity Therefore, the functions of the SLIP-long of the present invention (for example, caspase (especially lower caspases such as caspase 3 and caspase 7) inhibitory activity, apoptosis inhibitory activity, TRAF (Tumor Necrosis Receptor Associated Factor) binding activity, RIP Compounds that inhibit (Receptor Interacting Protein) binding activity, RPR (apotosis-inducing protein of Drosophia, Reaper) binding activity, etc.) or salts thereof can be used for various cancers (eg, uterine body cancer, endometrial
  • the function of the SLIP-long of the present invention eg, caspase (especially lower caspases such as caspase 3 and caspase 7) inhibitory activity, apoptosis inhibitory activity, TRAF (Tumor Necrosis Receptor Associated Factor) binding activity, RIP (Receptor Interacting Protein)
  • a compound that promotes binding activity, RPR (apotosis-inducing protein of Drosophia, Reaper) binding activity, etc.) or a salt thereof can be used as a medicament such as a therapeutic or prophylactic agent for apoptotic disorder.
  • the compound or a salt thereof which promotes the function of the SLIP-ring of the present invention can be used for various cancers (eg, endometrial cancer) , Endometrial tumor, breast cancer, colorectal cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.).
  • the compound or a salt thereof that inhibits the function of the SUP-ring of the present invention can be used as a medicament such as an agent for treating or preventing apoptosis disorder.
  • a polypeptide of the invention in particular a SLIP-long or SLIP-ring or an amide or an ester or a salt thereof, or a DNA of the invention, in particular a DNA or SLIP-ring encoding a SLIP-long.
  • the encoding DNA is also useful as a probe for screening a compound or a salt thereof that promotes or inhibits the function of the polypeptide of the present invention.
  • the present invention provides a method for screening a compound having an activity of promoting or inhibiting the function of the polypeptide of the present invention, which comprises using the polypeptide of the present invention.
  • a method for screening a compound having an activity of promoting or inhibiting the function of the polypeptide of the present invention which comprises using the polypeptide of the present invention.
  • Cells that have the ability to express the gene A DNA encoding the polypeptide of the present invention, or a DNA encoding the polypeptide of the present invention or its complementary DNA or a partial DNA thereof, and measuring the amount of mRNA encoding the polypeptide of the present invention.
  • a method for screening for a compound having an activity of promoting or inhibiting the function of the polypeptide of the present invention or a salt thereof more specifically, (2) (i) culturing cells capable of expressing the gene of the polypeptide of the present invention
  • the expression level of mRNA of the polypeptide of the present invention in the case of the above, and the polypeptide of the present invention when cells having the ability to express the U gene of the polypeptide of the present invention are cultured in the presence of the test compound.
  • a method for screening for a compound having activity to promote or inhibit the function of the polypeptide of the present invention or a salt thereof which comprises comparing the amount of mRNA with the amount of mRNA.
  • Examples of cells having the ability to express the polypeptide gene of the present invention include the above-mentioned known warm-blooded animal cells and animal cells into which the polypeptide gene of the present invention has been introduced and transformed. Animal cells into which the polypeptide gene of the present invention has been introduced and transformed can be produced by the above-described method.
  • Culture of cells having the ability to express the gene of the polypeptide of the present invention is performed in the same manner as in a known animal cell culture method.
  • MEM medium containing about 5 to 20% of fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959) ))), RPMI 1640 medium [The Journal of the American Medical Association, 1999, 519 (1967)], 199 medium [Procedure. Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)].
  • the pH is about 6-8.
  • the cultivation is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and if necessary, aeration or stirring may be added.
  • the amount of the mRNA encoding the polypeptide of the present invention is measured by measuring the RNA extracted from the cells according to a known method and the DNA encoding the polypeptide gene of the present invention or its complementary DNA or This is carried out by contacting the partial DNA and measuring the amount of mRNA bound to the gene DNA of the polypeptide of the present invention or its complementary DNA.
  • the mRNA bound to the complementary DNA of the gene DNA of the polypeptide of the present invention can be obtained by labeling the complementary DNA of the gene DNA of the polypeptide of the present invention or a partial DNA thereof with, for example, a radioisotope or a dye. Can be easily measured.
  • Radioactive isotopes include, for example, 32 P and 3 H
  • dyes include, for example, nuorescein, FAM (manufactured by PE Biosystems), J ⁇ E (manufactured by PE Biosystems), TAMRA (manufactured by PE Biosystems) , R ⁇ X (manufactured by PE Biosystems), Cy5 (manufactured by Amersham), Cy3 (manufactured by Amersham) and the like are used.
  • the amount of mRNA of the polypeptide of the present invention is determined by converting RNA extracted from cells into cDNA with reverse transcriptase, and then encoding the gene of the polypeptide of the present invention or its complementary DNA or a part thereof. It can be performed by measuring the amount of cDNA to be amplified by PCR using DNA as a primer.
  • the complementary DNA of the gene DNA of the polypeptide of the present invention used for measuring the amount of mRNA of the polypeptide of the present invention includes a sequence complementary to the gene DNA (upper chain) of the polypeptide of the present invention. DNA (lower strand).
  • DNA lower strand
  • the partial DNA of the gene DNA of the polypeptide of the present invention for example, in the nucleotide sequence of the DNA gene of the polypeptide of the present invention, 10 to 2200 contiguous, preferably 10 to 300, more preferably A base sequence composed of 10 to 30, particularly preferably 10 to 20 bases is exemplified.
  • Examples of the partial DNA of the DNA complementary to the gene DNA of the polypeptide of the present invention include a DNA having a sequence complementary to the partial DNA of the DNA encoding the polypeptide of the present invention. That is, for example, in the nucleotide sequence of the DNA gene of the polypeptide of the present invention, the polypeptide is composed of 10 to 2200 K, preferably 10 to 300, more preferably 10 to 30, particularly preferably 10 to 20 bases. DNA having a sequence complementary to the base sequence I can do it. More specifically, the measurement of the mRNA amount of the polypeptide of the present invention can be specifically performed as follows.
  • mice Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc., more specifically, obese mice, arteriosclerotic mice) , Arteriosclerosis, egrets, cancer-bearing mice, etc.), drugs (eg, blood pressure lowering drugs, anti-cancer drugs, anti-obesity drugs, anti-hyperlipidemic drugs, etc.) or physical stress (eg, flooding stress, electric shock, light and darkness) After a certain period of time, blood or specific organs (eg, brain, liver, kidney, etc.) or tissues or cells isolated from the organs are obtained.
  • non-human mammals eg, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc., more specifically, obese mice, arteriosclerotic mice
  • drugs eg, blood pressure lowering drugs, anti-cancer drugs, anti-obesity drugs, anti-hyperlipidemic drugs
  • the mRNA of the polypeptide of the present invention contained in the obtained cells can be quantified by, for example, extracting mRNA from cells or the like by a usual method and using a technique such as TaciManPCR, for example.
  • the analysis can also be performed by performing a Northern blot.
  • a transformant expressing the polypeptide of the present invention is prepared according to the method described above, and mRNA of the polypeptide of the present invention contained in the transformant can be quantified and analyzed in the same manner.
  • a test compound that increases the amount of mRNA of the polypeptide of the present invention can be selected as a compound having an activity of promoting the expression of the gene of the polypeptide of the present invention.
  • a test compound that reduces the amount of mRNA of the polypeptide of the present invention can be selected as a compound having the activity of inhibiting the expression of the gene of the polypeptide of the present invention.
  • Known promoter of the polypeptide of the present invention (2) Cells transformed with the DNA obtained by cloning the enhancer region from genomic DNA and ligating upstream of an appropriate repo overnight gene (eg, adipocyte, macrophage, skeleton) Cultivation in the presence of a test compound, and detecting the expression of Levo-Yuichi-Seniki instead of the expression of the polypeptide of the present invention.
  • Promote or A method for screening a compound having an inhibitory activity or a salt thereof is provided.
  • the reporter gene for example, a staining marker gene such as lac Z ( ⁇ 3-galactosidase gene) and the like are used.
  • a test compound that increases the amount of the reporter gene product can be used to determine the activity of the polypeptide of the present invention to promote gene expression.
  • a test compound that reduces the amount of a reporter gene product can be selected as a compound having the polypeptide gene of the present invention.
  • the cells can be cultured in the same manner as in the known animal cell culture described above.
  • the SLIP-long of the present invention was expressed in Escherichia coli and purified, and then the test compound was added together with the caspase activity of the SLIP-long of the present invention and (ii) the SLIP-long of the present invention.
  • the caspase activity in this case is measured using a commercially available caspase activity measurement kit (PharMingen) or the like according to the attached protocol, and the comparison is performed to promote the function of the SLIP-long of the present invention.
  • a method for screening a compound having an inhibitory activity or a salt thereof is provided.
  • test compound which increases the caspase inhibitory activity in the case of the above (ii) by about 20% or more, preferably 30% or more, and more preferably about 50% or more in comparison with the case of the above (i) can be used. It can be selected as a compound that promotes the function of SLIP-long.
  • the caspase inhibitory activity in case (ii) above is lower than that in case (i) by 20% or more, preferably 30% or more, more preferably 50% or more, particularly preferably 70% or more.
  • the test compound to be tested can be selected as a compound that inhibits the function of the SLIP-long polypeptide of the present invention.
  • the present invention relates to i) an apoptosis promoting activity when apoptosis is induced by etoposide stimulation in HEK cells expressing SLIP-ring, and ii) the SLEK-ring-expressing HEK cells.
  • Apo1 ⁇ -cis-stimulating activity by etoposide stimulation when a test compound is brought into contact with a test compound is measured and compared, and a compound having an activity of promoting or inhibiting the function of the SUP ring of the present invention or A method for screening the salt is provided.
  • ⁇ -Cis promoting activity is 20% or more, preferably 30% or more, more preferably 50% or more, particularly preferably, as compared with the case of the above U)).
  • a test compound that increases 70% or more can be selected as a compound that promotes the function of SL IP-ring of the present invention.
  • test compound which reduces the apoptosis-promoting activity in the case (ii) above by about 20% or more, preferably 30% or more, and more preferably about 50% or more as compared with the case of the above U). It can be selected as a compound that inhibits the function of the SLIP-ring polypeptide of the invention.
  • Apoptosis promoting activity can be determined by, for example, morphological analysis of apoptosis or biochemistry according to Cell Engineering Separate Volume Experimental Protocol Series, Apoptosis Experimental Protocol (published on December 20, 1994, Shujunsha). It can be measured by performing a statistical analysis. Examples of morphological analysis of apoptosis include apoptosis observation using an optical microscope (eg, observation using a phase contrast microscope, observation of floating or adherent cells by dye staining, observation by fluorescence staining, etc.).
  • Biochemical analysis of apoptosis includes, for example, analysis of DNA fragmentation (eg, agarose gel electrophoresis), cell death determination (eg, crystal violet, MTT, LDH) Etc.) are used.
  • a method for screening a compound having an activity of promoting or inhibiting the function of the polypeptide of the present invention or a salt thereof, and more specifically, 6 (i) a cell capable of expressing the gene of the polypeptide of the present invention; The expression level of the polypeptide of the present invention when cultured, and ( ⁇ i) the expression level of the polypeptide of the present invention when cells capable of expressing the gene of the polypeptide of the present invention are cultured in the presence of a test compound.
  • the activity of promoting or inhibiting the function of the polypeptide of the present invention is characterized in that the expression level is measured using the antibody of the polypeptide of the present invention and compared.
  • the antibody of the polypeptide of the present invention can be produced by the method described above.
  • the cells can be cultured in the same manner as in the known animal cell culture described above.
  • the expression level of the polypeptide of the present invention can be quantified according to the method for quantifying the polypeptide of the present invention shown in the following [3].
  • a cell having the ability to express the polypeptide gene of the present invention is cultured, and an antibody of the polypeptide of the present invention, the culture solution (test solution) and the labeled polypeptide of the present invention are isolated.
  • a cell having the ability to express the gene of the polypeptide of the present invention is cultured, and the culture solution (test solution) and an antibody of the polypeptide of the present invention insolubilized on a carrier, and (Ii) culturing cells capable of expressing the polypeptide gene of the present invention in the presence of a test compound, and reacting the cells with the antibody of the present invention simultaneously or successively.
  • Detection) and the activity of the labeling agent on the insolubilized carrier when the antibody of the polypeptide of the present invention insolubilized on the carrier and another labeled antibody of the present invention are reacted simultaneously or continuously.
  • a method for screening for a compound having the activity of promoting or inhibiting the function of the polypeptide of the present invention or a salt thereof characterized by measuring the following.
  • one antibody is an antibody that recognizes the N-terminal of the polypeptide of the present invention
  • the other antibody is an antibody that reacts with the C-terminal of the polypeptide of the present invention.
  • test compounds include, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and the like. Extract, animal tissue extract and the like. These compounds may be novel compounds or known compounds.
  • the screening kit of the present invention contains a cell capable of expressing the gene of the polypeptide of the present invention, a labeled polypeptide of the present invention, an antibody of the polypeptide of the present invention, and the like.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention may be a test compound as described above, for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, or a plant extract. Liquid, animal tissue extract, plasma, and the like, and a compound having an activity of promoting or inhibiting the function of the polypeptide of the present invention.
  • salt of the compound those similar to the aforementioned salts of the polypeptide of the present invention can be used.
  • the function of the SUP-long of the present invention (for example, caspase (especially lower caspases such as caspase 3 and caspase 7) inhibitory activity, apoptosis inhibitory activity, TRAF (Tumor Necrosis Receptor Associated Factor) binding activity, RIP (Receptor Interacting) Compounds having an activity to promote (Protein) binding activity, RPR (apotosis-inducing protein of Drosophia, Reaper) binding activity, and the like, for example, can be used for the treatment of abnormal apoptosis (eg, Alzheimer's disease, autoimmune disease) It can be used as a medicine such as a drug.
  • caspase especially lower caspases such as caspase 3 and caspase 7
  • apoptosis inhibitory activity for example, TRAF (Tumor Necrosis Receptor Associated Factor) binding activity, RIP (Receptor Interacting)
  • RIP Receptor Interacting
  • Compounds having an activity of inhibiting the function of SLIP-long of the present invention include, for example, various cancers (eg, endometrial cancer, endometrial tumor, breast cancer, colon cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer) , Neuroblastoma, bladder cancer, melanoma, etc.).
  • various cancers eg, endometrial cancer, endometrial tumor, breast cancer, colon cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer
  • Neuroblastoma e.g., endometrial tumor, breast cancer, colon cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer
  • Neuroblastoma e.g., melanoma, etc.
  • the compound having an activity of promoting the function of SLIP-ring (eg, an activity of promoting apoptosis by etoposide stimulation) of the present invention includes, for example, various cancers (eg, endometrial cancer, endometrial tumors). It can be used as a medicament for the treatment and prevention of diseases such as breast cancer, colon cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, and black thigh).
  • various cancers eg, endometrial cancer, endometrial tumors. It can be used as a medicament for the treatment and prevention of diseases such as breast cancer, colon cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, and black thigh).
  • Compounds having an activity of inhibiting the function of the SLIP-ring of the present invention include, for example, It can be used as a drug for treatment-prophylactic agent for treatment of monocis disorder (eg, Alzheimer's disease, autoimmune disease).
  • monocis disorder eg, Alzheimer's disease, autoimmune disease.
  • a compound obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out according to conventional means. For example, it is orally or parenterally administered as tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, etc. in the same manner as the above-mentioned pharmaceuticals containing the polypeptide of the present invention. be able to.
  • the preparations obtained in this way are safe and have low toxicity, for example, warm-blooded animals (eg humans, mice, rats, puppies, higgs, bush, puppies, pumas, birds, cats, cats) Dogs, monkeys, chimpanzees, etc.).
  • warm-blooded animals eg humans, mice, rats, puppies, higgs, bush, puppies, pumas, birds, cats, cats
  • the dose of the compound or a salt thereof varies depending on its action, target disease, subject of administration, route of administration, and the like.
  • a compound that inhibits the function of SLIP-long of the present invention for the purpose of treating cancer is orally administered.
  • the compound When administered, generally in adults (assuming a body weight of 6 O kg), the compound is present in an amount of about 0.1 to 10 Omg per day, preferably about 1.0 to 5 Omg, more preferably about 1.
  • Administer 0-2 Omg When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., for example, it inhibits the function of SLIP-1ong of the present invention for the purpose of treating cancer.
  • the compound to be administered When the compound to be administered is usually administered to an adult (as 60 kg) in the form of an injection, the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg. It is convenient to administer about 0.1 to 1 Omg by intravenous injection. In the case of other animals, the amount converted per 60 kg can be administered.
  • a compound that promotes the function of the SLIP-ring of the present invention for the purpose of treating cancer when orally administering a compound that promotes the function of the SLIP-ring of the present invention for the purpose of treating cancer, generally, in adults (assuming a body weight of 60 kg), the compound is used in an amount of about 0.1 g / day. 1 to: L 00 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • a compound that promotes the function of the SUP-ring of the present invention for the purpose of treating cancer may be administered as an injection.
  • the dose When usually administered to adults (as 60 kg) in the form of a compound, about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day Intravenous injection Conveniently for administration. For other animals, the dose can be administered in terms of 60 kg.
  • an antibody against the polypeptide of the present invention (hereinafter, may be abbreviated as the antibody of the present invention) can specifically recognize the polypeptide of the present invention, and thus can be used in a test solution. It can be used for quantification of peptides, particularly for quantification by sandwich immunoassay.
  • the antibody of the present invention is allowed to competitively react with a test solution and a labeled polypeptide of the present invention, and the ratio of the labeled polypeptide of the present invention bound to the antibody is measured.
  • a method for quantifying the polypeptide of the present invention in a test solution comprising:
  • one antibody may be an antibody that recognizes the N-terminal of the polypeptide of the present invention, and the other antibody may be an antibody that reacts with the C-terminal of the polypeptide of the present invention. desirable.
  • the polypeptide of the present invention can be quantified using a monoclonal antibody against the polypeptide of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
  • the antibody molecule itself may be used, or F (ab '),-,, Fab ⁇ or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the polypeptide of the present invention using the antibody of the present invention is not particularly limited. Any measurement method may be used as long as it is a measurement method in which the amount of the body is detected by chemical or physical means, and this is calculated from a curve prepared using a standard solution containing a known amount of antigen. .
  • nephrometry competition law
  • sandwich method described below from the viewpoints of sensitivity, specificity, in which the immunometric method and the sandwich method are preferably used.
  • a labeling agent used in a measuring method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C] used.
  • the enzyme is preferably a stable enzyme having a large specific activity. For example, 3-galactosidase, -dalcosidase, alkaline phosphatase, peroxidase, and lignoic acid dehydrogenase are used.
  • the fluorescent substance for example, fluorescein, fluorescein isothiosinate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • a test solution is allowed to react with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction By measuring the activity of the labeling agent on the insolubilized carrier, the amount of the polypeptide of the present invention in the test solution can be determined.
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent and the method of insolubilization can be the same as those described above.
  • the antibody used for the solid phase antibody or the antibody used for labeling is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is an antibody having different sites to which the polypeptide of the present invention binds. Is preferably used. That is, the first order
  • the antibody used in the primary and secondary reactions is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the polypeptide of the present invention, the antibody used in the primary reaction is preferably the C-terminal
  • an antibody that recognizes other than the N-terminal part is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like.
  • a competition method an antigen in a test solution and a labeled antigen are applied to the antibody. After reacting competitively, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (BZF separation), and the amount of labeling of either B or F is measured. Quantify the amount of antigen in the test solution.
  • a soluble antibody is used as an antibody
  • B / F separation is performed using a polyethylene glycol
  • a liquid phase method using a second antibody against the antibody or a solid phase antibody is used as the first antibody.
  • an immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.
  • the amount of insoluble sediment resulting from the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • the polypeptide of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • a decrease in the concentration of the SLIP-long of the present invention is detected by quantifying the concentration of the SLIP-long of the present invention using the SUP-long antibody of the present invention, for example, in a disease of abnormal apoptosis, It can be diagnosed as having or being likely to be affected in the future.
  • the concentration of the SLIP-long of the present invention is determined by quantifying the concentration of the SLIP-long of the present invention using the SLIP-long antibody of the present invention, an increase in the concentration of the SLIP-long of the present invention is detected.
  • Endometrial tumor, endometrial tumor, breast cancer, colorectal cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc. or will be affected in the future It can be diagnosed that the probability is high.
  • the concentration of the SLIP-ring of the present invention is quantified using the SLIP-ring antibody of the present invention, and an increase in the concentration of the SUP-ring of the present invention is detected, for example, in the case of an apoptosis abnormal disease It can be diagnosed as having or possibly in the future.
  • the concentration of the SUP-ring of the present invention is determined by quantifying the concentration of the SUP-ring of the present invention using the SLIP ring antibody of the present invention, for example, various cancers (eg, Disease, such as endometrial cancer, endometrial thigh ulcer, breast cancer, colorectal cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, nervous thigh, bladder cancer, ⁇ ⁇ etc.) It can be diagnosed that there is a high possibility of performing.
  • various cancers eg, Disease, such as endometrial cancer, endometrial thigh ulcer, breast cancer, colorectal cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, nervous thigh, bladder cancer, ⁇ ⁇ etc.
  • the SLIP-long antibody and SLIP-shor of the present invention are used to determine the concentration of the SLIP-long and SLIP-short of the present invention using a human body, whereby the SLIP-long to the SLIP-short of the present invention is determined. If an increased percentage is detected, for example, various cancers (eg, endometrial cancer, endometrial tumor, breast cancer, colorectal cancer, rectal cancer, prostate cancer, lung cancer, gastric cancer, kidney cancer, neuroblastoma, bladder cancer , Melanoma, etc.) or are likely to be affected in the future.
  • various cancers eg, endometrial cancer, endometrial tumor, breast cancer, colorectal cancer, rectal cancer, prostate cancer, lung cancer, gastric cancer, kidney cancer, neuroblastoma, bladder cancer , Melanoma, etc.
  • the antibody of the present invention can be used for detecting the polypeptide of the present invention present in a subject such as a body fluid or a tissue.
  • preparation of an antibody column used for purifying the polypeptide of the present invention, detection of the polypeptide of the present invention in each fraction during purification, and analysis of the behavior of the polypeptide of the present invention in test cells It can be used for such purposes.
  • the DNA of the present invention can be used, for example, as a probe to produce warm-blooded animals (eg, humans, rats, mice, guinea pigs, egrets, birds, higgies, dogs, dogs, cats, dogs, Abnormalities (genetic abnormalities) in the DNA or mRNA encoding the polypeptide of the present invention in monkeys, chimpanzees, etc.). It is useful as a diagnostic agent for a gene such as an increase or excessive expression of the DNA or mRNA.
  • warm-blooded animals eg, humans, rats, mice, guinea pigs, egrets, birds, higgies, dogs, dogs, cats, dogs, Abnormalities (genetic abnormalities) in the DNA or mRNA encoding the polypeptide of the present invention in monkeys, chimpanzees, etc.
  • the above-described genetic diagnosis using the DNA of the present invention includes, for example, the well-known Northern Hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), By Probed's The National Academy, Probedations of the National Academy of Sciences of the United States of America, Vol. 86, 2766-2770 (1989), etc. Can be implemented.
  • the expression of the SLIP-1ong of the present invention is reduced by Northern hybridization, or when the SUP long of the present invention is encoded by PCR-SSCP method. If a mutation in the DNA is detected, for example, it can be diagnosed that the possibility of an apoptosis disorder is high.
  • SLIP-long of the present invention when an increase in the expression of SLIP-long of the present invention is detected by Northern hybridization, for example, various cancers (eg, endometrial cancer, endometrial tumor, breast cancer, colorectal cancer, rectal cancer) , Prostate cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.).
  • various cancers eg, endometrial cancer, endometrial tumor, breast cancer, colorectal cancer, rectal cancer
  • Prostate cancer lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.
  • the decrease in the expression of the SLIP-ring of the present invention is detected by Northern hybridization, or when a mutation in the DNA encoding the SLIP-ring of the present invention is detected by the PCR-SSCP method.
  • various cancers eg, endometrial cancer, endometrial tumor, breast cancer, colon cancer, rectum cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.
  • an increase in the expression of SL IP-ring of the present invention is detected by Northern hybridization, it can be diagnosed that, for example, it is highly possible that the disease is an apoptosis abnormal disease.
  • the antisense polynucleotide of the present invention which binds complementarily to the polynucleotide (eg, DNA) of the present invention and can suppress the expression of the polynucleotide (eg, DNA), has low toxicity, Can suppress the function of the polypeptide of the present invention or the polynucleotide (eg, DNA) of the present invention in, for example, as a prophylactic or therapeutic agent for diseases caused by overexpression of the polypeptide of the present invention. Can be used.
  • the antisense-polynucleotide of the present invention for the DNA encoding the SLIPong of the present invention may be used, for example, for each polar cancer (eg, endometrial cancer, endometrial tumor, breast cancer). , Colorectal cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.).
  • polar cancer eg, endometrial cancer, endometrial tumor, breast cancer
  • the antisense polynucleotide of the present invention against the DNA encoding the SLIP-ring of the present invention can be used, for example, as a medicament such as a therapeutic or prophylactic agent for diseases of abnormal apoptosis (eg, Alzheimer's disease, autoimmune diseases).
  • a medicament such as a therapeutic or prophylactic agent for diseases of abnormal apoptosis (eg, Alzheimer's disease, autoimmune diseases).
  • the antisense polynucleotide can be formulated in the same manner as in the case of the above-mentioned polynucleotide of the present invention.
  • the formulations obtained in this way have low toxicity and are orally or non-toxic to humans or non-human mammals (eg, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). It can be administered orally.
  • non-human mammals eg, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.
  • the antisense polynucleotide can be administered as it is or together with a physiologically acceptable carrier such as an auxiliary for promoting uptake by a gene gun or a catheter such as a hydrogel catheter.
  • a physiologically acceptable carrier such as an auxiliary for promoting uptake by a gene gun or a catheter such as a hydrogel catheter.
  • the dosage of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like.
  • the antisense nucleotide for the DNA encoding the SLIP-long of the present invention for the treatment of cancer is used.
  • the dose is preferably about 0.1 to 100 mg per day for an adult (body weight 60 kg).
  • antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.
  • the present invention further provides
  • RNA containing a part of the RNA encoding the peptide of the present invention and its complementary RNA;
  • RNAi RNA interference method
  • ribozyme RNA interference method
  • the expression of the polynucleotide of the present invention eg, DNA
  • the polypeptide of the present invention or the polynucleotide of the present invention eg, DNA
  • Double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001). Ribozymes can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, 7, 221, 2001). For example, it can be produced by linking a known ribozyme to a part of RNA encoding the polypeptide of the present invention.
  • RNA encoding the polypeptide of the present invention examples include a portion (RNA fragment) close to a cleavage site on the RNA of the present invention which can be cleaved by a known ribozyme.
  • RNA fragment RNA fragment close to a cleavage site on the RNA of the present invention which can be cleaved by a known ribozyme.
  • the antibody of the present invention which has the activity of neutralizing the activity of the polypeptide of the present invention, can be used, for example, as a medicament for preventing or treating diseases or the like caused by overexpression of the polypeptide of the present invention. it can.
  • the antibody of the present invention having an activity of neutralizing the activity of SLIP long includes, for example, various cancers (eg, endometrial cancer, endometrial tumor, breast cancer, colorectal cancer, rectal cancer, prostate cancer, lung cancer, gastric cancer, It can be used as a medicament such as a therapeutic or prophylactic agent for diseases such as kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.
  • various cancers eg, endometrial cancer, endometrial tumor, breast cancer, colorectal cancer, rectal cancer, prostate cancer, lung cancer, gastric cancer
  • a medicament such as a therapeutic or prophylactic agent for diseases such as kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.
  • the antibody of the present invention having an activity to neutralize the activity of SUP-ring can be used as a drug such as a prophylactic agent for apoptosis disorder (eg, Alzheimer's disease, autoimmune disease) and the like. .
  • apoptosis disorder eg, Alzheimer's disease, autoimmune disease
  • the therapeutic / preventive agent for the above-mentioned diseases containing the antibody of the present invention can be directly used as a liquid, Or, as a pharmaceutical composition in an appropriate dosage form, it can be orally or non-humanly administered to humans or non-human mammals (eg, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). It can be administered orally.
  • the administration dose varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used in adults, the antibody of the present invention is usually used in a single dose of the antibody of the present invention.
  • the antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for the above administration contains the above or a salt thereof, and a pharmacologically acceptable carrier, diluent or vehicle. Such compositions are provided in dosage forms suitable for oral or parenteral administration.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). ), Syrups, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • compositions for parenteral administration for example, injections, suppositories, etc. are used.
  • Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Is included.
  • Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used, and a suitable solubilizing agent, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxythyl ene (50 mo1)) addu tof hydrogenated astor oil)].
  • a suitable solubilizing agent for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxythyl ene (50 mo1)) addu tof hydrogenated astor oil)].
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a so
  • each dosage unit dosage form is 5 to 500 mg, especially for injections.
  • the antibody contains 5 to 10 Omg, and 10 to 25 Omg of the above antibody in other dosage forms.
  • compositions contains other active ingredients as long as no undesired interaction occurs due to the combination with the above antibody.
  • the present invention relates to a DNA encoding an exogenous polypeptide of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). And a non-human mammal having the formula:
  • Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof may include unfertilized eggs, fertilized eggs, sperm and For germ cells containing progenitor cells, etc., preferably, at the stage of embryonic development in non-human mammal development (more preferably, at the stage of single cells or fertilized egg cells and generally before the 8-cell stage), the calcium phosphate method, Pulse method It can be produced by transferring the target DNA by the Boffexion method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, or the like.
  • the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like.
  • the DNA transgenic animal of the present invention can also be produced by fusing the cells with the above-mentioned germ cells by a known cell fusion method.
  • non-human mammals for example, porcupines, pigs, higgs, goats, magpies, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used.
  • a rat eg, Wistar, SD, etc.
  • the “mammal” in the recombinant vector that can be expressed in mammals includes humans and the like in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by a non-human mammal but to the DNA of the present invention once isolated and extracted from a mammal.
  • a mutation eg, mutation
  • a mutation in the base sequence of the original DNA of the present invention specifically, addition, deletion, or substitution of another base with another base
  • a DNA or the like in which an abnormality occurs is used, and also includes an abnormal DNA.
  • DNA that expresses the abnormal polypeptide of the present invention is used.
  • DNA that expresses a polypeptide that suppresses the function of the normal polypeptide of the present invention is used.
  • the exogenous DNA of the present invention may be derived from either the same gutter or a different kind of mammal as the target animal.
  • each gutter having the DNA of the present invention having homology with the human DNA DNA constructs e.g., eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, and the like
  • various promoters capable of expressing DNA derived therefrom e.g, The vector of the present invention can be produced by microinjecting a vector or the like into a fertilized egg of a target mammal, for example, a mouse fertilized egg.
  • Examples of the expression vector of the polypeptide of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacterium phage such as ⁇ phage, a retrovirus such as Moroni monoleukemia virus, a vaccinia virus or a baculovirus. Animal viruses such as viruses are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
  • promoters that regulate the expression of DN ⁇ ⁇ include: (1) a promoter of DNA derived from a virus (eg, simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); Promoters derived from various mammals (humans, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), such as albumin, insulin II, peroplacin II, erasose, erythropoietin, endothelin, muscle creature Gatin kinase, glial fibrillary acidic polypeptide, daltathione S-transferase, platelet-derived growth factor 3, keratin K1, 1 ⁇ 10 and 14, collagen I and II, cyclic AMP-dependent polypeptide kinase j3 I subunit, dystro Quinine, tartrate-resistant alkaline phosphatase, atrial sodium
  • I amyloid P component For example, promoters such as neuron, myoglobin, trobonin C, smooth muscle oactin, prebub enkephalin A, and vasopressin are used. Among them, a cytomegalovirus promoter that can be highly expressed throughout the whole body, a promoter of a human polypeptide chain elongation factor la (EF-1), a human and a nitractin promoter, and the like are preferable.
  • EF-1 human polypeptide chain elongation factor la
  • the vector preferably has a sequence that terminates transcription of a target messenger RN ⁇ in a DNA-transferred mammal (generally called terminator 1).
  • the sequence of DNA can be used, and preferably, Simian virus SV40 or the like is used.
  • the normal translation region of the polypeptide of the present invention includes liver, kidney, thyroid cells, and the like derived from various mammals (for example, humans, rabbits, dogs, cats, guinea pigs, Yasushi Yuichi, rats, mice, etc.). All or part of genomic DNA from fibroblast-derived DNA and various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cells, or fibroblast-derived RNA It can be obtained as a fee.
  • an exogenous abnormal DNA can produce a translation region obtained by mutating a normal polypeptide translation region obtained from the above cells or tissues by point mutagenesis.
  • the translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which it is ligated downstream of the above promoter and, if desired, upstream of the transcription termination site.
  • exogenous DNA of the present invention is provided at the fertilized egg cell stage in all germ cells and somatic cells of the target mammal.
  • the presence of the exogenous DNA of the present invention in the embryonic cells of the embryonated animal after the transfer of the DNA indicates that all the progeny of the animal to be produced, the germ-cells and the resting cells of the exogenous DNA of the present invention Hold A Means to do.
  • the offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.
  • the non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and should be subcultured as an animal having the DNA in a normal breeding environment. Can be done.
  • exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer indicates that all the offspring of the produced animal contain the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to have.
  • the progeny of this type of animal that inherited the exogenous DNA of the present invention obtains a homozygous animal that has the introduced DNA in excess of the exogenous DNA of the present invention in both its germ cells and somatic cells on both homologous chromosomes, By mating the male and female animals, it is possible to breed and passage so that all offspring have the DNA in excess.
  • the non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and eventually promotes the function of endogenous normal DNA, thereby finally obtaining the polypeptide of the present invention. May develop hyperfunction, and can be used as a model animal for the disease. For example, using the normal DNA-transferred animal of the present invention to elucidate the pathological mechanism of the polypeptide hyperactivity of the present invention and the disease associated with the polypeptide of the present invention, and the method of treating these diseases. It is possible to conduct a study.
  • the mammal to which the exogenous normal DNA of the present invention has been transferred since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released polypeptide of the present invention, it may be used as a therapeutic drug for a disease associated with the polypeptide of the present invention. It can also be used for testing.
  • non-human mammals having the exogenous abnormal DNA of the present invention are confirmed to stably retain the exogenous DNA by mating, and are reared as ordinary animals having the DNA in a normal breeding environment. I can do it.
  • the target exogenous DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
  • the DNA construct with the promoter can be prepared by the usual DNA: 1: chemical method. Fertilized egg Introduction of the abnormal DNA of the present invention at the cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germinal and somatic cells.
  • a homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing the male and female animals, it is possible to breed so that all offspring have the DNA.
  • the non-human mammal having the abnormal DNA of the present invention expresses the abnormal DNA of the present invention at a high level, and finally inhibits the function of the endogenous normal DNA, thereby finally producing the polypeptide of the present invention. May become functionally inactive refractory, and can be used as a disease model animal. For example, using the abnormal DNA transgenic animal of the present invention, it is possible to elucidate the pathological mechanism of the function inactive refractory of the polypeptide of the present invention and to examine a method for treating this disease.
  • the abnormal DNA-highly expressing animal of the present invention can be used for the production of a normal polypeptide by the abnormal polypeptide of the present invention in a functionally inactive refractory disease of the polypeptide of the present invention. It is a model for elucidating functional inhibition (dominant negative action).
  • the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released polypeptide of the present invention, it is also used in a therapeutic drug screening test for a functionally inactive refractory disease of the polypeptide of the present invention. Available.
  • ⁇ ⁇ Isolation and purification of the mutant polypeptide of the present invention and production of its antibody can be considered. Further, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of diseases related to the polypeptide of the present invention, including the inactive type refractory type of the polypeptide of the present invention, and the like. More detailed pathological findings in each organ of the disease model related to the polypeptide of the present invention can be obtained, and new therapeutic methods can be developed, and further, research and treatment of secondary diseases caused by the disease can be contributed. can do.
  • each organ is taken out from the DNA-transferred animal of the present invention, and after minced, the released DNA-transferred cells are obtained using a polypeptide-degrading enzyme such as tribcine, and the cultured cells or the cultured cells are systematized. It is possible. Furthermore, it is possible to examine the specificity of the polypeptide-producing cells of the present invention, the apoptosis, the relationship with differentiation or proliferation, or the signal transduction mechanism thereof, and the abnormality thereof, and the like. It is an effective research material for elucidating its action.
  • a polypeptide-degrading enzyme such as tribcine
  • DNA-transferred animal of the present invention to develop a therapeutic agent for a disease associated with the polypeptide of the present invention, including a functionally inactive type refractory type of the polypeptide of the present invention, has been described above. It is possible to provide an effective and rapid screening method for the therapeutic agent for the disease by using the test method and the quantification method. Further, using the DNA translocation product of the present invention or the exogenous DNA expression vector of the present invention, it is possible to study and develop a method for treating DNA associated with the polypeptide of the present invention.
  • the present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in the expression of the DNA of the present invention.
  • a non-human mammal deficient in expression of the DNA in which the DNA of the present invention is inactivated (7) the DNA is inactivated by introducing a reporter gene (eg, a -galactosidase gene derived from Escherichia coli);
  • a reporter gene eg, a -galactosidase gene derived from Escherichia coli
  • the non-human mammal according to (6), wherein the repo overnight gene can be expressed under the control of a promoter for DNA of the present invention.
  • a non-human mammal embryonic stem cell in which the DNA of the present invention has been inactivated is defined as a DNA that is non-human mammal has the DNA of the present invention artificially mutated to suppress the expression of the DNA, Alternatively, the DNA substantially does not have the ability to express the polypeptide of the present invention by substantially losing the activity of the polypeptide of the present invention encoded by the DNA (hereinafter referred to as the knockout of the present invention).
  • Non-human mammalian embryonic stem cells hereinafter abbreviated as ES cells).
  • non-human mammal those similar to the above can be used.
  • the method of artificially mutating the DNA of the present invention can be performed, for example, by deleting part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique.
  • the knockout DNA of the present invention may be prepared by, for example, shifting the codon reading frame or disrupting the function of the promoter overnight or exon.
  • non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated include, for example, The DNA of the present invention possessed by the non-human mammal described above is isolated and its exon portion is neomycin-resistant and hygromycin-resistant.
  • the exon function is destroyed by introducing a drug resistance gene represented by lacZ ( ⁇ 3-galactosidase gene) or a reporter gene represented by cat (chloramphenicylacetyltransferase gene), or Inserting a DNA sequence that terminates gene transcription (for example, a polyA addition signal) into the introns between exons and preventing the synthesis of complete messenger RNA may result in gene disruption.
  • a DNA strand having a DNA sequence constructed in the above (hereinafter abbreviated as a “getting vector”) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells are ligated with the DNA of the present invention.
  • Southern hybridization analysis using evening DNA sequence as a probe or evening gettin The DNA sequence on the vector and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector are analyzed by the PCR method as a primer, and the knockout ES cells of the present invention are obtained by selection. Can be.
  • ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like for example, those already established as described above may be used, or the methods described in the known methods of Evans and Kauima may be used. A newly established one may be used. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used. However, since the immunological background is not clear, an alternative pure immunogenic genetic background is used.
  • the BDF t mouse C 57 BLZ6 with a C 57 BLZ6 mouse or C 5 7 egg number of lack of BL / 6 improved by crossing with DBA / 2
  • a mouse established using F with DBA / 2 can also be used satisfactorily.
  • the mouse has a background of C57BL6 mice Therefore, the ES cells obtained using this can be used to backcross the C57BLZ6 mouse to the C57BLZ6 mouse when the disease model mouse is created by cross-crossing the C57BLZ6 mouse. Can be used advantageously.
  • male ES cells when establishing ES cells, generally use the blastocyst 3.5 days after fertilization; in addition, collect the 8-cell stage embryo and cultivate it to the blastocyst)! Thus, a large number of early embryos can be obtained efficiently.
  • male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.
  • a method for determining the sex of ES cells for example, a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR can be mentioned.
  • this method conventionally, for example G-banding method, requires about 10 6 cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture
  • the primary selection of ES cells in the early stage can be performed by discriminating between males and females, and the early stages of culture can be greatly reduced by enabling the selection of male cells at an early stage.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but they must be carefully subcultured because they tend to lose their ability to generate individuals.
  • a suitable feeder cell such as STO fibroblasts
  • CO2 incubator preferably 5% CO2, 95% air or 5%
  • LIF 1-1000 OU / ml
  • tribcinno-EDTA solution usually 001-0.5% tribcine Z0).
  • ES cells can be cultured in monolayers up to high density, or in suspension culture until cell clumps are formed, resulting in various types of head, muscle, visceral and cardiac muscle.
  • Cell types MJ Evans and MH aufm An, Nature, Vol. 292, pp. 154, 1981; GR Martin Proc. of National Academy of Sciences, Proc. Natl. Acad. Sci. USA, Vol. 78, 7634, 1981; C. Doetschman et al., Journal of Embryology 'and' Expermental Morphology, Vol. 87, p. 27, 1985], to differentiate ES cells of the present invention.
  • the DNA-deficient cells of the present invention obtained by the above method are useful in in vitro cell biology of the polypeptide of the present invention.
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
  • non-human mammal those similar to the aforementioned can be used.
  • the non-human mammal deficient in DNA expression of the present invention may be obtained, for example, by introducing the evening getter vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, and introducing the same into the present invention.
  • By knocking out the DNA of the present invention by homologous recombination of the DNA sequence in which the DNA has been inactivated replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. Can be done.
  • a cell in which the DNA of the present invention has been knocked out is subjected to Southern hybridization analysis using the DNA sequence on or near the DNA of the present invention as a probe or a DNA sequence on a gettering vector, and The DNA can be determined by PCR analysis using, as primers, the DNA sequence of a neighboring region other than the DNA of the present invention derived from the mouse used in the vector.
  • the cell line in which the DNA of the present invention has been inactivated is cloned by gene homologous recombination, and the cells are cloned at an appropriate time, for example, at the 8-cell stage.
  • the chimeric embryo is injected into a human ⁇ milk animal embryo or blastocyst, and is transplanted into the uterus of the pseudopregnant non-human mammal.
  • the produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially altered DNA locus of the present invention.
  • all of the populations obtained by crossing such a chimeric individual with a normal individual It can be obtained by selecting individuals composed of cells having the DNA locus of the present invention in which the tissue is artificially mutated, for example, by judging coat color or the like.
  • the individual thus obtained is usually an individual having a heterozygous expression of the polypeptide of the present invention, which is crossed with an individual having a heterozygous expression of the polypeptide of the present invention.
  • An individual lacking homo-expression of the peptide can be obtained.
  • a transgenic non-human mammal in which the evening vector is introduced into the chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by microinjection. Compared with these transgenic non-human mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination of the gene.
  • the germline can be obtained and maintained according to a standard method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained.
  • the obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and a plurality of homozygous animals are obtained.
  • homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated are very useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
  • the non-human mammal deficient in expressing the DNA of the present invention lacks various biological activities that can be induced by the polypeptide of the present invention, it may be caused by inactivation of the biological activity of the polypeptide of the present invention. It is useful for investigating the causes of these diseases and examining treatment methods.
  • a method for screening a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or the like of the present invention The non-human mammal deficient in DNA expression of the present invention is used for screening for a compound having a therapeutic / preventive effect against diseases (eg, abnormal apoptosis) caused by DNA deficiency or damage of the present invention. be able to.
  • diseases eg, abnormal apoptosis
  • the present invention is characterized in that a test compound is administered to a non-human mammal deficient in expression of the DNA of the present invention, and the change in the animal is observed and measured.
  • a method for screening a compound or a salt thereof having a therapeutic or preventive effect on a disease caused by the disease is provided.
  • Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include the same as described above.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.These compounds are novel compounds. Or a known compound.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are used as indices.
  • the test compound can be tested for its therapeutic and prophylactic effects.
  • test compound for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like.
  • the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
  • the compound obtained using the screening method of the present invention is a compound selected from the test compounds described above,
  • the compound of the present invention which has a therapeutic / prophylactic effect against a disease caused by SUP-long deficiency or damage (eg, apoptosis disorder) is a safe and low-toxic therapeutic / prophylactic agent against the disease.
  • a disease caused by SUP-long deficiency or damage eg, apoptosis disorder
  • the compounds of the present invention having a therapeutic / preventive effect on each gutter cancer caused by the loss or damage of SLIP-ring include safe and low-toxic treatment / prophylactic agents for each gutter cancer. It can be used as a medicine. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
  • the compound obtained by the screening method may form a salt
  • the salt of the compound may be a biorecognizable acid (eg, an inorganic acid or an organic acid) or a base (eg, an alkali metal )
  • physiologically acceptable acid addition salts are particularly preferable.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the polypeptide of the present invention.
  • the preparations obtained in this way are safe and have low toxicity, for example, mammals (eg, humans, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs, monkeys) Etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the target of administration, the route of administration, and the like.
  • the compound when the compound is orally administered for the purpose of treating a disorder of apoptosis, generally the adult (Assuming a body weight of 60 kg), the compound is administered in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • the single dose of the compound varies depending on the administration subject, the target disease and the like.
  • the compound is usually administered in the form of an injection for the purpose of treating an apoptosis disorder.
  • the compound When administered to an adult (as 60 kg), the compound is intravenously injected at about 0.01 to 3 Orag, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day. It is convenient to administer by the following. For other animals, the dose can be administered in terms of 60 kg.
  • the present invention provides a method of administering a test compound to a non-human babies deficient in DNA expression of the present invention, It is intended to provide a method for screening a compound or a salt thereof which promotes the activity of a promoter for DNA of the present invention, which comprises detecting the expression of a reporter gene.
  • the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention, wherein the DNA of the present invention introduces a repo allele gene.
  • the reporter gene which can be expressed under the control of the promoter for the DNA of the present invention is used.
  • test compound examples include the same compounds as described above.
  • reporter gene the same ones as described above can be used, and a galactosidase gene (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is suitable.
  • the reporter gene is coded because the repo overnight gene is under the control of the promoter for the DNA of the present invention.
  • the tissue originally expressing the polypeptide of the present invention may ] -Galactosidase is expressed instead of the polypeptide. Therefore, for example, the present invention can be easily carried out by staining with a reagent that is a substrate of 3-galactosidase such as 5-promote 4-monocloth-3-indolyl 3-galactopyranoside (X-gal). The state of expression of the polypeptide in animals can be observed.
  • a reagent that is a substrate of 3-galactosidase such as 5-promote 4-monocloth-3-indolyl 3-galactopyranoside (X-gal).
  • the polypeptide-deficient mouse of the present invention or a tissue section thereof is fixed with dartalaldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with a staining solution containing X-ga1. After reacting at room temperature or around 37 ° C. for about 30 minutes to 1 hour, the ⁇ -galactosidase reaction may be stopped by washing the tissue sample with a 1 mM EDT AZPBS solution, and the coloration may be observed. Alternatively, mRNA encoding 1 ac may be detected according to a conventional method.
  • the compound or a salt thereof obtained by the above screening method is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). Salts are used, especially the physiologically acceptable acid addition salts.
  • examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid
  • the compound or a salt thereof that promotes the promoter activity of the DNA encoding SLIP-long of the present invention can promote the expression of SUP-long of the present invention and promote the function of SLIP-long, for example, It is useful as a safe and low-toxic treatment / prophylactic agent for abnormal apoptosis.
  • the compound or a salt thereof that inhibits the promoter activity against the DNA encoding SLIP-1ong of the present invention can inhibit the expression of SLIP-long of the present invention and inhibit the function of SUP-long.
  • various cancers eg, endometrial cancer, endometrial tumor, breast cancer, colorectal cancer, rectum cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.
  • It is useful as a drug such as a safe and low-toxic treatment / prevention agent for the diseases of the disease.
  • the compound or a salt thereof that promotes the promoter activity of the DNA encoding the SUP-ring of the present invention can promote the expression of the SUP-ring of the present invention and promote the function of SLIP ring.
  • various cancers eg, endometrial cancer, endometrial tumor, breast cancer, colon cancer, rectal cancer, prostate cancer, lung cancer, stomach cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.
  • It is useful as a drug such as a safe and low-toxic treatment and prophylactic agent.
  • the compound or a salt thereof that inhibits the promoter activity of the DNA encoding SUP-ring of the present invention inhibits the expression of SL IP-ring of the present invention and impairs the function of SLIP-ring.
  • safety for apoptosis abnormal disease It is useful as a medicament such as a low-toxic therapeutic and prophylactic agent.
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the polypeptide of the present invention or a salt thereof.
  • the preparations obtained in this way are safe and have low toxicity, for example, mammals (e.g., humans, rats, mice, guinea pigs, egrets, higgs, bush, foxes, dogs, cats, dogs) , Monkeys, etc.).
  • mammals e.g., humans, rats, mice, guinea pigs, egrets, higgs, bush, foxes, dogs, cats, dogs
  • Monkeys etc.
  • the dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the route of administration, and the like.
  • the activity of the promoter for DNA encoding the SLIP-long of the present invention may be reduced.
  • the inhibitory compound is administered orally, generally in adults (with a body weight of 60 kg), the compound is administered in an amount of about 0.1-100 mg, preferably about 1.0-5 Omg, more preferably about 1.0-5 mg per day. Administer about 1.0 to 20 mg.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • the compound inhibits promoter activity for the DNA encoding SLIP-long of the present invention for the purpose of treating cancer.
  • the compound to be administered is usually administered to an adult (as 60 kg) in the form of an injection, the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 20 mg per day. It is convenient to administer about 0.1 to 10 mg by intravenous injection. In the case of other animals, the amount converted per 60 kg can be administered.
  • the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes the activity of the promoter of the DNA of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused by insufficiency or the development of preventive and therapeutic drugs.
  • genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal. Creating an offspring) will allow the protein to be synthesized specifically and its effects on the organism to be studied. Further, by binding an appropriate Levoi gene to the above-mentioned promoter part and establishing a cell line in which this is expressed, if the boripetide of the present invention itself is present in the body, It can be used as a search system for low molecular weight compounds that have the action of specifically promoting or suppressing the production ability. By analyzing the promoter portion, it is also possible to find a new cis element and a transcription factor binding thereto. In the present specification and drawings, when bases and amino acids are indicated by abbreviations,
  • DNA Deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger ribonucleic acid
  • a la Alanine
  • HONB I-hydroxy-5-norbornene-2,3-dicarboximide
  • DCC N, N, dicyclohexylcarbodiimide
  • FIG. 1 shows the amino acid sequence of the R NG-F ng er domain of the polypeptide of the present invention.
  • FIG. 1 shows the nucleotide sequence of DNA encoding SLIP-1ong of the present invention.
  • 1 shows the amino acid sequence of the SLIP-ring of the present invention.
  • the amino acid sequences of SLIP-long and SLIP-short partial peptides are shown.
  • the transformant Escherichia coli TOP10 / pTB2163 transformed with the DNA encoding SLIP-1 ong obtained in Example 1 (2) described below was obtained from December 14, 2000.
  • IFO Fermentation Research Institute
  • the DNA encoding the SLIP-short obtained in Example 1 (2) below The transformed transformant Escherichia coli TOP 10 / p TB 2164 has been available from December 14, 2000 at 1-1-1 Tsukuba East Higashi, Ibaraki Prefecture 1 Chuo No. 6 (Zip code 305-8566) Deposited at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH)) under the deposit number FERM BP—7397, and from November 14, 2000 ⁇ Deposited with the Fermentation Research Institute (IFO) under the deposit number IFO 16499.
  • AIST National Institute of Advanced Industrial Science and Technology
  • NIBH National Institute of Advanced Industrial Science and Technology
  • Example 2 The transformant Escherichia coli TOP10 / pTB2178 transformed with the DNA encoding the SLIP-ring obtained in Example 1 (2) described below was obtained from December 14, 2000. 1-chome, Tsukuba-Higashi, Ibaraki Pref. 1 Chuo No. 6 (Zip code 305-8566) National Institute of Advanced Industrial Science and Technology (AIST) Patent Depositary Depositary Center (formerly NIBH )) And deposit number FERM BP-7396, and from January 14, 2000, with the Fermentation Research Institute (IFO) under the deposit number IFO 16498.
  • IFO Fermentation Research Institute
  • Example 1 (1) Search for SLIP gene fragment using human gene information database Search for a sequence homologous to the amino acid sequence of Survivin in a private human gene information database (Celera) As a result, an amino acid sequence and a cDNA sequence having about 60% homology in the BIR domain were obtained.
  • Priraer 1 (5'-ATGGG ACCTAAAGACAGTGCCAAGTG-3 '; SEQ ID NO: 13) and Primer 2 (5, -CTAGGACAGGAAGGTG CGCACG-3, SEQ ID NO: 14) were prepared, and Multiple Tissue cDNA panel Hunan Expression was confirmed by the PGR method using I, Human II, Human Immune System, Human Fetal, and Human Tumor (GL0NTEGH) as a template. As a result Colon Adenoca Strong expression was observed in rcinoma (GX-1). This cDNA fragment was ligated to pT7 Blue T vector (Novagen), and the obtained plasmid DNA was introduced into E.
  • coli JM109 strain by a known method to obtain a transformant (clone). Five clones were randomly selected from the transformants, the plasmid was purified, and the nucleotide sequence of the insert was determined using Sequencer (Applied Biosystem; AB 1377).
  • Example 1 (2) Isolation of full-length SLIP gene (SLIP-long, SLIP-short, SLIP-ring) Primer 3 (5'-TCAGCAGTCAGCGGCCAGTCA TAG-3 based on the sequence obtained in Example 1 (1) '; SEQ ID NO: 15), Primer 4 (5'-GCCCCGGCGCCCTCCTCCTCTTC-3'; SEQ ID NO: 16), Primer 5 (5-GCGGCGGCTGCAGGAGGAGAGGAC-3 '; SEQ ID NO: 17), Primer 6 (5'- AGGAGAGGACGTGCAAGGTGTGCCTG-3 ′; SEQ ID NO: 18) was prepared, 5′- and 3′-RACE was performed, and full-length SLIP genes (SLIP-long, SLIP-short, SUP-ring) were isolated. ⁇ was performed using Marathon-ready human cololectal adenocarcinoma (C Xl) cDNA (CLONTECH) as immediate late.
  • C Xl cololectal adeno
  • 5'-ACE primary PCR is performed using Primer 3 and the Primer API (supplied with the Marathon cDNA template described above), and secondary PCR is performed using Primer 4 and Primer AP2 (supplied with the Marathon cDNA template described above). went.
  • 3'-RACE primary PCR was performed using Primer 5 and Primer API, and secondary PCR was performed using Primer 6 and Primer AP2. As a result, a cDNA fragment having a total nucleotide sequence of 1439 bp and 1260 bp was obtained.
  • a 723 bp ORF (Open Reading Frame) encoding 241 amino acids was found in the 1439 bp cDNA sequence.
  • SLIP contains a base substitution that is considered to be polymorphism (hereinafter also referred to as “polymorphism”), and it is not the sixth base force counted from the start codon A but the A type, and the 528th base is T
  • C type cDNA was also observed. None of these involved amino acid substitutions.
  • the cDM fragment having the i260 bp nucleotide sequence encodes 280 amino acids 840 A bp ORF was found.
  • This is different from SUP-long and SLIP-short because the translated protein has a RING-finger domain, so it was named SLIP-ring.
  • the SLIP-ring also has a base substitution thought to be pol yniorphism, and the 6th base, counted from the start codon i, has an A instead of G and a cDM of 528 instead of a base force. Was. None of these involved amino acid substitutions.
  • a cDNA fragment having 0RF of 723 bp among the obtained 1439 bp nucleotide sequence was cloned into Escherichia coli by the following procedure to obtain a transformant Escherichia coli JM109 / pcDNA3.1 (+)-SLIP-long.
  • a transformant having a cDNA fragment having 0RF of 672 bp and 840 bp Escherichia coli JM109 / pcDNA3.1 (+) -SLIP-short, Escherichia coli J109 / pcDNA3.1 (+)-SLIP-ring I got
  • Primer 7 (5-CC GGAATTCCAGACCGTGCATCATGGGGCCTAAAGACAGTGCCAAG) is complementary to the aforementioned 672 bp, 723 bp, and 840 bp base sequences and has different restriction enzyme recognition sites (Eco RI and Xho I).
  • SL IP A transformant transformed with long-coding DNA was named Escherichia coli TOP 10 / pTB 2163.
  • SL IP The transformant transformed with the long-encoding DNA was designated as Escherichia coli TOP 10 / pTB 2163.
  • the transformant transformed with the DNA encoding short rt was named Escherichia coli TOP I 0 / pTB 2164.
  • SLIP encodes the ring
  • the transformant transformed with the DNA was named Escherichia coli TOP 10 / pTB2178.
  • Example 1 (3) Expression tissue distribution of SUP gene (SLIP-long, SLIP-short, SLIP-ring)
  • Example 1 Using the Megaprime DNA Labeling System (Amers am Pharmacia Biotech), the cDNA fragment obtained in Example 1 (1) was labeled with 32 P-dCTP and used as a probe.
  • Figure 1 shows the results of Northern plotting using MTN Human, Human II, and Hunan Cancer Cell Line (CLONTECH). In FIG. 1, Normal indicates expression in normal tissues, and Tumor indicates expression in cancer tissues.
  • Lane 1 is heart, lane 2 is brain, lane 3 is placenta (placenta), lane 4 is lung (Lung), lane 5 is liver (Liver), lane 6 is skeletal muscle (Skeletal surface) scle), lane 7 is kidney (Kidney), lane 8 is knee (Pancreas), lane 9 is spleen (Spleen), lane 10 is thymus (Thymus), lane 11 is prostate (Prosta te), lane 12 is testicle (Testis), lane 13 is ovary (Ovary), lane 14 is small intestine (Small intestine), lane 15 is colon (Colon), lane 16 is leukocyte
  • Lane 7 is HL-60 cells (Leukemia (HL-60)
  • Lane 18 is HeLa S3 cells (Uterine cervical carcinoma (HeLa S3))
  • Lane 19 is L-562 cells
  • Lane 20 is MOLT-4 cells (Lymphoma (MOLT-4))
  • Lane 21 is Raji cells (Lymphoma (Raj i))
  • Lane 22 is SW480 cells (Colorectal aden ocarcinoma (SW480))
  • Lane 23 shows A549 cells (Lung carcinoma (A549))
  • lane 24 shows G-361 cells (Melanoma (G-361)).
  • the arrows on the left side of Fig. 1 indicate 2.4 kb, 1.35 kb, and 0.24 kb from the top.
  • FIG. 1 shows that strong expression was observed in the melanoma-derived cell line G-361 and weak expression was observed in the placenta.
  • FIG. 2 shows the results.
  • FIG. 2 (a) shows the expression of Survivin
  • FIG. 2 (b) shows the expression of the SUP gene (SLIP-long, SUP-short, SLIP-ring).
  • 2 (b) 1 represents a heart, 2 represents a brain, 3 represents a placenta, and 4 represents a lung.
  • 5 represents the liver (Liver)
  • 6 represents the skeletal muscle (Skeletal Muscle)
  • 7 represents the kidney (Kidney)
  • 8 represents the knee (Pancreas)
  • 9 represents the Represents the spleen (Spleen)
  • 10 represents the thymus (Thymus)
  • 11 represents the prostate gland (Prostate)
  • 12 represents the testis (Testis)
  • 13 represents the ovary (Ovary)
  • 14 Represents the small intestine
  • 15 represents the colon
  • 16 represents the leukocyte
  • 17 represents the spleen
  • 18 represents the lymph gland (Lymph Node).
  • 19 represents the thymus (Thymus), 20 represents the tonsils (Tonsil), 21 represents the bone marrow (Bone Marrow), 22 represents the fetal liver (Fetal Liver), and 23 represents the fetal liver (Fetal Liver).
  • 24 is fetal brain (Fe tal Brain)
  • 25 represents fetal lung (Fetal Lung)
  • 26 represents fetal liver (Fetal Liver)
  • 27 represents fetal kidney (Fetal Kidney)
  • 28 represents fetal heart (Fetal Heart)
  • 29 represents fetal spleen (Fetal Spleen)
  • 30 represents fetal thymus (Fetal Thy t)
  • 31 represents fetal skeletal muscle (Fetal Skeletal Muscle)
  • M represents marker (Marker).
  • Fig. 2 (a) Survivin is expressed in placenta, pancreas, testis, colon, bone marrow, bone marrow, fetal liver, fetal spleen ) It was found to be expressed in fetal thymus and others.
  • Fig. 2 (b) shows that the SUP gene is slightly expressed in the placenta (plaGenta), lung (lung), kidney (kidney), oversplen (spleen), lymph gland (lymph node), etc. I understood. From the above, it was confirmed that the SLIP genes (SLIP-long, SLIP-short, SLIP-ring) had a lower expression distribution in normal tissues and a lower expression level than normal survivin.
  • Example 1 (4) Apoptosis suppression experiment using SUP stably expressing cell line
  • LacZ (3-galactosidase gene) was used as a negative control gene.
  • Figure 3 shows the results.
  • Figure 3 shows cell lines transfected with CDNA3.1 (+) -SLIP-long, pcDNA3.1 (+)-SLIP-short, PGDNA3.1 (+)-SLIP-ring and pcDNA3.1 (+)-LacZ. Shows the percentage of DNA fragmentation when etoposide was added at 0 M, 0.5 M, 1 M, 2 M, 4 M, and 8 M, respectively.
  • the bar graph at each addition amount shows PGDNA3.1 (+)-SLIP-long, pcDNA3.1 (+) -SLIP- short, pcDNA3.1 (+)-SLIP-ring and PGDNA3.1 ( +) Indicates the amount of -LacZ DNA fragment.
  • FIG. 3 it was confirmed that the sensitivity to etoposide was reduced in the cells that strongly expressed SLIP-long, indicating that SLIP-long had an apoptotic inhibitory effect.
  • cells that strongly expressed SUP-ring showed increased sensitivity to etoposide, indicating that SLIP-ring had an apoptosis promoting effect. It can be seen that no clear change was observed in cells that strongly expressed SUP-short.
  • Example 1 Apoptosis experiment when the expression level of SLIP was suppressed using full-length antisense SLIP-ring
  • the pcDNA3.1 (+)-SUP-ring obtained in Example 1 (2) was digested with the restriction enzymes EcoR I and Xho I (hereinafter also referred to as “digestion”), and the incorporated SLIP-ring was digested. Refined. This was introduced into PCDNA3.1 (-) (manufactured by Invitrogen) digested with the same enzyme in advance to obtain PCDNA3.1 (-)-SLIP-ring. The gene expressed from this plasmid is an antisense SUP-ring.
  • G361 cells derived from Melaloma have RNA levels Expresses SLIP-long (short) and SLIP-ring.
  • the pcDM3.1 (-)-SLIP-ring or pcDNA3.1 (+)-LacZ plasmid prepared above was introduced into 4 ⁇ 10 5 cells. After transfection, each cell line was irradiated with ultraviolet light (intensity: 0, 20, 40 J / m 2 ) to induce apoptosis, and apoptosis was induced using Cell Death Detection ELISA plus (Roche) according to the attached protocol. The ratio of DNA fragmentation, which is an index of the above, was examined.
  • FIG. 4 shows the results.
  • Figure 4 shows the DNA fragments of the cell lines transfected with the pcDNA3.1 (-) -SLIP-ring, and pcDNA3.1 (+) -LacZ plasmids, in which the intensity of the UV light is OJ 20 J / m 2 and 40 J / ffl. It represents the amount of conversion.
  • the bar graph at each ultraviolet intensity shows the amount of DNA fragmentation of the cell strain into which the PGDNA3.1 H-SLIP-ring and pcDNA3.1 (+)-LacZ plasmids were introduced in order from the left.
  • Figure 4 shows that the cell line introduced with pcDM3.1 (-)-SUP-ring showed a decrease in the DM fragmentation rate, and suppression of endogenous SLIP suppressed apo! ⁇ -Cis.
  • Figure 4 confirms that From this, when SLIP-long (short) and SLIP-ring are expressed as in G361 cells, SLIP-ring that promotes promotion is more effective than SLIP-long that suppresses apoptosis. It was thought that the result was due to the decrease in only the SLIP-ring, because the ratio contributing to cis was large, or SLIP-long was difficult to be decomposed.
  • Example 1 Verification of subcellular localization of SLIP-long, SLIP-short, and SLIP-ring in forced expression
  • Plasmid pcDM3.1 (+)-SLIP-long obtained in Example 1 (2) was used as a template (te immediately late), and myc tag was incorporated.
  • primer 13 AGAGTGCCMG-3 ′; SEQ ID NO: 25), and
  • the PGR amplified product was digested with EGOR I and Xho I and inserted into pcDM3.1 (+) (Invitrogen), a plasmid vector digested with the same enzyme.
  • the plasmid vector PGDNA3.1 myc-SLIP-long was obtained. This was introduced into E. coli JM109 to obtain a transformant.
  • PCDNA3.1 myc-SLIP-short (using primer 13 and primer 15: (5'-CCGCTCGAGTCACCCAGGGGGTCCCGGGC-3 '; SEQ ID NO: 27)) and pcDNA3.1 myc-SL IP-ring (primer 13 and primer 16: (using 5′-CCGCTCGAGCTAGGACAGGAAG GTGCGCACG-3 ′: SEQ ID NO: 28)).
  • the plate was incubated with an anti-myc-tag antibody (manufactured by SIGMA) diluted with a blocking solution at room temperature for 2 hours, and then washed with PBS. Finally, the cells were incubated with a FITG-labeled anti-mouse IgG antibody (manufactured by SIGMA) diluted with a blocking solution at room temperature for 1 hour, washed with PBS, and observed with a fluorescence microscope.
  • an anti-myc-tag antibody manufactured by SIGMA
  • Figure 5 shows the results.
  • Figures 5 (a), 5 (b) and 5 (c) show the cells introduced with pcDNA3.1 my G-SLIP-long, pcDNA3.1 myc-SLIP-short and pcDNA3.1 myc-SLIP-ring, respectively.
  • 3 shows the results of microscopic examination with a fluorescence microscope.
  • Figure 5 shows that the cells in which the nucleus and cytoplasm, especially the nucleus, were strongly stained were observed in the cells transfected with pcDM3.1 myc-SLIP-long, pcDNA3.1 myc-SLIP-short and cDNA3.1 myc-SLIP-ring.
  • Fig. 5 It was suggested that SLIP-long, SLIP-short, and SLIP-ring proteins exist in the nucleus and cytoplasm.
  • Example 1 Examination of SUP gene expression level in human colon cancer, breast cancer and lung cancer tissues
  • FIG. 6 shows the results.
  • Figure 6 (a) shows SLIP ⁇ gene of human colon cancer Indicates the expression level.
  • FIG. 6 (b) shows the SLIP gene expression level in breast cancer.
  • FIG. 6 (c) shows the SUP gene expression level in lung cancer tissue.
  • the numbers in the figure represent the patient number, M represents the marker, and N represents the normal tissue. In addition, these samples were corrected in advance for the expression amount of the ⁇ -actin gene.
  • primer 17 (5′-GCGCCATGGGGGCTAAAGACAGTGCCAAG 3 ′; SEQ ID NO: 29) and prime r 18: (5′- A PCR reaction was performed using CCGCTCGAGGGACAGGAAGGTGCGCACGCGG-3 ′; SEQ ID NO: 30).
  • the PCR amplification product was digested with EGOR I and Xho I, and inserted into a plasmid vector PET 2 (Novagen) digested with the same enzyme to obtain a plasmid vector PET 21a-SLIP-ring. This was introduced into E. coli JM109 to obtain a transformant.
  • SLIP-ring protein prepared in Example 2
  • the immunized animals were male male egret KBL: JW (10-week-old, Oriental yeast) — wings, and sensitization was performed by subcutaneous injection on the back and repeated three times every 14 days.
  • the amount of recombinant protein used in one sensitization was 0.5 mg, and a complete Freund's adjuvant (Difco) suspension was used.
  • Difco complete Freund's adjuvant
  • Example 3 Western Blotting Using Egret Polyclonal Antibody A protein having the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, that is, SUP-long, SLIP-short, SLIP -Ring detection was performed using the egret polyclonal antibody prepared in Example 3 (1).
  • SUP produced in Example 1 (4) - long, SUP- short, SUP- ring stable expressing cell lines in 10 cm dish IX 10 6 cells were seeded and cultured overnight. The next day, the cells were detached and subjected to SDS-PAGE and Western blotting according to a conventional method.
  • Figure 7 shows the results.
  • the rabbit polyclonal antibody prepared in Example 3 (1) was used as the primary antibody, and the anti-rabbit IgG-HRP conjugate (manufactured by Juckson I Substitute Research, Inc.) was used as the secondary antibody. ) was used. Detection was performed using EGL plus (Amershani-Pharniacia) according to the attached manual.
  • FIGS. 7 (a), 7 (b), and 7 (c) show the results of Western blotting detecting SLIP-long, SLIP-short, and SUP-ring, respectively. From FIG. 7, it was confirmed that the egret polyclonal antibody recognizes all proteins of SLIP-long, SLIP-short, and SLIP-ring.
  • Example 4 (1) Preparation of peptide antibody
  • a partial peptide consisting of 10 amino acids (Glu-Pro-Gly-Ala-Gly-Pro-Gly-Pro-Pro-Pro- Gly; SEQ ID NO: 31) was synthesized.
  • This peptide was combined with keyhole limpet momosinin (KLH) as a carrier protein to obtain an antigen.
  • KLH keyhole limpet momosinin
  • the antigen sensitization was performed according to the method described in Example 3 (1) described above. 0.5 mg of antigen was used for one sensitization, and blood was collected on the 52nd day after the first sensitization to obtain serum. Purified IgG fraction was obtained from the whole amount in the same manner as in Example 3 (1).
  • Example 4 Detection of proteins having the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7, ie, SLIP-long and SLIP-short, was carried out using the peptide antibody prepared in Example 4 (1).
  • IX pieces of the cell lines stably expressing SLIP-long and SUP-short prepared in Example 1 (4) were seeded on a 10-cm dish and cultured overnight. The next day, the cells were detached and subjected to SDS-PAGE and Western blotting according to a conventional method.
  • Figure 8 shows the results.
  • Example 4 (1) the peptide antibody prepared in Example 4 (1) was used as a primary antibody, and an anti-Peagle IgG-HRP conjugate (Juckson, manufactured by Marunouchi Research) was used as a secondary antibody. Detection was performed using ECL plus Okie rsham-Pharmacia) according to the attached manual.
  • FIGS. 8 (a), 8 (b), and 8 (c) show the results of Western blotting detecting SLIP-long, SLIP-short, and SLIP-ring, respectively. From FIG. 8, it was confirmed that the peptide antibody recognizes only SL IP-1 ong and SLIP-short. Industrial applicability
  • the SUP-long of the present invention and a DNA encoding the same can be used, for example, as an agent for treating and / or preventing a disease associated with apoptosis.
  • cells having the ability to express the SLIP-Iong of the present invention or the SLIP-long gene of the present invention are capable of expressing the function of the SLIP-long of the present invention (for example, caspase inhibitory activity, apoptosis inhibitory activity, It is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits TRAF binding activity, RIP binding activity, RPR binding activity, etc.).
  • SLIP-ring can be used, for example, as a therapeutic or prophylactic agent for cancer.
  • the cell having the ability to express the SLIP-ring of the present invention or the IT gene of the SLIP-ring of the present invention functions as the SUP-iong of the present invention (for example, it promotes apoptosis by etoposide stimulation).
  • SUP-iong of the present invention for example, it promotes apoptosis by etoposide stimulation.
  • a compound or its salt that promotes or inhibits It is useful as a reagent for aging.
  • an antibody against the polypeptide of the present invention can specifically recognize the polypeptide of the present invention, it can be used for quantification of the polypeptide of the present invention in a test wave. For example, combining an antibody against SLIP-long and an antibody against SLIP-sh0rt of the present invention, or combining a DNA encoding SLIP-1ong and a DNA encoding SLIP-short of the present invention. Thereby, cancer diagnosis can be performed efficiently.

Abstract

L'invention porte sur des polypeptides analogues à la survivine et sur leurs ADN. Ces polypeptides ont une séquence d'acides aminés qui est identique ou pratiquement identique à la séquence d'acides aminées représentée par le NO ID SEQ : 5 ou le NO ID SEQ : 9, des amides, des esters ou des sels de ceux-ci. L'invention porte sur des ADN codant ces polypeptides ; sur des vecteurs de recombinaison contenant ces ADN ; sur des transformants transformés par ces vecteurs de recombinaison ; sur des médicaments contenant les polypeptides, amides, esters ou sels de ceux-ci précités ; sur des anticorps contre les polypeptides, amides, esters ou sels de ceux-ci précités et sur des procédé de criblage d'un composé ou de son sel ayant une activité inhibant l'activité de la caspase des polypeptides, amides, esters ou sels de ceux-ci précités.
PCT/JP2001/009071 2000-10-17 2001-10-16 Polypeptides analogues a la survivine et leurs adn WO2002033071A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001294267A AU2001294267A1 (en) 2000-10-17 2001-10-16 Survivin-like polypeptides and dnas thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2000-316721 2000-10-17
JP2000316721 2000-10-17
JP2000-386809 2000-12-20
JP2000386809 2000-12-20

Publications (1)

Publication Number Publication Date
WO2002033071A1 true WO2002033071A1 (fr) 2002-04-25

Family

ID=26602237

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2001/009071 WO2002033071A1 (fr) 2000-10-17 2001-10-16 Polypeptides analogues a la survivine et leurs adn

Country Status (2)

Country Link
AU (1) AU2001294267A1 (fr)
WO (1) WO2002033071A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1316037C (zh) * 2003-12-19 2007-05-16 叶尚勉 检测待测标本中生存素表达量的诊断试剂、检测方法及应用
US7687465B2 (en) 2003-04-11 2010-03-30 Kraeftens Bekaempelse Therapeutic cancer vaccine
AU2004228106B2 (en) * 2003-04-11 2010-05-13 Survac Aps Therapeutic cancer vaccine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008144A1 (fr) * 1998-07-31 2000-02-17 Schering Aktiengesellschaft Adn codant pour une proteine humaine inhibitrice de l'apoptose, hiap3
WO2000023589A2 (fr) * 1998-10-20 2000-04-27 Incyte Pharmaceuticals, Inc. Proteines associees a la proliferation et a l'apoptose
WO2000077201A1 (fr) * 1999-06-15 2000-12-21 Astrazeneca Ab Proteine-3 inhibiteur d'apoptose (iap-3), designee sous le nom de livine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008144A1 (fr) * 1998-07-31 2000-02-17 Schering Aktiengesellschaft Adn codant pour une proteine humaine inhibitrice de l'apoptose, hiap3
WO2000023589A2 (fr) * 1998-10-20 2000-04-27 Incyte Pharmaceuticals, Inc. Proteines associees a la proliferation et a l'apoptose
WO2000077201A1 (fr) * 1999-06-15 2000-12-21 Astrazeneca Ab Proteine-3 inhibiteur d'apoptose (iap-3), designee sous le nom de livine

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7687465B2 (en) 2003-04-11 2010-03-30 Kraeftens Bekaempelse Therapeutic cancer vaccine
AU2004228106B2 (en) * 2003-04-11 2010-05-13 Survac Aps Therapeutic cancer vaccine
CN1316037C (zh) * 2003-12-19 2007-05-16 叶尚勉 检测待测标本中生存素表达量的诊断试剂、检测方法及应用

Also Published As

Publication number Publication date
AU2001294267A1 (en) 2002-04-29

Similar Documents

Publication Publication Date Title
WO2002053738A1 (fr) Nouvelles proteines et adn correspondant
WO2003055506A1 (fr) Medicaments pour la prevention et le traitement du cancer
WO2002033071A1 (fr) Polypeptides analogues a la survivine et leurs adn
US7053053B2 (en) Humanin-like peptide and use thereof
WO2004028558A1 (fr) Agents préventifs/remèdes contre les maladies neurodégénératives
JP4530631B2 (ja) 新規タンパク質および癌の予防・治療剤
JP4320151B2 (ja) 新規ポリペプチドおよびその用途
WO2001048203A1 (fr) Nouvelle proteine et adn associe
JP4353697B2 (ja) がんの予防・治療剤
JP2001029090A (ja) 新規ポリペプチド
JP2004217634A (ja) がんの予防・治療剤
JP2001299363A (ja) 新規タンパク質およびそのｄｎａ
JP2004089182A (ja) がんの予防・治療剤
JP2001228146A (ja) 疾患関連遺伝子の用途
JP2003189873A (ja) がんの予防・治療剤
JP2002355062A (ja) サバイビン類似ポリペプチドおよびそのｄｎａ
JP2005015460A (ja) Ｓｇｌｔホモログ用途
JP2001299364A (ja) 新規タンパク質およびそのｄｎａ
WO2004058969A1 (fr) Medicaments preventifs/remedes pour le cancer
JP2004059502A (ja) ホルモン非依存性癌の予防・治療剤
WO2003087155A1 (fr) Nouvelle proteine et son adn
WO2004039407A1 (fr) Inducteur d'apoptose
JP2004075675A (ja) 骨・関節疾患の予防・治療剤
JP2004173677A (ja) 新規タンパク質およびそのｄｎａ
WO2004018678A1 (fr) Medicaments destines a la prevention et au traitement du cancer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP