JP4488720B2 - Apoptosis-related proteins and uses thereof - Google Patents

Description

  The present invention relates to a functional fragment of a protein that binds to and activates ASK1, and to various uses of the protein and the fragment, in particular pharmaceutical uses.

  Apoptosis plays a role in the removal of cells and abnormal cells that are no longer necessary during development, homeostasis, and the defense function that removes damaged cells. The mechanism at the molecular level is becoming increasingly clear. ing. Abnormalities in these molecules and disruption of control mechanisms impair the physiological functions of apoptosis, and become the cause of onset and exacerbation of various diseases. For example, if apoptosis is excessively suppressed, cells that should be removed will proliferate abnormally and induce tumorous diseases and autoimmune diseases. Death leads to neurodegenerative diseases.

  Mitogen-activated protein (MAP) kinase cascade is a MAP kinase kinase kinase activated by inflammatory cytokines such as physicochemical stress, tumor necrosis factor-α (TNF-α), and interleukin-1 (IL-1). MAPKKK) is a signal transduction mechanism that sequentially activates MAP kinase kinase (MAPKK) and MAP kinase (MAPK). In response to these stimuli, cells have phenotypes such as survival, proliferation, differentiation, and death (apoptosis). Show. c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38) are known as MAPKs that are responsible for part of the signal transduction pathway that induces apoptosis (see, for example, Gastrocnemius Patent Document 1). Furthermore, it is also involved in the induction of an inflammatory response by inducing the production of inflammatory cytokines.

  JNK and p38 are activated by MAPKKs MKK4 / 7 and MKK3 / 6, respectively. These MAPKKs are activated by one MAPKKKK called Apoptosis signal-regulating kinase 1 (ASK1) (Patent Document 1, Non-Patent Document 2). Many MAPKKKKs have been reported in addition to ASK1, but ASK1 is characterized by having the ability to induce apoptosis in cells through signaling through activation of JNK and / or p38. Recently, it has been suggested that activation of ASK1 is also involved in cell differentiation such as keratinocyte differentiation and neurite outgrowth of PC12 cells, and ASK1 plays an important role not only in apoptosis but also in cell fate control It has become clear. Furthermore, it has also been revealed that it is involved in the induction of an inflammatory response by inducing the production of inflammatory cytokines.

Thus, since ASK1 is an important molecule that determines the subsequent fate of cells, various factors are involved in its activation, and it is considered that ASK1 is under complicated control. Until now, it has been reported that the activation of ASK1 requires the formation of homo-oligomers between ASK1 and the subsequent phosphorylation of threonine in the activation loop, and this phosphorylation is mainly self-induced by ASK1. Although it depends on phosphorylation, the existence of another kinase has also been suggested (Non-patent Document 3). On the other hand, protein phosphatase 5 (PP5) is considered to bind directly to ASK1 under H ₂ O ₂ stimulation and return activated ASK1 to an inactive state by dephosphorylating threonine (Non-patent Document). 4). Furthermore, thioredoxin, a redox regulator, binds constitutively to the N-terminal domain of ASK1 in the absence of oxidative stress and acts as an activation inhibitor of ASK1, and leaves ASK1 upon application of oxidative stress, thereby causing ASK1 Is activated (Non-patent Document 5), and when ASK1 is activated by TNF-α, TNF receptor-associated factor 2 (TRAF2) binds to the C-terminal domain of ASK1 to activate ASK1. (Non-patent Document 6), 14-3-3 protein has also been reported to inhibit the activation of ASK1 by binding to the C-terminal domain (Non-patent Document 7).

When ASK1 knockout mouse cells were treated with an endoplasmic reticulum stress inducer, apoptosis was significantly suppressed compared to wild-type mouse cells, so ASK1 is closely related to the induction of apoptosis by endoplasmic reticulum stress, and thus , ASK1 inhibitors such as thioredoxin and 14-3-3 protein, ASK1 dominant negative mutants, ASK1 antisense oligonucleotides, etc., are endoplasmic reticulum stress-related diseases such as neurodegenerative diseases (eg, polyglutamine diseases). It has been suggested that it is effective for the prevention and treatment of (patent document 2). Nishigami et al. Found that accumulation of abnormal proteins in polyglutamine disease induced endoplasmic reticulum stress, and as a result, IRE1, TRAF2, and ASK1 as stress sensor molecules formed a ternary complex, and ASK1 was activated. It has been shown that apoptosis (= neural cell death) is induced through activation (Non-patent Document 8).
JP-A-10-93 International Publication No. 02/38179 Pamphlet “Science” (USA), Vol. 270, p. 1326, 1995 “Science” (USA), Vol. 275, p. 90-94, 1997 “Journal of Cellular Physiology” (USA), Vol. 191, p. 95-104, 2002 “EMBO Journal”, (UK), Volume 20, p. 6028-6036, 2001 “EMBO Journal” (UK), Vol. 17, p. 2596-2606, 1998 “Molecular Cell” (USA), Vol. 2, p. 389-395, 1998 “Proceedings of National Academy of Sciences, USA”, (USA), Vol. 96, p. 8511-8515, 1999 “Jeans Development”, Volume 16, p. 1345-1355, 2002

As described above, the physiological significance of ASK1 and its relation to diseases are gradually becoming clear. However, the complexity of the mechanism of ASK1 activation control and apoptosis induction / inflammatory reaction induction via ASK1 is also complicated. There are still many unclear points, and further research progress is desired.
Therefore, an object of the present invention is to provide new knowledge about the activation mechanism of ASK1 and the mechanism of apoptosis induction / inflammatory reaction induction via the activation mechanism. That is, the first object of the present invention is to identify a novel ASK1-binding protein that has not been known so far, and to clarify the mechanism of ASK1 activation control by the protein. Another object of the present invention is to provide a novel preventive / therapeutic means for various diseases involving ASK1, based on the interaction between the protein and ASK1.

In order to achieve the above object, the present inventors screened an expression library derived from human fetal brain using the human ASK1 full-length cDNA as a bait by the yeast two-hybrid method. unknown function of a protein consisting of 127 amino acids, newly succeeded in cloning as ASK1-binding protein, a SK1 B inding P rotein 1 ( hereinafter, abbreviated as "ABP1") was named. Furthermore, the present inventors have found that the protein not only binds to ASK1, but also activates ASK1 and its downstream JNK and p38, and induces caspase-dependent apoptosis. As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
[1] A peptide or salt thereof containing an amino acid sequence identical or substantially identical to a part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and capable of activating ASK1;
[2] The peptide of the above-mentioned [1] or a salt thereof, comprising the same or substantially the same amino acid sequence as the partial amino acid sequence consisting of about 60 amino acids or more in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4,
[3] The peptide or salt thereof according to [2] above, wherein the partial amino acid sequence is an N-terminal sequence,
[4] A polynucleotide containing a base sequence encoding the peptide of [1] above,
[5] The polynucleotide according to [4] above, which comprises the same or substantially the same base sequence as part of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3,
[6] A recombinant vector containing the polynucleotide according to [4] above,
[7] A transformant obtained by transforming a host with the recombinant vector according to [6] above,
[8] A method for producing the peptide or salt thereof, comprising culturing the transformant according to [7] and collecting the peptide or salt thereof according to [1] from the obtained culture.
[9] A peptide or salt thereof containing an amino acid sequence identical or substantially identical to a part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and capable of not activating or inactivating ASK1;
[10] The peptide according to the above [9] or a salt thereof, comprising the same or substantially the same amino acid sequence as the partial amino acid sequence consisting of about 35 amino acids or less in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4,
[11] The peptide of the above-mentioned [10] or a salt thereof, wherein the partial amino acid sequence is an N-terminal sequence,
[12] ASK1 activation promotion comprising a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the peptide of [1] above, or a salt thereof Agent,
[13] The agent according to [12] above, which is an apoptosis inducer,
[14] A pharmaceutical comprising a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the peptide of [1] above, or a salt thereof,
[15] The medicament according to [14] above, which is a prophylactic / therapeutic agent for diseases in which apoptosis is effective in preventing or treating,
[16] The above [15], wherein the disease is selected from the group consisting of cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ hyperplasia, post-angioplasty restenosis and recurrence after cancer resection The medicines described,
[17] A protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a polynucleotide containing a base sequence encoding the peptide of [1] above An ASK1 activation promoter,
[18] The agent according to [17] above, which is an apoptosis inducer,
[19] A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a polynucleotide containing a base sequence encoding the peptide of [1] above Medicines,
[20] The medicament according to the above [19], which is a prophylactic / therapeutic agent for diseases in which apoptosis is effective in preventing / treating apoptosis,
[21] The above [20], wherein the disease is selected from the group consisting of cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ hyperplasia, post-angioplasty restenosis and recurrence after cancer resection The medicines described,
[22] Apoptosis comprising a nucleotide sequence encoding a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a polynucleotide containing a part thereof, or Diagnostic agent for inflammation-related diseases,
[23] The disease is cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ dysplasia, restenosis after angioplasty, recurrence after cancer resection, transplant organ rejection, graft-versus-host disease, Immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, UV damage, toxic disease, nutritional disorder, inflammatory disease, ischemic neuropathy, diabetic neuropathy, vascular disease, respiratory disease and cartilage disease The diagnostic agent according to [22], selected from the group consisting of:
[24] A polynucleotide containing a base sequence complementary to a base sequence encoding a protein containing the same or substantially the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 4, or a part thereof. An ASK1 activation inhibitor comprising:
[25] The agent according to the above [24], which is an inhibitor of apoptosis or inflammatory cytokine production,
[26] A polynucleotide containing a base sequence complementary to a base sequence encoding a protein containing the same or substantially the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 4, or a part thereof. A pharmaceutical comprising,
[27] The medicament according to the above [26], which is a prophylactic / therapeutic agent for a disease in which apoptosis or inflammation is effectively prevented / treated,
[28] The disease is viral infection, endocrine disease, blood disease, organ dysplasia, transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, UV damage, toxicity The medicament according to [27] above, selected from the group consisting of diseases, nutritional disorders, inflammatory diseases, ischemic neuropathy, diabetic neuropathy, vascular diseases, respiratory diseases and cartilage diseases,
[29] An amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, characterized in that the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 can be specifically recognized An antibody against a protein or a salt thereof containing
[30] A diagnostic agent for an apoptosis or inflammation-related disease comprising an antibody against a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a salt thereof,
[31] The disease is cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ dysplasia, restenosis after angioplasty, recurrence after cancer resection, transplant organ rejection, graft-versus-host disease, Immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, UV damage, toxic disease, nutritional disorder, inflammatory disease, ischemic neuropathy, diabetic neuropathy, vascular disease, respiratory disease and cartilage disease The diagnostic agent according to the above [30], selected from the group consisting of:
[32] An ASK1 activation inhibitor comprising an antibody against a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a salt thereof,
[33] The agent according to the above [32], which is an inhibitor of apoptosis or inflammatory cytokine production,
[34] A medicament comprising an antibody against a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a salt thereof,
[35] The medicament according to the above [34], which is a prophylactic / therapeutic agent for a disease in which apoptosis or inflammation is effectively prevented / treated.
[36] The disease is viral infection, endocrine disease, blood disease, organ dysgenesis, transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, ultraviolet ray damage, toxicity The medicament according to [35] above, selected from the group consisting of diseases, nutritional disorders, inflammatory diseases, ischemic neuropathy, diabetic neuropathy, vascular diseases, respiratory diseases and cartilage diseases,
[37] Use of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, the peptide of the above [1], a salt thereof, or a cell producing the same. A screening method for an ASK1 activation modulator,
[38] The method according to [37] above, further comprising using ASK1 or a partial peptide thereof including the N-terminal activation control domain, or a salt thereof, or a cell producing the same.
[39] A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the peptide or salt thereof according to [1] above, and ASK1 or N-terminal activation control domain The method according to [38] above, wherein the binding to a partial peptide containing the same or a salt thereof is measured;
[40] A protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the peptide of the above [1], a salt thereof, or a cell producing the same , A kit for screening for an ASK1 activation modulator,
[41] The kit according to [40] above, further comprising ASK1 or a partial peptide thereof including the N-terminal activation control domain or a salt thereof or a cell producing the same.
[42] Activation of ASK1 or a partial peptide thereof or a salt thereof in a cell producing ASK1 or an N-terminal activation control domain thereof and a partial peptide thereof or a salt thereof containing a kinase domain, (1) SEQ ID NO: 2 or SEQ ID NO: 4 In the presence of a protein containing the same or substantially the same amino acid sequence as shown in the above, or the peptide according to [1] above or a salt thereof, and (2) the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. The method according to [38] above, wherein the protein comprising the same or substantially the same amino acid sequence as above, the peptide according to [1] above or a salt thereof and a test substance are compared.
[43] (1) a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the peptide or salt thereof according to [1] above, and (2) ASK1 or Comparing the activation of ASK1 or a partial peptide or a salt thereof in a cell producing a partial peptide or a salt thereof containing an N-terminal activation control domain and a kinase domain in the presence or absence of a test substance The method according to [38] above,
[44] A protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the protein or the peptide in a cell producing the peptide according to [1] above or a salt thereof Or a method for screening an ASK1 activation regulator, comprising comparing the expression of the salt in the presence and absence of the test substance,
[45] A polynucleotide containing a base sequence encoding a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a part thereof, or SEQ ID NO: 2 or a sequence The method according to [44] above, wherein an antibody against a protein containing the same or substantially the same amino acid sequence as shown in No. 4 or a salt thereof is used,
[46] A polynucleotide containing a nucleotide sequence encoding a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a part thereof, or SEQ ID NO: 2 or a sequence A kit for screening an ASK1 activation modulator comprising an antibody against a protein containing the same or substantially the same amino acid sequence as shown in No. 4 or a salt thereof,
[47] An apoptosis-inducing agent comprising a substance that increases the expression or activity of a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a salt thereof,
[48] A medicament comprising a substance that increases the expression or activity of a protein containing the same or substantially the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 4, or a salt thereof,
[49] The medicament according to [48] above, which is a prophylactic / therapeutic agent for a disease in which apoptosis is effective in preventing or treating apoptosis,
[50] The above [49], wherein the disease is selected from the group consisting of cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ hyperplasia, post-angioplasty restenosis and recurrence after cancer resection The medicines described,
[51] Apoptosis or inflammatory cytokine containing a substance that decreases the expression or activity of a protein containing the same or substantially the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 4 or a salt thereof Production inhibitor,
[52] A medicament comprising a substance that decreases the expression or activity of a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4, or a salt thereof,
[53] The medicament according to the above [52], which is a prophylactic / therapeutic agent for a disease effective in suppressing apoptosis or inflammation, and [54] the disease is a viral infection, endocrine disease, blood disease, Organ dysplasia, transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, UV damage, addictive disease, nutritional disorder, inflammatory disease, ischemic neuropathy, diabetic The pharmaceutical according to [53] above, which is selected from the group consisting of neuropathy, vascular disease, respiratory disease and cartilage disease.

Furthermore, the present invention provides
[55] The same or substantially the same amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4, comprising an ASK1 partial peptide containing ASK1 N-terminal activation control domain and no kinase domain, or a salt thereof An inhibitor of a protein or salt thereof containing the same amino acid sequence,
[56] The agent described in [55] above, which is an inhibitor of apoptosis or inflammatory cytokine production,
[57] A medicament comprising an ASK1 partial peptide containing ASK1 N-terminal activation control domain and not including a kinase domain, or a salt thereof,
[58] The medicament according to the above [57], which is a prophylactic / therapeutic agent for a disease that is effective for the prevention / treatment of suppressing apoptosis or inflammation,
[59] The disease is viral infection, endocrine disease, blood disease, organ dysplasia, transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, UV damage, toxicity The medicament according to [58] above, selected from the group consisting of diseases, nutritional disorders, inflammatory diseases, ischemic neuropathy, diabetic neuropathy, vascular diseases, respiratory diseases and cartilage diseases,
[60] An ASK1 activation inhibitor comprising the peptide of [9] above or a salt thereof,
[61] The agent described in [60] above, which is an inhibitor of apoptosis or inflammatory cytokine production,
[62] A medicament comprising the peptide of [9] above or a salt thereof,
[63] The medicament according to the above [62], which is a prophylactic / therapeutic agent for a disease that is effective for preventing / treating apoptosis or inflammation, and [64] the disease is a viral infection, endocrine disease, blood disease, Organ dysplasia, transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, UV damage, toxic disease, nutritional disorder, inflammatory disease, ischemic neuropathy, diabetic The pharmaceutical according to the above-mentioned [63], which is selected from the group consisting of neuropathy, vascular disease, respiratory disease and cartilage disease.

  The ABP1, the polynucleotide encoding the same, and the like of the present invention have effects such as inducing apoptosis in cells and inducing production of inflammatory cytokines by activating the ASK1 cascade. On the other hand, the ABP1 inhibitor (eg, anti-ABP1 antibody, ABP1 antisense polynucleotide, etc.) of the present invention inhibits ABP1 expression or activity, thereby inhibiting ASK1 activation and producing apoptosis or inflammatory cytokines. There is an effect such as suppression.

The protein used in the present invention (hereinafter referred to as “ABP1 of the present invention” or simply “ABP1”) is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or 4 It is.
The ABP1 of the present invention is a cell (for example, human, mouse, rat, guinea pig, hamster, rabbit, sheep, goat, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) Hepatocytes, spleen cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibroblasts, myocytes , Adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, Bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells or cancer cells) or any of those cells Tissue, for example, brain, brain parts (eg, olfactory bulb, amygdala, basal sphere, hippocampus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, Gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus It may be a protein derived from bone, joint, skeletal muscle or the like, or may be a protein synthesized by chemical synthesis or a cell-free translation system. Alternatively, it may be a recombinant protein produced from a transformant introduced with a polynucleotide having a base sequence encoding the amino acid sequence.

As the “substantially identical amino acid sequence” to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is about 70% or more, preferably about 80% or more, More preferred is an amino acid sequence having a homology of about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more.
Examples of the “protein containing substantially the same amino acid sequence” as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 are substantially the same as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. And a protein having substantially the same quality of activity as a protein containing the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

Examples of “substantially the same activity” include activation promoting activity of ASK1 or a group of kinases located downstream thereof (eg, MKK4 / 7, MKK3 / 6, JNK, p38, etc.), cell apoptosis-inducing activity, etc. Is mentioned. Substantially homogeneous indicates that their properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that the activities such as ASK1 cascade activation promotion are equivalent, but quantitative factors such as the degree of these activities and the molecular weight of the protein may be different (for example, about 0.01 To 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times).
ASK1 cascade activation promoting activity is measured by a known method, for example, using a labeled phosphate group donor, or a kinase group located downstream of ASK1 (eg, MKK4 / 7, MKK3 / 6, JNK, p38, etc. ), And the apoptosis-inducing activity can be measured by measuring cell death induction rate, morphological observation of cells, detection of DNA fragmentation, and the like.

The ABP1 of the present invention includes, for example, 1) 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10) in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, More preferably, the amino acid sequence in which several (1 to 5) amino acids are deleted, 2) 1 or 2 (preferably about 1 to 30) in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, Preferably about 1 to 10, more preferably a number (1 to 5) amino acids are added. 3) One or more amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4 (preferably 1 to 30 amino acids, preferably about 1 to 10 amino acids, more preferably a number (1 to 5)) of amino acid sequences, 4) in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 1 of Or an amino acid sequence in which 2 or more (preferably about 1 to 30, preferably about 1 to 10, more preferably (1 to 5) amino acids) are substituted with other amino acids, or 5) So-called muteins such as proteins containing amino acid sequences obtained by combining them are also included.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the activity of the protein is maintained.

  The ABP1 of the present invention is preferably human ABP1 having the amino acid sequence shown in SEQ ID NO: 2 (hABP1) or mouse ABP1 having the amino acid sequence shown in SEQ ID NO: 4 (mABP1), or other warm-blooded animals (for example, Rat, guinea pig, hamster, rabbit, sheep, goat, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.). Although hABP1 is a protein consisting of 127 amino acids encoded by a known gene derived from human T cells (GenBank accession number: AF116272) named PGR1, no report has been made on its function. Mouse ABP1 is a protein consisting of 125 amino acids encoded by mouse brain-derived cDNA (GenBank accession number: AB041651) named clone MNCb-1039, but there is no report regarding its function.

In the proteins described in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide designation. The ABP1 of the present invention, including a protein containing the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, has a C-terminal at the carboxyl group (—COOH), carboxylate (—COO ⁻ ), amide (—CONH ₂ ) Or ester (—COOR).
Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; C _6-12 aryl groups such as α-naphthyl; phenyl-C _1-2 alkyl groups such as benzyl and phenethyl; C _7- such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl; ₁₄ aralkyl group; pivaloyloxymethyl group and the like are used.
When ABP1 has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in ABP1 of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Furthermore, in ABP1 of the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) is a protecting group (eg, C _1-6 acyl such as C _1-6 alkanoyl such as formyl group, acetyl group). Group, etc.), an N-terminal glutamine residue produced by cleavage in vivo is pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, Amino group, imidazole group, indole group, guanidino group, etc.) protected with an appropriate protecting group (for example, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group, acetyl group, etc.) Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.

The present invention provides a peptide having the above-mentioned partial amino acid sequence of ABP1 and having substantially the same quality of activity as ABP1. Here, “substantially the same quality of activity” has the same meaning as described above. The “substantially the same quality of activity” can be measured in the same manner as described above. In the present specification, the partial peptide is hereinafter referred to as “the activated peptide of the present invention”.
The activation peptide of the present invention is not particularly limited as long as it has the above-mentioned properties. For example, it is about 60 amino acids or more in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, preferably about 60 to about 100 amino acids, more preferably Includes peptides containing the same or substantially the same amino acid sequence as the partial amino acid sequence consisting of about 60 to about 80 amino acids. The partial amino acid sequence may be the N-terminal sequence of ABP1, the C-terminal sequence, or an internal sequence. Or the combination of those partial arrangement | sequences may be sufficient.
Preferably, the activation peptide of the present invention has about 60 amino acids or more on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, more preferably about 60 to about 100 amino acids, particularly preferably about 60 to about Contains a partial amino acid sequence of 80 amino acids.
In a particularly preferred range, the activation peptide of the present invention may exhibit higher activity (eg, ASK1 cascade activation promoting activity, apoptosis inducing activity, etc.) than the full-length protein.

On the other hand, the partial peptides of ABP1 include those that can function as (competitive) inhibitors of ABP1 or “the activating peptide of the present invention”. Examples of such partial peptides include those that have binding activity to ASK1, but cannot activate the kinase. In the present specification, the partial peptide is hereinafter referred to as “inhibitory peptide of the present invention”.
That is, the inhibitory peptide of the present invention contains the same or substantially the same amino acid sequence as part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and can not activate or inactivate ASK1. It is a peptide. Examples of the inhibitory peptide include those containing a partial amino acid sequence consisting of about 35 amino acids or less in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, preferably a partial amino acid sequence on the N-terminal side.

The partial peptide of ABP1 of the present invention (including both of the activation peptide of the present invention and the inhibitory peptide of the present invention; hereinafter may be simply abbreviated as “partial peptide of the present invention”) has a C-terminal carboxyl group Any of (—COOH), carboxylate (—COO ⁻ ), amide (—CONH ₂ ), and ester (—COOR) may be used. Here, examples of R in the ester include the same as those described above for ABP1. When these peptides have a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Further, in the partial peptide of the present invention, as in the case of ABP1, the amino group of the N-terminal methionine residue is protected with a protecting group, and the Gln produced by cleaving the N-terminal side in vivo is pyroglutamic acid. In which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.

  Examples of the salt of ABP1 or a partial peptide thereof of the present invention include physiologically acceptable salts with acids or bases, and 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). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The ABP1 or a salt thereof of the present invention can be prepared from the aforementioned warm-blooded animal cells or tissues by a known protein purification method. Specifically, the tissue or cells of warm-blooded animals are homogenized, and the soluble fraction is separated and purified by chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. Can be manufactured.

The ABP1 of the present invention or a partial peptide thereof or a salt thereof (hereinafter may be generically referred to as “ABP1 of the present invention”) can also be produced according to known peptide synthesis methods.
The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. When the partial peptide or amino acid capable of constituting ABP1 is condensed with the remaining part and the product has a protecting group, the target protein can be produced by removing the protecting group.
Here, the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the methods described in the following 1) to 5).
1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
5) Supervised by Yajima Haruaki, Development of follow-up drugs, Volume 14, Peptide synthesis, Hirokawa Shoten

The ABP1s thus obtained can be isolated and purified by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When the protein (peptide) obtained by the above method is a free form, the free form can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, the protein (peptide) is converted into a salt. When obtained, the salt can be converted into a free form or other salt by a known method or a method analogous thereto.

  For the synthesis of ABP1 of the present invention, commercially available resin for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected, according to the sequence of the target protein or peptide (hereinafter sometimes collectively referred to as “protein etc.”) According to various known condensation methods, condensation is carried out on the resin. At the end of the reaction, proteins and the like are cut out from the resin, and at the same time, various protecting groups are removed, and an intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein or the amide form thereof.

  For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide, and the like. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. Can then be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range known to be usable for protein bond forming reaction, and is usually selected appropriately from a range of about −20 ° C. to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

The protection of the functional group that should not be involved in the reaction of the raw material, the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
Examples of the protecting group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.

  Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 Addition of a cation scavenger such as -butanedithiol, 1,2-ethanedithiol, etc. is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

  Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.

As another method for obtaining amides such as proteins, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide (protein) chain is extended to the desired chain length on the amino group side. Thereafter, a protein or the like from which only the protecting group of the α-amino group at the N-terminal of the peptide chain is removed and a protein from which only the protecting group of the carboxyl group at the C-terminal is removed are prepared. In a mixed solvent. The details of the condensation reaction are the same as described above. After purifying the protected protein and the like obtained by condensation, all the protecting groups are removed by the above method to obtain the desired crude protein and the like. This crude protein or the like can be purified by making use of various known purification means, and the main fraction can be freeze-dried to obtain an amide form of a desired protein or the like.
To obtain an ester such as a protein, for example, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired ester such as a protein is obtained in the same manner as the amide such as a protein. You can get a body.

  The partial peptide of the present invention or a salt thereof can also be produced by cleaving ABP1 or a salt thereof with an appropriate peptidase.

Furthermore, the ABP1 of the present invention can also be produced by culturing a transformant containing DNA encoding ABP1 or a partial peptide thereof, and separating and purifying the ABP1 from the resulting culture.
Examples of DNA encoding ABP1 of the present invention or a partial peptide thereof include genomic DNA, genomic DNA library, human or other warm-blooded animals (eg, human, mouse, rat, guinea pig, hamster, rabbit, sheep, goat, pig). , Cows, horses, birds, cats, dogs, monkeys, chimpanzees, etc.) (eg splenocytes, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, Endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes ), Megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or these cells Progenitor cells, stem cells or cancer cells), blood cells, or any tissue in which these cells exist, such as the brain, brain regions (eg, olfactory bulb, buccal nucleus, basal sphere, hippocampus, thalamus) , Hypothalamus, hypothalamic nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, parietal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver , Gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cell, prostate, testis, testis , CDNA derived from the ovary, placenta, uterus, bone, joint, skeletal muscle, etc. (particularly each part of the brain or brain), cDNA library derived from the cells / tissues described above, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as “RT-PCR method”) using a total RNA or mRNA fraction prepared from the cells / tissues described above.

Examples of the DNA encoding ABP1 of the present invention include DNA containing the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under highly stringent conditions. Activity substantially the same as the protein containing the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 (eg, ASK1 cascade activation promoting activity, apoptosis inducing activity, etc.) DNA encoding a protein having
Examples of the DNA that can hybridize with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under highly stringent conditions include, for example, about 50% or more, preferably about 50%, of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. DNA containing a nucleotide sequence having a homology of 60% or more, more preferably about 70% or more, particularly preferably about 80% or more, and most preferably about 90% or more is used.

Hybridization should be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning Second Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to highly stringent conditions.
The highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is preferable.
The DNA encoding ABP1 of the present invention is preferably hABP1 DNA containing the base sequence shown in SEQ ID NO: 1 or mABP1 DNA containing the base sequence shown in SEQ ID NO: 3, or other warm-blooded animals (for example, rat , Guinea pigs, hamsters, rabbits, sheep, goats, pigs, cows, horses, birds, cats, dogs, monkeys, chimpanzees, etc.) and their homologues.

  The DNA encoding the partial peptide of the present invention is any DNA as long as it contains a base sequence encoding the same or substantially the same amino acid sequence as part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. There may be. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, it can be directly amplified by RT-PCR using mRNA fraction prepared from the cells / tissues described above.

Specifically, as the DNA encoding the partial peptide of the present invention, for example,
(1) DNA having a partial base sequence of DNA having the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or (2) highly stringent with DNA having the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. (2a) substantially the same activity as a protein containing an amino acid sequence encoded by the DNA (eg, ASK1 cascade activation promoting activity, apoptosis inducing activity, etc.), Or (2b) an activity that inhibits the activity of a protein containing the amino acid sequence encoded by the DNA (eg, ASK1 cascade activation inhibitory activity, apoptosis inhibitory activity, etc.)
For example, DNA encoding a peptide having
Examples of DNA that can hybridize with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under highly stringent conditions include, for example, about 60% or more, preferably about 70% or more with the corresponding portion in the base sequence, More preferably, a polynucleotide containing a base sequence having a homology of about 80% or more, most preferably about 90% or more is used.

  A DNA encoding ABP1 of the present invention or a partial peptide thereof is amplified by a PCR method using a synthetic DNA primer having a part of a base sequence encoding the protein or peptide, or a DNA incorporated into an appropriate expression vector. They can be cloned by hybridization with a DNA fragment or a synthetic DNA labeled that encodes part or all of the protein of the present invention. Hybridization can be performed, for example, according to the method described in Molecular Cloning Second Edition (described above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

The DNA base sequence can be determined using known kits such as Mutan ^™ -super Express Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, the Gapped duplex method, Conversion can be performed according to a method known per se such as the Kunkel method or a method analogous thereto.

  The cloned DNA can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

The DNA expression vector encoding ABP1 of the present invention or a partial peptide thereof can be obtained, for example, by cutting out a target DNA fragment from DNA encoding ABP1 and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. Can be manufactured.
As expression vectors, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19, pSH15); Bacteriophages; animal viruses such as retrovirus, vaccinia virus, baculovirus; pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like.
The promoter may be any promoter as long as it is appropriate for the host used for gene expression.
For example, when the host is an animal cell, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter and the like are used. Of these, CMV promoter, SRα promoter and the like are preferable.
When the host is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, T7 promoter and the like are preferable.
When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable.
When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can. Examples of selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ^r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r, G418 resistance). In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the target gene can be selected using a medium not containing thymidine.
If necessary, a signal sequence suitable for the host may be added to the N-terminal side of the protein of the present invention. When the host is Escherichia, PhoA signal sequence, OmpA, signal sequence, etc .; When the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc .; When the host is yeast When the host is an animal cell, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, and the like are used, respectively.

The transformant containing “DNA encoding ABP1 of the present invention or a partial peptide thereof” obtained as described above is obtained by transforming a host with an expression vector containing the DNA according to a known method. Can be manufactured.
Here, examples of the expression vector include those described above.
As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells and the like are used.

Examples of the genus Escherichia include, for example, Escherichia coli K12 • DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. , 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], and the like.
Examples of Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gen, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984). )] Etc. are used.
Examples of yeast include Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia Used.

Examples of insect cells include, for the virus AcNPV, cabbage armyworm larvae derived cell line (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from mid-intestine of Trichoplusia ni, High Five ^TM cell derived from an egg of Trichoplusia ni , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, moth-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used as insect cells. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)).
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

Transformation can be performed according to a known method depending on the type of host.
Examples of the genus Escherichia include, for example, Proceedings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 (1972) and Gene ( Gene), Vol. 17, 107 (1982) and the like.
Bacillus can be transformed according to the method described in, for example, Molecular & General Genetics, 168, 111 (1979).
Yeast is, for example, Methods in Enzymology, 194, 182-187 (1991), Proceedings of the National Academy of Sciences of USA ( Proc. Natl. Acad. Sci. USA), 75, 1929 (1978), and the like.
Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
Animal cells are, for example, cell engineering separate volume 8 new cell engineering experiment protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973).

The culture of the transformant can be performed according to a known method depending on the type of the host.
For example, when culturing a transformant whose host is an Escherichia or Bacillus genus, a liquid medium is preferable as a medium used for the culture. Moreover, it is preferable that a culture medium contains a carbon source, a nitrogen source, an inorganic substance, etc. which are required for the growth of a transformant. Here, as the carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc .; As the nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, Inorganic or organic substances such as potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added to the medium. The pH of the medium is preferably about 5-8.
As a medium for culturing a transformant whose host is an Escherichia bacterium, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in Molecular Genetics ( Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, a drug such as 3β-indolylacrylic acid may be added to the medium in order to make the promoter work efficiently.
The transformant whose host is Escherichia is usually cultured at about 15 to 43 ° C. for about 3 to 24 hours. If necessary, aeration or agitation may be performed.
The transformant whose host is a Bacillus genus is usually cultured at about 30 to 40 ° C. for about 6 to 24 hours. If necessary, aeration or agitation may be performed.
As a medium for culturing a transformant whose host is yeast, for example, a Burkholder minimum medium [Bostian, KL et al., Proceedings of the National Academy of Sciences of Science The USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Proceedings of the National -Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours. Aeration and agitation may be performed as necessary.
As a medium for culturing a transformant whose host is an insect cell or insect, for example, 10% bovine serum inactivated in Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)) What added the thing suitably is used. The pH of the medium is preferably about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days. You may perform ventilation | gas_flowing and stirring as needed.
As a medium for culturing a transformant whose host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)] is used. The pH of the medium is preferably about 6-8. The culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours. You may perform ventilation | gas_flowing and stirring as needed.
As described above, the ABP1 of the present invention or a partial peptide thereof or a salt thereof (ABP1s) can be produced inside or outside the transformant.

The ABP1 of the present invention can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
For example, when the ABP1 of the present invention is extracted from cultured cells or cells, the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and are subjected to ultrasound, lysozyme and / or freeze-thaw, etc. A method of obtaining a crude extract of soluble protein by centrifugation or filtration after disrupting cells or cells by the method is appropriately used. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ .
The ABP1 of the present invention contained in the soluble fraction thus obtained can be isolated and purified according to a method known per se. As such methods, methods utilizing the solubility such as salting out and solvent precipitation method; mainly utilizing differences in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis method. A method utilizing a difference in charge such as ion exchange chromatography, a method utilizing a specific affinity such as affinity chromatography, a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography, etc. A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.

When the ABP1 thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the ABP1 is obtained as a salt, the per se known The salt can be converted to a free form or other salt by the method of or a method analogous thereto.
The ABP1 produced by the transformant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of the ABP1 of the present invention thus obtained can be confirmed by enzyme immunoassay or Western blotting using a specific antibody.

  Furthermore, ABP1 of the present invention uses a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc., using RNA corresponding to DNA encoding ABP1 or a partial peptide thereof as a template. It can also be synthesized by in vitro translation. Alternatively, a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize DNA encoding ABP1 or a partial peptide thereof as a template.

The antibody against ABP1 of the present invention or a salt thereof (hereinafter sometimes abbreviated as “the antibody of the present invention”) is either a polyclonal antibody or a monoclonal antibody as long as it can recognize ABP1 or a salt thereof. Also good. An antibody against ABP1 or a salt thereof can be produced according to a method for producing an antibody or antiserum known per se, using the ABP1 of the present invention as an antigen. As the ABP1 of the present invention used as an antigen, any of the above ABP1 or a partial peptide thereof or a salt thereof may be used.
A monoclonal antibody or a polyclonal antibody against ABP1 or a salt thereof can be produced, for example, as follows.

[Production of monoclonal antibodies]
(A) Production of Monoclonal Antibody-Producing Cells The ABP1 of the present invention is administered to a mammal at a site where antibody production is possible by administration, or with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used.
For example, an individual with an antibody titer selected from a mammal immunized with an antigen, such as a mouse, is collected 2 to 5 days after the final immunization, and spleen or lymph nodes are collected, and antibody-producing cells contained therein are allogeneic or Monoclonal antibody-producing hybridomas can be prepared by fusing with myeloma cells from different animals. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with 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 Kohler and Milstein (Nature, 256, 495 (1975)). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.

Examples of myeloma cells include mammalian myeloma cells such as NS-1, P3U1, SP2 / 0, AP-1, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. Cell fusion can be carried out efficiently by incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes.
Monoclonal antibody-producing hybridomas are prepared, for example, by adding the hybridoma culture supernatant to a solid phase (eg, microplate) on which an antigen is adsorbed directly or with a carrier, and then anti-immunoglobulin antibody (cell When mouse used for fusion is mouse, anti-mouse immunoglobulin antibody is used) or protein A is added to detect monoclonal antibody bound to the solid phase; solid phase adsorbed with anti-immunoglobulin antibody or protein A The method can be screened by adding a hybridoma culture supernatant, adding a protein labeled with a radioactive substance or an enzyme, and detecting a monoclonal antibody bound to the solid phase.
The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. The selection of the monoclonal antibody can be usually performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. Any medium can be used for the selection and breeding of the monoclonal antibody as long as the hybridoma can grow. Examples of such a medium include RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal calf serum, and GIT medium containing 1 to 10% fetal calf serum (Wako Pure Chemical Industries, Ltd.). Or a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
The monoclonal antibody thus obtained can be obtained by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, , DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method to obtain antibody by dissociating the binding using active adsorbent such as protein A or protein G ] Can be separated and purified according to the above.

[Preparation of polyclonal antibody]
A polyclonal antibody against ABP1 or a salt thereof can be produced according to a method known per se. For example, an immunizing antigen (ABP1) itself or a complex thereof and a carrier protein are prepared, and a mammal is immunized in the same manner as the above-described monoclonal antibody production method. It can be produced by collecting and purifying the antibody. In addition to mammals, chickens and the like can also be used.
Regarding the complex of immune antigen and carrier protein used to immunize mammals, the type of carrier protein and the mixing ratio of carrier and hapten should be such that an antibody can be efficiently produced against a hapten immunized by crosslinking to a carrier. Any ratio may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to 20, preferably about 1 to about 1 by weight with respect to hapten 1. A method of coupling at a rate of 5 is used.
For coupling of the hapten and the carrier, various condensing agents such as glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and active ester reagent containing a dithiobilidyl group are used.
The condensation product is administered to a mammal at a site where antibody production is possible, together with the carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably from blood.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

  When a partial peptide of ABP1 is used as an antigen, the position on ABP1 is not particularly limited, and examples thereof include oligopeptides having partial amino acid sequences of regions well conserved among various warm-blooded animals. Specifically, a peptide having the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 that is completely conserved between human and mouse is preferably exemplified.

  A polynucleotide containing a base sequence complementary to the base sequence encoding ABP1 of the present invention or a part thereof (hereinafter sometimes abbreviated as “antisense polynucleotide of the present invention”) encodes ABP1. What is necessary is just to have the effect | action which has a base sequence completely complementary to a base sequence, a substantially complementary base sequence, or its part, and has the effect | action which suppresses translation of this protein from RNA which codes ABP1. As the “substantially complementary base sequence”, a base sequence encoding ABP1 or a partial peptide thereof and a base sequence capable of hybridizing under physiological conditions of a cell expressing the protein, more specifically, About 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more with respect to the overlapping portion between the complementary strand of the base sequence encoding ABP1 or a partial peptide thereof The nucleotide sequence having homology of

  The antisense polynucleotide of the present invention can be designed and synthesized based on the nucleotide sequence information of the polynucleotide of the present invention that has been cloned or determined. Such polynucleotides can inhibit the replication or expression of the gene encoding ABP1. That is, the antisense polynucleotide of the present invention can hybridize with RNA transcribed from a gene encoding ABP1, and can inhibit mRNA synthesis (processing) or function (translation into protein).

The target region of the antisense polynucleotide of the present invention is not particularly limited in length as long as the antisense polynucleotide hybridizes, and as a result, the translation of ABP1 is inhibited. RNA encoding ABP1 The short sequence may be about 15 bases, and the long sequence may be the entire mRNA or initial transcription product sequence. In view of ease of synthesis and antigenicity, an oligonucleotide consisting of about 15 to about 30 bases is preferable, but not limited thereto. Specifically, for example, 5 ′ end hairpin loop of gene encoding ABP1, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon The 3 ′ end untranslated region, 3 ′ end palindromic region, and 3 ′ end hairpin loop can be selected as the target region, but any region within the gene can be selected as the target. For example, it is also preferable to use the intron portion of the gene as a target region.
Furthermore, the antisense polynucleotide of the present invention not only hybridizes with mRNA or initial transcription product encoding ABP1 to inhibit translation into protein, but also binds to a gene encoding ABP1 which is a double-stranded DNA. It is also possible to form a triplex to inhibit RNA transcription.

  Antisense polynucleotides may be deoxyribonucleotides containing 2-deoxy-D-ribose, ribonucleotides containing D-ribose, other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or non-nucleotides. Other polymers with a nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers that contain special linkages (provided that the polymer is a base such as found in DNA or RNA) And a nucleotide having a configuration allowing the attachment of a pairing or base). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and also unmodified polynucleotides (or unmodified oligonucleotides), and more publicly known Modified, such as those with labels known in the art, capped, methylated, substituted one or more natural nucleotides with analogs, intramolecular nucleotides Modified, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) ), For example, proteins (nucleases, nuclease inhibitors, and toxins) , Antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelates Compounds containing compounds (for example, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) It may be. Here, “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other heterocyclic bases modified. 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, eg, one or more hydroxyl groups are replaced by halogens or aliphatic groups, or converted to functional groups such as ethers, amines, etc. May be.

  Antisense polynucleotides are RNA, DNA, or modified nucleic acids (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense polynucleotide of the present invention can be preferably designed according to the following policy. That is, make the antisense polynucleotide more stable in the cell, increase the cell permeability of the antisense polynucleotide, increase the affinity for the target sense strand, and if it is toxic This reduces the toxicity of the antisense polynucleotide. Many such modifications are known in the art, such as J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed ., Antisense Research and Applications, CRC Press, 1993, etc.

  Antisense polynucleotides can be altered or contain modified sugars, bases, linkages, donated in specialized forms such as liposomes, microspheres, applied or added by gene therapy. Can be given in different forms. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( For example, hydrophobic substances such as phospholipids and cholesterols). Preferred lipids to be added include cholesterol and derivatives thereof (for example, cholesteryl chloroformate, cholic acid, etc.). Such can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of the nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

  A ribozyme capable of specifically cleaving mRNA or gene initial transcript encoding ABP1 within the coding region (including intron portion in the case of the initial transcript) can also be included in the antisense polynucleotide of the present invention. . “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligo DNA having the base sequence of the enzyme active site also has a nucleic acid cleavage activity. As long as it has sequence-specific nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends adjacent to the part having the hammerhead structure (about 10 bases in total) are made into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the additional advantage of not attacking genomic DNA since it only uses RNA as a substrate. When the mRNA encoding ABP1 has a double-stranded structure by itself, the target sequence is made single-stranded by using a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked with a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

A double-stranded oligo RNA (siRNA) complementary to mRNA encoding ABP1 or a partial sequence within the coding region of the gene initial transcript (including an intron in the case of the initial transcript) is also included in the present invention. Of antisense polynucleotides. When a short double-stranded RNA is introduced into a cell, mRNA complementary to the RNA is degraded, so-called RNA interference (RNAi) has been known in nematodes, insects, plants, etc. Recently, it has been confirmed that this phenomenon also occurs in mammalian cells [Nature, 411 (6836): 494-498 (2001)], and has attracted attention as an alternative to ribozyme.
The antisense oligonucleotide and ribozyme of the present invention determine the target region of mRNA or initial transcription product based on the cDNA sequence or genomic DNA sequence information encoding the protein of the present invention, and use a commercially available DNA / RNA automatic synthesizer (Applied -Biosystems, Beckman, etc.) can be prepared by synthesizing a complementary sequence thereto. For siRNA, a sense strand and an antisense strand are respectively synthesized by a DNA / RNA automatic synthesizer, denatured in an appropriate annealing buffer at, for example, about 90 to about 95 ° C. for about 1 minute, and then about 30 to It can be prepared by annealing at about 70 ° C. for about 1 to about 8 hours. In addition, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide strands so as to alternately overlap, annealing them, and then ligating with ligase.

  Since ABP1 or the activation peptide of the present invention binds to and activates ASK1, it can promote apoptosis induction via the ASK1 kinase cascade. Therefore, 1) ABP1 or the activation peptide of the present invention, 2) a polynucleotide encoding ABP1 or the activation peptide of the present invention, 3) a polynucleotide containing a base sequence encoding ABP1 or a part thereof, 4) this The antibody of the invention, 5) the antisense polynucleotide of the present invention, and 6) the inhibitory peptide of the present invention has the following uses.

(1) ASK1 activation promoter / apoptosis inducer ABP1 has a function of promoting apoptosis induction via the ASK1 kinase cascade by binding to and activating ASK1. Therefore, ABP1 or the activation peptide of the present invention or a salt thereof is added to the cell, or a polynucleotide encoding ABP1 or the activation peptide of the present invention is introduced into the cell and expressed, thereby increasing the amount of ABP1 in the cell. As a result, the activation of intracellular ASK1 can be promoted or apoptosis can be induced in the cell. For example, it can be used as a reagent for studying apoptosis.
When ABP1 or the activation peptide of the present invention or a salt thereof is used as the above ASK1 activation promoter or apoptosis inducer, water or a suitable buffer (eg, phosphate buffer, PBS, Tris-HCl buffer, etc.) It can be prepared by dissolving in a suitable concentration. Moreover, you may mix | blend the preservative, stabilizer, reducing agent, tonicity agent, etc. which are used normally as needed.
On the other hand, when the polynucleotide encoding ABP1 or the activation peptide of the present invention is used as the above-mentioned ASK1 activation promoter or apoptosis inducer, the polynucleotide may be used alone or in retrovirus vector, adenovirus vector, adenovirus associate. After insertion into an appropriate vector such as a Ted virus vector, it can be introduced into cells using the transformation methods described above (eg, liposome method, electroporation method, etc.).

(2) Prophylactic / Therapeutic Agent for Diseases Related to Inhibition of Apoptosis As described above, ABP1 has a function of activating ASK1 and inducing apoptosis in cells. Therefore, ABP1 or a nucleic acid encoding it in vivo (eg, If the gene, mRNA, etc. are abnormal or deficient, or the expression level is abnormally decreased, cell apoptosis induction is excessively suppressed by some other factor. If so, for example, various diseases such as cancer, autoimmune diseases, viral infections, endocrine diseases, blood diseases, and organ hyperplasia develop.
Therefore, when there is a patient who cannot expect removal of unwanted cells or abnormal cells by apoptosis due to a decrease in ABP1 or other factors, a) ABP1 or the activated peptide of the present invention or a salt thereof is administered to the patient, and ABP1 Or b) (i) by administering to the patient a DNA encoding ABP1 or an activating peptide of the present invention and expressing it in the target cell, or (ii) After introducing and expressing DNA encoding ABP1 or the activating peptide of the present invention, the amount of ABP1 in the patient's body is increased by transplanting the cell into the patient, and the like, and abnormal cells become unnecessary Cells can be induced to undergo apoptosis via the ASK1 cascade.
Accordingly, a) ABP1 or an activation peptide of the present invention or a salt thereof, or b) a polynucleotide encoding ABP1 or an activation peptide of the present invention is a disease that is prophylactically or therapeutically effective in inducing apoptosis, for example, Cancer (eg, leukemia, esophageal cancer, stomach cancer, colon cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, osteosarcoma, brain tumor, etc.), autoimmunity Disease (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, insulin-dependent diabetes, psoriasis, ulcerative colitis, idiopathic thrombocytopenic purpura, Crohn's disease, glomerulonephritis Etc.), viral infections (eg, hemorrhagic fever, T cell leukemia, Kaposi's sarcoma, infectious mononucleosis, lymphoma, nasopharyngeal cancer, cervical cancer, skin cancer, hepatitis, liver cancer, etc.) Endocrine diseases (eg, hormonal excess, cytokines etc.), blood diseases (eg, hematocytosis, B-cell lymphoma, polycythemia, etc.), organ hyperplasia (eg, half-yin yang, retention testicles, teratomas, kidneys) Blast cell carcinoma, multiple cystic kidneys, cardiac / aortic malformations, finger joints, etc.), restenosis after angioplasty, and recurrence after cancer resection.

When ABP1 or the activated peptide of the present invention or a salt thereof is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to conventional means.
On the other hand, when the polynucleotide encoding ABP1 or the activation peptide of the present invention is used as the prophylactic / therapeutic agent, the polynucleotide may be used alone or as a suitable retrovirus vector, adenovirus vector, adenovirus associated virus vector or the like. After insertion into a simple vector, it can be formulated according to conventional means. The polynucleotide can be administered as it is or together with an auxiliary agent for promoting intake by a gene gun or a catheter such as a hydrogel catheter.
For example, a) ABP1 or an activated peptide of the present invention or a salt thereof, or b) a polynucleotide encoding ABP1 or an activated peptide of the present invention is a sugar-coated tablet, capsule, elixir, It can be used orally as a microcapsule or the like, or parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection such as a suspension. For example, a) ABP1 or an activating peptide of the present invention or a salt thereof, or b) a polynucleotide encoding ABP1 or an activating peptide of the present invention, a known physiologically recognized carrier, flavoring agent, excipient, It can be prepared by mixing with vehicles, preservatives, stabilizers, binders and the like in the unit dosage form generally required for the practice of the formulation. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The preventive / therapeutic agent includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, for example, humans and other warm-blooded animals (for example, rats, mice, hamsters, rabbits, sheep, goats, pigs, cows, horses, cats, dogs, Monkeys, chimpanzees, birds, etc.).
The dose of ABP1 or the activation peptide of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example, in cancer patients (60 kg) About 0.1 mg to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.
The dose of the polynucleotide encoding ABP1 or the activation peptide of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example, a cancer patient (as 60 kg) ) Is about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.

(3) Gene Diagnostic Agent A polynucleotide containing a base sequence encoding ABP1 or a part thereof (hereinafter referred to as “sense polynucleotide of the present invention”) and an antisense polynucleotide of the present invention are used as probes. Abnormalities in DNA or mRNA encoding ABP1 in humans or other warm-blooded animals (eg, rats, mice, hamsters, rabbits, sheep, goats, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, birds, etc.) Since (gene abnormality) can be detected, it is useful, for example, as a gene diagnostic agent for DNA damage or mutation, mRNA splicing abnormality or decreased expression, or amplification or excessive expression of mRNA. The polynucleotide containing a part of the base sequence encoding ABP1 is not particularly limited as long as it has a length necessary as a probe (for example, about 15 bases or more), and encodes a partial peptide of ABP1 ( That is, it is not necessary to be in frame.

The above gene diagnosis using the sense or antisense polynucleotide of the present invention includes, for example, per se known Northern hybridization, quantitative RT-PCR, PCR-SSCP method, allele-specific PCR, PCR-SSOP method, DGGE method, An RNase protection method, a PCR-RFLP method, or the like can be used.
For example, as a result of Northern hybridization or quantitative RT-PCR for the RNA fraction extracted from the cells of the test warm-blooded animal, a decrease in the expression of ABP1 is detected, or for the RNA fraction or the genomic DNA fraction, PCR- When a mutation of the ABP1 gene is detected as a result of performing the SSCP method, a disease associated with apoptosis or suppression of inflammatory cytokine production, such as cancer (eg, leukemia, esophageal cancer, stomach cancer, colon cancer, rectal cancer, Lung cancer, liver cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, osteosarcoma, brain tumor, etc.), autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Sjogren) Syndrome, multiple sclerosis, insulin-dependent diabetes mellitus, psoriasis, ulcerative colitis, idiopathic thrombocytopenic purpura, clone , Glomerulonephritis, etc.), viral infection (eg, hemorrhagic fever, T cell leukemia, Kaposi's sarcoma, infectious mononucleosis, lymphoma, nasopharyngeal cancer, cervical cancer, skin cancer, hepatitis, liver cancer, etc.), endocrine disease (Eg, hormonal excess, cytokine excess, etc.), hematological disorders (eg, hematocytosis, B cell lymphoma, polycythemia, etc.), organ hyperplasia (eg, semi-yin yang, retention testicles, teratomas, nephroblasts) Cancer, polycystic kidney disease, cardiac / aortic malformation, joint dysplasia, etc.), restenosis after angioplasty, recurrence after cancer resection, etc. can do.
On the other hand, if excessive expression of ABP1 is detected by Northern hybridization or quantitative RT-PCR, diseases associated with hyperapoptosis or inflammation, such as viral infections (eg, AIDS, influenza, unknown fever, etc.), endocrine secretion Disease (eg, hormone deficiency, cytokine deficiency, etc.), blood disease (eg, cytopenia, renal anemia, etc.), organ dysplasia (eg, thyroid atrophy, cleft palate, etc.), transplant organ rejection, graft-versus-host disease , Immunodeficiency, neurodegenerative diseases (eg, polyglutamine disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellar degeneration), ischemic heart disease (eg, angina pectoris, myocardial infarction) Etc.), radiation damage, UV damage (eg, sunburn, etc.), toxic diseases (eg, renal tubular cell damage due to heavy metals, hepatocyte damage due to alcohol, etc.), nutrition Harm (eg, thymus atrophy due to vitamins, trace element deficiency, etc.), inflammatory diseases (eg, acute pancreatitis, arthritis, periodontal disease, colitis, etc.), ischemic neuropathy, diabetic neuropathy, vascular disease ( (Eg, arteriosclerosis, etc.), respiratory diseases (eg, interstitial pneumonia, pulmonary fibrosis, etc.), cartilage diseases (eg, osteoarthritis, etc.), or may be affected in the future Can be diagnosed as high.

(4) Diagnostic method using the antibody of the present invention The antibody of the present invention can specifically recognize ABP1 and therefore can be used for detection of ABP1 in a test solution.
That is, the present invention
(I) In a test solution, wherein the antibody of the present invention is reacted competitively with a test solution and labeled ABP1, and the ratio of labeled ABP1 bound to the antibody is measured. And (ii) after reacting the test solution with the antibody of the present invention insolubilized on the carrier and another labeled antibody of the present invention simultaneously or successively A method for quantifying ABP1 or a salt thereof in a test solution, characterized by measuring the amount (activity) of a labeling agent on an insolubilized carrier.

In the above quantification method (ii), it is desirable that the two antibodies recognize different parts of ABP1. For example, if one antibody recognizes the N-terminal part of ABP1 (eg, a portion having the amino acid sequence shown in SEQ ID NO: 5), the other antibody has a C-terminal part (eg, SEQ ID NO: 6) of ABP1. Can be used that reacts with a portion having an amino acid sequence represented by
In addition, ABP1 can be quantified using a monoclonal antibody against ABP1, and detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′, or Fab fraction of the antibody molecule may be used.
The method for quantifying ABP1 or a salt thereof using the antibody of the present invention is not particularly limited, and an antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test solution (for example, the amount of ABP1) is not limited. Any measurement method may be used as long as it is a measurement method in which the amount is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin- (strept) avidin system can also be used for binding of an antibody or antigen to a labeling agent.

  In the insolubilization of the antigen or antibody, physical adsorption may be used, or a chemical bond usually used for insolubilizing / immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

In the sandwich method, the test solution is reacted with the insolubilized antibody of the present invention (primary reaction), and another labeled antibody of the present invention is reacted (secondary reaction), and then the labeling agent on the insolubilized carrier. By measuring the amount (activity) of ABP1, the amount of ABP1 in the test solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. The labeling agent and the insolubilizing method can be the same as those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving the measurement sensitivity. May be.
In the measurement method of ABP1 by the sandwich method, the antibodies of the present invention used in the primary reaction and the secondary reaction are preferably antibodies having different ABP1 binding sites. For example, as described above, when the antibody used in the secondary reaction recognizes the C end of ABP1, the antibody used in the primary reaction preferably recognizes the N end, for example, other than the C end. An antibody is used.

The antibody of the present invention can also be used in measurement systems other than the sandwich method, for example, competitive method, immunometric method, nephrometry and the like.
In the competition method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody), and a solid phase is used as the primary antibody. An antibody is used, or a primary antibody is soluble and a solid phase method using a solid phase antibody as a secondary antibody is used.
In the immunometric method, 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, or the antigen in the test solution is separated. Are reacted with an excess amount of labeled antibody, and then a solid phased 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 labeled amount of any phase is measured to quantify the amount of antigen in the test solution.
In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.

In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of ABP1 by adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Sequel Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) )) (Academic Press Issue) and the like can be referred to.
As described above, ABP1 or a salt thereof can be quantified with high sensitivity by using the antibody of the present invention.

Therefore, by using a biological sample (eg, blood, plasma, urine, biopsy, etc.) derived from a test warm-blooded animal as a test sample, and quantifying the concentration of ABP1 or a salt thereof in the test sample using the antibody of the present invention. , If a decrease in ABP1 concentration is detected, a disease associated with inhibition of apoptosis, such as cancer (eg, leukemia, esophageal cancer, stomach cancer, colon cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer, uterine cancer, Ovarian cancer, prostate cancer, melanoma, myeloma, osteosarcoma, brain tumor, etc.), autoimmune disease (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, insulin-dependent diabetes mellitus, psoriasis) , Ulcerative colitis, idiopathic thrombocytopenic purpura, Crohn's disease, glomerulonephritis, etc.), viral infections (eg, hemorrhagic fever, T cell leukemia, Kaposi's sarcoma, infectious mononucleosis, lymphoma, nasopharyngeal carcinoma) , Cervical cancer, skin cancer, hepatitis, liver cancer, etc.), endocrine diseases (eg, hormonal excess, cytokine excess etc.), blood diseases (eg, hematocytosis, B cell lymphoma, polycythemia, etc.), organ hyperplasia (For example, semi-yin yang, retention testicles, teratomas, nephroblastoma, multiple cystic kidneys, cardiac and aortic malformations, and digitosis), diseases such as restenosis after angioplasty, recurrence after cancer resection It can be diagnosed as affected or likely to be affected in the future.
On the other hand, if an increase in ABP1 concentration is detected, diseases associated with hyperapoptosis or inflammation, such as viral infections (eg, AIDS, influenza, unknown fever, etc.), endocrine diseases (eg, hormone deficiency, cytokine deficiency) Etc.), blood diseases (eg, cytopenias, renal anemia, etc.), organ dysplasia (eg, thyroid atrophy, cleft palate, etc.), transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative diseases (eg, , Polyglutamine disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellar degeneration, etc., ischemic heart disease (eg, angina pectoris, myocardial infarction, etc.), radiation damage, UV damage ( Eg, sunburn, etc.) Toxic diseases (eg, renal tubular cell damage due to heavy metals, hepatocyte damage due to alcohol, etc.), nutritional disorders (eg, thymus atrophy due to vitamin or trace element deficiency, etc.), flame Diseases (eg, acute pancreatitis, arthritis, periodontal disease, colitis, etc.), ischemic neuropathy, diabetic neuropathy, vascular diseases (eg, arteriosclerosis, etc.), respiratory diseases (eg, interstitial) It can be diagnosed that the patient is suffering from diseases such as pneumonia, pulmonary fibrosis, etc., cartilage disease (eg, osteoarthritis, etc.), or is likely to suffer in the future.

(5) ASK1 activation inhibitor / apoptosis inhibitor The antibody of the present invention inhibits the binding of ABP1 to ASK1 by specifically binding to ABP1, thereby promoting ASK1 activation and induction of apoptosis / inflammatory cytokine production. The action can be inactivated (ie neutralized). On the other hand, since the antisense polynucleotide of the present invention inhibits the expression of ABP1 and decreases the production amount of the protein, the effect of ABP1 on ASK1 activation and the promotion of apoptosis / inflammatory cytokine production induction is consequently reduced. Can do.
Therefore, by adding the antibody of the present invention to a cell to inactivate ABP1, or introducing the antisense polynucleotide of the present invention into a cell to reduce the amount of ABP1 in the cell, the activity of ASK1 in the cell Can be inhibited, and apoptosis / inflammatory cytokine production of the cells can be suppressed. For example, it can be used as a reagent for studying apoptosis and inflammatory reaction.
When the antibody of the present invention is used as the above-mentioned ASK1 activation inhibitor or apoptosis / inflammatory cytokine production inhibitor, in water or an appropriate buffer (eg, phosphate buffer, PBS, Tris-HCl buffer, etc.) It can be prepared by dissolving to an appropriate concentration. Moreover, you may mix | blend the preservative, stabilizer, reducing agent, tonicity agent, etc. which are used normally as needed.
On the other hand, when the antisense polynucleotide of the present invention is used as the above-mentioned ASK1 activation inhibitor or apoptosis inhibitor, the polynucleotide can be used alone or as a suitable retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc. After insertion into an appropriate vector, it can be introduced into cells using the transformation methods described above (eg, liposome method, electroporation method, etc.).

(6) Preventive / Therapeutic Agent for Diseases Related to Hyperapoptosis or Inflammation As described above, ABP1 activates ASK1 to induce apoptosis in cells, or has a function of inducing inflammation. If the nucleic acid encoding it (eg, gene, mRNA, etc.) is abnormal (appearance of a highly active mutant), or if its expression level is abnormally increased, or if there are other factors, When apoptosis induction or inflammatory cytokine production is abnormally increased, for example, viral infection, endocrine disease, blood disease, organ dysplasia, transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic Heart disease, radiation disorder, ultraviolet ray disorder, toxic disease, nutritional disorder, inflammation, ischemic neuropathy (brain ischemia), diabetic peripheral neuropathy, vascular disease Various diseases such as respiratory diseases and cartilage diseases develop.
Therefore, when there is a patient who has lost cells necessary for the living body due to apoptosis due to an increase in ABP1 or is suffering from an inflammatory disease, a) the antibody of the present invention is administered to the patient and ABP1 B) (i) the antisense polynucleotide of the present invention is administered to the patient and introduced (and expressed) into the target cell, or (ii) the target is isolated into the isolated target cell. After introducing and expressing the antisense polynucleotide of the invention, the amount of ABP1 in the patient's body is decreased by, for example, transplanting the cell into the patient, thereby suppressing the apoptotic death or inflammatory response of the cell that is originally required be able to.
Therefore, a) a disease according to the present invention or b) an antisense polynucleotide according to the present invention is effective in preventing apoptosis or inflammation in terms of prevention or treatment, such as a disease caused by overexpression of ABP1, specifically, Virus infection (eg, AIDS, influenza, unknown fever, etc.), endocrine disease (eg, hormone deficiency, cytokine deficiency, etc.), blood disease (eg, cytopenia, renal anemia, etc.), organ formation failure (eg, , Thyroid atrophy, cleft palate, etc.), transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative diseases (eg, polyglutamine disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellum) Degeneration, etc.), ischemic heart disease (eg, angina pectoris, myocardial infarction, etc.), radiation damage, UV damage (eg, sunburn, etc.) Toxic disease (eg, renal tubular cells caused by heavy metals) Harm, hepatocyte damage due to alcohol, etc.), nutritional disorders (eg, atrophy of the thymus due to vitamin and trace element deficiencies), inflammatory diseases (eg, acute pancreatitis, arthritis, periodontal disease, colitis, etc.), ischemic nerve Disorders such as disorders, diabetic neuropathy, vascular diseases (eg, arteriosclerosis, etc.), respiratory diseases (eg, interstitial pneumonia, pulmonary fibrosis, etc.), cartilage diseases (eg, osteoarthritis, etc.) It can be used as a prophylactic / therapeutic agent.
When the antibody of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-described pharmaceutical containing ABP1 or the activated peptide of the present invention or a salt thereof. In addition, when the antisense polynucleotide of the present invention is used as the prophylactic / therapeutic agent, it can be formulated in the same manner as the pharmaceutical containing the above-described ABP1 or the polynucleotide encoding the activating peptide of the present invention.
Since the preparation thus obtained is safe and has low toxicity, for example, humans and other warm-blooded animals (for example, rats, mice, hamsters, rabbits, sheep, goats, pigs, cows, horses, cats, dogs, Monkeys, chimpanzees, birds, etc.).

The dose of the antibody of the present invention varies depending on the administration subject, target organ, symptom, administration method, and the like, but in the case of oral administration, for example, generally in polyglutamine disease patients (60 kg) per day About 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in a polyglutamine disease patient (as 60 kg) It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.
Although the dosage of the antisense polynucleotide of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., in the case of oral administration, generally, for example, in a polyglutamine disease patient (as 60 kg), About 0.1 mg to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in a polyglutamine disease patient (as 60 kg) It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.

(7) Use of inhibitory peptide of the present invention As described above, the inhibitory peptide of the present invention can function as a (competitive) inhibitor of ABP1, that is, it can bind to ASK1 but does not activate it or is inactive Therefore, like the antibody of the present invention, it can be used as an ASK1 activation inhibitor, an apoptosis / inflammatory cytokine production inhibitor, or a prophylactic / therapeutic agent for diseases associated with increased apoptosis or inflammation.
When the inhibitory peptide of the present invention is used as an inhibitor of ASK1 activation inhibition / apoptosis or inflammatory cytokine production, it is the same as the ASK1 activation promoter / apoptosis inducer containing ABP1 or the activation peptide of the present invention or a salt thereof. Thus, the reagent can be prepared. In addition, when the inhibitory peptide of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the pharmaceutical containing ABP1 or the activated peptide of the present invention or a salt thereof.
Since the preparation thus obtained is safe and has low toxicity, for example, humans and other warm-blooded animals (for example, rats, mice, hamsters, rabbits, sheep, goats, pigs, cows, horses, cats, dogs, Monkeys, chimpanzees, birds, etc.).
The dose of the inhibitory peptide of the present invention varies depending on the administration subject, target organ, symptom, administration method, and the like, but in the case of oral administration, for example, generally in polyglutamine disease patients (60 kg), Per 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in a polyglutamine disease patient (as 60 kg) It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.

(8) Use of ASK1 partial peptide having no phosphorylation activity or a salt thereof As demonstrated in Examples below, the site involved in the binding of ASK1 to ABP1 is the N-terminal activation regulatory domain, A partial peptide of ASK1, which has the activation control domain but lacks the kinase domain responsible for the activation of MAPKK located downstream of the cascade, can function as a (competitive) inhibitor of ASK1. That is, since the peptide can bind to ABP1 but cannot be activated thereby, it can competitively inhibit the binding between ASK1 and ABP1 endogenous to cells and the activation of ASK1 thereby. Therefore, the partial peptide can be used as an ABP1 inhibitor, an inhibitor of apoptosis or inflammatory cytokine production, or a prophylactic / therapeutic agent for diseases associated with hyperapoptosis or inflammation.
As the kinase domain of ASK1, for example, in the case of human ASK1, the amino acid sequence represented by amino acid numbers 678 to 936 in the amino acid sequence represented by SEQ ID NO: 1 in Patent Document 1 (Japanese Patent Laid-Open No. 10-93) can be mentioned. Therefore, a partial peptide of ASK1 having an activation control domain on the N-terminal side and lacking the kinase domain (hereinafter sometimes abbreviated as “ASK1 partial peptide of the present invention”) is the above-mentioned human ASK1. The amino acid sequence contains the same or substantially the same amino acid sequence as the whole or a part of the amino acid sequence represented by amino acid numbers 1 to 677 (hereinafter collectively referred to as “ASK1-N sequence”), and ABP1 Peptides having the binding ability of Here, the “substantially identical amino acid sequence” refers to about 70% or more, preferably about 80% or more of all or part of the amino acid sequence represented by amino acid numbers 1 to 677 in the amino acid sequence of human ASK1. Preferred examples include amino acid sequences having homology of preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more.
The ASK1 partial peptide of the present invention includes, for example, 1) 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1) in the ASK1-N sequence. -5) amino acid sequence from which amino acids have been deleted, 2) 1 or 2 (preferably about 1-30, preferably about 1-10, more preferably a number (1) in the ASK1-N sequence -5) amino acid sequence added with 3 amino acids, 3) 1 or 2 (preferably about 1-30, preferably about 1-10, more preferably a number (1- 1) in the ASK1-N sequence 5) amino acid sequence in which amino acids are inserted, 4) one or more (preferably about 1-30, preferably about 1-10, more preferably a number (1) in the ASK1-N sequence ~ 5) amino acids are other The amino acid sequence are substituted with an amino acid, or 5) mutant peptide comprising an amino acid sequence that is a combination thereof are included.
The ASK1 partial peptide of the present invention can be prepared by the same method as the above-mentioned ABP1 partial peptide.
When the ASK1 partial peptide of the present invention is used as an ABP1 inhibitor, particularly an apoptosis or inflammatory cytokine production inhibitor, an ASK1 activation promoting / apoptosis inducing agent containing the aforementioned ABP1 or the activation peptide of the present invention or a salt thereof, The reagent can be prepared in the same manner. In addition, when the ASK1 partial peptide of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the pharmaceutical containing ABP1 or the activated peptide of the present invention or a salt thereof.
Since the preparation thus obtained is safe and has low toxicity, for example, humans and other warm-blooded animals (for example, rats, mice, hamsters, rabbits, sheep, goats, pigs, cows, horses, cats, dogs, Monkeys, chimpanzees, birds, etc.).
The dose of the ASK1 partial peptide of the present invention varies depending on the administration subject, target organ, symptom, administration method, and the like, but in the case of oral administration, for example, in a polyglutamine disease patient (60 kg), About 0.1 mg to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in a polyglutamine disease patient (as 60 kg) It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.

(9) Screening for ASK1 Activation Modulating Substance The present invention also provides a method for screening for an ASK1 activation regulating substance by using ABP1 or the activating peptide of the present invention or a salt thereof or a cell producing the same. The screening methods are roughly classified into (A) a method using the binding ability of ABP1 and ASK1, (B) a method using ASK1 activation as an index, and (C) a method using the expression level of ABP1 as an index. In the methods (A) and (B), a partial peptide that retains binding to ASK1 or ABP1 (that is, a partial peptide containing at least the N-terminal activation control domain) or a salt thereof or a cell that produces the same Further used.
(A) Screening method utilizing the binding property between ABP1 and ASK1 Since ABP1 can bind to and activate ASK1, the activation peptide of the present invention and the ASK1 or N-terminal activation control domain By constructing a binding assay system using that partial peptide, the screening of compounds having the same action as ABP1 or the activation peptide of the present invention, or the inhibition of the action of ABP1 or the activation peptide of the present invention Screening can be performed. That is, the present invention provides a screening method for an ASK1 activation regulator using ABP1 or the activation peptide of the present invention and ASK1 or a partial peptide containing an N-terminal activation control domain.
More specifically, the present invention provides:
(A) (1) When ABP1 or the activation peptide of the present invention is contacted with the partial peptide containing ASK1 or N-terminal side activation control domain, and (2) including ASK1 or N-terminal side activation control domain The partial peptide containing ASK1 or the N-terminal side activation control domain and ABP1 or the activation peptide of the present invention when the partial peptide is contacted with ABP1 or the activation peptide of the present invention and a test substance A method for screening an ASK1 activation regulator, comprising comparing the amount of binding;
(B) (1) When ABP1 or the activation peptide of the present invention is contacted with a cell that produces the partial peptide containing ASK1 or the N-terminal activation control domain, and (2) ASK1 or N-terminal activation. The ASK1 or the partial peptide containing the N-terminal side activation control domain and ABP1 or ABP1 or the test peptide when the activation peptide of the present invention and the test substance are contacted with a cell producing the partial peptide containing the control domain A method for screening an ASK1 activation modulator, comprising comparing the amount of binding with the activation peptide of the present invention, and (c) ASK1 or its partial peptide containing N-terminal side activation control domain and ABP1 or the present When the activated peptide of the invention is produced and the two are bound, transcription of the reporter gene is activated The expression level of the reporter gene in a cell, provides a screening method of ASK1 activation regulators and comparing in the presence and absence of the test substance.

ASK1 used in the screening method (a) or (b) is isolated and purified from cells of humans or other warm-blooded animals using a method known per se (eg, the same method as described above for ABP1). be able to. The partial peptide of ASK1 including the N-terminal activation control domain is not particularly limited as long as it has the amino acid sequence of the N-terminal activation control domain described above, and includes a part of the kinase domain or the C-terminal amino acid sequence. Also good. The partial peptide can be obtained by digesting ASK1 with an appropriate proteolytic enzyme. In addition, ASK1 and its partial peptide containing the N-terminal activation control domain can be recombinantly produced according to the above-described ABP1 expression method after cloning the DNA encoding it according to a known genetic engineering technique. .
ABP1 and the activation peptide of the present invention (hereinafter sometimes simply referred to as ABP1) can be prepared according to the method described above.
The ASK1 producing cell is not particularly limited as long as it is a human or other warm-blooded animal cell that expresses it, and examples thereof include HeLa cells and HEK293 cells. Examples of cells that produce ASK1 or a partial peptide containing the N-terminal activation control domain (hereinafter sometimes simply referred to as ASK1) include transformants prepared by the above-described genetic engineering techniques. .
Examples of test substances include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel. Alternatively, a known one may be used.
The amount of binding can be determined by, for example, Western blot analysis using labeled anti-ABP1 antibody and anti-ASK1 antibody, labeling either ABP1 or ASK1 (eg, [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like), a gel shift assay, or surface plasmon resonance (SPR).
In the above screening method, a compound that binds to ASK1 and inhibits the binding between ABP1 and ASK1 can be selected as an ASK1 activation regulator.

In this screening method, the reaction between ABP1 and ASK1 can usually be performed at about 37 ° C. for several hours.
For example, to carry out the above screening method by a binding assay using labeled ABP1, first, an ASK1 preparation is prepared by suspending ASK1 or cells producing it in a buffer suitable for screening. The buffer may be any buffer that does not inhibit the binding between ABP1 and ASK1, such as a phosphate buffer having a pH of about 4 to 10 (preferably about pH 6 to 8) and a Tris-HCl buffer. In addition, for the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin, deoxycholate and the like can be added to the buffer. Furthermore, protease inhibitors such as PMSF, leupeptin, bacitracin, aprotinin, E-64 (manufactured by Protein Research Institute), and pepstatin can be added for the purpose of suppressing degradation of ABP1 and ASK1 by protease.
On the other hand, when the cells are immobilized cells, ABP1 and ASK1 can be bound while being immobilized in the incubator, that is, in a state where the cells are grown or using cells fixed with glutaraldehyde or paraformaldehyde. it can. In this case, a medium or Hanks' solution is used as the buffer solution.
Then, a certain amount (for example, about 10,000 cpm to 1,000,000 cpm for 2000 Ci / mmol) of labeled ABP1 (for example, ABP1 labeled with [ ¹²⁵ I]) is added to 0.01 ml to 10 ml of the ASK1 preparation, At the same time, 10 ⁻⁴ M to ^{10 −10} M test substance is allowed to coexist. In order to know the non-specific binding amount (NSB), a reaction tube to which a large excess of unlabeled ABP1 is added is also prepared. The reaction is carried out at 0 ° C. to 50 ° C., preferably 4 ° C. to 37 ° C. for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, filtration through glass fiber filter paper (when using ASK1-producing cells) or B / F separation (when using purified ASK1), washing with an appropriate amount of the same buffer, and radiation remaining on the glass fiber filter paper or solid phase Activity (eg, amount of [ ¹²⁵ I]) is measured with a liquid scintillation counter or γ-counter. When the count (B ₀ -NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) when there is no antagonistic substance is 100%, the specific binding amount (B-NSB) is, for example, A test substance that is 50% or less of the count (B ₀ -NSB) can be selected as an ASK1 activation regulator.

The screening kit of the present invention contains ABP1, preferably ASK1 or cells that produce it.
Examples of the screening kit of the present invention include, but are not limited to, the following.
[Reagent for screening]
1) Measurement buffer and washing buffer
Hanks' Balanced Salt Solution (Gibco) plus 0.05% bovine serum albumin (Sigma). The solution may be sterilized by filtration through a 0.45 μm filter and stored at 4 ° C. or may be prepared at the time of use.
2) ASK1 standard
HeLa cells or HEK293 cells were subcultured at 5 × 10 ⁵ cells / well in a 12-well plate and cultured at 37 ° C., 5% CO ₂ , 95% air for 2 days.
3) Labeled ABP1 preparation ABP1 labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like.
4) ABP1 standard solution ABP1 was dissolved to 0.1 mM in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) and stored at -20 ° C.
[Measurement method]
1) ASK1-producing cells cultured in a 12-well tissue culture plate are washed twice with 1 ml of measurement buffer, and 490 μl of measurement buffer is added to each well.
2) After adding 5 μl of 10 ⁻³ to 10 ⁻¹⁰ M test substance solution, 5 μl of 5 nM labeled ABP1 is added and allowed to react at room temperature for 1 hour. In order to know the amount of non-specific binding, 5 μl of 10 ⁻⁴ M ABP1 is added instead of the test substance.
3) Remove the reaction solution and wash 3 times with 1 ml of washing buffer. The labeled ABP1 bound to the cells is dissolved in 0.5 ml of 0.2N NaOH-1% SDS and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries).
4) Measure radioactivity using a liquid scintillation counter (manufactured by Beckman), and determine the Percent Maximum Binding (PMB) by the following formula (Equation 1). When labeled with [ ¹²⁵ I], it can be directly measured with a gamma counter without mixing with a liquid scintillator.

[Equation 1]
PMB = 100 × (B−NSB) / (B ₀ −NSB)

PMB: Percent Maximum Binding
B: Binding amount when the specimen is added NSB: Non-specific binding (non-specific binding amount)
B ₀ : Maximum amount of binding

Cells used in the screening method of (c) above include (1) ASK1 or a partial peptide containing an N-terminal activation control domain and a transcription factor (eg, GAL4, VP16, etc.) DNA binding domain or transcriptional activation. DNA encoding a fusion protein with one of the domains, (2) DNA encoding a fusion protein of ABP1 or the activation peptide of the present invention and the other domain of the transcription factor, and (3) active by the transcription factor A cell containing a reporter gene under the control of the promoter to be activated, preferably a yeast cell, a mammalian cell and the like. Examples of the reporter gene include luciferase gene, β-galactosidase gene, alkaline phosphatase gene, peroxidase gene, chloramphenicol acetyltransferase gene, and green fluorescent protein (GFP) gene.
In this screening method, the above cells are cultured for about several hours to one day under culture conditions suitable for the host cells to be used, and then a cell extract or supernatant is obtained and the reporter gene is detected by a conventional method. Do.
In the above screening method, a compound that inhibits the expression of a reporter gene can be selected as an ASK1 activation regulator.

(B) Screening method using ASK1 activation as an index If the effect of a test substance on the interaction between ABP1 and ASK1 is examined by measuring ASK1 activation, the substance inhibits ASK1 activation. Or can be directly evaluated. That is, the present invention relates to the activation of the protein or the peptide in a cell that produces ASK1 or its partial peptide containing the N-terminal activation control domain and the kinase domain. Provided is a method for screening an ASK1 activation regulator by measurement and comparison in the presence of (2) ABP1 or the activation peptide of the present invention and a test substance.

In this screening method, cells that produce ASK1 or its partial peptide containing the N-terminal activation control domain and kinase domain (hereinafter sometimes simply referred to as ASK1-producing cells) are cells that endogenously produce ASK1 ( Examples, HeLa cells, HEK293 cells, etc.) and animal cells into which DNA encoding a partial peptide including ASK1 or N-terminal activation control domain and kinase domain has been introduced. A partial peptide of ASK1 containing an N-terminal activation control domain and a kinase domain is represented by amino acid numbers 1 to 936 in the amino acid sequence shown in SEQ ID NO: 1 in the above-mentioned Patent Document 1 (JP-A-10-93) Peptides that contain the same or substantially the same amino acid sequence as all or part of the amino acid sequence (hereinafter collectively referred to as “ASK1-NK sequence”) and that can be activated by binding to ABP1 It is done. Here, the “substantially identical amino acid sequence” is about 70% or more, preferably about 80% or more, more preferably about 90% or more, particularly preferably about 95% or more, most preferably, the human ASK1-NK sequence. Preferably, an amino acid sequence having about 98% or more homology is used.
Examples of the partial peptide include 1) 1 or 2 or more in the ASK1-NK sequence (preferably about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5) 2) ASK1-NK sequence with 1 or more amino acids (preferably about 1-30, preferably about 1-10, more preferably number (1-5) 3) Amino acid sequence added with 3 amino acids, 3) 1 or 2 (preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1 to 5) ASK1-NK sequence 4) 1 or 2 or more (preferably about 1-30, preferably about 1-10, more preferably number (1-5) in the ASK1-NK sequence) Amino acids) other amino acids The amino acid sequence are substituted with Amino Acids, or 5) mutant peptide comprising an amino acid sequence that is a combination thereof are included.
ABP1 or the activation peptide of the present invention (hereinafter sometimes simply referred to as ABP1) may be added to the cell from the outside, or may be produced by the ASK1-producing cell itself. In the latter case, ABP1 may be endogenously produced, or a transformant that is genetically engineered according to the above ABP1 expression method using ASK1-producing cells as a host.
Examples of test substances include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel. Alternatively, a known one may be used.
The activation of ASK1 includes autophosphorylation of ASK1, kinases located downstream of the ASK1 cascade (for example, MKK4 / 7, MKK3 / 6, JNK, p38, etc.) and other proteins or synthetic peptide phosphorous that can be substrates for ASK1. It can be evaluated by measuring oxidation, cell death induction rate, and the like.

Specifically, first, ASK1-producing cells are cultured in a multiwell plate or the like. In conducting the screening, the medium is replaced with a fresh medium or an appropriate buffer that is not toxic to the cells in advance, and a test substance (or ABP1 if the cells do not produce ABP1) is added and incubated for a certain period of time. Cells are extracted or the supernatant is collected, and the products and phenomena produced are quantified according to each method.
Phosphorylation of ASK1 and other proteins or peptides can be detected by Western blot analysis using a phosphorylated protein (peptide) specific antibody, incorporation of [ ³² P] -labeled ATP in a substrate protein (peptide) and gel electrophoresis and It can be performed by a method such as detection by autoradiography.
The cell death assay is performed in a system in which the expression of ABP1 can be confirmed (for example, a cell into which DNA encoding a fusion protein of ABP1 and a fluorescent protein is introduced, or a cell into which an ABP1 expression vector further containing a selection marker gene is introduced). The ratio of dead cells in all ABP1-expressing cells can be calculated using, for example, detachment from the skin or morphological observation (eg, membrane blebbing, fragmentation, etc.) as an index.

  In the screening method described above, when ASK1 is activated when a test substance is added to ASK1-producing cells, the substance can be selected as an ASK1 activation promoting substance. On the other hand, when ASK1 is inactivated when a test substance is added, the substance can be selected as an ASK1 activation inhibitor.

(C) Screening method for substances that change the expression level of ABP1 By using the sense and antisense polynucleotides of the present invention as probes, or by using the antibodies of the present invention, screening for substances that change the expression level of ABP1 can do.
That is, the present invention provides, for example, (i) non-human mammals a) blood, b) specific organs, c) tissues or cells isolated from organs, or (ii) ABP1 contained in transformants, etc. Provided is a screening method for a substance that changes the expression level of ABP1, and thus a substance that regulates ASK1 activation, by measuring the amount of mRNA or the amount of ABP1 protein.

For example, the measurement of the amount of ABP1 mRNA or protein is specifically performed as follows.
(I) A drug (eg, TNF-α, IL-1, Fas) against a normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) , Anticancer agents, etc.) or physicochemical stress (eg, UV, active oxygen, ischemia, etc.), etc., after a certain period of time, blood, or a specific organ (eg, brain, liver, kidney, etc.), or organ Obtain tissue or cells isolated from
The ABP1 mRNA contained in the obtained cells can be quantified by, for example, extracting mRNA from the cells by a normal method and using, for example, a technique such as RT-PCR, or a well-known Northern blot analysis. Can also be quantified. On the other hand, the amount of ABP1 protein can be quantified by, for example, Western blot analysis using a labeled anti-ABP1 antibody after extracting the protein from cells or the like by a normal method.
(Ii) A transformant expressing ABP1 or the activation peptide of the present invention is prepared according to the above method, and ABP1 or the activation peptide of the present invention contained in the transformant or its mRNA is quantified in the same manner. Can be analyzed.

Screening for compounds that alter the expression level of ABP1
(I) A predetermined time before applying a drug or physicochemical stress to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 Before 6 hours) or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours later), or simultaneously with drug or physicochemical stress A test substance is administered, and after a certain time has elapsed after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the amount of ABP1 mRNA contained in the cells, Or by quantifying and analyzing protein mass,
(Ii) When cultivating the transformant according to a conventional method, the test substance is mixed in the medium and cultured for a fixed time (after 1 day to 7 days, preferably after 1 day to 3 days, more preferably after 2 days to 3 days). Day later), the amount of mRNA or the amount of protein of ABP1 or the activation peptide of the present invention contained in the transformant can be determined and analyzed.
Examples of the test substance include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products and the like. These substances may be novel substances or known substances.
In the above screening method, a substance that increases the expression level of ABP1 can be selected as an ASK1 activation promoting substance, and a substance that decreases the expression level of ABP1 can be selected as an ASK1 activation inhibitor.

A substance that increases the expression or activity of ABP1 or a salt thereof obtained by using the screening methods (A) to (C) above (= ASK1 activation promoting substance (hereinafter, used synonymously in the present specification)), Signaling by the ASK1 cascade can be activated to induce apoptosis in cells. Therefore, by adding an ASK1 activation promoting substance to a cell, apoptosis can be induced in the cell, for example, it can be used as a reagent for studying apoptosis.
When ASK1 activation promoter is used as an apoptosis inducer, it is prepared by dissolving in water or an appropriate buffer (eg, phosphate buffer, PBS, Tris-HCl buffer, etc.) to an appropriate concentration. can do. Moreover, you may mix | blend the preservative, stabilizer, reducing agent, tonicity agent, etc. which are used normally as needed.

As described above, since the ASK1 activation promoting substance has a function of inducing apoptosis in cells, ASK1 can be used when there is a patient who cannot expect removal of unnecessary cells or abnormal cells due to apoptosis due to decrease in ABP1 or other factors. By activating ASK1 by administering an activation promoting substance to the patient, apoptosis via the ASK1 cascade can be induced in abnormal cells or unnecessary cells in the patient's body.
Therefore, ASK1 activation promoting substances can be prevented or treated effectively by inducing apoptosis such as cancer (eg, leukemia, esophageal cancer, stomach cancer, colon cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, Breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, osteosarcoma, brain tumor, etc.), autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, insulin) Dependent diabetes, psoriasis, ulcerative colitis, idiopathic thrombocytopenic purpura, Crohn's disease, glomerulonephritis, etc.), viral infections (eg, hemorrhagic fever, T cell leukemia, Kaposi's sarcoma, infectious mononucleosis, Lymphoma, nasopharyngeal cancer, cervical cancer, skin cancer, hepatitis, liver cancer, etc.), endocrine disease (eg, hormonal excess, cytokine excess etc.), blood disease (eg, hematocytosis, B cell lymphoma, polycythemia) etc , Organ hyperplasia (eg, semi-yin yang, retention testicles, teratomas, nephroblastoma, multiple cystic kidneys, cardiac / aortic malformation, finger syndrome, etc.), restenosis after angioplasty, recurrence after cancer resection It can be used as a preventive / therapeutic agent.

When an ASK1 activation inhibitor is used as the prophylactic / therapeutic agent, it can be formulated in the same manner as the above-described pharmaceutical containing ABP1 or the activating peptide of the present invention or a salt thereof.
Since the preparation thus obtained is safe and has low toxicity, for example, humans and other warm-blooded animals (for example, rats, mice, hamsters, rabbits, sheep, goats, pigs, cows, horses, cats, dogs, Monkeys, chimpanzees, birds, etc.).

  The dose of the ASK1 activation promoting substance varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally in cancer patients (60 kg), it is about The amount is 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.

On the other hand, a substance that decreases the expression or activity of ABP1 or a salt thereof obtained by using the screening methods of (A) to (C) above (= ASK1 activation inhibitor (hereinafter used synonymously in this specification)) Can inhibit cell apoptosis and production of inflammatory cytokines by inhibiting signal transduction by the ASK1 cascade. Therefore, by adding an ASK1 activation inhibitor to a cell, apoptosis / inflammatory cytokine production of the cell can be suppressed, and for example, it can be used as a research reagent for apoptosis, inflammatory reaction and the like.
When an ASK1 activation inhibitor is used as an inhibitor of apoptosis or inflammatory cytokine production, it should be adjusted to an appropriate concentration in water or an appropriate buffer (eg, phosphate buffer, PBS, Tris-HCl buffer, etc.). It can be prepared by dissolving. Moreover, you may mix | blend the preservative, stabilizer, reducing agent, tonicity agent, etc. which are used normally as needed.

As described above, since the ASK1 activation inhibitor has a function of suppressing apoptosis of cells and production of inflammatory cytokines, cells necessary for the living body are lost due to apoptosis due to an increase in ABP1 or the like. When there is a patient who has a disease, ASK1 activation inhibitor is administered to the patient to inhibit the activation of ASK1, thereby suppressing originally necessary apoptotic death or inflammatory reaction of cells. .
Therefore, ASK1 activation inhibitor is effective in preventing or treating apoptosis or inflammation, such as viral infection (eg, AIDS, influenza, unknown fever, etc.), endocrine disease (eg, hormone deficiency, Cytokine deficiency, etc.), blood diseases (eg, cytopenias, renal anemia, etc.), organ dysplasia (eg, thyroid atrophy, cleft palate, etc.), transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative diseases (Eg, polyglutamine disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellar degeneration), ischemic heart disease (eg, angina pectoris, myocardial infarction, etc.), radiation damage, ultraviolet light Disorders (eg, sunburn, etc.) Toxic diseases (eg, renal tubular cell damage due to heavy metals, hepatocyte damage due to alcohol, etc.), nutritional disorders (eg, vitamin, trace element deficiency of thymus) Shrinkage, etc.), inflammatory diseases (eg, acute pancreatitis, arthritis, periodontal disease, colitis, etc.), ischemic neuropathy, diabetic neuropathy, vascular diseases (eg, arteriosclerosis, etc.), respiratory diseases ( For example, interstitial pneumonia, pulmonary fibrosis, etc.), cartilage diseases (eg, osteoarthritis, etc.) and the like.

When an ASK1 activation inhibitor is used as the prophylactic / therapeutic agent, it can be formulated in the same manner as the above-described pharmaceutical containing ABP1 or the activating peptide of the present invention or a salt thereof.
Since the preparation thus obtained is safe and has low toxicity, for example, humans and other warm-blooded animals (for example, rats, mice, hamsters, rabbits, sheep, goats, pigs, cows, horses, cats, dogs, Monkeys, chimpanzees, birds, etc.).

  Although the dose of the ASK1 activation inhibitor varies depending on the administration subject, target organ, symptom, administration method, etc., in the case of oral administration, for example, in general, in polyglutamine disease patients (60 kg), Per 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in a polyglutamine disease patient (as 60 kg) It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.

(10) Animal introduced with DNA encoding ABP1 and use thereof The present invention relates to exogenous DNA encoding ABP1 (hereinafter abbreviated as exogenous DNA of the present invention) or mutant DNA thereof (exogenous of the present invention). The present invention provides a novel use of a non-human mammal having an abbreviated mutant DNA).
Non-human mammals used in the present invention are:
[1] A non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
[2] The animal according to [1], wherein the non-human mammal is a rodent
[3] The animal according to [2], wherein the rodent is a mouse or a rat.
A non-human mammal having an exogenous DNA of the present invention or a mutant DNA thereof (hereinafter abbreviated as a DNA-transferred animal of the present invention) can be used for embryos including unfertilized eggs, fertilized eggs, spermatozoa and primordial cells thereof. Preferably, at the stage of embryonic development in non-human mammal development (more preferably at the single cell or fertilized egg cell stage and generally prior to the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, aggregation method, It can be produced by transferring the target DNA by the microinjection method, particle gun method, DEAE-dextran method or the like. Further, by the DNA transfer method, the target exogenous DNA of the present invention can be transferred to somatic cells, living organs, tissue cells, etc., and can be used for cell culture, tissue culture, etc. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned embryo cells with a cell fusion method known per se.
Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like. Among them, rodents, particularly mice (for example, C57BL / 6 strain, DBA2 strain, etc. as pure strains) are relatively short in terms of ontogeny and biological cycle from the viewpoint of creating pathological animal model systems, and are easy to reproduce. As the system, B6C3F ₁ line, BDF ₁ line, B6D2F ₁ line, BALB / c line, ICR line, etc.) or rats (for example, Wistar, SD, etc.) are preferable.
Examples of the “mammal” in the recombinant vector that can be expressed in the mammal include humans and the like in addition to the above non-human mammals.

The exogenous DNA of the present invention refers to DNA encoding ABP1 once isolated and extracted from mammals, not DNA encoding ABP1 originally possessed by non-human mammals.
The mutated DNA of the present invention is one in which a mutation (for example, mutation, etc.) has occurred in the base sequence of the DNA encoding the original ABP1, specifically, addition of a base, deletion, substitution to another base, etc. DNA in which stagnation occurs is used, and abnormal DNA is also included.
The abnormal DNA means DNA that expresses abnormal ABP1, and for example, DNA that expresses abnormal ABP1 that suppresses the function of normal ABP1 is used.
The exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal. In transferring the exogenous DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct bound downstream of a promoter that can be expressed in animal cells. For example, when DNA encoding human ABP1 is transferred, DNA derived from various mammals (for example, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having ABP1 DNA having high homology with this is expressed. DNA encoding ABP1 is microinjected into a fertilized egg of a target mammal, for example, a mouse fertilized egg, by ligating a DNA construct (eg, a vector, etc.) bound with DNA encoding human ABP1 downstream of various promoters. Highly expressed DNA transfer mammals can be generated.

As an expression vector for ABP1, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus, etc. are used. It is done. Of these, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis or plasmids derived from yeast are preferably used.
Examples of promoters that regulate DNA expression include 1) promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), 2) various types Promoters derived from mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, Glutathione S-transferase, platelet-derived growth factor β, keratin K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophin, liquor Isartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA Metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin, α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin , Pre Russia enkephalin A, such as a promoter, such as vasopressin is used. Among these, the cytomegalovirus promoter capable of being highly expressed throughout the body, the human peptide chain elongation factor 1α (EF-1α) promoter, the human and chicken β-actin promoters, and the like are suitable.
The vector preferably has a sequence (generally referred to as a terminator) that terminates the transcription of the target mRNA in a DNA-transferred mammal. For example, the sequences of DNAs derived from viruses and various mammals are used. Preferably, simian virus SV40 terminator or the like is used.

In addition, the splicing signal of each DNA, enhancer region, a part of intron of eukaryotic DNA, etc. 5 ′ upstream of the promoter region, between the promoter region and the translation region or between the translation regions for the purpose of further expressing the target foreign DNA It is also possible to connect it 3 'downstream of the target.
Normal ABP1 translation regions include liver, kidney, thyroid cell, fibroblast-derived DNA and various commercially available genomes derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). Complementary DNA prepared by a known method can be obtained as a raw material from a DNA library as all or part of genomic DNA or from RNA derived from liver, kidney, thyroid cells, fibroblasts and the like. Further, exogenous abnormal DNA can produce a translation region obtained by mutating a normal ABP1 translation region obtained from the cells or tissues described above by a point mutagenesis method.
The translation region can be prepared as a DNA construct that can be expressed in a metastasized animal by an ordinary DNA engineering technique in which it is linked downstream of the promoter and optionally upstream of the transcription termination site.
The transfer of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the subject mammal. The presence of the foreign DNA of the present invention in the embryo cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the foreign DNA of the present invention in all the germ cells and somatic cells. means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the foreign DNA of the present invention in all germ cells and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as a DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. .
The transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be excessively present in all germ cells and somatic cells of the target mammal. Excessive exogenous DNA of the present invention is present in the embryo cells of the produced animal after the DNA transfer. This means that all the offspring of the produced animal have the exogenous DNA of the present invention in all the germ cells and somatic cells. Means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has an excess of the foreign DNA of the present invention in all germ cells and somatic cells.
By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA in excess.

In the non-human mammal having the exogenous normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and finally develops the function of ABP1 by promoting the function of the endogenous normal DNA. It can be used as a model animal for the disease state. For example, by using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathologic mechanism of ABP1 hyperfunction and ABP1-related diseases and to examine methods for treating these diseases.
Further, since the mammal to which the exogenous normal DNA of the present invention is transferred has an increased symptom of ABP1, diseases associated with hyperfunction of ABP1, such as viral infections (eg, AIDS, influenza, unknown fever, etc.) ), Endocrine diseases (eg, hormone deficiency, cytokine deficiency, etc.), blood diseases (eg, cytopenia, renal anemia, etc.), organ dysplasia (eg, thyroid atrophy, cleft palate, etc.), transplant organ rejection, graft Anti-host disease, immunodeficiency, neurodegenerative disease (eg, polyglutamine disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellar degeneration), ischemic heart disease (eg, angina pectoris) , Myocardial infarction, etc.), radiation damage, UV damage (eg, sunburn, etc.), toxic diseases (eg, renal tubular cell damage due to heavy metals, hepatocyte damage due to alcohol, etc.), nutritional disorders (eg, vitamins, Thymic atrophy due to lack of quantitative elements), inflammatory diseases (eg, acute pancreatitis, arthritis, periodontal disease, colitis, etc.), ischemic neuropathy, diabetic neuropathy, vascular diseases (eg, arteriosclerosis, etc.) It can also be used in screening tests for prophylactic / therapeutic agents for diseases such as respiratory diseases (eg, interstitial pneumonia, pulmonary fibrosis, etc.) and cartilage diseases (eg, osteoarthritis, etc.).

  On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. Furthermore, the target foreign DNA can be incorporated into the aforementioned plasmid and used as a raw material. A DNA construct with a promoter can be prepared by a normal DNA engineering technique. The abnormal DNA transfer of the present invention at the fertilized egg 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 embryo cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the foreign abnormal DNA of the present invention in all of the germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA.

The non-human mammal having the exogenous abnormal DNA of the present invention has a high expression of the abnormal DNA of the present invention, and finally inhibits the function of the endogenous normal DNA, thereby finally inhibiting the function inactive type of ABP1. Response (eg, cancer (eg, leukemia, esophageal cancer, stomach cancer, colon cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, osteosarcoma, brain tumor, etc.) ), Autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, insulin-dependent diabetes mellitus, psoriasis, ulcerative colitis, idiopathic thrombocytopenic purpura, Crohn's disease , Glomerulonephritis, etc.), viral infection (eg, hemorrhagic fever, T cell leukemia, Kaposi's sarcoma, infectious mononucleosis, lymphoma, nasopharyngeal cancer, cervical cancer, skin cancer, hepatitis, liver cancer, etc.), endocrine disease (E.g. Hormo Hyperemia, hypercytosis, etc.), blood diseases (eg, hematocytosis, B-cell lymphoma, polycythemia, etc.), organ hyperplasia (eg, semi-middle yang, retention testicles, teratomas, nephroblastoma, multiple) Cystic kidneys, heart / aortic malformations, finger claws and the like)), and can be used as a disease state model animal. For example, by using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of ABP1 functional inactive refractory and to examine a method for treating this disease.
Further, as a specific applicability, the abnormal DNA highly expressing animal of the present invention becomes a model for elucidating the function inhibition (dominant negative action) of normal ABP1 by abnormal ABP1 in the functional inactive refractory disease of ABP1.
Moreover, since the mammal which transferred the foreign abnormal DNA of this invention has the increase symptom of abnormal ABP1, it can be utilized for the therapeutic drug screening test with respect to the functional inactive type refractory of ABP1.
Furthermore, in order to develop a prophylactic / therapeutic agent for diseases related to ABP1, including the functional inactive refractory of ABP1, using the DNA-transferred animal of the present invention, the above-described test methods and quantitative methods are used. Thus, it is possible to provide an effective and rapid screening method for a drug for preventing or treating the disease. It is also possible to study and develop a gene therapy method for diseases associated with ABP1 using the DNA-transferred animal of the present invention or the exogenous DNA expression vector of the present invention.

(11) Knockout animal ABP1 DNA expression deficient non-human mammal embryonic stem cell and ABP1 DNA expression deficient non-human mammal used in the present invention are:
[1] A non-human mammalian embryonic stem cell in which DNA encoding ABP1 is inactivated,
[2] The embryonic stem cell according to item [1], wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli),
[3] The embryonic stem cell of [1], which is neomycin resistant,
[4] The embryonic stem cell of [1], wherein the non-human mammal is a rodent
[5] The embryonic stem cell according to item [4], wherein the rodent is a mouse,
[6] The DNA expression-deficient non-human mammal in which the DNA encoding ABP1 is inactivated,
[7] The DNA can be inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli), and the reporter gene can be expressed under the control of a promoter for DNA encoding ABP1 [6 A non-human mammal according to claim,
[8] The non-human mammal according to item [6], wherein the non-human mammal is a rodent.
[9] The non-human mammal according to item [8], wherein the rodent is a mouse.
The non-human mammal embryonic stem cell in which the DNA encoding ABP1 is inactivated refers to suppressing the expression ability of the DNA by artificially adding mutation to the ABP1 DNA of the non-human mammal, or By substantially losing the activity of ABP1 encoded by the DNA, the DNA has substantially no ability to express ABP1 (hereinafter sometimes referred to as the knockout DNA of the present invention). An embryonic stem cell (hereinafter abbreviated as ES cell).
As the non-human mammal, the same ones as described above are used.
As a method for artificially adding mutation to DNA encoding ABP1, it can be carried out, for example, by deleting a part or all of the DNA sequence and inserting or replacing other DNA by genetic engineering techniques. With these mutations, for example, the knockout DNA of the present invention may be prepared by shifting the reading frame of codons or destroying the function of the promoter or exon.

Specific examples of non-human mammalian embryonic stem cells in which the DNA encoding ABP1 is inactivated (hereinafter abbreviated as the DNA inactivated ES cell of the present invention or the knockout ES cell of the present invention) include, for example, ABP1 DNA possessed by a non-human mammal is isolated and a neomycin resistance gene, a drug resistance gene typified by a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyltransferase) in its exon part Insert a reporter gene typified by a gene) to disrupt the function of the exon, or insert a DNA sequence that terminates gene transcription (for example, a polyA addition signal) into the intron between exons. By making it impossible to synthesize Thus, an ES cell obtained by introducing a DNA strand (hereinafter abbreviated as a targeting vector) having a DNA sequence constructed so as to destroy the gene into the animal chromosome by, for example, homologous recombination. The analysis was performed by Southern hybridization analysis using the DNA sequence on or near the ABP1 DNA as a probe, or by the PCR method using the DNA sequence on the targeting vector and the DNA sequence in the region other than the ABP1 DNA used for the preparation of the targeting vector as primers. It can be obtained by selecting the knockout ES cell of the present invention.
In addition, as the original ES cell for inactivating the DNA encoding ABP1 by homologous recombination method or the like, for example, those already established as described above may be used, or the known Evans and Kaufman method may be used. Similarly, a newly established one may be used. For example, in the case of mouse ES cells, currently 129 ES cells are generally used, but since the immunological background is unclear, an alternative and purely immunological genetic background For example, C57BL / 6 mice and BDF ₁ mice (C57BL / 6 and DBA / 2 which have improved the number of eggs collected from C57BL / 6 by crossing with DBA / 2) are obtained. Those established using F ₁ ) can also be used favorably. BDF ₁ mice have the advantage of having a large number of eggs collected and strong eggs, and also have C57BL / 6 mice in the background, so ES cells obtained using these mice can be used to create pathological model mice. The genetic background can be advantageously replaced with C57BL / 6 mice by backcrossing with C57BL / 6 mice.
In addition, when establishing ES cells, blastocysts on the 3.5th day after fertilization are generally used, but in addition to this, many blastocysts can be efficiently obtained by collecting 8-cell embryos and culturing them to blastocysts. Early embryos can be obtained.
Although both male and female ES cells may be used, male ES cells are usually more convenient for producing germline chimeras. In addition, it is desirable to discriminate between males and females as soon as possible in order to reduce troublesome culture work.

One example of a method for determining the sex of an ES cell is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. If this method is used, the number of ES cells of about 1 colony (about 50) is sufficient as compared with the case where about 10 ⁶ cells were conventionally required for karyotype analysis. It is possible to perform primary selection of ES cells in the early stage by discriminating between males and females, and by allowing male cells to be selected at an early stage, labor in the initial stage of culture can be greatly reduced.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of the obtained ES cell is preferably 100% of the normal number. However, if it is difficult due to physical operations at the time of establishment, the gene of the ES cell is knocked out, and then normal cells (for example, chromosomes in mice) It is desirable to clone again into cells where the number is 2n = 40.
The embryonic stem cell line obtained in this way is usually very proliferative, but it tends to lose its ontogenic ability, so it needs to be subcultured carefully. For example, in a carbon dioxide incubator in the presence of LIF (1-10000 U / ml) on a suitable feeder cell such as STO fibroblast (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% Culturing is carried out by a method such as culturing at about 37 ° C. with carbon dioxide gas and 90% air, and at the time of passage, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, Preferably, the cells are converted into single cells by treatment with about 0.1% trypsin / 1 mM EDTA, and seeded on newly prepared feeder cells. Such passage is usually carried out every 1 to 3 days. At this time, cells are observed, and if morphologically abnormal cells are found, it is desirable to abandon the cultured cells.
ES cells can be differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by monolayer culture to high density or suspension culture until a cell conglomerate is formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embryology and Experimental Morphology, 87, 27, 1985], ES cells of the present invention. The DNA expression-deficient cells of the present invention obtained by differentiation are useful in cell biology of ABP1 in vitro.

The DNA expression deficient non-human mammal of the present invention can be distinguished from normal animals by measuring the mRNA level of the animal using a known method and comparing the expression level indirectly.
As the non-human mammal, those similar to the above can be used.
The DNA expression-deficient non-human mammal of the present invention has a DNA sequence in which, for example, a targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the ABP1 DNA of the targeting vector is inactivated by the introduction. By homologous recombination that replaces ABP1 DNA on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination, the DNA encoding ABP1 can be knocked out.
Cells in which the DNA encoding ABP1 has been knocked out include those other than the ABP1 DNA derived from the Southern hybridization analysis or targeting vector using the DNA sequence on or near ABP1 DNA as a probe, and the mouse-derived ABP1 DNA used for the targeting vector. It can be determined by analysis by PCR method using a DNA sequence in the vicinity region as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA encoding ABP1 has been inactivated by gene homologous recombination is cloned, and the cells are cultured at a suitable time, for example, non-human at the 8-cell stage. It is injected into a mammalian embryo or blastocyst, and the produced chimeric embryo is transplanted into the uterus of the non-human mammal that is pseudopregnant. The produced animal is a chimeric animal composed of both cells having a normal ABP1 locus and cells having an artificially mutated ABP1 DNA locus.
When some of the germ cells of the chimeric animal have a mutated ABP1 DNA locus, all tissues were artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of cells having the ABP1 DNA locus, for example, by determining the coat color. The individual thus obtained is usually an ABP1 hetero-expression deficient individual, and ABP1 hetero-expression deficient individuals can be mated to obtain ABP1 homo-expression deficient individuals from their offspring.

In the case of using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by a microinjection method. Compared to mammals, it can be obtained by selecting those having mutations in the ABP1 DNA locus by gene homologous recombination.
In this way, an individual in which ABP1 DNA is knocked out can be reared in a normal breeding environment after confirming that the animal individual obtained by mating also has the DNA knocked out.
In addition, germline acquisition and retention may be followed in accordance with conventional methods. That is, a homozygous animal having the inactivated DNA in both homologous chromosomes can be obtained by mating male and female animals possessed by the inactivated DNA. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in a state where there are 1 normal individual and multiple homozygous animals. By crossing male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and passaged.
Non-human mammal embryonic stem cells in which ABP1 DNA has been inactivated are very useful in producing the non-human mammal deficient in DNA expression of the present invention.
In addition, since the non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by ABP1, it can be a model for diseases caused by inactivation of the biological activity of ABP1. It is useful for investigating the cause of diseases and examining treatment methods.

(11a) Method for screening compound having therapeutic / preventive effect on diseases caused by ABP1 DNA deficiency or damage The DNA expression deficient non-human mammal of the present invention is a disease caused by ABP1 DNA deficiency or damage. , For example, cancer (eg, leukemia, esophageal cancer, stomach cancer, colon cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, osteosarcoma, brain tumor, etc.) , Autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, insulin-dependent diabetes, psoriasis, ulcerative colitis, idiopathic thrombocytopenic purpura, Crohn's disease, Glomerulonephritis, etc.), inflammation (eg, arthritis, nephritis, etc.), viral infection (eg, hemorrhagic fever, T cell leukemia, Kaposi's sarcoma, infectious mononucleosis, lymphoma, nasopharyngeal cancer, uterus Cancer, skin cancer, hepatitis, liver cancer, etc.), endocrine diseases (eg, hormonal excess, cytokine excess, etc.), blood diseases (eg, hematocytosis, B cell lymphoma, polycythemia, etc.), organ hyperplasia (eg, , Hemi-Yin Yang, Suspended Testicles, Teratomas, Renal Blast Cancer, Multiple Cystic Kidneys, Cardiac / Aortic Malformations, Finger Syndrome, etc.) can be used for screening compounds having therapeutic / prophylactic effects.
That is, the present invention relates to a disease caused by ABP1 DNA deficiency or damage, which comprises administering a test compound to a non-human mammal deficient in DNA expression of the present invention and observing and measuring changes in the animal. A method for screening a compound having a therapeutic / prophylactic effect or a salt thereof is provided.
Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.
Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. These compounds are novel compounds. It may be a known compound.
Specifically, the non-human mammal deficient in DNA expression of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptom, etc. of the animal as an index. The therapeutic / prophylactic effect of the compound can be tested.
As a method for treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dosage of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

In the screening method, when a test compound is administered to a test animal (for example, a cancer-bearing animal), for example, the symptom of the test animal is about 10% or more, preferably about 30% or more, more preferably about 50% or more. In the case of improvement, the test compound can be selected as a compound having a therapeutic / prophylactic effect on the above-mentioned diseases.
The compound obtained by using the screening method is a compound selected from the above-mentioned test compounds, and is used as a pharmaceutical agent such as a safe and low-toxic therapeutic / preventive agent for the above-mentioned diseases caused by ABP1 deficiency or damage. be able to. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals). And the like, and physiologically acceptable acid addition salts are particularly preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
A medicament containing the compound obtained by the screening method or a salt thereof can be produced and used in the same manner as the aforementioned medicament containing ABP1 or the activation peptide of the present invention.

(11b) Method for screening a compound that promotes or inhibits the activity of a promoter for ABP1 DNA The present invention is characterized in that a test compound is administered to a non-human mammal deficient in DNA expression of the present invention to detect the expression of a reporter gene. A method for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for ABP1 DNA is provided.
In the above screening method, the non-human mammal deficient in DNA expression of the present invention is inactivated by introducing a reporter gene into the ABP1 DNA among the non-human deficient DNA expression of the present invention. A gene that can be expressed under the control of a promoter for ABP1 DNA is used.
Examples of the test compound are the same as described above.
As the reporter gene, the same ones as described above are used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene, or the like is preferable.
In the non-human mammal deficient in DNA expression of the present invention in which ABP1 DNA is replaced with a reporter gene, since the reporter gene exists under the control of the promoter for ABP1 DNA, by tracing the expression of the substance encoded by the reporter gene, Promoter activity can be detected.
For example, when a part of the DNA region encoding ABP1 is replaced with a β-galactosidase gene (lacZ) derived from E. coli, β-galactosidase is expressed instead of ABP1 in a tissue that originally expresses ABP1. Therefore, for example, by staining with a reagent that is a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal), ABP1 The expression state in the animal body can be observed. Specifically, an ABP1-deficient mouse or a tissue section thereof was fixed with glutaraldehyde, washed with phosphate buffered saline (PBS), and then stained with X-gal at about room temperature or around 37 ° C. After reacting for 30 minutes to 1 hour, the tissue specimen is washed with a 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the coloration. Moreover, you may detect mRNA which codes lacZ according to a conventional method.
The compound obtained by using the screening method or a salt thereof is a compound selected from the above-described test compounds, and is a compound that promotes or inhibits promoter activity against ABP1 DNA.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). In particular, physiologically acceptable acid addition salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.

Since a compound or a salt thereof that promotes promoter activity for ABP1 DNA can promote the expression of ABP1 and promote the function of ABP1, for example, a pharmaceutical agent such as a prophylactic / therapeutic agent for diseases associated with ABP1 dysfunction Can be used as Specifically, the compound is, for example, cancer (eg, leukemia, esophageal cancer, stomach cancer, colon cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, bone marrow. Tumor, osteosarcoma, brain tumor, etc.), autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, insulin-dependent diabetes mellitus, psoriasis, ulcerative colitis, idiopathic platelets) Reduced purpura, Crohn's disease, glomerulonephritis, etc.), viral infections (eg hemorrhagic fever, T cell leukemia, Kaposi's sarcoma, infectious mononucleosis, lymphoma, nasopharyngeal cancer, cervical cancer, skin cancer, hepatitis , Liver cancer, etc.), endocrine diseases (eg, hormonal excess, cytokine excess, etc.), blood diseases (eg, hematocytosis, B cell lymphoma, polycythemia, etc.), organ hyperplasia (eg, half-yin yang, retention testicles) , Teratoma, nephroblastoma, multiple sac Kidney, heart, aorta malformations, syndactyly, etc.), restenosis after angioplasty, low toxicity, such as prophylactic or therapeutic agents, such as cancer recurrence after resection can be used as a safe medicine.
On the other hand, a compound or a salt thereof that inhibits the promoter activity for ABP1 DNA can inhibit the expression of ABP1 and inhibit the function of ABP1, and thus, for example, a preventive / therapeutic agent for diseases associated with excessive expression of ABP1 It is useful as a pharmaceutical. Specifically, the compound is, for example, viral infection (eg, AIDS, influenza, unknown fever, etc.), endocrine disease (eg, hormone deficiency, cytokine deficiency, etc.), blood disease (eg, cytopenia, renal disease) Anemia, etc.), organ dysplasia (eg, thyroid atrophy, cleft palate), transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative diseases (eg, polyglutamine disease, Alzheimer's disease, Parkinson's disease, muscle atrophy) Lateral sclerosis, prion disease, cerebellar degeneration, etc., ischemic heart disease (eg, angina pectoris, myocardial infarction, etc.), radiation damage, UV damage (eg, sunburn, etc.) Toxic disease (eg, kidney due to heavy metals) Tubular cell injury, hepatocyte injury due to alcohol, etc.), nutritional disorders (eg, atrophy of the thymus due to vitamin and trace element deficiencies), inflammatory diseases (eg, acute pancreatitis, arthritis, periodontal disease, colitis, etc.), Ischemic neuropathy , Prevention of diseases such as diabetic neuropathy, vascular diseases (eg, arteriosclerosis, etc.), respiratory diseases (eg, interstitial pneumonia, pulmonary fibrosis, etc.), cartilage diseases (eg, osteoarthritis, etc.) -It can be used as a low-toxic and safe medicine such as a therapeutic agent.
Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
A medicament containing the compound obtained by the screening method or a salt thereof can be produced and used in the same manner as the aforementioned medicament containing ABP1 or the activation peptide of the present invention.

As described above, the non-human mammal deficient in DNA expression of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for ABP1 DNA. It can greatly contribute to the investigation of disease causes or the development of preventive / therapeutic drugs.
In addition, by using DNA containing the promoter region of the ABP1 gene, genes encoding various proteins are ligated downstream thereof, and this is injected into the egg cell of an animal to produce a so-called transgenic animal (transgenic animal). For example, the protein can be synthesized in a tissue and / or time-specific manner, and the action in the living body can be examined. Furthermore, if a suitable reporter gene is bound to the above promoter part and a cell line in which it is expressed is established, a search system for low molecular weight compounds having an action of specifically promoting or suppressing the production ability of ABP1 itself in the body Can be used as

In the present specification and drawings, bases, amino acids, and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: deoxycytidine triphosphate ATP: adenosine triphosphate EDTA: ethylenediaminetetraacetic acid SDS: sodium dodecyl sulfate Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: threonine CyrM: threonine : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid *: Corresponding to the stop codon Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Azoyl group TC: Thiol group TC: ) -Carboxamide group

In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyl Oxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenol Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy -4-oxo-
1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
The base sequence of the amino acid coding region of human ABP1 cDNA is shown.
[SEQ ID NO: 2]
The amino acid sequence of human ABP1 is shown.
[SEQ ID NO: 3]
The base sequence of the amino acid coding region of mouse ABP1 cDNA is shown.
[SEQ ID NO: 4]
The amino acid sequence of mouse ABP1 is shown.
[SEQ ID NO: 5]
The amino acid sequence of the peptide used as an antigen for preparation of anti-ABP1 antibody (ELA antibody) is shown.
[SEQ ID NO: 6]
1 shows the amino acid sequence of a peptide used as an antigen for producing an anti-ABP1 antibody (LVR antibody).
The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited by these examples.

Cloning of ABP1 cDNA In order to identify ASK1 binding protein, ASK1-KR in which lysine (K) at the ATP binding site (position 709) in the kinase domain of human ASK1 was replaced with arginine (R) was used as a bait. Brent's system [Zervos et al., Cell, Vol. 72, p.223-232, 1993; Gyuris et al., Cell, vol. 75, p. 791- 803, 1993] was screened by the yeast two-hybrid method. As a result, about 1200 positive clones were obtained. One of them was a part of the protein coding region of the gene named “PGR1” (a portion corresponding to amino acids 35 to 127) when database search was performed based on the obtained base sequence. It was. The protein encoded by this gene consists of 127 amino acids, but the information on the database relates only to the structure of the gene, and there has been no report on its function. Therefore, the full length cDNA encoding the protein of this gene was cloned by PCR. Since this protein was identified as an ASK1-binding protein, it was named ASK1 binding protein 1 (ABP1). Homology search revealed that ABP1 has similar molecules in mammals such as mice, but no molecules with high homology were found in flies and nematodes. Also, no motifs or functional domains existed even after motif search or domain search. FIG. 1 shows an alignment of human and mouse ABP1 amino acid sequences.

Expression distribution of ABP1 mRNA
Using Multiple Tissue Northern Blot (CLONTECH) Mouse (# 7762-1) and Mouse Embryo (# 7763-1), Northern blotting was performed according to the instructions for the above product using the full length of human ABP1 cDNA as a probe. As a result, the length of ABP1 mRNA was about 1.7 kb, and was ubiquitously expressed throughout the tissue (FIG. 2A). In addition, ABP1 mRNA was also expressed from a relatively early stage of embryonic day 7 in mouse fetal blots, and no significant change was observed during the course of embryogenesis (FIG. 2B).

Production of ABP1 protein in various animal cells Next, a rabbit polyclonal antibody against ABP1 was prepared. Based on the amino acid sequence information obtained from the base sequence of ABP1 cDNA, two types of peptide antibodies having ABP1-specific peptides (SEQ ID NO: 5 and SEQ ID NO: 6) as antigens are made, and named ELA antibody and LVR antibody, respectively. did. Both antibodies were purified using affinity by an antigen peptide.
HeLa cells and HEK293 cells were cultured in Dulbecco's modified Eagle medium (DMEM; SIGMA) containing high-concentration glucose (4.5 mg / ml) in the presence of 5% CO ₂ . 10% fetal bovine serum (FBS) and 100 units / ml penicillin were added to the culture solution. Porcine aortic endothelium (PAE) cell culture was performed by adding 10% FBS, 10 mM HEPES, 100 units / ml penicillin to F12 culture medium (Invitrogen).
Each cell was lysed using a lysis buffer [150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% Triton X-100, 1% deoxycholate, 1.5% aprotinin, 1 mM PMSF]. The supernatant is obtained by centrifuging the solution, SDS sample buffer [100 mM Tris-HCl (pH 8.8), 0.01% bromophenol blue, 36% glycerol, 4% SDS] is added, and SDS polyacrylamide gel electrophoresis ( SDS-PAGE) was performed. After transferring the protein from the gel to a PVDF membrane and blocking with 5% skim milk-containing TBS-T [150 mM NaCl, 50 mM Tris-HCl, (pH 8.0), 0.05% Tween 20] at room temperature for 3 hours. And reacted with each antibody. Detection was performed using the ECL system (Amersham Pharmacia Biotech).
When the cultured cell extract of HEK293 cells was subjected to immunoblot analysis using ELA antibody and LVR antibody, a band identified by both antibodies was observed at about 17 kDa. The detection position of this band was completely consistent between the two antibodies. Further, a plasmid expressing ABP1 untagged to HEK293 cells [prepared by subcloning the ABP1 cDNA obtained by PCR into pcDNA3 (Invitrogen, Inc.)] was introduced into a gene [FuGENE6 (Roche Diagnostics KK). When the ABP1 protein was overexpressed, the detection intensity increased depending on the amount of plasmid (FIG. 3). This band disappeared when peptide blocking was performed by adding an antigenic peptide during the primary antibody treatment.
Therefore, these antibodies recognized ABP1 and could detect the endogenous ABP1 protein. Similar results were obtained with HeLa cells and PAE cells.

Intracellular interaction between ABP1 and ASK1 Two fusion proteins, Flag-ABP1 and Myc-ASK1, were co-expressed in HEK293 cells. Cells are lysed using lysis buffer (described above), the lysate is centrifuged, and the supernatant is collected. After reaction with anti-Flag antibody (Clone M2; SIGMA), Protein A-sepharose 4B (Zymed Laboratories) is added and incubated for 30 minutes. After washing 3 times with lysis buffer, SDS sample buffer (described above) was added, and the binding of both proteins was confirmed by the same procedure as in the immunoblotting method (FIG. 4A). As the antibody, anti-Flag antibody (described above) and anti-Myc antibody (Clone 9E10, Calbiochem) were used. This binding was enhanced by treatment with H ₂ O ₂ which is an activator of ASK1. Next, when CFP-ABP1 and Myc-ASK1 plasmids were also coexpressed in HEK293 cells and immunoprecipitated with an anti-Flag antibody, the binding of both was confirmed (FIG. 4B). As the antibody, anti-Flag antibody (described above) and anti-GFP antibody (Medical & Biological Laboratories CO.) Were used. This binding was also enhanced by H ₂ O ₂ treatment. From this, it was found that ABP1 and ASK1 were also bound in mammalian cells, and the binding was enhanced by H ₂ O ₂ treatment.

Identification of binding site of ASK1 to ABP1 The binding of various ASK1 deletion mutants with HA tag shown in FIG. 5A and ABP1 was co-expressed with Flag-ABP1 as in Example 3, followed by immunoprecipitation with anti-Flag antibody. Searched at. As the antibody, anti-HA antibody (Clone 3F10, Roche Diagnostics KK) and anti-Flag antibody (Clone M2, SIGMA) were used. As the expression vector for ASK1, the one described in Saitoh et al. As a result, ABP1 coprecipitated with ASK1-NT and ASK1-ΔC, but did not show coprecipitation with ASK1-ΔN and ASK1-K (FIG. 5B). This revealed that ABP1 binds to the N-terminal domain of ASK1.

Induction of cell death by ABP1 (1) CFP-ABP1 plasmid expressing a protein in which CFP was fused to the N-terminus of ABP1 used for immunoprecipitation experiments in HeLa cells to investigate the intracellular localization and function of ABP1 [PCR The ABP1 cDNA obtained in this way was subcloned into pECFP-C1 (Clontech)] and was transiently expressed by gene transfer [using FuGENE6 (Roche Diagnostics KK) as described in the instructions for use]. The fluorescence of was observed over time with a fluorescence microscope. Under the fluorescence microscope, among the cells (500 cells) showing CFP fluorescence, the ratio of cells that peeled off the plate or showed cell death morphology such as membrane blebbing or fragmentation was calculated. Calculated for CFP positive cells.
As a result, the fluorescence by the CFP-ABP1 fusion protein was present in the entire cell including both the cytoplasm and the nucleus, as in the case where only the CFP protein was expressed. If observation was continued further, cells expressing CFP-ABP1 fusion protein began to show signs of cell death one after another, starting from 24 hours after gene transfer, such as peeling off from the culture plate or blebbing of the membrane. (FIG. 6A). When this cell death was quantified, it reached about 70% of cells expressing the CFP-ABP1 fusion protein 36 hours after gene introduction (FIG. 6B). Such a phenomenon was not observed even when the CFP protein was expressed alone. Further, intracellular localization and cell death induction were the same even when a protein in which CFP was fused to the C-terminus of ABP1 was expressed.
From this, it was considered that ABP1 is a protein having the ability to induce cell death.

(2) For the purpose of clarifying the mechanism of cell death by ABP1, a cell line (PAE-ABP1 cell) capable of inducing expression of ABP1 in a tetracycline-dependent manner in PAE cells was established. PAE-ABP1 cells were produced by partially modifying the method of Takeda et al. (J. Biol. Chem., 275, 9805-9813, 2000). Unlike the method of Takeda et al., The entire ABP1 cDNA with Myc tag added was subcloned into the pTet-Splice-neo vector, and the pTet-tTAk-hyg plasmid was simultaneously introduced into PAE cells, and 500 ng / ml tetracycline (Sigma ), 400 units / ml hygromycin B (Wako), 240 mg / ml Neomycin (Geneticin, Life Technologies, Inc.) was added in culture medium and selected, and the surviving and colonized cells were treated with PAE-ABP1. Cells were used. Cell maintenance culture was performed by adding 500 ng / ml tetracycline, 200 units / ml hygromycin B, and 30 mg / ml neomycin to the aforementioned culture medium for PAE cell maintenance.
The PAE-ABP1 cells do not express ABP1 when cultured in the presence of tetracycline. However, in the absence of tetracycline, the expression of ABP1 protein with Myc tag can be confirmed by immunoblot analysis after about 6 hours. Thereafter, expression of ABP1 is observed stably. When immunostaining with an anti-Myc antibody (Clone 9E10, Calbiochem) was performed, the expression of Myc-ABP1 was stably observed in most cells. When this cell was cultured with tetracycline removed, cell death was induced with the expression of the ABP1 protein, as in the case of transiently expressing the ABP1 protein in HeLa cells (FIG. 7A). When the proportion of dead cells in all cells (the proportion of cells showing the form of cell death among all cells was calculated under a microscope. The results are shown as an average value of three fields) was quantified 48 hours after removal of tetracycline. About 55% was reached (FIG. 7B). In this experimental system, ABP1 was confirmed to induce cell death.

(3) Membrane blebbing and cell fragmentation, which are morphological features of cell death by ABP1 in (1) and (2) above, are often observed during apoptosis. Therefore, whether or not cell death in PAE-ABP1 cells is apoptotic was examined based on the presence or absence of DNA fragmentation.
PAE-ABP1 cells (2 × 10 ⁶ cells) were lysed with 200 ml of lysis buffer [20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.5% Triton X-100]. The lysate was centrifuged and the supernatant was collected and treated with 0.2 mg / ml protein K at 42 ° C. for 1 hour. Next, DNA was purified by phenol-chloroform extraction method and ethanol precipitation method, and this was dissolved again in TE buffer (10 mM Tris-HCl, 1 mM EDTA) containing 0.2 mg / ml ribonuclease A, and 2% agarose gel was obtained. After electrophoresis, the electrophoresis pattern was photographed by staining with ethidium bromide.
As a result, the PAE-ABP1 cells formed a DNA ladder along with the expression of the ABP1 protein in agreement with the time of appearance of cell death when observed with a microscope (FIG. 8). From this, it was found that cell death by ABP1 is so-called apoptosis accompanied by DNA fragmentation. This could also be confirmed by TUNEL staining of PAE-ABP1 cells.

(4) DNA fragmentation during apoptosis usually follows caspase activation. Thus, caspase-3 activity was measured using PAE-ABP1 cells during induction of ABP1 protein expression. The measurement was performed using a synthetic fluorescent peptide DEVD-7-amino-4-trifluoromethyl coumarine (AFC) using CPP32 / caspase-3 fluorometric protease assay kit (MBL). The liberated AFC was measured for fluorescence intensity using a fluorescence spectrophotometer at an excitation wavelength of 360 nm and a fluorescence wavelength of 530 nm. Each sample was measured twice. The result was expressed as a relative value with a value of 1 when tetracycline was not removed.
As a result, caspase-3 activity increased before and after cell death (FIG. 9A). To examine whether cell death by ABP1 is caspase-dependent, the same PAE-ABP1 cells were treated with zVAD-fmk (Peptide Institute, Inc.), a caspase inhibitor, at the same time as tetracycline removal. In the early time zone of 18 hours after removal, cell death was almost suppressed (FIG. 9B). From this, it was concluded that cell death by ABP1 is caspase-dependent apoptosis.

Identification of cell death-inducing region of ABP1 As described above, ABP1 does not have a functional domain of an existing apoptosis-related factor. Therefore, in order to investigate the domain necessary for apoptosis by ABP1, plasmids expressing fusion proteins of various ABP1 deletion mutants and CFP shown in FIG. 10A were prepared and expressed in HeLa cells in the same manner as in Example 6 (1). The presence or absence of cell death was examined. An ABP1 expression vector was prepared by subcloning ABP1 cDNA obtained by PCR or ABP1-deficient mutant cDNAA into pECFP-C1 (Clontech). Gene transfer of each plasmid into the cells was performed using FuGENE6 (Roche Diagnostics KK) as per the instructions.
As a result, ΔC (1-72) shows a stronger cell death inducing ability than the wild type, but as N-terminal is deleted, ΔN (34-127), ΔN (65-127) and cell death inducing ability are increased. Decreased (FIG. 10B). From this, it was considered that the N-terminal side of ABP1 is important for the cell death inducing ability.

Effect of ABP1 on ASK1 signaling system
Using PAE-ABP1 cells, the activation of JNK and p38 when ABP1 expression was induced was examined using the respective anti-phosphorylated protein antibodies. Anti-phosphorylated JNK antibody (Cell Signaling), anti-phosphorylated p38 antibody (Cell Signaling), and anti-phosphorylated ASK1 antibody (Tobiume et al., EMBO Rep., 2, 222-228, 2001) were used as antibodies. The cells were lysed using a lysis buffer (described above) in the same manner as in Example 4. The lysate was centrifuged, and the supernatant was collected and reacted with an anti-Myc antibody (Clone 9E10, Calbiochem), followed by Protein A-sepharose 4B (Zymed Laboratories). ) Was added, and the mixture was incubated for 30 minutes, washed with lysis buffer three times, SDS sample buffer (described above) was added, and the same procedure as in the immunoblotting method was performed.
Prior to the marked cell death associated with the expression of ABP1 protein due to the removal of tetracycline, activation of JNK and p38 was observed 12 hours later, and this persisted thereafter (FIG. 11A). Thus, when the activation of ASK1 was similarly examined using PAE-ABP1 cells, activation of ASK1 was also observed 12 hours after removal of tetracycline, similar to JNK and p38 (FIG. 11B). From this, it was considered that the ASK1 signal transduction system is activated by overexpression of ABP1.

  The ABP1 of the present invention has an effect of activating the ASK1 cascade to induce apoptosis in cells or induce production of inflammatory cytokines. Therefore, ABP1, the polynucleotide encoding the same, and the like of the present invention are useful as a prophylactic / therapeutic agent for diseases that can be expected to have a prophylactic / therapeutic effect by inducing apoptosis in cells. On the other hand, the ABP1 inhibitor of the present invention (for example, anti-ABP1 antibody, ABP1 antisense polynucleotide, etc.) suppresses apoptosis and production of inflammatory cytokines, and therefore, a disease that can be expected to have a preventive / therapeutic effect by suppressing apoptosis. Or, it is useful as an agent for preventing or treating inflammatory diseases. Furthermore, by using ABP1 and ASK1 of the present invention, a means for screening a novel preventive / therapeutic agent for apoptosis or inflammation-related diseases is provided.

It is a figure which shows the alignment of the ABP1 amino acid sequence of a human (upper row) and a mouse | mouth (lower row). The partial sequences used as antigen peptides for the preparation of the two antibodies (ELA antibody and LVR antibody) of the present invention are indicated by boxes. It is a figure which shows the tissue distribution (A) of ABP1 mRNA expression in a mouse tissue, and the daily change (B) of ABP1 mRNA expression in a mouse fetus tissue. It is a figure which shows the increase in detection intensity dependent on the amount of plasmids when ABP1 protein is overexpressed by introducing a plasmid expressing ABP1 with no tag added into HEK293 cells. Cells were collected 24 hours after gene introduction, each cell extract was divided into two and subjected to SDS-PAGE, and immunoblot (IB) was performed using ELA antibody and LVR antibody. ABP1-pcDNA3 (-) indicates untransformed HEK293 cells, and the gradient indicates that the amount of plasmid increases from left to right. It is a figure which shows the result of the binding test of ABP1 and ASK1. (A) Flag-ABP1 and Myc-ASK1 plasmids were introduced into HEK293 cells, cells were collected 24 hours later, immunoprecipitated with anti-Flag antibody, and then subjected to immunoblot analysis with anti-Myc antibody. (B) Flag-ASK1 and CFP-ABP1 plasmids were introduced into HEK293 cells, and the cells were collected 24 hours later, immunoprecipitated with anti-Flag antibody, and then subjected to immunoblot analysis with anti-GFP antibody. CFP is a variant of GFP and can be recognized by an anti-GFP antibody. In both lanes on the right side of both (A) and (B), H ₂ O ₂ treatment (0.5 mM, 1 hour) was also performed. The lower panel shows a part of each cell lysate before immunoprecipitation, which was separately migrated. IP represents immunoprecipitation and IB represents an immunoblot. FIG. 2 is a schematic diagram (A) of an ASK1 deletion mutant and a diagram (B) showing the results of a binding test between ABP1 and an ASK1 deletion mutant. (A) Each plasmid containing an ASK1-deficient mutant has an HA tag added to the N-terminal side. Amino acid numbers 678 to 936 (shaded area) indicate the kinase domain. (B) The lower part is a part of each cell lysate before immunoprecipitation, which is separately migrated. IP represents immunoprecipitation and IB represents an immunoblot. It is a figure which shows the cell death induction by ABP1 in a HeLa cell. (A) A fluorescence microscopic image (upper row) and a differential interference image (lower row) of the same visual field at 36 hours after gene introduction of CFP or CFP-ABP1 plasmid into HeLa cells are shown. (B) Shows the percentage (%) of cell death 36 hours after gene transfer. It is a figure which shows the cell death induction by ABP1 in a PAE-ABP1 cell. (A) Phase-contrast microscopic images of 36 hours after culture of PAE-ABP1 cells in the presence (+) and absence (-) of tetracycline (Tet) are shown. (B) Percentage of cell death (%) after culturing in the presence of tetracycline (0h), tetracycline removal 24 hours and 48 hours. It is a figure which shows cell death induction (DNA fragmentation) by ABP1 in a PAE-ABP1 cell. After removing tetracycline from PAE-ABP1 cells, DNA fragmentation at each time was examined by agarose gel electrophoresis (upper). Both ends indicate molecular weight markers (M), and the right end indicates size. The lower panel confirms the Myc-ABP1 protein expression in each cell by immunoblot analysis using an anti-Myc antibody. It is a figure which shows the caspase dependence of the cell death by ABP1. (A) PAE-ABP1 cells were cultured with tetracycline removed, and caspase-3 activity was measured after 0, 12, 24, and 36 hours. The result was expressed as a relative value with a value of 1 when tetracycline was not removed. (B) PAE-ABP1 cells were treated with zVAD-fmk (50 μM) at the same time as tetracycline removal, and the percentage (%) of dead cells was quantified. However, the cell death assay was performed 18 hours after removal of tetracycline. Tet represents tetracycline. It is a figure which shows the cell death induction by an ABP1 deletion mutant. (A) A schematic diagram of an ABP1-deficient mutant is shown. In the same manner as in the wild type, a CFP tag was added to the N-terminal side. (B) shows the percentage (%) of cell death 36 hours after gene transfer in cells into which ABP1-deficient mutants have been introduced. It is a figure which shows activation of ASK1, JNK, and p38 by ABP1. (A) PAE-ABP1 cells were cultured after removing tetracycline, and endogenous JNK and p38 were activated by immunoblot analysis using anti-phosphorylated protein antibodies. (B) PAE-ABP1 cells were cultured with tetracycline removed, and the activation of endogenous ASK1 was examined by immunoblot analysis with an anti-phosphorylated protein antibody. IB represents an immunoblot.

[SEQ ID NO: 5]
An oligopeptide designed to function as an antigen for producing an anti-ABP1 antibody.
[SEQ ID NO: 6]
An oligopeptide designed to function as an antigen for producing an anti-ABP1 antibody.

Claims (13)

Protein Shitsuma other ASK1 activation accelerator comprising a salt thereof comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.   The agent according to claim 1, which is an apoptosis inducer. An ASK1 activation promoter comprising a polynucleotide comprising a base sequence encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.   The agent according to claim 3, which is an apoptosis inducer. An ASK1 activation inhibitor comprising a polynucleotide containing an antisense polynucleotide to a base sequence encoding a protein containing the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.   The agent according to claim 5, which is an inhibitor of apoptosis or inflammatory cytokine production.   An ASK1 activation inhibitor comprising an antibody against a protein containing the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a salt thereof.   The agent according to claim 7, which is an inhibitor of apoptosis or inflammatory cytokine production. Protein Shitsuma other salts thereof comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or which comprises using a cell producing it, ASK1 screening method of activating modulators. SEQ ID NO: 2, or proteins Shitsuma other containing the amino acid sequence shown in SEQ ID NO: 4 is a salt or comprising the cells producing it, ASK1 screening kit activation modulators. Comparing expression of the protein or the peptide or a salt thereof protein Shitsuma other containing the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in cells producing a salt thereof, in the presence and absence of the test substance A screening method for an ASK1 activation regulator. Antibodies against the protein or its salt containing the amino acid sequence shown in the polynucleotide or SEQ ID NO: 2 or SEQ ID NO: 4, contains a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 The method according to claim 11 , wherein: Antibodies against the protein or its salt containing the amino acid sequence shown in the polynucleotide or SEQ ID NO: 2 or SEQ ID NO: 4, contains a nucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 A kit for screening for an ASK1 activation-regulating substance, comprising:

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