JP2003113111A - Medicine for treating diabetes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both a medicine having actions of identifying a ligand of activin receptor-like kinase 7 (ALK7) specifically expressible in the pancreas and promoting the insulin secretion from the pancreas and a method for screening the medicine. SOLUTION: This medicine for treatment and/or prophylaxis of diabetes comprises a protein selected from activin AB, activin B and the ALK7 as an active ingredient. The medicine for the treatment and/or prophylaxis of the diabetes comprises a DNA encoding the protein as an active ingredient. The method for screening the medicine for treatment and/or prophylaxis of the diabetes comprises utilizing the protein or DNA. A diagnostic agent and a diagnostic kit for the diabetes are obtained by using an antibody specifically recognizing the DNA or protein. A method for transporting the medicine for treating the diabetes comprises using the antibody specifically recognizing the ALK7 to the kidney.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to orphan receptor ALK7, activin AB which is a ligand protein of the receptor, activin B, DNA encoding the receptor, or diabetes containing the DNA encoding the ligand protein. And a diagnostic agent for the treatment and / or prevention of Further, a method for screening a drug for treating and / or preventing diabetes using the receptor, the ligand protein, a DNA encoding the receptor, or a DNA encoding the ligand protein, and a drug obtained by the screening method Regarding

[0002]

2. Description of the Related Art Diabetes is a type of lifestyle-related disease that has become the most serious problem in developed countries. Diabetes is a disease caused by an absolute deficiency of insulin, a hormone secreted from pancreatic β cells (type 1 diabetes), or a relative deficiency / insufficiency of action (type 2 diabetes). The pathological condition of type 2 diabetes is composed of two factors: insufficient insulin secretion from β cells and impaired insulin action (insulin resistance) in insulin target cells, and is a multifactorial disease in which multiple genetic factors and environmental factors are involved in a complex manner. Is known to be. In human type 2 diabetes, there is also a report that pancreatic β-cells are reduced to about 60% compared with healthy people [Tohoku J. Exp. Med., 12
9 , 273-283, (1979)], it has been suggested that a decrease in the number of β cells is associated not only with type 1 but also with type 2 diabetic patients.

Pancreatic transplantation has been carried out since the 1960s as a method of fundamentally recovering insulin secretory ability. In addition to the difficulty of securing donors and transplant materials, pancreatic transplantation requires a very advanced technique. The problem is that it is a necessary transplant [Nature Med., 6 , 250-251, 2000].

On the other hand, in recent years, technically easier pancreatic Langerhans islet transplantation has been attempted in comparison with pancreatic transplantation, but the 1-year positive dressing rate is as low as 27% and the pancreas of an individual is considered. It is said that it is difficult to obtain a sufficient number of Langerhans Island.

Pancreatic islets of Langerhans are not only insulin-secreting β cells but also glucagon-producing α cells, PP
It is composed of four types of cells, PP cells that produce and δ cells that produce somatostatin. These pancreatic endocrine cells are highly terminally differentiated cells, and almost no neoplastic phenomenon is observed in adults, and their cell division ability is extremely low. However, it has been mainly demonstrated in animal models that β-cell regeneration is observed under very special circumstances, particularly in the case of pancreatic destruction / Langerhans islet destruction (Diabetes, 45 , 941-945 (1996). ), D
iabetes, 46 , 1281-1290 (1997), Endocrinology, 138 ,
1750-1762 (1997), Development, 118 , 33-46, (199
3)]. Therefore, at least in rodents, the adult pancreas has the potential to proliferate and regenerate endocrine cells, and it is considered that adult pancreatic stem cells are present particularly in the pancreatic duct.

In humans, the above-mentioned phenomenon of adult β-cell renewal observed in rodents has not been observed, but in autopsy of a nondiabetic dead person, insulin consisting of several cells or less near the pancreatic conduit Positive cell (single beta cell
unit) is frequently observed. This s
The ingle beta cell unit has a smaller morphology than β cells that form islets of Langerhans, so it is more undifferentiated β
It is considered to be a cell, and it has been suggested that β cells may be regenerated in adult humans [Diabetologia, 4
1 , 629-633 (1998)].

[0007] As an alternative method for β-cell replacement to pancreatic transplantation, administration of a specific substance causes proliferation and β-cell proliferation in vivo.
As a substance that induces nascent birth, a poly ADP ribose synthetase inhibitor nicotinamide [Biomed.
s., 18 , 171 (1997), DiabetesRes. Clin. Pract., 41 ,.
1 (1998)], Reg protein [Proc. Natl. Acad. Sci. US
A, 91 , 3589 (1994)]. However, nicotinamide has a long-lasting insulin-secreting effect only for about 1 year in patients who have some insulin-secreting ability after oral administration after the onset of diabetes (Molecula.
r Medicine, 38 , 62-67 (2001)], and in transgenic mice overexpressing Reg, β cells were decreased in number and insulin content was decreased. As a result, impaired glucose tolerance was observed and 38% developed diabetes. Most of the causes of death in mice that died up to 100 weeks of age were pancreatic ducts, malignant tumors in the ovaries, and lymphoma formation [2000, Annual Meeting of the Diabetes Society of Japan]. There is also a report showing that the expression of the reg gene is associated with carcinogenesis [Brit. J. Cancer, 83 ,
188-1 (2000)], nicotinamide, and Reg protein are considered to be difficult to use for treating human diabetes.

[0008] In recent years, the existence of adult stem cells has been clarified in all tissues of adults, and the possibility of regenerative therapy for various organ failures and diseases is expected. If it is possible to efficiently induce differentiation of adult pancreas, such as adult pancreatic stem cells that are thought to be lurking mainly in pancreatic ducts, into β-cells that secrete insulin, in patients with type 2 diabetes as well as type 2 diabetes. It is thought that it will lead to the radical treatment of diabetes by compensating for the decrease in the number of pancreatic β cells and the decrease in function, but such a technique has not been established.

On the other hand, in recent analysis of the early stage of pancreatic development, it has been clarified that activin receptor-like kinase 7 (ALK7) is specifically expressed in pancreatic cells [Biochem. Biophys. Res. Comm. ., 254 , 707-712
(1999)]. ALK7 is a molecule belonging to the TGF-β receptor family [Mol. Cell Neurosci., 7 , 467-478 (199
6); J. Biol. Chem., 271 , 30603-30609 (1996)], I
Although it is classified as a type receptor, its ligand has not been clarified so far.

In general, it has been revealed that the functions of the receptors belonging to the TGF-β receptor family are carried by different molecules of type I and type II, and the ligand first binds to the type II receptor, The complex between the ligand and the type II receptor is I
A signal is transduced into the cell by forming a complex with the type I receptor and activating the type I receptor [Endocrino
l., 141 , 2281-2284 (2000)].

Table 10-510143 includes rat-derived AL
K7 was obtained and the amino acid and cDNA of the ALK7
Of the ALK7 mRNA, and analysis of the expression distribution of the ALK7 mRNA in each rat organ revealed that
7 is estimated to be effective in the treatment of neurological diseases.
There is no description that the ligand of 7 was specifically identified and obtained.

[0012] On the other hand, activins are cytokines belonging to the TGF-β superfamily and have a dimeric structure like TGF-β and the like, but there are five types of subunits (called β subunits) constituting them. Are known to exist, and it has been clarified that βC and βE exist in addition to βA and βB in mammals [Biochem. Biophys. Res.
Commun., 228 , 669-674 (1996); Biochim. Biophys. A
cta., 1307 , 145-148 (1996)]. The activins that have been confirmed to be expressed as proteins in mammals so far are activin A (βA-βA) and activin AB (β.
A-βB) and activin B (βB-βB). The receptor constitution of activins (activin A, activin AB, activin B) is typical of the TGF-β receptor family as described above, and it binds to the type II receptor (ActRIIA or ActRIIB), and its complex is Type I receptor (AL
It is considered that the signal is transmitted by activating K4) [Endocrinol., 141 , 2281-2284 (2
000)]. In addition, according to the current knowledge, activin A, AB, B
No clear difference in activity was observed between the two.

Analysis of mutant type II activin receptor gene deficient in enzymatic activity and constitutively active type I activin receptor gene-introduced mice pointed out that activins are important for functional differentiation of pancreatic β cells. However, [J. Clin.
Invest., 102 , 294-301 (1998)], the effect of function redundancy with other molecules belonging to the TGF-β family was not determined, and the details of the function of activins were not clarified. No functional difference between activins has been suggested. So far, βB has been expressed in the pituitary, ovary, etc.
It has been clarified that it is relatively localized in comparison with (Proc. Natl. Acad. Sci. USA, 85 , 247-251 (198
8)]. Since then, it has been reported that both βA and βB are expressed in the pancreas [Endocrinology, 133 , 624-630 (1
993); FEBS Letters, 319 , 217-220 (1993); J. Gastro
intest. Surg., 4 , 269-275 (2000)], the results of these studies only show studies on mRNA expression, and it is not pointed out that activin A, AB, and B have functional differences. .

As described above, regarding activins, it has been suggested that they have some action on pancreatic function and the like, but there is no finding suggesting a functional difference between each molecular species, and activin A, Occurrence of pancreas between AB and B
It is not expected that different functions such as differentiation and function control are carried. As described above, the search for a pancreatic regenerating factor has attracted attention as a therapeutic drug having a novel pancreatic function improving action, but a molecule applicable to actual medical treatment has not been specified. Further, it has been clarified that ALK7 is expressed in pancreatic β cells, and it has been suggested that ALK7 plays some role in functional control of the pancreas, but its specific function and its ligand are known. Not not.

[0015]

[Problems to be Solved by the Invention] Adult pancreatic stem cells, which are considered to be present in the adult pancreas, can be induced to differentiate into functionally mature pancreatic β cells by secreting insulin, and patients with type 1 and type 2 diabetes mellitus. It will lead to the radical treatment of diabetes by compensating for the decrease in the number of pancreatic β-cells and the decrease in function in pancreas.
It is desired to identify a molecule having such activity and to develop a drug using it to improve pancreatic function.

That is, the object of the present invention is to provide a substance exhibiting an action of promoting glucose-dependent insulin secretion as a medicine for treating and / or preventing diabetes.

[0017]

[Means for Solving the Problems] The present inventors have conducted extensive studies to solve the above-mentioned problems, and have been shown to be a type I of the TGF-β receptor family known to be specifically expressed in pancreatic β cells. It was identified that the ligands of ALK7 belonging to the type I receptor are known activins AB and B. Furthermore, it was clarified that information is transmitted through the type I receptor ALK7 and the type II receptor ActRIIA expressed in pancreatic β cells. Furthermore, they have found that the administration of activin AB to a pancreatic β-cell-like model cell has a glucose-dependent insulin secretion promoting action, and completed the present invention. That is, the present invention provides the following (1) to (35)
Is provided.

(1) Activin AB, Activin B,
And a medicament for treating and / or preventing diabetes, which comprises a protein selected from the group consisting of ALK7 and ALK7 as an active ingredient. (2) A medicament for treating and / or preventing diabetes, which contains any of the following proteins as an active ingredient. [A] A dimer protein consisting of the amino acid sequence represented by SEQ ID NO: 1 and the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; [b] represented by SEQ ID NO: 1 A dimer composed of a polypeptide consisting of an amino acid sequence and a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acid residues are deleted, substituted, inserted and / or added. A protein having a function as a ligand for ALK7; [c] from an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1 And a dimeric protein consisting of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, A protein having a function as a ligand of LK7; or [d] a polypeptide comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1, And a dimeric protein consisting of a polypeptide consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2, Protein that functions as a ligand:

(3) A medicament for treating and / or preventing diabetes which comprises any of the following proteins as an active ingredient. [A] a dimeric protein composed of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; [b] a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and represented by SEQ ID NO: 2 A dimeric protein composed of a polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence, the protein having a function as a ligand of ALK7; Or [c] a dimeric protein composed of a polypeptide consisting of an amino acid sequence having one or more amino acid residues deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2 , A protein having a function as a ligand for ALK7:

(4) A medicine for the treatment and / or prevention of diabetes, which contains any of the following proteins as an active ingredient. [A] c containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein encoded by DNA; or [b] c containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein having an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in an amino acid sequence encoded by DNA, and has a function as a receptor protein for activin AB or activin B protein:

(5) A medicament for treating and / or preventing diabetes, which comprises any of the following polypeptides as an active ingredient. [A] a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 2; or [b] an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in SEQ ID NO: 1 or 2. A polypeptide which comprises a protein having a function as a ligand of ALK7,

(6) The drug according to any one of (1) to (5) above, wherein the drug for treating and / or preventing diabetes has a pancreatic function improving action. (7) The medicine according to any one of (1) to (5) above, wherein the medicine for treating and / or preventing diabetes has an action of promoting insulin secretion from pancreatic β cells. (8) The medicine according to any one of (1) to (5) above, wherein the medicine for treating and / or preventing diabetes has an action of promoting the regeneration of exocrine pancreatic cells and endocrine pancreatic cells. (9) Activin AB, Activin B, and ALK
An insulin secretagogue comprising a protein selected from the group consisting of 7 as an active ingredient.

(10) Activin βA chain, activin βB
A gene therapeutic agent for treating and / or preventing diabetes, which comprises a chain or a DNA encoding ALK7. (11) A gene therapeutic agent for treating and / or preventing diabetes, which comprises any of the following DNAs. [A] DNA encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2; [b] One or more amino acid residues deleted or substituted in the amino acid sequence represented by SEQ ID NO: 1 or 2. Insert and /
Or a DNA comprising a polypeptide consisting of an added amino acid sequence and encoding a protein that constitutes a protein having a function as a ligand for ALK7; or [c] containing the nucleotide sequences represented by SEQ ID NOs: 3 and 4 D
NA:

(12) A gene therapeutic agent for treating and / or preventing diabetes, which comprises any of the following DNAs: [A] c containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
DNA encoding a protein encoded by DNA; or [b] c containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein having an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in an amino acid sequence encoded by DNA, and has a function as a receptor protein for activin AB or activin B DNA encoding a protein:

(13) The gene therapeutic agent according to any one of the above (10) to (12), wherein the gene therapeutic agent for treating and / or preventing diabetes has a pancreatic function improving action. (14) The gene therapeutic agent according to any one of (10) to (12) above, wherein the gene therapeutic agent for treating and / or preventing diabetes has an action of promoting insulin secretion from pancreatic β cells. (15) The gene therapeutic agent for treating and / or preventing diabetes has an action of promoting regeneration of pancreatic exocrine cells and pancreatic endocrine cells (10) to (1) above.
The gene therapeutic agent according to any one of 2). (16) An insulin secretagogue, which comprises a DNA encoding activin βA chain, activin βB chain, or ALK7.

(17) A diagnostic agent for diabetes which comprises the DNA, the oligonucleotide or the derivative of the oligonucleotide according to any of the following [a] to [c]. [A] activin βA chain, activin βB chain, or AL
DNA encoding K7, or DNA that is a partial fragment of these DNAs; [b] DNA consisting of the nucleotide sequences represented by SEQ ID NO: 3 or 4, cDNA containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6, or DNA that is a partial fragment of those DNAs
DNA selected from [c] cDNA containing the nucleotide sequence represented by SEQ ID NO: 3 or 4, or the nucleotide sequence represented by SEQ ID NOS: 5 and 6.
Having a sequence consisting of consecutive 5 to 60 bases in the base sequence of, an oligonucleotide having a sequence complementary to the oligonucleotide, or a derivative of the oligonucleotide.

(18) A diagnostic agent for diabetes containing an antibody that recognizes a protein selected from the group consisting of activin AB, activin B and ALK7. (19) A diagnostic kit for diabetes, which comprises the DNA described in (17) above, an oligonucleotide or a derivative of the oligonucleotide, or the antibody described in (18).

(20) (i) The expression level of the polypeptide or protein in cells expressing the polypeptide or protein according to any one of the following [a] to [d], and (i)
i) A cell expressing the polypeptide or protein is compared with the expression level of the polypeptide or protein when the test sample is contacted, and the following [a] to [d] are compared with the test sample.
13. A method for screening a drug for treating and / or preventing diabetes, which comprises selecting a compound that increases the expression level of the polypeptide or protein according to any one of 1. [A] Activin AB, Activin B, and ALK7
A protein selected from the following: [b] a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, and a polypeptide selected from a protein encoded by a cDNA containing the nucleotide sequences represented by SEQ ID NO: 5 or 6; Protein: [c] A polypeptide comprising an amino acid sequence in which one or more amino acid residues have been deleted, substituted, inserted and / or added in the amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 or 2, And a polypeptide constituting a protein having a function as a ligand for ALK7; or [d] containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence encoded by DNA, and has activity as activin AB or activin B receptor protein protein:

(21) (i) Function of cells expressing the polypeptide or protein described in any of [a] to [d] below, and (ii) test with cells expressing the polypeptide or protein. Screening of a drug for treatment and / or prevention of diabetes, which comprises comparing a cell function when contacted with a sample and selecting a compound having an activity of controlling the cell function from a test sample Method. [A] Activin AB, Activin B, and ALK7
A protein selected from the following: [b] a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, and a polypeptide selected from a protein encoded by a cDNA containing the nucleotide sequences represented by SEQ ID NO: 5 or 6; Protein: [c] A polypeptide comprising an amino acid sequence in which one or more amino acid residues have been deleted, substituted, inserted and / or added in the amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 or 2, And a polypeptide constituting a protein having a function as a ligand for ALK7; or [d] containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence encoded by DNA, and has activity as activin AB or activin B receptor protein protein:

(22) The above-mentioned (20) or (2), wherein the cell is a cell expressing a type II activin receptor.
The screening method described in 1). (23) The cell according to (2) above, wherein the cell is a pancreatic β cell type cell.
The screening method according to any one of 0) to (22). (24) A DNA in which a reporter gene is linked downstream of a region that controls transcription of a gene encoding the polypeptide or protein described in any of [a] to [d] below.
A compound which brings a test sample into contact with a transformant transformed with a DNA containing DNA and increases the expression of a gene encoding the polypeptide or protein described in any one of [a] to [d] below from the test sample A method for screening a medicine for the treatment and / or prevention of diabetes, which comprises: [A] Activin AB, Activin B, and ALK7
A protein selected from the following: [b] a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, and a polypeptide selected from a protein encoded by a cDNA containing the nucleotide sequences represented by SEQ ID NO: 5 or 6; Protein: [c] A polypeptide comprising an amino acid sequence in which one or more amino acid residues have been deleted, substituted, inserted and / or added in the amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 or 2, And a polypeptide constituting a protein having a function as a ligand for ALK7; or [d] containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence encoded by DNA, and has activity as activin AB or activin B receptor protein protein:

(25) The method according to any one of (20) to (24), wherein the drug for treating and / or preventing diabetes is a compound having a pancreatic function improving action. (26) The method according to any of (20) to (24) above, wherein the drug for treating and / or preventing diabetes is a compound having an action of promoting insulin secretion from pancreatic β cells. (27) The method according to any of (20) to (24) above, wherein the drug for treating and / or preventing diabetes is a compound having an action of promoting regeneration of exocrine pancreatic cells or endocrine pancreatic cells. (28) A compound obtained by the method according to any one of (20) to (24) above, or a pharmaceutically acceptable salt thereof.

(29) The compound described in (28) above,
Alternatively, a medicament for the treatment and / or prevention of diabetes containing a pharmacologically acceptable salt thereof. (30) A medicament for treating and / or preventing diabetes, which is obtained by binding the antibody recognizing ALK7 and the compound according to (28) above or a pharmaceutically acceptable salt thereof. (31) Treatment and / or treatment of diabetes according to the above (29) or (30), which is a drug having a pancreatic function improving action.
Or medicine for prevention. (32) The medicament for treating and / or preventing diabetes according to (29) or (30) above, which is a medicament having an action of promoting insulin secretion from pancreatic β cells. (33) The above-mentioned (29) or (3) which is a drug having an action of promoting the regeneration of pancreatic exocrine cells or pancreatic endocrine cells.
A medicament for treating and / or preventing diabetes according to 0).

(34) Antibodies that recognize ALK7 and AL
A pharmaceutical composition for treating and / or preventing diabetes, which comprises selectively accumulating a fusion antibody obtained by binding with a pharmaceutical agent for treating and / or preventing diabetes that acts on K7 in the pancreas. Transportation method. (35) The method according to (34) above, wherein the medicament for treating and / or preventing diabetes is the compound according to (28) above or a pharmaceutically acceptable salt thereof.

According to still another aspect of the present invention, a therapeutically and / or prophylactically effective amount of the protein or polypeptide according to any one of (1) to (5) above is administered to mammals including humans. And / or treatment of diabetes, including
Alternatively, a method for prevention is provided. According to still another aspect of the present invention, there is provided use of the protein or polypeptide according to any one of (1) to (5) above in the manufacture of a medicament for treating and / or preventing diabetes. .

According to still another aspect of the present invention, a therapeutically and / or prophylactically effective amount of the above (10) to (12).
There is provided a method for treating and / or preventing diabetes, which comprises administering the DNA according to any of the above 1 to a mammal including a human. According to still another aspect of the present invention, there is provided use of the DNA according to any one of (10) to (12) above in the production of a gene therapeutic agent for treating and / or preventing diabetes.

[0036]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of proteins contained in the medicament of the present invention include activin AB, activin B, and ALK7. Activin AB, Activin B
May be a protein derived from a natural product or a protein produced by a genetic engineering method as long as it is a protein having an activity as a ligand protein of ALK7. Examples of the naturally derived protein include human and monkey. , Pig, cow,
AL derived from all mammals including sheep, horses and rats
The ligand protein of K7 can be mentioned. Specific examples of activin AB include a dimeric protein composed of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 and a polypeptide having the amino acid sequence represented by SEQ ID NO: 2. A specific example of activin B is a dimeric protein composed of a polypeptide having the amino acid sequence represented by SEQ ID NO: 2. ALK7 is a protein produced by a genetic engineering method as long as it is a protein that has an activity as ALK7, that is, an protein that is activated by activin AB or activin B and promotes insulin secretion of cells, even if it is a naturally derived protein. For example, the naturally-occurring protein may be ALK7 derived from any mammal such as human, monkey, pig, cow, sheep, horse and rat. A specific example of ALK7 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 9.

The protein contained in the medicament of the present invention includes (a) one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 1, deleted, substituted, inserted and / or
Alternatively, a protein which is a dimeric protein composed of a polypeptide consisting of an added amino acid sequence and a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 and having an activity as an ALK7 ligand, (b) SEQ ID NO: 1 In the amino acid sequence represented by SEQ ID NO: 1, one or more amino acid residues are deleted, substituted, inserted and / or added to the polypeptide, and in the amino acid sequence represented by SEQ ID NO: 2, one or more amino acid residues are A dimeric protein composed of a polypeptide consisting of a deleted, substituted, inserted and / or added amino acid sequence, and ALK
7. A protein having activity as a ligand, (c) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 and one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 2 deleted, substituted, inserted and / Or a protein which is a dimeric protein composed of a polypeptide consisting of an added amino acid sequence and has an activity as an ALK7 ligand, (d) one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 2. Is a dimeric protein composed of a polypeptide consisting of a deleted, substituted, inserted and / or added amino acid sequence, and ALK7
A protein having activity as a ligand, (e) one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence encoded by the cDNA containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6. Also included are proteins consisting of different amino acid sequences and having ALK7 activity.

The polypeptides contained in the medicament of the present invention include activin βA chain and activin β.
The B chain can be mentioned. Specifically, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 can be mentioned. Furthermore, a protein which is a polypeptide comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1 and which has an activity as activin AB Constituent polypeptide, SEQ ID NO: 2
A polypeptide comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence represented by
Alternatively, a polypeptide constituting a protein having an activity as activin B is also included as the polypeptide contained in the drug of the present invention.

Furthermore, a fusion protein with the above protein or polypeptide obtained by adding various polypeptides to the above protein or polypeptide is also a protein or polypeptide contained in the medicament of the present invention. Examples of the fusion protein include a fusion protein in which maltose binding protein, oligohistidine, antibody peptide epitope, human immunoglobulin constant region, protein A and the like are bound. The fusion protein can also have a sequence that can be specifically cleaved by a known method.

One or more amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1 or 2 or the amino acid sequence encoded by the cDNA containing the nucleotide sequences represented by SEQ ID NO: 5 and 6. And / or a protein having an added amino acid sequence and having activity as activin AB, activin B or ALK7, or a polypeptide constituting a protein having activity as activin AB or activin B is Molecu
lar Cloning, A Laboratory Manual, Second Edition,
Cold Spring Harbor Laboratory Press (1989) (hereinafter,
Molecular cloning 2nd edition), Current
Protocols in Molecular Biology, John Wiley & Sons
(1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acid
s Research, 10 , 6487 (1982), Proc. Natl. Acad. Sc
i. USA, 79 , 6409 (1982), Gene, 34 , 315 (1985), Nucl
eic Acids Research, 13 , 4431 (1985), Proc. Natl. A
cad. Sci. USA, 82 , 488 (1985) and the like, using the site-directed mutagenesis method, for example, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, or SEQ ID NOS: 5 and 6 It can be obtained by introducing a site-specific mutation into the DNA encoding the protein encoded by the cDNA containing the represented nucleotide sequence.

The number of amino acids to be deleted, substituted, inserted and / or added is 1 or more and the upper limit is not particularly limited, but deletion, substitution or addition can be carried out by a well-known method such as the above site-specific mutation method. It is a number of about 1 to several tens, preferably 1 to 20, more preferably 1 to 1.
The number is 0, and more preferably 1 to 5.

In the amino acid sequence of the protein or polypeptide contained in the drug of the present invention, one or more amino acid residues are deleted, substituted, inserted and / or added,
Deletion of one or more amino acid residues at any and one or more amino acid sequence positions in the same sequence,
Meaning that there are substitutions, insertions and / or additions, deletions, substitutions, insertions and / or additions may occur at the same time, and the amino acid residues that are substituted, inserted or added may be of natural or unnatural type. It doesn't matter. Natural amino acid residues include L-alanine, L-asparagine, L-aspartic acid,
L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like can be mentioned.

Examples of amino acid residues that can be mutually substituted are shown below. Amino acid residues included in the same group can be mutually substituted. Group A: Leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid C group: asparagine, glutamine D group: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid E group: proline, 3 -Hydroxyproline, 4-hydroxyproline F group: serine, threonine, homoserine G group: phenylalanine, tyrosine

Further, the protein or polypeptide contained in the medicament of the present invention is a protein having activity as activin AB or activin B or a polypeptide constituting a protein having activity as activin AB or activin B. Preferably has at least 60% or more, usually 80% or more, and particularly 95% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 2. In addition, in order for the protein contained in the drug of the present invention to have an activity as ALK7, SEQ ID NO: 5
It is preferable that the amino acid sequence has at least 60% or more, usually 80% or more, and particularly 95% or more identity with the amino acid sequence encoded by the cDNA containing the nucleotide sequences represented by 6 and 6.

The identity of amino acid sequences and base sequences is Karl.
in and Altschul's algorithm BLAST [Pro. Natl.
Acad. Sci. USA, 90 , 5873 (1993)] and FASTA [Methods
Enzymol., 183 , 63 (1990)]. Based on this algorithm BLAST, BLASTN and BLA
A program called STX is being developed [J. Mol. B
iol., 215 , 403 (1990)]. When the nucleotide sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. When analyzing the amino acid sequence by BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters for each program are used.
Specific methods of these analysis methods are known (http: /
/www.ncbi.nlm.nih.gov.).

As a method for confirming whether the protein or polypeptide contained in the medicament of the present invention has activity as a ligand for ALK7, for example, a group expressing the type II activin receptor and ALK7 can be used. Method for measuring the response of a cell when a modified cell is brought into contact with the protein, or insulin secretion from the cultured cell when brought into contact with a cultured cell having the trait of pancreatic β cell and the polypeptide or protein A method for measuring the amount can be given.

Examples of cell responses include arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, and intracellular c.
AMP production, intracellular cAMP reduction, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, cell growth activity, melanin pigment aggregation or diffusion, Alternatively, the expression level of the reporter gene can be raised.

The protein contained in the medicine of the present invention is AL
As a method for confirming whether or not it has activity as K7, the response of the recombinant cell expressing the type II activin receptor and the protein to the response of the cell when activin AB or activin B is contacted, or Examples thereof include a method of measuring the amount of insulin secreted from the cells.
The above-mentioned response can be mentioned as an example of a cell response.

1. Method for obtaining activin AB, activin B and ALK7 Like activin AB and activin B, full-length c
When the DNA is known, based on the full-length cDNA,
If necessary, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.

When the full-length cDNA is not known like human ALK7, the rat-derived ALK7 gene having the nucleotide sequence represented by SEQ ID NO: 10 or known human A
A cDNA encoding human ALK7 can be isolated by using, for example, a human pancreas cDNA library by using EST (expressed sequence tag) of LK7 as a probe. If the obtained cDNA is not full-length cDNA, screening of a cDNA library using this clone as a probe, or RAC
Method E [rapid amplification of cDNA ends; Frohman MA
et al., Proc. Natl. Acad. Sci. USA, 85 , 8998 (19
88)] makes it possible to obtain full-length cDNA.

The nucleotide sequence of the obtained cDNA clone is D
It is determined using an NA sequencer or the like, and its base sequence is translated into an amino acid sequence in each frame, and then compared with the amino acid sequence of known rat-derived ALK7, whereby the obtained cDNA is a DNA encoding human ALK7. You can check if there is. Using the obtained DNA encoding human ALK7, a DNA fragment having an appropriate length is prepared as necessary as described above.

A recombinant expression vector of the protein is prepared by inserting the DNA fragment or the full-length cDNA downstream of the promoter in the expression vector. Specifically, for example, an expression vector into which a cDNA encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2 or a full-length cDNA containing the nucleotide sequences represented by SEQ ID NO: 5 and 6 is inserted. To make. Alternatively, cDNA encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and cDNA encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2
A and A may be inserted into the same expression vector arranged so that the expression is regulated by an independent promoter.

The recombinant expression vector is introduced into a host cell suitable for the expression vector. As the host cell, it can be used all it can express the DNA of interest, e.g., Escherichia (Escherichia) genus Serratia (Serratia) genus Corynebacterium (Corynebacter
ium) genus Brevibacterium (Brevibacterium) genus,
Pseudomonas (Pseudomonas) genus Bacillus (Bacillu
s ) genus, bacteria belonging to Microbacterium genus, Kluyveromyces genus,
Saccharomyces (Saccharomyces) sp., Schizosaccharomyces (Shizosaccharomyces) genus Trichosporon (Trichosporon) genus, Shiwaniomisesu (Schwanniomyce
s ) Yeast belonging to the genus or the like, animal cells, insect cells and the like can be used.

As the expression vector, one that can autonomously replicate in the host cell or can be integrated into the chromosome and contains a promoter at a position where the DNA can be transcribed is used.

When bacteria are used as host cells, the D
The NA recombinant expression vector is preferably a recombinant expression vector capable of autonomous replication in the bacterium and at the same time composed of a promoter, a ribosome binding sequence, the DNA of the polypeptide and a transcription termination sequence. The vector may contain a gene that controls the promoter.

Examples of the expression vector include pBTrp2,
pBTac1, pBTac2 (both manufactured by Boehringer Mannheim), pKK233-2 (manufactured by Amersham Pharmacia Biotech),
pSE280 (Invitrogen), pGEMEX-1 (Promega), p
QE-8 (manufactured by QIAGEN), pKYP10 [JP-A-58-110600], pKY
P200 〔Agricultural Biological Chemistry, 48 , 669
(1984)], pLSA1 [Agric. Biol. Chem., 53 , 277 (198
9)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82 , 4306.
(1985)], pBluescript II SK (-) (manufactured by Stratagene),
pGEX (Amersham Pharmacia Biotech), pET-3 (Nov
agen), pTerm2 (USP4686191, USP4939094, USP5160
735), pSupex, pUB110, pTP5, pC194, pEG400 (J. Bact
eriol., 172 , 2392 (1990)] and the like.

As an expression vector, an appropriate distance (for example, 6 to 18 bases) is provided between the Shine-Dalgarno sequence which is a ribosome binding sequence and the initiation codon.
It is preferable to use the one adjusted to.

Any promoter may be used as long as it functions in the host cell. For example, trp
Promoter (Ptrp), lac promoter (Plac), P
_L promoter, P _R promoter, promoters derived from Escherichia coli or phage such as T7 promoter, SPO1
Examples thereof include a promoter, SPO2 promoter, penP promoter and the like. In addition, a promoter in which two Ptrp are connected in series (Ptrpx2), a tac promoter,
An artificially designed and modified promoter such as letI promoter [Gene, 44 , 29 (1986)] and lacT7 promoter can also be used.

Activin AB, Activin B, and A
The production rate of the desired polypeptide or protein can be improved by substituting the nucleotide sequence of the DNA encoding the LK7 so that the codon is optimal for the expression in the host cell. A transcription termination sequence is not necessarily required for the expression of the DNA encoding the above-mentioned polypeptide or protein, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

Examples of the host cell include microorganisms belonging to the genera Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., such as Escherichia coli XL1.
-Blue, Escherichia coli XL2-Blue, Escherichia coli
DH1, Escherichia coli MC1000, Escherichia coli KY
3276, Escherichia coli W1485, Escherichia coli JM1
09, Escherichia coli HB101, Escherichia coli No.4
9, Escherichia coli W3110, Escherichia coli NY49,
Bacillus s ubtilis , Bacillus amyloliquefaciens , Bre
vibacterium ammoniagenes , Brevibacterium immarioph
ilum ATCC14068, Brevibacterium saccharolyticum ATC
C14066, Corynebacterium glutamicum ATCC13032, Cory
nebacteriumglutamicum ATCC14067, Corynebacterium g
lutamicum ATCC13869, Corynebacte rium acetoacidophi
lum ATCC13870, Microbacterium ammoniaphilum ATCC15
354, Pseudomonas sp. D-0110 and the like.

As a method for introducing the recombinant expression vector, any method can be used as long as it is a method for introducing DNA into the above-mentioned host cell. For example, a method using calcium ion [Proc. Natl. Acad. Sci. USA, 69 , 2110 (197
2)], protoplast method [JP-A-63-248394], or G
ene, 17 , 107 (1982) and Molecular & General Genetics,
168 , 111 (1979) and the like.

When yeast is used as a host cell, examples of expression vectors include YEp13 (ATCC3711).
5), YEp24 (ATCC37051), YCp50 (ATCC3741)
9), pHS19, pHS15 and the like.

Any promoter may be used so long as it functions in yeast. For example, PHO5
Promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, g
Examples thereof include al 10 promoter, heat shock protein promoter, MFα1 promoter, CUP 1 promoter and the like.

As host cells, Saccharomyces cerevisiae , Schizosaccharomyces pombe , Kluyveromyces lactis ,
Trichosporon pullulan
s ), Schwanniomyces arvius
alluvius ) and the like.

As a method for introducing the recombinant expression vector, any method can be used as long as it is a method for introducing DNA into yeast. For example, the electroporation method [Methods.
inEnzymol., 194 , 182 (1990), spheroplast method [Proc. Natl. Acad. Sci., USA, 75 , 1929 (1978)],
Lithium acetate method [J. Bacteriol., 153 , 163 (1983)],
The method described in Proc. Natl. Acad. Sci. USA, 75 , 1929 (1978) can be mentioned.

When an animal cell is used as a host cell, an expression vector such as pcDNA1.1 (In
vitrogen), pCDM8 (Invitrogen), pA
GE107 [JP-A-3-22979; Cytotechnology, 3 , 133
(1990)], pAS3-3 (JP-A-2-227075), pcDN
A1.1 / Amp (manufactured by Invitrogen), pREP4 (In
Vitrogen), pAGE103 [J. Biochem., 101 ,
1307 (1987)], pAGE210 and the like.

Any promoter can be used as long as it functions in animal cells.
IE of cytomegalovirus (human CMV)
date early) gene promoter, SV
40 early promoters, retrovirus promoters, metallothionein promoters, heat shock protein promoters, SRα promoters and the like can be mentioned. In addition, the enhancer of the IE gene of human CMV may be used together with the promoter.

As the host cells, human cells, Namalwa cells, monkey cells, COS cells,
Chinese hamster cells, CHO cells, H
BT5637 [JP-A-63-299] and the like can be mentioned.

As a method for introducing the recombinant expression vector, any method capable of introducing DNA into animal cells can be used. For example, the electroporation method [Cytotech
nology, 3 , 133 (1990)], calcium phosphate method (JP-A-2-227075), lipofection method [Proc. Natl. Aca
d. Sci., USA, 84 , 7413 (1987), Virology, 52 , 456
(1973)] and the like can be used.

When insect cells are used as host cells, for example, Baculovirus Expression Vectors, A Laboratory Manual (Baculoviru) is used.
Expression Vectors, A Laboratory Manual), Current Protocols in Molecular Biology Supplement 1-38 (1987-1997), Bio / Technolog
A polypeptide or protein can be expressed by the method described in y, 6 , 47 (1988) or the like.

That is, a recombinant gene transfer vector and a baculovirus are co-introduced into an insect cell to obtain a recombinant virus in the insect cell culture supernatant, and the recombinant virus is further infected with the polypeptide or The protein can be expressed. Examples of the gene transfer vector include pVL1392, pVL1393, and pBI.
ueBacIII (both manufactured by Invitrogen) and the like can be mentioned. Examples of the baculovirus include, for example, a virus that infects night-thief moth insects, Outgrapha californica nucleia polyhedrosis virus.
(Autographa californica nuclear polyhedrosis viru
s) etc. can be used.

Insect cells include Spodoptera frugiperd
Sf9 is a ovarian cells, Sf21 [Baculovirus Exp
ression Vectors, A Laboratory Manual, WHFreeman
andCompany, New York, (1992)], Trichoplusia ni ovary cells High 5 (manufactured by Invitrogen) and the like can be used.

Examples of the method for co-introducing the above-mentioned recombinant gene transfer vector and the above-mentioned baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method [JP-A-2-227075] and the lipofection method [Proc.
Natl. Acad. Sci., It is possible to increase the USA, 84, 7413 (1987)] and the like.

As a gene expression method, in addition to direct expression, secretory production, fusion protein expression and the like can be performed according to the method described in Molecular Cloning 2nd Edition. When expressed in yeast, animal cells or insect cells, a sugar- or sugar-chain-added polypeptide or protein can be obtained.

Recombinant D incorporating the DNA of the protein
The NA-bearing transformant is cultured in a medium, the desired polypeptide or protein is produced and accumulated in the culture, and the desired polypeptide or protein is collected from the culture to obtain the desired polypeptide or protein. It can be manufactured. Activin AB, Activin B or A
The method for culturing the transformant for producing LK7 in a medium can be carried out according to the usual method used for culturing host cells.

When the transformant uses a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host cell, the medium for culturing these transformants is a carbon source that can be assimilated by the host cell.
Either a natural medium or a synthetic medium may be used as long as it is a medium containing a nitrogen source, an inorganic substance and the like and capable of efficiently culturing a transformant.

Any carbon source may be used as long as it can be assimilated by each host cell, and glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, propionic acid, etc. Organic acids, alcohols such as ethanol and propanol can be used.

Examples of the nitrogen source include ammonium salts of various inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract and corn steep. Liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof are used.

As the inorganic substance, potassium dibasic phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.

The culture is carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is preferably 15 to 40 ° C., and the culturing time is usually 16 hours to 7 days. The pH is maintained at 3.0 to 9.0 during the culture. To adjust the pH,
It is performed using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. If necessary, antibiotics such as ampicillin and tetracycline may be added to the medium during the culturing.

When culturing a transformant using an expression vector having an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a transformant using an expression vector having a lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like is added,
When culturing a transformant using an expression vector having a trp promoter, indole acrylic acid (IA
A) and the like may be added to the medium.

As a medium for culturing the transformant obtained by using animal cells as host cells, R which is generally used is used.
PMI1640 medium [The Journal of the American Me
dical Association, 199 , 519 (1967)], Eagle's MEM medium [Science, 122 , 501 (1952)], Dulbecco's modified MEM medium [Virology, 8 , 396 (1959)], 199.
Medium (Proceeding of the Society for the Biological
Medicine, 73 , 1 (1950)] or a medium obtained by adding fetal bovine serum or the like to these mediums.

The culture is usually carried out at pH 6-8, 30-40 ° C.
It is performed for 1 to 7 days under conditions such as the presence of 5% CO ₂ . Also,
If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.

As a medium for culturing a transformant obtained by using insect cells as host cells, T is generally used.
NM-FH medium (Pharmingen), Sf-900 I
ISFM medium (Life Technologies), ExCel
1400, ExCell405 (both JRH Bioscien
ces), Grace's Insect Medium (Grace, TCC, Na
ture, 195 , 788 (1962)] and the like can be used. Culturing is usually carried out under the conditions of pH 6 to 7, 25 to 30 ° C.
Do ~ 5 days. If necessary, an antibiotic such as gentamicin may be added to the medium during the culture.

In order to isolate and purify the desired polypeptide or protein from the culture of the transformant, conventional methods for isolating and purifying the polypeptide or protein may be used. For example, when the polypeptide or protein is produced in a dissolved state in the cells, after the culture is completed, the cells are collected by centrifugation and suspended in an aqueous buffer solution, followed by ultrasonic crusher, French press, Mantongaurin homogenizer. The cells are crushed with, for example, Dynomill to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract,
A conventional method for isolating and purifying a polypeptide or protein, that is,
Solvent extraction method, salting-out method with ammonium sulfate, desalting method, precipitation method with organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), etc. , S-Sepharos
e FF (Amersham Pharmacia Biotech) cation exchange chromatography method using a resin, butyl sepharose, hydrophobic chromatography method using a resin such as phenyl sepharose, gel filtration method using a molecular sieve,
A method such as an affinity chromatography method, a chromatofocusing method, or an electrophoretic method such as isoelectric focusing can be used alone or in combination to obtain a purified preparation of a polypeptide or protein.

When the polypeptide or protein is produced by forming an insoluble substance in cells, the cells are recovered, then crushed and centrifuged to recover the insoluble substance of the polypeptide or protein as a precipitate fraction. To do. The recovered insoluble body of polypeptide or protein is solubilized with a protein denaturing agent. After diluting or dialyzing the solubilized solution to reduce the concentration of the protein denaturant in the solubilized solution, the structure of the polypeptide or protein is restored to a normal three-dimensional structure, and then the same isolation and purification method as described above is performed. A purified preparation of polypeptide or protein is obtained.

When the polypeptide or protein or its sugar modified product is secreted extracellularly, the polypeptide or protein or its sugar modified product can be recovered from the culture supernatant. That is, a purified preparation can be obtained by recovering the culture supernatant from the culture by a method such as centrifugation and using the same isolation and purification method as described above from the culture supernatant.

Examples of the polypeptide or protein thus obtained include, for example, polypeptides having the amino acid sequences represented by SEQ ID NOS: 1 and 2 and polypeptides having the amino acid sequences represented by SEQ ID NOS: 1 and 2. Constituent dimeric protein, dimer protein composed of a polypeptide having the amino acid sequence represented by SEQ ID NO: 2, and protein encoded by cDNA containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6, etc. Can be raised.

Further, the polypeptide or protein can be labeled with F
moc method (fluorenylmethyloxycarbonyl method), t
It can also be produced by a chemical synthesis method such as the Boc method (t-butyloxycarbonyl method). Also, US Advanced
ChemTech, Perkin-Elmer, Amersham Pharmaci
a Biotech, USA Protein Technology Instrument, USA Synthecell-Vega, USA PerSeptive,
It can also be synthesized using a peptide synthesizer manufactured by Shimadzu Corporation.

2. Preparation of antibody that specifically recognizes activin AB, activin B or ALK7 Purified preparation of partial fragment of polypeptide or protein obtained in the above 1 or amino acid sequence represented by SEQ ID NO: 1 or 2, SEQ ID NO: 5 and By using the amino acid sequence of the protein encoded by the cDNA containing the nucleotide sequence represented by 6, it is possible to prepare an antibody that recognizes activin AB, activin B or ALK7, such as a polyclonal antibody or a monoclonal antibody.

(1) Preparation of Polyclonal Antibody Activin AB, activin B or ALK7 or a purified preparation of a partial fragment polypeptide of the protein, or a peptide having a partial amino acid sequence of the protein is used as an antigen and administered to animals. By doing so, a polyclonal antibody can be prepared. As the animal to be administered, rabbit, goat, rat, mouse, hamster and the like can be used.

The dose of the antigen is 50 to 1 per animal.
00 μg is preferred. When using a peptide, the peptide is keyhole limpet haemocy.
It is desirable to use an antigen that is covalently bound to a carrier protein such as anin) or bovine thyroglobulin. The peptide used as an antigen can be synthesized by a peptide synthesizer.

The antigen is administered 1-2 times after the first administration.
Do 3-10 times every week. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with the antigen used for immunization is confirmed by enzyme immunoassay [enzyme immunoassay (ELIS
Law A): Published by Medical Shoin (1976), Antibodies-A Lab
oratory Manual, Cold Spring Harbor Laboratory (198
8)] and so on.

Polyclonal antibodies can be obtained by obtaining serum from a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum. As a method of separation and purification,
Centrifugation, salting out with 40-50% saturated ammonium sulfate, caprylic acid precipitation [Antibodies, A Laboratory manua
l, Cold Spring Harbor Laboratory, (1988)], or DEAE-Sepharose column, anion exchange column,
Examples include a method in which chromatography using a protein A or G-column, a gel filtration column, or the like is used alone or in combination.

(2) Preparation of Monoclonal Antibody (a) Preparation of Antibody-Producing Cells Rats whose sera showed a sufficient antibody titer against the partial fragment polypeptide of the protein used for immunization were used as a source of antibody-producing cells. To serve. Spleens are extracted 3 to 7 days after the final administration of the antigenic substance to the rats showing the antibody titer.

The spleen is cut into small pieces in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers and centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded. The splenocytes of the obtained precipitate fraction were treated with Tris-ammonium chloride buffer (pH 7.6).
After treatment with 5) for 1 to 2 minutes to remove red blood cells, the spleen cells obtained are washed 3 times with MEM medium and used as antibody-producing cells.

(B) Preparation of myeloma cells As myeloma cells, cell lines obtained from mouse or rat are used. For example, 8-azaguanine resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (hereinafter,
Abbreviated as P3-U1) [Curr. Topics. Microbiol. Immuno
l., 81 , 1 (1978), Europ. J. Immunol., 6 , 511 (197
6)), SP2 / 0-Ag14 (SP-2) (Nature, 276 , 269 (197
8)], P3-X63-Ag8653 (653) [J. Immunol., 123 , 1548.
(1979)], P3-X63-Ag8 (X63) (Nature, 256 , 495 (197
5)] etc. can be used. These cell lines are 8-
Azaguanine medium [RPMI-1640 medium with glutamine (1.5 mmol / l), 2-mercaptoethanol (5 x
10 ^-5 mol / l), gentamicin (10 μg / ml)
And fetal calf serum (FCS) (CSL, 10%)-added medium (hereinafter referred to as normal medium) to which 8-azaguanine (15 μg / ml) was added] The cells are cultured in a normal medium 3 to 4 days before, and 2 × 10 ⁷ or more of the cells are used for fusion.

(C) Preparation of hybridoma The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) were mixed with MEM medium or PBS (disodium phosphate 1.83 g, monopotassium phosphate 0.21 g, Salt 7.6
Wash thoroughly with 5 g, distilled water 1 liter, pH 7.2),
The cells are mixed so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1, the mixture is centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.

The cells of the obtained precipitate fraction are thoroughly loosened,
To the cell group, while stirring, at 37 ° C., 10 ⁸ antibody-producing cells per polyethylene of glycol -1000 (P
EG-1000) 2g, MEM 2ml and dimethylsulfoxide (DMSO) 0.7ml mixed solution are added 0.2-1ml, and MEM medium 1-2ml is added several times every 1-2 minutes.

After the addition, MEM medium was added to bring the total volume to 50 m.
Prepare to be l. 5 times the prepared solution at 900 rpm
After centrifuging for minutes, discard the supernatant. The cells of the obtained precipitate fraction are loosely loosened and then gently sucked with a measuring pipette and gently blown out to blow HAT medium [hypoxanthine (10 ⁻⁴ mol / l) and thymidine (1.5 × 10 ^{− 5} m
ol / l) and aminopterin (4 × 10 ⁻⁷ mol / l) -containing medium] 100 ml.

100 ml of the suspension was added to a 96-well culture plate.
Dispense μl / well each, in a 5% CO ₂ incubator,
Incubate at 37 ° C. for 7 to 14 days.

After culturing, a part of the culture supernatant is taken and the antibodies [Antibodies, A Laboratory manual, Cold Sprin
g Harbor Laboratory, Chapter 14 (1988)] and the like, a hybridoma that specifically reacts with activin AB, activin B or a partial fragment polypeptide of ALK7 is selected.

The following methods may be mentioned as specific examples of the enzyme immunoassay method. During immunization, an appropriate plate is coated with activin AB, activin B or partial fragment polypeptide of ALK7 used as an antigen, and the hybridoma culture supernatant or the purified antibody obtained in (d) described below is reacted as a first antibody. Further, as a second antibody, an anti-rat or anti-mouse immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound or the like is further reacted, and then a reaction according to the labeling substance is performed to give activin AB, activin B or ALK7. Those that react specifically are selected as hybridomas that produce the monoclonal antibody of the present invention.

Using the hybridoma, cloning was repeated twice by the limiting dilution method [the first time was HT medium (medium obtained by removing aminopterin from HAT medium), the second time was using normal medium], and stable. Those showing a strong antibody titer are selected as hybridoma strains producing the monoclonal antibody of the present invention.

(D) Preparation of Monoclonal Antibody Pristane-treated mice of 8 to 10 weeks of age treated with 0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane, intraperitoneally and raised for 2 weeks) or Nude mice are intraperitoneally injected with 5 to 20 × 10 ⁶ cells / mouse of the monoclonal antibody-producing hybridoma cells obtained in (c). The hybridoma becomes ascites tumor in 10 to 21 days.

Ascites was collected from the mouse with ascites tumor,
Centrifuge at 3,000 rpm for 5 minutes to remove solids. From the obtained supernatant, a monoclonal antibody can be purified and obtained by the same method as that used for the polyclonal antibody.

The subclass of the antibody is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The amount of polypeptide is calculated by the Lowry method or the absorbance at 280 nm.

3. Activin AB, activin B or activin AB using DNA encoding ALK7,
Method for detecting activin B or ALK7 gene mRNA The DNA used in the detection method includes, for example, a DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4 or a nucleotide sequence represented by SEQ ID NO: 5 and 6. cDN
A or a DNA fragment obtained from them or the like can be mentioned. Hereinafter, these are also referred to as DNAs used in the present invention. Examples of the method for detecting the mRNA expression level or structural change of the gene include (1) Northern blotting (2)
in situ hybridization method, (3) quantitative PCR method, (4) differential hybridization method, (5) DNA chip method, (6) RNase
Examples include protection assay methods and the like.

Samples to be analyzed by the above method include pancreatic tissues obtained from diabetic patients and healthy subjects,
MRNA or total RNA obtained from a biological sample such as serum or saliva, or a primary culture cell sample obtained by culturing cells from the biological sample in an appropriate medium in a test tube (hereinafter, the mRNA and total RNA are used. From sample R
Referred to as NA). In addition, the tissue obtained from the biological sample,
It is also possible to use those isolated as paraffin or cryostat sections.

By Northern blotting, RNA derived from the specimen was used.
Was separated by gel electrophoresis, transferred to a support such as a nylon filter, and hybridized and washed with a labeled probe prepared from the DNA used in the present invention to specifically bind to the gene mRNA. By detecting the band, changes in the expression level and structure of the gene mRNA can be detected. When performing hybridization, probe and sample-derived RN
Incubate under conditions where the gene mRNA in A forms a stable hybrid. In order to prevent false positives, it is desirable to carry out the hybridization and washing steps under highly stringent conditions. This condition is determined by a number of factors such as temperature, ionic strength, base composition, probe length, and formamide concentration. These factors are described, for example, in Molecular Cloning, Second Edition.

Labeled probes used in the Northern blotting method include, for example, radioactive isotopes, biotin, fluorescent groups, chemiluminescent groups, etc., by known methods (nick translation, random priming or quinaging),
It can be prepared by incorporating it into the DNA used in the present invention or an oligonucleotide designed from the sequence of the DNA. The amount of the labeled probe bound is the gene mRN.
Since the expression level of A is reflected, the expression level of the gene mRNA can be quantified by quantifying the amount of the bound labeled probe. Further, by analyzing the labeled probe binding site, the structural change of the gene mRNA can be known.

The in situ hybridization method uses the above-mentioned labeled probe and a tissue obtained from a living body, which is isolated as a paraffin or cryostat section, to carry out the steps of hybridization and washing to thereby analyze the mRNA of the gene. This is a method of detecting the expression level. In order to prevent false positives in the in situ hybridization method, it is desirable that the hybridization and washing steps be performed under highly stringent conditions. This condition is determined by a number of factors such as temperature, ionic strength, base composition, probe length, and formamide concentration. These factors are described, for example, in Current Protocols in Molecular Biology.

The method for detecting the mRNA of the gene such as the quantitative PCR method, the differential hybridization method or the DNA chip method is carried out by using oligo d
It can be performed by a method based on synthesizing cDNA using T primer or random primer and reverse transcriptase (hereinafter, the cDNA is used as a sample-derived cDNA.
Referred to as A). When the sample-derived RNA is mRNA, any of the above-mentioned primers can be used, but when the sample-derived RNA is total RNA, an oligo dT primer is used.

In the quantitative PCR method, the sample-derived cDNA was used as a template, and activin βA chain and activin βB were used.
By performing PCR using a chain or a primer designed based on the nucleotide sequence of the DNA encoding ALK7,
It is a method for amplifying a DNA fragment derived from the mRNA of the gene. Since the amount of the amplified DNA fragment reflects the expression amount of the mRNA of the gene, actin or G3PDH (glyceral dehyde)
de 3-phosphate dehydrogenase) etc.
As an internal control, the mRN of the gene
It is possible to quantify the amount of A. Also, amplified DNA
By separating the fragments by gel electrophoresis, the change in the structure of the mRNA of the gene can be known. In this detection method, it is desirable to use an appropriate primer that specifically and efficiently amplifies the target sequence. Appropriate primers can be designed based on conditions such as binding between the target cDNAs at the annealing temperature without binding between the primers or binding within the primers, and deviating from the target cDNAs under denaturing conditions. It is necessary to quantify the amplified DNA fragment in a PCR reaction in which the amplification product increases exponentially. Such PCR reaction can be known by recovering the amplified DNA fragment produced in each reaction and quantitatively analyzing by gel electrophoresis.

Differential hybridization method [Trends in Genetics, 7 , 314-317 (1991)] and DN
A-chip method [Genome Research, 6 , 639-645 (1996)]
Using the sample-derived cDNA as a probe, the gene m is obtained by performing hybridization and washing on a filter or a slide glass or a substrate such as silicon on which the DNA used in the present invention is immobilized.
This is a method of detecting a change in the expression level of RNA. Both methods use actin or G3PD on the filter or substrate.
By immobilizing an internal control such as H, the difference in mRNA expression of the gene between the control sample and the target sample can be accurately detected. In addition, labeled cDNA synthesis is performed using different labeled dNTPs based on RNAs derived from the control sample and the target sample, and two labeled cDNA probes are simultaneously hybridized to a filter or a substrate to obtain an accurate mRNA expression level of the gene. Can be quantified.

The RNase protection assay is performed by the following method. First, a promoter sequence such as T7 promoter and SP6 promoter is ligated to the 3'end of the DNA used in the present invention, and in
Using rNTP labeled by a transcription system in vitro ,
Synthesize labeled antisense RNA. The labeled antisense RNA is bound to the sample-derived RNA to form RNA-
After forming an RNA hybrid, it is digested with RNase, and the RNA fragment protected from digestion is detected by forming a band by gel electrophoresis. By quantifying the obtained band, the expression level of mRNA of the gene can be quantified.

4. Immunological detection or quantification method using an antibody that specifically recognizes activin AB, activin B, or ALK7 protein An antibody (polyclonal antibody or monoclonal antibody) that specifically recognizes activin AB, activin B, or ALK7 protein As a method for immunologically detecting and quantifying activin AB, activin B, or ALK7 protein expressed intracellularly or extracellularly of microorganisms, animal cells or insect cells or tissues, fluorescent antibody method, enzyme immunoassay can be used. Method (ELISA method), radioactive substance-labeled immunoantibody method (RIA), immunohistochemical staining method (ABC method, CSA method, etc.) such as immunohistological staining method and immunocyte staining method, Western blotting method, dot blotting method, immunity Precipitation method, sandwich ELISA method [monoclonal antibody Test manual (Kodansha Scientific) (1987), continue Biochemical Experiment Lecture 5 immune biochemical research method (Tokyo Kagaku Dojin) (1986)] and the like.

The fluorescent antibody method is a method in which the antibody of the present invention is reacted with a microorganism, animal cell or insect cell or tissue in which activin AB, activin B, or ALK7 protein is expressed intracellularly or extracellularly.
This is a method in which an anti-mouse IgG antibody labeled with a fluorescent substance such as isothiocyanate (FITC) or a fragment thereof is reacted and then a fluorescent dye is measured with a flow cytometer.

The enzyme-linked immunosorbent assay (ELISA) is a method in which the antibody of the present invention is reacted with a microorganism, animal cell, insect cell or tissue in which activin AB, activin B, or ALK7 protein is expressed intracellularly or extracellularly. ,
Further, it is a method of reacting an anti-mouse IgG antibody or a binding fragment labeled with an enzyme such as peroxidase or biotin, and then measuring a coloring dye with an absorptiometer.

The radioactive substance-labeled immuno-antibody method (RIA) is a method in which the antibody of the present invention is reacted with a microorganism, an animal cell, an insect cell or a tissue in which activin AB, activin B, or ALK7 protein is expressed intracellularly or extracellularly. And reacting with a radiolabeled anti-mouse IgG antibody or fragment thereof, and then measuring with a scintillation counter or the like.

Immunohistochemical staining such as immunocytostaining or immunohistostaining refers to microorganisms, animal cells or insect cells or tissues in which activin AB, activin B or ALK7 protein is expressed intracellularly or extracellularly. The method of reacting with the antibody of the present invention, further reacting with a fluorescent substance such as FITC, an enzyme-labeled anti-mouse IgG antibody such as peroxidase or biotin, or a fragment thereof, and then observing using a microscope.

The Western blotting method is an SDS-polyacrylamide gel electrophoresis [Antibodies-] electrophoresis of an extract of a microorganism, animal cell, insect cell or tissue expressing activin AB, activin B, or ALK7 protein intracellularly or extracellularly. A Laboratory Manual,
Cold Spring Harbor Laboratory, (1988)] and then blotting the gel on a PVDF membrane or a nitrocellulose membrane, and reacting the membrane with the antibody of the present invention,
Further, it is a method in which a fluorescent substance such as FITC, an anti-mouse IgG antibody labeled with an enzyme such as peroxidase or biotin, or a fragment thereof is reacted and then confirmed.

The dot blotting method is a method in which an activin AB, activin B, or ALK7 protein intracellularly or extracellularly expressed microorganism, animal cell, or insect cell or tissue extract is blotted on a nitrocellulose membrane, After reacting with an antibody that specifically recognizes activin AB, activin B, or ALK7 protein, and further reacting with a fluorescent substance such as FITC, an anti-mouse IgG antibody or a binding fragment labeled with an enzyme such as peroxidase or biotin, Here's how to check.

The immunoprecipitation method means activin AB, activin B, or an activin AB, activin B, or A extract of a microorganism, animal cell, insect cell, or tissue expressing intracellular or extracellular ALK7 protein.
After reacting with an antibody that specifically recognizes LK7 protein,
In this method, a carrier having an ability to specifically bind immunoglobulin such as protein G-sepharose is added to precipitate the antigen-antibody complex. The sandwich ELISA method is an antibody that specifically recognizes activin AB, activin B, or ALK7 protein. Of two antibodies with different antigen recognition sites, one antibody is adsorbed on the plate in advance and the other antibody is used. Labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase or biotin, and a microorganism, an animal cell or an insect cell that expresses activin AB, activin B or ALK7 protein inside or outside the cell on an antibody adsorption plate, or This is a method in which a tissue extract is reacted, a labeled antibody is reacted, and the bound labeling substance is used for detection.

5. Activin βA chain, activin βB
Method for identifying mutation of strand or DNA encoding ALK7 As the DNA used in this method, for example, a DNA having a nucleotide sequence represented by SEQ ID NO: 3 or 4 or a nucleotide sequence represented by SEQ ID NOs: 5 and 6 is used. Including cDN
A, or a DNA fragment obtained from them or the like.

The most definitive tests for assessing the presence of activin βA chain, activin βB chain, or diabetes-causing mutations present in the ALK7 locus are:
Direct comparison of genes from a control population with genes from diabetics.

Specifically, from diabetic patients and healthy people,
Human biological samples such as pancreatic tissue, serum, saliva, or samples derived from primary culture cells established from the biological samples are collected, and DNA is extracted from the biological samples and the primary culture cell-derived samples.
Is extracted (hereinafter, the DNA is referred to as a sample-derived DNA). The sample-derived DNA is amplified directly or using a DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4 or a primer designed based on the nucleotide sequence of cDNA containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6. The DNA encoding the activin βA chain, activin βB chain, or ALK7 can be used as the sample DNA. Alternatively, a primer designed using the sample-derived cDNA as a template based on the DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4 or the nucleotide sequence of the cDNA containing the nucleotide sequences represented by SEQ ID NO: 5 and 6 DNA fragment amplified by performing PCR with
It can be used as NA.

As a method for detecting whether or not there is a mutation causing diabetes in the DNA encoding activin βA chain, activin βB chain, or ALK7, a DNA chain having a wild-type allele and a DN having a mutant allele
A method for detecting a heteroduplex formed by hybridization with the A chain can be used.

The method for detecting the heteroduplex includes:
(1) Heteroduplex detection method by polyacrylamide gel electrophoresis [Trends Genet., 7 , 5 (1991)], (2) Single-stranded conformation polymorphism analysis method [Genomics, 16 , 32
5-332 (1993)], (3) Mismatch chemical cleavage method (CC
M, chemical cleavage of mismatches) [Human Genetic
s (1996), Tom Strachan and Andrew P. Read, BIOS Sc
ientific Publishers Limited], (4) Enzymatic cleavage method of mismatch [Nature Genetics, 9 , 103-104 (1996)],
(5) Denaturing gel electrophoresis [Mutat. Res., 288 , 103-11
2 (1993)] and the like.

The heteroduplex detection method by polyacrylamide gel electrophoresis uses the sample-derived DNA or the sample-derived cDNA as a template for DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4, or SEQ ID NOS: 5 and 6. By PCR using a primer designed based on the nucleotide sequence of the cDNA containing the represented nucleotide sequence, 2
As a fragment smaller than 00 bp, a DNA fragment encoding activin βA chain, activin βB chain, or ALK7 was amplified, and the amplified DNA fragment was electrophoresed on polyacrylamide gel to migrate with a homoduplex having no mutation. It is a method of comparing degrees. When a heteroduplex is formed by a mutation in the DNA encoding activin βA chain, activin βB chain, or ALK7, the mobility of the gel is slower than that of a homoduplex having no mutation, and they are homoduplexes. It can be detected as a band different from the strand. For the electrophoresis, a commercially available gel [Hydro-link, MDE
(Manufactured by FMC) and the like] can also be used. Also, 2
The heteroduplex detection method using a DNA fragment smaller than 00 bp can detect insertion, deletion and substitution of one base. Heteroduplex analysis is preferably performed on a single gel in combination with the single-stranded conformational polymorphism analysis described below.

Single-stranded conformation polymorphism analysis (SS
CP analysis; single strand conformation polymorphism
analysis) is sample-derived DNA or sample-derived cDNA
200 bp from the DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4 or the cDNA designed containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6 by PCR using the primers as a template. Is a method of denaturing a DNA encoding activin βA chain, activin βB chain or ALK7 amplified as a small fragment, and then performing electrophoresis using a non-denaturing polyacrylamide gel. When the DNA is amplified, the primer is labeled with a radioactive isotope or a fluorescent dye, or the unlabeled amplification product is stained with silver to obtain the amplified activin βA chain, activin βB chain, or ALK7-encoding DNA. It can be detected as a band. In order to clarify the difference from the wild-type migration pattern, a control sample can also be electrophoresed at the same time to detect a fragment having a mutation in the nucleotide sequence from the difference in mobility.

Mismatch chemical cleavage method (CCM method)
Was designed based on the sample-derived DNA or the sample-derived cDNA as a template, based on the DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4, or the cDNA including the nucleotide sequences represented by SEQ ID NO: 5 and 6. Activin βA chain, activin βB chain, or A
A DNA fragment encoding LK7 is amplified, and the amplified DN
A fragment is hybridized with a DNA having the base sequence represented by SEQ ID NO: 3 or 4, or a labeled DNA obtained by incorporating a radioisotope or a fluorescent dye into a cDNA containing the base sequence represented by SEQ ID NO: 5 or 6. In this method, soybeans are treated with osmium tetroxide to cleave one strand of the DNA at the mismatched position to detect nucleotide sequence mutations. The CCM method is one of the most sensitive detection methods, and can be applied to a specimen having a length of kilobase.

In place of osmium tetroxide, enzymes such as T4 phage resolvase and endonuclease VII that are involved in repair of mismatches in cells and RNas
By combining with eA, the mismatch can be cleaved enzymatically.

Denaturing gel electrophoresis (denaturing gradien)
t gel electrophoresis: DGGE method) is a sample-derived DNA
Alternatively, using the sample-derived cDNA as a template, DNA having the base sequence represented by SEQ ID NO: 3 or 4, or cDNA containing the base sequence represented by SEQ ID NO: 5 and 6
In this method, a DNA fragment encoding activin βA chain, activin βB chain, or ALK7 amplified with a primer designed based on the nucleotide sequence of A is electrophoresed using a gel having a concentration gradient or a temperature gradient of a chemical denaturant. . The amplified DNA fragment moves to a position where it is denatured into a single strand in the gel, and does not move after the denaturation. Activin βA chain,
DNA encoding activin βB chain or ALK7
Since the mobility of the amplified DNA in the gel differs depending on whether or not there is a mutation, it is possible to detect the presence of the mutation. It is also possible to attach a poly (G: C) terminal to each primer used for PCR to increase the detection sensitivity.

As another method for detecting the causative gene of diabetes, a protein truncation test (P) is used.
TT method) [Genomics, 20 , 1-4 (1994)]. The test can specifically detect frameshift mutations, splice site mutations, and nonsense mutations that produce protein deficiencies. SEQ ID NO: 3 for PTT method
Alternatively, a primer having a T7 promoter sequence and a eukaryotic translation initiation sequence connected to the 5'end of the DNA having the nucleotide sequence represented by 4, or the cDNA containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6, Activin βA chain, activin βB chain or ALK by reverse transcription PCR (RT-PCR) method from RNA derived from a sample using the primer
A cDNA encoding 7 is prepared. A protein is produced by in vitro transcription and translation using the cDNA.
If the protein is subjected to polyacrylamide electrophoresis and the migration position of the protein is at a position corresponding to the full-length protein, there is no mutation that causes a deletion. The protein is electrophoresed,
The degree of the defect can be known from the position.

DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4 for determining the nucleotide sequences of the sample-derived DNA and the sample-derived cDNA, or SEQ ID NOS: 5 and 6
It is possible to use a primer designed based on the cDNA containing the nucleotide sequence represented by By analyzing the determined nucleotide sequence, it is possible to determine whether or not the sample-derived DNA or the sample-derived cDNA has a mutation that causes diabetes.

Mutations other than the activin βA chain, activin βB chain, or ALK7 gene coding region can be detected by examining noncoding regions, such as introns and regulatory sequences near or within the gene. . Diabetes caused by mutations in non-coding regions
It can be detected by a method similar to the method described above.

Regarding the gene in which the presence of a mutation in the non-coding region was suggested in this manner, the DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4 or the nucleotide sequences represented by SEQ ID NOs: 5 and 6 Can be cloned by using a cDNA containing Mutations in the non-coding region can be searched according to any of the methods described above.

The mutations found are in the Handbook of Human Ge
netics Linkage. The John HopkinsUniversity Press,
By performing statistical processing according to the method described in Baltimore (1994), SNPs (single
Nucleotide polymorphism). In addition, a causative gene of diabetes can be identified by obtaining DNA from a family member with a history of diabetes according to the method described above and detecting a mutation.

6. Activin βA chain, activin βB
Diagnosis Method of Diabetes Using Chain or DNA Encoding ALK7 The DNA used in the above method is, for example, a DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4, or represented by SEQ ID NOs: 5 and 6. CDNA containing nucleotide sequences
A, or a DNA fragment obtained from them or the like. The cause of diabetes can be confirmed by detecting a gene mutation in any tissue of human. For example, if there is a germline mutation, individuals who inherit the mutation may be more likely to develop diabetes. The mutation can be detected by testing DNA extracted from any tissue of the individual's body. For example, from cells of blood collected from humans, D
Diabetes can be diagnosed by extracting NA and detecting a gene mutation using the DNA. In addition, fetal cells, placental cells, or amniotic cells are collected to DN
Prenatal diabetes can be diagnosed by extracting A and detecting a gene mutation using the DNA.

Further, by obtaining DNA from the living tissue at the lesion site of a patient who has developed diabetes and detecting the mutation of the gene, it is possible to diagnose the type of diabetes and use it for the selection of the drug to be administered. . DNA from living tissue
Is a tissue isolated from the surrounding normal tissue at the lesion site, treated with trypsin, etc., the obtained cells are cultured in an appropriate medium, and the chromosomal DNA and m
It can be obtained by extracting RNA.

Hereinafter, the DNA obtained from any of the above-mentioned methods from a human sample for the purpose of diagnosis is a DNA derived from a diagnostic sample.
Called. Further, a cDNA synthesized from RNA obtained from a human sample by any of the above methods for the purpose of diagnosis is referred to as a diagnostic sample-derived cDNA. Using a DNA having the base sequence represented by SEQ ID NO: 3 or 4, or a cDNA containing the base sequence represented by SEQ ID NO: 5 and 6, and a diagnostic specimen-derived DNA or a diagnostic specimen-derived cDNA,
Diabetes can be diagnosed by a method similar to the method for detecting a mutation in the DNA encoding activin βA chain, activin βB chain, or ALK7 in the above 5, but the above method 5 causes a causal relationship with diabetes. As a method for diagnosing whether or not the found mutation has a diagnostic sample-derived DNA or a diagnostic sample-derived cDNA, (1) detection of a restriction enzyme site, (2) allele-specific oligonucleotide probe is used. How to use (ASO: allele specif
(3) PCR using allele-specific oligonucleotides (ARMS:
amplification refractory mutation system), (4)
Oligonucleotide ligation assay (OLA: oli
gonucleotide ligation assay), (5) PCR-PHFA method (P
CR-preferential homoduplex formation assay),
(6) A method using an oligo DNA array [protein nucleic acid enzyme, 43 , 2004-2011 (1998)] and the like can be mentioned.

Detection of restriction enzyme sites can be carried out according to the method described below. That is, when the restriction enzyme site disappears or occurs due to a change in a single base, the diagnostic sample-derived DNA or the diagnostic sample-derived cDNA is a DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4, or SEQ ID NO: 5 and CDNA containing the nucleotide sequence represented by 6
PCR using primers designed based on the nucleotide sequence of A
After being amplified by the method 1, the restriction enzyme digestion is carried out, and the obtained restriction enzyme-cut DNA fragment is compared with that of a normal person, whereby the mutation can be easily detected. More specifically, as a method for diagnosing a base change, DNA having the base sequence represented by SEQ ID NO: 3 or 4, or SEQ ID NOS: 5 and 6 is used.
An oligonucleotide probe is designed by combining the nucleotide sequence information of the cDNA containing the nucleotide sequence represented by and the information of the mutation identified by the above method 5, and the oligonucleotide probe is bound to a filter to hybridize. A method of detecting a mutation by a reverse dot blot method using soybean can be mentioned.

The method using an allele-specific oligonucleotide probe (ASO) is a method utilizing the characteristic that a short synthetic DNA probe hybridizes only with a perfectly matched nucleotide sequence, and a mutation of 1 nucleotide is used. It can be easily detected. Specifically, SEQ ID NO: 3 or 4
DNA having a nucleotide sequence represented by, or SEQ ID NO: 5
The nucleotide sequence of the cDNA containing the nucleotide sequences represented by 1 and 6 and the oligonucleotide designed based on the mutation of the nucleotide identified in 5 above are bound to a filter, and the diagnostic sample-derived DNA or the diagnostic sample-derived cDNA is used as a template. , DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4, or obtained by PCR using a dNTP labeled with a primer designed using the nucleotide sequence of cDNA containing the nucleotide sequences represented by SEQ ID NOS: 5 and 6. A reverse dot blotting method in which the amplified DNA fragment obtained is used as a probe for hybridization is exemplified.

The reverse dot blot is an oligonucleotide designed based on a mutation of the DNA, which is separately identified with a nucleotide sequence of the mutation-free DNA corresponding to the DNA to be measured, directly on a substrate such as a glass slide or silicon. This is a method of synthesizing and reacting a small amount of diagnostic sample-derived DNA or diagnostic sample-derived cDNA with a DNA chip which is a high-density array to detect various mutations more easily. This method is a mutation detection method suitable for large-scale diagnostic purposes.

The base mutation can also be detected by the method shown below by the oligonucleotide ligation assay (OLA) method. Activin βA to be investigated
Chain, activin βB chain, or D encoding ALK7
An oligonucleotide of about 20 bases having a mutation site of NA at its 3'end, and an oligonucleotide of about 20 bases following the mutation site having the base sequence represented by SEQ ID NO: 3 or 4, or SEQ ID NO: 5 It is designed and synthesized based on the nucleotide sequence of cDNA containing the nucleotide sequences represented by and 6. At this time, for example, biotin is added to the 5'end of the former oligonucleotide, and a different label such as digokeshigenin is added to the 3'end of the latter oligonucleotide. Next, using the diagnostic sample-derived DNA or the diagnostic sample-derived cDNA as a template, the DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4, or the cDNA containing the nucleotide sequence represented by SEQ ID NO: 5 and 6
PCR using primers designed based on the nucleotide sequence of
Depending on activin βA chain, activin βB chain, or A
Amplify the DNA encoding LK7. Next, the amplified DN
The A fragment and the above two oligonucleotides are hybridized. After hybridization, the two oligonucleotides are ligated with DNA ligase. After ligation, biotin-labeled single-stranded D obtained by heat denaturing double-stranded DNA
NA is recovered, for example, as DNA that binds to avidin. Since the above-mentioned two kinds of oligonucleotides hybridized with the amplified DNA fragment having the displacement site are ligated by the above-mentioned ligation reaction, it is obtained as a DNA having digokeshigenin at its 3'end, and the bound DNA can be easily detected. You can Therefore, the DNA encoding the activin βA chain having a displacement, the activin βB chain, or ALK7 can be detected rapidly and easily. OLA is a mutation detection method suitable for effectively diagnosing a large number of samples in a short period of time because it does not require electrophoresis or centrifugation.

Further, a minute amount of mutant gene can be quantitatively and easily detected by the following PCR-PHFA method. PCR-
PHFA methods include gene amplification (PCR), liquid phase hybridization with very high specificity, and ELISA.
ED-PCR (enzymatic
detection of PCR product). Using a primer set labeled with dinitrophenyl (DNP) and biotin, PCR is carried out using the DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4 or the cDNA containing the nucleotide sequence represented by SEQ ID NO: 5 and 6 as a template. Then, an amplified DNA fragment labeled with both ends is prepared. Next, using an unlabeled primer set having the same base sequence as the labeled primer, an unlabeled amplified DNA fragment obtained by amplifying the diagnostic sample-derived DNA or diagnostic sample-derived cDNA as a template is mixed with the labeled amplified DNA fragment. To do. At this time, the unlabeled amplified DNA fragment is used in a large excess of 20 to 100 times that of the labeled amplified DNA fragment. Then, after heat denaturing the mixture, 1 ° C / 5 min-10
The mixture is cooled with a gradual temperature gradient of about 1 minute to preferentially form a completely complementary strand. Labeled DNA thus reformed
Is detected and adsorbed on the streptavidin-immobilized well via biotin, bound to the enzyme-labeled anti-DNP antibody via DNP, and detected by a color reaction by the enzyme. When the gene having the same sequence as that of the labeled DNA is not present in the sample, the original double-stranded labeled DNA is preferentially reformed and shows a color. On the other hand, when genes having the same nucleotide sequence are present, replacement of complementary strands occurs randomly, so that the amount of labeled DNA that is reformed is reduced, resulting in a marked decrease in color development. This makes it possible to detect / quantify known mutation / polymorphic genes.

7. Diagnosis method of diabetes using an antibody that specifically recognizes activin AB, activin B or ALK7 protein Change and expression of activin AB, activin B or ALK7 protein in human biological samples and human primary culture cells It is useful to identify the structural change of the existing protein in order to know the risk of developing diabetes and the cause of the already deteriorated pancreatic function.

Activin AB, activin B, or A
As a method for diagnosing by detecting the expression level or structural change of LK7 protein, the fluorescent antibody method, enzyme immunoassay method (ELISA method), radioactive substance-labeled immunoantibody method (R
IA), immunohistochemical staining methods such as immunohistochemical staining method and immunocytochemical staining method (ABC method, CSA method, etc.), Western blotting method, dot blotting method, immunoprecipitation method, sandwich ELISA method and the like.

As the sample to be used for diagnosis by the above method, the tissue of the pancreatic lesion site obtained from the patient, the biological sample itself such as blood, serum, urine, stool, saliva, or the cells and cell extract obtained from the biological sample are used. Is used. Alternatively, a tissue obtained from a biological sample and isolated as a paraffin or cryostat section may be used.

8. Activin AB, Activin B or ALK7 protein, DNA encoding the protein, or a method for screening a therapeutic agent for diabetes using an antibody that recognizes any of the proteins. The protein used in the screening method of the present invention is activin AB, Mention may be made of activin B and ALK7. Activin AB and Activin B are
As long as it is a protein having an activity as a ligand protein of ALK7, it may be a protein derived from a natural product or a protein produced by a genetic engineering method. For example, the naturally-derived protein may be human, monkey or pig. , ALK7 from all mammals including cows, sheep, horses and rats
The ligand protein of Activin A
Specific examples of B include a dimeric protein composed of a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 and a polypeptide having the amino acid sequence represented by SEQ ID NO: 2, and activin A specific example of B is a dimeric protein composed of a polypeptide having the amino acid sequence represented by SEQ ID NO: 2. ALK7 is a protein produced by a genetic engineering method, even if it is a protein derived from a natural product, as long as it is a protein having an activity as ALK7, that is, an activity that is activated by activin AB or activin B and promotes insulin secretion of cells. The naturally-occurring protein may be, for example, human, monkey, pig, cow, sheep, horse,
ALK7 derived from any mammal such as rat can be mentioned. As a specific example of ALK7, SEQ ID NO: 5
And a protein encoded by a cDNA containing the nucleotide sequence represented by 6 and the protein consisting of the amino acid sequence represented by SEQ ID NO: 9. The DNA used in the screening method of the present invention includes activin AB,
Mention may be made of DNA encoding activin B and ALK7. The DNA may be any DNA as long as it encodes the protein used in the screening method of the present invention. Specific examples of activin AB or DNA encoding activin B include SEQ ID NO: 3
Alternatively, a DNA having the nucleotide sequence represented by 4 can be used. Specific examples of the DNA encoding ALK7 include cDNA containing the nucleotide sequences represented by SEQ ID NOS: 5 and 6, and DNA having the nucleotide sequence represented by SEQ ID NO: 10. Examples of the antibody used in the screening method of the present invention include an antibody that recognizes the protein used in the screening method of the present invention or a polypeptide fragment thereof.

(1) Screening for compounds that act specifically on activin AB, activin B or ALK7 protein Microorganisms, animal cells or insect cells transformed to express the ALK7 protein together with the type II activin receptor, and purified Activin AB, activin B, or ALK7 protein is activin AB, activin B
Alternatively, it is useful for screening a compound that acts specifically on the ALK7 protein. The type II activin receptor may be any protein having an activity as a type II activin receptor, for example, Cell, 65 , 97
Examples include ActRIIA described in 3-982 (1991). The compound obtained by the screening is useful as a therapeutic drug for diabetes.

One method of the above screening is AL
To select a compound that specifically induces ALK7 activation in an animal cell transformed to produce a K7 protein and a type II activin receptor (hereinafter referred to as a transformant for search). Examples of the method for detecting the activation of ALK7 include a method for measuring the cellular response of the transformant for search.

Specific cell responses include, for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cAMP reduction, intracellular cGMP production, inositol phosphate production, cell membrane potential. Fluctuation, phosphorylation of intracellular proteins, c-fos activation, pH
Decrease, cell proliferation activity, melanin pigment aggregation or diffusion, or reporter gene expression fluctuation [J. Biol. Chem., 271 , 1857 (1996), Scie.
nce, 268 , 98 (1995), Journal of Pharmacology and
Experimental Therapeutics, 275 , 1274 (1995), J. Bi
ol. Chem., 272 , 1822 (1997), Journal of Receptor a
nd Signal Transduction Research, 17 , 57 (1997), End
ocrinology 138 , 1400 (1997), Endocrinology 138 , 14
71 (1997), Nat.Biotechnol., 16 , 1334 (1998), Bio
Chem. Biophys. Res. Commun., 251 , 471 (1998), Bri
t. J. Pharmacol., 125 , 1387 (1998), Trends Biotech
nol., 15 , 487 (1997), Anal. Biochem., 252 , 115 (19
97), Nature, 358 , 325 (1992), Nature, 393 , 272 (19
98), Cell, 92 , 573 (1998), J. Biol. Chem., 272 , 274.
97 (1997), Trends Pharmacol. Sci., 18 , 430 (199
7), Trends Pharmacol.Sci., 20 , 370 (1999), WO98 / 46.
995].

When a cell response is monitored using a reporter gene, for example, the transformant for search is
ALK7 activation can be measured by the expression of the reporter gene by introducing DNA into which a suitable reporter gene is ligated downstream of the promoter sequence of the gene whose expression is induced by the activation of LK7. Examples of the promoter include ICAM-1 gene promoter, c-fos promoter, Krox-24 promoter [Biochem. J., 320 , 145 (1996)], insulin gene promoter, choline acetyltransferase gene promoter, A promoter for the tyrosine hydroxylase gene can be used. Also,
The promoter may be an artificial promoter consisting of an appropriate transcription factor binding sequence and a basic promoter. Examples of binding sequences for transcription factors include CRE (CREB binding
lement), TRE (TPA responsive element), SRE
(Serum responsive element), SBE-lux promoter [J. Biol. Chem., 273 , 21145-21152 (1998)] and the like can be used. As the reporter gene, chloramphenicol acetyltransferase gene, β
-Glucuronidase gene, β-galactosidase gene, β-lactamase gene, aequorin gene, luciferase gene and Green Fluorescent
Protein genes etc. can be used.

Further, the purified activin AB, activin B or ALK7 protein, or a polypeptide constituting a part of the protein is activin AB or activin B.
Alternatively, it can be used to select a target compound that specifically binds to the ALK7 protein. For example, after immobilizing the protein on a solid phase carrier or the like and contacting a test sample,
The target compound can be selected by thoroughly washing the plate and releasing the compound bound to the activin AB, activin B or ALK7 protein from the protein.

As another method of the above-mentioned screening, a large number of peptides constituting a part of activin AB, activin B or ALK7 protein are synthesized at high density on a plastic pin or a certain kind of solid support, and the peptide is synthesized. There is a method for efficiently screening a compound or protein that selectively binds to (WO84 / 03564).

(2) Activin βA chain, activin βB
Method for screening compound that regulates transcription or translation of chain or ALK7-encoding DNA Activin β A chain, activin β in pancreatic cell line
A compound having an activity of promoting the expression of B chain or ALK7 gene mRNA or activin AB, activin B or ALK7 protein is also useful as a therapeutic agent for diabetes. As the pancreatic-derived cell line, mouse pancreatic β cell type MIN6 cell [Endocrinology, 127 , 126-132 (199
0)], mouse insulinoma cells NIT-1 cells (AT
CC CRL-2055), mouse pancreatic α cell type αTC1 cell (ATC
C CRL-2350), rat pancreatic duct epithelial cell ARIP cell (ATCC
CRL-1674) and the like.

[0159] The expression level of activin βA chain, activin βB chain or mRNA of ALK7 gene in the cell line derived from the pancreas was contacted with various test samples and in the case where the test sample was not contacted, By comparison, activin βA chain, activin βB chain or ALK
A substance that suppresses or promotes the transcription of 7 genes can be screened. The expression level of activin βA chain, activin βB chain or mRNA of ALK7 gene can be detected by the above-mentioned PCR method 3, Northern blotting method and RNase protection assay method.

Further, the expression levels of activin AB, activin B or ALK7 protein in the cell line derived from the pancreas and in the case where various test samples are contacted and in the case where the test sample is not contacted are measured and compared. Thus, a substance that suppresses or promotes the transcription or translation of activin AB, activin B and ALK7 gene can be screened. The expression level of activin AB, activin B or ALK7 protein is the same as that of activin A in 4 above.
B, activin B or ALK7 protein using a fluorescent antibody method and an enzyme immunoassay method (ELI).
SA method), radioactive substance-labeled immunoantibody method (RIA), immunohistochemical staining methods such as immunohistological staining method and immunocytochemical staining method (ABC method, CSA method, etc.), Western blotting method, dot blotting method, immunoprecipitation method , Sandwich ELISA method.

Furthermore, activin βA chain and activin β
In a case where various test samples are contacted with an animal cell transformed with a recombinant DNA in which a reporter gene is linked to the downstream of the promoter of the B chain or the ALK7 gene, and the test sample is not contacted By measuring and comparing the expression level of the reporter gene, a substance that suppresses or promotes the expression of activin βA chain, activin βB chain or ALK7 gene can be screened. When the base sequence of the activin βA chain, activin βB chain or ALK7 gene is known, an animal-derived chromosomal DNA was used as a template with an oligonucleotide that can be designed and synthesized according to the sequence. It can be obtained by PCR. When the promoter sequence is not known, a known method using the DNA or oligonucleotide of the above-mentioned gene as a probe [The University of Tokyo, Institute of Medical Science, Cancer Research Division,
New Cell Engineering Experiment Protocol, Shujunsha (1993)]
Can be used to obtain the promoter region of the gene.

The expression level of the reporter gene in the cells can be determined by a known method [New Research Center for Cancer Research, Institute of Medical Science, University of Tokyo, New Cell Engineering Experimental Protocol, Shujunsha (1993), Biotechnique.
s, 20 , 914 (1996), J. Antibiotics, 49 , 453 (1996).
, Trends in Biochemical Sciences, 20 , 448 (199
5), Cell Engineering, 16 , 581 (1997)].

Examples of the reporter gene include a chloramphenicol acetyltransferase gene, a β-galactosidase gene, a β-lactamase gene, a luciferase gene, and a green fluorescent protein (GFP) gene.

The compound obtained by the above method was NOD
(Non-Obese Diabetes) Administered as a drug to a diabetes model animal such as a mouse, and measuring the insulin secretion amount and blood glucose level of the animal according to a known method,
It is possible to evaluate the therapeutic effect of the compound on diabetes.

9. Activin AB, activin B, or a medicine containing ALK7 protein as an active ingredient for the treatment and / or prevention of diabetes Activin AB, activin B, or ALK7 protein is used in various types of diabetes caused by reduced pancreatic function in the pancreas. It can be used to reconstruct the structure and function of and to promote insulin secretion from the pancreas.

Activin AB and activin B are A
If the LK7 ligand protein is naturally derived,
It may be a recombinant protein produced by a genetic engineering method. Examples of naturally-occurring ALK7 ligand proteins include ALK derived from all mammals such as human, monkey, pig, cow, sheep, horse, mouse and rat.
7 ligand protein can be mentioned, but when used as a drug for treating and / or preventing human diabetes, that is, when used as a pancreatic function improving agent or a pancreatic regeneration promoting agent, the amino acid sequence is identical to that of a human-derived ALK7 ligand protein. It is preferable to use proteins.

Activin AB, activin B, or A
The medicine for treating and / or preventing diabetes which contains LK7 protein as an active ingredient may contain only the protein as an active ingredient, but usually one or a pharmacologically acceptable one thereof is used. Desirably, it is mixed with the above carriers and provided as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics.
Preferably water, or salt, glycine, glucose,
An aseptic solution dissolved in an aqueous carrier such as an aqueous solution of human albumin is used. Further, pharmacologically acceptable additives such as buffering agents and isotonicity agents for bringing the formulation solution closer to physiological conditions, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, citric acid. Sodium or the like can also be added. Alternatively, it may be lyophilized and stored, and may be dissolved in an appropriate solvent before use.

It is desirable to use the most effective route of administration for the treatment. Oral administration or buccal administration,
Parenteral administration such as intratracheal, rectal, subcutaneous, intramuscular and intravenous administration can be mentioned. Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders and granules. Liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, and p-hydroxybenzoate. Preservatives such as acid esters and flavors such as strawberry flavor and peppermint can be used as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, etc., starch, disintegrating agents such as sodium alginate, etc.
It can be produced by using a lubricant such as magnesium stearate and talc, a binder such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin, a surfactant such as fatty acid ester and a plasticizer such as glycerin as additives.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. For example,
It is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid. Also,
The propellant is prepared using the polypeptide itself or a carrier which does not irritate the oral cavity and airway mucosa of the recipient and disperses the polypeptide in the form of fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders are possible depending on the properties of the polypeptide and the carrier used. Further, also in these parenteral preparations, the components exemplified as the additive in the oral preparation can be added.

The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight and the like, but is usually 10 μg / kg to 8 mg / kg per day for an adult.

10. Pharmaceuticals for the treatment and / or prevention of diabetes which contain an antibody that specifically recognizes activin AB, activin B or ALK7 as an active ingredient Contain an antibody that specifically recognizes activin AB, activin B or ALK7 as an active ingredient In the medicine for the treatment and / or prevention of diabetes mellitus, it is possible to administer the active ingredient alone, but usually the active ingredient is combined with one or more pharmaceutically acceptable carriers. Desirably, they are mixed together and provided as a pharmaceutical formulation prepared by any of the methods well known in the pharmacy art. Preferred pharmaceutical formulation forms, administration methods, etc.
As in 9 above.

11. Medicament for treating and / or preventing diabetes containing the compound obtained by the above-mentioned 8 screening method as an active ingredient Treatment and / or prevention for diabetes containing the compound obtained by the above-mentioned 8 screening method as an active ingredient Although it is possible to administer the active ingredient alone, usually, the active ingredient is mixed with one or more pharmaceutically acceptable carriers to prepare a pharmaceutical preparation. It is desirable to provide it as a pharmaceutical preparation manufactured by any method well known in the technical field. The preferable pharmaceutical preparation form and the administration method are as described in 9 above.

12. Activin βA chain, activin βB
Chain or a gene therapeutic agent for diabetes containing DNA encoding ALK7 protein activin βA chain, activin βB chain, or ALK7
As a method of using a DNA encoding a protein as a gene therapy drug for diabetes, the DNA is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, The method of formulation, formulation and administration according to the conventional method described in 9, or the method of administration by non-viral gene transfer method can be mentioned.

The recombinant viral vector can be prepared according to the method described below. Activin βA chain,
Activin βB chain or full-length cDNA of ALK7 protein
Based on NA, a DNA fragment having an appropriate length containing a portion encoding the protein is prepared, if necessary. The DNA
A recombinant viral vector is constructed by inserting a fragment or full-length cDNA into the downstream of the promoter in the viral vector.

In the case of RNA virus vector, R homologous to activin AB and activin B full-length cDNA
Recombinant virus is constructed by preparing NA fragments and inserting them downstream of the promoter in the viral vector. In addition to the double strand, the RNA fragment selects either the sense strand or the antisense strand depending on the type of the viral vector. For example, in the case of a retrovirus vector, RNA homologous to the sense strand is selected, and in the case of a sense virus vector, RNA homologous to the antisense strand is selected.

The recombinant viral vector is introduced into packaging cells compatible with the vector. The packaging cell may be any cell as long as it can supplement the defective protein of the recombinant viral vector lacking at least one gene encoding the protein required for viral packaging. For example, human kidney-derived HEK293 cells, mouse fibroblasts NI
H3T3 or the like can be used. In the case of retrovirus vector, gag, pol derived from mouse retrovirus,
In the case of lentiviral vector such as env, gag, pol, env, vpr, vp derived from HIV virus
Adenovirus-derived E1A, E in the case of adenovirus vector such as u, vif, tat, rev, nef
For adeno-associated viruses such as 1B, Rep (p5,
p19, p40) and proteins such as Vp (Cap).

As a viral vector, a recombinant virus can be produced in the above packaging cells, and activin βA chain, activin βB chain, or AL can be produced in target cells.
Those containing a promoter at a position where the DNA encoding K7 can be transcribed are used. As a plasmid vector, MFG [Proc. Natl. Acad. Sci. USA, 92 , 67
33-6737 (1995)], pBabePuro [Nucleic Acids
Res., 18 , 3587-3596 (1990)], LL-CG, CL-C
G, CS-CG, CLG [Journal of Virology, 72 , 815
0-8157 (1998)], pAdex1 [Nucleic Acids Res., 2
3 , 3816-3821 (1995)] and the like are used. Any promoter can be used as long as it functions in human tissues, and examples thereof include a cytomegalovirus (human CMV) IE (immediate early) gene promoter, an SV40 early promoter, and a retrovirus promoter. Examples thereof include metallothionein promoter, heat shock protein promoter, SRα promoter and the like. In addition, the enhancer of the IE gene of human CMV may be used together with the promoter. As a method for introducing a recombinant viral vector into a packaging cell, for example, the calcium phosphate method [JP-A-2-227075]
Publication], lipofection method [Proc. Natl. Acad. Sc
i. USA, 84 , 7413 (1987)] and the like.

As a method for administering the above recombinant viral vector, in addition to the method described in 9 above, the viral vector is directed to the pancreatic lesion by combining with direct in vivo gene transfer using liposome delivery. You can choose to let them do it. That is, a DNA encoding an activin βA chain, an activin βB chain, or ALK7 having an appropriate size is combined with a polylysine-conjugate antibody specific for an adenovirus hexon protein to prepare a complex, and the obtained complex is adenovirus. A viral vector can be prepared by binding to a viral vector.
The viral vector stably reaches the target cell, is taken up into the cell by the endosome, is decomposed in the cell, and the gene can be efficiently expressed.

Activin AB, activin B, or A
The LK7 gene DNA can also be delivered to the lesion by non-viral gene transfer methods. Non-viral gene transfer methods known in the art include calcium phosphate coprecipitation method.
[Virology, 52 , 456-467 (1973); Science, 209 , 1414-
1422 (1980)], Microinjection method [Proc. Nat
l. Acad. Sci. USA, 77 , 5399-5403 (1980); Proc. Nat
l. Acad. Sci. USA, 77 , 7380-7384 (1980); Cell, 27 ,
223-231 (1981); Nature, 294 , 92-94 (1981)], liposome-mediated membrane fusion-mediated transfer [Proc. Natl. Acad. S]
ci. USA, 84 , 7413-7417 (1987); Biochemistry, 28 , 9
508-9514 (1989); J. Biol. Chem., 264 , 12126-12129
(1989) ； Hum. Gene Ther., 3 , 267-275, (1992) ； Scienc
e, 249 , 1285-1288 (1990); Circulation, 83 , 2007-20
11 (1992)] or direct DNA uptake and receptor-
Mediated DNA Transfer Method [Science, 247 , 1465-1468 (1990); J.
Biol. Chem., 266 , 14338-14342 (1991); Proc. Natl.
Acad. Sci. USA, 87 , 3655-3659 (1991); J. Biol. Ch
em., 264 , 16985-16987 (1989) ； BioTechniques, 11 , 4
74-485 (1991); Proc. Natl. Acad. Sci. USA, 87 , 341.
0-3414 (1990); Proc. Natl. Acad. Sci. USA, 88 , 425
5-4259 (1991); Proc. Natl. Acad. Sci. USA, 87 , 4033.
-4037 (1990); Proc. Natl. Acad. Sci. USA, 88 , 8850.
-8854 (1991); Hum. Gene Ther., 3 , 147-154 (1991)]
Etc. can be given.

In tumor-related studies, the liposome-mediated membrane fusion-mediated transfer method allows local gene uptake and expression of the tissue by direct administration of the liposome preparation to the target tissue. Reported [Hum. Gene Ther. 3 , 399-410 (1992)]. Therefore, a similar effect is expected in pancreatic lesions. For direct targeting of DNA to pancreatic lesions, direct DNA uptake techniques are preferred. Receptor-mediated DNA transfer is carried out, for example, by conjugating DNA (usually in the form of a covalently closed supercoiled plasmid) to a protein ligand via polylysine. Ligands are selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell or tissue. The ligand-D
NA conjugates can be injected directly into blood vessels, if desired, and can be directed to target tissues where receptor binding and DNA-protein complex internalization occurs. DN
To prevent intracellular destruction of A, adenovirus can also be co-infected to disrupt endosomal function.

13. Method for specifically transporting drug to pancreas using antibody specifically recognizing ALK7 protein (drug delivery method) The antibody used for the drug delivery method is AL
Any antibody may be used as long as it specifically recognizes the K7 protein, but it is particularly preferable to use a humanized antibody.

Humanized antibodies include human chimeric antibodies,
Human type CDR (Complementary Determining Region; complementarity determining region; hereinafter referred to as CDR) transplant antibody and the like. A human chimeric antibody is a non-human animal antibody heavy chain variable region (hereinafter, heavy chain is also referred to as H chain, variable region is also referred to as V region, HV or VH) and antibody light chain variable region (hereinafter, light chain An antibody consisting of a human antibody heavy chain constant region (hereinafter also referred to as LV or VL as the L chain) (hereinafter, the constant region is also referred to as CH as the C region) and a human antibody light chain constant region (hereinafter also referred to as CL) means. As the non-human animal, any mouse, rat, hamster, rabbit or the like can be used as long as a monoclonal antibody-producing hybridoma can be produced.

CDNAs encoding VH and VL were obtained from a hybridoma producing a monoclonal antibody that binds to the ALK7 protein, and human antibody CH and human antibody CL were obtained.
Can be inserted into an expression vector for animal cells having a gene encoding the above to construct a human chimeric antibody expression vector, and introduced into animal cells for expression and production.

As the CH of the human chimeric antibody, any CH can be used as long as it belongs to human immunoglobulin (hereinafter referred to as hIg), and hIgG class is preferable.
Furthermore, any of subclasses such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class can be used. As the CL of the human chimeric antibody, any CL can be used so long as it belongs to hIg, and a κ class or a λ class can be used.

The human CDR-grafted antibody is prepared by comparing the amino acid sequences of CDRs of VH and VL of a nonhuman animal antibody with the VH of a human antibody.
And the antibody grafted into the appropriate position of the VL. Human CDR-grafted antibody reacts with ALK7 protein
VH and VL of non-human animal antibodies that bind to proteins
CDNA sequences encoding the V regions in which the CDR sequences of any human antibody were replaced with the CDR sequences of any human antibody with the CDR sequences of It can be produced by constructing a human CDR-grafted antibody expression vector by inserting each, introducing into human cells, and expressing.

[0187] As the CH of the human CDR-grafted antibody, any CH may be used as long as it belongs to hIg, but hIgG class is preferable, and hIgG1, hIgG2, hIgG belonging to hIgG class are preferable.
Any of the subclasses such as 3 and hIgG4 can be used. Further, as the CL of the human CDR-grafted antibody, any CL may be used as long as it belongs to hIg, and a κ class or a λ class can be used.

The human antibody originally means an antibody naturally existing in the human body, but a human antibody phage library prepared by the recent advances in genetic engineering, cell engineering, and developmental engineering techniques. And antibodies and the like obtained from human antibody-producing transgenic animals.

The antibody existing in the human body can be obtained, for example, by the following method. Human peripheral blood lymphocytes are isolated, infected with EB virus and the like to immortalize, and then cloned. The obtained lymphocytes that produce the desired antibody can be cultured and the antibody can be obtained from the culture.

The human antibody phage library is human B
This is a library in which antibody fragments such as Fab and single chain antibody are expressed on the surface of phage by inserting the antibody gene prepared from cells into the phage gene. Phage expressing an antibody fragment having a desired antigen-binding activity can be recovered from the library by using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment is
Furthermore, it can be converted into a fully human antibody by a genetic engineering technique.

Human antibody-producing transgenic animals are
It means an animal in which a human antibody gene is integrated into a cell.
Specifically, by introducing a human antibody gene into mouse ES cells,
A human antibody-producing transgenic animal can be prepared by transplanting the ES cell into an early embryo of another mouse and then allowing the embryo to develop. As a method for producing a human antibody from a human antibody-producing transgenic animal, a human antibody-producing hybridoma is obtained by a hybridoma production method that is generally performed in mammals other than humans, and the human antibody is cultured in the human antibody. There is a method of producing and accumulating.

Antibody fragments include Fab, Fab ′, F (ab ′) ₂ ,
Single chain antibody, disulfide-stabilized V region fragment (dsFv),
Examples include peptides containing CDR. Fab is a fragment obtained by treating IgG with the proteolytic enzyme papain (cleaved at the 224th amino acid residue of the H chain). It is a bound antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity. Fab is activin AB, activin B, or A
It can be obtained by treating an antibody that specifically reacts with the LK7 protein with the proteolytic enzyme papain. In addition, the antibody
A Fab can be obtained by inserting a DNA encoding Fab into a prokaryotic expression vector or a eukaryotic expression vector, introducing the vector into a prokaryotic organism or a eukaryotic organism, and expressing the DNA.

F (ab ') ₂ is a fragment obtained by treating IgG with the proteolytic enzyme pepsin (cleaved at the 234th amino acid residue of the H chain), and Fab has a disulfide bond in the hinge region. It is an antibody fragment having a molecular weight of about 100,000 and having an antigen-binding activity, which is slightly larger than that bound via the antibody. F
(ab ') ₂ is activin AB, activin B, or AL
It can be obtained by treating an antibody that specifically reacts with the K7 protein with the proteolytic enzyme pepsin. Also, the F of the antibody
After inserting the DNA encoding (ab ') ₂ into a prokaryotic expression vector or a eukaryotic expression vector, the vector is introduced into a prokaryotic organism or a eukaryotic organism, and the DNA is expressed to obtain F (ab ') You can get ₂ .

Fab ′ is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of F (ab ′) ₂ above. Fab ′ can be obtained by treating an antibody that specifically reacts with activin AB, activin B, or ALK7 protein by treating with a reducing agent dithiothreitol. In addition, after inserting a DNA encoding the Fab 'fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, introducing the vector into a prokaryote or a eukaryote, and expressing the DNA, Fab 'can be obtained.

A single-chain antibody (hereinafter, also referred to as scFv) is prepared by combining one VH and one VL with an appropriate peptide linker (hereinafter,
VH-P-VL or VL-P
-VH polypeptide is shown. VH and VL contained in scFv used in the present invention are activin AB, activin B,
Alternatively, an antibody that specifically reacts with the ALK7 protein, for example, a humanized antibody or an antibody derived from a human antibody can be used.

The single chain antibody can be obtained by the following method. VH of an antibody that specifically reacts with ALK7 protein and
After obtaining the cDNA encoding VL, the DNA encoding the single chain antibody is constructed. The DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryotic organism or a eukaryotic organism.
By expressing A, a single chain antibody can be obtained.

Disulfide-stabilized V region fragment (hereinafter dsF
Also referred to as v) refers to a polypeptide in which one amino acid residue in each of VH and VL is replaced with a cysteine residue, and the polypeptides are bound via a disulfide bond between the cysteine residues. Reit is the amino acid residue that replaces the cysteine residue.
er et al. [Protein Engineering, 7 , 697
(1994)], the selection can be made based on the prediction of the three-dimensional structure of the antibody. As VH and VL contained in dsFv used in the present invention, an antibody that specifically reacts with ALK7, for example, a humanized antibody or one derived from a human antibody can be used.

The disulfide stabilized V region fragment (dsFv) is
It can be obtained by the following method. C that encodes VH and VL of an antibody that specifically reacts with ALK7 protein
After obtaining the DNA, a DNA encoding dsFv is constructed.
The dsFv can be obtained by inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, introducing the expression vector into a prokaryotic organism or a eukaryotic organism, and expressing the DNA. The peptide containing CDR can be produced by a chemical synthesis method such as Fmoc method or tBoc method.

A fusion antibody prepared from an antibody that specifically recognizes the ALK7 protein can be used for drug delivery, which specifically conveys a drug or protein to a lesion of the pancreas. The fusion antibody is an antibody that specifically reacts with the ALK7 protein, for example, a humanized antibody, a human antibody or an antibody fragment thereof, which is chemically or genetically engineered with a drug such as a protein or a low molecular weight compound. Say. The fused antibody is the N-terminal side or C-terminal side of the H chain or L chain of an antibody or antibody fragment that specifically reacts with ALK7 protein, an appropriate substituent or side chain in the antibody or antibody fragment, and further It can be produced by chemically or genetically coupling a drug such as a protein or a low molecular weight compound to the sugar chain in the antibody fragment.

Examples of the low molecular weight compound include the compounds obtained by the above-mentioned screening method 8. The compound was prepared by a chemical method [Antibody Introduction (Osamu Kanemitsu, 19
1994, Jishin Shokan)]]. The protein is preferably an activating cytokine that enhances the function of the pancreas, and examples thereof include human beta-cellulin, human activin AB, human activin B,
Examples include human HGF (Hepatocyte Growth Factor).

A fusion antibody with a protein can be obtained by the following method. CDNA encoding an antibody or antibody fragment
A cDNA encoding a protein is ligated to and a DNA encoding a fusion antibody is constructed. A fusion antibody can be obtained by inserting the DNA into a prokaryotic or eukaryotic expression vector, introducing the expression vector into a host cell such as a prokaryotic or eukaryotic cell, and expressing the DNA. . The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the examples.

[0202]

Examples Example 1: Analysis of ligand-dependent transcriptional activity in cells overexpressing ALK7 Human renal epithelial cell HE that does not endogenously express ALK7
K293 cells (ATCC CRL-1573) with 10% fetal bovine serum, 2m
D-ME containing mol / L glutamine and 1g / L glucose
Suspend in M medium and add 6 x 10 ⁴ to 12-well cell culture plate.
Cells / well were seeded and cultured at 37 ° C. in a carbon dioxide incubator (5% carbon dioxide, 95% air). The next day, (1) a plasmid containing the full-length cDNA of rat ALK7 [Mol. Cell Neurosci., 7 , 467-478 (1
996)] Sma I- Xba I fragment and Ec of expression plasmid pcDNA1 for animal cells (manufactured by Invitrogen).
o A plasmid prepared by ligation with an RV- Xba I fragment [Mol. Cell Neurosci., 7 , 46
7-478 (1996)] 1.5 μg, (2) a plasmid containing the mouse type II activin receptor ActRIIA cDNA [Cell, 65 ,
973-982 (1991)] the restriction enzymes Bam and digested blunt-ended DNA fragments by HI, known animal cell expression plasmid pcDLSR-α [Mol. Cell. Biol., 8, 466-472
(1988)] and digested with Xho I and ligated with a blunt-ended DNA fragment [J. Biol. Chem., 272 , 13835-13842 (1997)] 30.
0 ng, (3) SBE-lux luciferase reporter plasmid [J. Biol. Chem., 273 , 21145-21152 (1998), Ludw
Transferred from ig Institute for Cancer Research] 500ng,
(4) Beta-galactosidase is CMV in animal cells
300 ng of plasmid pCMVβ (manufactured by Clontech), which is designed to be expressed under the control of a promoter, was introduced into the cells obtained by the above-mentioned culture by the calcium phosphate method, and further, 10% fetal bovine serum, 2 mmol / L glutamine, Using D-MEM medium containing 1 g / L glucose, the cells were cultured at 37 ° C. in a carbon dioxide gas incubator to obtain transformed cells. The next day, the transformed cells were washed with PBS buffer and suspended in the above medium, followed by J. Biol. Che.
m., 267 , 16385-16389 (1992), 20 ng / ml of activin A, AB or B purified from bovine follicular fluid was added. 16 at 37 ℃ in carbon dioxide incubator
After culturing for a period of time, the transformed cells were washed with PBS buffer, and 100 μL of cell lysate (1% TritonX-100, 15 mmol / L magnesium sulfate, 4 mmol / L EGTA, 1 mmol / L dithiothreitol, 25 mmol / L Glycylglycine, pH 7.8) was suspended in 50 μL for luciferase assay and 40 μL for beta-galactosidase assay.

The luciferase activity was measured using the above 50 μm.
Lysate of L cells and 5 μL of 30 mmol / L ATP solution,
It was performed by mixing 100 μL of 0.5 mmol / L luciferin solution and measuring the amount of luminescence with a luminometer (Bertold Co.) [Anal. Biochem., 171 , 404-408 (198
8)], The activity of beta-galactosidase was measured by o-nit
It was performed as previously reported by using rophenyl-β-D-galactopyranoside (ONPG) as a substrate [Bitechniqu
es, 7 , 576-579 (1989)]. The activity value of luciferase was standardized by the activity value of beta-galactosidase [Endo
crinology, 136 , 5493-5503 (1995)].

As shown in FIG. 1, a marked increase in transcription activity was observed in ALK7 gene-transfected HEK293 cells by activin AB and activin B.

Example 2 Analysis of Ligand-Dependent Transcriptional Activity in Endogenous ALK7-Expressing Pancreatic β-Cells In order to search for cell lines that endogenously express ALK7,
DNA having the nucleotide sequences represented by SEQ ID NOs: 7 and
Was synthesized and the DNA was used as a primer set to prepare mouse pancreatic β cell type MIN6 cells [Endocrinology, 127 , 126-
132 (1990)], human renal epithelial cells HEK293 cells (ATC
C CRL-1573), mouse insulinoma cell NIT-1 cell (ATCC CRL-2055), mouse pancreatic α cell type αTC1 cell (ATCC CRL-2350), rat pancreatic duct epithelial cell ARIP cell (ATCC CRL-1674), etc. A PCR reaction was performed using a single-stranded DNA prepared based on total RNA as a template.
As a result, MIN6 cells, which are used as a model system for mouse pancreatic β cells, were found to have higher expression than other cells.

Next, using this cell, measurement of transcription activity,
Mouse dominant negative ALK7 receptor cDNA
The inhibitory effect of signal transduction by introduction was examined. MIN6 cells, which are mouse pancreatic β-cell type cells, are treated with 10% fetal bovine serum,
D-containing 2 mmol / L glutamine and 4.5 g / L glucose
Suspend in MEM medium and place on a 12-well cell culture plate.
The cells were seeded at 10 ⁴ cells / well and cultured at 37 ° C in a carbon dioxide gas incubator. Next day, (1) SBE-
lux luciferase reporter plasmid 1 μg, (2)
Transfected cells were obtained by introducing 500 ng of pCMVβ into the cells obtained by the above culture by the lipofection method and further culturing at 37 ° C. in a carbon dioxide gas incubator. The next day, the transformed cells were washed with PBS buffer, suspended in the above medium, and then 60 ng / ml of activin A,
AB or B was added. After culturing at 37 ° C. for 16 hours in a carbon dioxide gas incubator, the cells were washed with PBS buffer, suspended in 100 μL of cell lysate, 50 μL was used for luciferase assay, and 40 μL was used for beta-galactosidase assay. The activity value of luciferase was standardized by the activity value of beta-galactosidase.

Next, using the DNAs having the base sequences represented by SEQ ID NOS: 7 and 8 as a primer set, rat ALK7 having the base sequence represented by SEQ ID NO: 10 was prepared.
Plasmid containing cDNA [Mol. Cell Neurosci., 7 ,
467-478 (1996)] as a template for PCR reaction.
Then, the amplified DNA fragment in the reaction product is treated with a restriction enzyme.
Digested with Eco RI and Xho I. After completion of the reaction, the reaction product was separated by agarose gel electrophoresis to recover a DNA fragment of about 500 base pairs. Ec of the obtained DNA fragment and plasmid pcDNA3 (manufactured by Invitrogen)
The RI- Xho I fragment was ligated with DNA ligase. Transform the E. coli using the reaction,
Incubate at 37 ° C on an agar plate containing ampicillin,
A clone that acquired ampicillin resistance was selected. After the emerged colonies were cultured in a medium containing ampicillin, the plasmid was recovered, the nucleotide sequence of the inserted DNA fragment was confirmed, and a clone having the desired nucleotide sequence was selected.

By the above procedure, a plasmid containing a dominant negative ALK7 receptor DNA lacking the intracellular kinase region and encoding 167 amino acid residues from the N-terminus was obtained.

MIN6 cells, which are mouse pancreatic β cell type cells,
Suspend in D-MEM medium containing 10% fetal bovine serum, 2 mmol / L glutamine, 4.5 g / L glucose, and inoculate on a 12-well cell culture plate at 8 × 10 ⁴ cells / well, and then in a carbon dioxide incubator. It was cultivated at 37 ° C. The next day, (1) SBE-lux luciferase reporter plasmid 1 μg, (2) Beta-galactosidase plasmid (Clontech) 500n designed to be expressed in animal cells under the control of CMV promoter
g, (3) Dominant negative type AL prepared above
3 μg of K7 receptor DNA was introduced into MIN6 cells by the lipofection method and cultured at 37 ° C. in a carbon dioxide incubator to obtain a transformant. The next day, the transformed cells were washed with PBS buffer, suspended in the above medium, and 60 ng / ml of activin A, AB, or B was added. 37 in the carbon dioxide incubator
After culturing at 0 ° C. for 16 hours, the transformed cells were washed with PBS buffer, suspended in 100 μL of cell lysate, 50 μL was used for luciferase assay, and 40 μL was used for beta-galactosidase assay. The activity value of luciferase was standardized by the activity value of beta-galactosidase.

As shown in FIG. 2, an increase in transcription activity was observed in endogenous ALK7-expressing MIN6 cells due to activin AB and activin B, and the dominant negative ALK7cD was observed.
The introduction of NA inhibited the increase in transcription activity.

Example 3 Promotion of Insulin Secretion from Mouse Pancreatic β Cell Type Cells MIN6 confirmed to express an endogenous ALK7 receptor and have reactivity to activin AB and activin B
Cells are 10% fetal bovine serum, 2 mmol / L glutamine, 4.5 g / L
The cells were suspended in D-MEM medium containing glucose, seeded on a 24-well cell culture plate at 7 × 10 ⁴ cells / well, and cultured at 37 ° C. in a carbon dioxide incubator. Wash cells with PBS buffer after 2 days and 40 or 8
The cells were suspended in 300 μL of a culture medium containing 0 ng / ml activin AB. After culturing at 37 ° C in a carbon dioxide incubator for another 2 days, collect the supernatant after culturing, and use 30 μL of the supernatant to measure the amount of insulin secreted by the ELISA method using a commercially available insulin assay kit (Morinaga Biochemical Laboratories). Was measured. As shown in FIG. 3, activin AB increased insulin secretion in endogenous ALK7-expressing MIN6 cells.

[0212]

INDUSTRIAL APPLICABILITY According to the present invention, activin AB, activin B and activin receptor-like kinase 7 (ALK
7) An antidiabetic agent containing a protein selected from 7) as an active ingredient, an antidiabetic agent containing a DNA encoding the protein as an active ingredient, a screening method for an antidiabetic agent using the protein or the DNA, the DNA or Diabetes diagnostic agent and diagnostic kit using an antibody that specifically recognizes the protein, and a method for delivering a drug to the kidney for the treatment and / or prevention of diabetes using an antibody that specifically recognizes ALK7 Will be provided.

[0213]

[Sequence list free text]

SEQ ID NO: 7-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 8-Description of Artificial Sequence: Synthetic DNA

[0214]

[Sequence list] SEQUENCE LISTING <110> KYOWA HAKKO KOGYO CO., LTD. <120> A DRUG FOR DIABETES <130> A11334MA <160> 10

[0215] <210> 1 <211> 116 <212> PRT <213> Homo sapience <400> 1 Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys Lys Gly Phe   1 5 10 15 Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro              20 25 30 Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile          35 40 45 Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn      50 55 60 His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys  65 70 75 80 Cys Tyr Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp                  85 90 95 Gly Gly Asn Ile Ile Lys Lys Asp Ile Gly Asn Met Ile Val Glu Glu             100 105 110 Cys Gly Cys Ser         115

[0216] <210> 2 <211> 115 <212> PRT <213> Homo sapience <400> 2 Gly Leu Glu Cys Asp Gly Arg Thr Asn Leu Cys Cys Arg Gly Gly Phe   1 5 10 15 Phe Ile Asp Phe Arg Leu Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro              20 25 30 Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser Cys Pro Ala Tyr Leu          35 40 45 Ala Gly Val Pro Gly Ser Ala Ser Ser Phe His Thr Ala Val Val Asn      50 55 60 Gly Tyr Arg Met Arg Gly Leu Asn Pro Gly Thr Val Asn Ser Cys Cys  65 70 75 80 Ile Pro Thr Lys Leu Ser Thr Met Ser Met Leu Tyr Phe Asp Asp Glu                  85 90 95 Tyr Asn Ile Val Lys Arg Asp Val Pro Asn Met Ile Val Glu Glu Cys             100 105 110 Gly Cys Ala         115

[0217] <210> 3 <211> 348 <212> DNA <213> Homo sapience <400> 3 ggc ttg gag tgt gat ggc aag gtc aac atc tgc tgt aag aaa cag ttc 48 Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe   1 5 10 15 ttt gtc agt ttc aag gac atc ggc tgg aat gac tgg atc att gct ccc 96 Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro              20 25 30 tct ggc tat cat gcc aac tac tgc gag ggt gag tgc ccg agc cat ata 144 Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile          35 40 45 gca ggc acg tcc ggg tcc tca ctg tcc ttc cac tca aca gtc atc aac 192 Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn      50 55 60 cac tac cgc atg cgg ggc cat agc ccc ttt gcc aac ctc aaa tcg tgc 240 His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys  65 70 75 80 tgt gtg ccc acc aag ctg aga ccc atg tcc atg ttg tac tat gat gat 288 Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp                  85 90 95 ggt caa aac atc atc aaa aag gac att cag aac atg atc gtg gag gag 336 Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu             100 105 110 tgt ggg tgc tca 348 Cys Gly Cys Ser         115

[0218] <210> 4 <211> 345 <212> DNA <213> Homo sapience <400> 4 ggc ctg gag tgc gat ggc cgg acc aac ctc tgt tgc agg caa cag ttc 48 Gly Leu Glu Cys Asp Gly Arg Thr Asn Leu Cys Cys Arg Gln Gln Phe   1 5 10 15 ttc att gac ttc cgc ctc atc ggc tgg aac gac tgg atc ata gca ccc 96 Phe Ile Asp Phe Arg Leu Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro              20 25 30 acc ggc tac tac ggc aac tac tgt gag ggc agc tgc cca gcc tac ctg 144 Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser Cys Pro Ala Tyr Leu          35 40 45 gca ggg gtc ccc ggc tct gcc tcc tcc ttc cac acg gct gtg gtg aac 192 Ala Gly Val Pro Gly Ser Ala Ser Ser Phe His Thr Ala Val Val Asn      50 55 60 cag tac cgc atg cgg ggt ctg aac ccc ggc acg gtg aac tcc tgc tgc 240 Gln Tyr Arg Met Arg Gly Leu Asn Pro Gly Thr Val Asn Ser Cys Cys  65 70 75 80 att ccc acc aag ctg agc acc atg tcc atg ctg tac ttc gat gat gag 288 Ile Pro Thr Lys Leu Ser Thr Met Ser Met Leu Tyr Phe Asp Asp Glu                  85 90 95 tac aac atc gtc aag cgg gac gtg ccc aac atg att gtg gag gag tgc 336 Tyr Asn Ile Val Lys Arg Asp Val Pro Asn Met Ile Val Glu Glu Cys             100 105 110 ggc tgc gcc 345 Gly Cys Ala         115

[0219] <210> 5 <211> 345 <212> DNA <213> Homo sapience <400> 5 agcggggtca ccgcccggct gcggggccag tggcaggagc gccacgcacc gccagccgca 60 gggggcgtgg gatgggggcg gccggggagg ggggcgccca cactgactag agccaaccgc 120 gcacttcaaa agggtgtcgg tgccgcgctc ccctcccgcg gcccgggaac ttcaaagcgg 180 gccgtgctgc cccggctgcc tcgctctgct ctggggcctc gcagccccgg cgcggccgcc 240 tggtggcgat gacccgggcg ctctgctcag cgctccgcca ggctctcctg ctgctcgcag 300 cggccgccga gctctcgcca ggactgaagt gtgtatgtct tntgtgtgat tcttcaaact 360 ttacctgcca aacagaagga gcatgtnggg catcagtcat gctaaccaat ggaaaagagc 420 aggtgatcaa atcctgtgtc tcccttccag aactga 456

[0220] <210> 6 <211> 927 <212> DNA <213> Homo sapience <400> 6 tttacctaag gcagaggcct gggcaatggc gggcgcccct cccccagtct cgttgccgcc 60 ttgcagttga tctcagactg ctgtgctagc aatcagcgag actccctggg cgtaggaccc 120 tctgagccag gtgtgggata tagtctcgtg gtgcgccgtt tcttaagccg gtctgaaaag 180 cgcaatattc gggtgggagt gacccgattt tccagcatca ccaaatgccc caaaacttgg 240 acccatggag ctggccatca ttattactgt gcctgtttgc ctcctgtcca tagctgcgat 300 gctgacagta tgggcatgcc agggtcgaca gtgctcctac aggaagaaaa agagaccaaa 360 tgtggaggaa ccactctctg agtgcaatct ggtaaatgct ggaaaaactc tgaaagatct 420 gatttatgat gtgaccgcct ctggatctgg ctctggtcta cctctgttgg ttcaaaggac 480 aattgcaagg acgattgtgc ttcaggaaat agtaggaaaa ggtagatttg gtgaggtgtg 540 gcatggaaga tggtgtgggg aagatgtggt gtgaaaatat tctcctccag agatgaaaag 600 atcttggttt cgtgaggcag aaaatttacc agacggtcat gctgcgaaat gaaaaccatc 660 ttggtttcat gggctgctga ccaccaagat aatggactgt ggctccactt ggggtggtat 720 ttgaatttca tgacagggtc ttatatgact atcgcataga attatggcgg gtgagagaca 780 aggggcccat gcggggtggg cctcttgggt gggcgcccag ggagccggtg gccgggccaa 840 accggaatct cgaacggatg gccccgccgt ggggcgacag gcaaccccat cgggacagat 900 tgcccgcgga tatctccctc cgtgacc 927

[0221] <210> 7 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 7 cctaagcttg atgaccccag cgcgccgctc cgcactga 38

[0222] <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> ccgctcgagc taagatctga gggtttttcc agcattgaca agg 43

[0223] <210> 9 <211> 493 <212> PRT <213> Rattus norvegicus <400> 9 Met Thr Pro Ala Arg Arg Ser Ala Leu Ser Leu Ala Leu Leu Leu Val   1 5 10 15 Ala Leu Ala Ser Asp Leu Ala Ala Gly Leu Lys Cys Val Cys Leu Leu              20 25 30 Cys Asp Ser Ser Asn Phe Thr Cys Gln Thr Glu Gly Ala Cys Trp Ala          35 40 45 Ser Val Met Leu Thr Asn Gly Lys Glu Gln Val Ser Lys Ser Cys Val      50 55 60 Ser Leu Pro Glu Leu Asn Ala Gln Val Phe Cys His Ser Ser Asn Asn  65 70 75 80 Val Thr Lys Thr Glu Cys Cys Phe Thr Asp Phe Cys Asn Asn Ile Thr                  85 90 95 Gln His Leu Pro Thr Ala Ser Pro Asp Ala Pro Arg Leu Gly Pro Thr             100 105 110 Glu Leu Thr Val Val Ile Thr Val Pro Val Cys Leu Leu Ser Ile Ala         115 120 125 Ala Met Leu Thr Ile Trp Ala Cys Gln Asp Arg Gln Cys Thr Tyr Arg     130 135 140 Lys Thr Lys Arg His Asn Val Glu Glu Pro Leu Ala Glu Tyr Ser Leu 145 150 155 160 Val Asn Ala Gly Lys Thr Leu Lys Asp Leu Ile Tyr Asp Ala Thr Ala                 165 170 175 Ser Gly Ser Gly Ser Gly Pro Pro Leu Leu Val Gln Arg Thr Ile Ala             180 185 190 Arg Thr Ile Val Leu Gln Glu Ile Val Gly Lys Gly Arg Phe Gly Glu         195 200 205 Val Trp His Gly Arg Trp Cys Gly Glu Asp Val Ala Val Lys Ile Phe     210 215 220 Ser Ser Arg Asp Glu Arg Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln 225 230 235 240 Thr Val Met Leu Arg His Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp                 245 250 255 Asn Lys Asp Asn Gly Thr Trp Thr Gln Leu Trp Leu Val Ser Glu Tyr             260 265 270 His Glu Gln Gly Ser Leu Tyr Asp Tyr Leu Asn Arg Asn Ile Val Thr         275 280 285 Val Ala Gly Met Val Lys Leu Ala Leu Ser Ile Ala Ser Gly Leu Ala     290 295 300 His Leu His Met Glu Ile Val Gly Thr Gln Gly Lys Pro Ala Ile Ala 305 310 315 320 His Arg Asp Ile Lys Ser Lys Asn Ile Leu Val Lys Lys Cys Asp Thr                 325 330 335 Cys Ala Ile Ala Asp Leu Gly Leu Ala Val Lys His Asp Ser Ile Met             340 345 350 Asn Thr Ile Asp Ile Pro Gln Asn Pro Lys Val Gly Thr Lys Arg Tyr         355 360 365 Met Ala Pro Glu Met Leu Asp Asp Thr Met Asn Val Asn Ile Phe Glu     370 375 380 Ser Phe Lys Arg Ala Asp Ile Tyr Ser Val Gly Leu Val Tyr Trp Glu 385 390 395 400 Ile Ala Arg Arg Cys Ser Val Gly Gly Leu Val Glu Glu Tyr Gln Leu                 405 410 415 Pro Tyr Tyr Asp Met Val Pro Ser Asp Pro Ser Ile Glu Glu Met Arg             420 425 430 Lys Val Val Cys Asp Gln Lys Leu Arg Pro Asn Leu Pro Asn Gln Trp         435 440 445 Gln Ser Cys Glu Ala Leu Arg Val Met Gly Arg Ile Met Arg Glu Cys     450 455 460 Trp Tyr Ala Asn Gly Ala Ala Arg Leu Thr Ala Leu Arg Val Lys Lys 465 470 475 480 Thr Ile Ser Gln Leu Cys Val Lys Glu Asp Cys Lys Ala                 485 490

[0224] <210> 10 <211> 1562 <212> DNA <213> Rattus norvegicus <220> <221> CDS <222> (32) ... (1510) <400> 10 ccccgaagcc ttgcaccgcc gcggggtggc c atg acc cca gcg cgc cgc tcc 52                                    Met Thr Pro Ala Arg Arg Ser                                      1 5 gca ctg agc ctg gcc ctc ctg ctg gtg gca ctg gcc tcc gac ctt gcg 100 Ala Leu Ser Leu Ala Leu Leu Leu Val Ala Leu Ala Ser Asp Leu Ala          10 15 20 gca gga ctg aag tgt gtg tgt ctt ttg tgt gat tcc tca aac ttt acc 148 Ala Gly Leu Lys Cys Val Cys Leu Leu Cys Asp Ser Ser Asn Phe Thr      25 30 35 tgc caa acc gaa gga gca tgc tgg gcc tct gtc atg cta acc aac ggg 196 Cys Gln Thr Glu Gly Ala Cys Trp Ala Ser Val Met Leu Thr Asn Gly  40 45 50 55 aaa gaa cag gtg agc aaa tcg tgc gtg tcc ctc ccg gaa cta aat gct 244 Lys Glu Gln Val Ser Lys Ser Cys Val Ser Leu Pro Glu Leu Asn Ala                  60 65 70 cag gtc ttc tgt cac agt tcc aac aac gtg acc aag acc gaa tgt tgc 292 Gln Val Phe Cys His Ser Ser Asn Asn Val Thr Lys Thr Glu Cys Cys              75 80 85 ttc aca gac ttc tgc aac aac atc act cag cac ctt ccc aca gca tct 340 Phe Thr Asp Phe Cys Asn Asn Ile Thr Gln His Leu Pro Thr Ala Ser          90 95 100 cca gat gcc cct aga ctt ggc ccc aca gag ctg aca gtt gtt atc act 388 Pro Asp Ala Pro Arg Leu Gly Pro Thr Glu Leu Thr Val Val Ile Thr     105 110 115 gta cct gtt tgc ctc ctg tcc atc gca gcc atg cta acg ata tgg gcc 436 Val Pro Val Cys Leu Leu Ser Ile Ala Ala Met Leu Thr Ile Trp Ala 120 125 130 135 tgc cag gac cgc cag tgc aca tac agg aag acc aag aga cac aat gtg 484 Cys Gln Asp Arg Gln Cys Thr Tyr Arg Lys Thr Lys Arg His Asn Val                 140 145 150 gag gaa cca ctg gca gag tac agc ctt gtc aat gct gga aaa acc ctc 532 Glu Glu Pro Leu Ala Glu Tyr Ser Leu Val Asn Ala Gly Lys Thr Leu             155 160 165 aaa gat ctg att tat gat gcc act gcc tcg ggc tca gga tct ggc ccg 580 Lys Asp Leu Ile Tyr Asp Ala Thr Ala Ser Gly Ser Gly Ser Gly Pro         170 175 180 cct ctt ttg gtt caa aga acc atc gca agg aca att gta ctt caa gaa 628 Pro Leu Leu Val Gln Arg Thr Ile Ala Arg Thr Ile Val Leu Gln Glu     185 190 195 atc gta gga aaa ggt cgg ttt ggg gaa gtg tgg cac gga aga tgg tgt 676 Ile Val Gly Lys Gly Arg Phe Gly Glu Val Trp His Gly Arg Trp Cys 200 205 210 215 gga gaa gat gtg gct gtg aaa ata ttc tcc tcc aga gat gag aga tct 724 Gly Glu Asp Val Ala Val Lys Ile Phe Ser Ser Arg Asp Glu Arg Ser                 220 225 230 tgg ttc cgt gag gca gaa att tat cag acg gta atg ctg aga cat gag 772 Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr Val Met Leu Arg His Glu             235 240 245 aat att ctc ggt ttc atc gcg gcc gac aac aaa gat aat gga acc tgg 820 Asn Ile Leu Gly Phe Ile Ala Ala Asp Asn Lys Asp Asn Gly Thr Trp         250 255 260 act cag ctt tgg ctt gtg tca gag tat cac gag cag ggc tcc tta tat 868 Thr Gln Leu Trp Leu Val Ser Glu Tyr His Glu Gln Gly Ser Leu Tyr     265 270 275 gac tat ttg aat aga aac ata gtg acc gtg gct gga atg gtc aag ttg 916 Asp Tyr Leu Asn Arg Asn Ile Val Thr Val Ala Gly Met Val Lys Leu 280 285 290 295 gcg ctt tca ata gcg agt ggt ctg gct cac cta cac atg gag atc gtg 964 Ala Leu Ser Ile Ala Ser Gly Leu Ala His Leu His Met Glu Ile Val                 300 305 310 ggc act caa ggt aag cct gct att gct cac cga gat ata aag tca aag 1012 Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Ile Lys Ser Lys             315 320 325 aat atc tta gtc aaa aag tgt gac act tgt gcc ata gct gac tta ggg 1060 Asn Ile Leu Val Lys Lys Cys Asp Thr Cys Ala Ile Ala Asp Leu Gly         330 335 340 ctg gct gtg aaa cat gat tct atc atg aac act ata gat ata ccc cag 1108 Leu Ala Val Lys His Asp Ser Ile Met Asn Thr Ile Asp Ile Pro Gln     345 350 355 aat cct aaa gtg gga acc aag agg tat atg gct ccc gaa atg ctt gat 1156 Asn Pro Lys Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Met Leu Asp 360 365 370 375 gat aca atg aac gtc aac atc ttt gag tcc ttc aag cga gct gac atc 1204 Asp Thr Met Asn Val Asn Ile Phe Glu Ser Phe Lys Arg Ala Asp Ile                 380 385 390 tat tcg gtg ggg ctg gtt tac tgg gaa ata gct cga agg tgt tca gtt 1252 Tyr Ser Val Gly Leu Val Tyr Trp Glu Ile Ala Arg Arg Cys Ser Val             395 400 405 gga gga ctt gtt gaa gag tac cag ttg cct tat tat gac atg gtg cct 1300 Gly Gly Leu Val Glu Glu Tyr Gln Leu Pro Tyr Tyr Asp Met Val Pro         410 415 420 tca gat cct tcc ata gag gaa atg agg aag gtc gtt tgt gat cag aaa 1348 Ser Asp Pro Ser Ile Glu Glu Met Arg Lys Val Val Cys Asp Gln Lys     425 430 435 ctg cga cca aat ctc cca aac cag tgg caa agc tgt gag gcg ctc cgg 1396 Leu Arg Pro Asn Leu Pro Asn Gln Trp Gln Ser Cys Glu Ala Leu Arg 440 445 450 455 gtc atg gga aga ata atg cgt gag tgc tgg tat gcc aac ggg gca gct 1444 Val Met Gly Arg Ile Met Arg Glu Cys Trp Tyr Ala Asn Gly Ala Ala                 460 465 470 cgc ctg acc gcc ctg cgc gtg aag aag acc att tct cag ctg tgt gtc 1492 Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Ile Ser Gln Leu Cys Val             475 480 485 aag gaa gac tgt aag gcc taaggataca ggcgacggga aagccctcac 1540 Lys Glu Asp Cys Lys Ala         490 cactctcttt catgtctcct gc 1562

[Brief description of drawings]

FIG. 1 shows the results of analysis of ligand-dependent transcriptional activity in ALK7-overexpressing cells. (-) Indicates that activin was not added, ALK7 (-) indicates a cell line into which the ALK7 gene has not been introduced, and ALK7 (+) indicates ALK7.
The transgenic strain is shown.

FIG. 2 is a diagram showing the analysis results of ligand-dependent transcriptional activity in endogenous ALK7-expressing pancreatic β cells. The filled columns show the results using MIN6 cells, and the open columns show the results using dominant negative ALK7-expressing MIN6 cells. Further, (-) indicates that activin was not added.

FIG. 3 is a diagram showing the results of measuring the amount of insulin secreted from mouse pancreatic β-cell tumor cells by the addition of activin.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 45/00 A61K 48/00 4C087 48/00 A61P 1/18 4H045 A61P 1/18 3/10 3/10 5/48 5/48 5/50 5/50 C12Q 1/02 C12Q 1/02 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33/53 D 33/53 M 33/566 33/566 C07K 14/52 // C07K 14/52 14/715 14/715 A61K 37/02 ZNA C12N 15/09 C12N 15/00 AF term (reference) 2G045 AA34 AA35 AA40 BA11 BB50 DA12 DA13 DA14 DA36 FB02 4B024 AA01 AA11 BA56 BA63 CA04 DA02 EA02 EA04 FA02 GA11 HA11 HA17 4B063 QA01 QA18 QQ01 QQ08 QQ13 QR33 QR59 QR77 QR80 QS05 QS36 QX02 4C084 AA01 AA02 AA13 AA17 BA01 BA02 BA08 BA21 BA44 CA18 DC50 NA13 NA14 ZA661 ZC351 ZC422 4C085 AA13 AA14 AA16 AA21 BB11 BB50 CC02 CC03 CC 04 CC07 CC08 CC23 DD23 DD62 DD63 4C087 AA01 AA02 BC83 CA12 NA13 NA14 ZA66 ZC35 ZC42 4H045 AA10 AA20 AA30 BA10 CA40 DA01 DA51 EA20 EA50 FA72 FA74

Claims (35)

[Claims] 1. A medicine for the treatment and / or prevention of diabetes, which comprises a protein selected from the group consisting of activin AB, activin B, and ALK7 as an active ingredient. 2. A medicament for treating and / or preventing diabetes, which comprises any of the following proteins as an active ingredient. [A] A dimer protein consisting of the amino acid sequence represented by SEQ ID NO: 1 and the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; [b] represented by SEQ ID NO: 1 A dimer composed of a polypeptide consisting of an amino acid sequence and a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acid residues are deleted, substituted, inserted and / or added. A protein having a function as a ligand for ALK7; [c] from an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1 And a dimeric protein consisting of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, A protein having a function as a ligand of LK7; or [d] a polypeptide comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1, And a dimeric protein consisting of a polypeptide consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2, Protein that functions as a ligand: 3. A medicament for treating and / or preventing diabetes, which comprises any of the following proteins as an active ingredient. [A] a dimeric protein composed of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; [b] a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and represented by SEQ ID NO: 2 A dimeric protein composed of a polypeptide having an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence, the protein having a function as a ligand of ALK7; Or [c] a dimeric protein composed of a polypeptide consisting of an amino acid sequence having one or more amino acid residues deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2 , A protein having a function as a ligand for ALK7: 4. A medicine for the treatment and / or prevention of diabetes which comprises any of the following proteins as an active ingredient. [A] c containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein encoded by DNA; or [b] c containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein having an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in an amino acid sequence encoded by DNA, and has a function as a receptor protein for activin AB or activin B protein: 5. A medicament for treating and / or preventing diabetes, which comprises any of the following polypeptides as an active ingredient. [A] a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 2; or [b] an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in SEQ ID NO: 1 or 2. A polypeptide which comprises a protein having a function as a ligand of ALK7, 6. The medicine according to any one of claims 1 to 5, wherein the medicine for treating and / or preventing diabetes has a pancreatic function improving action. 7. The medicine according to any one of claims 1 to 5, wherein the medicine for treating and / or preventing diabetes has an action of promoting insulin secretion from pancreatic β cells. 8. The medicine according to any one of claims 1 to 5, wherein the medicine for treating and / or preventing diabetes has an action of promoting regeneration of pancreatic exocrine cells and pancreatic endocrine cells. . 9. An insulin secretagogue comprising a protein selected from the group consisting of activin AB, activin B, and ALK7 as an active ingredient. 10. An activin βA chain, an activin βB chain,
Alternatively, a gene therapeutic agent containing the DNA encoding ALK7 for the treatment and / or prevention of diabetes. 11. A gene therapeutic agent for treating and / or preventing diabetes, which comprises any of the following DNAs. [A] DNA encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2; [b] One or more amino acid residues deleted or substituted in the amino acid sequence represented by SEQ ID NO: 1 or 2. Insert and /
Or a DNA comprising a polypeptide consisting of an added amino acid sequence and encoding a protein that constitutes a protein having a function as a ligand for ALK7; or [c] containing the nucleotide sequences represented by SEQ ID NOs: 3 and 4 D
NA: 12. A gene therapeutic agent for treating and / or preventing diabetes, which comprises any of the following DNAs. [A] c containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
DNA encoding a protein encoded by DNA; or [b] c containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein having an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in an amino acid sequence encoded by DNA, and has a function as a receptor protein for activin AB or activin B DNA encoding a protein: 13. The gene therapeutic agent according to any one of claims 10 to 12, wherein the gene therapeutic agent for treating and / or preventing diabetes has a pancreatic function improving action. 14. The gene therapeutic agent according to any one of claims 10 to 12, wherein the gene therapeutic agent for treating and / or preventing diabetes has an action of promoting insulin secretion from pancreatic β cells. . 15. The gene therapeutic agent for treating and / or preventing diabetes has an action of promoting regeneration of exocrine pancreatic cells and pancreatic endocrine cells.
13. The gene therapeutic agent according to any one of 12. 16. An activin βA chain, an activin βB chain,
Alternatively, an insulin secretagogue containing a DNA encoding ALK7. 17. A diagnostic agent for diabetes which comprises the DNA, the oligonucleotide or the derivative of the oligonucleotide according to any one of the following [a] to [c]. [A] activin βA chain, activin βB chain, or AL
DNA encoding K7, or DNA that is a partial fragment of these DNAs; [b] DNA consisting of the nucleotide sequences represented by SEQ ID NO: 3 or 4, cDNA containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6, or DNA that is a partial fragment of those DNAs
DNA selected from [c] cDNA containing the nucleotide sequence represented by SEQ ID NO: 3 or 4, or the nucleotide sequence represented by SEQ ID NOS: 5 and 6.
Having a sequence consisting of consecutive 5 to 60 bases in the base sequence of, an oligonucleotide having a sequence complementary to the oligonucleotide, or a derivative of the oligonucleotide. 18. Activin AB, activin B, AL
A diagnostic agent for diabetes containing an antibody that recognizes a protein selected from the group consisting of K7. 19. A diagnostic kit for diabetes, which comprises the DNA according to claim 17, an oligonucleotide or a derivative of the oligonucleotide, or the antibody according to claim 18. 20. (i) the expression level of the polypeptide or protein in cells expressing the polypeptide or protein according to any one of the following [a] to [d], and (ii) the polypeptide or By comparing the expression level of the polypeptide or protein when cells expressing the protein are contacted with a test sample, the polypeptide or protein of any one of the following [a] to [d] is compared with the test sample. A method for screening a medicine for treating and / or preventing diabetes, which comprises selecting a compound that increases the expression level. [A] Activin AB, Activin B, and ALK7
A protein selected from the following: [b] a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, and a polypeptide selected from a protein encoded by a cDNA containing the nucleotide sequences represented by SEQ ID NO: 5 or 6; Protein: [c] A polypeptide comprising an amino acid sequence in which one or more amino acid residues have been deleted, substituted, inserted and / or added in the amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 or 2, And a polypeptide constituting a protein having a function as a ligand for ALK7; or [d] containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence encoded by DNA, and has activity as activin AB or activin B receptor protein protein: 21. (i) the function of cells expressing the polypeptide or protein according to any one of the following [a] to [d], and (ii) cells expressing the polypeptide or protein and a test sample. The method for screening a drug for the treatment and / or prevention of diabetes, which comprises comparing a cell function when contacted with a cell and selecting a compound having an activity of controlling the cell function from a test sample. . [A] Activin AB, Activin B, and ALK7
A protein selected from the following: [b] a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, and a polypeptide selected from a protein encoded by a cDNA containing the nucleotide sequences represented by SEQ ID NO: 5 or 6; Protein: [c] A polypeptide comprising an amino acid sequence in which one or more amino acid residues have been deleted, substituted, inserted and / or added in the amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 or 2, And a polypeptide constituting a protein having a function as a ligand for ALK7; or [d] containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence encoded by DNA, and has activity as activin AB or activin B receptor protein protein: 22. The screening method according to claim 20, wherein the cell is a cell expressing a type II activin receptor. 23. The screening method according to any one of claims 20 to 22, wherein the cell is a pancreatic β cell type cell. 24. Transformation with a DNA containing a DNA linked with a reporter gene downstream of a region that controls the transcription of the gene encoding the polypeptide or protein according to any of the following [a] to [d]: The selected transformant is brought into contact with a test sample, and a compound that enhances the expression of the gene encoding the polypeptide or protein described in any of [a] to [d] below is selected from the test sample. A method for screening a medicine for treating and / or preventing diabetes. [A] Activin AB, Activin B, and ALK7
A protein selected from the following: [b] a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, and a polypeptide selected from a protein encoded by a cDNA containing the nucleotide sequences represented by SEQ ID NO: 5 or 6; Protein: [c] A polypeptide comprising an amino acid sequence in which one or more amino acid residues have been deleted, substituted, inserted and / or added in the amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 1 or 2, And a polypeptide constituting a protein having a function as a ligand for ALK7; or [d] containing the nucleotide sequences represented by SEQ ID NOs: 5 and 6
A protein consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted and / or added in the amino acid sequence encoded by DNA, and has activity as activin AB or activin B receptor protein protein: 25. The method according to any one of claims 20 to 24, wherein the drug for treating and / or preventing diabetes is a compound having a pancreatic function improving action. 26. The method according to any one of claims 20 to 24, wherein the drug for treating and / or preventing diabetes is a compound having an action of promoting insulin secretion from pancreatic β cells. 27. The method according to any one of claims 20 to 24, wherein the drug for treating and / or preventing diabetes is a compound having an action of promoting regeneration of exocrine pancreatic cells or pancreatic endocrine cells. 28. A compound obtained by the method according to any one of claims 20 to 24, or a pharmaceutically acceptable salt thereof. 29. A medicament for treating and / or preventing diabetes, which comprises the compound according to claim 28, or a pharmaceutically acceptable salt thereof. 30. An antibody that recognizes ALK7.
A drug for the treatment and / or prevention of diabetes, which is obtained by combining the compound according to 1. or a pharmaceutically acceptable salt thereof. 31. The medicine for treating and / or preventing diabetes according to claim 29 or 30, which is a medicine having a pancreatic function improving action. 32. The medicine for treating and / or preventing diabetes according to claim 29 or 30, which is a medicine having an action of promoting insulin secretion from pancreatic β cells. 33. The medicine for treating and / or preventing diabetes according to claim 29 or 30, which is a medicine having an action of promoting regeneration of exocrine pancreatic cells or pancreatic endocrine cells. 34. Diabetes, characterized in that a fusion antibody obtained by combining an antibody recognizing ALK7 and a drug for treating and / or preventing diabetes that acts on ALK7 is selectively accumulated in the pancreas. A method for delivering a medicine for the treatment and / or prevention of 35. The method according to claim 34, wherein the medicament for treating and / or preventing diabetes is the compound according to claim 28 or a pharmaceutically acceptable salt thereof.