JP2000228989A - New protein and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein which has a specific amino acid sequence, has affinity to an activin receptor and/or activin intracellular signal transducer, and can be used for developing a medicine for Alzheimer's disease, Parkinson's disease, Huntington's chorea, and so on. SOLUTION: This is a new protein (or its salt) which has the amino acid sequence which is identical or substantially identical to the amino acid sequence shown by the formula, has affinity for an activin receptor and/or activin intracellular signal transducer, is used for screening a compound which inhibits or promotes binding this protein with its binding protein and so on, and is useful for developing a medicine for preventing and/or treating Alzheimer's disease, Parkinson's disease, epilepsy, Huntington's chorea, and so on. This protein is obtained by cloning a human genomic DNA by amplifying by PCR using synthetic DNA primers having base sequences coding for partial sequences of the protein, followed by incorporating the obtained gene into a vector to express the gene in a host cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel protein having a specific amino acid sequence, a PDZ domain and / or a WW domain and specifically expressed in the brain, and a DNA region encoding the protein. The present invention relates to a DNA, a method for producing the protein, and uses of the protein and the DNA.

[0002]

2. Description of the Related Art Conventionally, many physiologically active substances have been isolated and identified, and their functions have been elucidated. It is known that some of them exhibit various activities in various organs or cells. Various biological activities in various organs or cells are usually embodied via a receptor to which the physiologically active substance binds, but a combination of the physiologically active substances that bind to the receptor is present in all organs and cells. Whether they are the same or specific to each organ / cell,
There are many cases that have not been elucidated. Some bioactive proteins are P
Some have a DZ domain or a WW domain. These domains are relatively recently discovered protein binding domain modules. Therefore, the distribution, function, control mechanism, and the like of a protein having a PDZ domain or a WW domain in a living body are still unknown. A protein having a PDZ domain plays an important role in the formation of a network of a cell membrane lining structure, a cytoskeleton, and a network of intracellular signal transduction developed in an intracellular surface layer through a function of protein binding. it is conceivable that. In the nervous system, it is indispensable for the assembly of protein clusters at synaptic sites, such as involved in the formation of complexes such as neurotransmitter receptors and ion channels. Recently, a PDZ protein whose expression is regulated by synaptic plasticity has been found. PDZ protein may be involved in plasticity-related changes in synaptic morphology through receptor rearrangement. It is also considered to be involved in network construction and higher-order functions of the living brain. The PDZ domain shares some common features with known peptide binding domains, but also has distinct features. The PDZ domain is a domain widely conserved from bacteria to higher plants and animals. In many cases, the PDZ domain recognizes a short amino acid sequence at the C-terminus of a target protein, and these target proteins are transmembrane receptors. Often a body or channel. P
DZ domains are often found in the same protein in a form that is repeated about 2 to 6 times, and, unlike other domain modules, some form homodimers. These are important features for the PDZ domain to be involved in the formation of a protein cross-linking network and microdomain in cell-cell adhesion, such as cell surface structures such as synapses and tight junctions. On the other hand, W
About 40 W domains are well conserved from yeast to mammals.
PY, a domain composed of amino acids, rich in proline
Recognizes and binds to relatively short amino acid sequences called motifs. Examples of proteins having a WW domain include ubiquitin protein ligase, dystrophin, and formin-binding protein. These proteins include 1-4 WWs
Domains exist in series. Many proteins having a WW domain have a domain involved in other protein-protein interactions or have an enzyme active domain such as protein phosphatase or rasGAP, suggesting that the WW domain has a very wide variety of biological functions. Activin is a follicle-stimulating hormone (F) from the anterior pituitary gland.
It is a regulator that promotes the secretion of SH). Conventionally, it was thought that steroid hormones produced in the gonads were mainly responsible for the regulation of gonadotropin secretion from the pituitary gland. It is of interest as a novel mechanism of pituitary-reproductive system hormone regulation. As the bioactivity analysis of activin progressed, this system
In addition to regulating FSH secretion, it has been found that it has various physiological activities such as the activity of inducing or inhibiting the differentiation of cells of the blood system and reproductive organs, and the activity of maintaining survival of nerve cells, but the detailed mechanism has not yet been elucidated There are many.

[0003]

The present invention relates to a protein having a PDZ domain and / or a WW domain expressed in the brain, a receptor capable of binding to the protein (eg, an activin receptor), and intracellular information. The novel protein is used as a means for elucidating the physiological activity of a transmission molecule (for example, Smad) and elucidating the detailed molecular mechanism of the activin-activin receptor system, such as inhibition of cell differentiation in nervous system tissue and neurotrophic factor-like activity. , Detection method, DNA containing the novel protein gene, method for producing protein encoded by the novel protein gene, and DNA
It is another object of the present invention to provide a use of the protein.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above problems, and as a result, SEQ ID NO:
Using a yeast two-hybrid method using the intracellular region of the mouse activin IIA-N receptor protein represented by No. 1 as a bait, a binding protein was searched from a mouse brain cDNA library, and C
A cDNA clone in which binding to the activin IIA-N receptor was confirmed even in OS7 cells was obtained. Further, a cDNA clone containing the full length of the gene encoded by this clone was isolated and analyzed. As a result, 5 PDZ domains and 2
This gene was found to contain a region encoding WW domains. The protein encoded by this gene is a protein factor having a plurality of protein-protein interaction domains, of which, via a PDZ domain,
1) binding to the intracellular region of the activin receptor, thereby inhibiting the transmission of activin into the cell;
2) It did not bind to the intracellular domain of receptors for other cytokines and found that it did not affect their intracellular signaling. In addition, they have found that they bind to an activin intracellular signaling molecule via another WW domain and inhibit activin signaling into cells.
Further, the present inventors have found that various organs of mouse
(A) ⁺ RNA was extracted and SEQ ID NO: 2, SEQ ID NO: 3
Alternatively, the expression of the novel protein of the present invention was examined by Northern hybridization using the DNA shown in SEQ ID NO: 4 as a probe, and as shown in FIG.
In particular, it was found that its expression is frequently observed in the brain. This suggests that the activin-activin receptor signaling system to which the novel protein has been added is responsible for controlling the proliferation and differentiation of brain cells, and is useful for diagnosis and treatment of brain and nervous system diseases. I found something that could be applied. We have:
As a result of further study based on these findings, the present invention has been completed.

That is, the present invention provides: 1. a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5, or a salt thereof; 2. a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 6, or a salt thereof; A first which has a PDZ domain and a WW domain, is specifically expressed in the brain, and has a binding ability to an activin receptor and / or an activin intracellular signaling molecule;
3. the protein according to item 2 or 4; 4. The protein according to item 3, wherein the activin intracellular signaling molecule is Smad3; 5.5, which has five PDZ domains and two WW domains, is specifically expressed in the brain,
5. The protein according to item 1 or 2, which has an ability to bind to mad3. 6. a partial peptide of the protein according to item 1, a partial peptide of the protein according to item 2, or a salt thereof; A recombinant DN containing a DNA having a nucleotide sequence encoding the protein according to item 1 or the protein according to item 2.
A, 8. 8. The DNA according to item 7, which has a nucleotide sequence represented by SEQ ID NO: 7, a nucleotide sequence represented by SEQ ID NO: 8 or a nucleotide sequence that hybridizes with the nucleotide sequence under high stringency conditions, 9. A recombinant DNA containing a DNA having a nucleotide sequence encoding the partial peptide according to item 6, 10. A recombinant vector containing the DNA according to item 7, 11. A transformant carrying the recombinant vector according to item 10, 11. The protein according to item 1, wherein the transformant according to item 11 is cultured to produce and accumulate the protein according to item 1 or the protein according to item 2, and the collected protein is collected. 12. The method for producing the protein or a salt thereof according to the above, The protein according to item 1, the protein according to item 2,
13. an antibody against the partial peptide or a salt thereof according to the above item; A test solution containing the protein according to item 1, the protein according to item 2, the partial peptide according to item 6, or a salt thereof, and the labeled antibody according to item 1, with respect to the antibody according to item 13. 7. The protein according to item 1, wherein the protein according to item 1, the protein according to item 2, or the partial peptide according to item 6 or a salt thereof is reacted competitively.
Method for quantifying the partial peptide or a salt thereof according to the above,

[0006] 15. 7. The protein according to item 1, the protein according to item 2, or the protein according to item 6, wherein the protein according to item 1, the protein according to item 2, the partial peptide according to item 6, or a salt thereof is used. 15. A method for determining a protein that binds to a partial peptide or a salt thereof, A fusion between a gene encoding a test protein and a transcription activation region, with an expression vector obtained by fusing the protein according to item 1, the protein according to item 2, or the partial peptide according to item 6 to a DNA binding region of a transcription factor. Library and
The transcription factor binding region is introduced into a host cell having a reporter gene retained on a promoter, and the protein described in Item 1, the protein described in Item 2, or the partial peptide described in Item 6, and a test protein are used. Measuring a change in the expression level of a reporter gene increased by the binding;
16. The determination method according to item 5, 17. A protein or a salt thereof, which is obtained by the method of claim 15, which binds to the protein of claim 1, the protein of claim 2, the partial peptide of claim 6, or a salt thereof. The protein according to item 1, the protein according to item 2,
17. The protein according to item 1, the protein according to item 2, the partial peptide according to item 6, or a salt thereof, and the protein according to item 17 or a salt thereof. 18. a method for screening a compound or a salt thereof that inhibits or promotes binding to a salt, an activin receptor or an activin intracellular signaling molecule, Item 4. The labeled protein according to item 1, the protein according to item 2, the partial peptide according to item 6, or a salt thereof is contacted with an activin receptor or an activin intracellular signaling molecule, and the labeled item 1. The protein according to claim 1, wherein the protein according to item 2, the partial peptide according to item 6, or a salt thereof and a test compound are brought into contact with an activin receptor or an activin intracellular signaling molecule, Measuring the binding amount of the protein according to item 2, the partial peptide according to item 6, or a salt thereof to an activin receptor or an activin intracellular signaling molecule,
Inhibiting or promoting the binding of the protein according to item 1, the protein according to item 2, the partial peptide according to item 6, or a salt thereof to an activin receptor or an activin intracellular signaling molecule, which is compared. 19. a method for screening a compound or a salt thereof; The protein according to item 1, the protein according to item 2, the partial peptide according to item 6, or a salt thereof contacted with the protein according to item 17, or the labeled protein according to item 1. A protein according to item 1, a protein according to item 2, or a protein according to item 6, wherein the protein, protein or salt thereof according to item 2 and a test compound are brought into contact with the protein according to item 17 or salt thereof. 9. The protein according to claim 1, wherein the partial peptide or the salt thereof is bound to the protein or the salt thereof according to item 17, and the amount is compared. 20. A screening method for a compound or a salt thereof that inhibits or promotes the binding between the partial peptide or a salt thereof and the protein or a salt thereof according to Item 17. Item 17. The protein according to Item 1, the protein according to Item 2, the protein according to Item 2, the partial peptide according to Item 6, or a salt thereof to cells expressing the protein or salt thereof, activin receptor or activin intracellular signaling molecule according to item 17. The protein according to item 1, the protein according to item 2, the protein according to item 2, and the cell according to item 6, wherein the protein according to item 17 or a salt thereof, an activin receptor or an activin intracellular signaling molecule is expressed.
Item 17. The protein according to Item 1, the protein according to Item 2, the protein according to Item 2, or the partial peptide or salt thereof according to Item 17, wherein the partial peptide or the salt thereof and the test compound are introduced. The protein according to claim 1, wherein the amount of binding to a protein or a salt thereof, an activin receptor or an activin intracellular signaling molecule is measured and compared, and the protein according to claim 2, the protein according to claim 2, and the portion according to claim 6. 18. A method for screening a compound or a salt thereof that inhibits or promotes the binding of a peptide or a salt thereof to the protein or a salt thereof according to claim 17, an activin receptor or an activin intracellular signaling molecule,

22. A labeled protein according to item 1,
The protein according to item 6, the partial peptide according to item 6, or a salt thereof is contacted with the membrane fraction of a cell that has expressed the protein or salt thereof according to item 17, an activin receptor or an activin intracellular signaling molecule. Item 17. The labeled protein of item 1, the protein of item 2, the partial peptide of item 6, or a salt thereof and a test compound, wherein the protein or salt thereof of item 17, activin receptor or activin intracellular signal transmission. The labeled protein according to claim 1, which is in contact with a membrane fraction of a cell expressing the molecule,
3. The labeled protein according to item 1, wherein the amount of the protein according to item 6, the partial peptide according to item 6, or a salt thereof to the membrane fraction of the cell is measured and compared. 23. A method for screening a compound or a salt thereof that inhibits or promotes the binding of a protein, a partial peptide according to Item 6, or a salt thereof to the protein or salt thereof, an activin receptor or an activin intracellular signaling molecule according to Item 17, . Item 17. A protein according to item 1, a protein according to item 2, or a salt thereof is introduced into cells expressing the protein or salt thereof, an activin receptor or an activin intracellular signaling molecule according to item 17, and The protein according to item 1, the protein according to item 2, the protein according to item 2, the partial peptide according to item 6, or a salt thereof and a test compound, in a cell that has expressed the protein or salt thereof, the activin receptor or the activin intracellular signaling molecule according to claim 1. Seventeenth when introduced
The protein according to claim 1, wherein the protein or salt thereof, the activin receptor or the cell stimulating activity mediated by an activin intracellular signaling molecule is measured and compared. 23. A screening method for a compound or a salt thereof that inhibits or promotes the binding of the partial peptide or a salt thereof according to item 6 to the protein or a salt thereof, an activin receptor or an activin intracellular signaling molecule according to item 17, 25. The method for determining a protein according to item 16 or the screening method according to any one of items 18 to 23, wherein a two-hybrid method is used. The protein according to item 1, the protein according to item 2,
The protein according to claim 1, which comprises the partial peptide or the salt thereof according to claim 1, the protein according to claim 2,
26. A screening kit for a compound or a salt thereof that inhibits or promotes binding of the partial peptide or a salt thereof according to Item 6 to the protein or a salt thereof, an activin receptor or an activin intracellular signaling molecule according to Item 17, . Item 27. The protein according to Item 1, the protein according to Item 2, or the portion according to Item 6, which is obtained by using the screening method according to any one of Items 18 to 23 or the screening kit according to Item 25. A peptide or a salt thereof;
26. The protein or a salt thereof, a compound or a salt thereof which inhibits or promotes binding to the protein or a salt thereof, an activin receptor or an activin intracellular signaling molecule, 27. 27. a medicine comprising the protein according to item 17, the compound according to item 26 or a salt thereof, and No. 27 which is a prophylactic / therapeutic agent for Alzheimer's disease, Parkinson's disease, epilepsy or Huntington's chorea
3. The medicament according to the above item is provided.

[0008] More specifically, the present invention provides: 26. The protein according to Item 17 for a mammal, 26.
18. The prevention or treatment of a neuronal abnormality or a brain disease associated with the protein or a salt thereof, an activin receptor or an activin intracellular signaling molecule according to claim 17, wherein the compound or the salt thereof is administered in an effective amount. Method, 30. Item 17. The protein according to Item 17, for producing a prophylactic / therapeutic agent for a neuronal abnormality or brain disease associated with the protein according to Item 17 or a salt thereof, an activin receptor or an activin intracellular signaling molecule, Item 26. 30. Use of the compound of the above or a salt thereof; The protein is one or two or more (preferably two or more) in the amino acid sequence represented by SEQ ID NO: 5 or in the amino acid sequence represented by SEQ ID NO: 5 other than SEQ ID NO: 6; 1) in the amino acid sequence of SEQ ID NO: 5 except for the amino acid sequence of SEQ ID NO: 5
Or an amino acid sequence to which two or more (preferably two or more and ten or less) amino acids have been added or inserted, or one of the amino acid sequences represented by SEQ ID NO: 5 in the amino acid sequence other than SEQ ID NO: 6 Alternatively, a first amino acid sequence containing an amino acid sequence in which two or more (preferably two or more and ten or less) amino acids are substituted with another amino acid
32. the proteins and salts thereof described in the above item; 32. The two-hybrid method according to item 24, wherein yeast is used. 33. a compound or a salt thereof obtained by the screening method according to any one of items 19 to 23; 34. A pharmaceutical composition comprising the compound according to item 33 or a salt thereof, The exogenous DNA according to item 7, or its mutant DN
36. a non-human mammal having A; 36. The non-human mammal according to paragraph 35, wherein the non-human mammal is a rodent. No. 35 wherein the rodent is a mouse or a rat
38. The non-human mammal according to the above item, and The exogenous DNA according to item 7, or its mutant DN
A containing a recombinant vector that can be expressed in mammals.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 of the present invention include humans and warm-blooded animals (for example, Cells of guinea pigs, rats, mice, chickens, rabbits, pigs, sheep, cows, monkeys, etc. [eg, hepatocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermis 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, etc.), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, or stromal cells, or Progenitor cells of these cells, stem cells or cancerous cells, etc.] or any tissue in which those cells are present [eg, brain, each part of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus,
Thalamus, hypothalamus, cerebral cortex, medulla, cerebellum, etc.) spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine) Blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bones, joints, skeletal muscle, etc.] or blood cells or their cell lines (especially brain) ) Or a synthetic protein.

A protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 is defined as a protein having an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6. 50% or more, preferably about 70%
Above, more preferably about 90% or more, most preferably about 95% or more amino acid sequences having homology. The protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 of the present invention is represented by, for example, the aforementioned SEQ ID NO: 5 or SEQ ID NO: 6 A protein having substantially the same amino acid sequence as the amino acid sequence and having substantially the same physiological activity as the protein containing the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 is preferred. Examples of the substantially identical physiological activity include qualitative factors such as receptor affinity, signal transduction ability, and specificity of organ expression distribution. Substantially identical indicates that their bioactivity is biologically or physiologically equivalent. Therefore, the activities such as the strength of receptor affinity are equivalent (for example, about 0.1 to 20 times, preferably about 0.5 to 2 times).
However, the quantitative factors such as the intensity of these activities and the molecular weight of the protein may be different. For example, the measurement of receptor affinity can be performed according to a method known per se. For example, it can be measured according to a screening method described later. Examples of the protein of the present invention include, for example, SEQ ID NO: 5 or SEQ ID NO:
6 or more (preferably about 1 to 30, more preferably 1 to 10) in the amino acid sequence represented by 6.
One or more (preferably several) amino acids in the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6, in which one or more (preferably several) amino acids are deleted.
About 1 to 30, more preferably about 1 to 10,
More preferably, several or more amino acids are added or inserted, or one or more (preferably about 1 to 30, more preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 Also includes so-called muteins such as proteins containing an amino acid sequence in which about 1 to 10 (more preferably several) amino acids have been substituted with other amino acids, or an amino acid sequence obtained by combining them.

According to the protein used in the present specification, the left end is N-terminal (amino terminal) and the right end is C
Terminal (carboxyl terminal). The protein of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6, usually has a carboxyl group (-COOH) or a carboxylate (-
COO ⁻ ), but the C-terminal may be an amide (—CONH ₂ ) or an ester (—COOR). Here, as R in the ester, for example, methyl, ethyl,
C _1-6 alkyl groups such as n-propyl, isopropyl or n-butyl, for example, C _3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl, for example, C _6-12 aryl groups such as phenyl, α-naphthyl, for example And C _6-12 such as benzyl, phenethyl and α-naphthylmethyl
In addition to the aryl-C _1-2 alkyl group, pivaloyloxymethyl ester widely used as an oral ester and the like are used. When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes a carboxyl group amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Furthermore, the proteins of the present invention include those in which the amino group of the N-terminal methionine residue is protected with a protecting group (for example, a C _1-6 acyl group such as a formyl group or an acetyl group), N-terminal glutamic acid residue formed by pyroglutamination, on the side chain of an amino acid in the molecule, for example, OH, COOH, NH ₂ , S
H is protected by a suitable protecting group (for example, a C _1-6 acyl group such as a formyl group and an acetyl group);
Alternatively, a complex protein such as a so-called glycoprotein to which a sugar chain is bound is also included. As a specific example of the protein of the present invention, a protein containing the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 is used. The protein of the present invention has a PDZ domain or / and WW domain, preferably 5 PDZ domains or / and 2 WW domains, and is specifically expressed in the brain. The protein of the present invention binds to an activin receptor isolated as a serine / threonine kinase type receptor via a PDZ domain, and binds to an activin intracellular signaling molecule via a WW domain. The activin receptor may be any subtype of the activin receptor, but in particular the activin receptor IIA
-N and the like are preferably used. Activin intracellular signaling molecules include, for example, Smad1,2,3,
Smads such as 4,5,6,7 and the like can be mentioned, and the protein of the present invention binds particularly strongly to Smad3. In particular, the proteins of the present invention include five PDZ domains and two
A protein having two WW domains, specifically expressed in the brain, and capable of binding to the activin receptor or (and) Smad3 is preferably used.

The partial peptide of the protein of the present invention includes
A partial peptide of the protein of the present invention described above, the biological activity of the protein of the present invention, for example, receptor affinity,
Any substance may be used as long as it has activity such as signal information transmission ability, specificity of organ expression distribution, and the like. For example, 100 or more, preferably 250 or more, more preferably 35 or more of the constituent amino acid sequences of the protein of the present invention.
Having an amino acid sequence of 0 or more, more preferably 500 or more, and most preferably 800 or more,
Peptides having signal information transmission ability and the like are used. In the partial peptide of the present invention, one or more (preferably about 1 to 10, more preferably several) amino acids in the amino acid sequence are deleted, or 1 or 2 amino acids are added to the amino acid sequence. At least one (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably several) amino acids are added, or one or two or more amino acids (preferably To about 10, more preferably about several, and even more preferably about 1 to 5) amino acids may be substituted with another amino acid. The partial peptide of the present invention usually has a carboxyl group (—COOH) or a carboxylate (—COO ⁻ ) at the C-terminus, but the amide (—CON)
H ₂ ) or an ester (—COOR).
Further, the partial peptide of the present invention has a structure in which the amino group of the N-terminal methionine residue is protected with a protecting group, similarly to the above-described protein of the present invention, and is formed by cleavage of the N-terminal side in vivo. Also included are those in which the glutamyl group is pyroglutaminated, those in which the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, and those in which a sugar chain is bonded, such as a so-called glycopeptide. The partial peptide of the present invention preferably has the ability to bind to the activin receptor or / and the aforementioned activin intracellular signaling molecule (particularly, Smad3).

The salt of the protein of the present invention or its partial peptide is preferably a physiologically acceptable acid addition salt. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid). In addition, inorganic bases (eg, alkali metals such as sodium and potassium,
Salts with alkaline earth metals such as calcium and magnesium, aluminum and ammonium, and organic bases (eg, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, Dicyclohexylamine, N, N '
-Dibenzylethylenediamine, etc.). The protein of the present invention or a salt thereof can be produced from the aforementioned human or warm-blooded animal cells or tissues by a method known per se or by culturing a transformant containing a DNA encoding the protein described below. Can also be manufactured. Further, it can be produced according to the peptide synthesis method described later. When producing from human or mammalian tissues or cells, after homogenizing human or mammalian tissues or cells, extraction with an acid or the like is performed, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be isolated and purified.

For the synthesis of the protein of the present invention, its partial peptide, their salts or their amides, commercially available resins for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylphenyl. Acetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenylhydroxymethyl) phenoxy resin,
4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin.
Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various condensation methods known per se. At the end of the reaction, the protein is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein, its partial peptide or an amide thereof. For the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Carbodiimides include DC
C, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide and the like are used. Activation by these involves adding a protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt), or symmetrical acid anhydride or HOBt ester or HOOBT
The ester can be added to the resin after activation of the protected amino acid in advance. The solvent used for activating the protected amino acid or condensing 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, and dimethyl sulfoxide. Examples thereof include sulfoxides, ethers such as pyridine, 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 a protein bond formation reaction, and is usually about -20 ° C.
To 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protecting group for the amino group of the starting material include Z, Boc, tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, and the like are used. Carboxyl groups can be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, ter
linear, branched or cyclic alkyl esterification such as t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc., aralkyl esterification (for example, benzyl ester, 4-
Nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide and the like. . The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for the esterification, for example, a lower alkanoyl group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group, and the like are used. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a tert-butyl group. As the protecting group for the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tert-butyl and the like are used.
As a protecting group for imidazole of histidine, for example,
Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bu
m, Boc, Trt, Fmoc, etc. are used. Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-
Trichlorophenol, 2,4-dinitrophenol,
Cyanomethyl alcohol, paranitrophenol, HO
NB, N-hydroxysuccinimide, ester with HOBt)] and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.

The method for removing (eliminating) the protecting group includes, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or the like. Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, diisopropylethylamine, triethylamine,
Base treatment with piperidine, piperazine, or the like, reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above 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, dimethylsulfide, 1,1,2
It is effective to add a cationic scavenger such as 4-butanedithiol, 1,2-ethanedithiol and the like. In addition, 2,4 used as an imidazole protecting group of histidine is used.
The dinitrophenyl group is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol,
In addition to deprotection by an acid treatment in the presence of 4-butanedithiol or the like, it is also removed by an alkali treatment with a dilute sodium hydroxide solution, dilute ammonia or the like. The protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means. As another method for obtaining an amide form of a protein or a partial peptide thereof, for example, first, after amidating and protecting the α-carboxyl group of the carboxyl terminal amino acid, a peptide (protein) chain having a desired chain length is attached to the amino group side. After that, a protein (partial peptide) from which only the N-terminal α-amino group protecting group was removed from the peptide chain and a protein (partial peptide) from which only the C-terminal carboxyl group protecting group was removed were produced. The two proteins (partial peptides) are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by condensation, remove all protecting groups by the above method,
A desired crude protein (partial peptide) can be obtained.
The crude protein (partial peptide) is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein (partial peptide). To obtain an ester of a protein or a partial peptide thereof, for example, after condensing the α-carboxyl group of the carboxyl terminal amino acid with a desired alcohol to form an amino acid ester, in the same manner as the amide of a protein (partial peptide), An ester of the desired protein (partial peptide) can be obtained.

The partial peptide of the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving the protein of the present invention with an appropriate peptidase. As a peptide synthesis method, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion and, when the product has a protecting group, removing the protecting group. Known condensation methods and elimination of protecting groups include, for example, the following methods. M. Bodanszky and M.E. A. Ondett
i, Peptide Synth
esis), Interscience Publis
hers, New York (1966) Schroeder and Luebke, The Peptide, Academic Pr.
ess, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten The protein of the present invention or its partial peptide can be isolated and purified by a combination of purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the protein or its partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when the protein or a partial peptide thereof is obtained by a salt, the free form is obtained by a known method. Can be converted to

The DNA encoding the protein of the present invention may be any DNA containing the above-described nucleotide sequence encoding the protein of the present invention. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, isolated from the above-mentioned cDNA library derived from the cells / tissues by a method known per se, or synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, using a total RNA fraction or an mRNA fraction prepared from the cells / tissues described above,
ever Transcriptase Polym
erase Chain Reaction (hereinafter, RT)
-PCR method). Examples of the DNA encoding the protein of the present invention include a DNA containing the nucleotide sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8, or a nucleotide sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8. It has a nucleotide sequence that hybridizes under high stringent conditions and has the same physiological activity as a protein having the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6, such as receptor affinity,
Any DNA may be used as long as it encodes a protein having an activity such as signal signal transduction ability and specificity of organ expression distribution. Examples of a DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8 under high stringency conditions include, for example, SEQ ID NO:
7 or about 70% or more, preferably about 80% or more, more preferably about 90% or more of the nucleotide sequence represented by SEQ ID NO: 8.
% Or more, and most preferably about 95% or more. Hybridization is performed by a method known per se or a method analogous thereto, for example, molecular cloning (Mol
eco Cloning) 2nd (J. Samb)
Look et al. , Cold Spring Har
borLab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions. High stringency conditions include, for example, a sodium concentration of about 1
A condition of 9 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically,
The DNA encoding the protein having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 includes SEQ ID NO: 7
Or a DNA having the base sequence represented by SEQ ID NO: 8
Are used.

DNA encoding the partial peptide of the present invention
May be any as long as it contains a base sequence encoding the above-described partial peptide of the present invention. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, isolated from the above-mentioned cDNA library derived from the cells / tissues by a method known per se, or synthetic DNA.
Examples of the DNA encoding the partial peptide of the present invention include a DNA containing the nucleotide sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8, or the nucleotide sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8 Partial nucleotide sequence of a DNA encoding a protein having a nucleotide sequence that hybridizes under high stringent conditions with a protein having the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 And the like having DNA. The same hybridization method and high stringency conditions as described above are used.

DN encoding the protein of the present invention or a partial peptide thereof (hereinafter abbreviated as the protein of the present invention)
As a means for cloning A, DNA amplified by a PCR method using a synthetic DNA primer having a nucleotide sequence encoding a partial sequence of the protein of the present invention, or incorporated into an appropriate vector, genomic DNA,
A genomic DNA library, a cDNA derived from the above-described cell / tissue, or a DNA isolated from the above-described cell / tissue-derived cDNA library by a method known per se, a DNA encoding a part or the whole region of the protein of the present invention It can be isolated by hybridization with fragments or those labeled with synthetic DNA. Hybridization methods include, for example, molecular cloning (Molecular Cloning) 2
nd (J. Sambrook et. al., Cold
Spring Harbor Lab. Press, 19
89) and the like. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. Conversion of the base sequence of DNA can be performed by a known kit, for example, Mutan.
Gapped Duplex using t ^™ -G (Takara Shuzo Co., Ltd.), Mutant ^™ -K (Takara Shuzo Co., Ltd.) and the like.
The method can be carried out according to a method known per se, such as the method or the Kunkel method, or a method analogous thereto.

The cloned DNA encoding the protein of the present invention can be used as it is depending on the purpose, or may be used after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter. The expression vector of the protein of the present invention includes, for example,
(A) A target DNA fragment is cut out from a DNA encoding the protein of the present invention, and (b) the DNA fragment is ligated downstream of a promoter in an appropriate expression vector. As a vector, a plasmid derived from E. coli (eg, pBR322, pBR325, pBR325,
pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, and animal viruses such as retrovirus, vaccinia virus, and baculovirus. PA1-11, pXT1, pRc / CMV,
pRc / RSV, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as a host, SV40-derived promoters, retroviral promoters, metallothionein promoters, heat shock promoters, cytomegalovirus promoters, SRα promoters and the like can be mentioned. Among these, it is preferable to use a cytomegalovirus promoter, SRα promoter, or the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP
_When the host is a bacterium belonging to the genus Bacillus, the _L promoter, the lpp promoter, etc.
When the host is yeast, such as 2 promoter and penP promoter, pH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

[0022] In addition to the above, an expression vector optionally containing an enhancer, a splicing signal, a poly-A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like may be used. Can be used. As a selection marker, for example, dihydrofolate reductase (hereinafter, dhfr)
Gene [sometimes referred to as methotrexate (M
TX) resistance], an ampicillin resistance gene (hereinafter referred to as Amp
^r ), neomycin resistance gene (hereinafter sometimes Neo, G418 resistance)
And the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr ⁻ ) cells, the transformant containing the target gene can be selected using a thymidine-free medium. Further, if necessary, a signal sequence suitable for the host can be added to the N-terminal side of the protein of the present invention. When the host is Escherichia, an alkaline phosphatase signal sequence, an OmpA signal sequence, and the like are used. When the host is a Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, and the like are used. When the host is an animal cell, for example, insulin signal sequence, α-interferon signal sequence, antibody molecule, etc.
A signal sequence or the like can be used. Using the vector containing the DNA encoding the protein of the present invention thus constructed, a transformant can be produced.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include
Escherichia col
i) K12 DH1 [Procedings of the National Academy of Sciences of
The USA (Proc. Natl. Acad. S
ci. USA), 60, 160 (1968)], JM
103 [Nukuleic Acids Research, (Nucl
eic Acids Research), 9, 309
(1981), JA221 [Journal of Molecule Biology (Journal of Molecule)
color Biology)], 120, 517
(1978)], HB101 [Journal of Molecular Biology, 41, 459 (196)
9)], C600 [Genetics (Genetic
s), 39, 440 (1954)]. Examples of Bacillus spp. Include, for example, Bacillus Subtilis MI114.
[Gene, 24, 255 (1983)], 207-2
21 [Journal of Biochemistry (Jou
rnal of Biochemistry), 95 vol.
87 (1984)]. As yeast,
For example, Saccharomyces cerevisiae (Saccha
romyces cerevisiae) AH22, A
^{H22R -, NA87-11A, DKD-5D} , 20B
-12, Schizosaccharomyces pombe (Schizo
saccharomyces pombe) NCYC1
913, NCYC2036, Saccharomyces picj.
ia pastoris). As the insect cell, for example, when the virus is AcNPV, a cell line derived from a larva of night-moth (Spodoptera fr
uiperda cell; Sf cells), Trich
MG1 cells derived from the midgut of Oplusia ni, Tri
High Five derived from E. choplusia ni eggs
^{A TM} cell, a cell derived from Mamestra brassicae, a cell derived from Estigmena acrea, or the like is used. When the virus is BmNPV, silkworm-derived cell lines (Bombyx mori N cells; BmNV
Cells) are used. Examples of the Sf cell include Sf9 cell (ATCC CRL1711), Sf2
1 cell (above, Vaughn, JL, et al., In vivo, 13, 213-217, 197).
7) is used. As insects, for example, silkworm larvae are used [Maeda et al., Nature (Nat
ure), 315, 592 (1985)]. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO, and DHFR gene-deficient Chinese hamster cells CHO (dhfr ^- CHO).
Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.

In order to transform Escherichia, for example, Procesings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. US)
A), Vol. 69, 2110 (1972) and Gene (Gen)
e), Vol. 17, 107 (1982). For transformation of Bacillus sp., For example, Molecular & General Genetics (Molecular & General)
Genetics), Vol. 168, 111 (1979). To transform yeast, for example, Methods in Enzymology,
194, 182-187 (1991), Proceedings of the National Academy of Sciences of the USA (Proc. Na)
tl. Acad. Sci. USA) 75, 1929
(1978). To transform insect cells or insects, for example,
Bio / Technology (Bio / Technology)
y), 6, 47-55 (1988). To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha),
Virology, 52, 456
(1973).
Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.

When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for cultivation, and a liquid medium necessary for growth of the transformant is contained therein. A carbon source, a nitrogen source, an inorganic substance and the like are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. Also,
Yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8. As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller (Mille
r), Journal of Experiments in Molecular Genetics (Journal of
Experiments in Molecular G
enetics), 431-433, Cold Spr.
ing Harbor Laboratory, NewY
ork 1972]. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, culturing is usually carried out for about 15 minutes.
To 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring may be applied. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added. When culturing a transformant in which the host is yeast, for example, Burkholder (Burkholde) may be used as a medium.
r) Minimal medium [Bostian, K. et al. L. Et al., The Prossings of the National Academy of
Sciences of the USA (Proc.
Natl. Acad. Sci. USA), 77, 45.
05 (1980)] or S containing 0.5% casamino acid
D medium [Bitter, G .; A. La Prossings
Of the National Academy of Sciences of the USA (Proc. Natl.
Acad. Sci. USA), 81, 5330 (19)
84)]. Preferably, the pH of the medium is adjusted to about 5 to 8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and / or stirring are added.

When culturing an insect cell or a transformant whose host is an insect, Grace's In
Section Medium (Grace, TCCC, Nature, 195, 788 (196)
For example, those obtained by appropriately adding an additive such as 10% bovine serum immobilized in 2)) are used. Preferably, the pH of the medium is adjusted from about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and stirring are added as necessary. When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122
Volume, 501 (1952)], DMEM medium [Virology, Vol. 396 (195
9)], RPMI 1640 medium [Journal of
The American Medical Association (Jo
urnal of the American Medic
al Association) 199 vol. 519 (1
967)], 199 medium [Proceding of the.
Society for the Biological Medicine (Proceeding of the Society)
y for the Biological Medical
ne), Vol. 73, 1 (1950)].
Preferably, the pH is between about 6 and 8. Culture is usually about 3
Perform at 0 ° C to 40 ° C for about 15 to 60 hours, adding aeration and stirring as needed. As described above, the protein of the present invention can be produced in a culture medium or a transformant.

The protein of the present invention can be separated and purified from the above culture by, for example, the following method. When extracting the protein of the present invention from cultured cells or cells, after culturing, cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After disrupting the cells or cells by centrifugation, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (trade name). When the protein is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. Purification of the protein contained in the culture supernatant or extract obtained in this manner is performed by:
The separation and purification methods known per se can be appropriately combined and carried out. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis,
Specific methods such as ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis that mainly use differences in molecular weight, methods that use differences in charge such as ion-exchange chromatography, and affinity chromatography A method using affinity, a method using a difference in hydrophobicity such as reverse phase high performance liquid chromatography, a method using a difference in isoelectric point such as isoelectric focusing, and the like are used. When the protein thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se or a method analogous thereto By
It can be converted to a free form or other salts. The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The activity of the thus produced protein of the present invention or a salt thereof can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.

An antibody against the protein of the present invention, its partial peptide or a salt thereof may be an antibody capable of recognizing the protein of the present invention, its partial peptide or a salt thereof.
Any of a polyclonal antibody and a monoclonal antibody may be used. An antibody against the protein of the present invention, its partial peptide or a salt thereof (hereinafter, abbreviated as the protein of the present invention) can be produced by using the protein of the present invention as an antigen according to a known method for producing an antibody or antiserum. Can be. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a site capable of producing an antibody by being administered to a warm-blooded animal itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually 2 to 6
It is performed once every week, two to ten times in total. Examples of warm-blooded animals used include monkeys, rabbits, dogs,
Guinea pigs, mice, rats, sheep, goats, chickens and the like can be mentioned, and mice and rats are preferably used. When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having a recognized antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. By fusing the contained antibody-producing cells with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion procedure is known in the art, for example the method of Kohler and Milstein [Nature, 256, 495].
(1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include NS-1,
P3U1, SP2 / 0, AP-1 and the like,
P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably P
EG1000 to PEG6000) at a concentration of about 10 to 80%, and 20 to 40 ° C., preferably 30
By incubating at 1 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently. Various methods can be used for screening anti-protein antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (for example, a microplate) on which a protein antigen is directly or adsorbed together with a carrier, and then the radioactive substance is added. To detect anti-protein monoclonal antibody bound to solid phase by adding anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used when the cell used for cell fusion is mouse) or protein A A method of adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding the protein labeled with a radioactive substance or an enzyme, and detecting an anti-protein monoclonal antibody bound to the solid phase. Is mentioned. Selection of the anti-protein monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture Medium (SFM-10
1, Nissui Pharmaceutical Co., Ltd.) can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant is
The measurement can be performed in the same manner as the measurement of the anti-protein antibody titer in the antiserum.

(B) Purification of Monoclonal Antibody Separation and purification of the anti-protein monoclonal antibody can be performed by a method known per se, for example, a separation and purification method of immunoglobulin [for example,
Salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption / desorption method using ion exchanger (for example, DEAE), ultracentrifugation method, gel filtration method, antigen-bound solid phase or protein A or protein G, etc. Specific purification method in which an antibody alone is collected with the active adsorbent and the bond is dissociated to obtain the antibody].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced by a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (protein antigen) and a carrier protein is formed, a warm-blooded animal is immunized in the same manner as in the above-described monoclonal antibody production method, and an antibody-containing substance against the protein of the present invention is collected from the immunized animal. Then, the antibody can be produced by separating and purifying the antibody. Regarding the complex of the immunizing antigen and the carrier protein used to immunize a warm-blooded animal, the type of the carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by cross-linking the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are used in a weight ratio of about 0.1 to 20, preferably about 1 to 1, relative to hapten. A method of coupling at a rate of 5 to 5 is used.
For coupling the hapten and the carrier, various condensing agents can be used. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithiopyridyl group are used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually about 2 to 6
It is performed about once every week, about 3 to 10 times. The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The polyclonal antibody can be separated and purified according to the same method for separating and purifying immunoglobulins as in the above-described method for separating and purifying a monoclonal antibody.

The antisense DNA having a base sequence substantially complementary to the DNA or mRNA encoding the protein or partial peptide of the present invention includes the DNA or mRN encoding the protein or partial peptide of the present invention.
Any oligonucleotide or derivative thereof having a nucleotide sequence substantially complementary to the nucleotide sequence of A or a partial nucleotide sequence thereof and having an action of suppressing the expression of the protein or partial peptide may be used. It may be sense DNA. The nucleotide sequence substantially complementary to the DNA or mRNA is, for example, about 70% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA or mRNA (that is, the complementary strand of the DNA or mRNA). % Or more, preferably about 80% or more, more preferably about 90%
Above, more preferably, a base sequence having about 95% or more homology. In particular, of the entire base sequence of the complementary strand of the DNA or mRNA of the present invention, the complementary sequence to the complementary sequence of the base sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, the base sequence near the start codon). 70
% Or more, preferably about 80% or more, more preferably about 9%.
Antisense DNA having a homology of 0% or more, more preferably about 95% or more, is suitable. These antisense DNAs can be produced using a known DNA synthesizer or the like.

The protein of the present invention, a partial peptide thereof or a salt thereof (hereinafter, may be abbreviated as the protein of the present invention), a DNA encoding the protein of the present invention or a partial peptide thereof (hereinafter referred to as a DNA of the present invention) Abbreviated), antibodies against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) and antisense DNA
Is a method for determining a binding protein to the protein of the present invention,
Construction of recombinant protein expression system, two-hybr
Development of an assay system using the id method and screening of candidate compounds, implementation of drug design based on comparison with structurally similar ligands / receptors, use as a reagent in the preparation of probes and PCR primers in gene diagnosis, etc. And can be used as a drug for gene therapy and the like. In particular, two
Obtaining a binding protein to the protein of the present invention using the -hybrid method, and furthermore, a human or the like by a binding assay system using the protein of the present invention and an activin receptor, another obtained receptor or an activin intracellular signaling molecule. Can be screened for a promoter or inhibitor of a signal transduction system specific to a warm-blooded animal, and the promoter or inhibitor can be used as a prophylactic / therapeutic agent for various diseases and the like. Will be described.

(1) Method for Determining Binding Protein to Protein of the Present Invention The protein of the present invention is useful as a reagent for searching for or determining a binding protein to the protein of the present invention. That is, the present invention provides a method for determining a binding protein to the protein of the present invention, which comprises screening a binding protein interacting with the protein of the present invention. Specifically, the method for determining the binding protein of the present invention comprises a live fusion of a vector obtained by fusing a protein of the present invention to a DNA binding region of a transcription factor, a test protein and a transcription activation region on a host cell expression vector. Rally is introduced into a host cell having a reporter gene carrying the transcription factor binding region on a promoter, and interacts with the protein by a change in the expression level of the reporter gene which is increased by the binding of the two proteins. This is a method for determining a protein or a salt thereof. The method for determining a binding protein of the present invention is characterized by using a two-hybrid method for detecting an interaction between a protein of the present invention and a test protein by converting the interaction into expression of a specific reporter gene. A specific description of the method for determining a binding protein of the present invention will be given below. First, as the DNA encoding the protein of the present invention used in the method for determining a binding protein, a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 of the present invention is used. Any nucleotide may be used as long as it contains the nucleotide sequence encoding the nucleotide sequence encoding the partial sequence thereof. Genomic DNA of warm-blooded animals (eg, humans), genomic DNA library of warm-blooded animals (eg, humans), cDNA derived from tissues and cells of warm-blooded animals (eg, humans), warm-blooded animals It may be DNA isolated from a cDNA library derived from tissues (eg, humans) or cells by a method known per se, or synthetic or semi-synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. On the other hand, mRNA fractions can be isolated directly from tissues and cells by RT-PCR using a prepared mRNA fraction, or by partially synthesizing a partial base sequence and linking them. It can also be manufactured.

As the test protein library to be screened, cDNAs derived from various organs of various commercially available animals are used.
Library (MATCHMA manufactured by Clontech)
KER cDNA). Examples of vectors for expressing a fusion protein of a DNA binding region and a test protein include pAS2-1, pGBT9, pKAD-09, and pAS2-1.
pSD09, pEG202, pBTM116 and the like are used. Vectors expressing a fusion protein of a transcription factor activation region and a protein include pGAD424 and pAC
T2, pKT10Gal-VP, pJG4-5, pVP
16 or the like is used. As transcription factors, GAL4,
LexA, SRF and the like are used. As reporter genes, b-galactosidase gene (LacZ),
The histidine gene (HIS3), luciferase gene, chloramphenicol acetyltransferase gene and the like are used. As a host cell, yeast (Saccha)
romyces cerevisiae), Escherichia coli and the like, among which CG-1945, Y190, Y
187, HF7c, SFY526, L40, EGY4
8, HIS / L1, 62L yeast strain and the like are used. Specifically, a method for determining a protein that binds to the protein includes a DNA fragment containing a nucleotide sequence encoding the protein and a DNA fragment containing a nucleotide sequence encoding a DNA binding region on a plasmid (for example, pAS2-1). A plasmid bound to the same open frame as the fragment, and a plasmid (eg, pA
CT2), a cDNA library that binds to a DNA fragment containing a nucleotide sequence encoding the transcriptional activation region of a transcription factor (eg, GAL4) and is expressed in the form of a fusion protein is prepared, for example, by molecular cloning (Molec
ultra Cloning) 2nd (J. Sambro)
ok et.al., Cold Spring Harbo
r Lab. Press, 1989).

In order to introduce the above two plasmids into a host cell (eg, Saccharomyces cerevisiae Y190), they may be transformed at the same time, or one plasmid may be introduced first, and then the other plasmid may be introduced. May be successively introduced, for example, Methods in Enzymology,
194, 182-187 (1991), Proceedings of the National Academy of Sciences of the USA (Proc. Na)
tl. Acad. Sci. USA) 75, 1929
(1978). The expression can be detected by converting the expression of the reporter gene of the transformant, the amount of reporter enzyme coloring or fluorescent substance produced or the amount of luminescence, and the like. More specifically, when the host cell is yeast, the expression of the reporter gene (eg, β-galactosidase activity) is determined by a replica plate method or a filter method, preferably by a blue / white color screening using a filter method. Can be detected. Colonies colored (eg, blue) by the filter method can be used, for example, by the A Practical approach (A Practicing).
cal Approach) (Bartel, P.L.
et al. , Oxford University P
ress, Oxford; 153-179, 1993
A single colony of a positive clone can be isolated by treating according to the method described in a) or the like. From the isolated single transformant, for example, Gene, 57, 267 (1987),
Technics (BioTechniques), 14
Volume, 552 (1993), etc., plasmid DNA is recovered, and the receptor for the protein of the present invention can be determined by determining the nucleotide sequence of the DNA. Also, commercially available kits such as MATCHMA
KER GAL4 Two-Hybrid System
s (manufactured by Clontech) and the like can also be used to determine the binding protein to the protein of the present invention, and in that case, it can be performed according to the method described in the attached instruction manual.

(2) Agent for Prevention / Treatment of Protein Deficiency of the Present Invention In the above method (1), if a protein binding to the protein of the present invention is identified, the expression site of the protein and the activity of the protein via the binding protein are determined. The effects of the protein of the present invention can be clarified. Based on these findings, the DNA encoding the protein of the present invention can be used as an agent for preventing or treating a disease mediated by the binding protein. Further, since the protein of the present invention exhibits a binding activity to an activin receptor or an activin intracellular signaling molecule, it can be used as an agent for preventing or treating a disease mediated by an activin receptor or an activin intracellular signaling molecule. it can. For example, since the gene of the protein of the present invention is specifically expressed in the brain, patients with neuronal abnormalities or brain diseases associated with the binding protein, activin receptor or activin intracellular signaling molecule, etc. (A) administering the DNA encoding the protein of the present invention to the patient and expressing it, or (ii) inserting and expressing the DNA encoding the protein of the present invention in brain cells or the like, By transplanting the brain cells into the patient, the effect of the protein of the present invention on the brain cells of the patient can be sufficiently exerted. Therefore, the DNA encoding the protein of the present invention can be used as a safe and low-toxicity preventive / therapeutic agent for diseases mediated by the protein binding protein of the present invention, activin receptor or activin intracellular signaling molecule. . When the DNA of the present invention is used as the above-mentioned therapeutic agent, the DNA may be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, followed by a routine procedure. it can. For example, tablets, capsules, elixirs, microcapsules, and the like, optionally coated with sugar, aseptic, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections. For example, the DN of the present invention
A can be produced by mixing A with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, in the unit dosage form generally required for the practice of formulations. it can. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

Examples of additives which can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. And a lubricating agent such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, reddish oil or cherry, and the like. When the preparation unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. Suitable solubilizing agents, for example, You may use together with alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol), nonionic surfactant (for example, polysorbate 80 (brand name), HCO-50). Oily liquids include sesame oil and soybean oil,
You may use together with benzyl benzoate, benzyl alcohol, etc. as a solubilizing agent. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage Agents (eg, benzyl alcohol,
Phenol, etc.), antioxidants and the like.
The prepared injection is usually filled in a suitable ampoule. The preparations obtained in this way are safe and have low toxicity, so that, for example, warm-blooded mammals (eg, rat, rabbit,
Sheep, pigs, cattle, cats, dogs, monkeys, humans, etc.). The dose of the DNA varies depending on the symptoms and the like. However, in the case of oral administration, generally, in an adult (as 60 kg), the amount is about 0.5 mg / day.
1 to 100 mg, preferably about 1.0 to 50 mg,
More preferably, it is about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. For example, in the case of an injection, usually in an adult (as 60 kg),
About 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably 0.1 to 1 mg per day
It is convenient to administer about 0 mg by intravenous injection. In the case of other animals, the dose can be administered per 60 kg.

(3) A method for screening a compound that inhibits or promotes the binding between the protein of the present invention and its binding protein. The protein of the present invention or a salt thereof is used, or a recombinant binding protein expression system is constructed. Compounds that inhibit or promote the binding of the protein of the present invention to its binding protein by using a protein competitive binding assay system using the expression system (eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation production Or a salt thereof. Furthermore, in a transformant into which a DNA encoding the protein of the present invention has been introduced, a reporter gene expression system (t
Compounds (eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or compounds thereof that inhibit or promote the binding of the protein of the present invention to its binding protein can also be obtained by using the (wo-hybrid method). Salts can be screened. Such compounds include compounds having a cell stimulating activity (for example, an activity of promoting or suppressing growth promotion, phosphorylation of intracellular protein, etc.) via a binding protein (a so-called agonist).
And compounds having no cell stimulating activity (so-called antagonists). As the protein that binds to the protein of the present invention, the protein determined in (1) above, the activin receptor, the above-described activin intracellular signaling molecule (particularly, Smad such as Smad3) and the like are used. Hereinafter, these may be abbreviated as binding proteins to the protein of the present invention.

That is, the present invention relates to 1) the case where the protein of the present invention or a salt thereof is contacted with (i) the protein binding to the protein of the present invention, and (ii) the protein binding to the protein of the present invention. Or 2) (i) a transformant into which a protein encoding the protein of the present invention and a DNA encoding the protein binding to the protein of the present invention have been introduced; (Ii) a compound or a salt thereof that inhibits or promotes the binding of the protein of the present invention to a protein binding to the protein of the present invention, which is compared with the transformant when brought into contact with a test compound. A screening method is provided. In the screening method of the present invention, 1) (i) a case where the protein of the present invention or a salt thereof is brought into contact with a protein binding to the protein of the present invention; and (ii) a protein binding to the protein of the present invention. When the protein or a salt thereof and a test compound are brought into contact,
For example, the binding amount of the protein or a salt thereof to the binding protein of the protein of the present invention, the cell stimulating activity and the like are measured and compared, or 2) (i) binding of the protein of the present invention and the protein of the present invention It is characterized in that, for example, the degree of expression of a reporter gene based on the degree of coloration is compared between the transformant into which the DNA encoding the protein has been introduced and (ii) the transformant contacted with the test compound.

More specifically, the present invention relates to a case where a labeled protein of the present invention or a salt thereof is brought into contact with a protein binding to the protein of the present invention, and a case where a labeled protein of the present invention or a salt thereof and a test compound are tested. Is contacted with a protein binding to the protein of the present invention, the amount of binding of the labeled protein of the present invention or a salt thereof to the binding protein is measured and compared with the protein of the present invention or a salt thereof. A method for screening a compound or a salt thereof that inhibits or promotes the binding to the binding protein, wherein the binding protein to the protein of the present invention is a membrane-bound protein (eg, a transmembrane receptor, a channel, etc.)
The labeled protein of the present invention or a salt thereof was contacted with a cell containing a protein binding to the protein of the present invention or a membrane fraction of the cell, and the labeled protein of the present invention or a salt thereof and a test compound were compared with those of the present invention. The amount of the labeled protein of the present invention or a salt thereof bound to the cell or the membrane fraction when the cell is contacted with a cell containing the protein binding to the invention or a membrane fraction of the cell is measured and compared. A method for screening a compound or a salt thereof that inhibits or promotes the binding of the protein of the present invention or a salt thereof to the binding protein, and a method of binding a labeled protein of the present invention or a salt thereof to a protein of the present invention. A culture containing a transformant containing the DNA encoding was contacted with a binding protein expressed in cells by culturing the transformant. A labeled book obtained when the protein of the present invention or a salt thereof and a test compound are brought into contact with a binding protein expressed in cells by culturing a transformant containing a DNA encoding the binding protein to the protein of the present invention. A method for screening a compound or a salt thereof that inhibits or promotes the binding between the protein or a salt thereof and the binding protein, comprising measuring and comparing the amount of binding of the protein or a salt thereof to the binding protein. And a host cell into which a plasmid capable of expressing the gene of the protein of the present invention and a plasmid capable of expressing the gene of the protein binding to the protein of the present invention have been introduced, and the host when the host cell has been contacted with a test compound. Intracellular interaction of the protein of the present invention and a binding protein to the protein of the present invention Comparing the expression of a group of genes whose expression is more controlled or the physiological response (eg, secretion of FSH when the binding protein is an activin receptor) with the protein of the present invention or a salt thereof. A method for screening a compound or a salt thereof that inhibits or promotes the binding to the binding protein is provided.

The specific description of the screening method of the present invention is as follows. First, any binding protein used in the screening method of the present invention may be used as long as it contains the binding protein or a salt thereof, but an extract of a warm-blooded animal organ is preferred. . However, since a human-derived organ is particularly difficult to obtain, a binding protein or a salt thereof that has been expressed in a large amount using a gene recombination technique is suitable for screening. The above-mentioned method is used for producing the binding protein. For example, it can be carried out by expressing DNA encoding the protein in mammalian cells or insect cells. Complementary DNA is included in the DNA fragment encoding the target portion.
Is used, but the present invention is not limited thereto. For example, a gene fragment or a synthetic DNA may be used.
In order to introduce a DNA fragment encoding the binding protein into host animal cells and express them efficiently, the DN
Nuclear polyhedrosis virus (nuclear polyhedrosis) belonging to baculovirus using fragment A as an insect host
It is preferable to incorporate the gene into the downstream of a polyhedrin promoter of ossis virus (NPV), a promoter derived from SV40, a retrovirus promoter, a metallothionein promoter, a human heat shock promoter, a cytomegalovirus promoter, and an SRα promoter. The amount and quality of the expressed binding protein can be examined by a method known per se. For example, in the literature [Nambi. P. Et al., The Journal of Biological Chemistry (J. Biol. Che)
m. ), 267, 19555-19559, 199.
2 years]. Therefore, in the screening method of the present invention, the protein containing a binding protein or a salt thereof may be a binding protein or a salt thereof purified according to a method known per se, or may be obtained by using a cell containing the protein. Well,
When the protein is a membrane-bound protein, a membrane fraction of cells containing the protein may be used. In the screening method of the present invention, when using a cell containing a binding protein,
The cells may be fixed with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a method known per se.

The cell containing the binding protein refers to a host cell that has expressed the binding protein. Examples of the host cell include Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like. The membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. As a method for disrupting cells, Potter-
A method of crushing cells with an Elvehjem homogenizer, a Waring blender or a polytron (Kinell)
(manufactured by Matica Inc.), crushing by ultrasonic waves, crushing by ejecting cells from a thin nozzle while applying pressure by a French press or the like. For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is centrifuged at a low speed (500 to 3000 rpm) for a short time (typically about 1 to 10 minutes), and the supernatant is further centrifuged at a high speed (15000 to 30000 rpm) for usually 30 minutes to 2 hours. This is the membrane fraction. The membrane fraction is rich in the expressed binding protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the binding protein in cells or membrane fractions containing the binding protein is preferably from 10 ² to 10 ⁸ molecules per cell, and more preferably from 10 ⁵ to 10 ⁷ molecules per cell. The higher the expression level, the higher the binding activity (specific activity) with the protein of the present invention per cell extract or membrane fraction, which not only enables the construction of a highly sensitive screening system, but also allows the same lot to be used. A large amount of sample can be measured. In order to screen for a compound that inhibits the binding between the protein of the present invention and the binding protein to the protein of the present invention, a suitable binding protein fraction and a labeled protein of the present invention are required to carry out the above-mentioned steps. . The binding protein fraction is preferably a natural binding protein fraction or a recombinant binding protein fraction having an activity equivalent thereto. Here, the equivalent activity indicates an equivalent binding activity to the protein of the present invention.

As the labeled protein of the present invention, a labeled protein of the present invention, a labeled protein analog compound of the present invention and the like are used. For example, [ ³ H], [ ¹²⁵ I], [
Labeled proteins of the present invention, such as [ ¹⁴ C] and [ ¹³⁵ S], can be used. Specifically, to screen for a compound that inhibits the binding between the protein of the present invention and a binding protein to the protein of the present invention, first, cells containing the binding protein or a membrane fraction of the cells are subjected to screening. Prepare a binding protein preparation by suspending it in a suitable buffer. The buffer has a pH of 4 to 10
Any buffer may be used as long as it does not inhibit the binding of the protein of the present invention and the protein binding to the protein of the present invention, such as a phosphate buffer (preferably pH 6 to 8) and a Tris-HCl buffer. For the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 (trade name) (manufactured by Kao-Atlas Co., Ltd.), digitonin, and deoxycholate can be added to the buffer. Furthermore, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories), pepstatin and the like can be added for the purpose of suppressing the degradation of the binding protein and the protein of the present invention by the protease. 0.0
To 1 to 10 ml of the binding protein solution, add a fixed amount (500
(0 to 500,000 cpm) of the labeled protein of the present invention is added, and at the same time, 10 ^-4 to 10 ^-10 M test compound is allowed to coexist. A reaction tube containing a large excess of the unlabeled protein of the present invention is also prepared to determine the non-specific binding amount (NSB). The reaction is carried out at 0 to 50 ° C, preferably 4 to 37 ° C.
C. for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. When the count (B0-NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B0) when there is no antagonistic substance is 100%, the specific binding amount (B-NSB) is, for example, 50% or less. Can be selected as a candidate substance having competitive inhibitory ability, while the specific binding amount (B-NSB) is, for example, 150
% Of the test compound can be selected as a candidate compound having a binding promoting ability.

In order to carry out the above-mentioned method for screening a compound which inhibits or promotes the binding between the protein of the present invention and a protein binding to the protein of the present invention, a plasmid expressing the gene of the protein of the present invention and a plasmid of the present invention are used. Expression of a gene of the present invention and a group of genes whose expression is controlled by the interaction of the protein of the present invention and the binding protein to the protein of the present invention in a host cell into which a plasmid for expressing the gene of the protein binding to the protein of the present invention has been introduced, or Physiological responses (eg, FSH secretion when the binding protein is an activin receptor) can be measured using known methods. Specifically, when the host cell is yeast, first, a plasmid that allows the yeast cell to express a protein obtained by fusing the protein of the present invention with the DNA binding region of a transcription control factor, a binding protein to the protein of the present invention, and transcription control A transformant into which a plasmid capable of expressing a protein fused with an activation region of a factor has been introduced is prepared in the same manner as described above. In performing screening, the transformant is cultured at 30 ° C. for 2 to 4 days on an agar medium containing 10 ⁻⁴ to 10 ⁻¹⁰ M test compound.
As agar medium, tryptophan / leucine / histidine-deficient SD medium, tryptophan / leucine-deficient SD medium
A medium or the like is used. Then, in order to detect the expression of the reporter gene as the color of the colony due to the β-galactosidase activity, the expression is detected using a filter method or the like. Detection was performed on the filter to which the transformant was attached,
Whatman # 5 filter or VWR grade 410 moistened with buffer / X-gal (Clontech)
After placing the filter and incubating at 30 ° C. for 30 minutes to 8 hours, the color of the colonies on the filter is compared with the degree of color development with the transformant as a control containing no test compound. At this time, the test compound added to the culture medium of the colony showing a darker color as a candidate compound having a binding promoting ability compared to the control, and the test compound added to the culture medium of the colony showing a lighter color The compound can be selected as a candidate compound having the ability to inhibit binding. Alternatively, the expression of the reporter gene can be quantified as β-galactosidase activity by measuring the amount of o-nitrophenol generated by decomposition of the substrate. First, the transformant is cultured with shaking at 30 ° C. for 8 to 24 hours on a liquid medium containing 10 ⁻⁴ to 10 ⁻¹⁰ M test compound. As a liquid medium, tryptophan / leucine / histidine-deficient SD medium, tryptophan / leucine-deficient SD medium, and the like are used. Preferably, after culturing overnight, a part of the culture solution is inoculated into YPD medium, and cultured with shaking at 30 ° C. for 3 to 5 hours so that OD ₆₀₀ is 0.5 to 1.0. An o-nitrophenylgalactoside solution (Sigma) is added to the Z buffer / β-mercaptoethanol mixture of the residue obtained by centrifuging a part of the culture solution and reacted. The reaction is carried out at 0 to 50 ° C. for 3 minutes to 24 hours, desirably at 30 ° C. for 30 minutes to 15 hours, and the absorbance at 420 nm (OD ₄₂₀ ) of the yellow-colored supernatant is measured. β-galactosidase activity is calculated from the obtained absorbance (OD ₄₂₀ ) as a Miller unit using the following formula.

By adding a test compound to the medium,
When the β-galactosidase activity value increases by about 10% or more, preferably about 20% or more, more preferably about 30% or more, and most preferably about 50% or more, the test compound is compared with the protein of the present invention and the protein of the present invention. Can be selected as a candidate compound capable of promoting the binding to a binding protein. On the other hand, by adding the test compound to the medium, the β-galactosidase activity value is about 10% or more, preferably about 20% or more, more preferably about 30% or more.
Most preferably, when the decrease is about 50% or more, the test compound can be selected as a candidate compound capable of inhibiting the binding between the protein of the present invention and a binding protein to the protein of the present invention. In order to perform screening by measuring the expression activity of the reporter gene, a DNA encoding a binding protein to the protein of the present invention is required. The DNA encoding the binding protein to the protein of the present invention is preferably a DNA containing the DNA or a DNA that hybridizes with the DNA under high stringent conditions. Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.

A kit for screening a compound or a salt thereof which inhibits or promotes the binding of the protein of the present invention to a protein binding to the protein of the present invention comprises a protein of the present invention or a salt thereof, a partial peptide thereof or a salt thereof, Cells containing a binding protein to the protein of the present invention, an extract fraction of cells containing a binding protein to the protein of the present invention,
Alternatively, a plasmid that expresses a protein in which the protein of the present invention is fused with the DNA binding region of the transcription control factor and a plasmid that expresses a protein in which the protein binding to the protein of the present invention is fused with the activation region of the transcription control factor are introduced. It contains the transformed transformant. Examples of the screening kit of the present invention include the following. 1. Screening Reagent Transformant Plasmid Expressing a Protein Fused with the Protein of the Present Invention and the DNA Binding Region of Transcription Regulator and Activin IIA
A yeast Y190 strain transformed with a -N receptor protein and a plasmid that expresses a protein obtained by fusing the activation region of a transcriptional regulator was transformed. Liquid culture medium Tryptophan / leucine / histidine-deficient SD medium:
After sterilizing an aqueous solution of yeast nitrogen source (Difco) in an autoclave, L-isoleucine, L-valine, L-adenine hemisulfate, L-arginine hydrochloride, L-lysine hydrochloride, L-methionine, L-phenylalanine, L- Add the dropout solution sterilized in an autoclave consisting of threonine, L-tyrosine and L-uracil and stored at 4 ° C. Further, a 40% dextrose / stock solution (Sigma) sterilized by filtration through a filter was added, and 2%
And stored at 4 ° C., or prepared at the time of use. YPD medium: After sterilizing an aqueous solution of the yeast extract in Difco peptone with an autoclave, adding 40% dextrose filtered and sterilized with a filter to adjust the final concentration to 2%, and storing at 4 ° C or preparing at the time of use. Good. Buffer Z _{buffer: Na 2 HPO 4 · 7H 2} O, NaH 2 PO 4 · H 2
After containing O, KCl, and MgSO ₄ .7H ₂ O and adjusting the pH to about 7, it may be sterilized in an autoclave and stored at 4 ° C., or may be prepared at the time of use. Z buffer / β-mercaptoethanol: Z buffer 100
To which β-mercaptoethanol was added at a rate of 0.27. β-galactosidase substrate o-nitrophenylgalactoside solution (Sigma)
Is dissolved in Z buffer and adjusted to a concentration of 4 mg / ml before use. Stop solution Use 1M sodium carbonate solution stored at 4 ° C, or
Alternatively, it may be prepared at the time of use.

2. Assay Method The yeast transformant is shake-cultured overnight at 30 ° C. in 1 ml of SD medium lacking tryptophan / leucine to which 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M is added. 0.4 ml of the culture solution is added to 1.6 ml of YPD medium, and cultured with shaking at 30 ° C. for 3 to 5 hours so that the OD ₆₀₀ is 0.5 to 1.0. 0.3 ml of the culture is centrifuged at 14000 rpm for 30 seconds, the residue is suspended in 0.3 ml of Z buffer, and after centrifugation again, the precipitate is suspended in 0.1 ml of Z buffer. After temporarily freezing in liquid nitrogen, thaw at 37 ° C. for 30 seconds to 1 minute. 0.7 ml of a Z buffer / β-mercaptoethanol mixture and 0.16 ml of o-nitrophenylgalactoside solution are added, and the mixture is kept warm at 30 ° C. until the solution turns yellow. Add. Centrifuge at 14000 rpm for 10 minutes.
The absorbance at 0 nm is measured, and the β-galactosidase activity is determined by the following formula (Equation 1) as a Miller unit.

[Equation 1] β-galactosidase activity = 1000 × OD ₄₂₀ / (t
× V × OD ₆₀₀ ) OD420: Absorbance at 420 nm t: Reaction time (minutes) V: Amount of suspension of transformant in Z buffer used for reaction ×
Dilution ratio

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a protein of the present invention (in particular, having five PDZ domains or (and) two WW domains, and particularly expressed in the brain). And a compound that inhibits or promotes the binding of an activin receptor or (and / or a protein that binds to an activin intracellular signaling molecule) to a protein that binds to the protein of the present invention (hereinafter, a promoting compound). Compounds that inhibit the binding of the protein of the present invention to the binding protein to the protein of the present invention include a compound that binds to the protein of the present invention by binding to the binding protein to the protein of the present invention. By binding to a compound or a salt thereof (so-called agonist), which itself has cell stimulating activity via a binding protein, and a binding protein to the protein of the present invention, thereby binding the protein of the present invention to the protein of the present invention. A compound or a salt thereof (a so-called antagonist) that inhibits binding to a protein but does not itself have cell-stimulating activity via the binding protein;
A compound that inhibits the binding between the protein of the present invention and the binding protein to the protein of the present invention without binding to the protein binding to the protein of the present invention (hereinafter abbreviated as inhibitory compound)
Or a salt thereof. The presence or absence of the binding property between the compound obtained by using the screening method or the screening kit of the present invention or a salt thereof and the binding protein to the protein of the present invention can be confirmed according to the above-described method for measuring the binding activity. The cell stimulating activity via the binding protein can be measured according to a method known per se or a method analogous thereto.

Compounds obtained using the screening method or screening kit of the present invention include peptides, proteins, non-peptidic compounds, synthetic compounds,
Fermentation products and the like may be mentioned, and these compounds may be novel compounds or known compounds. Since the agonist and the promoting compound have the same action as the biological activity of the protein of the present invention or have the action of enhancing the biological activity, a drug that is safe and low toxic depending on the protein activity The composition is particularly useful as a prophylactic / therapeutic agent for neuronal abnormalities or brain diseases associated with the binding protein or activin receptor (eg, Alzheimer's disease, Parkinson's disease, epilepsy, Huntington's chorea, etc.). Conversely, the antagonist or inhibitory compound can suppress the physiological activity of the protein of the present invention, so that it is a safe and low toxic pharmaceutical composition that suppresses the protein activity, particularly the binding protein or activin receptor. It is useful as a prophylactic / therapeutic agent for related neuronal abnormalities or brain diseases (eg, Alzheimer's disease, Parkinson's disease, epilepsy, Huntington's chorea, etc.).

When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method. For example, tablets or capsules coated with sugar, capsules, elixirs, microcapsules, or the like, or sterile solution or suspension with water or other pharmaceutically acceptable liquids, if necessary. It can be used parenterally in the form of injections such as liquids. For example, the compound or a salt thereof is mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dosage form required for generally accepted pharmaceutical practice. Can be manufactured. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained. tablet,
Examples of additives that can be mixed with capsules include binders such as gelatin, corn starch, tragacanth, and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, and alginic acid. And lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry. When the preparation unit form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Examples of aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.)
And a suitable solubilizing agent such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 ^™ , HCO-5).
0) and the like. As an oily liquid, for example,
Sesame oil, soybean oil, and the like are used, and may be used in combination with benzyl benzoate, benzyl alcohol, or the like as a solubilizing agent. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage Agents (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, it can be administered to mammals (for example, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, humans, etc.). . The dose of the compound or a salt thereof varies depending on the symptoms and the like.
(As Kg) usually about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose depends on the subject, target organ,
Although it depends on the administration method of the symptoms and the like, for example, in the form of an injection, it is usually about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 30 mg per day for an adult (as 60 kg). Is about 0.1
It is convenient to administer from about 10 mg to about 10 mg by intravenous injection. In the case of animals other than humans, an amount equivalent to 60 kg can be administered.

(4) Quantification of the Protein of the Present Invention, Partial Peptides or Salts Thereof Since the protein antibody of the present invention can specifically recognize the protein or the like of the present invention, it can be used in a test solution. Can be used for quantification of proteins and the like, particularly quantification by sandwich immunoassay. That is, the present invention provides (i) an antibody that reacts with the protein or the like of the present invention, and a test solution and a labeled protein or the like of the present invention competitively reacted with each other to bind to the labeled antibody or the like. A method for quantifying the protein or the like of the present invention in a test solution, which comprises measuring the proportion of the protein or the like of the present invention; (ii) the antibody of the present invention insolubilized on the test solution and a carrier; A method for quantifying the protein or the like of the present invention in a test solution, comprising reacting the antibody of the present invention with the antibody of the present invention simultaneously or continuously, and then measuring the activity of the labeling agent on the insolubilized carrier. Is an antibody that recognizes the N-terminal or C-terminal of the protein of the present invention, and the other antibody is an antibody that reacts with the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. The present invention provides a method for quantifying the protein or the like of the present invention therein. In addition to measuring the protein of the present invention using a monoclonal antibody that recognizes the protein or the like of the present invention (hereinafter sometimes referred to as anti-protein antibody), detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, and F (ab ′) ² , Fab ′ or Fab of the antibody molecule may be used.
Fractions may be used. The method for measuring the protein or the like of the present invention using the antibody of the present invention is not particularly limited,
The amount of an antibody, an antigen or an antibody-antigen complex corresponding to the amount of the antigen (for example, the amount of the protein of the present invention) in the liquid to be measured is detected by chemical or physical means, and this is detected as a standard solution containing a known amount of the antigen. Any measurement method may be used as long as it is a measurement method calculated from a standard curve prepared by using. For example,
Nephrometry, a competitive method, an immunometric method, and a sandwich method are suitably used, but the sandwich method described below is particularly preferable in terms of sensitivity and specificity.

The labeling agent used in the measurement method using a labeling substance includes a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C], etc., and as the above enzyme, those having a stable and high specific activity are preferable. -Glucosidase,
Alkaline phosphatase, peroxidase, malate dehydrogenase and the like, fluorescent substances include fluorescamine and fluorescein isothiocyanate, and luminescent substances include luminol, luminol derivatives, luciferin, lucigenin and the like. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent. For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and 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; and glass. In the sandwich method, the test solution is reacted with the insolubilized anti-protein antibody (primary reaction), and further reacted with the labeled anti-protein antibody (secondary reaction), and then the activity of the labeling agent on the insolubilized carrier is measured. By doing so, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may. In the method for measuring the protein or the like of the present invention by the sandwich method of the present invention, an anti-protein antibody used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the protein or the like of the present invention. That is, the antibody used in the primary reaction and the secondary reaction is used in the primary reaction, for example, when the antibody used in the secondary reaction recognizes the C-terminal or N-terminal of the protein or the like of the present invention. As the antibody, an antibody that recognizes the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 is preferably used.

An antibody against the protein or the like of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry. In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody, or A solid-phase method using a soluble first antibody and a solid-phased antibody as the second antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. Is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated.
Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution. In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephelometry utilizing laser scattering is preferably used.

In applying these individual immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measuring system of the protein etc. of the present invention by adding the ordinary technical considerations of those skilled in the art to the ordinary conditions operating method in each method. For details of these general technical means, it is possible to refer to reviews, books, and the like [for example, "Radio Immunoassay" edited by Hiro Irie (published by Kodansha, 1974), "Radio Immunoassay" edited by Hiro Irie. (Kodansha, published in 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. (Medical Publishing, published in 1978), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. (2nd edition) (Medical Publishing, Showa 57) Eiji Ishikawa et al. “Enzyme immunoassay” (3rd edition) (Medical Publishing, published in 1987) “Me
articles in Enzymology, Vol. 70
(ImmunochemicalTechniques
(Part A)), ibid., Vol. 73 (Immuno
chemical Technologies (Part
B)), ibid., Vol. 74 (Immunochemical)
al Technologies (Part C), ibid. V
ol. 84 (Immunochemical Tech)
niques (Selected Immunoass
ays (Part D)), ibid., Vol. 92 (Imm
unchemical Chemicals (Mo
noclonal Antibodies and Gen
eral Immunoassay Methods (P
art E)), ibid., Vol. 121 (Immunoc
chemical Technologies (Hybrid
oma Technology and Monoclo
nal Antibodies (Part I)) (see Academic Press). As described above, the protein or the like of the present invention can be quantified with high sensitivity by using an antibody against the protein or the like of the present invention. Furthermore, by quantifying the concentration of the protein or the like of the present invention using an antibody against the protein or the like of the present invention, for example, a disease associated with the protein or the like of the present invention can be diagnosed. Further, the antibody of the present invention can be used for detecting the protein of the present invention or the like present in a subject such as a body fluid or a tissue. Further, preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction during purification, analysis of the behavior of the protein of the present invention in the test cells, etc. Can be used for

(5) Gene Diagnostic Agent The DNA of the present invention can be used, for example, as a probe to produce a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow,
Gene, which encodes the protein of the present invention or a partial peptide thereof in horses, cats, dogs, monkeys, chimpanzees, etc.).
It is useful as a gene diagnostic agent for A mutation or abnormal accumulation or decrease of mRNA. D of the present invention
The above-described genetic diagnosis using NA can be performed, for example, by Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, 874) known per se.
879 (1989), The Prossings of
The National Academy of Sciences
Proceedings of
he National Academy of Scie
nces of the United States o
f America), Vol. 86, 2766-2770
(1989)).

(6) DN containing antisense DNA
A. An antisense DNA capable of binding to a DNA or mRNA encoding the protein or the like of the present invention complementarily and suppressing transcription or translation of the mRNA suppresses abnormal expression of a gene encoding the protein or the like of the present invention. can do. Therefore, the antisense DNA can be used, for example, as an agent for preventing or treating a disease caused by abnormal expression of a gene encoding the protein or the like of the present invention. When the antisense DNA is used as the above-mentioned prophylactic / therapeutic agent, it can be produced in the same manner as the above-mentioned drug containing the DNA of the present invention. For example, the antisense D
NA can be administered to humans or warm-blooded animals alone or after being inserted into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus-associated virus vector, or the like, in a conventional manner. The antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting eating, and can be administered using a gene gun or a catheter such as a hydrogel catheter. Further, the antisense DNA can be used for the DN of the present invention in tissues and cells.
It can also be used as a diagnostic oligonucleotide probe for examining the presence of A and its expression status.

(7) Preparation of DNA-Transferred Animal The present invention relates to a DNA encoding an exogenous protein of the present invention.
(Hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). That is, the present invention relates to (i) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof, and (ii) the non-human mammal according to (i), wherein the non-human mammal is a rodent. (Iii) the non-human mammal according to (ii), wherein the rodent is a mouse; (iv) the non-human mammal according to (ii), wherein the rodent is a rat; It is intended to provide a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in mammals. Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including sperm and their progenitor cells, and the like. Preferably, at the stage of embryonic development in non-human mammal development (more preferably, at the stage of single cells or fertilized eggs and generally before the 8-cell stage), calcium phosphate method, electric pulse method, lipofection method, agglutination method, It can be produced by transferring a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. Further, by the DNA transfer method, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a cell fusion method known per se.

As the non-human mammal, for example, rat, mouse, guinea pig, hamster, rabbit, sheep, pig, cow, cat, dog and the like are used. Above all, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, C57BL as a pure strain,
/ 6 line, DBA2 line, etc.
F ₁ system, BDF ₁ system, B6D2F ₁ system, BALB /
c strain, ICR strain, etc.) or rat (eg, Wi
and the like are preferable. "Mammals" in a recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals. The exogenous DNA of the present invention is not a DNA of the present invention originally possessed by a non-human mammal, but a DN encoding a protein of the present invention once isolated and extracted from a mammal.
A. As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA or the like is used, and also includes abnormal DNA. The abnormal DNA means a DNA containing a gene that expresses an abnormal protein of the present invention. For example, a DNA that contains a gene that expresses a protein that suppresses the function of the normal protein of the present invention is used. . The exogenous DNA of the present invention may be derived from a mammal which is the same or different from the animal of interest. In transferring the DNA encoding the protein of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when transferring the DNA of the present invention, DNAs derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto are expressed. DNA that highly expresses the DNA of the present invention by microinjecting a DNA construct (eg, a vector or the like) having the DNA of the present invention downstream of various promoters into a fertilized egg of a target mammal, for example, a mouse fertilized egg Metastatic mammals can be created.

Examples of the vector carrying the construct include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as phage λ, a retrovirus such as Moloney leukemia virus, a vaccinia virus or a baculovirus. Animal viruses and the like are used. Among them, a plasmid derived from Escherichia coli or a plasmid derived from yeast is preferably used. Examples of the promoter for controlling the DNA expression include, for example, viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus,
Promoters derived from JC virus, breast cancer virus, poliovirus, etc., various mammals (human, rabbit,
Dogs, cats, guinea pigs, hamsters, rats, mice, etc.) include albumin, insulin I
I, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase,
Platelet-derived growth factor β, keratin K1, K10 and K
14. Collagen type I and type II, cyclic AMP
-Dependent protein kinase βI subunit, dystrophin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine triphosphate oxidase (Na, K-ATPas)
e), 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, polypeptide chain elongation factor 1α (EF-1α), β
Actin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin basic protein, thioglobulin, Thy
1, promoters such as immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A, vasopressin and the like are used. Capable of cytomegalovirus promoter, human polypeptide chain elongation factor 1α
(EF-1α) promoter, human and chicken β
An actin promoter or the like can be used.

The above-described vector preferably has a sequence (generally called a terminator) that terminates transcription of a target mRNA in a DNA-transferred mammal. A sequence can be used, and preferably, a simian virus SV40 terminator or the like is used. In addition, a splicing signal of each DNA, an enhancer region,
It is also possible to link a part of an intron of a eukaryotic DNA 5 ′ upstream of the promoter region, between the promoter region and the translation region, or 3 ′ downstream of the translation region. The normal translation region of the protein of the present invention includes genomic DNA derived from liver, kidney, thyroid cells, fibroblasts derived from various mammals (eg, rabbit, dog, cat, guinea pig, hamster, rat, mouse, human, etc.). All or part of genomic DNA from various commercially available genomic DNA libraries, or liver, kidney, thyroid cells,
It can be obtained by a method known per se using a complementary DNA prepared from a fibroblast-derived RNA by a known method as a raw material. In addition, the exogenous abnormal DNA can be obtained from the above-described cells or tissues that have developed a disease caused by mutation of the protein of the present invention. In addition, a translation region obtained by mutating a translation region of a normal protein obtained from the above cells or tissues by a point mutagenesis method can be prepared. The translation region is a DNA construct that can be expressed in a transgenic animal, and is a normal DN that is ligated downstream of the above-mentioned promoter and optionally upstream of a transcription termination site.
A It can be produced by an engineering technique. Transfer of the exogenous 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 target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germ cells and somatic cells. means. The offspring of such animals that have inherited the exogenous DNA of the present invention have the DNA of the present invention in all of their germinal and somatic cells.

The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as a DNA-bearing animal after confirming that the non-human mammal stably retains the DNA by mating. it can. Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germinal and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all of the offspring of the produced animal have the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means The progeny of this type of animal that has inherited the exogenous DNA of the present invention has the exogenous DN of the present invention in all of its germinal and somatic cells.
A in excess. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess. Normal DNA of the present invention
Non-human mammals having the expression of the normal DNA of the present invention are highly expressed, and may eventually develop hyperactivity of the protein of the present invention by promoting the function of endogenous normal DNA. Can be used as a disease model animal. For example, using the normal DNA transgenic animal of the present invention, it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible. In addition, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the protein of the present invention, it can be used for a screening test for a therapeutic drug for a disease associated with the protein of the present invention. On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Further, the desired exogenous DNA can be incorporated into the above-mentioned plasmid and used as a raw material. DN with promoter
The A construct can be prepared by a general genetic engineering technique. The transfer of the abnormal 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 target mammal. Abnormal DNA of the present invention in germ cells of a produced animal after DNA transfer
Means that the offspring of the produced animal have the abnormal DNA of the present invention in all of its germinal and somatic cells. The progeny of such animals that have inherited the DNA will have the abnormal DNA of the invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed so that all offspring have the DNA.

In the non-human mammal having the abnormal DNA of the present invention, the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is ultimately reduced by inhibiting the function of endogenous normal DNA. Inactive refractory disease may occur, and it can be used as a disease state model animal.
For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease. In addition, specific applicability includes the abnormal DNA of the present invention.
The highly expressing animal serves as a model for elucidating the function inhibition (dominant negative action) of the normal protein by the abnormal protein of the present invention in the function inactive refractory of the protein of the present invention. In addition, since the mammal into which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the protein of the present invention, it can be used for a therapeutic drug screening test for inactive refractory of the protein of the present invention. . Other possible uses of the two DNA-transferred animals of the present invention include, for example, use as a cell source for tissue culture, and direct analysis of mRNA in the tissue of the DNA-transferred mammal of the present invention. Analyzing the expressed protein tissue for analysis of the relationship with the protein specifically expressed or activated by the protein of the present invention, to enhance or suppress the function of the cell by using the cell described above. Screening of various drugs, isolation and purification of the mutant protein of the present invention, and production of its antibody are considered. Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention. More detailed pathological findings in each organ of the relevant disease model were obtained, development of new treatment methods,
Furthermore, it can contribute to research and treatment of secondary diseases caused by the disease. In addition, it is possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a protease such as trypsin to obtain the released DNA-transferred cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to examine the specificity of the protein-producing cell of the present invention, its relationship with differentiation or proliferation, or its signal transduction mechanism, and its abnormalities. It is an effective research material. Furthermore, in order to develop a therapeutic agent for a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, using the DNA-transferred animal of the present invention, It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease by using a method or the like. Further, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a gene therapy method for a disease associated with the protein of the present invention.

In the present specification and drawings, when bases, amino acids and the like are represented by abbreviations, IUPAC-IUB
Commission on Biochemical N
This is based on the abbreviation by omenclature or the abbreviation commonly used in the art, examples of which are described below. When amino acids may have optical isomers,
Unless otherwise specified, L-form is shown. 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 triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate EIA: Enzyme immunoassay Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Serin: Isoleucine Serine Cys: cysteine Met: methionine Glu: glutamic acid Asp: aspartic acid Lys: lysine Arg: al Nin His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGl: Pyroglutamic acid Me: Methyl group Et: Ethyl group Bu: ThiR phenyl group PhC: ThiP phenyl group -Carboxamide group

The 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-dicarbodiimide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] ba used for two-hybrid method
1 shows the nucleotide sequence of a DNA fragment encoding the intracellular domain of the activin IIA-N receptor protein as an it plasmid. [SEQ ID NO: 2] Shows the base sequence of a DNA fragment as a probe used for Northern hybridization. [SEQ ID NO: 3] This shows the base sequence of DNA fragment as a probe used for Northern hybridization. [SEQ ID NO: 4] This shows the base sequence of DNA fragment as a probe used for Northern hybridization. [SEQ ID NO: 5] This shows the amino acid sequence of the protein of the present invention. [SEQ ID NO: 6] This shows the amino acid sequence of the protein of the present invention. [SEQ ID NO: 7] This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 5 of the present invention. [SEQ ID NO: 8] This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 6 of the present invention. The transformant Escherichia coli DH5α / p obtained in Example 1 described later.
BSYN3-6 has been deposited with the National Institute of Advanced Industrial Science and Technology (NIBH) on December 2, 1998 under the deposit number FERM BP-6592 on November 18, 1998. (IFO) and deposit number IFO 16221.

[0067]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described more specifically below with reference to examples, but the present invention is not limited thereto. In addition, two-hybrid using yeast
The operation method of the d method is a commercially available kit (manufactured by Clontech)
According to the method described in the manual attached thereto, and the genetic manipulation method using Escherichia coli, the method described in Molecular cloning (Molecular cloning) was followed.

[0068]

【Example】

Example 1 Cloning of cDNA encoding the protein of the present invention (1) Activin IIA- by two-hybrid method
Screening for Proteins Interacting with the N Receptor Protein Activin IIA-N
For screening for a protein that interacts with the receptor protein, MATCHMAKER ^™ Two-Hy is used as a kit.
brid System 2 (Cat. No. K160)
4-1: Clontech) was used. According to the above method, the EcoRI of the DNA binding vector pAS2-1
The site and the BamHI site were cleaved with appropriate restriction enzymes, treated with alkaline phosphatase and purified. afterwards,
A DNA fragment encoding the intracellular domain of the activin IIA-N receptor protein represented by SEQ ID NO: 1 is ligated, and the desired GAL4 DNA binding domain and the entire intracellular domain of the activin IIA-N receptor are used as a fusion protein. Expression plasmid (pAS-IIA
-N). As the GAL4 transcription activation region fusion library plasmid, commercially available mouse brain MATCHMA
KER cDNA library (Clontech
Was used. A single colony of yeast strain Y190 (2 to 3 mm in diameter) was inoculated into 20 ml of tryptophan-deficient SD medium and cultured with shaking at 30 ° C. for 18 hours. 10 ml of this culture was added to 300 ml of YPD medium and OD ₆₀₀ =
The cells were planted at 0.2 to 0.3 and cultured with shaking at 30 ° C. for 3 hours. Transfer the culture to a sterile centrifuge tube, 1000x
and centrifuged at room temperature for 5 minutes at room temperature. Discard the supernatant and remove the cells 25m
The suspension was suspended in 1 liter of sterilized water and centrifuged again at 1,000 × g for 5 minutes at room temperature. The supernatant was discarded, and the cells were added to 1.5 ml of TE buffer (0.01 M Tris-HCl, 1 mM EDTA,
pH 7.5) /0.1 M lithium acetate solution (pH 7.5)
The resultant was suspended in the mixed solution of 5) and used for the next transformation. 10 μg of the previously prepared plasmid (pAS-IIA-N) and 2
mg herring testis carrier DNA (Clontech)
Was added to 1 ml of the cell suspension prepared above and mixed well. Further, 6 ml of PEG (40% PEG 40
A mixture of (00) / TE buffer / 0.1 M lithium acetate solution was added and mixed with a vortex mixer. 30 ° C,
After shaking at 200 rpm for 30 minutes, 700 μl of DMSO
(Final concentration 10%) and gently stirred. Thereafter, the mixture was heated at 42 ° C. for 15 minutes while shaking occasionally, and the container was ice-cooled and centrifuged at 1,000 × g for 5 minutes. Discard the supernatant,
The cells were suspended in 0.5 ml of TE buffer. 100 μl of the obtained cell suspension was spread on a tryptophan-deficient SD medium and cultured at 30 ° C. for 4 days to obtain a strain stably retaining the plasmid. Subsequently, a GAL4 transcription activation region fusion library plasmid was also introduced into the yeast strain in the same manner. 0.2 ml of the resulting transformant was spread on a tryptophan / leucine / histidine-deficient SD medium and cultured at 30 ° C. for 8 days, and then His ⁺ colonies were streaked on a tryptophan / leucine / histidine-deficient agar plate.

In a Petri dish, 5 ml of a Z buffer (Na ₂ HP) was added.
_{O 4 · 7H 2 O, NaH} 2 PO 4 · H 2 O, KCl, MgS
O ₄ · 7H ₂ O, the pH 7) / X-gal solution (5-bromo-4-chloro-3-indolyl -β-D- 2% DMF solution galactoside) / beta-mercaptoethanol mixture placed and sterilized The Whatman # 5 filter was wetted. After placing another filter on the agar plate with the transformant colony, remove this filter,
The colonies were frozen with liquid nitrogen facing up. The filter was taken out of liquid nitrogen, thawed at room temperature, and then placed on the wetted filter, colony side up.
The petri dish was closed and incubated at 30 ° C. for 1 hour to isolate about 100 positive blue colonies. Each of the obtained positive clones was inoculated in 3 ml of a leucine-deficient SD liquid medium, and cultured for 2 days. Then, this culture solution was diluted 10,000 times, spread on a leucine-deficient SD plate, and kept at 30 ° C. for 3 days. Twenty to thirty colonies were picked with a sterile toothpick and replicated on tryptophan / leucine deficient SD plates and leucine deficient SD plates. Here, colonies showing tryptophan auxotrophy were selected from the above positive colonies, their β-galactosidase activity was assayed, and two colonies showing no activity were selected. The obtained true positive colonies were inoculated in 2 ml of a YPD liquid medium and cultured at 30 ° C. overnight. The culture was centrifuged at room temperature for 5 seconds, the supernatant was discarded, and 0.2 ml of a yeast lysis solution (2% Triton X-10) was removed.
0.1% SDS, 100 mM NaCl, 10 mM Tr
Is-HCl (pH 8.0), 1 mM EDTA) was added and suspended. 0.2 ml of phenol / chloroform / isoamyl alcohol (25: 24: 1) and acid washed glass beads were added and stirred with a vortex mixer for 2 minutes. After centrifugation at 14000 rpm for 5 minutes at room temperature, 1/10 volume of a 3 M sodium acetate solution (pH 5.2) and 2.5 volumes of ethanol were added to the separated supernatant. After washing the obtained precipitate with 70% ethanol,
Dissolved in 20 μl of sterile water. After 1 μl of this was introduced into E. coli HB101 by electroporation, the plasmid DNA was purified by the usual miniprep method to obtain the desired cDNA (YN3). further,
Full-length cDNA containing the entire coding region of the base sequence of YN3
In order to obtain, the full length insert of the obtained YN3 was labeled with ³² P by a random prime method. Using this labeled YN3 as a probe, mouse brain Lambd
a cDNA library (Lambda ZAPII
Vector: Stratagene) was screened by normal plaque hybridization. Using the full length insert of the obtained clone as a probe, the same screening was repeated to obtain a clone which is considered to have the longest full length insert including the entire coding region of the protein of the present invention. One of the obtained positive phages,
Phagemid ((pBluescript SK) (-) vector) according to the method of Stratagene
And its base sequence was determined. The full-length cDNA (YN3-6) of the protein of the present invention is 5156 bp, and is a polypeptide composed of 1161 amino acids represented by SEQ ID NO: 5 [FIGS. 1 to 6] or 1 represented by SEQ ID NO: 6.
A polypeptide consisting of 112 amino acids [FIGS.
2]. SEQ ID NO: 5 or SEQ ID NO:
Plasmid pBS containing a full-length cDNA (YN3-6) encoding a polypeptide consisting of the amino acid represented by No. 6
YN3-6 was transformed into Escherichia coli DH5α to obtain a transformant: Escherichia coli DH5α / pBSYN3-6.

[0070]

Example 2 Poly (A) ⁺ R derived from various organs of mouse
Detection of Expression by Northern Hybridization Method Using NA The DNA fragments represented by SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 in the YN3-6 insert were
IG-PCR probe synthesis kit (Boehring
er) and labeled with digoxigenin and used as a probe. In addition, brain, liver, spleen, embryo, kidney, heart, testis, ovary, and skeletal muscle were excised from Balb / c mouse,
Tot with RIzol reagent (GIBCO BRL)
alRNA was extracted. After that, PolyAT tra
ct mRNA Isolation System (P
poly (A) ⁺ RNA was purified using Romega). Using 1 μg of each poly (A) ⁺ RNA, 1% agarose gel electrophoresis was performed by a formalin gel method, and a blotting device (Amersham-P) was used.
HybondN (Amersham-Ph) by a vacuum blotting method using H.A.
armacya). This was mixed with a hybridization buffer (5 × SSC, 0.1% N-laur) containing 5 ng / ml of each of the previously prepared probe DNAs.
oylsarcosine, 0.02% SDS, 0.5
% Blocking reagent (Boehrin
ger), 100 μg / ml salmon testis DNA)
Incubated at 65 ° C. overnight. Thereafter, the plate was washed three times with 0.1 × SSC and 0.1% SDS at 65 ° C. for 20 minutes. Further, a solution (0. 1) containing an alkaline phosphatase-labeled anti-digoxigenin antibody (Boehringer).
1 M Tris-HCl pH 7.5, 0.15 M Na
Cl, 150 mU / ml antibody) and 0.1
M Tris-HCl (pH 7.5), 0.15 M N
Washed three times for 15 minutes with aCl and 0.1% Tween 20. Finally, Lumi-Phos 530
(Wako Pure Chemical Industries, Ltd.) as a substrate for chemiluminescence.
As a result of exposure to X-ray film and detection, it was confirmed that the protein of the present invention was particularly highly expressed in the brain [FIG.
3].

[0071]

Example 3 Interaction between ARIP1 and Smad3 Full-length Smad fused to GAL4 DNA binding domain
Smad in a mammalian two-hybrid system in CHO cells transduced with the protein of the present invention (ARIP1) fused to 3DNA and the VP16 activation domain
The specific interaction with ARIP1 with 3 was examined. DNA constructs for mammalian two-hybrid screening were prepared as follows. That is, GAL4
The plasmid pBIND was used for expression of the fusion protein of the DNA binding domain and Smad3, and the full-length cDNA encoding human Smad3 was ligated to pBIND to prepare pBIND-Smad3. In addition, plasmid pACT was used for expression of a fusion protein of the VP16 activation domain and ARIP1. An AR prepared by ligating a fragment obtained by PCR to a fragment prepared from plasmid pBS-ARIP1-short having a partial base sequence (1187 to 4446) of full-length cDNA encoding ARIP1
The partial nucleotide sequence of the full-length cDNA encoding IP1 (92
3 to 4446) were subcloned into pACT, and pACT-ARIP1
Was prepared. For mammalian two-hybrid assays,
CheckMateMammalian as a kit
The test was performed using Two-Hybrid System (Promega) according to the protocol. Plasmids pBIND-Smad3 and pACT-A obtained above
RIP1, βg derived from cytomegalovirus promoter
al (CMV-βgal) and a reporter plasmid pG5luc which induces a luciferase gene under the control of a GAL4 responsive promoter were introduced into CHO cells. The strength of the interaction between Smad3 and ARIP1 in CHO cells was determined by using the activity of luciferase expressed by the luciferase gene as an index. The luciferase activity was determined by a method known per se (Endocrinology) as in the case of β-galactosidase activity.
y), 136, 5493-5503 (1995)). As a result, the protein of the present invention (ARIP1) was found to be Smad in mammalian cells in the same manner as activin receptor IIA in mammalian cells.
A direct interaction with 3 was shown [FIG. 14].

[0072]

The protein of the present invention, its partial peptide or a salt thereof (hereinafter abbreviated as the protein of the present invention), a DNA encoding the protein of the present invention, an antibody against the protein of the present invention, and an antisense DNA are described in Determination of a binding protein to the protein of the invention, construction of a recombinant protein expression system, binding assay system using the expression system, and two-
Development of an assay system using the hybrid method and screening of candidate compounds, implementation of drug design based on comparison with structurally similar ligands / receptors, use as a reagent in the preparation of probes and PCR primers in gene diagnosis, etc. Can also
It can be used as a drug for gene therapy and the like. In particular, elucidation of the structure and properties of the protein of the present invention will lead to the development of unique drugs acting on these systems.

[0073]

[Sequence List] [SEQUENCE LISTING] <110> Takeda Chemical Industries, Ltd. <120> Novel Protein And Its Use <130> A99246 <150> JP 10-323199 <151> 1998-11-13 <150> JP 10- 346925 <151> 1998-12-07 <160> 8 <210> 1 <211> 1466 <212> DNA <213> Mouse <400> 1 AGACATCACA AGATGGCCTA CCCTCCTGTA CTTGTTCCTA CTCAACACGC CTTTCATATA 60 ATGATAGAGG ACCCAGGACC ACCCCCACCT TCCCCATTAC TAGGGTTGAA GCCATTGAGGATGATGAGCGATGAGTAGAGCCATAGAGAG GGTTGTGTCT GGAAAGCCCA GTTGCTCAAT 180 GAATATGTGG CTGTCAAAAT ATTTCCAATA CAGGACAAAC AGTCCTGGCA GAATGAATAT 240 GAAGTCTATA GTCTACCTGG AATGAAGCAT GAGAACATAC TACAGTTCAT TGGTGCAGAG 300 AAAAGAGGCA CCAGTGTGGA TGTGGACCTG TGGCTAATCA CAGCATTTCA TGAAAAGGGC 360 TCACTGTCAG ACTTTCTTAA GGCTAATGTG GTCTCTTGGA ATGAACTTTG TCATATTGCA 420 GAAACCATGG CTAGAGGATT GGCATATTTA CATGAGGATA TACCTGGCTT AAAAGATGGC 480 CACAAGCCTG CAATCTCTCA CAGGGACATC AAAAGTAAAA ATGTGCTGTT GAAAAACAAT 540 CTGACAGCTT GCATTGCTGA CTTTGGGTTG GCCTTAAAGT TCGAGGCTGG CAAGTCTGCA 600 GGTGACACCC ATGGGCAGGT TGGTACCCGG AGGTATATGG CTCCAGAGGT GTTGGAGGGT 660 GCTATAAACT TCCAAAGGGA CGCATTTCTG AGGATAGATA TGTACGCCAT GGGATTAGTC 720 CTATGGGAAT TGGCTTCTCG TTGCACTGCT GCAGATGGAC CCGTAGATGA GTACATGTTA 780 CCATTTGAGG AAGAAATTGG CCAGCATCCA TCTCTTGAAG ATATGCAGGA AGTTGTTGTG 840 CATAAAAAAA AGAGGCCTGT TTTAAGAGAT TATTGGCAGA AACATGCAGG AATGGCAATG 900 CTCTGTGAAA CGATAGAAGA ATGTTGGGAT CATGATGCAG AAGCCAGGTT ATCAGCTGGA 960 TGTGTAGGTG AAAGAATTAC TCAGATGCAA AGACTAACAA ATATCATTAC TACAGAGGAC 1020 ATTGTAACAG TGGTCACAAT GGTGACAAAT GTTGACTTTC CTCCCAAAGA ATCTAGTCTA 1080 TGATGGTGGC ACCGTCTGTA CACACTGAGG ACTGGGACTC TGAACTGGAG CTGCTAAGCT 1140 AAGGAAAGTG CTTAGTTGAT TTTCTGTGTG AAATGAGTAG GATGCCTCCA GGACATGTAC 1200 GCAAGCAGCC CCTTGTGGAA AGCATGGATC TGGGAGATGG ATCTGGGAAA CTTACTGCAT 1260 CGTCTGCAGC ACAGATATGA AGAGGAGTCT AAGGGAAAAG CTGCAAACTG TAAAGAACTT 1320 CTGAAAATGT ACTCGAAGAA TGTGGCCCTC TCCAAATCAA GGATCTTTTG GACCTGGCTA 1380 ATCAAGTATT TGCAAAACTG ACATCAGATT TCTTAATGTC TGTCAGAAGA CACTAATTCC 1440 TTAAATGAAC TACTGCTATT TTTTTT 1466 <210> 2 <211> 1391 <212> DNA <213> Mouse <400> 2 TCGCCGCCAC GACGCGCCCA GCACCTCCGA GCGACTGACC GACCTCCACG CGCGTCCCGA 60 ACACACTGCC ACCGCCGCCG CCGCCGCGCG CGCTCGCGCC GCACTCCCTC GCACGTCACC 120 ACGTGCGCTG CCGCCAACGC CTCCCGGCCG CTTCCGGCTC TGATGCCTGA GCGAATCACA 180 GGCGAGCTCC CGGGAAGATC CCGCTCTGAG GCTCCGCCCC CGGACAGGGC CCCGCCCACC 240 TCATAGCTCT TTTCCTCAGC CGCCCCCTCC TTCCTTCTCG GCTCAACTAG GTCAGCGCAA 300 GGTGATCCCG GAGAGCGGGG CGGCGGGGAC CGCTCCTCCT GTTACTTATC GAGCGCGCGC 360 TCCCTCCCGA GCCTCACACC CTCGCTTCGC CCTTTTTTTT CCACTGTCCA GGAACTGGTT 420 CCCTCCTTCC TCTTCCACCT GCCCTACCTT CTCCAGAGAT CCGACGTGGC GATTAGAGTT 480 CTCAGCGTCA CACTGACTTC TAGGCAACTA GCCTAGACTG GAGCTGCGTG TTGTGGGAAC 540 CCCGCGGCAG TAGTTGAGCA TCAGGCTCTT ACCTTGGAGG TGGAGGGGTG AGAAGAATAG 600 AGGAAGAAGG GATAAGTCAG AGGAGGGCCT GAACAACTAG CCCCTCTATT GGCCTGCTTT 660 GGGTGAGCAT TCAGTGAGTG TGTTTAAAAA AAAAAAGGGA GGGAAAACAA AAGACCTCAG 720 GAGCAGTTTT GTGTTGCTGT GTCTGGCTTC AAGAAGAAAA TTCTAGACAT TTATGCCGGC 780 AAGACCAAAG CTCAGCTAAG ACTACTTCTC CCAAGAAGAT AATTGTATCA GAGGATGGGT 840 TGGATCAGTA CAGGTGGTTT GAGGAGACGC TGACAGAGGA CCATGGAAAG GTGGGAGAGG 900 ACGCGCGGCT CCTGGGCTTC CTCTGAGCTC AGCTCCAGGC ACCACAAGGC CACATAAGGA 960 GGGTGAGGTC CCTGGAGTGG ACTACATTTT CATAACCGTT GAGGAGTTTA TGGAATTGGA 1020 GAAAAGTGGT GCTCTCCTAG AAAGCGGGAC CTATGAAGAC AACTACTACG GTACCCCGAA 1080 GCCTCCAGCT GAACCAGCAC CATTATTAAA TGTAACAGAC CAGATACTTC CGGGAGCTAC 1140 TCCAAGTGCT GAGGGGAAGC GGAAAAGAAA TAAGTCAGTG ACCAACATGG AGAAAGCAAG 1200 TATAGAGCCT CCAGAGGAGG AAGAAGAAGA AAGGCCTGTA GTCAATGGAA ACGGCGTGGT 1260 CATAACCCCA GAATCCAGTG AACATGAAGA CAAAAGTGCA GGTGCCTCAG GGGAGACACC 1320 CTCCCAGCCT TACCCTGCAC CCGTGTACAG CCAGCCCGAA GAGCTCAAGG ACCAGATGGA 1380 CGATACAAAG C 1391 <210> 3 <211> 1431 <212> DNA <213> Mouse <400> 3 CAGTTGAAGG GAACGTTCCT CAGCACCACC CTCAAAAAGA GCAACATGGG CTTTGGGTTT 60 ACCATAATTG GTGGAGACGA GCCGGATGAG TTTCTACAGG TGAAAAGTGT GATCCCGGAT 120 GGGCCTGCCG CACAGGATGG GAAAATGGAG ACAGGTGATG TCATTGTCTA TATTAATGAA 180 GTTTGTGTCC TTGGACACAC TCATGCAGAT GTTGTCAAAC TTTTCCAGTC TGTTCCTATT 240 GGTCAGAGTG TCAACTTGGT GTTGTGTCGT GGCTACCCTT TGCCCTTTGA CCCTGAAGAT 300 CCTGCTAACA GCATGGTGCC ACCCCTTGCA ATAATGGAGA GGCCACCTCC GGTGATGGTC 360 AATGGAAGAC ATAACTATGA AACATACTTG GAATACATTT CTCGGACCTC ACAGTCGGTC 420 CCAGATATTA CAGACCGGCC ACCTCATTCT TTGCACTCCA TGCCAGCTGA CGGCCAGCTA 480 GATGGCACGT ATCCACCACC CGTCCATGAC GACAATGTGT CTATGGCTTC GTCTGGAGCC 540 ACTCAAGCTG AACTTATGAC CTTAACCATT GTGAAAGGTG CCCAGGGATT TGGCTTTACT 600 ATTGCCGACA GTCCCACGGG ACAGCGGGTG AAACAAATCC TTGACATTCA GGGATGCCCT 660 GGGCTGTGTG AAGGAGACCT CATTGTTGAG ATCAACCAAC AGAATGTACA GAACCTGAGC 720 CATACAGAAG TAGTGGATAT ACTTAAGGAC TGCCCCGTTG GAAGTGAGAC TTCTTTAATC 780 ATCCATCGAG GAGGTTTCTT TTCTCCATGG AAAACTCCAA AGCCTATGAT GGACCGATGG 840 GAGAACCAAG GCAGTCCACA AACAAGTTTA TCTGCTCCGG CCGTCCCACA GAACCTGCCC 900 TTCCCACCTG CCCTTCACAG GAGCTCCTTT CCTGATTCAA CAGAGGCCTT TGACCCACGG 960 AAGCCTGACC CATATGAGCT CTACGAGAAA TCGAGAGCCA TTTATGAAAG TAGGCAACAA 1020 GTGCCACCCA GGACCAGTTT TCGAATGGAT TCCTCTGGTC CAGATTATAA GGAACTGGAT 1080 GTTCACCTTC GGAGGATGGA GTCTGGATTT GGCTTTAGAA TCCTTGGGGG AGATGA ACCT 1140 GGACAGCCTA TTTTGATCGG AGCCGTCATT GCCATGGGCT CAGCTGACAG AGACGGCCGT 1200 CTACACCCAG GAGATGAGCT TGTCTATGTC GATGGGATCC CAGTGGCTGG CAAGACCCAC 1260 CGCTATGTCA TCGACCTCAT GCACCACGCG GCCCGCAATG GGCAGGTTAA CCTCACTGTG 1320 AGAAGAAAGG TGCTATGTGG AGGGGAGCCC TGCCCAGAGA ATGGGAGGAG TCCAGGCTCT 1380 GTATCAACTC ACCACAGCTC TCCGCGCAGT GACTATGCCA CCTACTCCAA C 1431 <210> 4 <211> 1085 <212> DNA < 213> Mouse <400> 4 ACCATAACTG TGCCCCATAA AATTGGACGA ATCATTGATG GGAGCCCTGC AGATCGCTGT 60 GCCAAACTCA AAGTGGGCGA CCGTATCTTA GCAGTCAACG GCCAGTCTAT CATCAACATG 120 CCTCACGCTG ACATTGTGAA GCTCATCAAG GACGCCGGTC TCAGTGTCAC CCTTCGCATC 180 ATTCCTCAGG AGGAGCTCAA CAGCCCAACA TCAGCACCCA GTTCAGAGAA ACAGAGCCCC 240 ATGGCCCAGC AGCACAGCCC TCTGGCCCAG CAGAGTCCTC TGGCCCAGCC AAGCCCCGCC 300 ACCCCCAACA GCCCAGTCGC ACAGCCAGCT CCTCCCCAAC CTCTCCAGCT GCAAGGACAC 360 GAAAATAGTT ACAGGTCAGA AGTTAAAGCG AGGCAAGATG TGAAGCCAGA CATCCGGCAG 420 CCTCCCTTCA CAGACTACAG GCAGCCCCCG CTGGACTACA GGCAGCCCCC GGGAGGAGAC 480 TACTCACAGC CCCCACCCTT GGACTACAGG CAGCACTC TC CAGACACCAG GCAGTACCCT 540 CTGTCAGACT ACAGGCAGCC ACAGGATTTT GATTATTTCA CTGTGGACAT GGAGAAAGGA 600 GCCAAAGGAT TTGGATTCAG CATTCGTGGA GGAAGGGAAT ACAAGATGGA TCTGTATGTG 660 TTGAGATTGG CAGAGGATGG GCCAGCCATA AGGAACGGCA GGATGAGGGT AGGAGATCAG 720 ATCATTGAAA TAAATGGGGA AAGCACACGA GACATGACCC ACGCCAGAGC AATAGAACTC 780 ATCAAGTCTG GAGGAAGAAG AGTGCGGCTG CTGCTGAAGA GAGGCACGGG GCAGGTCCCG 840 GAGTATGGAA TGGTACCTTC CAGCCTCTCC ATGTGCATGA AAAGTGACAA GCATGGGTCC 900 CCATATTTCT ACTTACTGGG CCACCCTAAA GACACGACGA ACCCCACGCC TGGAGTGCTG 960 CCGCTGCCGC CGCCCCAGGC CTGCCGGAAG TAGGCGTCTC CCTCGAAGAC ATCCTCTCTC 1020 CATTCTCTTC ATCACATCCA GCCCCACCCT CCGACCCTTC CCACCAGATA GGCCCAGACC 1080 CAACT 1085 <210> 5 <211> 1161 <212> Ply GPT AspGlyply Arg Gly Arg Ala Ala 1 5 10 15 Pro Gly Leu Pro Leu Ser Ser Ala Pro Gly Thr Thr Arg Pro His Lys 20 25 30 Glu Gly Glu Val Pro Gly Val Asp Tyr Ile Phe Ile Thr Val Glu Glu 35 40 45 Phe Met Glu Leu Glu Lys Ser Gly Ala Leu Leu Glu Ser Gly Thr Tyr 50 55 60 Glu Asp Asn Tyr Tyr Gly Thr Pro lys Pro Pro Ala Glu Pro Ala Pro 65 70 75 80 Leu Leu Asn Val Thr Asp Gln Ile Leu Pro Gly Ala Thr Pro Ser Ala 85 90 95 Glu Gly Lys Arg Lys Arg Asn Lys Ser Val Thr Asn Met Glu Lys Ala 100 105 110 Ser Ile Glu Pro Pro Glu Glu Glu Glu Glu Glu Arg Pro Val Val Asn 115 120 125 Gly Asn Gly Val Val Ile Thr Pro Glu Ser Ser Glu His Glu Asp Lys 130 135 140 Ser Ala Gly Ala Ser Gly Glu Thr Pro Ser Gln Pro Tyr Pro Ala Pro 145 150 155 160 Val Tyr Ser Gln Pro Glu Glu Leu Lys Asp Gln Met Asp Asp Thr Lys 165 170 175 Pro Thr Lys Pro Glu Glu Asn Glu Asp Ser Asp Pro Leu Pro Asp Asn 180 185 190 Trp Glu Met Ala Tyr Thr Glu Lys Gly Glu Val Tyr Phe Ile Asp His 195 200 205 Asn Thr Lys Thr Thr Ser Trp Leu Asp Pro Arg Leu Ala Lys Lys Ala 210 215 220 Lys Pro Pro Glu Glu Cys Lys Glu Asn Glu Leu Pro Tyr Gly Trp Glu 225 230 235 240 Lys Ile Asp Asp Pro Ile Tyr Gly Thr Tyr Tyr Val Asp His Ile Asn 245 250 255 Arg Arg Thr Gln Phe Glu Asn Pro Val Leu Glu Ala Lys arg Lys Leu 260 265 2 70 Gln Gln His Asn Met Pro His Thr Glu Leu Gly Ala Lys Pro Leu Gln 275 280 285 Ala Pro Gly Phe Arg Glu Lys Pro Leu Phe Thr Arg Asp Ala Ser Gln 290 295 300 Leu Lys Gly Thr Phe Leu Ser Thr Thr Leu Lys Lys Ser Asn Met Gly 305 310 315 320 Phe Gly Phe Thr Ile Ile Gly Gly Asp Glu Pro Asp Glu Phe Leu Gln 325 330 335 Val Lys Ser Val Ile Pro Asp Gly Pro Ala Ala Gln Asp Gly Lys Met 340 345 350 Glu Thr Gly Asp Val Ile Val Tyr Ile Asn Glu Val Cys Val Leu Gly 355 360 365 His Thr His Ala Asp Val Val Lys Leu Phe Gln Ser Val Pro Ile Gly 370 375 380 Gln Ser Val Asn Leu Val Leu Cys Arg Gly Tyr Pro Leu Pro Phe Asp 385 390 395 400 Pro Glu Asp Pro Ala Asn Ser Met Val Pro Pro Leu Ala Ile Met Glu 405 410 415 Arg Pro Pro Pro Val Met Val Asn Gly Arg His Asn Tyr Glu Thr Tyr 420 425 430 430 Leu Glu Tyr Ile Ser Arg Thr Ser Gln Ser Val Pro Asp Ile Thr Asp 435 440 445 Arg Pro Pro His Ser Leu his Ser Met Pro Ala Asp Gly Gln Leu Asp 450 455 460 Gly Thr Tyr Pro Pro Pro Val His Asp Asp Asn Val Ser Met Ala Ser 465 470 475480 Ser Gly Ala Thr Gln Ala Glu Leu Met Thr Leu Thr Ile Val Lys Gly 485 490 495 Ala Gln Gly Phe Gly Phe Thr Ile Ala Asp Ser Pro Thr Gly Gln Arg 500 505 510 Val Lys Gln Ile Leu Asp Ile Gln Gly Cys Pro Gly Leu Cys Glu Gly 515 520 525 Asp Leu Ile Val Glu Ile Asn Gln Gln Asn Val Gln Asn Leu Ser His 530 535 540 Thr Glu Val Val Asp Ile Leu Lys Asp Cys Pro Val Gly Ser Glu Thr 545 550 555 560 Ser Leu Ile Ile His Arg Gly Gly Phe Phe Ser Pro Trp Lys Thr Pro 565 570 575 Lys Pro Met Met Asp Arg Trp Glu Asn Gln Gly Ser Pro Gln Thr Ser 580 585 590 Leu Ser Ala Pro Ala Val Pro Gln Asn Leu Pro Phe Pro Pro Ala Leu 595 600 605 His Arg Ser Ser Phe Pro Asp Ser Thr Glu Ala Phe Asp Pro Arg Lys 610 615 620 Pro Asp Pro Tyr Glu Leu Tyr Glu Lys Ser Arg Ala Ile Tyr Glu Ser 625 630 635 640 Arg Gln Gln Val Pro Pro Arg Thr Ser Phe Arg Met Asp Ser Ser Gly 645 650 655 655 Pro Asp Tyr Lys Glu Leu Asp Val His Leu Arg Arg Met Glu Ser Gly 660 665 670 Phe Gly Phe Arg Ile Leu Gly Gly Asp Glu Pro Gly Gln Pro Ile Leu 675 680 685Ile Gly Ala Val Ile Ala Met Gly Ser Ala Asp Arg Asp Gly Arg Leu 690 695 700 His Pro Gly Asp Glu Leu Val Tyr Val Asp Gly Ile Pro Val Ala Gly 705 710 715 715 720 Lys Thr His Arg Tyr Val Ile Asp Leu Met His His Ala Ala Arg Asn 725 730 735 735 Gly Gln Val Asn Leu Thr Val Arg Arg Lys Val Leu Cys Gly Gly Glu Glu 740 745 750 Pro Cys Pro Glu Asn Gly Arg Ser Pro Gly Ser Val Ser Thr His His 755 760 765 Ser Ser Pro Arg Ser Asp Tyr Ala Thr Tyr Ser Asn Ser Asn His Ala 770 775 780 Ala Pro Ser Ser Asn Ala Ser Pro Pro Glu Gly Phe Ala Ser His Ser 785 790 795 800 Leu Gln Thr Ser Asp Val Val Ile His Arg Lys Glu Asn Glu Gly Phe 805 810 815 Gly Phe Val Ile Ile Ser Ser Leu Asn Arg Pro Glu Ser Gly Ala Thr 820 825 830 Ile Thr Val Pro His Lys Ile Gly Arg Ile Ile Asp Gly Ser Pro Ala 835 840 845 Asp Arg Cys Ala Lys Leu Lys Val Gly Asp Arg Ile Leu Ala Val Asn 850 855 860 860 Gly Gln Ser Ile Ile Asn Met Pro His Ala Asp Ile Val Lys Leu Ile 865 870 875 880 880 Lys Asp Ala Gly Leu Ser Val Thr Leu Arg Ile Ile Pro Gln Glu Glu 885 890 890 895Leu Asn Ser Pro Thr Ser Ala Pro Ser Ser Glu Lys Gln Ser Pro Met 900 905 910 Ala Gln Gln His Ser Pro Leu Ala Gln Gln Ser Pro Leu Ala Gln Pro 915 920 925 Ser Pro Ala Thr Pro Asn Ser Pro Val Ala Gln Pro Ala Pro Pro Gln 930 935 940 Pro Leu Gln Leu Gln Gly His Glu Asn Ser Tyr Arg Ser Glu Val Lys 945 950 955 960 Ala Arg Gln Asp Val Lys Pro Asp Ile Arg Gln Pro Pro Phe Thr Asp 965 970 975 Tyr Arg Gln Pro Pro Leu Asp Tyr Arg Gln Pro Pro Gly Gly Asp Tyr 980 985 990 Ser Gln Pro Pro Pro Leu Asp Tyr Arg Gln His Ser Pro Asp Tyr Arg 995 1000 1005 Gln Tyr Pro Leu Ser Asp Tyr Arg Gln Pro Gln Asp Phe Asp Tyr Phe 1010 1015 1020 Thr Val Asp Met Glu Lys Gly Ala Lys Gly Phe Gly Phe Ser Ile Arg 1025 1030 1035 1040 Gly Gly Arg Glu Tyr Lys Met Asp Leu Tyr Val Leu Arg Leu Ala Glu 1045 1050 1055 Asp Gly Pro Ala Ile Arg Asn Gly Arg Met Arg Val Gly Asp Gln Ile 1060 1065 1070 Ile Glu Ile Asn Gly Glu Ser Thr Arg Asp Met Thr His Ala Arg Ala 1075 1080 1085 Ile Glu Leu Ile Lys Ser Gly Gly Arg Arg Val Arg Leu Leu Leu Ly s 1090 1095 1100 Arg Gly Thr Gly Gln Val Pro Glu Tyr Gly Met Val Pro Ser Ser Leu 1105 1110 1115 1120 Ser Met Cys Met Lys Ser Asp Lys His Gly Ser Pro Tyr Phe Tyr Leu 1125 1130 1135 Leu Gly His Pro Lys Asp Thr Thr Asn Pro Thr Pro Gly Val Leu Pro 1140 1145 1150 Leu Pro Pro Pro Gln Ala Cys Arg Lys 1155 1160 1161 <210> 6 <211> 1112 <212> PRT <213> Mouse <400> 6 Met Glu Leu Glu Lys Ser Gly Ala Leu Leu Glu Ser Gly Thr Tyr Glu 5 10 15 Asp Asn Tyr Tyr Gly Thr Pro lys Pro Pro Ala Glu Pro Ala Pro Leu 20 25 30 Leu Asn Val Thr Asp Gln Ile Leu Pro Gly Ala Thr Pro Ser Ala Glu 35 40 45 Gly Lys Arg Lys Arg Asn Lys Ser Val Thr Asn Met Glu Lys Ala Ser 50 55 60 Ile Glu Pro Pro Glu Glu Glu Glu Glu Glu Arg Pro Val Val Asn Gly 65 70 75 80 Asn Gly Val Val Ile Thr Pro Glu Ser Ser Glu His Glu Asp Lys Ser 85 90 95 Ala Gly Ala Ser Gly Glu Thr Pro Ser Gln Pro Tyr Pro Ala Pro Val 100 105 110 Tyr Ser Gln Pro Glu Glu Leu Lys Asp Gln Met Asp Asp Thr Lys Pro 115 120 125 Thr Lys Pro Glu Glu Asn Glu Asp Ser Asp Pro Leu Pro As p Asn Trp 130 135 140 Glu Met Ala Tyr Thr Glu Lys Gly Glu Val Tyr Phe Ile Asp His Asn 145 150 155 160 Thr Lys Thr Thr Ser Trp Leu Asp Pro Arg Leu Ala Lys Lys Ala Lys 165 170 175 Pro Pro Glu Glu Cys Lys Glu Asn Glu Leu Pro Tyr Gly Trp Glu Lys 180 185 190 Ile Asp Asp Pro Ile Tyr Gly Thr Tyr Tyr Val Asp His Ile Asn Arg 195 200 205 Arg Thr Gln Phe Glu Asn Pro Val Leu Glu Ala Lys arg Lys Leu Gln 210 215 220 Gln His Asn Met Pro His Thr Glu Leu Gly Ala Lys Pro Leu Gln Ala 225 230 235 240 Pro Gly Phe Arg Glu Lys Pro Leu Phe Thr Arg Asp Ala Ser Gln Leu 245 250 255 Lys Gly Thr Phe Leu Ser Thr Thr Leu Lys Lys Ser Asn Met Gly Phe 260 265 270 Gly Phe Thr Ile Ile Gly Gly Asp Glu Pro Asp Glu Phe Leu Gln Val 275 280 285 Lys Ser Val Ile Pro Asp Gly Pro Ala Ala Ala Gln Asp Gly Lys Met Glu 290 295 300 Thr Gly Asp Val Ile Val Tyr Ile Asn Glu Val Cys Val Leu Gly His 305 310 315 320 Thr His Ala Asp Val Val Lys Leu Phe Gln Ser Val Pro Ile Gly Gln 325 330 335 Ser Val Asn Leu Val Leu Cys Arg Gly Tyr Pro Leu Pro Ph e Asp Pro 340 345 350 Glu Asp Pro Ala Asn Ser Met Val Pro Pro Leu Ala Ile Met Glu Arg 355 360 365 Pro Pro Val Met Val Asn Gly Arg His Asn Tyr Glu Thr Tyr Leu 370 375 380 Glu Tyr Ile Ser Arg Thr Ser Gln Ser Val Pro Asp Ile Thr Asp Arg 385 390 395 400 400 Pro Pro His Ser Leu his Ser Met Pro Ala Asp Gly Gln Leu Asp Gly 405 410 415 Thr Tyr Pro Pro Pro Val His Asp Asp Asn Val Ser Met Ala Ser Ser 420 425 430 Gly Ala Thr Gln Ala Glu Leu Met Thr Leu Thr Ile Val Lys Gly Ala 435 440 445 Gln Gly Phe Gly Phe Thr Ile Ala Asp Ser Pro Thr Gly Gln Arg Val 450 455 460 Lys Gln Ile Leu Asp Ile Gln Gly Cys Pro Gly Leu Cys Glu Gly Asp 465 470 475 480 Leu Ile Val Glu Ile Asn Gln Gln Asn Val Gln Asn Leu Ser His Thr 485 490 495 Glu Val Val Asp Ile Leu Lys Asp Cys Pro Val Gly Ser Glu Thr Ser 500 505 510 Leu Ile Ile His Arg Gly Gly Phe Phe Ser Pro Trp Lys Thr Pro Lys 515 520 525 Pro Met Met Asp Arg Trp Glu Asn Gln Gly Ser Pro Gln Thr Ser Leu 530 535 540 Ser Ala Pro Ala Val Pro Gln Asn Leu Pro Phe Pro Pro Ala Le u His 545 550 555 560 Arg Ser Ser Phe Pro Asp Ser Thr Glu Ala Phe Asp Pro Arg Lys Pro 565 570 575 Asp Pro Tyr Glu Leu Tyr Glu Lys Ser Arg Ala Ile Tyr Glu Ser Arg 580 585 590 Gln Gln Val Pro Pro Arg Thr Ser Phe Arg Met Asp Ser Ser Gly Pro 595 600 605 Asp Tyr Lys Glu Leu Asp Val His Leu Arg Arg Met Glu Ser Gly Phe 610 615 620 620 Gly Phe Arg Ile Leu Gly Gly Asp Glu Pro Gly Gln Pro Ile Leu Ile 625 630 635 640 Gly Ala Val Ile Ala Met Gly Ser Ala Asp Arg Asp Gly Arg Leu His 645 650 655 Pro Gly Asp Glu Leu Val Tyr Val Asp Gly Ile Pro Val Ala Gly Lys 660 665 670 Thr His Arg Tyr Val Ile Asp Leu Met His His Ala Ala Arg Asn Gly 675 680 685 Gln Val Asn Leu Thr Val Arg Arg Lys Val Leu Cys Gly Gly Glu Glu Pro 690 695 700 Cys Pro Glu Asn Gly Arg Ser Pro Gly Ser Val Ser Thr His His Ser 705 710 710 715 720 Ser Pro Arg Ser Asp Tyr Ala Thr Tyr Ser Asn Ser Asn His Ala Ala 725 730 735 Pro Ser Ser Asn Ala Ser Pro Pro Glu Gly Phe Ala Ser His Ser Leu 740 745 750 Gln Thr Ser Asp Val Val Ile His Arg Lys Glu Asn Glu Gly Ph e Gly 755 760 765 Phe Val Ile Ile Ser Ser Leu Asn Arg Pro Glu Ser Gly Ala Thr Ile 770 775 780 Thr Val Pro His Lys Ile Gly Arg Ile Ile Asp Gly Ser Pro Ala Asp 785 790 795 800 Arg Cys Ala Lys Leu Lys Val Gly Asp Arg Ile Leu Ala Val Asn Gly 805 810 815 Gln Ser Ile Ile Asn Met Pro His Ala Asp Ile Val Lys Leu Ile Lys 820 825 830 Asp Ala Gly Leu Ser Val Thr Leu Arg Ile Ile Pro Gln Glu Glu Leu 835 840 845 Asn Ser Pro Thr Ser Ala Pro Ser Ser Glu Lys Gln Ser Pro Met Ala 850 855 860 Gln Gln His Ser Pro Leu Ala Gln Gln Ser Pro Leu Ala Gln Pro Ser 865 870 875 880 Pro Ala Thr Pro Asn Ser Pro Val Ala Gln Pro Ala Pro Pro Gln Pro 885 890 895 Leu Gln Leu Gln Gly His Glu Asn Ser Tyr Arg Ser Glu Val Lys Ala 900 905 910 Arg Gln Asp Val Lys Pro Asp Ile Arg Gln Pro Pro Phe Thr Asp Tyr 915 920 925 Arg Gln Pro Pro Leu Asp Tyr Arg Gln Pro Pro Gly Gly Asp Tyr Ser 930 935 940 Gln Pro Pro Pro Leu Asp Tyr Arg Gln His Ser Pro Asp Tyr Arg Gln 945 950 955 960 Tyr Pro Leu Ser Asp Tyr Arg Gln Pro Gln Asp Phe Asp Tyr Ph e Thr 965 970 975 Val Asp Met Glu Lys Gly Ala Lys Gly Phe Gly Phe Ser Ile Arg Gly 980 985 990 Gly Arg Glu Tyr Lys Met Asp Leu Tyr Val Leu Arg Leu Ala Glu Asp 995 1000 1005 Gly Pro Ala Ile Arg Asn Gly Arg Met Arg Val Gly Asp Gln Ile Ile 1010 1015 1020 Glu Ile Asn Gly Glu Ser Thr Arg Asp Met Thr His Ala Arg Ala Ile 1025 1030 1035 1040 Glu Leu Ile Lys Ser Gly Gly Arg Arg Val Arg Leu Leu Leu Lys Arg 1045 1050 1055 Gly Thr Gly Gln Val Pro Glu Tyr Gly Met Val Pro Ser Ser Leu Ser 1060 1065 1070 Met Cys Met Lys Ser Asp Lys His Gly Ser Pro Tyr Phe Tyr Leu Leu 1075 1080 1085 Gly His Pro Lys Asp Thr Thr Asn Pro Thr Pro Gly Val Leu Pro Leu 1090 1095 1100 Pro Pro Pro Gln Ala Cys Arg Lys 1105 1110 1112 <210> 7 <211> 3483 <212> DNA <213> Mouse <400> 7 GGAGACGCTG ACAGAGGACC ATGGAAAGGT GGGAGAGGAC GCGCGGCTCC TGGGCTTCAGCTCCCTGAGCGCAGCAGCAG GTGAGGTCCC TGGAGTGGAC 120 TACATTTTCA TAACCGTTGA GGAGTTTATG GAATTGGAGA AAAGTGGTGC TCTCCTAGAA 180 AGCGGGACCT ATGAAGACAA CTACTACGGT ACCCCGAAGC CTCCA GCTGA ACCAGCACCA 240 TTATTAAATG TAACAGACCA GATACTTCCG GGAGCTACTC CAAGTGCTGA GGGGAAGCGG 300 AAAAGAAATA AGTCAGTGAC CAACATGGAG AAAGCAAGTA TAGAGCCTCC AGAGGAGGAA 360 GAAGAAGAAA GGCCTGTAGT CAATGGAAAC GGCGTGGTCA TAACCCCAGA ATCCAGTGAA 420 CATGAAGACA AAAGTGCAGG TGCCTCAGGG GAGACACCCT CCCAGCCTTA CCCTGCACCC 480 GTGTACAGCC AGCCCGAAGA GCTCAAGGAC CAGATGGACG ATACAAAGCC AACAAAGCCT 540 GAGGAGAACG AGGACTCTGA TCCATTGCCT GATAACTGGG AAATGGCCTA CACAGAGAAG 600 GGGGAAGTCT ACTTCATTGA CCATAACACA AAGACAACAT CATGGCTGGA TCCGCGACTT 660 GCGAAAAAGG CTAAACCTCC AGAAGAGTGC AAAGAAAATG AGCTTCCATA TGGCTGGGAA 720 AAAATCGATG ATCCTATATA TGGCACTTAC TATGTTGACC ACATAAATAG AAGAACACAG 780 TTTGAAAACC CTGTCCTGGA AGCAAAAAGG AAGCTACAGC AACATAACAT GCCCCACACA 840 GAACTTGGAG CAAAGCCCCT GCAGGCCCCA GGTTTCCGAG AAAAGCCACT CTTCACCCGG 900 GATGCATCCC AGTTGAAGGG AACGTTCCTC AGCACCACCC TCAAAAAGAG CAACATGGGC 960 TTTGGGTTTA CCATAATTGG TGGAGACGAG CCGGATGAGT TTCTACAGGT GAAAAGTGTG 1020 ATCCCGGATG GGCCTGCCGC ACAGGATGGG AAAATGGAGA CAGGTGATGT CATTGTCTAT Ten 80 ATTAATGAAG TTTGTGTCCT TGGACACACT CATGCAGATG TTGTCAAACT TTTCCAGTCT 1140 GTTCCTATTG GTCAGAGTGT CAACTTGGTG TTGTGTCGTG GCTACCCTTT GCCCTTTGAC 1200 CCTGAAGATC CTGCTAACAG CATGGTGCCA CCCCTTGCAA TAATGGAGAG GCCACCTCCG 1260 GTGATGGTCA ATGGAAGACA TAACTATGAA ACATACTTGG AATACATTTC TCGGACCTCA 1320 CAGTCGGTCC CAGATATTAC AGACCGGCCA CCTCATTCTT TGCACTCCAT GCCAGCTGAC 1380 GGCCAGCTAG ATGGCACGTA TCCACCACCC GTCCATGACG ACAATGTGTC TATGGCTTCG 1440 TCTGGAGCCA CTCAAGCTGA ACTTATGACC TTAACCATTG TGAAAGGTGC CCAGGGATTT 1500 GGCTTTACTA TTGCCGACAG TCCCACGGGA CAGCGGGTGA AACAAATCCT TGACATTCAG 1560 GGATGCCCTG GGCTGTGTGA AGGAGACCTC ATTGTTGAGA TCAACCAACA GAATGTACAG 1620 AACCTGAGCC ATACAGAAGT AGTGGATATA CTTAAGGACT GCCCCGTTGG AAGTGAGACT 1680 TCTTTAATCA TCCATCGAGG AGGTTTCTTT TCTCCATGGA AAACTCCAAA GCCTATGATG 1740 GACCGATGGG AGAACCAAGG CAGTCCACAA ACAAGTTTAT CTGCTCCGGC CGTCCCACAG 1800 AACCTGCCCT TCCCACCTGC CCTTCACAGG AGCTCCTTTC CTGATTCAAC AGAGGCCTTT 1860 GACCCACGGA AGCCTGACCC ATATGAGCTC TACGAGAAAT CGAGAGCCAT TTATGAAAGT 1920 AGG CAACAAG TGCCACCCAG GACCAGTTTT CGAATGGATT CCTCTGGTCC AGATTATAAG 1980 GAACTGGATG TTCACCTTCG GAGGATGGAG TCTGGATTTG GCTTTAGAAT CCTTGGGGGA 2040 GATGAACCTG GACAGCCTAT TTTGATCGGA GCCGTCATTG CCATGGGCTC AGCTGACAGA 2100 GACGGCCGTC TACACCCAGG AGATGAGCTT GTCTATGTCG ATGGGATCCC AGTGGCTGGC 2160 AAGACCCACC GCTATGTCAT CGACCTCATG CACCACGCGG CCCGCAATGG GCAGGTTAAC 2220 CTCACTGTGA GAAGAAAGGT GCTATGTGGA GGGGAGCCCT GCCCAGAGAA TGGGAGGAGT 2280 CCAGGCTCTG TATCAACTCA CCACAGCTCT CCGCGCAGTG ACTATGCCAC CTACTCCAAC 2340 AGCAACCACG CCGCCCCCAG CAGCAATGCC TCACCTCCTG AAGGCTTTGC CTCACACAGC 2400 TTGCAGACCA GTGATGTGGT CATTCACCGC AAAGAAAACG AAGGGTTTGG CTTCGTCATC 2460 ATCAGCTCTC TGAACAGGCC TGAGTCTGGA GCCACCATAA CTGTGCCCCA TAAAATTGGA 2520 CGAATCATTG ATGGGAGCCC TGCAGATCGC TGTGCCAAAC TCAAAGTGGG CGACCGTATC 2580 TTAGCAGTCA ACGGCCAGTC TATCATCAAC ATGCCTCACG CTGACATTGT GAAGCTCATC 2640 AAGGACGCCG GTCTCAGTGT CACCCTTCGC ATCATTCCTC AGGAGGAGCT CAACAGCCCA 2700 ACATCAGCAC CCAGTTCAGA GAAACAGAGC CCCATGGCCC AGCAGCACAG CCCTCTGGCC 2760 CAGCAGAGT C CTCTGGCCCA GCCAAGCCCC GCCACCCCCA ACAGCCCAGT CGCACAGCCA 2820 GCTCCTCCCC AACCTCTCCA GCTGCAAGGA CACGAAAATA GTTACAGGTC AGAAGTTAAA 2880 GCGAGGCAAG ATGTGAAGCC AGACATCCGG CAGCCTCCCT TCACAGACTA CAGGCAGCCC 2940 CCGCTGGACT ACAGGCAGCC CCCGGGAGGA GACTACTCAC AGCCCCCACC CTTGGACTAC 3000 AGGCAGCACT CTCCAGACAC CAGGCAGTAC CCTCTGTCAG ACTACAGGCA GCCACAGGAT 3060 TTTGATTATT TCACTGTGGA CATGGAGAAA GGAGCCAAAG GATTTGGATT CAGCATTCGT 3120 GGAGGAAGGG AATACAAGAT GGATCTGTAT GTGTTGAGAT TGGCAGAGGA TGGGCCAGCC 3180 ATAAGGAACG GCAGGATGAG GGTAGGAGAT CAGATCATTG AAATAAATGG GGAAAGCACA 3240 CGAGACATGA CCCACGCCAG AGCAATAGAA CTCATCAAGT CTGGAGGAAG AAGAGTGCGG 3300 CTGCTGCTGA AGAGAGGCAC GGGGCAGGTC CCGGAGTATG GAATGGTACC TTCCAGCCTC 3360 TCCATGTGCA TGAAAAGTGA CAAGCATGGG TCCCCATATT TCTACTTACT GGGCCACCCT 3420 AAAGACACGA CGAACCCCAC GCCTGGAGTG CTGCCGCTGC CGCCGCCCCA GGCCTGCCGG 3480 AAG 3483 <210> 8 <211> 3336 <212> DNA <213 > Mouse <400> 8 ATGGAATTGG AGAAAAGTGG TGCTCTCCTA GAAAGCGGGA CCTATGAAGA CAACTACTAC 60 GGTACCCCGA AGCCTCCAGC TGAACCAGCA CC ATTATTAA ATGTAACAGA CCAGATACTT 120 CCGGGAGCTA CTCCAAGTGC TGAGGGGAAG CGGAAAAGAA ATAAGTCAGT GACCAACATG 180 GAGAAAGCAA GTATAGAGCC TCCAGAGGAG GAAGAAGAAG AAAGGCCTGT AGTCAATGGA 240 AACGGCGTGG TCATAACCCC AGAATCCAGT GAACATGAAG ACAAAAGTGC AGGTGCCTCA 300 GGGGAGACAC CCTCCCAGCC TTACCCTGCA CCCGTGTACA GCCAGCCCGA AGAGCTCAAG 360 GACCAGATGG ACGATACAAA GCCAACAAAG CCTGAGGAGA ACGAGGACTC TGATCCATTG 420 CCTGATAACT GGGAAATGGC CTACACAGAG AAGGGGGAAG TCTACTTCAT TGACCATAAC 480 ACAAAGACAA CATCATGGCT GGATCCGCGA CTTGCGAAAA AGGCTAAACC TCCAGAAGAG 540 TGCAAAGAAA ATGAGCTTCC ATATGGCTGG GAAAAAATCG ATGATCCTAT ATATGGCACT 600 TACTATGTTG ACCACATAAA TAGAAGAACA CAGTTTGAAA ACCCTGTCCT GGAAGCAAAA 660 AGGAAGCTAC AGCAACATAA CATGCCCCAC ACAGAACTTG GAGCAAAGCC CCTGCAGGCC 720 CCAGGTTTCC GAGAAAAGCC ACTCTTCACC CGGGATGCAT CCCAGTTGAA GGGAACGTTC 780 CTCAGCACCA CCCTCAAAAA GAGCAACATG GGCTTTGGGT TTACCATAAT TGGTGGAGAC 840 GAGCCGGATG AGTTTCTACA GGTGAAAAGT GTGATCCCGG ATGGGCCTGC CGCACAGGAT 900 GGGAAAATGG AGACAGGTGA TGTCATTGTC TATATTAATG AAGTTTGTGTCCTTGGACAC 960 ACTCATGCAG ATGTTGTCAA ACTTTTCCAG TCTGTTCCTA TTGGTCAGAG TGTCAACTTG 1020 GTGTTGTGTC GTGGCTACCC TTTGCCCTTT GACCCTGAAG ATCCTGCTAA CAGCATGGTG 1080 CCACCCCTTG CAATAATGGA GAGGCCACCT CCGGTGATGG TCAATGGAAG ACATAACTAT 1140 GAAACATACT TGGAATACAT TTCTCGGACC TCACAGTCGG TCCCAGATAT TACAGACCGG 1200 CCACCTCATT CTTTGCACTC CATGCCAGCT GACGGCCAGC TAGATGGCAC GTATCCACCA 1260 CCCGTCCATG ACGACAATGT GTCTATGGCT TCGTCTGGAG CCACTCAAGC TGAACTTATG 1320 ACCTTAACCA TTGTGAAAGG TGCCCAGGGA TTTGGCTTTA CTATTGCCGA CAGTCCCACG 1380 GGACAGCGGG TGAAACAAAT CCTTGACATT CAGGGATGCC CTGGGCTGTG TGAAGGAGAC 1440 CTCATTGTTG AGATCAACCA ACAGAATGTA CAGAACCTGA GCCATACAGA AGTAGTGGAT 1500 ATACTTAAGG ACTGCCCCGT TGGAAGTGAG ACTTCTTTAA TCATCCATCG AGGAGGTTTC 1560 TTTTCTCCAT GGAAAACTCC AAAGCCTATG ATGGACCGAT GGGAGAACCA AGGCAGTCCA 1620 CAAACAAGTT TATCTGCTCC GGCCGTCCCA CAGAACCTGC CCTTCCCACC TGCCCTTCAC 1680 AGGAGCTCCT TTCCTGATTC AACAGAGGCC TTTGACCCAC GGAAGCCTGA CCCATATGAG 1740 CTCTACGAGA AATCGAGAGC CATTTATGAA AGTAGGCAAC AAGTGCCACC CAGGACC AGT 1800 TTTCGAATGG ATTCCTCTGG TCCAGATTAT AAGGAACTGG ATGTTCACCT TCGGAGGATG 1860 GAGTCTGGAT TTGGCTTTAG AATCCTTGGG GGAGATGAAC CTGGACAGCC TATTTTGATC 1920 GGAGCCGTCA TTGCCATGGG CTCAGCTGAC AGAGACGGCC GTCTACACCC AGGAGATGAG 1980 CTTGTCTATG TCGATGGGAT CCCAGTGGCT GGCAAGACCC ACCGCTATGT CATCGACCTC 2040 ATGCACCACG CGGCCCGCAA TGGGCAGGTT AACCTCACTG TGAGAAGAAA GGTGCTATGT 2100 GGAGGGGAGC CCTGCCCAGA GAATGGGAGG AGTCCAGGCT CTGTATCAAC TCACCACAGC 2160 TCTCCGCGCA GTGACTATGC CACCTACTCC AACAGCAACC ACGCCGCCCC CAGCAGCAAT 2220 GCCTCACCTC CTGAAGGCTT TGCCTCACAC AGCTTGCAGA CCAGTGATGT GGTCATTCAC 2280 CGCAAAGAAA ACGAAGGGTT TGGCTTCGTC ATCATCAGCT CTCTGAACAG GCCTGAGTCT 2340 GGAGCCACCA TAACTGTGCC CCATAAAATT GGACGAATCA TTGATGGGA GCCCTGCAGAT 2400 CGCTGTGCCA AACTCAAAGT GGGCGACCGT ATCTTAGCAG TCAACGGCCA GTCTATCATC 2460 AACATGCCTC ACGCTGACAT TGTGAAGCTC ATCAAGGACG CCGGTCTCAG TGTCACCCTT 2520 CGCATCATTC CTCAGGAGGA GCTCAACAGC CCAACATCAG CACCCAGTTC AGAGAAACAG 2580 AGCCCCATGG CCCAGCAGCA CAGCCCTCTG GCCCAGCAGA GTCCTCTGGC CCAGCCAAGC 26 40 CCCGCCACCC CCAACAGCCC AGTCGCACAG CCAGCTCCTC CCCAACCTCT CCAGCTGCAA 2700 GGACACGAAA ATAGTTACAG GTCAGAAGTT AAAGCGAGGC AAGATGTGAA GCCAGACATC 2760 CGGCAGCCTC CCTTCACAGA CTACAGGCAG CCCCCGCTGG ACTACAGGCA GCCCCCGGGA 2820 GGAGACTACT CACAGCCCCC ACCCTTGGAC TACAGGCAGC ACTCTCCAGA CACCAGGCAG 2880 TACCCTCTGT CAGACTACAG GCAGCCACAG GATTTTGATT ATTTCACTGT GGACATGGAG 2940 AAAGGAGCCA AAGGATTTGG ATTCAGCATT CGTGGAGGAA GGGAATACAA GATGGATCTG 3000 TATGTGTTGA GATTGGCAGA GGATGGGCCA GCCATAAGGA ACGGCAGGAT GAGGGTAGGA 3060 GATCAGATCA TTGAAATAAA TGGGGAAAGC ACACGAGACA TGACCCACGC CAGAGCAATA 3120 GAACTCATCA AGTCTGGAGG AAGAAGAGTG CGGCTGCTGC TGAAGAGAGG CACGGGGCAG 3180 GTCCCGGAGT ATGGAATGGT ACCTTCCAGC CTCTCCATGT GCATGAAAAG TGACAAGCAT 3240 GGGTCCCCAT ATTTCTACTT ACTGGGCCAC CCTAAAGACA CGACGAACCC CACGCCTGGA 3300 GTGCTGCCGC TGCCGCCGCC CCAGGCCTGC CGGAAG 3336

[0074]

[Brief description of the drawings]

FIG. 1 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 5).

FIG. 2 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 5). It is a continuation of FIG.

FIG. 3 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 5). It is a continuation of FIG.

FIG. 4 shows the entire nucleotide sequence of the cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 5). It is a continuation of FIG.

FIG. 5 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 5). FIG. 4 is a continuation of FIG. 4.

FIG. 6 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 5). It is a continuation of FIG.

FIG. 7 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 6).

FIG. 8 shows the entire nucleotide sequence of the cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 6). It is a continuation of FIG.

FIG. 9 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 6). It is a continuation of FIG.

FIG. 10 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 6). It is a continuation of FIG.

FIG. 11 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 6). It is a continuation of FIG.

FIG. 12 shows the entire nucleotide sequence of cDNA encoding the novel protein of the present invention and the amino acid sequence encoded thereby (SEQ ID NO: 6). It is a continuation of FIG.

FIG. 13 shows the results of analysis of expression by Northern hybridization.

FIG. 14 shows the results of examining the specific interaction between the novel protein of the present invention and Smad3. ARIP1 on the horizontal axis indicates the novel protein of the present invention. Relative Interaction on the vertical axis indicates the intensity of luciferase activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 25/14 A61P 25/16 25/16 25/28 25/28 C07K 14/47 C07K 14/47 14 / 72 14/72 16/18 16/18 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/00 Z C12P 21/02 G01N 33 / 15 Z C12Q 1/00 33/50 Z G01N 33/15 33/53 D 33/50 33/566 33/53 A61K 37/02 33/566 C12N 5/00 A

Claims (28)

[Claims] 1. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5, or a salt thereof. 2. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 6, or a salt thereof. 3. The method according to claim 1, which has a PDZ domain and a WW domain, is specifically expressed in the brain, and has a binding ability to an activin receptor and / or an activin intracellular signaling molecule. protein. 4. The activin intracellular signaling molecule is Smad.
The protein according to claim 3, which is 3. 5. The protein according to claim 1, which has five PDZ domains and two WW domains, is specifically expressed in the brain, and has an activin receptor and Smad3 binding ability. 6. A partial peptide of the protein according to claim 1, a partial peptide of the protein according to claim 2, or a salt thereof. 7. A recombinant DNA comprising a protein having the nucleotide sequence encoding the protein according to claim 1 or the protein according to claim 2. 8. The DNA according to claim 7, which has a nucleotide sequence represented by SEQ ID NO: 7, a nucleotide sequence represented by SEQ ID NO: 8, or a nucleotide sequence hybridizable thereto under high stringency conditions. 9. A recombinant DNA containing a DNA having a base sequence encoding the partial peptide according to claim 6. 10. A recombinant vector containing the DNA according to claim 7. [11] A transformant carrying the recombinant vector according to [10]. (12) culturing the transformant according to (11),
A method for producing the protein according to claim 1, wherein the protein according to claim 1 or the protein according to claim 2 is produced and accumulated, and collected. 13. An antibody against the protein according to claim 1, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof. (14) a test solution containing the protein according to (1), the protein according to (2), the partial peptide according to (6) or a salt thereof, and a labeled antibody; The protein according to claim 1, wherein the protein according to claim 1, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof is reacted competitively.
A method for quantifying the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof. 15. The protein according to claim 1, wherein the protein according to claim 1, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof is used. A method for determining a protein that binds to the partial peptide or a salt thereof according to claim 6. 16. The DNA binding region of a transcription factor according to claim 1.
And a fusion library of a gene encoding a test protein and a transcriptional activation region, and a fusion library of the protein described in claim 2, the protein described in claim 2, or the partial peptide described in claim 6. Is introduced into a host cell having a reporter gene retained on a promoter, and the reporter is increased by the binding of the protein of claim 1, the protein of claim 2, or the partial peptide of claim 6 to a test protein. The method according to claim 15, wherein a change in the expression level of the gene is measured. 17. Obtained by the method of claim 15,
A protein or a salt thereof, which binds to the protein of claim 1, the protein of claim 2, the partial peptide of claim 6, or a salt thereof. 18. The protein according to claim 1, wherein the protein according to claim 1, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof is used. A method for screening a compound or a salt thereof that inhibits or promotes the binding of the partial peptide or a salt thereof according to claim 6 to the protein or a salt thereof, an activin receptor or an activin intracellular signaling molecule according to claim 17. (19) The method according to (1), wherein the labeled protein according to claim 1, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof is contacted with an activin receptor or an activin intracellular signal transduction molecule. Claims wherein the protein according to claim 1, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof and a test compound are brought into contact with an activin receptor or an activin intracellular signaling molecule. The amount of binding of the protein of claim 1, the protein of claim 2, the partial peptide of claim 6, or a salt thereof to an activin receptor or an activin intracellular signaling molecule is measured and compared. The protein according to claim 2, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof and an activin receptor or activin. Method of screening a compound or its salt that inhibits or promotes the binding of intracellular signaling molecules. 20. The protein of claim 1, the protein of claim 2, the partial peptide of claim 6, or a salt thereof contacted with the protein of claim 17 or a salt thereof. The protein according to claim 1, the protein according to claim 2, or a salt thereof and a test compound.
When contacted with the described protein or a salt thereof,
The amount of binding of the labeled protein of claim 1, the protein of claim 2, the partial peptide of claim 6, or a salt thereof to the protein of claim 17 or a salt thereof is measured and compared. The protein of claim 1,
A method for screening a compound or a salt thereof that inhibits or promotes the binding of the protein of claim 2 or the partial peptide or a salt thereof according to claim 6 to the protein or a salt thereof according to claim 17. 21. The protein according to claim 1, wherein the protein or a salt thereof, an activin receptor or an activin intracellular signaling molecule is expressed in the cell according to claim 17.
The protein according to claim 1 or the partial peptide according to claim 6 or a salt thereof is introduced, and the protein according to claim 17 or a salt thereof, an activin receptor or a cell that expresses an activin intracellular signal transduction molecule. The protein according to claim 1, the protein according to claim 2, the protein according to claim 2, or the partial peptide according to claim 6, wherein the protein, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof and a test compound are introduced. 18. The protein according to claim 1, wherein the amount of binding of the salt to the protein according to claim 17 or the salt thereof, the activin receptor or the activin intracellular signaling molecule in the cell is measured and compared. The protein according to claim 2, the partial peptide according to claim 6, or a salt thereof, and the protein according to claim 17, a salt thereof, or activin receptor. Method of screening a compound or its salt that inhibits or promotes the binding of somatic or activin intracellular signaling molecules. 22. The protein according to claim 1, the protein according to claim 2, the partial peptide according to claim 6 or a salt thereof, and the protein according to claim 17 or a salt thereof, an activin receptor or activin intracellular information. The protein of claim 1, the protein of claim 2, the protein of claim 2, the partial peptide of claim 6, or a salt thereof and a test compound which are in contact with the membrane fraction of the cell expressing the transfer molecule. The protein according to claim 1, wherein the protein or the salt thereof, the activin receptor or the activin in the case of being brought into contact with a membrane fraction of a cell expressing the intracellular signaling molecule is labeled. The labeled amount of the partial peptide or the salt thereof according to claim 6, which is measured and compared with the membrane fraction of the cell.
Inhibiting or promoting the binding of the protein of claim 2, the protein of claim 2, the partial peptide of claim 6, or a salt thereof to the protein or salt thereof of claim 17, an activin receptor or an activin intracellular signaling molecule. A method for screening a compound or a salt thereof. 23. The protein according to claim 1, wherein the protein according to claim 17 or a salt thereof, an activin receptor or an activin intracellular signaling molecule is expressed in the cell.
2. The method according to claim 1, wherein the protein or a salt thereof is introduced.
7. The protein according to claim 1, the protein according to claim 2, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof, and a test compound, in a cell that has expressed the protein according to 7 or a salt thereof, an activin receptor or an activin intracellular signaling molecule. The protein according to claim 1, wherein a cell stimulating activity via a protein or a salt thereof, an activin receptor or an activin intracellular signal transduction molecule is measured and compared when the protein is introduced. A compound that inhibits or promotes the binding of the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof to the protein or salt thereof, an activin receptor or an activin intracellular signaling molecule, or a salt thereof. Screening method. 24. Two-hybrid
The method for determining a protein according to claim 16 or the screening method according to any one of claims 18 to 23, wherein the method d) is used. 25. The protein according to claim 1, comprising the protein according to claim 1, the protein according to claim 2, the partial peptide according to claim 6, or a salt thereof. A screening kit for a compound or a salt thereof that inhibits or promotes binding of a protein, a partial peptide according to claim 6 or a salt thereof, and a protein or a salt thereof according to claim 17, an activin receptor or an activin intracellular signaling molecule. . 26. The protein according to claim 1, obtained by using the screening method according to any one of claims 18 to 23, or the screening kit according to claim 25. 18. A compound or a salt thereof which inhibits or promotes the binding of the partial peptide or a salt thereof according to item 6 to the protein or a salt thereof, an activin receptor or an activin intracellular signaling molecule according to claim 17. (27) a pharmaceutical comprising the protein according to (17), the compound according to (26), or a salt thereof; 28. The medicament according to claim 27, which is an agent for preventing or treating Alzheimer's disease, Parkinson's disease, epilepsy or Huntington's chorea.