JPWO2001098493A1 - Insulin-like growth factor binding protein - Google Patents

Abstract

(57) [Abstract] By using the protein of the present invention, DNA encoding the protein, and an antibody that recognizes the protein, it is possible to provide a method for determining diseases associated with the novel insulin-like growth factor binding protein of the present invention, as well as diagnostic, preventive, and therapeutic agents for diseases associated with the protein of the present invention.

Description

Detailed Description of the InventionTechnical Field The present invention relates to a novel insulin-like growth factor binding protein, a D that encodes the protein,
NA and an antibody that recognizes said protein, and a method for determining a disease associated with said protein,
Related to diagnostic, prophylactic or therapeutic agents.Background technologyInsulin-like growth factor binding protein (insulin-like growth factor binding protein)
factor binding protein (hereinafter referred to as "IGFBP")
The insulin-like growth factor (insulin-like growth factor) in body fluids
IGF-1 exists as a macromolecular complex.
This is a group of molecules discovered through the study of IGFBP-1 to IGFBP-10.
The existence of molecules of this type has been reported, and it is known that they form a superfamily.
[Endocr. Rev.,18, 801 (1997), Prog.
Growth Factor Res. ，3, 243 (1991), Mol. R
eprod. Dev.,35, 368 (1993), Proc. Natl. Ac
ad. Sci. USA,94, 12981 (1997)]. The functions of IGFBPs include direct action by binding to integrins.
Although it has been suggested that it also acts as a steroid hormone, its main function is to bind to IGF and insulin and inhibit their activity and decomposition.
It is believed that this effect is exerted by regulating metabolism, etc. [Endocr. R
ev.,18, 801 (1997), Bio Science Terminology Library
Cytokines and Growth Factors, Revised Edition, pp. 14-17 (1988)], specifically, I
GFBPs transport IGF into and out of the bloodstream, inhibit its degradation, and bind to its receptor.
It is thought that they exert their functions through regulation. Of the IGFBP superfamily, there are six types, IGFBP-1 to IGFBP-6.
The molecule is structurally similar and binds to IGF with higher affinity than insulin.
Because they bind to IGF-1, they are classified into a subfamily of IGF-high affinity IGFBPs.
[Mol. Endocrinol.,2, 404 (1988), EMBO
J.,8, 2497 (1989), Mol. Endocrinol. ，2, 11
76 (1989), Mol. Endocrinol. ，4, 1806 (1990
), Biochem. Biophys. Res. Commun. ，176, 21
9 (1991), J. Biol. Chem. ，266, 9043 (1991),
J. Biol. Chem. ，266, 10646 (1991)]. The amino acid sequence homology between the IGF high affinity IGFBP subfamily molecules is
In humans, the ratio is 49% to 60%.
18 cysteine residues are conserved in the α- and β-glucans, of which the three at the N-terminus are Gly-
A homologous sequence represented by Cys-Gly-Cys-Cys-XX-Cys, where X is
Any amino acid sequence is represented, and the homologous sequence has an insulin-like growth factor binding motif.
(hereinafter also referred to as "IGFBP motif"), which binds IGF.
It has been shown that it is involved in the synthesis of
es.,3, 243 (1991)]. IGFBP-1 and 3 both inhibit and promote the action of IGF.
It is known that IGFBP-2, 4, and 6 are inhibitory IGFBPs.
IGF-5 is a stimulatory IGF-binding protein. IGF binds to the hepatic steroid hormone in a growth hormone-dependent manner.
, produced in bone tissue and other tissues, and functions as a growth factor that promotes physical growth.
It is expressed in large amounts in the central nervous system, bone tissue, etc. along with IGF-I, and is mainly involved in fetal growth.
IGF-1 and IGF-II are thought to play an important role, but
P-1, 3, and 4 exhibited equivalent binding activity to IGF-I and IGF-II.
BP-2, BP-5, and BP-6 are known to exhibit strong binding activity mainly to IGF-II.
In addition, the tissue distribution of IGF high-affinity IGFBP subfamily molecules varies depending on the molecule.
IGFBP-1 is mainly found in amniotic fluid and fetal serum, and IGFBP
IGF-2 is mainly found in fetal liver and adult brain, while IGFBP-3 is mainly found in liver and serum.
IGFBP-4 is mainly expressed in the renal glomeruli, skin, and intestinal epithelium, while IGFBP-5 is mainly expressed in the intestinal epithelium and
It is known that IGFBP-1 is present mainly in bones, while IGFBP-6 is present mainly in skin and heart. The following findings have been reported regarding the relationship between IGFBP and pathology:
In patients with dwarfism and acromegaly, changes in the expression of IGF and IGFBPs are directly related to the pathophysiology.
It is known that it is involved in chronic renal failure in children.
Although long-term damage occurs, the expression levels of growth hormone and IGF are normal, and most
It is known that the cause is IGF dysfunction due to an increase in GFBP-2 and GFBP-3.
[Miner. Electrolyte Meter.,18, 320 (19
IGFBP-4 and IGFBP-5 play important roles in bone metabolism and are known to be involved in osteoporosis.
In elderly women with rheumatoid arthritis, the expression level of IGFBP-5 is decreased and parathyroid hormone is elevated.
It has been reported that IGFBP-4 expression is elevated in patients with bone fractures.
In the compensatory hypertrophy after nephrectomy or small bowel resection, the paracrine action of IGF-I is enhanced.
However, the amount of IGF-I mRNA did not change, and the expression of IGFBP-3 did not change.
It has been reported that a decrease in IGF-I levels increases free IGF-I [J. Fuller,
er;Baillieres Clin. Endocrinol. Metab.
,8, 165 (1994)]. Furthermore, malignant endometrial tumors are more likely to develop than benign tumors.
It has been reported that IGFBP-1 expression is decreased in all
Regul.,3, 74, (1993)]. On the other hand, among the GFBP superfamily, four types, IGFBP-7 to IGFBP-10,
These molecules share the common property of being structurally similar and having low affinity for IGF.
Since it is thought to have the same properties as IGF, it is thought to be a separate subfamily of IGF low affinity IGFBPs.
It is classified as a genus of lactic acid bacteria [Proc. Natl. Acad. Sci. USA,9
4, 12981 (1997), Cancer Res. ，59, 2787 (19
99)]. Various physiological activities of IGFBP-7 have been reported, particularly in cancer and
Many reports have been published on the relationship between IGFBP-7 and its pathology. IGFBP-7 expression is elevated in senescent human epithelial cells [J. Cli
n. Endocrinol. Metab. ，4, 715 (1993)] On the other hand,
Since its expression is reduced in carcinoma cell lines, it is thought to be a tumor suppressor gene.
It is thought that it may have a function as a
ad. Sci. USA,92, 4472 (1995)]. Human IGFBP-7
The gene locus is 4q12, and treatment of human epithelial cells with retinoic acid
It is also known that its expression increases in the presence of α-glucan [Proc. Natl. Acad. S
ci. USA,92, 4472 (1995)]. In breast cancer tissue, LOH (loss of heterozygosity) was observed at the chromosome 4q12-13 position.
terozygosity) is observed at a frequency of approximately 50%, and IGFBP-
It has been confirmed that the expression of 7 is decreased [Oncogene,16, 24
59 (1998)]. IGFBP-7 expression was also observed at the mRNA level in prostate cancer tissues.
It has been reported that the expression level of IL-1 is decreased in prostate cancer cells and is not detected in cell lines derived from malignant prostate cancer.
It has been reported [J. Clin. Endocrinol. Metab.,83, 43
55 (1998)]. Furthermore, we investigated the effect of IGFBP-7 on prostate cancer cell lines with reduced IGFBP-7 expression.
Forced expression of this gene results in a prolongation of cell division time and a decrease in colony formation ability in soft agar medium.
Bottom: Decrease in tumorigenicity when transplanted into nude mice, and induction of apoptosis by drug treatment
An increased incidence of IGFBP-7 has been observed, and the relationship between IGFBP-7 expression and the malignancy of prostate cancer has been
It has been suggested that59, 2370 (1999)]. In leukemia patients, the concentrations of IGFBP-7 and IGFBP-3 in the cerebrospinal fluid were
It has been reported that the level of leukemia increases, and attention is being paid to its relationship with the pathology of leukemia [J.
Clin. Endocrinol. Metab. ，84, 1283 (1999)
In colon cancer, IGFBP-7 expression is elevated in colon cancer tissues and colon cancer cell lines.
Since the level is increasing, attention is being paid to its relationship with pathological conditions [J. Gastroen
terology,33, 213 (1998)]. In large uterine leiomyomas, IGFBP-7 expression was decreased, and gonadotropin activity was
Gonadotropin-releasing hormone
Increased expression of IGFBP-7 was observed in patients treated with steroid hormone.
It has been reported [J. Reprod. Immunol.,43, 53 (2000
) ). In mouse hepatoma cells induced by SV40 T antigen, the IGFBP-7 gene
The 5' upstream region is methylated and the expression level is reduced, suggesting that IG is involved in cancer transformation.
A gene methylation mechanism has been proposed for the regulation of FBP-7 expression [Bio
chem. Biophys. Res. Commun. ，267, 109 (200
0)]. The relationship between IGFBP-7 and diabetes has also been reported. A factor that acts on vascular endothelial cells to promote the production of prostacyclin PGI2 (P
GI2-stimulating factor (hereinafter referred to as "PSF")
, which has been shown to be identical to IGFBP-7 [Biochem. J.,303,
591, (1994)], which is expressed in vascular endothelial cells and smooth muscle cells.
The factor [Thromb Haemost.,74, 1407 (1995)],
In a type 1 diabetes model induced by treptozotocin, the disease occurred in the kidney and vascular lesions.
It has been reported that the level of45, S111 (
1996), J. Diabetes & its Complications
,12, 252 (1998)]. In addition, the coronary artery smooth muscle cells of type II diabetes patients
In the 1990s, a decrease in IGFBP-7 expression was observed at the protein level [Diab
etes,46, 1627 (1997)]. Furthermore, bovine arterial smooth muscle cells
The expression level of IGFBP-7 was decreased when cultured in a high-glucose medium.
A and protein levels [Diabetes,46, 1627 (1
997), Diabetologia,41, 134 (1998)]. TGF-β (Transforming growth factor-β)
, parathyroid hormone (PTH) and prostaglandin E2 (PGE2)
Therefore, IGFBP-7 expression is elevated in osteoblasts, and therefore IGFBP-
It has been suggested that 7 exerts a physiological effect on osteoblasts [Endocrinology
Logy,140, 1998 (1999)].
Treatment with α-I suppresses the expression of IGF-I while increasing the expression of IGFBP-7.
It has also been reported that it increases the blood sugar level [Endocrinology,140, 228
(1999)]. IGFBP-7 also suppresses the differentiation-promoting action of IGFs in skeletal muscle differentiation.
It has been suggested that this is due to the influence of23
7, 192 (1997), Endocrinology,141, 100 (20
00). IGFBP-7 interacts with PSF, a factor that promotes the production of prostacyclin PGI2.
Since they are the same molecule, some of the physiological functions of IGFBP-7 involve the activation of PGI2.
It is thought that PGI2, a type of prostaglandin, has a strong inhibitory effect on platelet aggregation and vasoconstriction.
It has a relaxing effect and acts antagonistically with TXA2, which has the opposite effect, and maintains homeostasis in the body.
It is known that it is involved in the maintenance of76,
3 (1982)], and in thrombosis and arteriosclerosis, the production of TXA2 and PGI2
It is known that an imbalance of these two hormones, especially a decrease in the production of PGI2, can lead to vascular disorders.
[Br. J. Pharmac.,76, 3 (1982)]. diabetic vasculopathy
In the onset and progression of inflammatory bowel disease, in addition to the increased production of platelet-derived TXA2, [Thr
mb. Res.,19, 211 (1980), J. Lab. Clin. Med.
,97, 87 (1981)], and the decrease in the production of PGI2 derived from blood vessels increases platelet aggregation.
It has been confirmed that diabetic patients and experimental diabetic animals have the potential to cause progression [Lanc
et,1, 325 (1979), Lancet,2, 1365 (1979), N.
Engl. J. Med.,300, 366 (1979), Life Sci.
,23, 351 (1978)]. PSF is also a factor present in the bloodstream that stimulates PGI2 production in the vascular wall.
Nature,271, 549 (1978)], and the factor
Bloody uremic syndrome [Lancet,2, 871 (1978)], thrombotic thrombocytopenia.
Oligopurpura [Lancet,2, 748 (1979)], sickle cell anemia [
Br. J. Haematol. ，48, 545 (1981)], acute myocardial infarction [
Coronary,2, 49 (1985)], diabetic vascular disease [Metabo
lism,38, 837 (1989), Haemostasis,16, 447
(1986), Diab. Res. Clin. Pract. ，3, 243 (19
87)], and it has been reported that blood levels are reduced in patients with arteriosclerotic diseases.
In other words, IGFBP-7 stimulates the production of PGI2 in vascular endothelial cells, increasing the amount of PGI in the blood.
Increasing the concentration of 2 has the effect of inhibiting platelet aggregation, relaxing smooth muscles, and suppressing gastric acid secretion
As mentioned above, factors belonging to the IGFBP superfamily act as inhibitors of IGF and insulin.
Regulation of thrombin function, pregnancy, compensatory role in wasting diseases, bone metabolism, skeletal muscle cells
differentiation, promotion of PGI2 production in vascular endothelial cells, platelet aggregation via PGI2
It is involved in various physiological phenomena such as suppression of blood flow, relaxation of vascular smooth muscle, relaxation of bronchial smooth muscle, and inhibition of gastric acid secretion.
It has also been shown to be involved in dwarfism, acromegaly, and chronic renal failure in children.
, osteoporosis, breast cancer, prostate cancer, acute leukemia, colon cancer, uterine leiomyoma, liver cancer, I
Type diabetes, type II diabetes, thrombosis, arteriosclerosis, hemolytic uremic syndrome, thrombotic blood
Thrombocytopenic purpura, sickle cell anemia, acute myocardial infarction, diabetic vascular disease, etc.
It has also been shown to be involved in diseases. Therefore, its activity as an IGFBP belonging to the IGFBP superfamily
A protein having the formula (I), a gene encoding the protein, antisense DNA, and the protein
The antibodies that recognize the disease are those associated with abnormal cell proliferation, vascular disorders, and abnormalities in bone metabolism.
Diseases associated with abnormalities, diseases associated with impaired IGF and growth hormone action, abnormal smooth muscle cells
diseases involving the differentiation and proliferation of abnormal skeletal muscle cells; diseases involving the differentiation and proliferation of abnormal gastric acid secretion
Identification, treatment or management of diseases associated with abnormal lymphocyte infiltration or inflammatory diseases
It is thought that it may be a preventive medicine, and it is a member of the IGFBP superfamily.
Factors belonging to the IGFBP superfamily are attracting much attention as useful targets for new drug development. There is also the possibility that new factors belonging to the IGFBP superfamily may exist.
If a novel IGFBP gene can be obtained, the amino acid sequence of the IGFBP can be determined.
and the amino acid sequence of a known IGFBP, or
By examining the expression distribution of these IGFBPs, we can predict their functions and develop drugs.
Furthermore, if a novel IGFBP gene can be obtained, the relevant information can be obtained.
It is now possible to screen for substances that suppress the expression or function of IGFBPs
The compounds obtained by this screening are expected to be useful pharmaceuticals.
.Disclosure of the InventionThe present invention relates to a novel insulin-like growth factor binding protein, a DNA encoding the protein, and
A. Antibodies that recognize the protein, methods for determining diseases associated with the protein, diagnostic agents, and prognostic agents.
To solve the above problems, the present inventors conducted extensive research and discovered that IGFBP-related drugs
Novel insulin-like growth factor binding protein belonging to the genus taurin and a gene encoding said protein
The present inventors have succeeded in obtaining DNA that binds to insulin-like growth factor receptor 1 (IGGF-1), which has been shown to be a novel nucleotide sequence encoding an insulin-like growth factor-binding protein (IGGF-1), comprising the amino acid sequence represented by SEQ ID NO: 1.
Protein. (2) An insulin-like growth factor binding protein consisting of the amino acid sequence represented by SEQ ID NO: 2
Protein. (3) A protein described in (A) or (B) below. (A) One or more amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 1.
, a substituted or added amino acid sequence, and is a protein according to (1).
(B) A protein having the same qualitative activity as the amino acid sequence represented by SEQ ID NO: 2, but lacking one or more amino acids.
, a substituted or added amino acid sequence, and is used in combination with the protein according to (2).
A protein having the qualitatively identical activity (4) A protein described in (A) or (B) below. (A) An amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 1.
A protein having substantially the same activity as the protein described in (1).
Protein (B) An amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2
A protein having substantially the same activity as the protein described in (2).
Protein (5) A protein described in (A) or (B) below: (A) a protein having amino acid numbers 75 to 82 in the amino acid sequence represented by SEQ ID NO: 1.
and (1) a protein comprising an amino acid sequence containing the amino acid sequence of (1)
(B) A protein having the same activity as amino acid numbers 75 to 82 in the amino acid sequence represented by SEQ ID NO: 2
and (2) a protein comprising an amino acid sequence containing the amino acid sequence of (1) or (2)
A protein having the same qualitative activity. (6) A protein described in (A) or (B) below. (A) A protein in which one or more amino acids are missing from the amino acid sequence described in (A) of (5).
The protein according to (1) comprises an amino acid sequence having a deletion, substitution or addition.
(B) A protein having substantially the same activity as (5) (B), in which one or more amino acids are missing from the amino acid sequence described in (B).
(2) A protein comprising an amino acid sequence having a deletion, substitution or addition.
Protein having substantially the same activity (7) A protein described in (A) or (B) below. (A) An amino acid sequence having 60% or more homology with the amino acid sequence described in (A) of (5).
and (1) has substantially the same activity as the protein described in (1).
Protein (B) An amino acid sequence having 60% or more homology with the amino acid sequence described in (B) of (5)
and (2) having substantially the same activity as the protein of (1).
Protein (8) A protein described in (A) or (B) below. (A) In the amino acid sequence represented by SEQ ID NO: 1, amino acids 171 to 173 are included.
A protein comprising a partial amino acid sequence containing the amino acid sequence of (1)
(B) a protein according to (A) of claim 5, which has substantially the same activity as a protein comprising amino acid numbers 171 to 173 in the amino acid sequence represented by SEQ ID NO: 2;
The protein according to (2), which comprises a partial amino acid sequence containing the amino acid sequence of position 97.
The protein according to claim 5 (B), which has substantially the same activity as the protein. (9) The protein according to the following (A) or (B): (A) The protein according to (8) (A), wherein one or more amino acids are missing from the amino acid sequence described in (A).
The protein according to (1) comprises an amino acid sequence having a deletion, substitution or addition.
(B) A protein having substantially the same activity as (8) (B), in which one or more amino acids are missing from the amino acid sequence described in (B).
(2) A protein comprising an amino acid sequence having a deletion, substitution or addition.
Protein having substantially the same activity (10) A protein described in (A) or (B) below. (A) An amino acid sequence having 60% or more homology with the amino acid sequence described in (A) of (8).
and (1) has substantially the same activity as the protein described in (1).
Protein (B) An amino acid sequence having 60% or more homology with the amino acid sequence described in (B) of (8).
and (2) having substantially the same activity as the protein of (1).
Protein (11) A polypeptide described in (A) or (B) below: (A) A polypeptide having at least 5 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 1.
(B) A polypeptide consisting of an amino acid sequence of at least 5 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2.
(12) A polypeptide comprising an amino acid sequence of at least 1 amino acid. (A) A polypeptide comprising amino acids 171 to 304 in the amino acid sequence represented by SEQ ID NO: 1.
A polypeptide consisting of an amino acid sequence of at least 5 consecutive amino acids.
(B) Amino acids 171 to 197 in the amino acid sequence represented by SEQ ID NO: 2
A polypeptide consisting of an amino acid sequence of at least 5 consecutive amino acids.
(13) The protein or polypeptide according to any one of (1) to (12).
(14) DNA comprising a coding region of the nucleotide sequence represented by SEQ ID NO: 3. (15) DNA comprising a coding region of the nucleotide sequence represented by SEQ ID NO: 4. (16) DNA described in (A) or (B) below. (A) DNA encoding the protein described in (1), or any of (3) to (12)
A DNA encoding the protein or polypeptide according to (A), or a sequence thereof
DNA containing the coding region of the base sequence represented by number 3 and stringent conditions
and (1) has substantially the same activity as the protein described in (1).
(B) DNA encoding the protein described in (2), or any of (3) to (12).
DNA encoding the protein or polypeptide according to (B) or a sequence thereof
DNA containing the coding region of the base sequence represented by number 4 and stringent conditions
and (2) has substantially the same activity as the protein described in (2).
(17) A DNA encoding a protein comprising the DNA according to any one of (13) to (16) incorporated into a vector.
(18) A trait obtained by introducing the recombinant DNA according to (13) into a host cell.
Transformant. (19) The host cell is a bacterium, yeast, insect cell, plant cell, or animal cell.
(18) The transformant according to (18), which is a cell selected from the group consisting of: (20) The transformant FERM BP-71 containing the DNA according to (14).
81. (21) FERM BP-71, a transformant containing the DNA described in (15).
80. (22) A transformant selected from the transformants described in any one of (18) to (21).
The transformant is cultured in a medium, and the culture is added with the compound according to any one of (1) to (12).
and extracting the protein or polypeptide from the culture.
The method according to any one of (1) to (12), characterized in that a peptide is collected.
(23) An antibody that recognizes the protein described in any one of (1) to (10). (24) A method for producing a protein or polypeptide described in (A) of any one of (1) or (3) to (10).
and (B) of any one of (2) or (3) to (10).
(25) An antibody that does not recognize the protein described in (B) of any one of (2) or (3) to (10).
and (A) of any one of (1) or (3) to (10).
(26) An antibody that does not recognize the protein described in (11)-(A) or (12)-(A).
A protein according to any one of (1) or (3) to (10) (A) used as an antigen.
(27) A method for producing an antibody that recognizes a protein. (27) A method for producing an antibody that recognizes a protein, the method comprising: immunizing the polypeptide described in (11)(B) or (12)(B).
A protein according to (B) of any one of (2) or (3) to (10) used as an antigen.
A method for producing an antibody that recognizes white matter. (28) (1) or (3) to (10) obtained by the method described in (26).
(29) An antibody that specifically recognizes the protein according to any one of (A) above. (2) or (3) to (10) obtained by the method according to (27).
(30) An antibody that specifically recognizes the protein described in any one of (B) above. (31) An antibody produced by a hybridoma having deposit number FERM BP-7603.
(31) A monoclonal antibody produced by a hybridoma having the deposit number FERM BP-7604.
(32) A monoclonal antibody according to any one of (23) to (25) and (28) to (31).
Immunologically detecting the protein according to any one of (1) to (10) using an antibody of
(33) A method for detecting or quantifying a linkage in the base sequence of the DNA according to any one of (13) to (16).
an oligonucleotide having a sequence consisting of 5 to 60 consecutive bases,
oligonucleotides having a sequence complementary to the oligonucleotides, or
A derivative of otide. (34) The DNA according to any one of (13) to (16), or (33).
as a probe.
Any of (1) to (10) above, comprising carrying out hybridization using
A method for detecting or quantifying the expression of a gene encoding the protein described in item 1. (35) The oligonucleotide or oligonucleotide derivative described in item (33).
Polymerase chain reaction using the antibody as a primer
A gene encoding the protein according to any one of (1) to (10),
A method for detecting or quantifying expression. (36) The DNA according to any one of (13) to (16), or (33).
The oligonucleotide or oligonucleotide derivative according to the present invention is used for hybridization.
(11) A method for producing a protein according to any one of (1) to (10), comprising the step of:
(37) A method for detecting a mutation in a gene encoding white matter. (37) The oligonucleotide or oligonucleotide derivative according to (33).
(1) to (3), which include carrying out a polymerase chain reaction using a nucleic acid sequence.
(10) A method for detecting a mutation in a gene encoding a protein according to any one of (10) above.
(38) A method for determining a disease described in (A) or (B) below. (A) A method for determining a disease described in any one of (1) to (10).
A method for diagnosing a disease characterized by detecting mutations or measuring expression levels and comparing them with those of healthy individuals.
(B) A method for detecting or detecting a mutation in a protein according to any one of (1) to (10).
A method for diagnosing a disease, comprising measuring the amount of a protein in a patient with a disease and comparing it with that of a healthy individual. (39) A method for diagnosing a disease described in (A) or (B) below. (A) A method for diagnosing a disease, comprising: (a) measuring the amount of a protein in a patient with a disease described in (A) of any one of (1) or (3) to (10);
The mutation or expression level of the DNA to be read and any of (2) or (3) to (12)
and comparing the mutation or expression level of the DNA encoding the protein described in (B).
(B) A method for determining a disease, comprising: (1) or (3) to (10) comprising:
and (B) of any one of (2) or (3) to (10).
A method for determining a disease, characterized by comparing protein mutations or expression levels (40) The disease is a disease accompanied by abnormal cell proliferation, a disease accompanied by vascular disorders, a disease accompanied by bone metabolism disorders,
Diseases involving abnormalities, diseases involving disorders of insulin-like growth factor and growth hormone action,
Diseases associated with abnormal differentiation and proliferation of smooth muscle cells, diseases associated with abnormal differentiation and proliferation of skeletal muscle cells
Diseases associated with abnormal gastric acid secretion or inflammatory diseases associated with abnormal lymphocyte infiltration
(41) The method for determining whether or not a disease accompanied by abnormal cell proliferation is acute myeloid leukemia, breast cancer, prostate cancer,
Colon cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumors or solid tumors, vascular
Disability-causing diseases include myocardial infarction, cerebral infarction, peripheral vascular occlusion, angina pectoris, high blood pressure, and hyperlipidemia.
disease, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombosis, hemolytic uremic syndrome
group, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic encephalopathy, heart failure, congestion
Or choroidal circulatory disorder, and the disease accompanied by abnormal bone metabolism is osteoporosis, and
Diseases associated with impaired function of growth hormone and steroid hormones include dwarfism, acromegaly, and
or pediatric chronic renal failure, and atherosclerosis is a disease accompanied by abnormal differentiation and proliferation of smooth muscle cells.
bronchial disease or restenosis, and diseases accompanied by abnormal differentiation and proliferation of skeletal muscle cells are serious.
Myasthenia gravis is a condition that involves abnormal gastric acid secretion, and gastric ulcers are a disease that involves abnormal lymphocytes.
Inflammatory diseases involving infiltration include microbial infections, chronic hepatitis B, rheumatoid arthritis, and sepsis.
graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammation
Inflammatory bowel disease, allergies, atopy, asthma, hay fever, airway hypersensitivity or autoimmune disease
The method for determining whether the patient is a patient with a pulmonary edema or a pulmonary edema according to (40), wherein the patient is a patient with a pulmonary edema or a pulmonary edema. (42) The method for determining whether the patient is a patient with a pulmonary edema or a pulmonary edema according to the method described in (A) or (B) below:
The determination method according to any one of (38) to (41). (A) The detection method according to (36) or (37). (B) The detection or quantification method according to (32), (34), or (35). (43) The protein or polypeptide according to any one of (1) to (12).
(44) A pharmaceutical comprising the DNA according to any one of (13) to (16), or (33).
A pharmaceutical agent containing the oligonucleotide or oligonucleotide derivative according to
(45) The medicine is a gene prevention vector or a gene therapy vector.
(44) The pharmaceutical composition according to (46) any one of (23) to (25) and (28) to (31).
(47) A pharmaceutical containing an antibody of the present invention. (47) A pharmaceutical containing an antibody of the present invention is used to treat diseases accompanied by abnormal cell proliferation, diseases accompanied by vascular disorders, diseases associated with bone metabolism,
Diseases involving abnormalities, diseases involving disorders of insulin-like growth factor and growth hormone action,
Diseases associated with abnormal differentiation and proliferation of smooth muscle cells, diseases associated with abnormal differentiation and proliferation of skeletal muscle cells
Diseases associated with abnormal gastric acid secretion or inflammatory diseases associated with abnormal lymphocyte infiltration
The pharmaceutical agent according to any one of (43) to (46), which is a diagnostic agent, a preventive agent, or a therapeutic agent for
(48) Diseases accompanied by abnormal cell proliferation include acute myeloid leukemia, breast cancer, prostate cancer, and thyroid cancer.
Intestinal cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumors or solid tumors with vascular disorders
Harmful diseases include myocardial infarction, cerebral infarction, peripheral vascular occlusion, angina pectoris, hypertension, and hyperlipidemia.
, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombosis, hemolytic uremic syndrome
, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic encephalopathy, heart failure, congestion
or choroidal circulatory disorders, osteoporosis is a disease accompanied by abnormalities in bone metabolism, and insulin
Diseases associated with impaired action of phospho-like growth factors and growth hormones include dwarfism, acromegaly, and
is a childhood chronic renal failure, and is a disease accompanied by abnormal differentiation and proliferation of smooth muscle cells, leading to arteriosclerosis,
Bronchial disease or restenosis, with abnormal skeletal muscle cell differentiation and proliferation, is severe
Myasthenia gravis, a disease accompanied by abnormal gastric acid secretion is gastric ulcer, and abnormal lymphocyte infiltration
Inflammatory diseases accompanied by inflammatory bowel disease include microbial infections, chronic hepatitis B, rheumatoid arthritis, and sepsis.
, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammation
bowel disease, allergies, atopy, asthma, hay fever, airway hypersensitivity or autoimmune disease
(49) (i) A cell expressing the protein according to any one of (1) to (10).
(ii) contacting a test sample with cells expressing the protein;
The expression level of the protein when the test sample was subjected to the above-mentioned procedure was compared with that when the test sample was subjected to the above-mentioned procedure (1) to (10).
The method is characterized by selecting a compound that controls the expression level of the protein according to any one of the above.
a compound for controlling the expression level of the protein according to any one of (1) to (10).
(50) (i) A method for screening a strain of a plant that expresses a protein according to any one of (1) to (10).
(ii) the function of the cell when the test sample is contacted with the cell expressing the protein.
and comparing the function of the cell with that of the test sample, and
(1) to (10) are characterized by selecting a compound that controls the function of a protein.
(51) A method for screening for a compound that controls the function of the protein described in any one of (1) to (10). (52) A gene encoding the protein described in any one of (1) to (10).
A plasmid containing DNA with a reporter gene linked downstream of the region that controls transcription of the
The transformant transformed with smid was contacted with a test sample, and the test sample was
2. A compound that controls the expression of a gene encoding a protein according to any one of the above 1) to 10).
(10) a protein according to any one of (1) to (10),
(52) A method for screening for a compound that controls the expression of a gene encoding the protein of any one of (1) to (10),
When a test sample is contacted with cells expressing the protein, (i)
(i) or (3) to (10) (A) of the expression level of the protein according to (A),
i) the protein according to any one of (2) or (3) to (10) (B) in the cell;
and comparing the expression amount of the target gene with that of the target gene, and
Screening for compounds that regulate splicing of protein-coding genes
(53) A method for screening a subject by the screening method according to any one of (49) to (52).
(54) A compound or a pharmacologically acceptable salt thereof according to (53) or a pharmaceutical composition containing the compound or a pharmacologically acceptable salt thereof.
(55) A drug for treating diseases accompanied by abnormal cell proliferation, diseases accompanied by vascular disorders, diseases associated with bone metabolism,
Diseases involving abnormalities, diseases involving disorders of insulin-like growth factor and growth hormone action,
Diseases associated with abnormal differentiation and proliferation of smooth muscle cells, diseases associated with abnormal differentiation and proliferation of skeletal muscle cells
Diseases associated with abnormal gastric acid secretion or inflammatory diseases associated with abnormal lymphocyte infiltration
(56) The pharmaceutical according to (54), which is a prophylactic or therapeutic agent for diseases associated with abnormal cell proliferation, such as acute myeloid leukemia, breast cancer, prostate cancer, and gastrointestinal tract cancer.
Intestinal cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumors or solid tumors with vascular disorders
Harmful diseases include myocardial infarction, cerebral infarction, peripheral vascular occlusion, angina pectoris, hypertension, and hyperlipidemia.
, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombosis, hemolytic uremic syndrome
, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic encephalopathy, heart failure, congestion
or choroidal circulatory disorders, osteoporosis is a disease accompanied by abnormalities in bone metabolism, and insulin
Diseases associated with impaired action of phospho-like growth factors and growth hormones include dwarfism, acromegaly, and
is a childhood chronic renal failure, and is a disease accompanied by abnormal differentiation and proliferation of smooth muscle cells, leading to arteriosclerosis,
Bronchial disease or restenosis, with abnormal skeletal muscle cell differentiation and proliferation, is severe
Myasthenia gravis, a disease accompanied by abnormal gastric acid secretion is gastric ulcer, and abnormal lymphocyte infiltration
Inflammatory diseases accompanied by inflammatory bowel disease include microbial infections, chronic hepatitis B, rheumatoid arthritis, and sepsis.
, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammation
bowel disease, allergies, atopy, asthma, hay fever, airway hypersensitivity or autoimmune disease
(57) The protein or polypeptide according to any one of (1) to (12).
and a test sample is contacted with the protein or polypeptide from the test sample.
13. The method according to claim 12, wherein a material is selected that is compatible with the method.
A method for screening for a substance that specifically binds to the protein or polypeptide described above. (58) (1) to (12) obtained by the screening method described in (57).
(59) A gene encoding the protein according to any one of (1) to (10).
(60) A knockout non-human animal in which the expression of the protein according to any one of (1) to (10) is reduced or completely suppressed.
or a knockout non-human animal in which the protein or polypeptide is completely suppressed. The proteins and polypeptides of the present invention include: (a) an insulin-like growth factor-binding protein having the amino acid sequence represented by SEQ ID NO: 1;
(b) an insulin-like growth factor-binding protein having the amino acid sequence represented by SEQ ID NO: 2
Protein (c) One or more amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 1
, a substituted or added amino acid sequence, and is a protein according to (a).
(d) A protein having the same qualitative activity as the amino acid sequence represented by SEQ ID NO: 2, in which one or more amino acids are deleted.
(b) a protein comprising a substituted or added amino acid sequence;
(e) A protein having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 1.
A protein having substantially the same activity as the protein described in (a).
(f) an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2
(b) a protein having substantially the same activity as the protein of (b)
(g) Amino acid numbers 75 to 82 in the amino acid sequence represented by SEQ ID NO: 1
The protein (a) comprises a partial amino acid sequence containing the amino acid sequence of
(h) a protein having substantially the same activity as that of the amino acid sequence represented by SEQ ID NO: 2, comprising amino acid numbers 75 to 82
The protein (b) comprises a partial amino acid sequence containing the amino acid sequence of
(i) a protein having substantially the same activity as the protein described in (g) above, wherein the amino acid sequence is one or more amino acids
The amino acid sequence of the present invention is an amino acid sequence in which one or more amino acids are deleted, substituted or added, and is the amino acid sequence described in (a) above.
(j) A protein having substantially the same activity as the protein described above. (h) A protein having one or more amino acids in the amino acid sequence representing the protein described above.
The amino acid sequence of the present invention is an amino acid sequence in which one or more amino acids are deleted, substituted or added, and is the amino acid sequence described in (b) above.
(k) A protein having substantially the same activity as the protein described above. (k) A protein having 60% or more homology with the amino acid sequence representing the protein described above in (g).
and the amino acid sequence is substantially the same as the protein described in (a) above.
(l) A protein having 60% or more homology with the amino acid sequence representing the protein described in (h) above.
and the amino acid sequence is substantially the same as that of the protein described in (b) above.
(m) a protein having the activity of amino acid numbers 171 to 30 in the amino acid sequence represented by SEQ ID NO: 1
A partial amino acid sequence containing the amino acid sequence of No. 4, and
(n) a protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 2, comprising amino acid numbers 171 to 19
7, and the partial amino acid sequence (b)
(o) a protein having substantially the same activity as the protein described in (m) above, in which one or more amino acids are deleted or substituted;
The protein (a) comprises an amino acid sequence substituted or added thereto, and is synthesized in the same manner as the protein (a).
(p) A protein having the same qualitative activity as the amino acid sequence described in (n) above, in which one or more amino acids are deleted or substituted.
The protein (b) is a protein having a substituted or added amino acid sequence.
A protein having the same qualitative activity as (q) an amino acid sequence having 60% or more homology with the amino acid sequence described in (m) above.
A protein having substantially the same activity as the protein described in (a) above.
(r) an amino acid sequence having 60% or more homology with the amino acid sequence described in (n) above
A protein having substantially the same activity as the protein described in (b) above.
Protein (s) At least 5 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 1
(t) a polypeptide consisting of an amino acid sequence of at least 5 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2
(u) a polypeptide consisting of an amino acid sequence of at least 171 amino acids; (u) a polypeptide consisting of amino acid numbers 171 to 304 in the amino acid sequence represented by SEQ ID NO: 1;
In the present invention, a polypeptide consisting of an amino acid sequence of at least 5 consecutive amino acids is
(v) amino acid numbers 171 to 197 in the amino acid sequence represented by SEQ ID NO: 2
In the present invention, a polypeptide consisting of an amino acid sequence of at least 5 consecutive amino acids is
Factors belonging to the insulin-like growth factor binding protein superfamily include IGF
Alternatively, it is a protein factor with an affinity for insulin, and is an insulin-like growth factor.
It has an insulin-binding motif (IGFBP motif).
Conserved N-terminal regions of proteins belonging to the growth factor-like binding protein superfamily
and is represented by Gly-Cys-Gly-Cys-Cys-XX-Cys.
It refers to a portion of a protein consisting of an amino acid sequence that is expressed by a specific amino acid (X represents any amino acid). In proteins belonging to the insulin-like growth factor binding protein superfamily, I
At least 10 cysteine residues are conserved around the GFBP motif.
, and exhibits affinity for IGF or insulin via the IGFBP motif portion.
In the amino acid sequence represented by SEQ ID NO: 1, amino acid numbers 75 to 82 are
In the amino acid sequence represented by SEQ ID NO: 2, the amino acid
The amino acid sequence represented by numbers 75 to 82 is the IGFBP motif. Proteins having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2
Examples of proteins having substantially the same activity as the protein include those represented by SEQ ID NO: 1 or SEQ ID NO:
A protein having an amino acid sequence represented by 2, and an integrin, an IGF,
Examples include proteins that are substantially identical to insulin or its receptor.
The term "same" means that the activity is essentially the same.
Quantitative factors such as the molecular weight of the protein may differ. In the amino acid sequence represented by SEQ ID NO: 1, which is the protein of the present invention, one or more amino acids may be present.
The amino acid sequence of SEQ ID NO: 1 is a sequence in which amino acids are deleted, substituted or added.
a protein having substantially the same activity as a protein having an amino acid sequence as shown in
or one or more amino acids are deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2
or an amino acid sequence added thereto, and the amino acid sequence is represented by SEQ ID NO: 2.
A protein having substantially the same activity as a protein having the sequence is
Cloning, A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory
Press (1989) (hereinafter referred to as "Molecular Cloning, 2nd Edition"
), Current Protocols in Molecular Bio
Logy, John Wiley & Sons (1987-1997) (hereinafter
Current Protocols in Molecular Biology (abbreviated as "Current Protocols in Molecular Biology"), N
ucleic acids research,10, 6487 (1982),
Proc. Natl. Acad. Sci. ,USA,79, 6409 (1982
), Gene,34, 315 (1985), Nucleic Acids Re.
search,13, 4431 (1985), Proc. Natl. Acad.
Sci USA,82, 488 (1985) and other methods.
A protein having the amino acid sequence shown in SEQ ID NO: 1 or 2 is isolated using the
It can be obtained by introducing site-specific mutations into the DNA to be loaded.
The number of amino acids to be deleted, substituted or added is one or more, and the number is not particularly limited.
However, deletions and substitutions can be made by well-known techniques such as the above-mentioned site-directed mutagenesis.
For example, 1 to several tens, preferably 1 to 20
, more preferably 1 to 10, and even more preferably 1 to 5.
In addition to such artificially created mutants, mutations occurring in nature are also included in the
Variants are also included. A protein having substantially the same activity as a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2.
To have this property, the amino acid sequence of SEQ ID NO: 1 or 2 must be at least 6
0% or more, preferably 70% or more, more preferably 80% or more, and even more preferably
is 90% or more, particularly preferably 95% or more, and most preferably 97% or more
It is preferable that the protein has an amino acid sequence having the following structure: The identity of the amino acid sequence or the nucleotide sequence can be determined by the method of Karlin and Altschu
The algorithm BLAST [Proc. Natl. Acad. Sci. US
A.90, 5873 (1993)] and FASTA [Methods Enzyme
ol.,183, 63 (1990)].
Based on the algorithm BLAST, programs called BLASTN and BLASTX are
A new method for the treatment of rhesus macular degeneration has been developed [J. Mol. Biol.,215, 403 (1990)
When analyzing a base sequence using BLASTN based on BLAST,
For example, the parameters are Score=100 and wordlength=12.
In addition, when analyzing amino acid sequences using BLASTX based on BLAST,
In this case, the parameters are, for example, score=50, wordlength=3, and
When using BLAST and Gapped BLAST programs,
The default parameters of the program are used.
The method is publicly known (http://www.ncbi.nlm.nih.gov.
). The protein of the present invention may have a partial amino acid sequence within the amino acid sequence of the protein of the present invention.
and having the amino acid sequence set forth in SEQ ID NO: 1 or 2.
This also includes proteins having substantially the same activity as the protein having the amino acid sequence set forth in SEQ ID NO: 1 or 2.
In order to have this property, the amino acid sequence represented by SEQ ID NO: 1 must be the amino acid sequence of amino acid number 7.
In the amino acid sequence of amino acids 5 to 82 or the amino acid sequence represented by SEQ ID NO: 2
a protein consisting of a partial amino acid sequence containing the amino acid sequence of amino acid numbers 75 to 82;
It is preferable that the protein is a protein. Also, the protein may have an amino acid sequence in which one or more amino acids are deleted, substituted, or added.
The protein of the present invention also includes the well-known proteins described above.
The number of amino acids that can be introduced by site-directed mutagenesis is deleted, substituted, or added.
and a protein having the amino acid sequence set forth in SEQ ID NO: 1 or 2.
Examples of proteins that have substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 or 2 of the protein of the present invention include proteins having the same activity as the amino acid sequence represented by SEQ ID NO: 1 or 2 of the present invention.
The above amino acids comprise the deleted amino acid sequence and are SEQ ID NO: 1 or 2.
As a protein having substantially the same activity as the protein consisting of the amino acid sequence shown in
Examples of such proteins include proteins from which the signal peptide has been removed. Furthermore, proteins having a similar structure to the proteins described in (g), (h), (m), or (n) above at 60% or more
and having the amino acid sequence set forth in SEQ ID NO: 1 or 2.
A protein having substantially the same activity as that of SEQ ID NO: 1 is also included in the present invention.
or 2, which has substantially the same activity as the protein having the amino acid sequence of
has a correlation with the amino acid sequence of the protein described in (g), (h), (m) or (n).
When the same sex is calculated using the above analysis software such as BLAST, it is found that at least 6
0% or more, preferably 70% or more, more preferably 80% or more, and even more preferably
The percentage of the polypeptide of the present invention is 90% or more, particularly preferably 95% or more, and most preferably 97% or more. The polypeptide of the present invention is a polypeptide that can be used to produce antibodies that recognize the above-mentioned protein of the present invention.
There are no particular limitations on the polypeptide as long as it can be used as an antigen polypeptide when
Specifically, the amino acid sequence of SEQ ID NO: 1 or 2 is a sequence of 5 consecutive amino acids.
10 or more amino acids, preferably 10 or more amino acids, more preferably 15 or more amino acids
In particular, the polypeptide represented by SEQ ID NO: 1 can be used.
and amino acid numbers 171 to 304 in the amino acid sequence shown in SEQ ID NO: 2.
The amino acid sequence of the present invention is
10 or more amino acids, preferably 10 or more amino acids, more preferably 15 or more amino acids
The polypeptide consisting of the amino acid is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1.
and the protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
The DNA of the present invention is useful for the production of: (1) DNA having the nucleotide sequence of SEQ ID NO: 3 (2) DNA having the nucleotide sequence of SEQ ID NO: 4 (3) DNA having the nucleotide sequences (a), (c), (e), (g), (i), (k), and (m) defined above
(4) DNA encoding the protein of the present invention described in (b), (d), (f), (h), (j), (l), or (n) as defined above
(5) A DNA encoding the protein of the present invention described in (3), (p), or (r). (6) A DNA encoding the protein of the present invention described in (3), or the base number of the base sequence represented by SEQ ID NO: 3.
DNA having a base sequence of 177 to 1088 is hybridized under stringent conditions.
and substantially identical to a protein having the amino acid sequence represented by SEQ ID NO: 1.
(6) DNA encoding a protein having the same activity as the DNA described in (4) or the base number of the base sequence represented by SEQ ID NO: 4
DNA having a base sequence of 926 to 1516 is hybridized under stringent conditions.
and substantially identical to a protein having the amino acid sequence represented by SEQ ID NO:2.
(7) DNA encoding a polypeptide described in (s) or (u) above.
A (8) A DNA encoding a polypeptide according to (t) or (v) defined above.
A Examples of DNA that hybridizes under stringent conditions include, for example, SEQ ID NO:
The DNA of the present invention, such as the DNA having the base sequence represented by 3 or 4, or
Using some DNA fragments as probes, colony hybridization and
Lark hybridization or Southern blot hybridization
Specifically, it refers to DNA obtained by colony or
Alternatively, a filter on which plaque-derived DNA was immobilized was used, and the resulting solution was diluted to 0.7 to 1.0%.
Hybridization was carried out at 65°C in the presence of 100 mol/L sodium chloride.
Then, a 0.1 to 2 times concentrated SSC solution (the composition of a 1 times concentrated SSC solution is 150 ml
mol/L sodium chloride, 15 mmol/L sodium citrate)
The DNA that can be identified by washing the filter at 65°C is listed below.
Hybridization can be performed using the Molecular Cloning Method II.
Current Protocols in Molecular Biology, DNA
Cloning 1: Core Techniques, A Practica
l Approach, Second Edition, Oxford Uni
This can be carried out in accordance with the method described in Versity (1995) etc.
Specific examples of hybridizable DNA include DNA fragments obtained by the above-mentioned BLAST analysis.
When calculated using analysis software, the base sequence represented by SEQ ID NO: 3 or 4 is
DNA having at least 60% or more homology, preferably 70% or more, more preferably
Preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more
, and most preferably DNA having a homology of 98% or more. The DNAs described in (7) and (8) above encode the polypeptides of the present invention.
The DNA is a DNA sequence corresponding to the polypeptide of the present invention, and is a sequence corresponding to the polypeptide of the present invention.
or 4. Detection of expression or expression of DNA containing the coding region of the base sequence
In particular, the above polypeptides (u) and (v) can be used to measure the amount of the
The DNA is a DNA containing the coding region of the base sequence shown in SEQ ID NO: 3 and a DNA containing the coding region of the base sequence shown in SEQ ID NO:
By comparing the expression levels of DNA containing the coding region of the base sequence shown in No. 4,
The expression ratio of the protein described in SEQ ID NO: 1 to the protein described in SEQ ID NO: 2 is normal.
These DNAs are useful for diagnosing diseases characterized by differences in the amount of nucleotides present in the mRNA. The present invention is described in detail below. 1. Preparation of the DNA of the present invention The DNA of the present invention is prepared by synthesizing mRNA derived from human tissue, for example, mRNA derived from the human small intestine.
is isolated, a cDNA library is prepared, and the cDNA library is then
Human small intestine mRNA can be prepared by screening the target clone. Human small intestine mRNA can be prepared by using commercially available products (e.g., Clontech).
Alternatively, it may be prepared from human small intestinal tissue as described below.
First, total RNA is prepared from small intestinal tissue, and mRNA is isolated from the total RNA.
One method for preparing total RNA from small intestinal tissue is guanidinium thiocyanate-thiol.
Cesium trifluoroacetate method [Methods in Enzymology,1
54, 3 (1987)], acidic guanidine thiocyanate, phenol, chloroform
The ALCOH (AGPC) method [Analytical Biochemistry,16
2, 156 (1987), Experimental Medicine,9, 1937 (1991)].
Poly(A) is synthesized from total RNA.⁺As a method for preparing mRNA as RNA,
For this purpose, oligo(dT)-immobilized cellulose column method (Molecular Cloning
2nd edition) or Fast Track mRNA Iso
lation Kit (Invitrogen), Quick Prep
mRNA Purification Kit (Pharmacia), etc.
mRNA can be prepared using this kit. A cDNA library is created from the prepared human small intestine tissue mRNA.
The A library preparation method is based on Molecular Cloning, 2nd Edition, Current
- Methods described in Protocols in Molecular Biology, etc.
Alternatively, commercially available kits, such as SuperScript Plasmid Sys
tem for cDNA Synthesis and Plasmid C
loning (Life Technologies), ZAP-cDNA
Methods using Synthesis Kit (Stratagene) and the like
Examples of cloning vectors for preparing cDNA libraries include:
Any vector that can replicate autonomously in the K12 strain of bacteria can be used as a phage vector or a plasmid vector.
Specifically, ZAP Express [STR
ATAGENE, Strategies,5, 58 (1992)], pBlu
escript II SK(+) [Nucleic Acids Resea
rch,17, 9494 (1989)], Lambda ZAP II (STR
ATAGENE), λgt10, λgt11 [DNA cloning, A
Practical Approach,1, 49 (1985)], λTri
plEx (Clontech), λExCell (Pharmacia)
), pT7T318U (Pharmacia), pcD2 [Mol. Cel
l. Biol.,3, 280 (1983)] and pUC18 [Gene,33, 103 (1985)]. Host microorganisms include bacteria belonging to the genus Escherichia.
Organisms, especially Escherichia coli (Escherichia coli,below,"
Any microorganism belonging to the Escherichia coli family can be used.
Physically,Escherichia coli XL1-Blue MRF' [
STRATAGENE, Strategies,5, 81 (1992)],E
scherichia coli C600 [Genetics,39, 440
(1954)],Escherichia coli Y1088 [Scien
ce,222, 778 (1983)],Escherichia coliY
1090 [Science,222, 778 (1983)],Escheric
hia coli NM522 [J. Mol. Biol. ，166, 1 (198
3) ],Escherichia coli K802 [J. Mol. Biol
．16, 118 (1966)] andEscherichia coliJ
M105 [Gene,38, 275 (1985)] or the like is used. This cDNA library may be used as is for the following analysis, but
To reduce the proportion of long cDNA and obtain full-length cDNA as efficiently as possible,
The oligo-capping method developed by Kanno et al. [Gene,138, 171 (1994)
, Gene,200, 149 (1997), Protein Nucleic Acid Enzymes,41, 603 (1
996), Experimental Medicine,11, 2491 (1993), cDNA cloning, sheep
(1996), Method for Gene Library Construction, Yodosha (1994)]
The cDNA library prepared by this method may be used for the following analysis. Each clone is isolated from the prepared cDNA library, and each clone is analyzed.
The base sequence of the cDNA is determined from the end by a commonly used base sequence analysis method, e.g.
For example, the dideoxy method of Sanger et al. [Proc. Natl. Aea
d. Sci. USA,74, 5463 (1977)] or ABIPRISM
Base sequence of 377 DNA sequencer (PE Biosystems) etc.
The DNA base sequence is determined by analyzing it using an analytical device. Whether each cDNA base sequence contains a novel sequence is determined by BLA.
GenBank, EMBL, and DD were used to search for homology using a homology search program such as ST.
By searching the base sequence database such as BJ, existing
It shows clear homology with the base sequence of the gene, which is considered to be a perfect match.
This can be confirmed by the absence of a base sequence. The base sequence of a cDNA containing a novel sequence obtained by the above method is, for example, the sequence
Examples include the base sequences represented by sequence numbers 3 and 4. The protein (sequence) obtained by translating DNA consisting of the base sequence represented by sequence number 3
The protein consisting of the amino acid sequence represented by sequence number 1 was analyzed by BLAST2 [Nuc.
Acid. Res. ，25In a homology analysis using
38% of the protein is human IGFBP-7, which belongs to the IGFBP superfamily.
The protein obtained by translating the DNA consisting of the base sequence represented by SEQ ID NO: 4 (sequence
The protein consisting of the amino acid sequence represented by sequence number 2 was compared using BLAST2.
In a homozygous analysis, human IGFB, a protein belonging to the IGFBP superfamily,
It has 35% homology with P-7. In the IGFBP superfamily, it binds to IGF or insulin.
The insulin-like growth factor binding motif and IGFBP motif, which are important in this case, are
The positions of at least 10 central cysteine residues are known to be conserved.
In the protein consisting of the amino acid sequences shown in SEQ ID NOs: 1 and 2,
However, the region corresponding to the insulin-like growth factor binding motif contains a representative insulin-like growth factor binding motif.
Gly-Cys-Gly-Cys-Cys-X, a cysteine-like growth factor binding motif
-X-Cys (where X represents any amino acid)
Ala-Gly-Gly-Cys-Cys-Trp-Glu-Cys
There is an amino acid sequence with 10 cysteine residues at the center of this motif.
Located at a position conserved among factors belonging to the IGFBP superfamily
Therefore, proteins consisting of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2
It has been found that the protein has activity as a protein belonging to the IGFBP superfamily.
Regarding the relationship between proteins belonging to the IGFBP superfamily and diseases,
Myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine leiomyoma, malignant
Diseases involving abnormal cell proliferation such as tumors and solid tumors, myocardial infarction, cerebral infarction, peripheral vascular occlusion
Cholecystitis, angina pectoris, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arterial
Sclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart disease,
Diseases accompanied by vascular disorders such as ischemic cerebral disease, heart failure, congestion, choroidal circulatory disorders, osteoporosis
Diseases associated with abnormalities in bone metabolism such as porositis, dwarfism, acromegaly, and childhood chronic renal failure
Diseases associated with impaired insulin-like growth factor and growth hormone action, arteriosclerosis, bronchial disease
Diseases involving abnormal differentiation and proliferation of smooth muscle cells, such as restenosis, and disorders such as myasthenia gravis
diseases accompanied by the differentiation and proliferation of skeletal muscle cells, diseases accompanied by abnormalities in gastric acid secretion such as gastric ulcers,
Bioinfection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease,
Insulin-dependent diabetes, nephritis, traumatic injury, inflammatory bowel disease, allergies,
Accompanied by abnormal lymphocyte infiltration in conditions such as eczema, asthma, hay fever, airway hypersensitivity, and autoimmune diseases
Since there have been reports of the protein of the present invention being effective against inflammatory diseases, etc.,
It is predicted that the protein is related to the above. From a comparison of the base sequence represented by SEQ ID NO: 3 and the base sequence represented by SEQ ID NO: 4,
It can be seen that the base sequences represented by SEQ ID NOs: 3 and 4 share the same sequence.
In such cases, these molecules may have arisen artificially during the construction of the cDNA library.
To confirm that this is not the case, a human genomic library was cloned using the sequence shown in SEQ ID NO: 3 or
or 4, and the obtained
This can be determined by determining the base sequence of the genomic clone. Human genomic libraries are commercially available (e.g., Research Gene
Alternatively, a BAC library prepared by tics may be used, or a library prepared by human cells or tissues may be used.
The method is well known in the art [Genomics,29, 413 (1995), Genomic
s,24A human genomic library may be prepared by the method described in, for example, [J., 527 (1994)].
As a method for screening using a suitable sequence, the method for screening using a suitable sequence is
PCR method using primers specific to the base sequence [PCR Protocol
s, Academic Press (1990)] and the sequences shown in SEQ ID NO: 3 or 4.
Colony hybridization using oligonucleotides specific to the target base sequence
ion and plaque hybridization (Molecular Cloning 1999).
2nd edition) etc. Genomes containing both the base sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 using the above method.
A DNA clone (e.g., a human genomic BAC clone) is obtained.
The DNA base sequence was determined and compared with the base sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4.
In comparison, the base sequence represented by SEQ ID NO: 3 has five parts, each of which has the same structure as SEQ ID NO: 4.
The base sequence is divided into seven parts and exists over approximately 7 kb on the same human genome.
That is, it can be seen that the protein having the amino acid sequence shown in SEQ ID NO: 1
The protein is composed of five exons and has the amino acid sequence shown in SEQ ID NO: 2.
consists of seven exons, the second and fourth exons of which are
However, the third exon of each gene is
Because the beginning of the 5th exon and the end of the 5th exon are different from each other, the sequence
Comparison of numbers 1 and 2 reveals the differences in the amino acid sequences observed.
Therefore, a protein having the amino acid sequence represented by SEQ ID NO: 1 and a protein having the amino acid sequence represented by SEQ ID NO: 2
Proteins with the amino acid sequence shown are proteins with amino acid sequences that differ from each other.
These are alternative splice forms of the same gene, each with the unique feature of
It can be seen that the product is a product of the following. DNA consisting of the base sequences shown in SEQ ID NOs: 3 and 4 is once obtained,
After the base sequence is determined, the base sequence is analyzed based on the 5' and 3' ends of the base sequence.
The primers were prepared and analyzed using the DNA fragments contained in tissues or cells such as the small intestine of a human or non-human animal.
cDNA or a cDNA library synthesized from mRNA contained in the
The PCR method [PCR Protocols, Academic Press (
The DNA of the present invention can be obtained by amplifying the DNA using the method described in the above.
In addition, the entire length or a part of the DNA represented by SEQ ID NO: 3 and 4 can be used as a probe.
and from mRNA contained in tissues or cells such as the small intestine of a human or non-human animal.
Colony hybridization to synthesized cDNA or cDNA library
fusion and plaque hybridization (Molecular Cloning No.
The DNA of the present invention can be obtained by carrying out the method (2nd edition). Based on the determined DNA base sequence, the DNA can be purified using the phosphoramidite method.
DNA synthesis was performed using a DNA synthesizer such as the Perkin-Elmer DNA synthesizer model 392.
The DNA of the present invention can also be obtained by chemical synthesis. The obtained DNA is then transfected into a host cell using a recombinant vector containing the DNA.
The protein is expressed using the transformant obtained by the above procedure, or the DNA
The amino acid sequence encoded by human IGFBP-1, human IGFBP-2, and human IGFBP-1
GFBP-3, human IGFBP-4, human IGFBP-5, human IGFBP-6
, human IGFBP-7, human IGFBP-8, human IGFBP-9 or human
Comparison of the amino acid sequence homology with IGFBP-10 and the position of cysteine residues
By doing so, the DNA has activity as an IGFBP superfamily member.
It can be confirmed that the DNA encodes a protein. Information regarding the base sequences of SEQ ID NOs: 3 and 4 or fragments thereof
Based on the information, the DNA salt of the present invention can be synthesized by conventional methods or using a DNA synthesizer.
A base sequence, for example, 5 to 6 consecutive base sequences of the base sequence represented by SEQ ID NO: 3 or 4
an oligonucleotide having a sequence corresponding to 0 bases, preferably 10 to 40 bases
or an oligonucleotide having a sequence complementary to said oligonucleotide (hereinafter
The oligonucleotides of the present invention can be prepared using oligonucleotides such as oligo-DNA and oligo-RNA.
oligonucleotides and derivatives of said oligonucleotides (hereinafter referred to as oligonucleotides)
Examples of such oligonucleotides or antisense oligonucleotides include:
For example, in the base sequence of a part of the mRNA to be detected,
a sense primer corresponding to the nucleotide sequence at the 3' end, and an antisense primer corresponding to the nucleotide sequence at the 3' end.
However, the salt corresponding to uracil in mRNA can be
The base is thymidine in oligonucleotide primers. For sense and antisense primers, the melting temperatures of both (
The oligonucleotides are those with a Tm and base number that do not change drastically, and are 5 to 60
The oligonucleotide derivatives include those having 10 to 50 bases, preferably 10 to 50 bases. The oligonucleotide derivatives include phosphodiesters in oligonucleotides.
an oligonucleotide derivative in which the diol bond is converted to a phosphorothioate bond;
The phosphodiester bond in a oligonucleotide is N3'-P5' phosphoramide
oligonucleotide derivatives converted to ribonucleotide bonds,
and oligonucleotides in which the phosphodiester bond was converted to a peptide nucleic acid bond.
Derivatives, uracil in oligonucleotides replaced with C-5 propynyluracil
The oligonucleotide derivatives, wherein uracil in the oligonucleotide is C-5 thiamin
azole-uracil substituted oligonucleotide derivatives, in oligonucleotides
An oligonucleotide derivative in which the cytosine is replaced with C-5 propynylcytosine
, the cytosine in the oligonucleotide is a phenoxazine-modified cytosine (phenoxazine-modified cytosine).
cytosine-modified cytosine-substituted oligonucleotides
ribose in oligonucleotide is 2'-O-propyl ribose
oligonucleotide derivatives substituted with hydroxyl groups or hydroxyl groups in oligonucleotides
Oligonucleotide derivatives in which the ribose is replaced with 2'-methoxyethoxyribose
Examples include cell engineering,16, 1463 (1997)]. 2. Production of the Protein or Polypeptide of the Present Invention The protein or polypeptide of the present invention can be produced by the method described in Molecular Cloning, 2nd Edition, or
As described in Current Protocols in Molecular Biology, etc.
The DNA of the present invention can be expressed in host cells using methods such as those described below.
Based on the full-length cDNA, it can be produced by adding a portion that encodes the protein, if necessary.
Prepare a DNA fragment of an appropriate length. Insert the DNA fragment or full-length cDNA under the promoter of an appropriate expression vector.
A recombinant vector is produced by inserting the vector into a host cell compatible with the expression vector.
Thus, a transformant that produces the protein of the present invention can be obtained. Host cells of interest include bacteria, yeast, animal cells, insect cells, and plant cells.
Any vector can be used as long as it can express the gene. Expression vectors include those that can replicate autonomously or are integrated into the chromosome in the host cells.
and inserting the promoter into a position where the DNA encoding the protein of the present invention can be transcribed.
When prokaryotes such as bacteria are used as host cells, a cell encoding the protein of the present invention is used.
The recombinant vector containing the DNA is capable of autonomous replication in prokaryotes.
Sometimes, a promoter, a ribosome binding sequence, a gene encoding the protein of the present invention
and a transcription termination sequence.
It may also contain a gene that controls the expression of the . Expression vectors include, for example, pBTrp2, pBTac1, and pBTac2.
(both commercially available from Boehringer Mannheim), pKK233-2 (Phar
macia), pSE280 (Invitrogen), pGEMEX
-1 (Promega), pQE-8 (QIAGEN), pKYP10
(JP 58-110600), pKYP200 [Agricultural
Biological Chemistry,48, 669 (1984)], p.
LSA1 [Agric. Biol. Chem. ，53, 277 (1989)],
pGEL1 [Proc. Natl. Acad. Sci. USA,82, 4306
(1985)], pBluescript II SK(-) (Stratag
ene), pTrs30[Escherichia coliJM109
/prepared from pTrS30 (FERM BP-5407)], pTrs32[Es
Cherichia coli JM109/pTrS32 (FERM BP-
5408)], pGHA2[Escherichia coliIG
HA2 (FERM B-400), JP-A-60-221091], pG
KA2 [Escherichia coli IGKA2 (FERM BP-6
798), JP-A-60-221091], pTerm2 (US 4,686
191, US4939094, US5160735), pSupex, pUB1
10, pTP5, pC194, pEG400 [J. Bacteriol. ，17
2, 2392 (1990)], pGEX (Pharmacia), pET
Examples of promoters include pSupex (Novagen), pSupex, etc. Any promoter can be used as long as it functions in the host cell.
Good. For example,trpPromoter (P_trp),lacPromoter, P_LP
Romotor, P_Rpromoter, T7 promoter, etc., for E. coli and phages
Examples of promoters include promoters derived from P_trpTwo series-connected
Motor (P_trp×2),tacpromoter, lacT7 promoter,
Artificially designed and modified promoters such as the let I promoter are also used.
It is possible to bind Shine-Dalgarno ribosomes.
o) A promoter with an appropriate distance (e.g., 6 to 18 bases) between the sequence and the start codon
It is preferable to use a plasmid. The base sequence of the portion encoding the protein of the present invention is determined by using codons optimal for expression in the host.
By substituting the bases so that the desired protein is produced, the production rate can be improved.
In the recombinant vector of the present invention, a transcription termination sequence is required for expression of the DNA of the present invention.
Although it is not necessary to place a transcription termination sequence immediately downstream of the structural gene,
Preferred host cells include those belonging to the genera Escherichia, Serratia, Bacillus, and Brevibacter.
genus Corynebacterium, genus Microbacterium, genus Pseudomonas, etc.
Microorganisms belonging to, e.g.Escherichia coliXL1-Blu
e.Escherichia coliXL2-Blue,Escheric
hia coliDH1,Escherichia coliMC1000
,Escherichia coliKY3276,Escherichia coliW1485,Escherichia coliJM109,E
scherichia coliHB101,Escherichia co
liNo. 49,Escherichia coliW3110,Esch
erichia coliNY49,Serratia ficaria,S
erratia Fonticola,Serratia liquidation
ens,Serratia marcescens,Bacillus sub
tilis,Bacillus amyloliquefacines,Bre
vibacterium immariophilumATCC14068,
Brevibacterium saccharolyticumATCC1
4066,Brevibacterium flavumATCCT4067
,Brevibacterium ammoniagenes,Brevib
Acterium lactofermentum ATCC13869,Co
rynebacterium glutamicum ATCC13032,C
orynebacterium acetoacidophilumATCC
13870,Microbacterium ammoniaphilumA
TCC15354,Pseudomonassp. D-0110 etc.
The recombinant vector can be introduced by introducing DNA into the host cell.
Any method using calcium ions can be used.
roc. Natl. Acad. Sci. USA,69, 2110 (1972)]
, protoplast method (Japanese Patent Application Laid-Open No. 63-248394), or Gene,17, 1
07 (1982) and Molecular & General Genetic
s,168, 111 (1979). When yeast is used as the host cell, the expression vector can be, for example, YEP
13 (ATCC37115), YEp24 (ATCC37051), YCp50
(ATCC37419), etc. Any promoter can be used as long as it functions in yeast strains.
For example, promoters of glycolytic genes such as hexose kinase,
PHO5 promoter, PGK promoter, GAP promoter, ADH promoter
Motor, gal 1 promoter, gal 10 promoter, heat shock
protein promoter, MFα1 promoter, CUP1 promoter, etc.
Host cells include Saccharomyces, Kluyveromyces, Trichosporus,
Microorganisms belonging to the genus Bacillus, Schwanniomyces, etc., for example,Saccharomyc
es cerevisiae,Schizosaccharomyces p.o.
mbe,Kluyveromyces lactis,Trichosporo
n pullulans,Schwanniomyces alluviusetc.
The recombinant vector can be introduced by any method that introduces DNA into yeast.
Any of these methods can be used, for example, electroporation [Methods
．． Enzymol. ，194, 182 (1990)], spheroplast method [P
roc. Natl. Acad. Sci. USA,84, 1929 (1978)]
, lithium acetate method [J. Bacteriology,153, 163 (1983
)], Proc. Natl. Acad. Sci. USA,75, 1929 (19
78) and the like. When animal cells are used as hosts, the expression vector can be, for example, pcD
NAI, pcDM8 (Funakoshi Co., Ltd.), pAGE107 [JP 3-22979
;Cytotechnology,3, 133, (1990)], pAS3-3
(Unexamined Japanese Patent Publication No. 2-227075), pCDM8 [Nature,329, 840, (
1987)], pcDNAI/Amp (Invitrogen), pREP
4 (Invitrogen), PAGE103 [J. Biochemist
ry,101, 1307 (1987)], pAGE210, etc.
Any promoter that functions in animal cells can be used.
For example, immediate encephalopathy (IE) of cytomegalovirus (CMV) can be
e early) gene promoter, SV40 early promoter, retro
Viral promoters, metallothionein promoters, heat shock promoters
Examples of the promoter include the I promoter of human CMV.
The enhancer of the E gene may be used together with the promoter. Host cells include human Namalwa cells, monkey cells, and the like.
COS cells, which are cells of the human body, CHO cells, which are cells of the Chinese hamster,
Examples of methods for introducing recombinant vectors include methods for introducing DNA into animal cells.
Any of these methods can be used, for example, electroporation [Cytot
technology,3, 133 (1990)], calcium phosphate method (Patent Publication No.
2-227075), lipofection method [Proc. Natl. Acad. S
ci. USA,84, 7413 (1987)]. When insect cells are used as hosts, for example, Current Protocols in
Molecular Biology, Baculovirus Expression
on Vectors, A Laboratory Manual, W. H. F
reeman and Company, New York (1992), Bi
o/Technology,6, 47 (1988) and the like.
, and proteins can be expressed. That is, the recombinant gene transfer vector and baculovirus are co-transfected into insect cells.
After obtaining the recombinant virus in the insect cell culture supernatant, the recombinant virus was further
The vector can be used to infect insect cells and express the protein. Examples of gene transfer vectors used in this method include pVL13
92, pVL1393, pBlueBac III (both from Invitrogen)
Examples of baculovirus include viruses that infect insects of the family Mythraeidae, such as
Autographa californica nuclear polyhedrosis virus (A
autographa californica nuclear polyhe
drossis virus), etc. can be used. Insect cells include:Spodoptera frugiperdaovarian cells
Sf9, Sf21 [Baculovirus Expression V
ectors, A Laboratory Manual, W. H. Free
and Company, New York (1992)],Trich
oplusia niHigh 5 (Invitrogen) ovarian cells
(manufactured by Epstein-Barr University Hospital) can be used. The above-mentioned recombinant gene transfer vector can be used to introduce into insect cells to prepare a recombinant virus.
Examples of methods for co-transfection of the vector and the baculovirus include the calcium phosphate method.
(JP-A-2-227075), lipofection method [Proc. Natl. Ac
ad. Sci. USA,84, 7413 (1987)] can be cited as examples.
When using plant cells as host cells, the expression vector can be, for example, T
Examples include iplasmid, tobacco mosaic virus vectors, etc. Any promoter can be used as long as it functions in plant cells.
For example, the 35S promoter of cauliflower mosaic virus (CaMV) may be present.
Examples of host cells include tobacco, potato, tomato, carrot, soybean, and oilseed.
Examples include plant cells such as rapeseed, alfalfa, rice, wheat, and barley.
The method for introducing a recombinant vector can be any method for introducing DNA into plant cells.
Any of these can be used, for example, Agrobacterium (Agrobacterium
erium) (JP-A-59-140885, JP-A-60-70080, WO94
/00977), electroporation method (Japanese Patent Laid-Open Publication No. 60-251887),
- Method using tickle gun (gene gun) (Patent No. 2606856, Patent No. 25
17813) and the like. When yeast, animal cells, insect cells, or plant cells are used as host cells,
Proteins or polypeptides to which sugars or sugar chains have been added can be obtained. The transformant obtained as described above is cultured in a medium, and the protein or polypeptide of the present invention is contained in the culture.
The protein or polypeptide is produced and accumulated in the culture, and then collected from the culture.
The transformant of the present invention can produce the protein or polypeptide of the present invention.
The method for culturing the host in the medium is carried out according to the usual method used for culturing the host.
Transformation can be achieved using prokaryotes such as E. coli or eukaryotes such as yeast as hosts.
The medium for culturing the organism contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the organism.
If the medium contains the necessary nutrients and can efficiently cultivate transformants, it can be a natural medium or a synthetic medium.
Any carbon source can be used as long as it can be assimilated by the organism, and examples include glucose, flocculation, and cellulose.
Sugars, sucrose, molasses containing these, starch or starch hydrolysates
Carbohydrates such as hydrolysates, organic acids such as acetic acid and propionic acid, ethanol, propanol
Alcohols such as these can be used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate.
ammonium salts of inorganic or organic acids such as ammonium and ammonium phosphate;
Other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn starch
Cheap liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermented
Bacterial bodies and their digested products can be used. Inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate,
Um, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate,
Calcium carbonate, etc. can be used. Cultivation is usually carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture.
The culture temperature is preferably 15 to 40°C, and the culture time is usually 16 hours to 7 days.
The pH of the mixture is maintained at 3.0 to 9.0. The pH can be adjusted by adding inorganic or organic acids,
This is done using potassium solution, urea, calcium carbonate, ammonia, etc. Also, antibiotics such as ampicillin and tetracycline may be used as needed during the culture.
It may be added to the medium. Transformation with a recombinant vector using an inducible promoter
When culturing the microorganism, an inducer may be added to the medium as needed.
For example,lacMicroorganisms transformed with recombinant vectors using promoters
When culturing organisms, isopropyl-β-D-thiogalactopyranoside, etc.
trpWhen a microorganism transformed with a recombinant vector using a promoter is cultured,
In some cases, indoleacrylic acid or the like may be added to the medium. The medium generally used for culturing transformants obtained using animal cells as hosts is
The RPMI 1640 medium used in
American Medical Association,199, 519
(1967)], Eagle's MEM medium [Science,122, 501 (
1952)], Dulbecco's modified MEM medium [Virology,8, 396 (1
959)], 199 medium [Proceedings of the Society
y for the Biological Medicine,73, 1 (1
950)] or a medium containing fetal bovine serum or the like can be used.
Culture is usually carried out at pH 6-8, 30-40°C, and 5% CO₂Under conditions such as in the presence of 1 to
The culture period is 7 days. If necessary, antibiotics such as kanamycin or penicillin may be added to the culture medium.
It may be added. Generally, the medium used to culture transformants obtained using insect cells as hosts is
TNM-FH medium (Pharmingen), Sf-900
II SFM medium (Life Technologies), ExCell
400, ExCell 405 (both manufactured by JRH Biosciences)
, Grace's Insect Medium [Grace, T. C. C. ，
Nature,195, 788 (1962)] can be used. Cultivation is usually carried out for 1 to 5 days under conditions such as pH 6 to 7 and 25 to 30°C. If necessary, antibiotics such as gentamicin can be added to the medium during cultivation.
Transformants obtained using plant cells as hosts can be used as cells or plant cells.
The transformant can be cultured by differentiating it into a cell or organ.
The commonly used Murashige and Skoog (MS) medium, White
(White) medium, or these media containing auxin, cytokinin, etc.,
A medium containing hormones can be used. Cultivation is usually carried out under conditions of pH 5-9 and 20-40°C for 3-60 days. If necessary, antibiotics such as kanamycin and hygromycin can be added during cultivation.
As described above, the DNA encoding the protein or polypeptide of the present invention may be added to the culture medium.
derived from microorganisms, animal cells, or plant cells carrying recombinant vectors
The transformant is cultured according to a conventional culture method, and the protein or polypeptide is extracted.
and collecting the protein or polypeptide from the culture.
The protein or polypeptide can be produced by the method of producing the protein or polypeptide of the present invention.
methods of secreting the protein outside the host cell, methods of producing it as a fusion protein, and
Alternatively, there is a method of producing the protein on the outer membrane of a host cell.
This method can be selected by changing the structure of the protein to be produced. When the protein of the present invention is produced inside a host cell or on the outer membrane of a host cell,
The method of Luzon et al. [J. Biol. Chem.,264, 17619 (1989)
], the method of Lowe et al. [Proc. Natl. Acad. Sci., USA,86,
8227 (1989), Genes Develop. ，4, 1288 (199
0)], or the methods described in JP-A-5-336963, WO94/23021, etc.
By applying the above method, the protein can be actively secreted outside the host cell.
That is, a protein containing the active site of the protein of the present invention is produced using a genetic recombination technique.
By expressing the gene with a signal peptide added before the target gene,
Proteins can be actively secreted outside the host cell. In addition, dihydrofolic acid can be secreted according to the method described in JP-A-2-227075.
It is also possible to increase production by using a gene amplification system that uses a reductase gene, etc.
Furthermore, by redifferentiating the cells of animals or plants into which genes have been introduced, the genes can be
An animal or plant into which a gene has been introduced (a transgenic non-human animal)
The protein of the present invention is produced using these individuals.
If the transformed organism is an animal or plant, it can be raised or cultivated in accordance with conventional methods.
and cultivating the animal or plant to produce and accumulate the protein, and extracting the protein from the animal or plant.
The protein can be produced by collecting the protein from the animal. Methods for producing the protein of the present invention using animal individuals include, for example, known methods.
[American Journal of Clinical Nutrit
ion,63, 639S (1996), American Journal o
f Clinical Nutrition,63, 627S (1996), B
io/Technology,9, 830 (1991)].
In the case of individual animals, for example, a method of producing the protein of the present invention in an animal by introducing DNA encoding the protein of the present invention into the animal is included.
a transgenic non-human animal is raised to produce and accumulate the protein in the animal;
The protein can be produced by collecting the protein from the animal.
The site of production and accumulation in the animal is, for example, the milk of the animal (Japanese Patent Application Laid-Open No. 63-1988).
309192), eggs, etc.
Any gene that can be expressed in animals can be used as the gene.
For example, the α-casein promoter and β-casein promoter, which are mammary cell-specific promoters,
Promoter, β-lactoglobulin promoter, whey acidic protein promoter
As a method for producing the protein of the present invention using a plant individual, for example,
The transgenic plants into which DNA encoding the protein has been introduced are prepared by known methods [tissue
culture,20(1994), Tissue Culture,21(1995), Trends in
Biotechnology,15, 45 (1997)], and the protein
Protein is produced and accumulated in the plant, and the protein is collected from the plant.
, and a method for producing the protein. The protein produced by the transformant of the present invention can be produced, for example, by the method of producing the protein of the present invention.
If the protein is expressed in a dissolved state within the cells, the cells are harvested by centrifugation after the culture is completed.
After suspending in an aqueous buffer solution, the mixture was homogenized using an ultrasonicator, a French press, or a Menton-Gaulin
The cells are disrupted using a homogenizer, dynomill, or the like to obtain a cell-free extract.
The supernatant obtained by centrifuging the cytosolic extract is purified by the usual enzyme isolation method.
That is, solvent extraction, salting out with ammonium sulfate, desalting, precipitation with organic solvents,
Ethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75
Anion exchange chromatography using resins such as S-S
Positive ion implants using resins such as epharose FF (Pharmacia)
ion exchange chromatography, butyl sepharose, phenyl sepharose, etc.
Hydrophobic chromatography using resin, gel filtration using molecular sieves,
Infinity chromatography, chromatofocusing, isoelectric focusing
To obtain a purified sample, electrophoresis or other methods are used alone or in combination.
Furthermore, if the protein is expressed by forming an insoluble body within the cell, it can also circulate the cell.
After harvesting, the cells are crushed and centrifuged to recover the insoluble protein as a precipitate fraction.
The recovered insoluble protein is solubilized with a protein denaturant. The solubilized solution is diluted.
Alternatively, the protein may be returned to its normal conformation by dialysis, and then the same procedure as above may be repeated.
A purified preparation of the protein can be obtained by the isolation and purification method described above. When the protein of the present invention or its derivatives such as glycosylated forms are secreted extracellularly,
In this case, the protein or its derivatives such as glycosylated products can be recovered from the culture supernatant.
That is, by treating the culture by the same method as above, such as centrifugation,
The soluble fraction is obtained by the same isolation and purification method as above.
A purified preparation can be obtained by this method. Proteins obtained in this way include, for example, SEQ ID NO: 1 or SEQ ID NO:
2. The protein of the present invention can also be synthesized by the Fmoc method (fluorenylmethyloxycarbonyl
It can also be synthesized by chemical synthesis methods such as the tBoc method (t-butyloxycarbonyl method)
It can also be manufactured by Advanced ChemTech, Inc., Parker
Jen-Elmer, Pharmacia, Protein Technology
y Instrument, Synthecell-Vega, PerSe
It can also be synthesized chemically using peptide synthesizers from companies such as Ptive and Shimadzu.
3. Preparation of antibodies that recognize the proteins of the present invention Purified preparations of the proteins of the present invention or partial fragment polypeptides of these proteins, or
Alternatively, a peptide having a partial amino acid sequence of the protein of the present invention is used as an antigen.
By this, polyclonal antibodies, monoclonal antibodies, etc., which recognize the protein of the present invention can be obtained.
It is possible to produce antibodies that recognize the proteins of the present invention. (1) Production of polyclonal antibodies Purified preparations of the proteins of the present invention or partial fragment polypeptides of those proteins, or
Alternatively, a peptide having a partial amino acid sequence of the protein of the present invention is used as an antigen,
Polyclonal antibodies can be produced by administering the antibody to animals. Rabbits, goats, rats, mice, hamsters, etc. are used as the animals to administer the antibody to.
The antigen can be administered at a dose of 50 to 100 μg per animal. When a peptide is used, the peptide is fused to keyhole limpet hemocyanin (keyhol
Carriers such as elimpet hemocyanin and bovine thyroglobulin
It is preferable to use a peptide as an antigen that is covalently bound to a protein.
It can be synthesized using a peptide synthesizer. The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration.
After the immunization, blood is collected from the venous plexus of the eyeground on the 3rd to 7th day, and the serum reacts with the antigen used for immunization.
This is based on the enzyme immunoassay method (ELISA method): published by Igaku Shoin (197
6), Antibodies-A Laboratory Manual, Co.
Spring Harbor Laboratory (1988) and others.
Confirm that the serum of a non-human mammal shows sufficient antibody titer against the antigen used for immunization.
The serum is then separated and purified to obtain polyclonal antibodies.
Methods for separation and purification include centrifugation and 40-50% saturated ammonium sulfate.
Salting out by caprylic acid precipitation [Antibodies, A Laboratory
y manual, Cold Spring Harbor Laborato
ry (1988)], or DEAE-Sepharose column, anion exchange column
Chromatography using a column, protein A or G, or gel filtration column, etc.
Examples of methods include the use of techniques such as fluoroscopy, either alone or in combination. (2) Production of Monoclonal Antibodies (a) Preparation of Antibody-Producing Cells The serum produced by the antibody-producing cells is sufficiently sensitive to the partial fragment polypeptide of the protein of the present invention used for immunization.
Rats that exhibit a sufficient antibody titer are used as a source of antibody-producing cells. Three to seven days after the final administration of the antigenic substance to the rats that exhibited the antibody titer, the spleens are removed.
The spleen was minced in MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and
After centrifugation at 200 rpm for 5 minutes, discard the supernatant. The resulting pelleted splenocytes were resuspended in Tris-ammonium chloride buffer (pH 7.65
) for 1 to 2 minutes to remove red blood cells, and then washed three times with MEM medium.
Spleen cells are used as antibody-producing cells. (b) Preparation of myeloma cells Myeloma cells are established cell lines obtained from mice or rats.
For example, the 8-azaguanine-resistant mouse (BALB/c-derived) myeloma cell line P3-
X63Ag8-U1 (hereinafter abbreviated as P3-U1) [Curr. Topics. M
icrobiol Immunol. ，81, 1 (1978), Europe. J
．． Immunol. ，6, 511 (1976)], SP2/0-Ag14 (SP
-2) [Nature,276, 269 (1978)], P3-X63-Ag8
653 (653) [J. Immunol. ，123, 1548 (1979)],
P3-X63-Ag8(X63) [Nature,256, 495 (1975)
These cell lines can be grown in 8-azaguanine medium [RPM
I-1640 medium containing glutamine (1.5 mmol/L), 2-mercaptoethanol
Roll (5 x 10⁻⁵mol/L), gentamicin (10 μg/ml) and
A medium containing fetal calf serum (FCS) (CSL, 10%) (hereinafter referred to as normal medium)
The cells were subcultured in a medium containing 8-azaguanine (15 μg/ml).
However, the cells were cultured in normal medium for 3 to 4 days before cell fusion, and 2 × 10⁷
(c) Hybridoma Production The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) are cultured in MEM medium or
or PBS (disodium phosphate 1.83 g, monopotassium phosphate 0.21 g,
The cells were washed thoroughly with 7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2, and the number of cells was measured by antibody.
The production cells and myeloma cells were mixed at a ratio of 5 to 10:1, and the mixture was stirred at 1,200 rpm for 5 minutes.
After centrifugation for 1 minute, discard the supernatant. The cells in the resulting precipitated fraction are thoroughly loosened and the cells are stirred at 37°C.
So, 10⁸Polyethylene glycol-1000 (PEG) per antibody-producing cell
2 g of 2-1000 mg of MEM, 2 ml of dimethyl sulfoxide (DMSO), and 0 ml of dimethyl sulfoxide (DMSO)
Add 0.2 to 1 ml of a solution mixed with 0.7 ml of MEM every 1 to 2 minutes.
Add 1-2 ml of medium several times. After the addition, add MEM medium to bring the total volume to 50 ml.
The prepared solution is centrifuged at 900 rpm for 5 minutes, and the supernatant is discarded.
The cells in the precipitated fraction were gently loosened and then separated using a measuring pipette.
Slowly add hypoxanthine (10% of normal medium) to HAT medium by suction and blowing.⁻⁴
mol/L), thymidine (1.5 × 10⁻⁵mol/L) and aminopterin
(4 x 10⁻⁷The suspension was then suspended in 100 ml of a medium containing 100 μl of ethanol (100 μL/L). The suspension was dispensed into a 96-well culture plate at 100 μl per well and incubated in 5% CO₂stomach
The cells are cultured in an incubator at 37°C for 7 to 14 days. After culturing, a portion of the culture supernatant is taken and used as an antibody.
Laboratory manual, Cold Spring Harbo
r Laboratory, Chapter 14 (1988) and others.
The partial fragment polypeptide of the protein of the present invention is specifically detected by the enzyme immunoassay method.
Hybridomas that react with the target protein are selected. The following method is a specific example of an enzyme immunoassay: During immunization, a partial fragment polypeptide of the protein of the present invention used as an antigen is added to an appropriate plate.
The hybridoma culture supernatant or the purified product obtained in (d) described below is coated on the membrane.
The antibody reacts as the first antibody, and then biotin, enzymes, or chemical activators are added as the second antibody.
Anti-rat or anti-mouse immunoglobulins labeled with fluorescent or radioactive compounds
After reacting with the phosphoantibody, a reaction according to the labeling substance is carried out to detect the specific
The hybridomas that react with the antibody of the present invention are identified as hybridomas that produce the monoclonal antibodies of the present invention.
Using the hybridomas, cloning is repeated twice by limiting dilution [
The first time was in HT medium (a medium in which aminopterin was removed from HAT medium), and the second time was in
A normal medium is used], and a stable and strong antibody titer is confirmed to be the monoclonal antibody of the present invention.
A hybridoma line producing a clonal antibody is selected. (d) Preparation of monoclonal antibodies Pristane treatment [2,6,10,14-tetramethylpentadecane (Pris)]
0.5 ml of ethanol was intraperitoneally administered to 8-10 week old mice and the mice were then reared for 2 weeks.
The monoclonal antibody against the protein of the present invention obtained in (c) was administered to a mouse or nude mouse.
5-20 x 10 human-producing hybridoma cells⁶Cells/animal are injected intraperitoneally.
The hybridomas develop into ascites tumors within 21 days. Ascites fluid is collected from the mice that have developed ascites tumors and centrifuged at 3000 rpm for 5 minutes.
The solids are removed by centrifugation. The resulting supernatant is used to isolate monoclonal antibodies using the same method as used for polyclonal antibodies.
Monoclonal antibodies can be purified and obtained. The subclass of the antibody can be determined using a mouse monoclonal antibody typing kit or
The protein amount is determined using a rat monoclonal antibody typing kit.
Calculations are made using the Lee method or absorbance at 280 nm. 4. Uses of the DNA, protein, or antibody of the present invention (1) Disease detection and quantification by detecting and quantifying the expression of DNA encoding the protein of the present invention.
Disease assessment method Northern hybridization is performed using the DNA or oligonucleotide of the present invention.
ion method (Molecular Cloning 2nd ed.), PCR method and RT (rev
PCR (trans-transcribed) method (both PCR Protocol
ols, Academic Press (1990)] (the above are collectively referred to as the PCR method)
and detecting the expression of the DNA encoding the protein of the present invention.
Acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine smooth muscle
Diseases involving abnormal cell proliferation such as tumours, malignant tumours and solid tumours, myocardial infarction, cerebral infarction, peripheral
Peripheral vascular occlusion, angina pectoris, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerular nephropathy
inflammation, arteriosclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic
Diseases accompanied by vascular disorders such as heart disease, ischemic cerebral disease, heart failure, congestion, and choroidal circulatory disorders
Diseases associated with abnormalities in bone metabolism such as osteoporosis, dwarfism, acromegaly, and childhood chronic renal failure
Diseases involving impaired insulin-like growth factors and growth hormone action, arteriosclerosis,
Bronchial diseases, diseases involving abnormal differentiation and proliferation of smooth muscle cells such as restenosis, myasthenia gravis
Diseases accompanied by abnormal differentiation and proliferation of skeletal muscle cells, such as gastric ulcers, and disorders of gastric acid secretion
Disease, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host
Main disease: insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, allergies
Abnormal lymphocyte infiltration in diseases such as rheumatoid arthritis, atopy, asthma, hay fever, airway hypersensitivity, and autoimmune diseases
Involvement of proteins belonging to the IGFBP superfamily in inflammatory diseases accompanied by inflammation
It is possible to diagnose diseases that have been reported. RT-PCR is a simple method, so it is particularly useful as a method for detecting DNA expression.
For example, the consecutive 100 to 1772 bases in the base sequence represented by SEQ ID NO: 3
DNA having the same sequence or a sequence complementary to said DNA, or
is the same sequence as the consecutive 100 to 2698 bases in the base sequence represented by SEQ ID NO: 4
or a DNA having a sequence complementary to said DNA is used as a probe.
Northern hybridization was performed using the DNA of SEQ ID NO: 3 or 4.
The expression level of the protein can be quantified and compared with that of healthy individuals to determine the above diseases.
Specific methods for this determination include, for example, the following: Total RNA (10-20 μg) derived from white blood cells or tissues of a subject and a healthy individual
or their mRNA (1-5 μg) was dissolved in a denaturing solution [50% (v/v) holophosphate
amide, 2.2 mol/L formaldehyde, 20 mmol/L MOPS [
3-(N-morpholino)propanesulfonic acid] (pH 7.0), 5 mmol/L
sodium acetate, 1 mmol/L EDTA] at 65°C for 5 minutes to denature
and electrophoresed on a 1% agarose gel containing 2.2 mol/L formaldehyde.
After electrophoresis, the RNA in the gel is transferred to a nitrocellulose filter (Optimal
BA-S85 (manufactured by Schleicher & Schuell)
The filter is then heated under reduced pressure at 80°C for 1 hour to fix the hybridization solution. The filter is then placed in hybridization solution [5x SSPE (750 mmol/L)].
/L NaCl, 50mmol/L NaH₂P.O.₄,5mmol/L EDT
A; pH 7.4), 5x Denhardt's solution (0.1% Ficoll, 0.1% Polyvinylpyrrolidone solution)
1% sodium dodecyl sulfate, 0.1% bovine serum albumin), 1% SDS (sodium dodecyl sulfate)
thorium), 0.2 mg/ml salmon sperm DNA (Pharmacia Bio
Prehybridization is performed by immersing the sample in a solution of 1000 kJ/ml (manufactured by tech). After prehybridization, the probe is added to the solution and hybridized at 65°C.
As a probe, for example, the DNA fragment set forth in SEQ ID NO: 3 or the DNA fragment set forth in SEQ ID NO: 4
The DNA fragments described above were subjected to multiprime DNA labeling using a multiprime DNA labeling system (Amersham).
It³²P-labeled filters can be used. After hybridization, wash the filter in the following order: (a) Wash with 2x SSC (300 mmol/L NaCl, 30
The cells are washed in a solution of 100 mmol/L sodium citrate at room temperature for 15 minutes.
Repeat several times. (b) 1x SSC (150 mmol/L NaCl, 15
1 mmol/L sodium citrate solution at 50-70°C for 15 minutes.
Repeat this process several times. (c) 0.1x SSC (15 mmol/L) containing 0.1% SDS at 50-70°C.
NaCl, 1.5 mmol/L sodium citrate solution at 50-70°C
Wash the filter with PBS for 15 minutes. Repeat this process several times. After washing the filter, perform autoradiography using an imaging plate.
The results were analyzed using a bioimaging analyzer BAS2000 (Fuji Photo Film Co., Ltd.).
) can detect and quantify the expression of the DNA set forth in SEQ ID NO: 3 or 4. Also, for example, a set of oligonucleotides specific to the DNA encoding the protein of the present invention can be used.
The nucleotides were used as primers to detect white blood cells or tissues from subjects and healthy individuals.
Total RNA derived from tissue, its mRNA or cDNA prepared from these RNAs
PCR was performed using the DNA as a template, and the amplified fragments were detected and quantified.
The above diseases can be diagnosed by comparing the expression levels of the oligonucleotides. The oligonucleotides used can be the oligonucleotides of the present invention.
The mRNA or total RNA used as a template for PCR can be obtained from various sources isolated and extracted from blood.
They can be extracted from white blood cells, or from tissues suspected of having a disease. White blood cells include polymorphonuclear leukocytes, monocytes, lymphocytes, T cells, B cells, etc.
Polymorphonuclear leukocytes and monocytes are extracted from the peripheral blood of subjects by Nycomed Pharma.
Polymorphr kit manufactured by Nycomed Pharma
ep^TMBy using this method, it is possible to separate and obtain the mononuclear cells. From the obtained mononuclear cells, J. Immunol.,130, 706 (1983) etc.
Monocytes and lymphocytes were isolated by the method described in Tissue Antigen,
9, 153 (1977), J. Immunol. ，11, 273 (1976),
According to the method described in the Nycomed manual for isolating blood cells,
, T cells and B cells can be isolated and obtained. T cells can be isolated using the nylon wool method [Eur. J. Immunol.,3, 645 (1
973)]. Also, T cells, B cells, monocytes/macrophages,
Magnetic beads (e.g., Dynal) conjugated with antibodies specific to each phage were used.
Each cell can be isolated and obtained using Dynabeads (manufactured by the company). Total RNA can be prepared from white blood cells or tissues using thiocyanate digestion.
Enzyme-mediated cesium trifluoroacetate method
l.,154, 3 (1987)]. Poly(A) synthesis from total RNA is also possible.⁺As a method for preparing RNA, oligo(dT) immobilization
Specific examples include the stabilized cellulose column method (Molecular Cloning, 2nd Edition).
Fast Track mRNA Isolation Kit (Fas
tTrack mRNA Isolation Kit; Invitroge
Quick Prep mRNA Purification Kit (Q
Uick Prep mRNA Purification Kit; Pharma
mRNA can also be prepared using a kit such as that manufactured by Cia. Single-stranded cDNA can be prepared from total RNA or mRNA using a single-stranded cDNA synthesis kit.
Superscript preamplification system (
The synthesis can be carried out using a kit (manufactured by BRL).
RT-PCR can be carried out according to the method described above using total RNA, mRNA, or cDNA prepared as described above.
CR method [PCR Protocols, Academic Press (199
0)], the expression level of the gene encoding the protein of the present invention can be quantified.
(2) Diagnosis of disease by detecting mutations in the gene encoding the protein of the present invention.
Method: Southern blot analysis of genomic DNA using the oligonucleotide of the present invention as a probe.
Hybridization method (Molecular Cloning 2nd Edition), PCR method, etc.
By carrying out the above steps, it is possible to detect mutations in the gene encoding the protein of the present invention.
This detection method is based on the idea that a protein belonging to the IGFBP family is involved.
There have been reports of acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, and
Diseases involving abnormal cell proliferation such as uterine leiomyoma, malignant tumors, solid tumors, myocardial infarction, brain
Infarction, peripheral vascular occlusion, angina pectoris, hypertension, hyperlipidemia, diabetes, diabetic retinopathy,
Glomerulonephritis, arteriosclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura
, ischemic heart disease, ischemic cerebral disease, heart failure, congestion, choroidal circulatory disorders, and other vascular disorders
Diseases accompanied by abnormalities in bone metabolism such as osteoporosis, dwarfism, acromegaly,
Diseases involving impaired insulin-like growth factor and growth hormone action, such as chronic renal failure,
Diseases involving abnormal differentiation and proliferation of smooth muscle cells, such as atherosclerosis, bronchial disease, and restenosis, and severe
Diseases involving abnormal differentiation and proliferation of skeletal muscle cells, such as myasthenia gravis, and abnormal gastric acid secretion, such as gastric ulcers
Common diseases, microbial infections, chronic hepatitis B, rheumatoid arthritis, sepsis, transplants
- host-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease
abnormal risk factors such as allergies, atopy, asthma, hay fever, airway hypersensitivity, and autoimmune diseases
This can be used to determine inflammatory diseases accompanied by lymphocyte infiltration. Specifically, for example, translation of DNA encoding the protein of the present invention carried by a patient can be used.
The translated region is amplified by PCR, the base sequence is determined, and the salt of the DNA present in healthy individuals is analyzed.
By comparing with the base sequence, it is possible to check for mutations in the coding region.
The cDNA used as a template for PCR can be obtained by the method described in (1).
The base sequence of the obtained cDNA was analyzed by DNA sequencing at Perkin-Elmer.
Sir377 and reaction kit (ABI Prism^TMBig Dye^TMTer
minator Cycle Sequencing Ready React
The ion kit (manufactured by Applied Biosystems) was used to determine the
(3) Disease Assessment Methods Using Immunological Detection and Quantitation Methods The antibodies of the present invention can be used to assess the presence or absence of the present invention in the blood or tissues of subjects and healthy individuals.
The expression of the protein is immunologically detected or quantified, and the results are compared with those of healthy individuals.
Disease can be diagnosed. Immunological detection methods include ELISA using microtiter plates.
Examples include the SA method, fluorescent antibody method, Western blotting method, and immunohistochemical staining method.
Immunological quantification methods include, for example, using a compound that reacts with the protein of the present invention in a liquid phase.
A sandwich antibody was prepared using two types of monoclonal antibodies with different epitopes.
Chi ELISA method,¹²⁵The protein of the present invention labeled with a radioisotope such as I and the protein
Examples of immunological methods include radioimmunoassays that use antibodies that recognize proteins. This immunological method has been reported to involve proteins belonging to the IGFBP family.
acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, and uterine smooth muscle
Diseases involving abnormal cell proliferation such as tumours, malignant tumours and solid tumours, myocardial infarction, cerebral infarction, peripheral
Peripheral vascular occlusion, angina pectoris, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerular nephropathy
inflammation, arteriosclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic
Diseases accompanied by vascular disorders such as heart disease, ischemic cerebral disease, heart failure, congestion, and choroidal circulatory disorders
Diseases associated with abnormalities in bone metabolism such as osteoporosis, dwarfism, acromegaly, and childhood chronic renal failure
Diseases involving impaired insulin-like growth factors and growth hormone action, arteriosclerosis,
Bronchial diseases, diseases involving abnormal differentiation and proliferation of smooth muscle cells such as restenosis, myasthenia gravis
Diseases accompanied by abnormal differentiation and proliferation of skeletal muscle cells, such as gastric ulcers, and disorders of gastric acid secretion
Disease, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host
Main disease: insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, allergies
Abnormal lymphocyte infiltration in diseases such as rheumatoid arthritis, atopy, asthma, hay fever, airway hypersensitivity, and autoimmune diseases
It can be used to determine diseases such as inflammatory diseases accompanied by inflammation. It also specifically binds to a protein consisting of the amino acid sequence represented by SEQ ID NO: 1.
An antibody that specifically binds to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2
By using antibodies that express these proteins, it is possible to identify the differences in the expression ratios of these proteins in the above diseases.
Diseases characterized by the above can be diagnosed by the above immunological method. (4) Diseases by analyzing the expression pattern of the DNA or protein of the present invention
Method for determining expression of the DNA or protein of the present invention described in any one of (1) to (3) above
The amount of ATP in the blood or tissues of subjects and healthy individuals was determined using a method to quantify the amount of ATP.
DNA encoding a protein consisting of the amino acid sequence represented by sequence number 1 and SEQ ID NO:
Quantitative expression of DNA encoding a protein consisting of the amino acid sequence shown in 2.
The expression ratio of the DNA or the protein is different from that of a healthy person.
The above-mentioned method of determination can be used to determine diseases in which proteins belonging to the IGFBP family have been reported to be involved.
Acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, and uterine leiomyoma
Diseases involving abnormal cell proliferation such as malignant tumors and solid tumors, myocardial infarction, cerebral infarction, peripheral
Vascular occlusion, angina pectoris, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis
, arteriosclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart
Diseases accompanied by vascular disorders such as ischemic brain disease, heart failure, congestion, and choroidal circulatory disorders
, diseases accompanied by abnormalities in bone metabolism such as osteoporosis, dwarfism, acromegaly, and chronic renal failure in children
Diseases involving impaired insulin-like growth factor and growth hormone action, arteriosclerosis,
Bronchial diseases, diseases involving abnormal differentiation and proliferation of smooth muscle cells such as restenosis, myasthenia gravis, etc.
Diseases accompanied by abnormal differentiation and proliferation of skeletal muscle cells, diseases accompanied by abnormal gastric acid secretion such as gastric ulcers
Disease, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host
Disease, insulin-dependent diabetes, nephritis, traumatic injury, inflammatory bowel disease, allergies
Abnormal lymphocyte infiltration in atopic dermatitis, asthma, hay fever, airway hypersensitivity, autoimmune diseases, etc.
(5) By inhibiting the transcription or translation of DNA encoding the protein of the present invention, the protein can be used to determine diseases such as inflammatory diseases accompanied by
Prevention and treatment of diseases Regarding the relationship between elevated IGFBP expression and disease, pediatric chronic renal failure is associated with increased IGFBP expression.
The cause is an increase in BP-2 and BP-3 [Electroiyte Mrtab.
,18, 320 (1992)], Fractures in elderly women with elevated parathyroid hormone
In patients with leukemia, the expression of IGFBP-4 was increased, and in cerebrospinal fluid of patients with leukemia, the expression of IGFBP-4 was increased.
Increased levels of IGFBP-7 and IGFBP-3 [J. Clin. En
docrinol. Metab., 84, 1361 (1996)], and in colon cancer
, IGFBP-7 expression is elevated in colon cancer tissues and colon cancer cell lines
J. Gastroenterology,33, 213 (1998)] etc.
It has been reported that TGF-β and PTHPGE2 increase the expression of IGFBP-7.
Endocrinology,140, 1998 (1999)], osteoblasts
Treatment of cells with glucocorticoids suppresses IGF-I expression, whereas IGF
Increasing the expression of BP-7 [Endocrinology,140, 22
8 (1999)], IGFBP-7 also promotes differentiation into skeletal muscle through the IGF differentiation-promoting effect.
It affects the cells by suppressing the237, 1
92 (1997), Endocrinology,141, 100 (2000)
], IGFBP-7 is also involved in diseases related to bone metabolism and skeletal muscle differentiation.
It is thought that an increase in the expression level of IGFBP genes or an increase in the function of IGFBPs
For the disease causing the disease, the amount of transcription of the DNA encoding the protein of the present invention is
Alternatively, reducing the translation amount is useful for the prevention and treatment of diseases.
Even in the case of diseases not directly caused by GFBP, the gene encoding the protein of the present invention can be used.
or suppressing the function of the protein of the present invention.
This can also be used to prevent or treat the above diseases symptomatically. In addition, increased expression of IGFBPs can enhance receptor-mediated physiological actions.
If there is a patient, the DNA, oligonucleotide or derivative thereof of the present invention is administered to the patient.
By administering it to the rats, the physiological action can be suppressed and the expression of IGFBPs can be suppressed.
Even if the increase in expression is not the direct cause of the disease, the DNA, oligonucleotide or
The DNA of the present invention can be used for diseases that can be treated symptomatically by administering its derivatives.
Oligonucleotides or their derivatives can be effective preventive and therapeutic agents. Methods for using such preventive or therapeutic agents include, for example, antisense RNA/
DNA Technology [Bioscience and Industry,50, 322 (1992),
Chemistry,46, 681 (1991), Biotechnology,9, 358 (
1992), Trends in Biotechnology,10, 87 (
1992), Trends in Biotechnology,10, 152
(1992), Cell Engineering,16, 1463 (1997)], Triple Helical
Trends in Biotechnology,10, 132 (1
992)], ribozyme technology [Current Opinion in Che
mical Biology,3, 274 (1999), FEMS Micro
biology Reviews,23, 257 (1999), Frontie
rs in Bioscience,4, D497 (1999), Chemis
Try & Biology,6, R33 (1999), Nucleic Ac.
ids Research,26, 5237 (1998), Trends In
Biotechnology,16, 438 (1998)], or decoy
DNA method [Nippon Rinsho-Japanese Journal
of Clinical Medicine,56, 563 (1998), Ci
rculation Research,82, 1023 (1998), Exp.
erimental nephrology,5, 429 (1997), Nip.
pon Rinsho-Japanese Journal of Clini
cal Medicine,54, 2583 (1996)] can be cited as an example.
By using this method, the expression of any gene can be suppressed. The DNA, oligonucleotide, or derivatives thereof of the present invention can be used as the above-mentioned preventive and therapeutic agents.
When used as a therapeutic agent, the DNA, oligonucleotide or derivative thereof of the present invention
The conductor can be used alone or in combination with a retroviral vector, adenoviral vector, or adenovirus vector.
After inserting it into an appropriate vector such as a virus-associated virus vector
, can be formulated, prescribed, and administered according to the standard methods described below. Viral vectors such as retroviruses and adenoviruses, and other gene therapy vectors
The gene therapy vectors incorporated into the vectors are used as gene therapy drugs or prophylactic drugs.
To use the gene therapy vector, the base used for the gene therapy agent is mixed with the vector.
It can be produced by the method described in Nature Genet.8, 42 (1
994). The base used for gene therapy agents can be any base normally used for injections.
It may be any of the following: distilled water, sodium chloride, or a mixture of sodium chloride and an inorganic salt.
salt solutions such as mannitol, lactose, dextran, glucose, etc.
solution, amino acid solution such as glycine, arginine, etc., organic acid solution or salt solution and glucose
In addition, in accordance with the usual method, these bases may be mixed with osmotic
pH adjuster, vegetable oil such as sesame oil or soybean oil, or lecithin or non-ionic surfactants
It is injected as a solution, suspension, or dispersion using auxiliary agents such as surfactants, etc.
These injections may be prepared for use by powdering, freeze-drying, etc.
The gene therapy agent of the present invention can also be prepared as a preparation for dissolution at the time of administration.
In the case of an individual, the gene therapy agent is placed in the above-mentioned base, which has been sterilized as necessary.
The agent for gene therapy of the present invention can be dissolved and used immediately before the treatment.
One method is to administer it locally so that it is absorbed into the treatment area of the patient.
It is also possible to deliver DNA to the desired treatment site using non-viral gene transfer methods.
Non-viral gene transfer methods known in the art include calcium phosphate co-precipitation [V
irology,52, 456-467 (1973); Science,209 , 1414-1422 (1980)], microinjection method [Proc
．． Natl. Acad. Sci. USA,77, 5399-5403 (1980
); Proc. Natl. Acad. Sci. USA,77, 7380-738
4 (1980); Cell, 27, 223-231 (1981); Nature
,294, 592-94 (1981)], membrane fusion-mediated transfer via liposomes
Law [Proc. Natl. Acad. Sci. USA,84, 7413-741
7 (1987); Biochemistry,28, 9508-9514 (19
89);J. Biol. Chem. ，264, 12126-12129 (198
9);Hum. Gene Ther. ，3, 267-275, (1992);S
science,249, 1285-1288 (1990);
on,83, 2007-2011 (1992)] or direct DNA uptake and
and receptor-mediated DNA transfer [Science,247, 1465-1468
(1990); J. Biol. Chem. ，266, 14338-14342 (
1991); Proc. Natl. Acad. Sci. USA,87, 3655
-3659 (1991); J. Biol. Chem. ，264, 16985-1
6987 (1989); BioTechniques,11, 474-485 (
1991); Proc. Natl. Acad. Sci. USA,87, 3410
-3414 (1990); Proc. Natl. Acad. Sci. USA,8
8, 4255-4259 (1991); Proc. Natl. Acad. Sci
.USA,87, 4033-4037 (1990); Proc. Natl. Ac
ad. Sci. USA,88, 8850-8854 (1991); Hum. Ge
ne Ther.,3, 147-154 (1991)] and the like can be mentioned.
Liposome-mediated membrane fusion-mediated delivery involves the use of liposome preparations to target cells.
By administering the gene directly to the tissue, local uptake and expression of the gene in the tissue can be achieved.
It has been reported in tumor studies that this is possible [Hum. Gen.
e Ther.,3, 399 (1992)]. (6) Prevention and treatment of diseases in which the expression level or function of the protein of the present invention is reduced. Regarding the relationship between reduced IGFBP expression and disease, IGFBP-5 is known to be a major cause of osteoporosis.
The expression of IGFBPs was decreased in the compensatory hypertrophy after nephrectomy and small intestinal resection.
The decrease in expression of IGF-3 was accompanied by an increase in free IGF-I [Baillier
es Clin. Endocrinol. Metab. ，8, 165 (1994
) ], and the expression of IGFBP-1 is decreased in malignant endometrial tumors [Gr
owth Regulation. ，3, 74 (1993)], and in breast cancer tissue, human staining
LOH was observed at the IGFBP-7 locus on the chromosome with a frequency of 50% or more.
A decrease in the expression of GFBP-7 was observed [Oncogene,16, 2459
(1996)], mRNA levels of IGFBP-7 expression in prostate cancer tissues
Decreased expression of IGFBP-7 was detected in malignant prostate cancer cell lines.
Not being released [J. Clin. Endocrinol. Metab.,83,
4355 (1998)], and in the case of large uterine leiomyomas, adjacent uterine smooth muscle cells
The volume of the tumor or cell is 120 cm³The m of IGFBP-7 compared to the small uterine smooth muscle
The expression of RNA is decreased [Am. J. Reprod. Immunol.
,43, 53 (2000)], in mouse hepatoma cells induced by SV40 T antigen.
In this case, the 5' upstream region of the IGFBP-7 gene is methylated, resulting in reduced expression.
Biochem. Biophys. Res. Commun.267, 1
09 (2000)], and in a streptozotocin-induced type 1 diabetes model, kidney
and that IGFBP-7 expression is reduced at the site of vascular injury [Diabet
es,45, S111 (1996), J. Diabetes & its Co
mplifications,12, 252 (1998)], coronary heart disease in type II diabetes patients
Decreased expression of IGFBP-7 was also observed at the protein level in smooth muscle cells of the pulmonary artery.
Diabetes,46, 1627 (1997)], bovine arterial
When smooth muscle cells were cultured in a high-glucose medium, the expression level of IGFBP-7 increased.
A and protein levels [Diabetes,46, 1627 (19
97), Diabetologia,41, 134 (1998)], and IGF
BP-7 stimulates PGI2 production in the vascular wall [Nature,271, 5
49 (1978)], and hemolytic uremic syndrome [Lancet,2, 871 (
1978)], thrombotic thrombocytopenic purpura [Lancet,2, 748 (197
9)], sickle cell anemia [Br. J. Haematol. ，48, 545 (
1981)], acute myocardial infarction [Coronavirus,2, 49 (1985)], sugar
Diabetic vascular disorders [Metabolism,38, 837 (1989), Haem
ostasis,16, 447 (1986), Diab. Res. Clin. P
ract.,3, 243 (1987)], and blood levels in arteriosclerotic diseases
It has been reported that IGFBP-7 expression is increased by TGF-β and PTHPGE2.
Endocrinology,140, 1998 (1999)], osteoblasts
Treatment of cells with glucocorticoids suppresses IGF-I expression, whereas IGF
Increasing the expression of BP-7 [Endocrinology,140, 22
8 (1999)], IGFBP-7 also promotes differentiation into skeletal muscle through the IGF differentiation-promoting effect.
It affects the cells by suppressing the237, 1
92 (1997), Endocrinology,141, 100 (2000)
], it is thought that IGFBP-7 is also involved in diseases related to bone metabolism and skeletal muscle differentiation.
It is thought that IGFBP-7 was forced to be expressed in prostate cancer cell lines with reduced IGFBP-7 expression.
When the cells are allowed to grow, the cell division time increases, the colony formation ability in soft agar medium decreases, and the growth of the cells is affected.
Decreased tumorigenicity when transplanted into mice, and increased apoptosis induction rate by drug treatment
[Cancer Res.,59, 2370 (1999)], p53
IGFBP-7 was forced to be expressed in osteosarcoma cell lines that lacked its function.
When the p53 gene is introduced, the proliferation of the cell line can be suppressed in the same way as when the p53 gene is introduced.
Therefore, the DNA of the present invention
A and protein expression are increased, and the decreased expression of IGFBPs is involved.
The above diseases can be prevented or treated.
also increases the transcription or translation amount of the gene encoding the protein of the present invention.
Alternatively, the prevention of the above diseases which can be treated symptomatically by increasing the amount of the protein of the present invention.
It is also possible to prevent or treat IGFBPs. Therefore, the physiological effects of IGFBPs are reduced due to decreased expression or function.
If there is a patient who is recovering, the DNA, oligonucleotide or derivative thereof of the present invention may be used.
By administering the conductor or the protein of the present invention to a patient, the physiological action is promoted.
It is also possible that decreased expression or function of IGFBPs is the direct cause of the disease.
For diseases that can be treated symptomatically even if they are not causative agents, the DNA and oligonucleotides of the present invention can be used.
The otide or its derivative, or the protein of the present invention is useful as a preventive agent for the above diseases and
These drugs are useful as therapeutic agents. Methods for administering these preventive and therapeutic agents include, for example, administering drugs to treat conditions in which the expression of the protein of the present invention is reduced.
In cases where there are patients for whom normal physiological effects cannot be expected due to a disease or mutation, (i)
(ii) administering to the patient a DNA encoding the protein of interest and expressing it;
After inserting DNA encoding the protein of the present invention into a cell, the cell is expressed.
or (iii) administering a protein of the present invention to the patient.
By using a method such as administering a therapeutic agent to a patient, the amount of the protein of the present invention in the patient's body is increased, and the protein
Therefore, the protein of the present invention can fully exert its physiological functions.
The DNA encoding the protein of the present invention is
a disease in which the function of the protein is reduced, or a disease caused by a functional abnormality due to a mutation in the protein,
or DNA encoding the protein of the present invention, even if not directly attributable to the protein of the present invention.
or for diseases that can be treated symptomatically by administering the protein of the present invention.
It is useful as a safe and low-toxicity preventive and therapeutic drug. The DNA encoding the protein of the present invention is used as the above preventive and therapeutic drug.
In this case, the DNA of the present invention may be used alone or in combination with a retroviral vector or adenovirus vector.
vector, adenovirus-associated virus vector, or other suitable vector.
After inserting the catheter into the catheter, it is formulated, prescribed and administered according to the conventional method described above in (5).
In addition, pharmaceuticals containing the protein of the present invention as an active ingredient can be administered by administering the active ingredient alone.
Although it is possible to administer the active ingredient in a pharmacologically acceptable form, it is usually
or more carriers, and methods well known in the art of pharmaceutical formulations.
It is desirable to provide it as a pharmaceutical preparation prepared by any method.
Water or aqueous solutions such as salt, glycine, glucose, human albumin, etc.
A sterile solution dissolved in a carrier is used.
pharmacologically acceptable additives, such as buffering agents and tonicity agents,
, sodium acetate, sodium chloride, sodium lactate, potassium chloride, citric acid
Sodium, etc. can also be added. It can also be stored after freeze-drying and then adjusted to the appropriate temperature when used.
It can also be used by dissolving it in a suitable solvent. The most effective route of administration for treatment is preferably oral.
or parenteral administration, such as oral, intratracheal, rectal, subcutaneous, intramuscular, and intravenous administration.
Examples of dosage forms include sprays, capsules, tablets, and granules.
, syrup, emulsion, suppository, injection, ointment, tape, etc. Formulations suitable for oral administration include emulsion, syrup, capsules, tablets, powder
Liquid preparations such as emulsions and syrups include:
Water, sucrose, sorbitol, fructose and other sugars, polyethylene glycol, propylene glycol
glycols such as ethylene glycol, oils such as sesame oil, olive oil, and soybean oil, p-
Preservatives such as hydroxybenzoates, strawberry flavor, peppermint
It can be produced using flavors such as cinnamon and other flavorings as additives.
Powders and granules contain excipients such as lactose, glucose, sucrose, and mannitol, and starch.
disintegrants such as cellulose, sodium alginate, magnesium stearate, talc, etc.
Lubricants, polyvinyl alcohol, hydroxypropyl cellulose, gelatin, etc.
Additives include binders, surfactants such as fatty acid esters, and plasticizers such as glycerin.
Preparations suitable for parenteral administration include injections, suppositories, sprays, etc.
For example, injections may contain a carrier such as a salt solution, a glucose solution, or a mixture of both.
Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats, or carboxylic acids.
The spray is prepared by spraying the protein itself or the oral cavity and
It does not irritate the respiratory tract mucosa and disperses the protein into fine particles, making it easy to absorb.
Specific examples of carriers include lactose and glycerin.
Depending on the properties of the protein and the carrier used, it may be used in the form of an aerosol, a dry powder, or the like.
In addition, these parenteral preparations can be used as oral preparations with additives.
The ingredients exemplified above can also be added. The dosage or frequency of administration should be determined based on the desired therapeutic effect, administration method, treatment period, age,
Although it varies depending on body weight, the usual daily dose for adults is 10 μg/kg to 8 mg/kg.
(7) Method for Obtaining the Promoter Region of a Gene Encoding the Protein of the Present Invention Using the DNA or oligonucleotide of the present invention as a probe, a known method (such as
Edited by the Department of Cancer Research, Institute of Medical Science, University of Tokyo, New Cell Engineering Experimental Protocol, Shujunsha (19
The promoter region of the gene can be obtained using the promoter sequence of the gene encoding the protein of the present invention in mammalian cells.
All promoter regions involved in the transcription of genes ...
A promoter involved in the transcription of the gene encoding the protein of the present invention in the small intestine of a mouse.
The promoter can be a region of the protein of the present invention described below in (8).
A method for screening substances that regulate the transcription or translation of DNA encoding protein
(8) A substance that controls the transcription or translation of a gene encoding the protein of the present invention.
Cleaning method Acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine leiomyoma
Diseases involving abnormal cell proliferation such as malignant tumors and solid tumors, myocardial infarction, cerebral infarction, peripheral
Vascular occlusion, angina pectoris, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis
, arteriosclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart
Diseases accompanied by vascular disorders such as ischemic brain disease, heart failure, congestion, and choroidal circulatory disorders
, diseases accompanied by abnormalities in bone metabolism such as osteoporosis, dwarfism, acromegaly, and chronic renal failure in children
Diseases involving impaired insulin-like growth factor and growth hormone action, arteriosclerosis,
Bronchial diseases, diseases involving abnormal differentiation and proliferation of smooth muscle cells such as restenosis, myasthenia gravis, etc.
Diseases accompanied by abnormal differentiation and proliferation of skeletal muscle cells, diseases accompanied by abnormal gastric acid secretion such as gastric ulcers
Disease, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host
Disease, insulin-dependent diabetes, nephritis, traumatic injury, inflammatory bowel disease, allergies
Abnormal lymphocyte infiltration in atopic dermatitis, asthma, hay fever, airway hypersensitivity, autoimmune diseases, etc.
Diseases such as inflammatory diseases accompanied by inflammatory bowel disease are characterized by the involvement of proteins belonging to the IGFBP superfamily.
It has been reported that one of the causes of the above diseases is fluctuations in the amount of transcription of DNA encoding IGFBPs,
In this case, the function of the protein of the present invention may be changed.
Controlling the amount of transcription or translation of DNA is effective in preventing and treating diseases.
Furthermore, even for diseases that are not directly caused by IGFBP, the protein of the present invention can be used to treat the disease.
By controlling the transcription or translation amount of the genes involved,
It is possible to prevent or treat diseases. Therefore, the transcription process of the gene encoding the protein of the present invention or the transcription product
Compounds that promote or inhibit the translation process from a protein to a target protein are useful for controlling the expression of the target protein.
By doing so, it is possible to control the cellular functions exerted via the protein.
This compound is useful as a safe and low-toxicity pharmaceutical composition. The compound can be obtained by the following methods (a) to (c): (a) [i] cells expressing the protein of the present invention; and [ii] cells expressing the protein of the present invention in the presence of a test substance.
The cells expressing the protein of the present invention are cultured for 2 hours to 1 week using the culture method described in 2 above.
Then, the amount of the protein in the cells is measured using the antibody of the present invention described in (3) above.
and comparing the results to select and obtain a compound having the activity of increasing or decreasing the amount of the protein.
As a measurement method using the antibody of the present invention, for example,
ELISA, fluorescent antibody technique, Western blotting, immunohistochemical staining, etc.
(b) [i] a cell expressing the protein of the present invention and [ii] a cell expressing the protein of the present invention in the presence of a test substance.
The cells expressing the protein of the present invention are cultured for 2 hours to 1 week using the culture method described in 2 above.
Then, the amount of the transcription product of the DNA encoding the protein in the cells is measured using the method described in (1) above.
Measurement and comparison are carried out using methods such as Northern hybridization or PCR.
and selecting and obtaining a compound having the activity of increasing or decreasing the amount of the transcript.
(c) A reporter gene is linked downstream of the promoter obtained in (7) above.
A plasmid incorporating the DNA is prepared by a known method, and the method described in 2 above is then carried out.
[i] The transformant is introduced into an animal cell according to the method described above to obtain a transformant.
ii] In the presence of a test substance, the transformant is cultured for 2 to 3 hours by the culture method described in 2 above.
After culturing for one week, the expression level of the reporter gene in the cells was measured by a known method [
Research Institute for Cancer Research, New Cell Engineering Experimental Protocols, Shujunsha (1993),
Biotechniques,20, 914 (1996), J. Antibio
tics,49, 453 (1996), Trends in Biochemi.
cal Sciences,20, 448 (1995), Cell Engineering,16, 58
1 (1997)] and compare the activity of increasing or decreasing the expression level.
A method for selecting and obtaining compounds having the following. Examples of reporter genes include chloramphenicol acetyltransferase (ATP).
isoformase gene, β-galactosidase gene, β-lactamase gene,
Luciferase gene, green fluorescent protein (GFP) gene
Examples of such cancers include acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, and uterine leiomyoma. (9) Method for screening for substances that regulate the function of the protein of the present invention.
Diseases involving abnormal cell proliferation such as malignant tumors and solid tumors, myocardial infarction, cerebral infarction, peripheral
Vascular occlusion, angina pectoris, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis
, arteriosclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart
Diseases accompanied by vascular disorders such as ischemic brain disease, heart failure, congestion, and choroidal circulatory disorders
, diseases accompanied by abnormalities in bone metabolism such as osteoporosis, dwarfism, acromegaly, and chronic renal failure in children
Diseases involving impaired insulin-like growth factor and growth hormone action, arteriosclerosis,
Bronchial diseases, diseases involving abnormal differentiation and proliferation of smooth muscle cells such as restenosis, myasthenia gravis, etc.
Diseases accompanied by abnormal differentiation and proliferation of skeletal muscle cells, diseases accompanied by abnormal gastric acid secretion such as gastric ulcers
Disease, microbial infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host
Disease, insulin-dependent diabetes, nephritis, traumatic injury, inflammatory bowel disease, allergies
Abnormal lymphocyte infiltration in atopic dermatitis, asthma, hay fever, airway hypersensitivity, autoimmune diseases, etc.
Inflammatory diseases accompanied by inflammatory bowel disease are caused by proteins belonging to the IGFBP superfamily.
It has been reported that altered IGFBP function is thought to be one of the causes of the above diseases.
Inhibiting or enhancing the function of this protein is effective in preventing and treating the disease.
In addition, even in the case of diseases that are not directly caused by IGFBP, the function of the protein of the present invention can be effectively utilized.
By inhibiting or enhancing the activity of the steroid hormone, the above diseases can be prevented or treated symptomatically.
Therefore, the physiological activity of cells can be reduced or enhanced due to the functional change of the protein of the present invention.
If there is a patient with a progressive disease, the compound of the present invention that controls the function can be administered to the patient.
The physiological effects can be controlled by the protein of the present invention, and functional changes of the protein of the present invention can be used to treat diseases.
Even if the disease is not the cause of the infection, it is possible to treat the symptoms by controlling the function of the protein of the present invention.
Diseases can also be prevented and treated by administering the compound. The compound can be obtained, for example, by the method described below. [i] Cells expressing the protein of the present invention; and [ii] Cells expressing the protein of the present invention in the presence of a test substance.
The cells expressing the protein are cultured for 2 hours to 1 week by the culture method described in 2 above,
The cellular responses resulting from the function of the protein are detected and compared, and the function of the protein is
A method for selecting and obtaining a compound having activity to inhibit or enhance a protein of the present invention. A method for detecting a cellular response caused by the function of the protein of the present invention is as follows:
For example, depending on insulin or insulin-like growth factors contained in the medium
These are publicly available technologies that detect changes in intracellular signal transduction, gene transcription, sugar uptake, proliferation, etc.
Also, known methods for measuring changes in prostaglandin production include
The method is also mentioned (Nature,263, 663, 1976; Clinical Science,17, 958, 1981). (10) Method for screening for substances that specifically bind to the protein of the present invention Substances that specifically bind to the protein of the present invention can be used to screen for diseases caused by the protein of the present invention.
For example, the protein of the present invention can be used in the development of preventive or therapeutic drugs.
The receptor binds to the protein of the present invention, transmits information into the cell, and exerts a physiological effect.
Since it has the function of inhibiting the activity of the receptor or the gene encoding the receptor,
The substance that controls transcription or translation of the gene is the protein of the present invention or a compound that controls the transcription or translation of the gene.
The proteins of the present invention, as well as substances that regulate the transcription or translation of genes encoding the proteins,
The substance is useful as a preventive or therapeutic agent for diseases caused by dysfunction of the substance.
Examples of the target protein include low molecular weight compounds and proteins. For example, a method for screening proteins that specifically bind to the protein of the present invention is
Examples include Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology, and Molecular Biology.
Molecular Biology, Science,255, 989 (1992
) and the like,
When a test sample is contacted with the protein of the present invention, a protein that specifically binds to the protein of the present invention is detected as the test sample.
These can be obtained by selecting from the following methods. Specifically, the following method can be used: Prepare cDNA from tissue. Introduce the cDNA into a promoter region of an appropriate expression vector.
By inserting the gene downstream of the cDNA library, a recombinant vector was created.
The recombinant vector is introduced into a host cell compatible with the expression vector.
By this, a transformant expressing the gene expressed in the tissue is obtained.
Transformants that produce a protein to which the protein of the present invention specifically binds are selected. The gene sequence encoded by the cDNA introduced into the selected transformants is determined.
By determining the specific binding site of the protein of the present invention, it is possible to obtain a protein that specifically binds to the protein of the present invention.
Methods for preparing a cDNA library include the method described in 1 above.
Methods for introducing recombinant vectors, methods for obtaining transformants, and methods for obtaining the transformants
The method for culturing the cells in a medium includes the method described in 2 above. The present invention uses the cells labeled with known protein labeling methods such as radioisotopes and biotinylation.
By co-culturing the protein of the present invention with the transformant, the labeled protein of the present invention and the specific
Transformants that produce gene products that specifically bind to the cDNA can be selected. The cDNA introduced into the selected transformants can be isolated using molecular
Cloning, 2nd Edition, Current Protocols in Molecular Biology
Biology, Mol. Cell. Biol.,8, 2837 (1988)
The method for recovering the phage vector or plasmid vector described above or the Hirt method
Examples of methods for determining the gene sequence of isolated cDNA include the method described in 1 above.
(11) Pharmaceuticals Containing the Antibodies of the Present Invention The antibodies of the present invention can be used as preventive and therapeutic drugs for diseases associated with the proteins of the present invention.
It is useful. Regarding the relationship between elevated IGFBP expression and disease, pediatric chronic renal failure is associated with increased IGFBP expression.
The cause is an increase in BP-2 and BP-3 [Electroiyte Mrtab.
,18, 320 (1992)], Fractures in elderly women with elevated parathyroid hormone
In patients with leukemia, the expression of IGFBP-4 is increased, and in cerebrospinal fluid of patients with leukemia, the expression of IGFBP-4 is increased.
Increased levels of IGFBP-7 and IGFBP-3 [J. Clin. En
docrinol. Metab. ，84, 1361 (1996)], and in colon cancer
, IGFBP-7 expression is elevated in colon cancer tissues and colon cancer cell lines
J. Gastroenterology,33, 213 (1998)] etc.
It has been reported that IGFBP-7 expression is increased by TGF-β, PTH, and PGE2.
Endocrinology140, 1998 (1999)],
Treatment of osteoblasts with glucocorticoids suppresses IGF-I expression, whereas
Increasing the expression of IGFBP-7 [Endocrinology,140, 228 (1999)], IGFBP-7 also promotes differentiation into skeletal muscle by IGF differentiation.
It affects the cell proliferation by suppressing the cell proliferation [Exp. Cell Res.,23
7, 192 (1997), Endocrinology,141, 100 (20
00)], it has been shown that IGFBP-7 is also involved in diseases related to bone metabolism and skeletal muscle differentiation.
Therefore, it is thought that diseases caused in part by increased expression or enhanced function of IGFBPs may be
In response to the above, the expression level of the protein of the present invention can be suppressed or the function can be inhibited.
It is effective in preventing and treating diseases that are not directly caused by IGFBPs.
However, the present invention can be countered by suppressing the expression level or inhibiting the function of the protein of the present invention.
It is also possible to prevent or treat the above-mentioned diseases for which treatment is possible. Therefore, it is possible to prevent or treat the above-mentioned diseases for which treatment is possible by increasing the expression level or hyperactivity of IGFBPs, which causes physiological changes in cells.
If there is a patient with an enhanced effect, an antibody that binds to the protein of the present invention is administered.
By doing so, the physiological action can be suppressed, and the protein of the present invention can directly treat the disease.
Diseases that can be treated symptomatically by suppressing the function of the protein of the present invention, even if they are not the cause of the disease
can also be prevented and treated by administering the antibody of the present invention. Medicines containing the antibody as an active ingredient are also suitable for use with the protein of the present invention described above in (6).
Any of the methods well known in the art of pharmaceuticals for the manufacture of pharmaceuticals containing the substance.
(12) A pharmaceutical preparation containing a compound obtained by the screening method of (8). A pharmaceutical preparation containing a compound obtained by the screening method of (8) can be provided as a pharmaceutical preparation produced by the method of (12). A pharmaceutical preparation containing a compound obtained by the screening method of (8) can be provided as a pharmaceutical preparation produced by the method of (8).
Compounds that promote or inhibit the translation process into proteins are safe and low-toxicity pharmaceutical compositions.
The compound obtained in (8) is useful as a pharmaceutical containing the protein of the present invention described in (6).
Manufactured by any method well known in the art of pharmaceutical science, according to the manufacturing method.
(13) Creation of knockout non-human animals using the DNA of the present invention A recombinant vector containing the DNA of the present invention can be used to create a knockout non-human animal of interest.
Embryonic stem cells from animals such as cattle, sheep, goats, pigs, horses, mice, and chickens (
In the embryonic stem cell, the protein of the present invention on the chromosome
The gene encoding the protein can be inserted into the host cell by a known homologous recombination technique [e.g., Nature,32
6, 6110, 295 (1987), Cell,51, 3, 503 (1987)
etc.] to prepare a mutant clone in which the sequence is inactivated or replaced with a desired sequence [e.g.
,Nature,350, 6315, 243 (1991)]. Mutation of embryonic stem cells
Using clones, chimeras are created by injecting animal fertilized eggs into blastocysts.
By using techniques such as the chimera method or the aggregation chimera method, it is possible to create a clone consisting of embryonic stem cell clones and normal cells.
A chimeric individual can be prepared. By crossing this chimeric individual with a normal individual,
By this, any mutation occurs in the gene encoding the protein of the present invention on the chromosome of cells in the whole body.
It is possible to obtain individuals with homologous chromosomes by crossbreeding these individuals.
From homozygotes with mutations in both the
A knockout non-human animal can be obtained as an individual in which expression is partially or completely suppressed.
It is also possible to introduce a mutation into any position in the gene encoding the protein of the present invention on the chromosome.
It is also possible to create knockout non-human animals by introducing
For example, a base may be substituted or deleted in the translation region of the gene encoding the protein of the present invention on the chromosome.
By introducing mutations such as deletions and insertions, the activity of the product can be altered.
It is also possible to introduce a similar mutation into the expression control region.
It is also possible to modify the level, timing, tissue specificity, etc. of expression.
By combining with the -loxP system, the expression time, expression site, expression amount, etc. can be more actively controlled.
It is also possible to regulate the expression of certain genes in specific regions of the brain.
An example of deleting a target gene only in the region using a promoter that is
l,87, 7, 1317 (1996)] or Cre-expressing adenovirus.
In this study, a target gene was deleted in an organ-specific manner at a desired time [Science
,278, 5335, (1997)] is known. Therefore, the gene encoding the protein of the present invention on the chromosome may also be subjected to such a procedure.
It is possible to control expression at any time and in any tissue, or to make any insertion, deletion, or substitution.
It is possible to create knockout non-human animals that have genes in the translation region or expression control region.
Knockout non-human animals can be produced at any time, to any extent, or in any location.
It is possible to induce symptoms of various diseases caused by the protein of the present invention. In this way, the knockout non-human animals of the present invention can induce symptoms of various diseases caused by the protein of the present invention.
This animal model is extremely useful in the treatment and prevention of various diseases.
It is extremely useful as a model for evaluating drugs, preventive medicines, functional foods, health foods, etc.
do.Best Mode for Carrying Out the InventionThe present invention will be described in more detail in the following examples, but the examples are not intended to be a mere representation of the present invention.
These are merely examples and are not intended to limit the scope of the present invention. Example 1: Preparation of a cDNA library derived from human small intestinal mucosal tissue A cDNA library was prepared from human small intestinal mucosal tissue using the method described in Molecular Cloning, 2nd Edition.
Furthermore, polyA(+)RNA was extracted using oligo-dT cellulose.
A was purified from each polyA(+)RNA by the oligocap method [Gen
e.138A cDNA library was constructed according to the method described in [O., 171 (1994)].
Ligo-cap linker (SEQ ID NO: 5) and Oligo dT pr
Using mer (SEQ ID NO: 6),41, 197 (19
96), Gene,200BAP was prepared according to the method described in [J., 149 (1997)].
(Bacterial Alkaline Phosphatase) treatment, T
AP (Tobacco Acid Phosphatase) treatment, RNA ligation
Gating, first strand cDNA synthesis and RNA removal were performed.
A sense primer (SEQ ID NO: 7) on the 3' end and an antisense primer on the 3' end
PCR (polymerase chain reaction) was carried out using two PCR primers (SEQ ID NO: 8).
The resulting DNA is converted into double-stranded cDNA by double chain reaction,SFI I. This PCR was performed using the commercially available Geneamp XL kit.
Using a PCR kit (Perkin Elmer), heat at 95°C for 5 minutes.
After treatment, the reaction cycle was repeated at 95°C for 1 minute, 58°C for 1 minute, and 72°C for 10 minutes.
This was repeated 12 times, and then the mixture was kept at 4°C.DraI
The vector pME18SFL3 (GeneBank AB0098
64, expression vector, 3392 bp) and the cDNA was ligated in a specific direction to form a recombinant
DNA is prepared, and the recombinant DNA is usedEscherichia coli A cDNA library was created by transforming DH5α.
For each plasmid DNA carried by the transformants, the 5' end of the cDNA and
The base sequence of the 3' end was determined using a DNA sequencing reagent (Dye Terminator
or Cycle Sequencing FS Ready Reactio
n Kit,dRhodamine Terminator Cycle Se
Quencing FS Ready Reaction Kit or BigD
ye Terminator Cycle Sequencing FS Re
A 100% ELISA kit (PE Biosystems) was used.
After performing the sequencing reaction according to the manual,
(ABI PRISM 377, manufactured by PE Biosystems)
Example 2: Identification of a novel protein belonging to the IGFBP superfamily The base sequences of each clone in the cDNA library were analyzed for protein amino acid sequence.
Acid database SWISSPROT or base sequence database GenBan
The known proteins belonging to the IGFBP superfamily registered in
Human IGFBP-1, human IGFBP-2, human IGFBP-3, human IGFBP
-4, human IGFBP-5, human IGFBP-6, human IGFBP-7, human I
10 molecules of human IGFBP-8, human IGFBP-9, or human IGFBP-10
Two clones, C-hs, which have homology with the amino acid sequences of these molecules, were used.
i13412 and C-kaia397 were selected. C-hsi1 was selected as SEQ ID NO: 1.
The amino acid sequence of 3412 is shown in SEQ ID NO: 3, and its base sequence is shown in SEQ ID NO: 2.
The amino acid sequence of C-kaia397 and its base sequence are shown in SEQ ID NO: 4. The amino acid sequence of C-hsi13412 was analyzed by homology analysis using BLAST2.
In this study, human insulin-like growth factor binding protein (IGF-binding protein)
IGFBP-7 (accession number: I52825) with a P value of 4.0 × 10
^-41Similarly, the amino acid sequence of C-kaia397 showed a significant homology of 38%.
The sequence was compared with that of insulin-like growth factor-binding protein (IGF-1) in a homology analysis using BLAST2.
Human IGFBP-7, a protein belonging to the fusion protein family, had a P value of 2 × 10^−
16showed a significant homology of 35%.
In the -family, it is important for binding to IGF-1, IGF-2, and insulin
There are 10 cysteine residues, of which 4 are cysteine residues.
A thrombin-like growth factor binding motif [GCGCCXXC (G: glycine residue, C: cysteine residue)]
The amino acid residues (X: any amino acid residue) are the main components of this family.
It is highly conserved among factors.
Amino acid sequences were compared with those of the insulin-like growth factor binding protein superfamily.
By comparison, the amino acid sequences shown in SEQ ID NOs: 1 and 2 also contain cis-
The position and number of tein residues are conserved, and the insulin-like growth factor binding motif is
The amino acid sequence of the N-terminal region was also highly conserved (Fig. 1).
The column also shows the binding of C-hsi13412 and C-kaia397 to insulin-like growth factors.
It was highly conserved among factors belonging to the synprotein superfamily (Figure 1). These results suggest that C-hsi13412 and C-kaia397 are insulin-specific.
It is a novel protein belonging to the insulin-like growth factor binding protein superfamily.
It has activity as a protein belonging to the glutamic acid-like growth factor binding protein superfamily.
It was revealed that IGFBP-1, IGFBP-2, and IGFBP-3 in Figure 1
GFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, IGFBP
-7 are human IGFBP-1, human IGFBP-2, and human IGFBP-
3. Human IGFBP-4, human IGFBP-5, human IGFBP-6, human IG
FBP-7. Also, C-hsi13412 and C-kaia397 and insulin-like growth factor
Conserved amino acid sequences among the binding protein superfamily are shown in white.
Cysteine residues that are highly conserved among families are marked with an asterisk. Based on the base sequence shown in SEQ ID NO: 3, the base sequence database GenBank
/EMBL/DDBJ was searched using BLAST2, and C-hsi1
It is thought to be an EST derived from the same gene as the base sequence encoding 3412 amino acids.
The GenBan search results for these ESTs were found to match two of the base sequences found in
The accession numbers are AI667734 and R30743.
Based on the base sequence shown in SEQ ID NO: 4, the base sequence database GenBank
/EMBL/DDBJ was searched using BLAST2, and C-kaia
It is thought to be an EST derived from the same gene as the base sequence encoding 397 amino acids.
The GenBank database for this EST was
The accession number is AI667734. These EST base sequences are
It does not cover the full length of C-hsi13412 and C-kaia397.
In addition, genes showing homology with the base sequences of each EST were searched for in GenBank and EM
Even when searching the BL and DDBJ databases using BLAST2,
It does not show significant homology to the phosphorylation-like growth factor binding protein superfamily.
C-hsi13412 and C-kaia397 were obtained for the first time by the present invention.
It was found to be a novel gene. A cDNA (sequence) encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 was identified.
A large intestine containing plasmid C-hsi13412 containing the entire base sequence of sequence number 3
Bacteria:Escherichia coli DH5α/pME18SFL3-C-
hsi13412 and a protein having the amino acid sequence represented by SEQ ID NO: 2
Plasmid C-kaia containing the cDNA encoding the
E. coli containing 397:Escherichia coliDH5α/pME1
8SFL3-C-kaia397 was obtained from FERM BP-7181 and F
ERM BP-7180, issued by the Independent Administrative Agency of Industrial Technology
National Institute of Biological Sciences, Patent Organism Deposit Center, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan
It is deposited at Chuo No. 6 (postal code 305-8566) (formerly known as the Ministry of International Trade and Industry
Agency of Industrial Science and Technology, National Institute of Bioscience and Human-Technology, Former address: 1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan
No. 3). Example 3: Analysis of the Nucleotide Sequences of C-hsi13412 and C-kaia397 C-hsi13412 shown in SEQ ID NO: 3 and C-kaia397 shown in SEQ ID NO: 4
Based on the base sequence of a397, the protein initiation codon prediction program ATGPr[
Bioinformatics,14, 384, (1998)] was used to start
The sequence around Don was analyzed. C-hsi13412 is located at positions 177 to 179.
The ATG at position 1089-1091 is the start codon, and the TAG at positions 1089-1091 is the stop codon.
The protein encoded by the ORF was found to consist of 304 amino acids.
C-kaia397 has an opening ATG at positions 926-928.
The start codon and TAG located at positions 1517 to 1519 were identified as the stop codon.
The protein encoded by the ORF was predicted to consist of 197 amino acids.
C-hsi13412 shown in sequence number 1 and C-kaia3 shown in sequence number 2
Based on the amino acid sequence encoded by 97, GENETYX-MAC7.3 (SO
A hydrophobic plate was prepared using a
As a result of preparing lots, the N-terminal regions of both genes contained hydrophobic residues characteristic of secretory proteins.
There were regions with high levels of C-hsi13412 and C-kaia397 (Figures 2 and 3). Example 4: Analysis of the genomic genes of C-hsi13412 and C-kaia397 The C-hsi13412 gene shown in SEQ ID NO: 3 and the C-kai gene shown in SEQ ID NO: 4 were analyzed.
Based on the base sequence of a397, GenomeWalker^TMkits (Cl
The DNA fragments were synthesized using the DNA fragments represented by SEQ ID NO: 3 and SEQ ID NO: 4 according to the attached protocol.
The nucleotide sequence information of the human genomic DNA region containing both of the nucleotide sequences was obtained.
In addition, the base sequence database GenBank/EMBL/DDBJ is used for BLAST.
2, and a search was performed to find a genome containing both the base sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4.
We obtained the base sequence information of genome clone AL133215. The obtained human genome gene sequence and the base sequence represented by SEQ ID NO: 3 and sequence number
From the comparison of the base sequence shown in SEQ ID NO: 4, the base sequence shown in SEQ ID NO: 3 is composed of five parts.
The base sequence represented by SEQ ID NO: 4 is divided into seven parts and is identical to the human 10th chromosome.
It was found that the gene is present in the genome over an area of approximately 7 kb (Fig. 4).
The protein having the amino acid sequence represented by SEQ ID NO: 1 is composed of five exons,
The protein with the amino acid sequence represented by the following is composed of seven exons:
The second and fourth exons were completely identical.
In C-kaia397, the beginning of the third exon and the fifth exon
The ends of the sequences are different from each other, so the difference in the sequences observed when comparing SEQ ID NOs: 1 and 2
It was found that there were differences in the amino acid sequence.
and a protein having the amino acid sequence represented by SEQ ID NO: 2.
Proteins have the unique characteristic of having different amino acid sequences.
These genes were found to be products of different splice forms derived from the same gene. Example 5: Organs expressing C-hsi13412 and C-kaia397 The sequence of C-hsi13412 matches a portion of the amino acid sequence encoding C-hsi13412.
The EST R30743 was isolated from fetal heart.
The nucleotide sequence of the 3'-terminal untranslated region of the cDNA clone encoding 13412 was also
In addition, BLAST searches the base sequence databases GenBank, EMBL, and DDBJ.
2, a search was performed to identify 11 base sequences that are thought to be ESTs derived from the same gene.
The GenBank accessions of these ESTs were
The license numbers are AI658885, W28828, AI377545, AA89
6981, AA688321, AW370932, AA126609, AQ43
1640, AQ742986, AI971557, AL030921.
Among them, AI658885, W28828, AI377545, AA89698
1, AA688321, and AW370932 are UniGene Hs. 1552
34. AI658885 and AA688321 are for prostate, W
28828 is the eye, AA896981 is the spleen, AW370932 is the mammary gland, AA12
6609 was isolated from the uterus of a pregnant woman. Therefore, it is possible that C-hsi13412 is present in fetal heart, prostate, eye, spleen, mammary gland, and pregnant woman.
Similarly, C-kaia397 was predicted to be expressed in the uterus of females.
The base sequence data is generated based on the base sequence of the 3'-end untranslated region of the cDNA clone to be downloaded.
The databases GenBank, EMBL, and DDBJ were searched using BLAST2.
As a result, it was found that the seven base sequences were identical to those thought to be ESTs derived from the same gene.
The GenBank accession numbers of these ESTs are AA
937577, AW080122, AA534966, AW374463, AQ
394346, F23541, F23538. AW080122 is the esophagus,
AA534966 was isolated from the large intestine, and F23538 from muscle.
It was estimated that ia397 is expressed in the esophagus, large intestine, and muscle. Example 6: C-hsi13412 and C-kaia39 using RT-PCR
Expression analysis of 7 Human organ polyA purchased from Clontech⁺4 μg of RNA, and
Cancer cell lines were isolated using the AGPC method [Analytical Biochemistry,
162, 156 (1987), Experimental Medicine,9, 1937 (1991)]
4 μg of the total RNA was used as a template, and the resulting mixture was purified using commercially available SUPER SCRIPT PREMIUM.
Plification System for first strand
cDNA Synthesis (GIBCO BRL) was used, and the attached manual
cDNA was synthesized according to the method described in [translate]. Human organ polyA⁺RNA from the brain, kidney, pancreas, pituitary gland, small intestine, and bone
Marrow, heart, liver, lungs, lymph nodes, mammary glands, placenta, prostate, skeletal muscles, spleen, stomach, testes
, thymus, thyroid, and uterus-derived polyA⁺RNA was used. As the cancer cell line, a T cell line (Jurkat (Riken Cell Bank)
k; RCB 086), Molt-3 (ATCC CRL-1552)], B
Cell strain [Namalwa KJM-1 (J. Biol. Chem.,268, 22
782, 1993), Daudi (ATCC CCL-213)], monocytic cells
strain [HL-60 (ATCC CCL-240)], vascular endothelial cell line [HUVEC
(Kurabo), IVEC (J. Cell. Physiol.,157, 41,
1993; N. T. L. FRANCE Co., Ltd.)], melanoma cell line [WM266-
4 (ATCC CRL-1676), WM115 (ATCC CRL-1675
)], neuroblastoma cell lines [SK-N-MC (ATCC HTB-10), SK
-N-SH (ATCC HTB-11)], lung cancer cell line [PC-9 (Immunobiological Laboratories)
Research Institute), HLC-1 (Osaka University Cancer Institute), QG90 (Aichi Cancer Center)],
Gastric cancer cell line [KATO-III (Immunobiological Research Institute)], pancreatic cancer cell line [Capa
n-1 (ATCC HTB-79), Capan-2 (ATCC HTB-80
)], colorectal cancer cell line [Colo 205 (ATCC CCL-222), SW1
116 (Aichi Cancer Center), LS180 (ATCC CCL-187)]
Next, PCR was performed using the synthesized cDNA as a template.
C-hsi13412 and C-kaia397, which are sequences specific to human β-actin
The synthesized cDNA was diluted 50-fold with sterile water using primers containing the nucleotide sequence.
Solution and Advantage^RcDNA PCR kit (clontec
After preparing a reaction solution using a standard method using a 100% ethanol solution (manufactured by Epson Corporation), the reaction was carried out at 94°C for 7 minutes, and then
32 cycles of 94°C for 1 minute and 68°C for 3 minutes were repeated, and finally 68°C
PCR was carried out under the conditions of 7 minutes at 20°C. The reaction solution was subjected to agarose gel electrophoresis.
By comparing the intensity of the DNA band specific to the primers used, expression
As a result of semi-quantitative comparison of the amount of C-hsi13412 and C-kaia397
It was found that the expression patterns differed between the two (Figure 5). In addition, in this PCR, the expression patterns of the nucleotide sequences represented by SEQ ID NO: 9 and SEQ ID NO: 10 were
The oligonucleotides were used as a primer set specific to C-hsi13412.
, oligonucleotides consisting of the base sequences represented by SEQ ID NO: 9 and SEQ ID NO: 11
as a primer set specific to C-kaia397, SEQ ID NO: 12 and SEQ ID NO:
The oligonucleotide consisting of the base sequence shown in sequence number 13 was used to ligate human β-actin.
The reaction was carried out using specific primer sets.
In the above RT-PCR reaction using the primers, a primer-specific DNA band was confirmed.
The size of the C-hsi13412, C-kaia397, and human β
For actin, the lengths were approximately 730 bp, 310 bp, and 600 bp, respectively. Furthermore, the differences in expression levels between human normal tissues and cancer tissues were investigated using single
Tumor Tissue Multi Sample Breast 1 (B
RT-PCR was carried out under the same conditions as above using a DNA fragment (manufactured by ioChain) as a template.
C-hsi13412 and C-kaia397 were expressed in both normal and cancer tissues.
However, a tendency for expression to decrease was observed in the breast cancer tissues examined (Part 1).
Figure 6). Example 7: Northern blotting of C-hsi13412 and C-k
Expression analysis of aia397 96 Dot Tumor/Normal Tissue Total RNA
A Dot Blot (BioChain) was used to detect C-hsi13412
The expression patterns of C-kaia397 and C-kaia397 in human cancer tissues and normal tissues are as follows:
The following was investigated. (1) Preparation of DNA probes The two clones prepared in Example 2, C-hsi13412 and C-kaia
397 as a template, and consisting of the base sequences represented by SEQ ID NO: 9 and SEQ ID NO: 14
Primer set, Ex Taq DNA Polymerase (TAKAR
A reaction mixture was prepared by a conventional method using a 10-µL ELISA kit manufactured by Company A, and then the mixture was reacted at 94°C for 10 minutes.
The reaction was repeated 35 times at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes.
Then, PCR was carried out at 72°C for 10 minutes.
The amplification product of about 300 bp was separated by electrophoresis, and C-hsi134
A DNA probe common to C-kaia397 and C-kaia397 was prepared. A primer set consisting of the base sequences represented by SEQ ID NO: 9 and SEQ ID NO: 14
are C-hsi13412 and C-kaia shown in SEQ ID NO: 3 and SEQ ID NO: 4.
The DNA probe was designed based on the common base sequence of the 397 translated region. (2) Preparation of labeled DNA probe The DNA probe prepared in (1) above was used for labeling using the random prime method [Anal. B
iochem.132, 6 (1983), Anal. Biochem.137,
266 (1984)], labeled with a radioisotope (hereinafter abbreviated as "RI")
75 ng of the DNA probe (1) above and [α-³²P] dCTP (NEN Co., Ltd.
5 μl, Oligolabelling Kit (Amersham Ph.
After preparing the reaction mixture according to the attached manual,
The reaction was carried out for 1 hour. After that, the unreacted labeled nucleotide was removed by Centri-Se.
p Spin Column (PRONCETON SEPARATIONS)
The RI was removed by centrifuging at 3,000 rpm for 2 minutes using a centrifuge (manufactured by RI Research Laboratories, Inc.).
A labeled DNA probe was prepared. (3) Hybridization The above-mentioned 96 Dot Tumor/Normal Tissue Total
The RNA Dot Blot membrane was immersed in prehybridization solution [6
×SSPE [6 × (0.15M NaCl, 8.65mM NaH₂P.O.₄・2
H₂0, 1.25 mM EDTA)], 2x Denhardt's solution (Nacalai Tesque, Inc.
(Manufactured by), 50% formamide, 0.5% SDS, 100 μg salmon sperm DNA (St
The plate was immersed in 5 ml of PBS (manufactured by Ratagene) and left to stand for 3 hours at 42°C with shaking.
During this time, the DNA prepared in (2) above was allowed to stand.
The labeled DNA probe was heat denatured at 94°C for 5 minutes to form a single strand.
After rehybridization, the membrane was transferred to a new prehybridization
The labeled DNA probe was immersed in 2 ml of the solution and denatured into a single strand.
x10⁶The mixture was left to stand for 20 hours with shaking at 42°C.
Hybridization was performed by placing the membrane in the hybridization solution. (4) Washing the membrane Remove the membrane from the hybridization solution and wash it with 0.1% SDS.
The solution was changed twice in 2×SSPE at 42°C for 10 minutes with shaking.
The membrane was washed by shaking it at 50°C for 30 minutes three times. (5) Signal detection After washing, the membrane was placed in an X-ray film cassette along with the X-ray film.
After leaving the plate at -80°C for 24 hours, autoradiography was performed.
Signal analysis revealed that normal tissues were mainly affected by the stomach, small intestine, gallbladder, thyroid gland, and kidneys.
Expression was observed in the bladder, thymus, prostate, and breast tissues.
Expression was observed in the pancreas, bladder, prostate, mammary gland, thyroid gland, and thymus.
Decreased expression was observed in cancer tissues compared to normal tissues in the bladder, small intestine, and thyroid gland.
However, no significant difference was observed in other tissues.
Furthermore, the expression patterns of C-hsi13412 and C-kaia397 vary depending on the tissue and cancer type.
Therefore, it is necessary to distinguish between C-hsi13412 and C-kaia397.
Expression analysis by Northern blotting using a new probe was also performed.
The importance of expression analysis that distinguishes between i13412 and C-kaia397 was suggested. Example 8: Expression of C-hsi13412 or C-kaia397 in animal cells as a host
(1) Expression of the Protein Encoded by (1) Construction of Recombinant Vector (i) pcDNA3-C-hsi13412 The clone C-hsi13412 prepared in Example 2 was used as a template to express the protein encoded by SEQ ID NO: 15
and a primer set consisting of the base sequence represented by SEQ ID NO: 16, KOD D
The reaction mixture was purified by a conventional method using NA Polymerase (manufactured by TOYOBO Co., Ltd.).
After preparation, the mixture was subjected to two cycles of 98°C for 15 seconds, 65°C for 2 seconds, and 74°C for 30 seconds.
PCR was carried out under the condition of repeating 5 cycles.
The primer set consisting of the base sequence represented by 16 is C-hsi13412
The resulting fragment was designed so that a FLAG marker peptide was added to the C-terminus.
The resulting amplification product of approximately 990 bp was cloned into pBluescript II SK-(Stra
(manufactured by Tagene)SamThe 5' side of C-hsi13412 was inserted into the I site of pBl
Script II SK-KpnI site side, 3 of C-hsi13412
' side is pBluescriptII SK-SacInsert so that it faces the I part
pSK-C-hsi13412 was created by this. Next,EcoRI-NotI fragment (900b
p) into the expression vector pcDNA3 (Invitrogen) for animal cells.Eco RI-NotI site to generate pcDNA3-C-hsi13412.
(ii) pcDNA3-C-kaia397 The clone C-kaia397 prepared in Example 2 was used as a template to generate the DNA fragments of SEQ ID NO: 15 and
and a primer set consisting of the base sequence represented by SEQ ID NO: 17, KOD DN
A reaction solution was prepared using A Polymerase (manufactured by TOYOBO Co., Ltd.) in a conventional manner.
After preparation, the mixture was subjected to 25 cycles of 98°C for 15 seconds, 65°C for 2 seconds, and 74°C for 30 seconds.
PCR was carried out under conditions of repeated cycles.
The primer set consisting of the base sequence shown in
The resulting approximately 6
The 20 bp amplification product was cloned into pBluescript II SK- (Strategy
ne Co., Ltd.)SamThe 5' side of C-kaia397 was inserted into pBluesc
iptII SK-KpnThe 3' side of C-kaia397 is pB1
Script II SK-SacBy inserting it so that it is on the I side
pSK-C-kaia397 was created using this. Next,EcoRI-NotI fragment (600 bp
) into the expression vector pcDNA3 (Invitrogen) for animal cells.EcoR
I-NotThe vector was inserted into the I site to create pcDNA3-C-kaia397. (2) Vector introduction into cells FuGENE was added to 100 μl of Opti-MEM (Gibco).^TM6 Transfer
Add 2 μl of injection reagent (Roche Diagnostics) and incubate at room temperature.
The mixture was left for 5 minutes. Then, the control plasmid pcDNA3, the above (1) (i
) C-hsi13412 expression plasmid pcDNA-3-C-hsi
13412, or the C-kaia397 expression plus prepared in (1)(ii) above
1 μg of midopcDNA3-C-kaia397 was added, and the mixture was incubated at room temperature for another 15 minutes.
During this time, 2 wells of DMEM medium containing 10% dFCS were added to the 6-well plate.
ml/well, and then COS-1 cells (Riken Cell Bank
;RCBO143) at 3 × 10⁵After leaving it for 15 minutes,
Sumido and FuGENE^TM6. Mixture of transfection reagents at 25 μl/well
Each well was added to a 6-well plate and incubated at 37°C in CO₂72 in the incubator
The transformants were cultured for 1 hour to obtain transient transformants.
The transformants prepared by introducing C-kaia3412 and C-kaia397 were then subjected to CO
S-1/mock strain, COS-1/C-hsi13412 strain, COS-1/C-
This strain is referred to as kaia397. (3) Confirmation of Expression (i) Preparation of Culture Supernatant The COS-1/mock strain and COS-1/C-hsi13 strain prepared in (2) above were used.
4 ml of culture supernatant of strain 412 and strain COS-1/C-kaia 397 was collected.
The solid matter was removed by centrifugation at 200 rpm for 5 minutes, and the culture supernatant was obtained.
The obtained culture supernatant was stored at -20°C and, if necessary, thawed and used in the following experiments.
(ii) Obtaining cell lysate The COS-1/mock strain and COS-1/C-hsi13 strain prepared in (2) above were used.
The cells of the 412 strain and the COS-1/C-kaia 397 strain were collected using a cell scraper (
After peeling from the 6-well plate using a phosphate buffer (Bakelite Co., Ltd.),
Buffered saline (pH 7.2) (hereinafter referred to as "PBS" (Gibber)
The mixture was suspended in 2 ml of PBS (manufactured by Co., Ltd.), centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed.
Next, 10 mL of a protease inhibitor cocktail for animal cells (Sigma) was added.
The cells were suspended in 1 ml of PBS containing 0 μl of PBS, and then disrupted by ultrasonication.
The cell lysate was stored at -20°C and used as needed.
After thawing, the cells were used in the following experiments. (iii) Detection by Western blotting 10 μl of the culture supernatant prepared in (3)(i) above or 10 μl of the culture supernatant prepared in (3)(ii) above was used.
5 μl of the prepared cell lysate was incubated with anti-FLA as a detection antibody in accordance with the procedure described in Example 14.
Western blotting using GM2 monoclonal antibody (Sigma)
was carried out. COS-1/C-hsi13412 strain and COS-1/C-kaia397 strain
The bands not observed in the COS-1/mock strain were observed in the cell lysate and culture supernatant.
Therefore, C-hsi1341 was detected in COS-1 cells.
Expression of the proteins encoded by 2 and C-kaia397 was confirmed (Figure 7).
Example 9: C-hsi13412 or C-kaia39 in animal cells as hosts
Expression of the protein encoded by 7 (2) (1) Construction of recombinant vectors (i) pAGE210-C-hsi13412 and pAGE210-C-hs
Preparation of i13412h Using pSK-C-hsi13412 prepared in Example 8 as a template,
and an oligonucleotide primer consisting of the base sequence represented by SEQ ID NO: 19
or consisting of the base sequences represented by SEQ ID NO: 18 and SEQ ID NO: 20
Oligonucleotide primer set and native pfu polymer
A reaction mixture was prepared using 9-HT20 RT-PCR (Stratagene) in a conventional manner.
Incubate at 4°C for 5 minutes, then at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes
The cycle between the two was repeated 25 times, and finally the reaction was carried out at 72°C for 7 minutes.
PCR was performed. The resulting amplification product of approximately 900 bp was then inserted into pBluescript II S
K-'sHindIII-XbaI site to give pSK-C-hsi13412
pSK-C-hsi13412c and pSK-C-hsi13412h were then constructed.
ofHindIII-XbaThe I fragment (900 bp) was cloned into the animal cell expression vector pA
GE210 [J. Biochem. ，101, 1307 (1987)]Hin dIII-XbaI site, and pAGE210-C-hsi13412 and
pAGE210-C-hsi13412h and pAGE210-C-kaia397 were constructed. (ii) pAGE210-C-kaia397 and pUC-C-kaia39
7h Using pSK-C-kaia397 prepared in Example 8 as a template,
and an oligonucleotide primer consisting of the base sequence represented by SEQ ID NO: 21
or an oligonucleotide set consisting of the base sequences represented by SEQ ID NO: 18 and SEQ ID NO: 22
PCR was carried out in the same manner as in (i) above using a set of oligonucleotide primers.
. Then, the resulting amplification product of approximately 600 bp was inserted into pBluescript II S
K-'sHindIII-XbaI site, and pSK-C-kaia397c
and pSK-C-kaia397h were constructed. Next, pSK-C-kaia397c and pSK-C-kaia397hH
indIII-XbaThe I fragment (600 bp) was cloned into pAGE210 and pUC18 (
(manufactured by Amersham Pharmacia)HindIII-XbaI site
and inserted into pAGE210-C-kaia397 and pUC-C-kaia3
97h was prepared. (2) Introduction of the vector into cells pAGE210-C-hsi1341 prepared in (1) (i) and (ii) above
2, pAGE210-C-hsi13412h, and pAGE210-C-ka
pUC-C-kaia397 and pUC-C-kaia397h were prepared by electroporation [C
ytotechnology,3, 133 (1990)] as follows:
CHO cells (Somatic Cell and Molecular Ge
netics,12, 555 (1986)). pAGE210, pAGE210-C-hsi13412, pAGE210-
C-hsi13412h, pAGE210-C-kaia397, pUC-C-
kaia397h isFspIt was cleaved with phenol-chloroform and linearized.
After extraction, ethanol precipitation was performed, and the resulting linearized plasmid was dissolved in TE solution.
On the other hand, CHO cells were lysed in 5% fetal bovine serum (Gibco), 0.09%
Sodium citrate (Gibco), 1% Penicillin-streptomycin (Gibco),
αMEM1900 medium (Gibco, hereinafter referred to as "medium A") was added.
CHO cells were passaged in K-PBS buffer (137 nmol/mL).
l/l potassium chloride, 2.7 nmol/l sodium chloride, 8.1 mmol/l
Disodium monohydrogen phosphate, 1.5 nmol/l monosodium dihydrogen phosphate, 4
8 × 10⁶Cells/ml
, 200 μl of cell suspension (1.6 × 10⁶(including cells) plus the linearization described above
The mixture was transferred to a cuvette (electrode distance 2 mm) and
A pulse voltage of 0.30 was applied using an enePulser II (BioRad) device.
The cuvette was placed on ice and allowed to stand.
After the incubation, the cell suspension in the cuvette was suspended in 15 ml of medium A, and 5 ml of the suspension was
The cells were divided into LASCO plates and cultured in a 5% CO₂ incubator at 37°C.
% fetal bovine serum, 0.09% sodium bicarbonate, 1% Penicillin-strep
mycin, 300 μg/ml hygromycin B (Gibco)
The medium was replaced with MEM2000 medium (hereinafter referred to as "medium B"), and the culture was continued.
The cells were subcultured while being diluted along the way, and approximately two weeks after gene transfection, hygromycin
A transformed cell line resistant to B was obtained. The transformed cell line was subcultured in B medium.
. Below, pAGE210, pAGE210-C-hsi13412, pAGE2
10-C-hsi13412h, pAGE210-C-kaia397, pUC
The transformed strains prepared by introducing -C-kaia397h were CHO/m
ock strain, CHO/C-hsi13412 strain, CHO/C-hsi13412h
strain, CHO/C-kaia397 strain, and CHO/C-kaia397h strain.
(3) Confirmation of expression CHO/mock strain and CHO/C-hsi13412 strain prepared in (2) above
, CHO/C-hsi13412h strain, CHO/C-kaia397 strain, CHO
The /C-kaia397h strain was statically cultured in medium B.
Cells seeded at one-fifth the cell density were 25 cm²The culture vessel was almost entirely filled with
The cells were grown until they became fuzzy, and the culture supernatant and cells were collected separately.
100 μl of the culture supernatant was subjected to organic solvent precipitation using acetone.
The supernatant was redissolved in 50 μl of 1 mmol/l Tris-HCl buffer (pH 7.5).
The cells were collected using a cell scraper and separated into 500 μl of 10
0 mmol/L Tris-HCl buffer (pH 7.5) and then ultrasonically disrupted.
The supernatant fraction and the cell fraction were each prepared at 11.25 μL.
After separating l by SDS polyacrylamide gel electrophoresis according to the Laemmli method,
Anti-C-hsi13412/C-kaia397 monoclonal antibody prepared in Example 13
Western blotting was performed using antibody KM2962 as described in Example 14.
CHO/C-hsi13412 strain, CHO/C-hsi13412h strain, CH
In either the O/C-kaia397 strain or the CHO/C-kaia397h strain
Both the culture supernatant and the cell fraction contained C-hsi13412 or C-kaia397.
The expression level was significantly higher in the cell fraction than in the culture supernatant of all the transformants.
Furthermore, the molecular weight of C expressed in COS-1 cells was higher.
In the case of strain OS-1/C-hsi13412 and strain COS-1/C-kaia397
The mobility was almost the same as that of C-hsi13412 and C-kaia397 in CHO cells.
Expression was confirmed (Figure 8). (4) Confirmation of glycosylation Next, 36 μl of the supernatant fraction or 36 μl of the cell fraction prepared in (3) above was added to 1
7.5 μl of 100% SDS was added and the mixture was heat denatured at 95° C. for 5 minutes. After denaturation, supernatant fraction 1
4.5 μl or 14.5 μl of the cell fraction was diluted with 5% Nonidet P-40 [N
P-40: Polyoxyethylene nonylphenyl ether (Sigma) 5 μl
25 mM/50 μl of Glycopeptidase F [N-glycosylation enzyme
(Takara Shuzo Co., Ltd.)] 2 μl, and 25 mM/50 μl of o-Glycoside
Add 1 μl of SE [o-glycosylation enzyme (manufactured by Roche Diagnostics)]
The reaction was carried out overnight at 37°C.
The reaction solution was added with 8 μl of mmol/L Tris-HCl buffer (pH 7.5).
After the reaction, 11.25 μl of the supernatant fraction or 11.25 μl of the cell fraction was used.
Western blotting analysis was performed in the same manner as in (3) above. The molecular weight changed due to the addition of the glycosylation enzyme, indicating that the transformation
C-hsi13412 and C-kaia397 produced by recombinant cells were
It was estimated that the protein encoded by C-hsi13412 was glycosylated (Figure 8). Example 10: Analysis of the protein encoded by C-hsi13412 using insect cells as a host
Expression Recombinant protein production in insect cells requires the use of a recombinant virus containing the target gene.
The production of a gene requires the following steps: (1) creating a cDNA encoding the target protein;
(2) the process of creating a special vector with baculovirus DNA and
The transfer vector is co-transfected into insect cells, and homologous recombination occurs.
(3) the process of producing and propagating the recombinant virus;
The process involves infecting cells with the virus and expressing the target protein.
(1) Preparation of a transfer vector The full-length C-hsi13412 (amino acids 1 to 304 of SEQ ID NO: 1)
Transfer vector pVL-C-hsi134 encoding the nucleotide sequence
12 to the plasmid pSK-C-hsi13412 described in Example 8.EcoRI
-BamThe HI fragment (900 bp) was cloned into the insect cell transfer vector pVL13
92 (manufactured by Pharmingen)EcoRI-BamInserted into the HI site
(2) Recombinant virus production The virus was cultured in ESF921 medium (Protein Expression).
Insect cells Sf9 (manufactured by Iwaki Glass Co., Ltd.) were transfected with linear baculovirus DNA (Baculovirus
Ludo baculovirus DNA (Pharmingen) and the above (1)
The transfer vectors prepared in the above were transfected into the cells using the lipofectin method [protein nucleic acid enzyme,37, 2701 (1992)] to create a recombinant baculovirus.
The details are as follows: pVL-C-hsi13412 and 15 ng of linear baculovirus DNA were mixed in 1
Dissolved in 2 μl of sterile distilled water, and then dissolved in 6 μl of Lipofectin and 6 μl of sterile distilled water.
The mixture was added to the Sf9 cells 1× and left at room temperature for 15 minutes.
10⁶The cells were suspended in 2 ml of ESF921 medium and placed in a cell culture plate with a diameter of 50 mm.
The plasmid DNA and linear baculoplasmic reticulum were then placed in a plastic petri dish.
The entire volume of the virus DNA and lipofectin mixture was added, and the cells were incubated at 27°C for 3 days.
After that, 1 ml of the culture supernatant containing the recombinant virus was collected.
1 ml of F921 medium was added, and the mixture was further cultured at 27°C for 3 days to obtain the recombinant virus-containing medium.
An additional 1.5 ml of culture supernatant containing the C-hsi13412 gene was collected. Next, the recombinant virus carrying the C-hsi13412 gene was amplified using the following procedure.
Sf9 cells were grown at 5 x 10 in 50 ml of ESF921 medium.⁵/ml
The mixture was then cultured in a 125 ml Erlenmeyer flask at 27°C with shaking at 125 rpm.
The cells were 2 x 10⁶When the recombinant virus was grown to an MOI of 1/ml,
The culture was then rotated at 1,200 rpm for 3 days.
Centrifuge for 10 minutes to remove cells and use the recombinant virus solution for protein expression.
The titer of the recombinant virus solution was measured using the following method. Sf9 cells 6 x 10⁵The pieces were suspended in 4 ml of ESF921 medium and
The cells were placed in a plastic petri dish for cell culture and left at room temperature for 1 hour to form a petri dish.
The supernatant was removed and 400 μl of ESF921 medium and 100 μl of ESF921 medium were added.
100 μl of the recombinant virus solution diluted with
The medium was removed, and 5 ml of 1% low-melting-point agarose [agar plaque agarose (
1 ml of sterile 5% agar plaque containing medium (manufactured by Pharmingen)
Agarose solution and 4 ml of TMN-FH insect medium (Far
The mixture was mixed with 100% ethanol (manufactured by Mingen Co., Ltd.) and kept at 42°C.
After leaving it at room temperature for 15 minutes, wrap vinyl tape around the dish to prevent it from drying out.
The petri dish was then placed in a sealable plastic container and cultured at 27°C for 5 days.
1 ml of PBS containing 0.01% neutral red was added to the dish.
After culturing for another day, the number of plaques that appeared was counted and the number was 0.5 to 2 × 10⁸/m
It was confirmed that a recombinant virus solution of 100 μg was prepared. (3) Protein Expression 5 x 10 Sf9 cells were cultured.⁵/ml in 100 ml of ESF921 medium
The culture was then cultured in a 250 ml Erlenmeyer flask at 27°C with shaking at 125 rpm.
3-4 x 10 cells⁶When the cells grew to 3 × 10⁷To become individuals
25 ml of ESF921 medium was added to the bottom surface of 182 cm²
The cells were allowed to stand at room temperature for 1 hour to allow them to adhere, and then the medium was removed.
The recombinant virus carrying the C-hsi13412 gene was then transfected at an MOI of 5.
ESF921 medium was added to make 10 ml, and the mixture was infected at room temperature for 1 hour.
Then, 20 ml of ESF921 medium was added, and the cells were cultured at 27°C for 3 days.
The target recombinant protein was expressed. Cell suspensions were prepared by infecting cells with recombinant viruses carrying the C-hsi13412 gene.
5 μl of the suspension and 20 μl of the culture supernatant were used as samples, and the anti-FL antibody was
Western blotting using AG M2 monoclonal antibody (Sigma)
We performed a screening test. The cell suspension and culture supernatant of cells infected with C-hsi13412 contained
A band with a molecular weight of approximately 33 kDa was detected, which was not observed in uninfected cells.
(Figure 9). Example 11 Protein encoded by C-hsi13412 or C-kaia397
Preparation of antigens for antibody production against Amino acid sequences of proteins encoded by C-hsi13412 and C-kaia397
The sequence was analyzed to identify the highly hydrophilic portion, N-terminus, C-terminus, secondary structure, turn structure, and
Among the parts with random coil structure, partial sequences that are considered suitable as antigens were selected.
Compound 1 (a polypeptide having the amino acid sequence represented by SEQ ID NO: 23)
Compound 2 (a polypeptide having the amino acid sequence represented by SEQ ID NO:24)
and Compound 3 (a polypeptide having the amino acid sequence represented by SEQ ID NO:25).
) was selected. The abbreviations for the amino acids and their protecting groups used in this invention are as follows:
IUPAC-IUB Joint Commission on Names
Mission on Biochemical Nomenclature
) recommendations [European Journal of Biochemistry (European Journal of Biochemistry)]
American Journal of Biochemistry, Vol. 138, p. 9
(1984)]. Unless otherwise specified, the following abbreviations represent the corresponding amino acids listed below: Ala: L-alanine Asn: L-asparagine Asp: L-aspartic acid Asx: L-aspartic acid or L-asparagine Arg: L-arginine Cys: L-cysteine Glu: L-glutamic acid Glx: L-glutamic acid Gly: glycine Leu: L-leucine Phe: L-phenylalanine Pro: L-proline Ser: L-serine Thr: L-threonine Trp: L-tryptophan Tyr: L-tyrosine Met: L-methionine Val: L-valine The following abbreviations represent the protecting groups and side chain-protected amino acids of the corresponding amino acids listed below.
Fmoc: 9-Fluorenylmethyloxycarbonyl tBu: t-Butyl Trt: Trityl Boc: t-Butyloxycarbonyl Pmc: 2,2,5,7,8-Pentamethylchroman-6-sulfonyl Fmoc-Arg(Pmc)-OH: N^α-9-Fluorenylmethyloxycarbonyl
Bonyl-N^g-2,2,5,7,8-pentamethylchroman-6-sulfonyl-
L-Arginine Fmoc-Asn(Trt)-OH:N^α-9-Fluorenylmethyloxycarbonyl
Bonyl-N^gamma-Trityl-L-asparagine Fmoc-Asp(OtBu)-OH:N^α-9-fluorenylmethyloxycarbonyl
Carbonyl-L-aspartic acid-β-t-butyl ester Fmoc-Cys(Trt)-OH:N^α-9-Fluorenylmethyloxycarbonyl
Bornyl-S-trityl-L-cysteine Fmoc-Glu(OtBu)-OH:N^α-9-fluorenylmethyloxycarbonyl
Carbonyl-L-glutamic acid-γ-t-butyl ester Fmoc-Ser(tBu)-OH:N^α-9-Fluorenylmethyloxycarbonyl
Bornyl-O-t-butyl-L-serine Fmoc-Thr(tBu)-OH:N^α-9-Fluorenylmethyloxycarbonyl
Bornyl-O-t-butyl-L-threonine Fmoc-Trp(Boc)-OH:N^α-9-Fluorenylmethyloxycarbonyl
Bonyl-N^ind-t-Butyloxycarbonyl-L-tryptophan Fmoc-Tyr(tBu)-OH:N^α-9-Fluorenylmethyloxycarbonyl
Bornyl-O-t-butyl-L-tyrosine The following abbreviations represent the corresponding reaction solvents, reaction reagents, etc. HBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetrahydrobenzoyl
Trimethyluronium hexafluorophosphate HOBt: N-hydroxybenzotriazole DIEA: diisopropylethylamine DMF: N,N-dimethylformamide TFA: trifluoroacetic acid In the following examples, the physicochemical properties of the compounds were measured by the following methods. Mass spectrometry was performed using a JEOL JMS-HX110A by FAB-MS.
Alternatively, a Bruker mass spectrometer REFLEX may be used for MALDI-TOFMS analysis.
Amino acid analysis was performed according to the method of Cohen, S.A. et al.
Analytical Biochemistry
story),222Hydrolysis was carried out in hydrochloric acid vapor.
The hydrolysis was carried out at 110°C for 20 hours, and the amino acid composition of the hydrolyzed product was determined by Waters Accu
Tag (Waters AccQ-Tag) amino acid analyzer (Waters)
(1) Compound 1 (peptide H-C consisting of the amino acid sequence represented by SEQ ID NO: 23)
ys-Arg-Pro-Ser-Pro-Gly-Pro-Asp-Tyr-L
eu-Arg-Arg-Gly-Trp-Met-Arg-Leu-NH₂)of
Synthesis Support resin (Rink amide) bound to 18 μmol of Fmoc-NH
30 mg of MBHA resin (Novabiochem) was added to an automatic synthesizer (Shimadzu)
Place in a reaction vessel (manufactured by the manufacturer), add 600 μl of DMF, stir for 3 minutes, and then drain the solution.
After that, the following operations were carried out according to the Shimadzu synthesis program: (a) 500 μl of 30% piperidine-DMF solution was added, and the mixture was stirred for 4 minutes.
The solution was then drained, and this procedure was repeated once more. (b) The support resin was washed with 600 μl of DMF for 1 minute, the solution was then drained, and this procedure was repeated once more.
This procedure was repeated five times. (c) Fmoc-Leu-OH (165 μmol), HBTU (165 μmol)
), HOBt monohydrate (165 μmol) and DIEA (330 μmol)
in DMF (660 μl) for 3 minutes, and the resulting solution was added to the resin to form a mixture.
The mixture was stirred for 60 minutes, and the solution was then drained. (d) The support resin was washed with 900 μl of DMF for 1 minute, after which the solution was drained. This was repeated five times.
This was repeated. Through these steps, Fmoc-Leu-NH was synthesized on the support. Next, after steps (a) and (b), Fmoc-Arg(Pmc) was synthesized in step (c).
-OH, followed by a washing step (d), to give Fmoc-Arg(
Fmoc-Met-OH, Fmoc-Trp (B
oc)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pmc)-OH,
Fmoc-Arg(Pmc)-OH, Fmoc-Leu-OH, Fmoc-Ty
r(tBu)-OH, Fmoc-Asp(OtBU)-OH, Fmoc-Pro
-OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Ser
(tBu)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pmc)-O
H, Fmoc-Cys(Trt)-OH, and repeating (a) to (d).
After that, the reaction mixture was deprotected and washed in the steps (a) and (b), and then the reaction mixture was deprotected in methanol, butyl ether, and the like.
and dried under reduced pressure for 12 hours to obtain the carrier resin to which the side chain-protected peptide was bound.
To this was added TFA (8) containing 2-methylindole at a concentration of 5 mg/mL.
2.5%), thioanisole (5%), water (5%), ethyl methyl sulfide (
3%), 1,2-ethanedithiol (2.5%) and thiophenol (2%)
1 ml of a mixed solution consisting of
In both cases, the peptide was cleaved from the resin. After filtering off the resin, the resulting solution was diluted with ether.
Approximately 10 ml of the solution was added, and the resulting precipitate was collected by centrifugation and decantation.
This crude product was dissolved in an aqueous acetic acid solution.
After dissolution, the solution was loaded onto a reverse-phase silica gel cartridge (YMC Dispo SPE C1
8) to adsorb the peptide, and then eluted with 0.1% TFA, 15% acetonitrile water
After washing with the solution, the compound was eluted with 0.1% TFA and 25% acetonitrile aqueous solution.
A fraction containing Compound 1 was obtained. This was lyophilized to obtain 28.9 mg of Compound 1. Mass spectrometry [FABMS]: m/z = 2058.9 (M+H⁺) Amino acid analysis: Asx 1.0 (1), Ser 1.0 (1), Gly 2.0
(2), Arg 3.9 (4), Pro 2.9 (3), Tyr 1.0 (1)
, Met 1.0 (1), Leu 2.1 (2), Cys 1.2 (1) (2) Compound 2 (peptide H-C consisting of the amino acid sequence represented by SEQ ID NO: 24)
ys-Arg-Pro-Pro-Ala-Phe-Thr-Pro-Arg-A
la-Pro-Asp-Apg-Val-Thr-Ser-Ile-OH)
Synthesis Support resin (Wang resin, No. 14.4 μmol) bound to Fmoc-Ile
Starting material was 30 mg of Fmoc-
Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Va
l-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Asp(OtBu)
-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Arg
(Pmc)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-O
H, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Pro-O
H, Fmoc-Pro-OH, Fmoc-Apg(Pmc)-OH, Fmoc-
After sequential condensation of Cys(Trt)-OH, the Fmoc group was removed, washed, and dried.
The support resin to which the side-chain protected peptide was bound was obtained.
), thioanisole (5%), water (5%), ethyl methyl sulfide (3%),
Composed of 1,2-ethanedithiol (2.5%) and thiophenol (2%)
Add 1 ml of the mixed solution, leave it at room temperature for 8 hours, remove the side chain protecting groups, and
The peptide was cleaved from the oil. After filtering off the resin, the resulting solution was added with about 10 ml of ether.
The precipitate formed was collected by centrifugation and decantation, and crude pellets were obtained.
29.2 mg of the peptide was obtained. This crude product was dissolved in an aqueous acetic acid solution,
The sample was passed through a reversed-phase silica gel cartridge (YMC Dispo SPE C18).
The peptides were adsorbed onto the column and washed with 0.1% TFA and 10% acetonitrile aqueous solution.
After washing, the column was eluted with 0.1% TFA and 25% acetonitrile in water.
This fraction was freeze-dried to obtain 24.5 mg of compound 2. Mass spectrometry [TOFMS]: m/z = 1883.6 (M+H⁺) Amino acid analysis: Asx 1.0 (1), Ser 1.0 (1), Arg 3.0
(3), Thr 1.9 (2), Ala 2.1 (2), Pro 4.0 (4)
, Val 1.0 (1), Ile 1.0 (1), Phe 1.0 (1), Cy
s 1.3(1) (3) Compound 3 (peptide H-C consisting of the amino acid sequence represented by SEQ ID NO: 25)
ys-Asn-Leu-Val-Pro-Glu-Glu-Glu-Ala-G
lu-Ser-Glu-Glu-Asn-Asp-Asp-Tyr-Tyr-O
Synthesis of Fmoc-Tyr(tBu), 18 μmol of which is bound to a support resin (SynPr
Starting with 30 mg of oPep Resin (manufactured by Shimadzu Corporation), (1)
Similarly, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(Ot
Bu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtB
u)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu
)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, F
moc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, F
moc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Va
l-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fm
After sequential condensation of oc-Cys(Trt)-OH, the Fmoc group was removed, washed, and dried.
After drying, the support resin to which the side chain protected peptide was bound was obtained.
%), thioanisole (5%), and 1,2-ethanedithiol (5%)
The mixture was added with 1 ml of the above mixture and left at room temperature for 2 hours to remove the side chain protecting groups.
The peptide was cleaved from the resin. After filtering off the resin, the resulting solution was added with about 1 mL of ether.
0 ml was added, and the resulting precipitate was collected by centrifugation and decantation.
32.5 mg of crude peptide was obtained. This crude product was purified by acetic acid, dithiothreitol, and thiazolinone.
The mixture was dissolved in a mixture of ethanol, DMF, and water, and the mixture was then placed on a reversed-phase silica gel packed column.
The peptides were adsorbed onto a column (YMC Dispo SPE C18).
After washing with 0.1% TFA and 10% acetonitrile aqueous solution,
Elution with 25% aqueous acetonitrile gave a fraction containing Compound 3.
The mixture was dried to obtain 17.1 mg of compound 3. Mass spectrometry [TOFMS]: m/z = 2148 (M + H⁺) Amino acid analysis: Asx 4.0 (4), Glx 6.2 (6), Ser 1.0
(1), Ala 1.1 (1), Pro 1.0 (1), Tyr 1.8 (2)
, Val 0.9 (1), Leu 0.9 (1), Cys 1.0 (1) Example 12 Protein encoded by C-hsi13412 or C-kaia397
Preparation of polyclonal antibodies that recognize (1) Preparation of immunogen Compounds 2 and 3 obtained in Example 11 were prepared as follows to enhance immunogenicity:
A conjugate with KLH (manufactured by Calbiochem) was prepared by the method described above, and used as an immunogen.
That is, KLH was dissolved in PBS to prepare a 10 mg/ml solution, and 1/10 volume of
Amount of 25 mg/ml MBS [N-(m-Maleimidobenzoylox
y) succinimide; manufactured by Nacalai Tesque Inc.] was added dropwise and stirred for 30 minutes.
The reaction was carried out on a Sephadex G-25 column (Amersham) previously equilibrated with PBS.
KLH-MB2. was obtained by removing free MBS using a method similar to that described above (manufactured by Sham Pharmacia).
5 mg of peptide dissolved in 0.1 M sodium phosphate buffer (pH 7.0)
The mixture was mixed with 1 mg of acetone and stirred at room temperature for 3 hours. After the reaction, the mixture was dialyzed against PBS.
The compound was used as an immunogen. (2) Immunization of Animals and Preparation of Antisera 100 μL of the KLH conjugates of Compound 2 and Compound 3 prepared in (1) above were used.
g of each was mixed with aluminum hydroxide adjuvant [Antibodies-AL
Laboratory Manual, Cold Spring Harbor
Laboratory, p99, 1988] 2 mg and pertussis vaccine (Chiba Prefecture
Serum Institute) 1 x 10⁹One female BALB/c mouse aged 6-8 weeks was placed in a 1000-well tube with the cells.
Two weeks after administration, 100 μg of each KLH conjugate was administered once a week.
The mice were administered once a day, a total of four times. Blood was collected from the carotid artery of the mice, and antisera were prepared. (3) Western blotting using immunized mouse antisera The COS-1/C-hsi13412 and COS-1/C strains obtained in Example 8 were
5 μl of cell lysate from strain C-kaia397 was added to C-hsi13412 and C-kai
a397 was used as a sample of the protein encoded by the a397 gene, and the results were analyzed according to the procedure described in Example 14.
As a detection antibody, 500 μl of the immunized mouse antiserum obtained in (2) above was used.
Estanblotting was performed. Mouse antisera obtained using Compound 2 and Compound 3 as immunogens were C-
Specifically recognizes the protein encoded by hsi13412 and C-kaia397
It was confirmed that this can be done (Figure 10). Example 13 Proteins encoded by C-hsi13412 or C-kaia397
Monoclonal antibody recognizing K (1) Preparation of immunogen Compound 1 obtained in Example 11 was purified by the same method as in Example 12(1).
A conjugate with KLH was prepared and used as an immunogen. (2) Immunization of Animals and Preparation of Antibody-Producing Cells 100 μg of the KLH conjugate of Compound 1 prepared in (1) above was added to each
Aluminum hydroxide adjuvant [Antibodies-A Laboratories
ory Manual, Cold Spring Harbor Labora
tory, p. 99, 1988] 2 mg and pertussis vaccine (manufactured by Chiba Prefecture Serum Research Institute)
) 1 x 10⁹The antibody was administered together with the cells to three female BALB/c mice aged 6 to 8 weeks.
Starting two weeks after administration, 100 μg of each KLH conjugate was administered once a week for a total of four times.
Blood was collected from the carotid artery of the mice, and the serum antibody titer was measured using the following oxygen immunization test.
The spleen was removed from mice that showed sufficient antibody titers three days after the final immunization.
The removed spleen was cultured in MEM (Minimum Essential Medium)
) medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and then the cells were chopped into small pieces, loosened with tweezers, and centrifuged (250×
The resulting precipitate was diluted with Tris-ammonium chloride buffer (pH
7.6) was added and treated for 1 to 2 minutes to remove red blood cells.
The precipitated fraction (cell fraction) was washed three times with MEM medium and used for cell fusion. (3) Enzyme-linked immunosorbent assay (binding ELISA) Compound 1 obtained in Example 11 was used as the antigen for the assay.
The preparation method used was a conjugation with a crosslinking agent (hereinafter referred to as "THY").
Instead of MBS, SMCC [4-(N-Maleimidomethyl)-
cyclohexane-1-carboxylic acid N-hydro
The same procedures were repeated except that xysuccinimido ester (Sigma) was used.
The same procedure as in 12 was carried out.
The conjugate (10 μg/ml) prepared as above was dispensed at 50 μl/well.
The plate was washed and then incubated with 1% bovine serum albumin.
BSA/Dulbecco's phosphate buffer solution
e buffered saline (PBS) was added at 100 ml/well,
The plate was left at room temperature for 1 hour to block any remaining active groups. After leaving the plate for 1 hour, the 1% BSA/PBS was discarded and the immunized mouse antiserum was applied to the plate.
50 μl/well of culture supernatant of monoclonal antibody or purified monoclonal antibody
The plate was then filled with 0.05% polyoxyethylene (
20) Sorbitan monolaurate [trade name: Span 20 (ICI Twee trademark)]
n20 equivalent: manufactured by Wako Pure Chemical Industries, Ltd.) / PBS (hereinafter abbreviated as "Tween-PBS"
After washing with ABTS substrate solution (2,2-azinobis(3-ethylbenzothiazolinone)
Ammonium benzoate (6-sulfonate), 1 mM ABTS/0.1 M citrate buffer
The color was developed by adding PH2 (pH 4.2) and measuring the absorbance at OD 415 nm.
Measurement was performed using a chromatograph (Emax; manufactured by Molecular Devices).
(4) Preparation of mouse myeloma cells 8-azaguanine-resistant mouse myeloma cell line P3X63Ag8U.1 [P3-U
1: purchased from ATCC] was mixed with normal medium [10% fetal calf serum (hereinafter referred to as "FCS"].
By culturing in RPMI medium supplemented with 2 × 10⁷Take more than 100 cells
The mouse splenocytes obtained in (2) above and the myeloma cells obtained in (4) above were used as parent lines for cell fusion. (5) Hybridoma Production The mouse splenocytes obtained in (2) above and the myeloma cells obtained in (4) above were used as parent lines for cell fusion.
The mixture was mixed at a ratio of 0:1 and centrifuged (250×g, 5 minutes).
After thoroughly loosening the cells in the sludge fraction, the cells were stirred at 37°C in polyethylene glycol.
2 g of col-1000 (PEG-1000), 2 ml of MEM medium, and dimethyl sulfoxide
A mixture of 0.7 ml of sulfoxide was added to 10⁸Add 0.5 ml per mouse splenocyte
After adding 1 ml of MEM medium to the suspension every 1 to 2 minutes,
The total volume was adjusted to 50 ml. The suspension was centrifuged (900 rpm, 5 minutes), and the cells in the resulting precipitate were collected.
After gently loosening the cells, the cells were gently removed by suction and removal with a measuring pipette.
HAT medium [RPMI medium supplemented with 10% FCS with HAT/Media Supp
Medium containing 100 ml of cereal (manufactured by Roche Diagnostics)
The suspension was then placed in a 96-well culture plate at 200 μl/well.
Dispense into aliquots and incubate in 5% CO₂The cells were cultured in an incubator at 37°C for 10 to 14 days. After the culture, the culture supernatant was examined by the enzyme immunoassay described above (3) to detect antigen peptides.
Select wells that react with the peptide but not with the control peptide, and measure the
The cells were cloned twice by limiting dilution method, and anti-C-hsi1341
A hybridoma producing the 2/C-kaia397 monoclonal antibody was established.
As a result, two types of anti-C-hsi13412/C-ka antibodies were detected using Compound 1 as an antigen.
ia397 monoclonal antibodies KM2961 and KM2962 were obtained.
2961 and KM2962 reacted specifically with the antigen peptide Compound 1.
(Figure 14). Anti-C-hsi13412/C-kaia397 monoclonal antibody KM296
The hybridoma cell lines producing KM2962 and KM1 were obtained on May 24, 2001.
National Institute of Advanced Industrial Science and Technology, Patent Organism Deposit Center (Ibaraki Prefecture, Japan)
Tsukuba City Higashi 1-1-1 Central 6: Postal Code 305-8566)
Deposited as FERM BP-7603 and FERM BP-7604
(6) Purification of monoclonal antibodies The monoclonal antibodies obtained in (5) above were purified by injecting them into pristane-treated 8-week-old nude female mice (BALB/c).
The hybridoma strains were cultured at 5-20 × 10⁶Cells were injected intraperitoneally per animal.
After 10 to 21 days, the hybridomas became ascites tumors, resulting in mice with ascites.
Ascites fluid was collected from the stool (1-8 ml/animal). The ascites fluid was centrifuged (1200 x g, 5 minutes) to remove solids. Purified Ig
The G monoclonal antibody was isolated by caprylic acid precipitation (Antibodies-A La
laboratory manual, Cold Spring Harbor L
The product was obtained by purification using the method described in the laboratory (1988).
The subclass of the clonal antibody was determined by ELISA using a subclass typing kit.
As a result, KM2961 was determined to be IgG1 and KM2962 was determined to be IgG3. Example 14: C-hsi13412 and KM2962 using KM2961 and KM2962
Western blotting analysis of the protein encoded by C-kaia397
COS-1/mock strain and COS-1/C-hsi134 strain prepared in Example 8
2.5 μl/lane of cell lysate from strains 12 and COS-1/C-kaia397
The culture supernatant was subjected to 15% SDS-polyacrylamide gel electrophoresis at 10 μl/lane.
Antibodies-A Laboratory Manual, Col.
d Spring Harbor Laboratory, 1988)
Thereafter, the mixture was blotted onto a PVDF membrane (manufactured by Millipore) according to a conventional method.
Blocking was performed with skim milk-PBS (hereinafter referred to as "blocking solution").
After rinsing, the membrane was coated with anti-C-hsi13412/C-kaia397 monoclonal antibody.
KM2961 or KM2962 was added to a concentration of 2 μg/ml.
The membrane was thoroughly washed with Tween-TBS and then incubated with a secondary antibody.
Peroxidase-labeled rabbit anti-mouse immunoglobulin diluted 2,000 times as a control.
Detection was performed using brin (DAKO). Anti-C-hsi13412/C-kaia397 monoclonal antibody KM296
1 and KM2962 were obtained from Ch-h cells expressed in COS-1 cells as described in Example 8.
Specifically recognizes the protein encoded by si13412 or C-kaia397.
It was found that (Figure 11). Example 15 Proteins encoded by C-hsi13412 or C-kaia397
Binding of IGF to proteins encoded by C-hsi13412 and C-kaia397
In order to examine the binding of COS-1/C-hsi13412 obtained in Example 8,
strain, COS-1/C-kaia397, and COS-1/m as a control.
A cell lysate of the ock strain was used for Far Western (Molecular Cloning).
ning A Laboratory Manual Third Editi
on Cold Spring Harbor Laboratory, Tampa
The study was conducted using the COS-1/mock strain, COS-1/C-hsi13412 strain, and COS-1
/C-kaia397 strain cell lysate was used in 5 μl/lane as in Example 14
SDS-PAGE was performed as described, transferred to a PVDF membrane, and blocked.
Then, human IGF-I (Peprotech) diluted with blocking solution was added.
2 μg/ml, human IGF-II (Peprotech) 2 μg/ml, and
Alternatively, 2 μg/ml of human insulin (Sigma) was enclosed together with the membrane.
The membrane was incubated overnight at room temperature.
antibody (Upstate Biotechnology), anti-human IGF-
II monoclonal antibody (Upstate Biotechnology)
, or anti-human insulin polyclonal antibody (Santa Cruz)
was added to the membrane at a concentration of 2 μg/ml, and the membrane was left standing at room temperature for 1 hour.
After thorough washing with TBS, the secondary antibody was diluted 2,000 times with peroxidase.
Detection was performed using enzyme-labeled rabbit anti-mouse immunoglobulin (DAKO). For anti-IGF-I antibody and anti-IGF-II antibody, C-hsi134123 C
-The molecular weights of the proteins encoded by kaia397 are approximately 33 kDa and approximately 2
A 2 kDa band was detected, but when anti-insulin antibody was used, these bands were not detected.
No band was detected. Therefore, C-hsi13412 or C-kaia3
The protein encoded by 97 binds to IGF-I and IGF-II but not to insulin.
It was shown that this protein may not bind to urin (Figure 12). Example 16: Proteins encoded by C-hsi13412 or C-kaia397
Effects on cancer cells Investigating the effects of C-hsi13412 and C-kaia397 on cancer cell proliferation
Therefore, the COS-1/mock strain and COS-1/C-hsi1 strain obtained in Example 8 were used.
The culture supernatants of the 3412 and COS-1/C-kaia 397 strains were used to investigate the effects of human colon cancer.
Effect on IGF-dependent proliferation of cell line HT-29 (ATCC HTB-38)
The effects were investigated as follows: Bovine serum albumin (Gibco) 200 μg/ml, human transferrin
(Gibco) 10 μg/ml, penicillin (Gibco) 50 units/ml
D-MEM/F supplemented with 50 μg/ml of streptomycin (Gibco)
1 × 10⁵HT-2 prepared at 100 cells/ml
The cells were seeded in a 96-well plate at 50 μl/well and incubated at 37°C in CO₂
The mixture was cultured in an incubator for 3 hours.
Human IGF-II (manufactured by Peprotech) was added at a final concentration of
The concentration of the β-glucan was 0.1 ng/ml.
strain OS-1/C-hsi13412 or strain COS-1/C-kaia397
The culture supernatant was added to the well at 5 μl/well, and the plate was further incubated at 37°C in CO₂in
After culturing in an incubator for 3 days, the viable cell count was determined using Cell Prolifera.
tion Reagent WST-1 (Roche Diagnostics)
) was measured. COS-1/C-hsi13412 and COS-1/C-kaia397
The culture supernatant of the strain inhibits the proliferation of IGF-dependent human colon cancer cell line HT-29 cells.
It was shown that this occurs (Figure 13).Industrial ApplicabilityAccording to the present invention, a novel insulin-like growth factor binding protein and a gene encoding the protein are
DNA that recognizes the protein, an antibody that recognizes the protein, and a method for determining a disease associated with the protein.
Diagnostic, preventive, and therapeutic drugs can be provided. "Sequence Listing Free Text" SEQ ID NO:5 - Description of Artificial Sequence: Synthetic RNA SEQ ID NO:6 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:7 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:8 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:9 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:10 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:11 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:12 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:13 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:14 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:15 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:16 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:17 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:18 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:19 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:20 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:21 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:22 - Description of Artificial Sequence: Synthetic DNA SEQ ID NO:23 - Description of Artificial Sequence: Synthetic Peptide SEQ ID NO:24 - Description of Artificial Sequence: Synthetic Peptide SEQ ID NO:25 - Description of Artificial Sequence: Synthetic Peptide

[Sequence List]

[Brief explanation of the drawings]

Figure 1: Comparison of the amino acid sequences of C-hsi13412 and C-kaia397 with IGFBP family factors. Figure 2: Hydrophobicity plot of the protein encoded by C-hsi13412. Figure 3: Hydrophobicity plot of the protein encoded by C-kaia397. Figure 4: Comparison of C-hsi13412 and C-kaia397 with the human genome sequence. Figure 5: Expression of C-hsi13412 and C-kaia397 by RT-PCR. (A) shows the results of expression analysis in each tissue, and (B) shows the results of expression analysis in each cell line. Figure 6: Expression of C-hsi13412 and C-kaia397 by RT-PCR. Figure 7: Expression of C-hsi13412 and C-kaia397 using COS-1 cells as a host.
Figure 8 shows the expression and glycosylation of proteins encoded by C-hsi13412 and C-kaia397 in CHO cells as hosts. In the figure, V indicates CHO/mo
ck strain, C-kaia397 strain CHO/C-kaia397 strain, C-kai
a397h is the CHO/C-kaia397h strain, and C-hsi13412 is the CH
O/C-hsi13412 strain, and C-hsi13412h strain was CHO/C-hsi
The graph shows the 13412h strain. In addition, + indicates the case where a glycosylase was added, and - indicates the case where 100 mmol/L Tris-HCl buffer (pH 7.0) was used instead of the glycosylase.
. 5) was added, and n indicates the case where the enzyme reaction was not performed. Figure 9 shows the expression of proteins encoded by C-hsi13412 and C-kaia397 in insect cells as hosts. Mock represents the control. Figure 10 shows the analysis by Western blotting of polyclonal antibodies that recognize proteins encoded by C-hsi13412 or C-kaia397. In the figure, the blot on the right shows the results using antibodies obtained using compound 2 as an immunizing antigen, and the blot on the left shows the results using antibodies obtained using compound 3 as an immunizing antigen. Figure 11 shows the detection by Western blotting of proteins encoded by C-hsi13412 or C-kaia397 using KM2961 and KM2962. Mock represents the control. Figure 12 shows the results of far-western analysis of C-hsi13412 or C-kaia397.
Figure 13 shows the binding of the protein encoded by C-hsi13412 to IGF. Mock indicates the control. Figure 14 shows the binding of C-hsi13412 to IGF-dependent proliferation of HT-29 cells.
or the effect of the protein encoded by C-kaia397.
14 shows the binding activity of KM2961 and KM2962 to antigen peptides. In the figure, KM2961 is the culture supernatant obtained by culturing a hybridoma producing monoclonal antibody KM2961 for 3 days;
The culture supernatant of a hybridoma producing M2961 cultured for 3 days is shown.
The open columns show the reaction between the plates not coated with the antigen peptide and these culture supernatants, and the black and hatched columns show the reaction between the plates coated with the antigen peptide and these culture supernatants.

───────────────────────────────────────────────────────── Continued from the front page (51) Int.Cl. ⁷ Identification Symbol FI A61K 48/00 A61P 1/00 A61P 1/00 1/04 1/04 1/16 1/16 3/06 3/06 3/10 3/10 5/00 5/00 7/00 7/00 9/00 9/00 9/04 9/04 9/10 9/10 9/12 9/12 11/00 11/00 11/06 11/06 13/08 13/08 13/12 13/12 15/00 15/00 17/00 17/00 19/08 19/08 19/10 19/10 21/04 21/04 27/02 27/02 29/00 29/00 101 101 31/00 31/00 31/04 31/04 31/12 31/12 35/00 35/00 35/02 35/02 37/02 37/02 37/08 37/08 C07K 14/47 C07K 14/47 16/18 16/18 C12N 1/19 C12N 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/02 15/02 1/68 A C12P 21/02 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/53 D G01N 33/15 M 33/50 33/566 33/53 C12P 21/08 C12N 5/00 A 33/566 B // C12P 21/08 15/00 C A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA(BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP(GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CO, CR, CU, CZ, DE , DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, I N, IS, JP, KE, KG, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, P T, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor: Norio Ota, Kyowa Hakko Kogyo Co., Ltd., Tokyo Research Laboratory, 3-6-6 Asahi-cho, Machida-shi, Tokyo (72) Inventor: Akiko Furuya, Kyowa Hakko Kogyo Co., Ltd., Tokyo Research Laboratory, 3-6-6 Asahi-cho, Machida-shi, Tokyo (72) Inventor: Takeshi Shibata, Irving, Texas, USA 5311 North MacArthur Boulevard #2027 (72) Inventor: Kobayashi Yuki, 3-6-6 Asahi-cho, Machida-shi, Tokyo, Japan Kyowa ▲ Hakko Kogyo Co., Ltd. Tokyo Research Laboratory (72) Inventor: Takebayashi Miho, 3-6-6 Asahi-cho, Machida-shi, Tokyo, Japan Kyowa ▲ Hakko Kogyo Co., Ltd. Tokyo Research Laboratory (72) Inventor: Nakamura Yusuke, 1-17-33 Azamino, Aoba-ku, Yokohama-shi, Kanagawa-ken (72) Inventor: Kanno Sumio, 4-8-13 Minami-Ogikubo, Suginami-ku, Tokyo (Note) This publication is based on the gazette published internationally by the International Bureau of Patents (WIPO). The effect of the international publication of the Japanese patent application (Japanese utility model registration application) related to this publication arises pursuant to Article 184-10, Paragraph 1 of the Patent Act (Article 48-13, Paragraph 2 of the Utility Model Act), and is unrelated to this publication.

Claims (60)

[Claims] 1. An insulin-like growth factor binding protein consisting of the amino acid sequence represented by SEQ ID NO:1. 2. An insulin-like growth factor binding protein consisting of the amino acid sequence represented by SEQ ID NO:2. [Claim 3] A protein according to either (A) or (B) below: (A) a protein consisting of an amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted or added, and having substantially the same activity as the protein according to claim 1; (B) a protein consisting of an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and having substantially the same activity as the protein according to claim 2. [Claim 4] A protein according to either (A) or (B) below: (A) a protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 1, and having substantially the same activity as the protein according to claim 1; (B) a protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2, and having substantially the same activity as the protein according to claim 2. 5. A protein according to the following (A) or (B): (A) a protein comprising amino acid numbers 75 to 82 in the amino acid sequence represented by SEQ ID NO: 1;
(B) a protein comprising an amino acid sequence containing the amino acid sequence of amino acid numbers 75 to 82 in the amino acid sequence represented by SEQ ID NO: 2, and having substantially the same activity as the protein of claim 1;
A protein comprising an amino acid sequence containing the amino acid sequence of claim 2 and having substantially the same activity as the protein of claim 2. [Claim 6] A protein according to the following (A) or (B): (A) A protein consisting of the amino acid sequence of claim 5 (A) in which one or more amino acids have been deleted, substituted or added, and having substantially the same activity as the protein of claim 1; (B) A protein consisting of the amino acid sequence of claim 5 (B) in which one or more amino acids have been deleted, substituted or added, and having substantially the same activity as the protein of claim 2. [Claim 7] A protein according to the following (A) or (B): (A) a protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence of claim 5 (A) and having substantially the same activity as the protein of claim 1; (B) a protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence of claim 5 (B) and having substantially the same activity as the protein of claim 2. 8. A protein according to the following (A) or (B): (A) a protein comprising amino acid numbers 171 to 371 in the amino acid sequence represented by SEQ ID NO: 1;
(B) a protein according to (A) of claim 5, which comprises a partial amino acid sequence containing the amino acid sequence of amino acid number 171 to 172 in the amino acid sequence represented by SEQ ID NO: 2, and has substantially the same activity as the protein according to claim 1;
The protein according to (B) of claim 5, which comprises a partial amino acid sequence containing the 97th amino acid sequence and has substantially the same activity as the protein according to claim 2. [Claim 9] A protein according to the following (A) or (B): (A) A protein consisting of the amino acid sequence of claim 8 (A) in which one or more amino acids have been deleted, substituted or added, and having substantially the same activity as the protein of claim 1; (B) A protein consisting of the amino acid sequence of claim 8 (B) in which one or more amino acids have been deleted, substituted or added, and having substantially the same activity as the protein of claim 2. [Claim 10] A protein according to the following (A) or (B): (A) a protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence of claim 8 (A) and having substantially the same activity as the protein of claim 1; (B) a protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence of claim 8 (B) and having substantially the same activity as the protein of claim 2. 11. A polypeptide according to either (A) or (B) below: (A) a polypeptide consisting of an amino acid sequence of at least 5 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 1; (B) a polypeptide consisting of an amino acid sequence of at least 5 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2. 12. A polypeptide according to either (A) or (B) below: (A) a polypeptide consisting of an amino acid sequence of at least five consecutive amino acids at amino acid positions 171 to 304 in the amino acid sequence represented by SEQ ID NO: 1; (B) a polypeptide consisting of an amino acid sequence of at least five consecutive amino acids at amino acid positions 171 to 197 in the amino acid sequence represented by SEQ ID NO: 2. 13. A DNA encoding the protein or polypeptide according to any one of claims 1 to 12. 14. A DNA comprising the coding region of the base sequence represented by SEQ ID NO:3. 15. A DNA comprising the coding region of the base sequence represented by SEQ ID NO:4. [Claim 16] A DNA set forth in either (A) or (B) below: (A) A DNA that hybridizes under stringent conditions with DNA encoding the protein of claim 1, DNA encoding the protein or polypeptide of any one of claims 3 to 12 (A), or DNA comprising the coding region of the nucleotide sequence represented by SEQ ID NO: 3, and that encodes a protein having substantially the same activity as the protein of claim 1; (B) A DNA that hybridizes under stringent conditions with DNA encoding the protein of claim 2, DNA encoding the protein or polypeptide of any one of claims 3 to 12 (B), or DNA comprising the coding region of the nucleotide sequence represented by SEQ ID NO: 4, and that encodes a protein having substantially the same activity as the protein of claim 2. 17. A recombinant DNA obtained by inserting the DNA according to any one of claims 13 to 16 into a vector. 18. A transformant obtained by introducing the recombinant DNA according to claim 13 into a host cell. 19. The transformant according to claim 18, wherein the host cell is a cell selected from the group consisting of a bacterium, a yeast, an insect cell, a plant cell, or an animal cell. 20. A transformant FERM B containing the DNA according to claim 14.
P-7181. 21. A transformant FERM B containing the DNA according to claim 15.
P-7180. [Claim 22] A method for producing a protein or polypeptide described in any one of claims 1 to 12, characterized by culturing a transformant selected from the transformants described in any one of claims 18 to 21 in a medium, producing and accumulating the protein or polypeptide described in any one of claims 1 to 12 in the culture, and recovering the protein or polypeptide from the culture. 23. An antibody that recognizes the protein according to any one of claims 1 to 10. 24. An antibody that recognizes the protein according to (A) of claim 1 or any one of claims 3 to 10, and the protein according to (A) of claim 2 or any one of claims 3 to 10.
An antibody that does not recognize the protein described in B). 25. An antibody that recognizes the protein according to (B) of claim 2 or any one of claims 3 to 10, and (1) or any one of (3) to (10)
An antibody that does not recognize the protein described in A). [Claim 26] A method for producing an antibody that specifically recognizes the protein described in claim 1 or any one of claims 3 to 10 (A), using a polypeptide described in claim 11 (A) or claim 12 (A) as an immunogen. [Claim 27] A method for producing an antibody that specifically recognizes the protein described in claim 2 or any one of claims 3 to 10 (B), using a polypeptide described in claim 11 (B) or claim 12 (B) as an immunogen. 28. An antibody that specifically recognizes the protein according to (A) of claim 1 or any one of claims 3 to 10, obtained by the method according to claim 26. 29. An antibody that specifically recognizes the protein according to (B) of claim 2 or any one of claims 3 to 10, obtained by the method of claim 27. 30. A monoclonal antibody produced by the hybridoma having deposit number FERM BP-7603. 31. A monoclonal antibody produced by the hybridoma having deposit number FERM BP-7604. 32. A method for immunologically detecting or quantifying the protein according to any one of claims 1 to 10, which comprises using the antibody according to any one of claims 23 to 25 and 28 to 31. 33. An oligonucleotide having a sequence consisting of 5 to 60 consecutive bases in the base sequence of the DNA described in any one of claims 13 to 16, an oligonucleotide having a sequence complementary to said oligonucleotide, or a derivative of said oligonucleotide. [Claim 34] A method for detecting or quantifying the expression of a gene encoding a protein described in any one of claims 1 to 10, which comprises performing hybridization using a DNA described in any one of claims 13 to 16, or an oligonucleotide or oligonucleotide derivative described in claim 33, as a probe. 35. A method for detecting or quantifying the expression of a gene encoding a protein described in any one of claims 1 to 10, comprising carrying out a polymerase chain reaction using the oligonucleotide or oligonucleotide derivative described in claim 33 as a primer. [Claim 36] A method for detecting a mutation in a gene encoding a protein described in any one of claims 1 to 10, comprising hybridization using a DNA described in any one of claims 13 to 16, or an oligonucleotide or oligonucleotide derivative described in claim 33. 37. A method for producing a polymerase chain reaction using the oligonucleotide or oligonucleotide derivative according to claim 33.
A method for detecting a mutation in a gene encoding the protein according to any one of claims 1 to 10. 38. A method for determining a disease according to (A) or (B) below: (A) A method for determining a disease, comprising detecting a mutation in DNA encoding the protein according to any one of claims 1 to 10 or measuring the expression level, and comparing the results with those of a healthy person; (B) A method for determining a disease, comprising detecting a mutation in the protein according to any one of claims 1 to 10 or measuring the expression level, and comparing the results with those of a healthy person. 39. A method for determining a disease according to (A) or (B) below: (A) A method for determining a disease, comprising comparing a mutation or expression level of a DNA encoding the protein according to (A) of claim 1 or any one of claims 3 to 10 with a mutation or expression level of a DNA encoding the protein according to (B) of claim 2 or any one of claims 3 to 10. (B) A method for determining a disease, comprising detecting a mutation or measuring the expression level of a protein according to any one of claims 1 to 10, and comparing the mutation or expression level of the protein according to (A) of claim 1 or any one of claims 3 to 10 with the mutation or expression level of the protein according to (B) of claim 2 or any one of claims 3 to 10. [Claim 40] A method for determining the disease described in claim 38 or 39, wherein the disease is a disease accompanied by abnormal cell proliferation, a disease accompanied by vascular disorders, a disease accompanied by abnormal bone metabolism, a disease accompanied by impaired action of insulin-like growth factor or growth hormone, a disease accompanied by abnormal differentiation and proliferation of smooth muscle cells, a disease accompanied by abnormal differentiation and proliferation of skeletal muscle cells, a disease accompanied by abnormal gastric acid secretion, or an inflammatory disease accompanied by abnormal lymphocyte infiltration. 41. The method according to claim 41, wherein the disease accompanied by abnormal cell proliferation is acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumor or solid tumor, and the disease accompanied by vascular disorder is myocardial infarction, cerebral infarction, peripheral vascular occlusion, angina pectoris, hypertension,
Hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic encephalopathy, heart failure,
The method of claim 40, wherein the disease is congestion or choroidal circulatory disorder, the disease accompanied by abnormal bone metabolism is osteoporosis, the disease accompanied by impaired insulin-like growth factor or growth hormone action is dwarfism, acromegaly or childhood chronic renal failure, the disease accompanied by abnormal smooth muscle cell differentiation and proliferation is arteriosclerosis, bronchial disease or restenosis, the disease accompanied by abnormal skeletal muscle cell differentiation and proliferation is myasthenia gravis, the disease accompanied by abnormal gastric acid secretion is gastric ulcer, and the inflammatory disease accompanied by abnormal lymphocyte infiltration is microbial infection, chronic hepatitis B, chronic rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, allergy, atopy, asthma, hay fever, airway hyperresponsiveness or autoimmune disease. 42. The method according to any one of claims 38 to 41, wherein the method is the method described in (A) or (B) below: (A) the detection method according to claim 36 or 37; (B) the detection or quantification method according to claim 32, 34 or 35. 43. A pharmaceutical comprising the protein or polypeptide according to any one of claims 1 to 12. 44. A medicine comprising the DNA according to any one of claims 13 to 16, or the oligonucleotide or oligonucleotide derivative according to claim 33. 45. The pharmaceutical according to claim 44, wherein the pharmaceutical is a vector for gene prevention or a vector for gene therapy. 46. A pharmaceutical comprising the antibody according to any one of claims 23 to 25 and 28 to 31. [Claim 47] A pharmaceutical described in claims 43 to 46, which is a diagnostic, preventive or therapeutic agent for diseases accompanied by abnormal cell proliferation, diseases accompanied by vascular disorders, diseases accompanied by abnormal bone metabolism, diseases accompanied by disorders of insulin-like growth factor or growth hormone action, diseases accompanied by abnormal smooth muscle cell differentiation and proliferation, diseases accompanied by abnormal skeletal muscle cell differentiation and proliferation, diseases accompanied by abnormal gastric acid secretion or inflammatory diseases accompanied by abnormal lymphocyte infiltration. 48. The method according to claim 48, wherein the disease associated with abnormal cell proliferation is acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumor, or solid tumor;
The diseases accompanied by vascular disorders are myocardial infarction, cerebral infarction, peripheral vascular occlusion, angina pectoris, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic encephalopathy, heart failure, congestion, or choroidal circulatory disorder, and the diseases accompanied by abnormalities in bone metabolism are osteoporosis,
Diseases accompanied by impaired insulin-like growth factor or growth hormone action include dwarfism, acromegaly, or childhood chronic renal failure; diseases accompanied by abnormal smooth muscle cell differentiation and proliferation include arteriosclerosis, bronchial disease, or restenosis; diseases accompanied by abnormal skeletal muscle cell differentiation and proliferation include myasthenia gravis; diseases accompanied by abnormal gastric acid secretion include gastric ulcers; inflammatory diseases accompanied by abnormal lymphocyte infiltration include microbial infections, chronic hepatitis B, and rheumatoid arthritis;
The pharmaceutical composition of claim 47, which is for treating sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, allergy, atopy, asthma, hay fever, airway hyperresponsiveness or an autoimmune disease. [Claim 49] A method for screening for a compound that controls the expression level of a protein described in any one of claims 1 to 10, characterized by: (i) comparing the expression level of the protein described in any one of claims 1 to 10 in a cell that expresses the protein with (ii) the expression level of the protein when the cell that expresses the protein is contacted with a test sample; and selecting a compound that controls the expression level of the protein described in any one of claims 1 to 10 from the test sample. 50. A method according to any one of claims 1 to 10, comprising comparing (i) the function of a cell expressing the protein according to any one of claims 1 to 10 with (ii) the function of the cell when the cell expressing the protein is contacted with a test sample, and selecting a compound that controls the function of the protein according to any one of claims 1 to 10 from the test sample.
A method for screening for a compound that controls the function of the protein according to any one of the above items. [Claim 51] A method for screening for a compound that controls the expression of a gene encoding a protein described in any one of claims 1 to 10, characterized by contacting a test sample with a transformant transformed with a plasmid containing DNA in which a reporter gene is linked downstream of a region that controls the transcription of the gene encoding the protein described in any one of claims 1 to 10, and selecting a compound that controls the expression of the gene encoding the protein described in claims 1 to 10 from the test sample. 52. A method for determining the expression level of a protein according to any one of claims 1 to 10 in a cell expressing the protein, the method comprising contacting the cell with a test sample, and determining (i) the expression level of the protein according to (A) of claim 1 or any one of claims 3 to 10 in the cell;
(ii) A method for screening a compound that controls the splicing of a gene encoding a protein described in any one of claims 1 to 10 from a test sample, by comparing the expression level of the protein described in (B) of claim 2 or any one of claims 3 to 10 in the cell with the expression level of the protein described in (B) of claim 2 or any one of claims 3 to 10 in the test sample. 53. A compound or a pharmacologically acceptable salt thereof obtained by the screening method according to any one of claims 49 to 52. 54. A medicine comprising the compound according to claim 53 or a pharmaceutically acceptable salt thereof. 55. The pharmaceutical according to claim 54, which is a preventive or therapeutic drug for diseases accompanied by abnormal cell proliferation, diseases accompanied by vascular disorders, diseases accompanied by abnormal bone metabolism, diseases accompanied by impaired action of insulin-like growth factor or growth hormone, diseases accompanied by abnormal differentiation and proliferation of smooth muscle cells, diseases accompanied by abnormal differentiation and proliferation of skeletal muscle cells, diseases accompanied by abnormal gastric acid secretion, or inflammatory diseases accompanied by abnormal lymphocyte infiltration. 56. The method of claim 56, wherein the disease associated with abnormal cell proliferation is acute myeloid leukemia, breast cancer, prostate cancer, colon cancer, liver cancer, myeloma, uterine leiomyoma, malignant tumor, or solid tumor;
The diseases accompanied by vascular disorders are myocardial infarction, cerebral infarction, peripheral vascular occlusion, angina pectoris, hypertension, hyperlipidemia, diabetes, diabetic retinopathy, glomerulonephritis, arteriosclerosis, thrombosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, ischemic heart disease, ischemic encephalopathy, heart failure, congestion, or choroidal circulatory disorder, and the diseases accompanied by abnormalities in bone metabolism are osteoporosis,
Diseases accompanied by impaired insulin-like growth factor or growth hormone action include dwarfism, acromegaly, or childhood chronic renal failure; diseases accompanied by abnormal smooth muscle cell differentiation and proliferation include arteriosclerosis, bronchial disease, or restenosis; diseases accompanied by abnormal skeletal muscle cell differentiation and proliferation include myasthenia gravis; diseases accompanied by abnormal gastric acid secretion include gastric ulcers; inflammatory diseases accompanied by abnormal lymphocyte infiltration include microbial infections, chronic hepatitis B, and rheumatoid arthritis;
56. The pharmaceutical composition of claim 55, wherein the treatment is sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, nephritis, traumatic injury, inflammatory bowel disease, allergy, atopy, asthma, hay fever, airway hyperresponsiveness or an autoimmune disease. 57. A method for screening for a substance that specifically binds to a protein or polypeptide described in any one of claims 1 to 12, comprising contacting a test sample with the protein or polypeptide described in any one of claims 1 to 12 and selecting a protein from the test sample that specifically binds to the protein or polypeptide. 58. A method according to claim 1 obtained by the screening method of claim 55.
13. A substance that specifically binds to the protein or polypeptide described in any one of claims 1 to 12. 59. A knockout non-human animal in which the expression of the gene encoding the protein according to any one of claims 1 to 10 is reduced or completely suppressed. 60. A knockout non-human animal in which the function of the protein according to any one of claims 1 to 10 is reduced or completely suppressed.