U.S. patent application number 10/476985 was filed with the patent office on 2005-07-21 for polypeptide serving as angiogenic marker and dna thereof.
Invention is credited to Ohta, Hideki, Sato, Yasufumi, Sonoda, Hikaru.
Application Number | 20050158719 10/476985 |
Document ID | / |
Family ID | 18983518 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158719 |
Kind Code |
A1 |
Sato, Yasufumi ; et
al. |
July 21, 2005 |
Polypeptide serving as angiogenic marker and dna thereof
Abstract
A marker for neovascularization, vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease and cancer, the method of detection
of the marker and a diagnosis kit of the diseases are provided.
Additionally, therapeutic agents of the diseases are provided. The
expression of KIAA1036 is enhanced in ovarian cancer and large
bowel cancer and KIAA1036 expresses in umbilical vein endothelial
cell and inhibits DNA synthesis in the cells, cell migrating and
lumen formation. Therefore KIAA1036 is useful as a marker for
neovascularization, vascular disease, inflammatory disease,
entoptic neovascular disease, reproductive system disease, central
nervous system disease or cancer. Additionally, KIAA1036 is useful
for screening of agonists, antagonists, DNA synthesis inhibitors,
cell migrating inhibitors and neovascular inhibitors. The
substances obtained by the screening, KIAA1036 and the antibodies
are useful as therapeutic agents the above disease.
Inventors: |
Sato, Yasufumi; (Sendai-shi,
JP) ; Sonoda, Hikaru; (Toyonaka-shi, JP) ;
Ohta, Hideki; (Toyonaka-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
18983518 |
Appl. No.: |
10/476985 |
Filed: |
January 28, 2005 |
PCT Filed: |
April 26, 2002 |
PCT NO: |
PCT/JP02/04251 |
Current U.S.
Class: |
435/6.14 ;
435/7.23; 530/350; 536/23.2 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
29/00 20180101; A61P 15/00 20180101; A61P 43/00 20180101; A61P
35/00 20180101; C12Q 1/6883 20130101; A61K 38/00 20130101; A61P
25/00 20180101; C12Q 2600/158 20130101; C12N 2799/026 20130101;
C07K 14/47 20130101; A61P 27/02 20180101 |
Class at
Publication: |
435/006 ;
435/007.23; 530/350; 536/023.2 |
International
Class: |
C12Q 001/68; G01N
033/574; C07H 021/04; C07K 014/705 |
Foreign Application Data
Date |
Code |
Application Number |
May 7, 2001 |
JP |
2001-136179 |
Claims
1. A marker for neovascularization, vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease or cancer, which contains a
polynucleotide having a base sequence as set forth in A of 386-C of
1480 of SEQ ID NO: 1 or fragment thereof.
2. A marker for neovascularization, vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease or cancer, which contains a
polynucleotide hybridizing under a stringent condition with a
polynucleotide having a base sequence as set forth in C of 1-C of
1480 of SEQ ID NO: 1 or fragment thereof.
3. A marker of claim 1, which contains a polynucleotide having a
base sequence as set forth in C of 1-C of 1480 of SEQ ID NO: 1 or
fragment thereof.
4. A marker of claim 1, which contains a polynucleotide having a
base sequence as set forth in C of 1-C of 5481 of SEQ ID NO: 1 or
fragment thereof.
5. A marker for neovascularization, vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease or cancer, which contains a
polypeptide comprising an amino acid sequence as set forth in Met
of 1-Val of 365 of SEQ ID NO: 2 or fragment thereof.
6. A marker for neovascularization, vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease or cancer, which contains a
polypeptide wherein 1 or more amino acid residue(s) has a
mutation(s) selected from deletion, substitution and addition in an
amino acid sequence as set forth in Met of 1-Val of 365 of SEQ ID
NO: 2 or fragment thereof.
7. A marker of claim 5, which contains a polypeptide consisting of
an amino acid sequence as set forth in Met of 1-Val of 365 of SEQ
ID NO: 2 or fragment thereof.
8. A method of detection of a marker of any one of claims 1-7 from
a sample derived from human.
9. A method of detection or assay of a marker of any one of claims
1-4, which comprises the following processes and using a
polynucleotide having a base sequence as set forth in A of 386-C of
1480 of SEQ ID NO: 1 or fragment thereof, (a) a process that the
polynucleotide and a sample are contacted, and (b) a process that a
polynucleotide binding with the polynucleotide is detected.
10. A method of detection or assay of a marker of any one of claims
1-4, which comprises the following processes and using a
polynucleotide having a base sequence as set forth in A of 386-C of
1480 of SEQ ID NO: 1 or fragment thereof, (a) a process that cDNA
is prepared from a sample, and (b) a process that cDNA is amplified
with the polynucleotide fragment and detected.
11. A method of detection or assay of a marker of any one of claims
5-7, which comprises the following processes and using an antibody
recognizing a polypeptide comprising an amino acid sequence as set
forth in Met of 1-Val of 365 of SEQ ID NO: 2 or fragment thereof,
(a) a process that the antibody and a sample are contacted, and (b)
a process that the binding between the antibody and a polypeptide
is detected.
12. A method of detection of a disease relating to a marker of any
one of claims 1-7, which comprises using a method of any one of
claims 9-11.
13. A method of detection of claim 12, wherein the disease is
vascular disease, inflammatory disease, entoptic neovascular
disease, reproductive system disease, central nervous system
disease or cancer.
14. A diagnostic kit of a disease relating to a marker of any one
of claims 1-7, which comprises using a method of any one of claims
9-11.
15. A diagnostic kit of claim 14, wherein the disease is vascular
disease, inflammatory disease, entoptic neovascular disease,
reproductive system disease, central nervous system disease or
cancer.
16. A screening method of a binding substance of a marker of any
one of claims 5-7, which comprises the following processes, (a) a
process that a marker of any one of claims 5-7 and a subject are
contacted, and (b) a process that the binding between the marker
and the subject is detected.
17. A kit for screening of a binding substance of claim 16, which
contains a polypeptide comprising an amino acid sequence as set
forth in SEQ ID NO: 2 or fragment thereof.
18. A screening method of a binding activity regulatory substance
which regulates a binding between a marker of any one of claims 5-7
and a binding substance of the marker, which comprises the
following processes, (a) a process that a marker of any one of
claims 5-7 and a binding substance of the marker are contacted in
the presence or absence of a subject, and (b) a process that a
binding activity of the marker and the binding substance is
compared in the presence or absence of a subject.
19. A kit for screening of a binding activity regulatory substance
of claim 18, which contains a polypeptide comprising an amino acid
sequence as set forth in SEQ ID NO: 2 or fragment thereof.
20. A screening method of an expression regulatory substance of a
marker of any one of claims 1-7, which comprises the following
processes, (a) a process that a cell, which can express a marker of
any one of claims 1-7, and a subject are contacted, and (b) a
process that a change of an expression level of the marker is
detected by using a method of any one of claims 9-11.
21. A kit for screening of an expression regulatory substance of a
marker of any one of claims 1-7, which comprises using the method
of claim 20.
22. A DNA synthesis inhibitor containing a polypeptide comprising
an amino acid sequence as set forth in Met of 1-Val of 365 of SEQ
ID NO: 2 or fragment thereof.
23. A cell migrating activity inhibitor containing a polypeptide
comprising an amino acid sequence as set forth in Met of 1-Val of
365 of SEQ ID NO: 2 or fragment thereof.
24. A pharmaceutical composition which contains at least one
selected from a DNA synthesis inhibitor of claim 22, a cell
migrating activity inhibitor of claim 23, a polynucleotide having a
base sequence as set forth in SEQ ID NO: 1 or fragment thereof, a
human gene therapeutic expression vector containing polynucleotide
having a base sequence as set forth in SEQ ID NO: 1 or fragment
thereof, a polypeptide comprising an amino acid sequence as set
forth in SEQ ID NO: 2 or fragment thereof and an antibody to the
polypeptide.
25. A pharmaceutical composition of claim 24, which is a
therapeutic agent for vascular disease, inflammatory disease,
entoptic neovascular disease, reproductive system disease, central
nervous system disease or cancer.
26. A method of treating vascular disease, inflammatory disease,
entoptic neovascular disease, reproductive system disease, central
nervous system disease or cancer, which comprises administrating a
pharmaceutical composition of claims 24 or 25.
27. Use of a polynucleotide having a base sequence as set forth in
SEQ ID NO: 1 and a polypeptide comprising an amino acid sequence as
set forth in SEQ ID NO: 2 to produce a pharmaceutical composition
of claim 24 or 25.
Description
TECHNICAL FIELD
[0001] The present invention relates to a polypeptide and a
polynucleotide useful as a marker for neovascularization, vascular
disease, inflammatory disease, entoptic neovascular disease,
reproductive system disease, central nervous system disease or
cancer. And the present invention relates to the treatment of those
diseases.
BACKGROUND ART
[0002] By a double adapter method, shotgun library of human genome
was constructed and a nucleotide sequence of AF055021 (SEQ ID NO:
3), EST clone, was determined with the DNA library (ANLITICAL
BIOCHEMISTRY, 236, 107-113 (1996).). Subsequently, a nucleotide
sequence of full-length cDNA of AF055021 (SEQ ID NO: 1) was
determined and named KIAA1036 (DNA Research, 6 (3) 197-205
(1999).). KIAA1036 polypeptide (SEQ ID NO: 2) is a polypeptide
consisting of 365 amino acids and the function has not been
elucidated yet.
[0003] Since cells need oxygen and nutriments provided from blood
to survive in vivo, neovascularization is vigorous in vigorously
propagating tissues. It is known that neovascularization relates
not only to the physiological regeneration of tissues but also to
diseases accompanied by the pathological cell propagation. As the
disease, vascular disease, inflammatory disease, entoptic
neovascular disease, reproductive system disease, central nervous
system disease and cancer were reported (Nature, 407, 249 (2000).).
Since neovascularization is also observed upon the propagation of
malignant neoplasma cell, human umbilical vein endothelial cells
(HUVEC) treated with vascular endothelial cell growth factor (VEGF)
were reported as a model of neovascularization caused by cancer
(Cancer Research, 55, 5296-5301 (1995).).
[0004] Though the research of genes relating to the above-mentioned
various diseases have been conducted, the mechanism of pathogenesis
has not been perfectly elucidated, there being a possibility of
unknown genes relating to the mechanism. Under this circumstance,
an identification of a new gene relating to the pathogenesis of the
above-mentioned diseases and a diagnostic procedure of diseases
with the gene have been desired.
DISCLOSURE OF INVENTION
[0005] The present inventors found that expression of KIAA1036
polypeptide was increased in ovarian cancer and colon cancer and
confirmed that the peptide was useful as a marker for cancer. As
results of further researches, they found that KIAA1036 polypeptide
was a new marker for neovascular and useful as a marker and a
therapeutic agent of various diseases such as vascular disease
relating to neovascularization, inflammatory disease, entoptic
neovascular disease, reproductive system disease, central nervous
system disease or cancer to accomplish the present invention.
[0006] The present invention is
[0007] (1) a marker for neovascularization, vascular disease,
inflammatory disease, entoptic neovascular disease, reproductive
system disease, central nervous system disease or cancer, which
contains a polynucleotide having a base sequence as set forth in A
of 386-C of 1480 of SEQ ID NO: 1 or fragment thereof,
[0008] (2) a marker for neovascularization, vascular disease,
inflammatory disease, entoptic neovascular disease, reproductive
system disease, central nervous system disease or cancer, which
contains a polynucleotide hybridizing under a stringent condition
with a polynucleotide having a base sequence as set forth in C of
1-C of 1480 of SEQ ID NO: 1 or fragment thereof,
[0009] (3) a marker of (1), which contains a polynucleotide having
a base sequence as set forth in C of 1-C of 1480 of SEQ ID NO: 1 or
fragment thereof,
[0010] (4) a marker of (1), which contains a polynucleotide having
a base sequence as set forth in C of 1-C of 5481 of SEQ ID NO: 1 or
fragment thereof,
[0011] (5) a marker for neovascuarlization, vascular disease,
inflammatory disease, entoptic neovascular disease, reproductive
system disease, central nervous system disease or cancer, which
contains a polypeptide comprising an amino acid sequence as set
forth in Met of 1-Val of 365 of SEQ ID NO: 2 or fragment
thereof,
[0012] (6) a marker for neovascularization, vascular disease,
inflammatory disease, entoptic neovascular disease, reproductive
system disease, central nervous system disease or cancer, which
contains a polypeptide wherein 1 or more amino acid residue(s) has
a mutation(s) selected from deletion, substitution and addition in
an amino acid sequence as set forth in Met of 1-Val of 365 of SEQ
ID NO: 2 or fragment thereof,
[0013] (7) a marker of (5), which contains a polypeptide comprising
an amino acid sequence as set forth in Met of 1-Val of 365 of SEQ
ID NO: 2 or fragment thereof,
[0014] (8) a method of detection of a marker of any one of (1)-(7)
from a sample derived from human,
[0015] (9) a method of detection or assay of a marker of any one of
(1)-(4), which comprises the following processes and using a
polynucleotide having a base sequence as set forth in A of 386-C of
1480 of SEQ ID NO: 1 or fragment thereof,
[0016] (a) a process that the polynucleotide and a sample are
contacted, and
[0017] (b) a process that a polynucleotide binding with the
polynucleotide is detected,
[0018] (10) a method of detection or assay of a marker of any one
of (1)-(4), which comprises the following processes and using a
polynucleotide having a base sequence as set forth in A of 386-C of
1480 of SEQ ID NO: 1 or fragment thereof,
[0019] (a) a process that cDNA is prepared from a sample, and
[0020] (b) a process that cDNA is amplified with the polynucleotide
fragment and detected,
[0021] (11) a method of detection or assay of a marker of any one
of (5)-(7), which comprises the following processes and using an
antibody recognizing a polypeptide comprising an amino acid
sequence as set forth in Met of 1-Val of 365 of SEQ ID NO: 2 or
fragment thereof,
[0022] (a) a process that the antibody and a sample are contacted,
and
[0023] (b) a process that the binding between the antibody and a
polypeptide is detected,
[0024] (12) a method of detection of a disease relating to a marker
of any one of (1)-(7), which comprises using a method of any one of
(9)-(11),
[0025] (13) a method of detection of (12), wherein the disease is
vascular disease, inflammatory disease, entoptic neovascular
disease, reproductive system disease, central nervous system
disease or cancer,
[0026] (14) a diagnostic kit of a disease relating to a marker of
any one of (1)-(7), which comprises using a method of any one of
(9)-(11),
[0027] (15) a diagnostic kit of (14), wherein the disease is
vascular disease, inflammatory disease, entoptic neovascular
disease, reproductive system disease, central nervous system
disease or cancer,
[0028] (16) a screening method of a binding substance of a marker
of any one of (5)-(7), which comprises the following processes,
[0029] (a) a process that a marker of any one of (5)-(7) and a
subject are contacted, and
[0030] (b) a process that the binding between the marker and the
subject is detected,
[0031] (17) a kit for screening of a binding substance of (16),
which contains a polypeptide comprising an amino acid sequence as
set forth in SEQ ID NO: 2 or fragment thereof,
[0032] (18) a screening method of a binding activity regulatory
substance which regulates a binding between a marker of any one of
(5)-(7) and a binding substance of the marker, which comprises the
following processes,
[0033] (a) a process that a marker of any one of (5)-(7) and a
binding substance of the marker are contacted in the presence or
absence of a subject, and
[0034] (b) a process that a binding activity of the marker and the
binding substance is compared in the presence or absence of a
subject,
[0035] (19) a kit for screening of a binding activity regulatory
substance of (18), which contains a polypeptide comprising an amino
acid sequence as set forth in SEQ ID NO: 2 or fragment thereof,
[0036] (20) a screening method of an expression regulatory
substance of a marker of any one of (1)-(7), which comprises the
following processes,
[0037] (a) a process that a cell, which can express a marker of any
one of (1)-(7), and a subject are contacted, and
[0038] (b) a process that a change of an expression level of the
marker is detected by using a method of any one of (9)-(11),
[0039] (21) a kit for screening of an expression regulatory
substance of a marker of any one of (1)-(7), which comprises using
the method of (20),
[0040] (22) a DNA synthesis inhibitor containing a polypeptide
comprising an amino acid sequence as set forth in Met of 1-Val of
365 of SEQ ID NO: 2 or fragment thereof,
[0041] (23) a cell migrating activity inhibitor containing a
polypeptide comprising an amino acid sequence as set forth in Met
of 1-Val of 365 of SEQ ID NO: 2 or fragment thereof,
[0042] (24) a pharmaceutical composition which contains at least
one selected from a DNA synthesis inhibitor of (22), a cell
migrating activity inhibitor of (23), a polynucleotide having a
base sequence as set forth in SEQ ID NO: 1 or fragment thereof, a
human gene therapeutic expression vector containing polynucleotide
having a base sequence as set forth in SEQ ID NO: 1 or fragment
thereof, a polypeptide comprising an amino acid sequence as set
forth in SEQ ID NO: 2 or fragment thereof and an antibody to the
polypeptide,
[0043] (25) a pharmaceutical composition of (24), which is a
therapeutic agent for vascular disease, inflammatory disease,
entoptic neovascular disease, reproductive system disease, central
nervous system disease or cancer,
[0044] (26) a method of treating vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease or cancer, which comprises
administrating a pharmaceutical composition of (24) or (25),
[0045] (27) use of a polynucleotide having a base sequence as set
forth in SEQ ID NO: 1 and a polypeptide comprising an amino acid
sequence as set forth in SEQ ID NO: 2 to produce a pharmaceutical
composition of (24) or (25).
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a schematic comparing an amino acid sequence of
KIAA1036 and that of AK022567.
[0047] Query: 614 shows an amino acid sequence of KIAA1036. Sbjct:
1 shows an amino acid sequence of AK022567.
[0048] FIG. 2 shows an expression level of KIAA1036 in human
umbilical vein endothelial cells by Northern blot.
[0049] Total RNA was prepared by treating human umbilical vein
endothelial cell with nM VEGF for 0, 0.5, 2, 6, 12 or 24 hours. And
then signal intensity of KIAA1036 gene against signal intensity of
.beta.-actin gene was calculated. A relative expression level is
shown when signal intensity in a control division (1 nM VEGF was
not added) is 1.
[0050] The expression was inhibited in 0.5 to 2 hours after VEGF
was added. But a 3.2-fold high gene expression was induced at 24
hours after VEGF was added.
[0051] FIG. 3 shows an expression level of KIAA1036 in human cancer
tissues by Dot blot.
[0052] By using a nylon membrane on which cDNA was immobilized,
wherein cDNA was derived from a cancer tissue obtained from a
patient with human ovarian cancer or large bowel cancer and a
normal tissue obtained from the same patient, signal intensity of
KIAA1036 gene against signal intensity of .beta.-actin gene was
calculated. The ratio of signal intensity in each cancer tissue
against in each normal tissue of the same patient was calculated.
And a change of expression level of the gene in each cancer tissue
was analyzed. As a result, in cancer tissue of all of the 4
patients with ovarian cancer and all of the 11 patients with large
bowel cancer, the expression of KIAA1036 gene was increased.
[0053] FIG. 4 shows a cytostatic effect of KIAA1036 polypeptide
against a vascular endothelial cell.
[0054] Cell propagation in human vascular endothelial cells was
calculated by measuring an uptake of bromodeoxyuridine (BrdU) in
DNA synthesis. Human vascular endothelial cells were divided in
M199 medium in which a concentration of KIAA1036 polypeptide was 0,
1 or 10 nM. BrdU reagent was added thereto and the cells were
cultured for 12 hours. And then the quantity of BrdU taken in cells
was measured (lane 1 to 3). The same operation was carried out with
M199 medium to which 1 nM VEGF was added and the quantity of BrdU
taken in cells was measured (lane 4 to 6).
[0055] As a result, regardless of the addition of VEGF, DNA
synthesis in cells was decreased, depending on the concentration of
added KIAA1036 polypeptide.
[0056] FIG. 5 shows a depression effect of KIAA1036 against
migrating activity of a vascular endothelial cell.
[0057] M199 medium, in which a concentration of KIAA1036
polypeptide was 0, 1 or 10 nM, was added in the lower layer of a
culture apparatus divided with a filter and vascular endothelial
cells were added in the upper layer of it. The cells were cultured
for 4 hours. Then cell population attached to the filter was
measured (lane 1 to 3). The same operation was carried out with
M199 medium to which 0.25 nM VEGF was added and cell population
attached to the filter was measured (lane 4 to 6).
[0058] As a result, regardless of the addition of VEGF, migrating
activity of cells was inhibited, depending on the concentration of
added KIAA1036 polypeptide.
BEST MODE FOR CARRYING OUT THE INVENTION
[0059] Preparation of KIAA1036 polynucleotide which is a marker of
the present invention, preparation of the polypeptide, a
recombinant vector, a transformant, a method of production thereof,
an antibody, the method of detection of polypeptide, a method of
detection of mRNA, the method of detection of neovascularization,
vascular disease, inflammatory disease, entoptic neovascular
disease, reproductive system disease, central nervous system
disease or cancer and a diagnostic kit, a therapeutic agent or a
method of treating for vascular disease, inflammatory disease,
entoptic neovascular disease, reproductive system disease, central
nervous system disease or cancer are explained below. In this
description, if there is no instruction, a gene recombinant
technique, a production engineering technique of a recombinant
polypeptide in animal cells, insect cells, yeast and Escherichia
coli, a molecular-biological method, the method of separation and
purification of expressed polypeptide, assay and immunological
method, which are well-known in this field, are adopted.
[0060] A marker of the present invention is "a polynucleotide
having a base sequence as set forth in A of 386-C of 1480 of SEQ ID
NO: 1 or fragment thereof" and a polynucleotide sequence of
KIAA1036. Additionally, a marker of the present invention includes
"a polynucleotide having a base sequence as set forth in C of 1-C
of 1480 of SEQ ID NO: 1 or fragment thereof" and "a polynucleotide
hybridizing with the present invention marker under a stringent
condition or fragment thereof". Hereinafter, a polynucleotide,
which is a marker of the present invention, is referred to as
polynucleotide A.
[0061] In this description, "neovascularization" means a phenomenon
that a new vasculature is developed from an existing vasculature. A
disease related to "neovascular" includes "vascular disease",
"inflammatory disease", "entoptic neovascular disease",
"reproductive system disease", "central nervous system disease" or
"cancer".
[0062] "Vascular disease" includes arterial sclerosis, hypertonia,
angina pectoris, obstructive arteriosclerosis, myocardial
infarction, cerebral infarction, diabetic angiopathy or vascular
malformation. "Inflammatory disease" includes hepatitis,
pneumonitis, glomerular nephritis, thyreoiditis, osteitis,
arthromeningitis, osteoclasia, chondrolysis, rheumatism, bronchial
asthma, sarcoidosis, Crow-Fukase syndrome, pannus, allergic oedema,
ulcers, hydroperitoneum, peritoneal screlosis or tissular
conglutination. "Entoptic neovascular disease" includes diabetic
retinopathy, occlusion of retinal vein or aging macular
degeneration. "Reproductive system disease" includes uterus
dysfunction, placental dysfunction, ovarian hyperergasia or
follicle cyst. "Central nervous system disease" includes retinosis,
cerebral apoplexy, vascular dementia or Alzheimer disease. "Cancer"
includes a malignant neoplasma such as solid cancer, angiomatous,
hemangioendothelioma, sarcomas, sarcoma or hematopoietic organic
ulcer, ovarian cancer or large bowel cancer. Additionally, it
includes metastatics of these cancers.
[0063] "A polynucleotide hybridizing under a stringent condition"
means a polynucleotide obtainable by a generally known and common
method in this field with using a fragment of polynucleotide A as a
probe, for example, colony hybridization, plaque hybridization or
Southern blotting hybridization. An example of the above
polynucleotide includes a polynucleotide obtained by hybridization
at 65.degree. C. in the presence of 0.7 to 1.0M NaCl by using a
membrane on which polynucleotides from colony or plaque are
immobilized and a washing of a membrane at 65.degree. C. with SSC
(Saline Sodium Citrate; 150 mM sodium chloride, 15 mM sodium
citrate) solution whose concentration is 0.1-fold to twice.
Hybridization can be performed as well as methods described in
Molecular Cloning: A Laboratory Manual, Second Edition (1989)(Cold
Spring Harbor Laboratory Press), Current Protocols in Molecular
Biology (1994)(Wiley-Interscience), DNA Cloning 1: Core Techniques,
A practical Approach, Second Edition (1995)(Oxford University
Press). Preferably, sequences comprising of only adenine (a) or
thymine (T) are excluded from sequences hybridizing under a
stringent condition. "A fragment of a polynucleotide" includes a
polynucleotide having a base sequence comprising 5 or more serial
bases in a base sequence of SEQ ID NO: 1, for example, a
polynucleotide having a base sequence comprising 5, 8, 10, 12, 15,
20, 25, 30, 50, 100, 500 or 1000 bases.
[0064] In this description, "a polynucleotide hybridizing" includes
a polynucleotide hybridizing with the other polynucleotide under
the above hybridizing condition. Examples of the above
polynucleotide includes a polynucleotide having a homology of at
least 60% or more, preferably 80% or more and more preferably 95%
or more with a base sequence of DNA encoding a polypeptide having
an amino acid sequence of SEQ ID NO: 1. And the homology is shown
as a score, for example, by using a search program BLAST with
algorithm developing by Altschul et al (The Journal of Molecular
Biology, 215, 403-410 (1990).).
[0065] A marker of the present invention includes "a polypeptide
comprising an amino acid sequence as set forth in Met of 1-Val of
365 of SEQ ID NO: 2 or fragment thereof", a polypeptide sequence of
KIAA1036 including their salts. A "salt" includes a salt
physiologically acceptable with an acid or a base. A salt with an
acid is especially preferable. The salt includes, for example, a
salt with inorganic acid such as hydrochloric acid, phosphoric
acid, hydrobromic acid or sulfuric acid, or a salt with organic
acid such as acetic acid, fomic acid, proprionic acid, fumaric
acid, maleic acid, succinic acid, tartaric acid, citric acid, malic
acid, oxalic acid, benzoic acid, methanesulfonic acid or
benzenesulfonic acid. "A fragment of polypeptide" includes a
polypeptide sequence comprising 5 or more serial amino acid
residues in an amino acid sequence of SEQ ID NO: 2, for example, a
polypeptide comprising an amino acid sequences comprising 5, 8, 10,
12, 15, 20, 25, 30, 50 or 100 amino acid residues.
[0066] A marker of the present invention includes "a polypeptide
having a mutation selected from deletion, substitution and addition
in 1 or a few amino acid residue(s) of an amino acid sequence as
set forth in Met of 1-Val of 365 of SEQ ID NO: 2 or fragment
thereof". Hereinafter, a polypeptide, which is a marker of the
present invention, is referred to as polypeptide B.
[0067] "1 or a few amino acid residue(s)" means amino acid
residues, which can delete, substitute or add by a site-specific
mutagenic method or the like. Namely, it means amino acid residues
having 50 or less, preferably 30 or less, more preferably 20 or
less, much more preferably 10 or less amino acid residues.
Furthermore, polypeptide B includes a polypeptide to be used as a
marker for neovascularization, vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease or cancer even after deletions,
substitutions or additions. It is, for example, a polypeptide
having a homology of at least 60% or more, preferably 80% or more
and more preferably 95% or more with an amino acid sequence of SEQ
ID NO: 2.
[0068] In this description, "a marker" includes a substance used to
detect neovascularization and all kinds of disease related to
neovascularization derived from organism samples, for example,
vascular disease, inflammatory disease, entoptic neovascular
disease, reproductive system disease, central nervous system
disease or cancer. And it includes polynucleotide A and polypeptide
B. mRNA or a protein can be a "marker" when the expression
increases or decreases under diseases. Examples of the above maker
include KIAA1036 polypeptide comprising an amino acid sequence as
set forth in Met of 1-Val of 365 of SEQ ID NO: 2, KIAA1036
polynucleotide having a base sequence as set forth in A of 386-C of
1480 of SEQ ID NO: 1, a polynucleotide having a complementary base
sequence thereof or a fragment thereof.
[0069] In this description, terms are used with usual meanings in
this field unless otherwise mentioned especially referred. The
terms especially used in this description are explained below.
[0070] An "antibody" means a general antibody in this field and
includes all of the antibodies, fragments thereof, derivatives,
conjugations or modified antibodies. An antibody recognizing
polypeptide B or fragment thereof is preferable, an antibody
recognizing specifically the above polypeptide is more preferable
and an antibody recognizing single specifically the polypeptide is
much more preferable. These antibodies include a polyclonal
antibody or a monoclonal antibody.
[0071] "A detection or a quantitation" of a marker of the present
invention can be accomplished by any appropriate method including
an immunological assay or a molecular-biological assay.
[0072] When a marker is polypeptide B, the above method includes,
for example, an immunological assay such as ELISA (Enzyme Linked
Immuno Sorbent Assay), RIA (Radio Immuno Assay), fluorescence
antibody technique, Western blot or an immune structure dyeing
method.
[0073] By using an antibody against polypeptide B or fragment
thereof, neovascularization, vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease or cancer can be detected or
quantitated. Because a marker of the present invention can be used
as a marker for above diseases, a method of detection or
quantitation of diseases with the above antibody can be
accomplished by any appropriate method including an immunological
assay as well as an above detection or quantitation of a
polypeptide.
[0074] The present invention relates to a diagnostic kit of
neovascularization, vascular disease, inflammatory disease,
entoptic neovascular disease, reproductive system disease, central
nervous system disease or cancer, characterized by including an
antibody against polypeptide B or fragment thereof. The above kit
includes at least an antibody against polypeptide B or fragment
thereof and a standard reagent of polypeptide B. Because
polypeptide B can be used as a marker for neovascularization,
vascular disease, inflammatory disease, entoptic neovascular
disease, reproductive system disease, central nervous system
disease or cancer, it can be immunologically measured as well as an
above mentioned method of detection or quantitation by an
immunological assay.
[0075] And when a marker is a polynucleotide such as mRNA, the
assay includes a molecular-biological assay, for example, Northern
blot, Dot blot or polymerase chain reaction (PCR).
[0076] mRNA can be detected or quantitated by using polynucleotide
A or fragment thereof as a probe or primer.
[0077] Because a marker of the present invention can be a marker of
above diseases, a method of detection or quantitation of diseases
with the probe or the primer is accomplished by an appropriate
method including a molecular-biological assay as well as the above
method for detection or quantitation of a polynucleotide.
[0078] A probe or a primer includes a nucleotide comprising serial
bases in a base sequence of SEQ ID NO: 1, for example, a base
sequence comprising 5, 8, 10, 12, 15, 20, 25, 30, 40, 50 or 60
bases. It includes an oligonucleotide having the same sequence, an
oligonucleotide having a sequence complementary to the sequence of
the oligonucleotide, and the derivative thereof. A derivative
thereof includes, for example, a oligonucleotide wherein a
phosphodiester bond in the oligonucleotide is transformed into a
phosphorothioate bond or a N3'-P5' phosphoamidite bond, a
oligonucleotide wherein a ribose and a phosphodiester bond are
transformed into a peptide bond, a oligonucleotide wherein a uracil
in the oligonucleotide is substituted with a C-5 propionyl uracil
or a C-5 thiazole uracil, a oligonucleotide wherein a cytosine in
the oligonucleotide is substituted with C-5 propionyl cytosine or
cytosine modified with phenoxazine or a oligonucleotide wherein a
ribose in DNA is substituted with 2'-O-propyl ribose,
2'-methoxyethoxy ribose or the like. Those polynucleotides are
useful, for example, as a gene marker, a primer for PCR or a probe
for hybridization.
[0079] The present invention relates to a part or all of the
polynucleotide encoding polypeptide B and a diagnostic kit of
vascular disease, inflammatory disease, entoptic neovascular
disease, reproductive system disease, central nervous system
disease or cancer including the polynucleotide as a standard
reagent. This kit includes at least a probe or a primer against
polynucleotide A or fragment thereof and a standard reagent of
polynucleotide A. Because polynucleotide A can be used as a marker
for neovascularization, vascular disease, inflammatory disease,
entoptic neovascular disease, reproductive system disease, central
nervous system disease or cancer, it is molecular-biologically
measured as well as the above method of detection or quantitation
by a molecular-biological assay.
[0080] A transcription of DNA or a translation of mRNA can be
inhibited by using polynucleotide A. Methods to inhibit
transcription of DNA or translation of mRNA of the polypeptide B
can be provided by appropriately preparing a polynucleotide having
a complementary base sequence to a base sequence of polynucleotide
A, a polynucleotide hybridizing with polynucleotide A or an
oligonucleotide and a derivative oligonucleotide having a
complementary sequence to an oligonucleotide having serial 5 to 60
base sequences in base sequence of polynucleotide A and using
antisense RNA/DNA techniques known in this field.
[0081] The present invention relates to "a screening method of a
binding substance" of a marker of the present invention. "A binding
substance" includes any substance binding to a marker of the
present invention. The binding substance includes, for example, a
low-molecular weight substance, a polynucleotide or a polypeptide
such as a receptor and an antibody. "A screening method of a
binding substance" can be accomplished by appropriately using
techniques widely known in this technical field. It includes, for
example, a bioassay and a binding assay.
[0082] "A kit for screening of a binding substance" of the present
invention includes at least a marker of the present invention. A
binding substance can be screened by biochemical method with the
above kit.
[0083] Additionally the present invention relates to "a screening
method of a binding activity regulatory substance". "A binding
activity regulation" means that a binding activity of a marker of
the present invention and the binding substance is enhanced or
reduced and "a binding activity" is evaluated by Percent Maximum
Binding (PMB). "A binding activity regulatory substance" includes a
substance that makes a binding activity of the binding substance
against a marker of the present invention enhanced or reduced, for
example, an agonist or an antagonist. The substance includes, for
example, a low-molecular weight substance, a polynucleotide or a
polypeptide. "A screening method of a binding activity regulatory
substance" can be accomplished by appropriately using techniques
widely known in this field. A binding activity regulatory substance
can be screened by a screening method of the present invention. The
method is, for example, a bioassay or a binding assay.
[0084] "A screening kit of a binding activity regulatory substance"
of the present invention includes at least a marker of the present
invention. A binding activity regulatory substance can be screened
by a biochemical method with a kit of the present invention.
[0085] And the present invention relates to "a screening method of
an expression regulatory substance". Expression level means the
amount of a marker of the present invention expressed in target
cells. Expression level can be evaluated by an appropriate method
including an immunological assay with an antibody against
polypeptide B, for example, ELISA, RIA, a fluorescence antibody
technique or an immune structure dyeing method or by a
molecular-biological assay measuring mRNA of polynucleotide A, for
example, Northern blot hybridization, Dot blot or RT-PCR.
"Expression regulation" means that expression level of a marker of
the present invention in protein or mRNA level evaluated by any
appropriate method including the above immunological or
molecular-biological assay is enhanced or reduced. "An expression
regulatory substance" includes a substance enhancing or reducing
expression level of a marker of the present invention in protein or
mRNA level, which can be evaluated by any appropriate method
including the above immunological or molecular-biological assay.
The above substance includes, for example, a low-molecular weight
substance, a polynucleotide or a polypeptide. "A screening method
of an expression regulatory substance" can be accomplished by
appropriately using techniques widely known in this field. An
expression regulatory substance can be screened by a screening
method of the present invention. The method includes, for example,
a bioassay or a binding assay.
[0086] "A screening kit of an expression regulatory substance" of
the present invention includes at least a marker of the present
invention. An expression regulatory substance can be screened by a
biochemical method based on a screening method of an expression
regulatory substance with a kit of the present invention.
[0087] Because polypeptide B inhibits a DNA synthesis in a vascular
endothelial cell and a migrating activity of a vascular endothelial
cell, polynucleotide A, a complementary polynucleotide of the above
polynucleotide, polypeptide B or/and an antibody against the above
polypeptide can be a DNA synthesis inhibitor or/and a cell
migrating activity inhibitor. Then, the present invention provides
a DNA synthesis inhibitor and a cell migrating inhibitor.
[0088] "A pharmaceutical composition" of the present invention
includes at least one selected from polypeptide B, an antibody
against the polypeptide, polynucleotide A, a complementary
polynucleotide of the polynucleotide, a DNA synthesis inhibitor, a
cell migrating activity inhibitor, a binding substance, a binding
activity regulatory substance and an expression regulatory
substance. It can be used for a therapeutic agent of a specific
disease, for example, vascular disease, inflammatory disease,
entoptic neovascular disease, reproductive system disease, central
nervous system disease or cancer. The substance includes, for
example, a low-molecular weight substance, a polynucleotide or a
polypeptide. Preferred is a polypeptide comprising an amino acid
sequence as set forth in Met of 1-Val of 365 of SEQ ID NO: 2 or
fragment thereof. And a pharmaceutical composition of the present
invention includes "a gene therapeutic expression vector". "A gene
therapeutic expression vector" includes an expression vector
inserted all or a part of polynucleotide A and a vector which
inserts a normal gene in a cell, repairs/modifies a defect of a
gene and regulates an expression of a gene by introducing a gene in
a cell/tissue. The vector includes a viral vector wherein a part or
all of a base sequence of a viral, which is lack of reproductive
capability, is replaced with a therapeutic gene. Then, the present
invention provides therapeutic agents or methods of treating for
these diseases.
[0089] "A method of treating" includes a method for treating of
specific diseases, for example, vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease or cancer relating to
neovascularization by a administration of the above pharmaceutical
composition.
[0090] Hereinafter, the Present Invention is Described in
Detail.
[0091] (1) An Preparation of KIAA1036 Polynucleotide
[0092] cDNA library is manufactured from human brain, heart,
skeletal muscle, spleen, kidney, liver, small intestine, placenta,
human normal cells from these tissues, human umbilical vein
endothelial cells or human ovarian cancer or human large bowel
cancer by a usual method.
[0093] A method which manufactures cDNA library is a method
described in Molecular Cloning: A Laboratory Manual, Second Edition
(1989) (Cold Spring Harbor Laboratory Press), Current Protocols in
Molecular Biology (1994) (Wiley-Interscience) or DNA Cloning 1:
Core Techniques, A Practical Approach, Second Edition (1995)
(Oxford University Press) or the method that a kit on the market,
for example, SuperScript Plasmid System for cDNA Synthesis and
Plasmid Cloning (Invitrogen) or ZAP-cDNA Synthesis Kits
(STRATAGENE) is used.
[0094] cDNA obtained by the method is, for example, DNA encoding a
polypeptide of SEQ ID NO: 2. Examples of the above cDNA include a
DNA comprising a base sequence of SEQ ID NO: 1. A plasmid including
DNA of SEQ ID NO: 1 is, for example, a plasmid described in the
following examples.
[0095] The obtained DNA is inserted to an appropriate expression
vector and an expression plasmid is constructed. The expression
vectors are introduced to appropriate hosts and then transformants
can be obtained. The expression vector is any vector in which cDNA
is inserted and which express in animal cells. The vector is, for
example, pcDNA1.1, pcDNA1.1/Amp, pCDM8, pREP (Invitrogen), pHM6,
pHB6 (Roche Diagnostics), pKK223-3, pGEX (Amersham Pharmacia
Bioteque), pET-3, pET-11, pBluescriptII SK(+), pBluescriptII SK(-)
(STRATAGENE), pUC19, pTrxFus (Invitrogen), pUC118, pSTV28 (TaKaRa),
pMAL-c2X (New England BioLabs), pAGE107 (Cytotechnology, 3 (2),
133-140 (1990).; JP1991-22979), pAGE103 (The Journal of
Biochemistry, 101 (5), 1307-1310 (1987).), pAMo, pAMoA (The Journal
of Biological Chemistry, 268 (30), 22782-22787 (1993).), pAMoPRSA
(JP1993-336963) or pAS3-3 (JP1990-227075).
[0096] The expression vectors with a cDNA insert introduce a target
cDNA into optional animal cells and transformed cells are obtained.
Any method for introducing DNA can be used as a method for
introducing the expression vectors a host. When a host is animal
cells, it is, for example, an electroporation (Cytotechnology, 3
(2), 133-140 (1990).), a calcium phosphate method (JP1990-227075)
or a lipofection method (Proceedings of the National Academy of
Sciences USA, 84, 7413 (1987).; Virology, 52, 456 (1973).).
[0097] Appropriate cells or tissue for expression vectors can be
used as a host. It is, for example, animal cells. An animal cell as
a host is, for example, Namalwa (Burkitt lymphoma, ATCC: CRL-1432)
and the subline NamalwaKJM-1 which is an established cell from
human, HCT-15 (human large bowel cancer cell, ATCC: CCL-225),
COS-1(African green monkey's nephrocyte (SV40 transformed cell),
ATCC: CRL-1650) and COS-7 (African green monkey's nephrocyte (SV40
transformed cell), ATCC: CRL-1651) which is an established cell
from monkey or CHO-K1 (Chinese hamster ovary cell, ATCC: CCL-61)
and HBT5637 (JP1987-299) which is an established cell derived from
hamster. Preferred is Namalwa cell, NamalwaKJM-1 cell or HCT-15
cell.
[0098] Transformants of the present invention are cultured by a
generally known and common method in this field. It can be
accomplished with a medium appropriate to a transforming host and a
liquid medium is appropriate as a medium for a culturing liquid
medium. For example, MEM medium (Science, 130, 432 (1959).), D-MEM
medium (Virology, 8, 396 (1959).), PRMI 1640 medium (The Journal of
the American Medical Association, 199, 519 (1967).), YT medium or
BEM medium can be used.
[0099] When transformants is prepared by using animal cells as a
host are cultured, for example, a medium wherein cattle fetal serum
(FCS) is appropriately added into MEM medium, D-MEM medium or PRIM
medium can be used. A medium can optionally include a substance
promoting transcription activity to enhance transcription activity
of a promoter of an expression vector. For example,
isopropyl-1-thio-.beta.-D-galactopyranosin (IPTG) can be used.
[0100] A medium includes nutriments needed to grow up
transformants, for example, glucose, amino acid, peptone, vitamin,
hormone or serum, preferably, FCS, calcium chloride or magnesium
chloride. The medium like this, which has any composition, can be
used and a medium available on the market can be used. A
cultivation is held in pH 6.0 to 8.0 at 25 to 40.degree. C. in the
presence of 5% CO.sub.2.
[0101] The obtained transformed cells are cultured by a usual
method. For example, they can be cultured by the following culture
method. When transformants are animal cells, examples of a medium
for culturing the cells includes commonly used RPMI1640 medium (The
Journal of the American Medical Association, 199, 519 (1967).), MEM
medium of Eagle (Science, 122, 501 (1952).), D-MEM medium
(Virology, 8, 396 (1959).), 199 medium (Proceeding of the Society
for the Biological Medicine, 73, 1 (1950).) or a medium added fetal
calf serum (FCS).
[0102] Cultivation is usually carried out in pH 6 to 8, at 25 to
40.degree. C., in the presence of 5% CO.sub.2 for 1 to 7 days. An
antibiotic such as kanamycin, penicillin or streptomycin can
optionally be added during cultivation.
[0103] DNAs encoding KIAA1036 which is a neovascular marker can be
obtained from human brain, heart, skeletal muscle, spleen, kidney,
liver, lung, placenta, human normal cells from these tissues, human
umbilical vein endothelial cells or human ovarian cancer or human
large bowel cancer by the above-mentioned.
[0104] On the basis of an amino acid sequence, DNA encoding
KIAA1036 can be prepared by a chemical synthesis. A chemical
synthesis of DNA can be accomplished with a DNA synthesizer using a
thiophosphite method (Shimazu Corporation) or a DNA synthesizer
model 392 using a phosphoamidite method (PerkinElmer, Inc.).
[0105] The following oligonucleotide is used as a sense primer (SEQ
ID NO: 4) or an antisense primer (SEQ ID NO: 5) and cDNA prepared
from mRNA in cells expressing complementary mRNA of these DNA is
used as a template. Target DNA can be prepared by PCR under this
condition.
[0106] (2) The Production of KIAA1036 Polypeptide
[0107] KIAA1036 can be produced by a method described in Molecular
Cloning: A Laboratory Manual, Second Edition (1989)(Cold Spring
Harbor Laboratory Press) or Current Protocols in Molecular Biology
(1994)(Wiley-InterScience). For example, KIAA1036 polynucleotide is
made to express in host cells by the following method and KIAA1036
can be produced.
[0108] On a basis of a full length of DNA encoding KIAA1036
polypeptide, an appropriate length of DNA fragment including a part
encoding the polypeptide is optionally prepared. And DNA whose
bases substituted is prepared, as a base sequence encoding the
polypeptide is the most suitable codons for expression in a host.
This DNA is useful to enhance productivity of the polypeptide. By
inserting the DNA fragment or a full length of DNA to the
downstream of a promoter of an appropriate expression vector, a
recombinant DNA (a recombinant vector) is produced. By the
recombinant vectors are produced in host cells adapting to the
expression vector, transformants producing KIAA1036 polypeptide can
be obtained.
[0109] Any cell such as a prokaryotic cell, yeast, an animal cell,
a plant cell or a insect cell can be used as a host cell, if a
target gene can be expressed in the cells. As an expression vector,
the vector which can autonomously replicate in the above host cells
or be inserted into a chromosome and which includes a promoter at
the appreciate position for the transcription of KIAA1036
polypeptide gene.
[0110] (i) The case that prokaryote is used as a host.
[0111] An expression vector of KIAA1036 can autonomously replicate
in prokaryote as well as is preferably constructed with a promoter,
a ribosome binding sequence, KIAA1036 and a transcription
termination sequence. A gene regulating a promoter can be
included.
[0112] An expression vector is, for example, pBTrp2, pBTac1, pBTac2
(Roche Diagnostics), BluescriptII SK(+), pBluescriptII SK(-)
(STRATAGENE), pSTV28, pUC118, pUC19 (TaKaRa), pKK233-2 (Pharmacia),
pSE280, pSupex, pUB110, pTP5, pC194, pTrxFus (Invitrogen), pGEMEX-1
(Promega), pQE-8 (QIAGEN), pGEX (Pharmacia), pETsystem (Novagen),
pMAL-c2 (New England BioLabs), pKYP10 (JP1982-110600), pKYP200
(Agricultural Biological Chemistry, 48, 669 (1984).), pLSA1
(Agricultural Biological Chemistry, 53, 277 (1989).), pGEL1
(Proceedings of the National Academy of Sciences USA, 82,
4306(1985).), pEG400 (Journal of Bacteriology, 172, 2392(1990).),
pTrs30 (FERM BP-5407), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400),
pGKA2 (FERM BP-6798), pPA1 (JP1987-233798) or pTerm2 (JP1990-22979,
U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat. No.
5,160,735).
[0113] Any promoter which can express in a host cell such as
Escherichia coli can be used. For example, it is a promoter from
Escherichia coli or a phage such as trp promoter (Ptrp), lac
promoter (Plac), PL promoter, PR promoter or PSE promoter, SPO1
promoter, SPO2 promoter or penP promoter. And a promoter
artificially modified such as a promoter which two Ptrps is
connected in series (Ptrp.times.2), tac promoter, lacT7 promoter or
letI promoter can be used.
[0114] Preferred is a plasmid that the distance between
Shine-Dalgarno sequence which is a ribosome binding sequence and an
initiation codon is regulated to an appropriate distance, for
example, 6 to 18 bases. A transcription termination sequence is not
always needed for an expression of KIAA1036 polynucleotide but
preferred is that a transcription termination sequence is
positioned at the right downstream of a structural gene.
[0115] A host cell is, for example, a prokaryote of Escherichia
genus, Serratia genus, Bacillus genus, Brevibacterium genus,
Corynebacterium genus, Microbacterium genus or Pseudomonas genus,
for example, XL1-Blue strain, XL2-Blue strain, DH1 strain, MC1000
strain, KY3276 strain, W1485 strain, JM109 strain, HB101 strain,
No. 49 strain, W3110 strain, NY49 strain, BL21 (DE3) strain, BL21
(DE3) pLysS strain, HMS174 (DE3) strain or HMS174 (DE3) pLysS
strain of E. coli as Escherichia genus, S. ficaria strain, S.
fonticola strain, S. liquefaciens strain or S. marcescens strain as
Serratia genus, B. subtilis strain or B. amyloliquefaciens strain
as Bacillus genus, B. ammoniagenes strain, B. Immariophilum (ATCC:
14068) strain or B. saccharolyticum (ATCC: 14066) strain as
Brevibacterium genus, C. glutamicum (ATCC: 13032) strain, C.
glutamicum (ATCC: 14067) strain, C. glutamicum (ATCC: 13869) strain
or C. acetoacidophilum (ATCC: 13870) strain as Corynebacterium
genus, M. ammoniaphilum (ATCC: 15354) strain as Microbacterium
genus or S. mephitica strain as Pseudomonas genus.
[0116] Any method to introduce DNA to the above host cell can be
used as an introduction method of a recombinant vector, for
example, an electroporation (Nucleic Acids Research, 16, 6127
(1988).), calcium phosphate method (Proceedings of the National
Academy of Sciences USA, 69, 2110 (1972).), a protoplast method
(JP1987-2483942) or the method described in Gene, 17, 107 (1982),
or Molecular & General Genetics, 168, 111 (1979).
[0117] (ii) The Case that Yeast is Used as a Host.
[0118] When yeast is used as a host, an expression vector is, for
example, YEp13 (ATCC:37115), YEp24 (ATCC:37051), YCp50
(ATCC:37419), pHS19 or pHS15.
[0119] Any promoter which express in yeast can be used as a
promoter, for example, ADH1 (alcohol dehydrogenase) promoter, PHO5
(acid phosphatase) promoter, PGK1 (phosphoglycerate kinase)
promoter, GAPDH (glyceraldehyde3-phosphate dehydrogenase) promoter,
GAL1 (galactose kinase) promoter, GAL10 (UDP galactose 4-epimerase)
promoter, MF .alpha. 1 (.alpha. pheromone) promoter or CUP1
(metallothionein) promoter.
[0120] A host is, for example, S. cerevisiae species of
Saccharomyces genus, S. pombe species of Schizosaccharomyces genus,
K. lactis species of Kluyveromyces genus, T. pullulans species of
Trichosporon genus, S. alluvius species of Schwanniomyces genus or
P. pastoris species of Pichia genus.
[0121] Any method to introduce DNA into a host can be used as a
introduction method of a recombinant vector, for example, an
electroporation (Methods in Enzymology, 194, 182 (1990).), a
spheroplast method (Proceedings of the National Academy of Sciences
USA, 84, 1929 (1978).) or a lithium acetate method (Journal of
Bacteriology, 153, 163 (1983), or Proceedings of the National
Academy of Sciences USA, 75, 1929 (1978).).
[0122] (iii) The Case that an Animal Cell is Used as a Host.
[0123] When an animal cell is used as a host, an expression vector
is, for example, pcDNA1/Amp, pcDNA1, pCDM8, pREP4 (Invitrogen),
pAGE 107 (Cytotechnology, 3, 133 (1990).), pAGE 103 (The Journal of
Biochemistry, 101, 1307 (1987).), pAMo, pAMoA (pAMoPRSA) (The
Journal of Biological Chemistry, 268, 22782-22787 (1993).) or
pAS3-3 (JP1990-22705).
[0124] Any promoter that can express in a host can be used as a
promoter, for example, a promoter of IE (Imediate-early) gene of
human cytomegalovirus (hCMV), an early promoter of SV40, Long
Terminal Repeat Promoter of Moloney Murine Leulemia Virus, a
promoter of retrovirus, HSP promoter, SR .alpha. promoter or a
promoter of metallothionein. An enhancer of IE gene of human CMV
can be used with a promoter.
[0125] An animal cell as a host is, for example, HEK293 (a human
fetal nephrocyte, ATCC:CRL-1573), Namalwa (Burkitt lymphoma,
ATCC:CRL-1432), HeLa (a cell of carcinoma of uterine cervix,
ATCC:CCL-2), HBT5637 (a leukemia cell, JP1987-299), BALL-1 (a
leukemia cell) or HCT-15 (a large bowel cancer cell) of an
established cell from a human, Sp2/0-Ag14 (a mouse myeloma cell,
ATCC:CRL-1581) or NSO (a mouse myeloma cell) of an established cell
from a mouse, COS-1 (African green monkey nephrocyte (SV40
transformed cell), ATCC:CRL-1650) or COS-7 (African green monkey
nephrocyte (SV40 transformed cell), ATCC:CRL-1651) of an
established cell from a monkey, CHO-K1 (Chinese hamster ovary cell,
ATCC:CCL-61) or BHK-21 (C-13) (Sicilian hamster kidney cell,
ATCC:CCL-10) of an established cell from a hamster, PC12 (an
adrenal pheochromocytoma, ATCC:CRL-1721) or YB2/0 (a rat myeloma
cell, ATCC:CRL-1662) of an established cell from a rat.
[0126] Any method to introduce DNA into a host can be used as an
introduction method of a recombinant vector, for example, an
electroporation (Cytotechnology, 3, 133, (1990).), a calcium
phosphate method (JP1990-22705) or a lipofection method
(Proceedings of the National Academy of Sciences, USA, 84, 7413
(1987), or Virology, 52, 456 (1973).).
[0127] (iv) The Case that a Plant Cell is Used as a Host.
[0128] When a plant cell or a plant is used as a host, a
polypeptide can be produced as well as a well-known method (The
Tissue Culture, 20 (1994)., The Tissue Culture, 21 (1995), or
Trends in Biotechnology, 15, 45 (1997).). An expression vector is,
for example, Ti plasmid or Tobacco mosaic virus vector. Any
promoter that can express in a plant cell can be used as a promoter
for a gene expression, for example, 35S promoter of Cauliflower
mosaic virus (CaMV) or Rice actin 1 promoter. And expression
productivity of a gene can be enhanced by inserting intron 1 of an
alcohol dehydrogenase gene of maize between a promoter and an
expressed.
[0129] A host is, for example, a plant cell such as potato,
tobacco, maize, rice, rape, soybean, tomato, carrot, wheat, barley,
rye, alfalfa or flax.
[0130] Any method to introduce DNA into a host can be used as a
introduction method of a recombinant vector, for example, a method
with Agrobacterium (JP1983-140885, JP1984-70080 or WO94/00977), an
electroporation (JP1984-251887) or a particle gun (gene gun) method
(JP2606856, JP2517813).
[0131] (v) The Case that an Insect Cell is Used as a Host.
[0132] When an insect cell is used as a host, a transfer vector is,
for example, pVL1392, pVL1393 or pBlueBacIII (Invitrogen) and a
virus for infection is, for example, a Vaculovirus which infects
insects of Mamestra brassicoe family; Autographa california nuclear
polyhedrosis virus (AcMNPV) Bac-N-Blue DNA. A transformation method
of an insect cell is, for example, a method described in
Baculovirus Expression Vector: A Laboratory Manual (1992) (W.H.
Freeman and Company), Molecular Cloning: A Laboratory Manual,
Second Edition (1989) (Cold Spring Harbor Laboratory Press),
Current Protocols in Molecular Biology (1994) (Wiley-InterScience)
or Biotechnology, 6, 47 (1988).
[0133] A transfer vector including a target gene and baculovirus
DNA for infection to an insect cell are added into a culture and a
virus expressing a target gene produced by recombinant infects an
insect cell to be expressed a polypeptide.
[0134] An insect cell as a host is, for example, an established
cell from Spodoptera frugiperda (Mamestra brassicoe) or an
established cell from Trichoplusia ni. For example, a cell from S.
frugiperda includes Sf9 (ATCC: CRL-1711, an ovary cell) or Sf21 (an
ovary cell) and a cell strain from T.ni is, for example, High Five
or BTI-TN-5B1-4 (an egg cell, Invitrogen).
[0135] Any method to introduce into a host can be used as an
introduction method of a recombinant vector, for example, a calcium
phosphate method (JP1990-22705) or a lipofection method
(Proceedings of the National Academy of Sciences USA, 84, 7413
(1987).). And an electroporation (Cytotechnology, 3, 133 (1990).)
can be used as well as an animal cell.
[0136] (vi) A Culture Method
[0137] When a transformant having a recombinant vector with an
inserted DNA encoding KIAA1036 polypeptide of the present invention
is a cell such as Escherichia coli, yeast, an animal cell or a
plant cell, a cultivation by a usual culture method suited to all
kinds of hosts is held. And the polypeptides are produced,
accumulated and collected from transformants or a culture solution
to produce the polypeptide. When transformants are animals or
plants, they are cultured by a usual growth method suited to all
kinds of hosts. And the polypeptides are produced, accumulated and
collected from animals or plants to produce the polypeptide.
[0138] When a host is an animal, for example, a nonhuman transgenic
animal having polynucleotides of the present invention is cultured.
And KIAA1036 polypeptides encoded by the recombinant DNA are
produced and accumulated in the animal and they are collected from
an animal to produce KIAA1036 polypeptides. A
production/accumulation place in an animal is, for example, milk,
sputum or egg of the animal.
[0139] When a host is a plant, for example, a transgenic plant
having KIAA1036 polynucleotides of the present invention is
cultured. And KIAA1036 polypeptides encoded by the recombinant DNA
are produced or accumulated in a plant and collected from a plant
to produce KIAA1036 polypeptides.
[0140] When a host is a prokaryote such as Escherichia coli or a
eucaryote such as yeast, for example, transformants having
polynucleotides of the present invention are cultured in a medium.
And KIAA1036 polypeptides encoded by the recombinant DNA are
produced or accumulated in a culture solution and collected from
the culture to produce polypeptides of the present invention.
[0141] The method that transformants of KIAA1036 of the present
invention are cultured in a medium is accomplished by a usual
method for cultivation.
[0142] When a host is prokaryote such as Escherichia coli or a
eucaryote, a natural medium or a synthetic medium can be used as a
medium that obtained transformants are cultured if it has a carbon
source, a nitrogen source and mineral that a host can assimilate
and it is a medium that cultivation of transformants is held
efficiently. For example, YT medium including bactotryptone, yeast
extract and sodium chloride is preferable as a medium when
transformants that hosts are Escherichia coli are cultured.
[0143] Any carbon source that each microorganism can assimilate can
be used, for example, glucose, fructose, sucrose, syrup including
them, carbohydrate such as starch or starch hydrolysate, an organic
acid such as acetic acid or proprionic acid or alcohol such as
ethanol or propanol.
[0144] A nitrogen source is, for example, all kinds of inorganic
acids such ammonia, ammonium chloride, ammonium sulfate, ammonium
acetate, ammonium phosphate, ammonium salts of organic acids, other
nitrogenous substances, peptone, meat extract, yeast extract, Corn
Steep Liquor, casein hydrolysate, soybean cake, soybean cake
hydrolysate, all kinds of fermentative bacteria or the digest.
[0145] A mineral is, for example, potassium phosphate monobasic,
potassium phosphate dibasic, magnesium phosphate, magnesium
sulfate, sodium chloride, ferrous sulfate, manganese sulfate,
copper sulfate or calcium carbonate. Cultivation is held under an
aerobic condition such as a shaking culture or a submerged
culture.
[0146] An incubation temperature is preferably 15 to 40.degree. C.
and a culture time is usually 5 hours to 7 days. During
cultivation, pH is kept from 3.0 to 9.0. Adjustment of pH is held
with mineral or organic acid, alkali solution, urea, calcium
carbonate or ammonia. And an antibiotic such as ampicillin or
tetracycline can be optionally added into a medium during
cultivation.
[0147] When microorganisms transforming with an expression vector
with an inductive promoter are cultured, inducers can be optionally
added into a medium. For example, when transformants transforming
with an expression vector with lac promoter are cultured, isopropyl
.beta.-D-thiogalactopyra- noside can be added into a medium. When
transformants transforming with an expression vector with trp
promoter are cultured, indoleacrylic acid can be added into a
medium. Cells and organs of plants introduced genes can do a mass
culture with jar fermenter. A culture medium is, for example,
popularly used Murashige & Skoog (MS) medium, White medium, or
a medium that plant hormones such as oxine or cytokinin are added
into these mediums.
[0148] When transformants for production of KIAA1036 polypeptide
are animal cells, a medium that cells are cultured is popularly
used RPMI1640 medium (The Journal of the American Medical
Association, 199, 519 (1967).), MEM medium (Science, 130, 432
(1959).), D-MEM medium (Virology, 8, 396 (1959).), 199 medium
(Proceedings of the Society for the Biological Medicine, 73, 1
(1950).) or a medium that fetal calf serum (FCS) is added into
these mediums.
[0149] Cultivation held usually in pH 6 to 8, at 25 to 40.degree.
C., in the presence of 5% CO.sub.2 for 1 to 7 days. And an
antibiotic such as kanamycin, penicillin or streptomycin can be
optionally added into a medium during cultivation.
[0150] When transformants are insect cells, a culture medium is
popularly used TNM-FH medium (Pharmingen), Sf-900II SFM medium
(Invitrogen), ExCell400, ExCell405 (JRH Biosciences Inc.) or
Grace's Insect Medium (Nature, 195, 788 (1962).).
[0151] (vii) The Method of Production
[0152] Transformants are cultured and KIAA1036 polypeptides are
isolated and purified from a culture to produce KIAA1036
polypeptides. A method of isolation/purification of KIAA1036
polypeptides can be usual method widely known in this field, for
example, a method of isolation/purification of an enzyme or a
method of purification of transglucosylase by Sandler (Methods in
Enzymology, 83, 458).
[0153] When KIAA1036 polypeptides are produced and accumulated as
dissolved polypeptides, a culture solution that transformants are
cultured as mentioned above is separated to cells or fungus body
and a medium, for example, by a centrifugal separation. When
KIAA1036 polypeptides exist in host cells, after cells or fungus
bodies extracted are washed with an appropriate buffer such as STE
solution and broken into pieces by ultrasonic waves, French press,
Manton Gaulin homogenizer or Dynomill, KIAA polypeptides can be
obtained as an acellular solution by a centrifugal separation or a
filtration.
[0154] The suitable quantity of surfactant can be included in a
buffer for separation/purification of KIAA1036 polypeptide. For
example, sodium lauryl sulfate (SDS) or Sodium
N-Dodecanoylsalcosinate (salcosiyl) can be used.
[0155] A method of separation/purification of target proteins
included in an obtained crude material can be accomplished with the
combination of all kinds of well-known methods of
separation/purification. The well-known method is, for example, a
solvent extraction method, a salting-out method with ammonium
sulfate, a dialysis, an sedimentation with an organic solvent, an
ultrafiltration method, a gel filtration, all kinds of
chromatography such as a diethylaminoethyl (DEAE)-sepharose
chromatography, an anion chromatography or an ion exchange
chromatography using lysine such as DIAION HPA-75 (Mitsubishi
Chemical Corporation), a cation chromatography using lysine such as
S-Sepharose FF (Pharmacia), a hydrophobic chromatography or an
affinity chromatography such as butylsepharose or all kinds of
electrophoresis such as a SDS-polyacrylamide gel electrophoresis or
an electro-focussing electrophoresis. An affinity chromatography
can be accomplished by using antibodies against KIAA1036
polypeptide.
[0156] When KIAA1036 polypeptides are produced and accumulated as
insoluble polypeptides, cells or fungus bodies are separated as
mentioned above and broken into pieces by an appropriate method.
Then a division including the polypeptides is collected. A
collected sample is solubilized with a solubilizer like a
surfactant such as sodium lauryl sulfate (SDS) or Sodium
N-Dodecanoylsalcosinate (salcosiyl). After the solubilized solution
is diluted or dialyzed to the concentration that a solubilizer is
not or almost not included and the polypeptide is constructed to a
normal stereo structure, a purification sample can be obtained by a
method of separation/purification as mentioned above.
[0157] And KIAA1036 polypeptides can be produced as fusion proteins
with the other proteins and purified by an affinity chromatography
with substances having affinity with the fusion proteins (Yamakawa
Akio, "Experimental Medicine", 13, 469-474 (1995).). An addition
protein used as a fusion protein is, for example, protein A or FLAG
(Proceedings of the National Academy of Sciences USA, 86, 8227
(1989)., Genes Development, 4, 1288 (1990)., JP1993-336963 or
JP1994-823021). When protein A are used, fusion proteins with
KIAA1036 polypeptides and protein A can be produced and purified by
an affinity chromatography with immunoglobulin G. When FLAG
peptides are used, fusion proteins with KIAA1036 polypeptides and
FLAG can be produced and purified by an affinity chromatography
with anti-FLAG antibodies.
[0158] KIAA1036 polypeptides can be produced as well as a
well-known method with in vitro transcription/translation system
(Journal of Biomolecular NMR, 6, 129-134 (1995)., Science, 242,
1162-1164 (1988), or The Journal of Biochemistry, 110, 166-168
(1991).).
[0159] KIAA1036 polypeptides can be chemosynthesized on the basis
of the amino acid sequence by a chemical synthesis method such as
Fmo method (Fluorenylmethyl oxycarbonyl method) or tBoc method
(t-butyl oxycarbonyl method), or by a peptide synthetic equipment
on the market such as, for example, APEX396 (Advanced Chemtech),
433A (Applied Biosystems), PS3 (Protein Technologies), 9050
(Perseptive) or PSSM-8 (Shimazu corporation).
[0160] A structural analysis of KIAA1036 polypeptide can be
accomplished by a usual method in the field of protein chemistry,
for example, a method described in "A protein structural analysis
for gene cloning" (Hisashi Hirano, TOKYO KAGAKU DOZIN Co., LTD.,
1993). The chordin activity of a polypeptide of the present
invention can be measured as well as a well-known assay (The
Journal of Biological Chemistry, 258, 9893-9898 (1983)., The
Journal of Biological Chemistry, 262, 15649-15658 (1987)., The
Journal of Biological Chemistry, 273, 58-65 (1998)., The Journal of
Biological Chemistry, 273, 433-440 (1998)., The Journal of
Biological Chemistry, 273, 12770-12778 (1998)., Archives of
Biochemistry and Biophysics, 270, 630-646 (1989)., Archives of
Biochemistry and Biophysics, 274, 14-25 (1989), or
JP1994-181759).
[0161] (3) A Method of Production of a Variant Polypeptide
Mutation
[0162] A deletion, a substitution or an addition of an amino acid
of KIAA1036 polypeptide is accomplished by a widely known
technique, a site-specific potentially mutagenic method. A
deletion, a substitution or an addition of 1 or few amino acid(s)
can be prepared as well as the method described in Molecular
Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring
Harbor Laboratory Press.), Current Protocols in Molecular Biology
(1994) (Wiley-InterScience), Nucleic Acids Research, 10, 6487
(1982)., Proceedings of the National Academy of Sciences USA, 79,
6409 (1982)., Gene, 34, 315 (1985)., Nucleic Acids Research, 13,
4431 (1985)., Proceedings of the National Academy of Sciences USA,
82, 488(1985)., Proceedings of the National Academy of Sciences
USA, 81, 5662 (1984)., Science, 224, 1431 (1984)., WO85/00817 or
Nature, 316, 601 (1985).
[0163] (4) A Production of an Antibody Against KIAA1036
Polypeptide
[0164] (i) A Production of a Polyclonal Antibody
[0165] Antibodies can be produced by giving all length of KIAA1036
polypeptides, a part of the peptides or polypeptides including a
part of the peptides as antigens to a mammal. The peptide itself
and a carrier, for example, a carrier combined with cattle serum
albumin (BSA), keyhole limpet hemocyanin (KLH) or bovine
thyroglobulin (BTG) can be used as an antigen. To enhance immune
reactions with antigens, for example, a complete Freund adjuvants
(CFA) and an incomplete Freund adjuvants (IFA) can be given. A
mouse, a rat, a rabbit, a goat or a hamster can be used as a mammal
to immunize.
[0166] Polyclonal antibodies can be produced, for example, as well
as the method by Lane et al. (Antibodies: A Laboratory Manual,
Second Edition (1989)(Cold Spring Harber Laboratory Press)).
[0167] After the first immunization, a mammal is immunized by an
appropriate antigen 3 to 10 times at 1- to 2-week intervals. And
then a serum from the mammal is extracted and purified to produce
the antibodies.
[0168] The antigens are given 3 to 10 times at 1- to 2-week
intervals. A preferable dosage of the antigens is 50 to 100 .mu.g
at one time per an animal. When peptides are used, peptides
covalently bonded to appropriate carriers are preferably used as
antigens. Peptides as antigens can be synthesized by a method of
genetic engineering or a peptide synthesizer. Three to seven days
after immunization, the blood from venous plexus of eyegrounds is
collected and the responsiveness of the serum against the antigens
can be measured by an enzyme-liked-immunosorbent assay
(Enzyme-liked-immunosorbent assay (ELISA): Igaku-Shoin Ltd. (1976),
Antibodies: A Laboratory Manual, Second Edition (1989)(Cold Spring
Harbor Laboratory Press).
[0169] Blood is collected from a mammal immunized and an antibody
titer is measured. Blood is collected when the immunized animal
shows a sufficient antibody titer, and then polyclonal antibodies
can be prepared from the serum. Separation and purification of
polyclonal antibodies can be accomplished by an individual or a
combination of all kinds of chromatography such as a centrifugal
separation, salting-out with ammonium sulfate, precipitation with
caplyric acid (Antibodies: A Laboratory Manual, Second Edition
(1989)(Cold Spring Harbor Laboratory Press), DEAE-sepharose column,
anion exchange column, protein A column or G-column or a gel filter
column.
[0170] (ii) A Production of a Monoclonal Antibody
[0171] (a) A Preparation of an Antibody Producing Cell
[0172] After a sufficient antibody titer in (i) is obtained, spleen
or lymph node is extracted from the mammal. And then a monoclonal
antibody producing hybridoma can be obtained by fusing an
antibody-producing cell from the spleen or lymph node with a
myeloma cell. As for the myeloma cell, cell strains established
from a mouse or a rat can be used. Cell fusion can be done
according to an already known method, for example, a method by
Kohler and Milstein (Nature, 256, 495-497 (1975).).
[0173] KIAA1036 polypeptides, a part of the peptides or
polypeptides including the part of the peptides are immunized to a
rat. Three to seven days after a rat showed a sufficient antibody
titer, the rat is immunized with the antigen for the last time, and
its spleen is extracted as antibody producing cells. The spleen is
cut to pieces in MEM medium (Nissui Pharmaceutical Co. Ltd.) and
untied with tweezers. And then a precipitant is obtained after a
centrifugation at 1,200 rpm for 5 minutes. Splenocytes is separated
by treating the precipitant with Tris-ammonium chloride buffer (pH
7.65) for 1 to 2 minutes to remove red blood cells. The splenocytes
are washed with MEM medium 3 times and are used as antibody
producing cells.
[0174] (b) A Preparation of a Myeloma Cell
[0175] An established cell line from mouse or rat is used as a
myeloma cell. For example, it is a myeloma cell of 8-azaguanine
resistance mouse (from BALB/c), P3-X63Ag8-U1 strain (described blow
as P3-U1) (Current Topics Microbiological Immunology, 81, 1 (1978),
or European Journal of Immunology, 6, 511 (1976).), SP2/0-Ag14
strain (described blow as SP-2) (Nature, 276, 269 (1978).),
P3-X63-Ag8653 strain (described blow as 653) (Journal of
Immunology, 123, 1548 (1979).) or P3-X63-Ag8 (described blow as
X63) (Nature, 256, 495 (1975).). These cell strains are subcultured
in a 8-azaguanine medium (a normal medium including 15 .mu.g/ml
8-azaguanine (RPMI1640 medium including 1.5 mM glutamine,
5.times.10.sup.-5M 2-mercaptoethanol, 10 .mu.g/ml gentamysin and
10% FCS made by CSL)) and cultured in a normal medium for 3 to 4
days before cell fusion. 2.times.10.sup.7 or more cells are
prepared for cell fusion.
[0176] (c) A Production of Hybridoma
[0177] Antibody producing cells prepared in (a) and myeloma cells
prepared in (b) are washed with MEM medium or PBS (per 1 L; 1.83 g
sodium phosphate dibasic, 0.21 g monobasic potassium phosphate,
7.65 g NaCl, pH 7.2) and mixed as the number of antibody producing
cells is 5 to 10 times larger than that of the myeloma cells. After
a centrifugal separation at 1,200 rpm for 5 minutes, a precipitant
is obtained. The precipitated cells are well untied and 0.2 to 1 ml
of polyethylene glycol solution (2 g polyethylene glycol-1000
(PEG-1000), 2 ml MEM medium, 0.7 ml dimethyl sulfoxide (DMSO)) per
10.sup.8 antibody producing cells is added to the cells with
stirring at 37.degree. C. And then 1 to 2 ml of MEM medium is added
for several times every 1 to 2 minutes. The solution is prepared
with MEM medium to 50 ml in total. After a centrifugal separation
at 900 rpm for 5 minutes, a precipitant is obtained. 100 ml of HAT
medium (normal medium including 10.sup.-4 M hypoxanthine,
1.5.times.10.sup.-5 M thymidine and 4.times.10.sup.-7 M
aminopterin) is added to a precipitant and the precipitant is
untied slowly and suspended.
[0178] The suspension is poured into the 96-well culture plate at
100 .mu.l per well and cultured at 37.degree. C. in the presence of
5% CO.sub.2 for 7 to 14 days. By the method described in "enzyme
immunoassay" (Antibodies: A Laboratory Manual, Second Edition
(1989)(Cold Spring Harbor Laboratory Press)), hybridomas producing
antibodies specifically reacting with KIAA1036 polypeptides are
selected.
[0179] (5) an Immunoassay of KIAA 1036 Polypeptide
[0180] The method to measure polypeptides or a part of the peptides
by using antibodies that recognize the polypeptides or a part of
the peptides and are directly or indirectly bonded to enzymes,
fluorescent substances, radioisotopes or latexes can be used as an
immunoassay of KIAA 1036 polypeptides. The assay is, for example,
ELISA or a chemiluminescence method detecting enzyme activities
such as horseradish peroxidase or alkaline phosphatase, FITC method
detecting fluorescent tags such as luminol or GFP (Green
Fluorescence Protein), RIA method detecting radioisotope tags such
as 125I or a latex agglutination method detecting binding with
latex.
[0181] And the assay is, for example, Western blotting or an immune
structure dyeing method.
[0182] Furthermore, KIAA1036 polypeptides or a part of peptides can
be quantitated by the assay.
[0183] Antibodies for an immunoassay can be immobilized to a solid
phase carrier and the trapped polypeptides can be detected by using
secondary antibodies with a reporter group or using reagents. A
competitive method that KIAA1036 polypeptides are labeled with a
reporter group, reacted with antibodies and a sample and bonded
with immobilized antibodies can be used to detect. The level of
inhibition the binding between the labeled polypeptides and
antibodies by KIAA1036 polypeptides of a sample is shown by
reactivity with immobilized antibodies of a sample and the
concentration of KIAA1036 polypeptides in a sample can be
calculated. Any substance, to which antibodies can attach and which
is widely known to a parson having ordinary skill in the art, can
be used as a solid phase carrier. The substance includes, for
example, a microtitre plate, a membrane such as a nitrocellulose
membrane, bead, disk, glass, glass fiber, plastic material such as
latex, polystyrene or polyvinyl chloride. Magnetic particles or
fiber optical sensors (U.S. Pat. No. 5,359,681) can be used. A
well-known method used in this field can be used as a method which
antibodies are immobilized to a carrier. In this description,
"solid phase" means immobilization by a physical method such as
adsorption or a chemical binding by a covalent bond between an
antibody and a functional group on a carrier. An antibody and a
functional group on a carrier can be bonded directly or through a
cross-linking agent.
[0184] Immobilization by a physical method can be accomplished by
appropriately diluted antibodies contacted with a carrier,
preferably, a microtiter plate or a membrane in an appropriate
buffer for an appropriate time. The contact time varies depending
on the temperature, but it is typically between about 1 hour and 1
day. About 10 ng to 1 .mu.g, preferably, about 100 to 200 ng of
antibodies is added and immobilized on each well of a microtiter
plate made of plastic such as polystyrene or polyvinyl
chloride.
[0185] Immobilization by a chemical method can be accomplished by a
reaction of a carrier and functional groups of antibodies, for
example, a reaction of a carrier and a two-functional reagent that
reacts with both hydroxyl groups and amino groups and a carrier.
For example, antibodies can be immobilized to a carrier having an
appropriate polymer coat with a covalent bond by using benzoquinone
or a condensation between aldehyde groups on a carrier and an amine
or an active hydrogen on a combination partner. The method can be
accomplished by using for example, Pierce immunotechnology Catalog
and Handbook (1991) A12 to A13 as a reference.
[0186] A carrier-immobilized antibody is treated to inhibit
physical adsorption of other polypeptides by a well-known method
for a parson having ordinary skill in the art with an appropriate
blocking reagent, for example, cattle serum albumin or Tween 20
(Sigma-Aldrich).
[0187] A carrier-immobilized antibody is reacted with a sample and
polypeptides of the present invention and antibodies are combined.
A sample can be appropriately diluted with an appropriate diluent,
for example, phosphate buffered saline solution (PBS). A reaction
time of a sample and antibodies should be enough to detect the
presence of polypeptides of the present invention in a sample
obtained from an individual having a cancer, preferably, a time to
achieve at least 95% of binding level compared to the level at
which bound and not-bound polypeptides are equilibrated. A time to
reach to equilibrium can be easily decided by measuring the binding
level by the time. Substances other than bound polypeptides can be
removed by washing a solid carrier with an appropriate buffer, for
example, PBS (including 0.1% Tween 20). Labeled secondary
antibodies are reacted with a solid carrier. The labels are
preferably enzymes such as horseradish peroxidase, ground
substances, supplemental elements, inhibitors, pigments,
radioisotopes, coloring substances or fluorescent substances. The
binding between antibodies and labels can be accomplished by a
well-known method for a parson having ordinary skill in the art.
The secondary antibodies are reacted for a sufficient time to bind
to complexes, which include immobilized antibodies and polypeptides
of the present invention. An appropriate time can be easily decided
by measuring binding level by the time. The non-binding secondary
antibodies can be removed by washing a solid carrier with an
appropriate buffer, for example, PBS (including 0.1% Tween 20). The
method of detection of labels of the secondary antibodies is
different by a kind of labels. When radioisotopes are used as
labels, detection by a scintillation counter or an autoradiography
can be used. When pigments, coloring substances or fluorescent
substances are used as labels, detection by a spectrophotometer can
be used. When enzymes are used as labels, ground substances against
the enzymes are added and reacted for a fixed time and the products
are detected by a spectrophotometer. Labels and secondary
antibodies can bind directly or indirectly by an avidin-biotin
method. When they bind indirectly, one part of the avidin-biotin is
bound to a secondary antibody and another is bound to a label.
KIAA1036 polypeptides can be detected by a flow through test or a
strip test. In a flow through test, a sample is added to a
nitrocellulose membrane on which antibodies are immobilized, and
when a sample passes through the membrane, polypeptides bind to the
immobilized antibodies to form immune complexes. And when a
solution including labeled secondary antibodies pass through the
membrane, it binds to the immune complexes. In a strip test, once a
sample is added, the sample pass through a region including labeled
antibodies, and polypeptides bind to labeled antibodies to form
immune complexes. When a sample pass through a region including a
solid phase antibodies, polypeptides bind to the immune complexes.
The quantity of secondary antibodies detected in the region with
immobilized antibodies shows the presence or absence of cancer.
Labels in the detection part can be visualized for confirmation. If
it can not be confirmed, it shows a negative result. A quantity of
antibodies immobilized to a membrane is preferably about 25 ng to 1
.mu.g and more preferably about 50 ng to 1 .mu.g.
[0188] (6) An Assay of mRNA
[0189] Using oligonucleotides prepared from KIAA1036
polynucleotides, mRNAs are quantitated by Northern hybridization or
RT-PCR, and then expression level of a gene encoding KIAA1036
polypeptide can be quantitated.
[0190] When mRNAs are quantitated with normal or disease model
animals, for example, mice, rats, rabbits, sheep, pigs, cattle,
cats, dogs or monkeys, an agent such as an anticancer agent is
optionally given. After a fixed time, organs or tissues such as
blood, brain, stomach or kidney is isolated and cells are prepared.
mRNAs can be extracted from obtained cells by a widely known and
usual method in this field and quantitated/analyzed by RT-PCR or
Northern blot hybridization.
[0191] When mRNAs from transformants, which express KIAA1036
polypeptides or a part of the peptides, are quantitated, mRNAs can
be extracted from the transformants by a widely known and usual
method in this field and quantitated/analyzed by RT-PCR or Northern
blot hybridization.
[0192] (7) Identification of a Structure of the Gene
[0193] Recently, a lot of sequences of human genome whose functions
are unknown are registered on databases. Therefore, by comparing
KIAA1036 polynucleotide sequence and a sequence of human genome
registered on databases, human gene encoding KIAA1036 polypeptide
can be identified and a structure of the gene can be clarified. If
a genomic sequence, which coincides with cDNA sequence, is
registered, a promoter region and a structure of exon and intron of
the gene encoding a polypeptide of the present invention can be
screened by comparing cDNA sequence and a sequence of the gene. By
using KIAA1036 polynucleotide as a probe and a widely known and
usual method in this field (Division of Oncology, Institute of
Medical Science, Univ. of Tokyo, New Protocol for Molecular and
Cellular Biology, Shujunsha (1993).), a promoter region of the gene
can be obtained. Promoter regions are all of the promoter regions
concerned with transcription of a gene encoding KIAA1036
polypeptide in mammal cells.
[0194] (8) A Method of Detection of Disease
[0195] KIAA1036 polypeptides can be used for diagnosis of diseases
relating to neovaucularization, for example, vascular disease,
inflammatory disease, entoptic neovascular disease, reproductive
system disease, central nervous system disease or cancer because
the expression is increased in angiogenesis models. Furthermore,
because the expression of the polypeptides is increased in ovarian
cancer and large bowel cancer tissue, it can be used for diagnosis
of these cancers. Polypeptides of the present invention or
polynucleotides such as mRNAs are specifically detected from
biological samples, for example, cancer or normal tissue specimen,
dissected tissue, blood, lymph node, serum, urine or other tissues
obtained from a patient and their homogenates or extracts. Then
presence or absence of cancer in a patient can be decided by
comparing the expression level in a patient with a level in a
normal person or a normal tissue.
[0196] The method of detection of disease includes, for example, an
immunoassay or a molecular-biological method of detection of the
polypeptides.
[0197] An immunoassay of the polypeptides includes, for example,
sandwich ELISA. In this method, a pair of antibodies recognizing
different epitopes of the polypeptide are used and one antibody is
labeled directly or indirectly with an enzyme. Another method is a
competitive RIA by using the polypeptides labeled with
radioisotopes such as .sup.125I and antibodies recognized them.
[0198] A molecular-biological method of detection includes, for
example, in situ hybridization or PCR. PCR can be used to amplify
cDNA prepared from mRNA isolated from a biological sample from a
patient. A primer for PCR can be chemically synthesized on the
basis of DNA sequence of SEQ ID NO: 1. Preferred is Primer1 which
is an oligonucleotide sequence of SEQ ID NO:
4,5'-GTTCAGGACTGTCTTTCAGC-3', or Primer2 which is an
oligonucleotide sequence of SEQ ID NO:
5,5'-GTCAATACTGATGGACTTGC-3'. cDNA amplified by PCR can be
separated by gel electrophoresis and then turned visible by the
method widely known to a parson having ordinary skill in the art.
Usually, samples are appropriately prepared from a pair of tissues,
which are a cancer tissue and a normal tissue from the same
individual or different individuals and subjected to PCR. An
amplification reaction by PCR is preferably held with a serial two
orders dilution of template cDNAs. In some dilution points, more
than two-fold increase in expression of a cancer sample compared to
a normal sample is considered as a positive result.
[0199] The presence or absence of cancer is judged by measuring
markers of the present invention detected in a normal sample and a
patient sample and comparing. Generally, a mean value of markers
from a healthy person without cancer is considered as a cut-off
value and the cut-off value is compared to detected value of
markers from a patient sample. Generally, when the detected value
is over three times of the standard deviation plus a cut-off value,
the sample is considered as a positive about cancer. And a cut-off
value is decided by Receiver Oparator Curve described in Sackett et
al., Clinical Epidemiology: A Basic Science for Clinical Medicine
106-107, (1985)(Little Brown and Co).
[0200] (9) A Diagnostic Kit
[0201] KIAA1036 polynucleotides can be used for a diagnosis of
neovascularization related disease, for example, vascular disease,
inflammatory disease, entoptic neovascular disease, reproductive
system disease, central nervous system disease or cancer such as
ovarian cancer or large bowel by Northern hybridization or PCR. And
a diagnosis of neovascularization related disease, for example,
vascular disease, inflammatory disease, entoptic neovascular
disease, reproductive system disease, central nervous system
disease or cancer such as ovarian cancer or large bowel can be held
by an immunologic method with antibodies against KIAA1036
polypeptides. Therefore, in the case of a diagnosis with
polynucleotides, labeled KIAA1036 polynucleotides are contained in
a kit. And in the case of a diagnosis with antibodies, KIAA1036
polypeptides are contained as standard antigens in addition to
antibodies against KIAA1036 polypeptides. In a kit, a standard
curve can be contained.
[0202] (10) The Method to Inhibit mRNA Translation
[0203] Giving KIAA1036 polynucleotides can inhibit the production
of KIAA1036 polypeptides. Namely, by using KIAA1036
polynucleotides, transcription of DNA encoding KIAA1036 polypeptide
and translation of mRNA encoding the polypeptide can be
inhibited.
[0204] (11) The Screening Method of Binding Substances
[0205] As a reagent for search or screening binding substances to
markers of the present invention, KIAA1036 polypeptides are useful.
Namely, the screening method of binding substances to markers of
the present invention, which comprises contact between markers and
subjects, is provided. Subjects include, for example, chemical
substances synthesized naturally or chemically, proteins, tissue
extracts of a mammal such as human, mouse, rat, pig, cattle, sheep
or monkey or cell culture. These subjects are added to markers of
the present invention and the mixture is separated with measuring
DNA synthesis activity of vascular endothelial cells. Finally,
single ligands can be obtained.
[0206] For example, the screening method of binding substances of
the present invention includes a method by using KIAA1036
polypeptides or the partial peptides, by constructing an expression
system of recombinant polypeptides and using a binding assay with
the expression system, by binding to KIAA1036 polypeptides and
measuring cell stimulatory activity, for example, activity to
promote or inhibit DNA synthesis by bringing BrdU
(bromodeoxyuridine) or cell migrating activity or by measuring
visually network formation of vascular endothelial cells with
microscope.
[0207] At first, any polypeptide comprising polypeptide B can be
used as polypeptides for the screening method of the binding
substances but polypeptides expressed massively with animal cells
is preferable. To produce polypeptide B, the above mentioned
expression method is used, but the method is preferable when it
includes expressing DNA encoding the polypeptide in mammal cells or
insect cells. Generally cDNA is used as a fragment which encodes a
protein to be expressed, but it should not be always cDNAs but
fragment from genomic DNA or synthetic DNA can be used. To
introduce DNA fragments encoding KIAA1036 polypeptides into host
animal cells and effectively express them, it is preferable that
the DNA fragments are inserted downstream of a polyhedrin promoter
of nuclear polyhedrosis virus (NPV) of baculovirus which use
insects as a host, a promoter from SV40, a promoter of retrovirus,
a metallothionein promoter, a human heat-shock promoter, a
cytomegalovirus promoter or a SR .alpha. promoter. The examination
of quantity and quality of expressed polypeptides can be done by a
well-known method. For example, it is the method described in The
Journal of Biological Chemistry, 267, 19555 to 19559 (1992).
[0208] Therefore, in a screening method of binding substances of
the present invention, polypeptides or the partial peptides
purified by the well-known method, cells including the polypeptides
or their supernatants can be used as a thing including KIAA1036
polypeptides or the partial peptides. In a screening method of
binding substances of the present invention, when cells including
KIAA1036 polypeptides are used, that cells can be immobilized to
agarose gel. The method of immobilization can be held by the
well-known method. Cells including KIAA1036 polypeptides mean host
cells with expressing KIAA1036 polypeptides. For example,
Escherichia coli, Bacillus subtilis, yeast, insect cells and animal
cells are used as the host cells.
[0209] For the screening of binding substances to KIAA1036
polypeptides, appropriate polypeptide fractions and labeled test
substances are needed. As a polypeptide fraction, for example, a
natural polypeptide fraction or a recombinant polypeptide fraction
having the same activity as a natural one is preferable. The same
activity means, for example, the same ligand binding activity or
the same signal transduction activity. The labeled test substances
are, for example, substances labeled with .sup.3H, .sup.125I,
.sup.14C or .sup.35S.
[0210] For example, for a screening method of binding substances to
KIAA1036 polypeptides, at first, a polypeptide sample is prepared
by suspending cells including KIAA1036 polypeptides or a membrane
fraction of the cells in a buffer which is suitable for a screening
method. Any buffer, which does not inhibit combining a ligand and a
polypeptide, for example, a phosphoric acid buffer that is pH 4 to
10, preferably pH 6 to 8 or a Tris-HCl buffer is used as a buffer.
And to decrease non-specific binding, a surfactant, for example,
CHAPS, Tween-80 (Kao-Atlas), digitonin or deoxycolate or all kinds
of protein, for example, cattle serum albumin or gelatin can be
added into the buffer. Furthermore, to repress degradation of
receptors and ligands by protease, a protease inhibitor, for
example, PMSF, leupeptin, E-64 (PEPTIDE INSTITUTE, INC.) or
pepstatin can be added. Test substances are labeled with, for
example, constant quantity, preferably 5000 cpm to 500000 cpm of
3H, .sup.125I, .sup.14C or .sup.35S are coexistence in 0.01 to 10
ml of the polypeptide solution. To measure the quantity of
non-specific binding (NSB), a reaction tube with excessive amount
of unlabeled test substances is also prepared. The reaction is held
at 0.degree. C. to 50.degree. C., preferably at 4.degree. C. to
37.degree. C., for 20 minutes to 24 hours, preferably for 30
minutes to 3 hours. After the reaction, the solution is filtered by
glass fiber filter papers and washed with a suitable quantity of
the same buffer. The radioactivity remaining on the glass fiber
filter paper is measured by a liquid scintillation counter or a
.gamma.-counter. Test substances that the count (B-NSB), the result
obtained by subtracting quantity of non-specific binding (NSB) from
all quantity of binding (B), is over 0 cpm can be selected as
binding substances to markers of the present invention.
[0211] For the screening of binding substances to markers of the
present invention, cell stimulatory activity through the
polypeptides, for example, activity to promote or inhibit DNA
synthesis by bringing BrdU (bromodeoxyuridine) or cell migrating
activity can be measured by well-known method or a measuring kit on
the market. And the activity can be measured by measuring visually
network formation of vascular endothelial cells with microscope.
For example, at first, cells including polypeptides are cultured on
the multiwell plate. When the screening of binding substances is
held, in advance, medium or buffer changes to fresh medium or
appropriate buffer, which is non-toxic to cells and test substances
are added. After incubation for a fixed time, cells are extracted
or their supernatants are collected and the products are
quantitated by the method for them.
[0212] (12) A Kit for Screening of Binding Substances
[0213] A kit for screening of binding substances bonded to markers
of the present invention is a kit comprising cells expressing
polypeptide B or the polypeptides or their supernatant fraction
including the polypeptides. The examples of a kit for screening of
binding substances are the followings.
[0214] (i) A Reagent for Screening of Binding Substances
[0215] As a screening solution and washing solution, a solution
that Hank's Balanced Salt Solution (Invitrogen) added 0.05% cattle
serum albumin (SIGMA-Aldrich) is sterilized by filtration with 45
.mu.m filter, and it is kept at 4.degree. C. or prepared just
before use.
[0216] (a) COS-7 Cells Expressing Polypeptides of the Present
Invention are Cultured on a 12-well Plate with 5.times.10.sup.5
Cells/well and Cultured at 37.degree. C. for 2 Days.
[0217] (b) A Labeled Subject
[0218] A solution containing commercially available subjects
labeled with .sup.3H, .sup.125I, .sup.14C or .sup.35S is kept at
4.degree. C. or 20.degree. C., and diluted to 1 .mu.M with assay
buffer just before use. Subjects with the low solubility to water
are dissolved in dimethyl formamide, DMSO or methyl alcohol.
[0219] (c) A Non-Labeled Subject
[0220] The same things as labeled substances are prepared at 100 to
1000 times higher concentration.
[0221] (ii) Assay
[0222] (a) COS-7 cells expressing polypeptides of the present
invention cultured on a 12-well plate for tissue culture are washed
twice with assay buffer (iml) and then assay buffer is added (490
.mu.l per well).
[0223] (b) Labeled subjects (5 .mu.l) are added and the solution is
reacted at a room temperature for 1 hour. Non-labeled subjects (5
.mu.l) are added to measure the quantity of unspecific binding.
[0224] (c) The reaction solution is removed and the remaining cells
are washed with a washing solution (1 ml) 3 times. Labeled subjects
bound to cells are dissolved with 0.2M NaOH (included 1% SDS) and
the solution is mixed with liquid scintillator A (4 ml) (Wako Pure
Chemical Industries, Ltd.).
[0225] (d) The radioactivity is measured by liquid scintillation
counter (Beckman Coulter).
[0226] (13) A Screening Method of Binding Activity Regulatory
Substances
[0227] A marker of the present invention is useful as a reagent to
search or screen binding activity regulatory substances to the
markers. As a screening method of binding activity regulatory
substances of the markers and binding substances, namely,
substances changing the binding activity, a recombinant expression
system expressing markers of the present invention is constructed
and bioassay system or binding assay system is used. Then
substances changing the binding activity between binding substances
and markers of the present invention, for example, peptides,
proteins, nonpeptide substances, synthetic substances or ferment
products can be efficiently screened. The substances comprise a
substance which can enhance or reduce (a) cell stimulatory activity
between binding substances and a marker of the present invention,
for example, an activity to promote or inhibit DNA synthesis
monitored by BrdU (bromodeoxyuridine) incorporation or an activity
to promote or inhibit of cell migrating activity, or (b) the
binding activity between a binding substance and a marker of the
present invention.
[0228] Namely, the present invention provides with a screening
method of binding activity regulatory substances between binding
substances and markers of the present invention characterized by
comparing the quantity of binding of binding substances when (i)
markers of the present invention and binding substances are
contacted and (ii) markers of the present invention, binding
substances and subjects are contacted.
[0229] A screening method of binding activity regulatory substances
of the present invention is characteristic of comparing the
quantity of binding between the markers and binding substances by
measuring, for example, cell stimulatory activity in the case of
(i) and (ii).
[0230] A screening method of binding activity regulatory substances
of the present invention is, for example, (a) a method to screen
binding activity regulatory substances which can change the binding
activity between binding substances and KIAA1036 polypeptides,
comprising measuring and comparing the quantity of binding of
labeled substances to the polypeptides when labeled binding
substances are contacted to KIAA1036 polypeptides and when labeled
binding substances and subjects are contacted, (b) a method to
screen binding activity regulatory substances which can change the
binding activity between binding substances and polypeptides of the
present invention comprising measuring and comparing the quantity
of binding of label binding substances to the polypeptides in the
cells or cell culture when cells expressing KIAA1036 polypeptides
or the cell culture solution and labeled binding substances are
contacted and when labeled binding substances and subjects are
contacted or (c) a method to screen binding activity regulatory
substances which can change the binding activity between binding
substances and KIAA1036 polypeptides comprising measuring and
comparing the quantity of binding of label binding substances to
the polypeptides when polypeptides secreted in cell culture
solutions by culturing transformants having KIAA1036
polynucleotides and label binding substances are contacted and when
label binding substances and subjects are contacted.
[0231] (14) A Kit for Screening of Binding Activity Regulatory
Substances
[0232] A kit for screening of binding activity regulatory
substances changing the binding activity between KIAA1036
polypeptides and binding substances is a kit comprising cells
including the polypeptides or a cell culture solution. The examples
of a kit for screening of the present invention are the
followings.
[0233] (i) A Reagent for Screening of Binding Activity Regulatory
Substances
[0234] As a screening solution and washing solution, a solution
that Hank's Balanced Salt Solution (Invitrogen) added 0.05% cattle
serum albumin (SIGMA-Aldrich) is sterilized by filtration with 45
.mu.m filter, and it is kept at 4.degree. C. or prepared just
before use.
[0235] (a) KIAA1036 Polypeptide
[0236] COS-7 cells expressing KIAA1036 polypeptides are cultured on
a 12 well plate with 5.times.10.sup.5 cells/well and cultured at
37.degree. C. for 2 days.
[0237] (b) A Labeled Subject
[0238] A solution containing commercially available subjects
labeled with .sup.3H, .sup.125I, .sup.14C or .sup.35S is kept at
4.degree. C. or 20.degree. C., and diluted to 1 .mu.M with assay
buffer and prepared just before use. Subjects with the low
solubility to water are dissolved in dimethyl formamide, DMSO or
methyl alcohol.
[0239] (c) A Non-Labeled Subject
[0240] The same things with labeled substances are prepared at 100
to 1000 times higher concentration.
[0241] (ii) An Assay
[0242] (a) COS-7 cells expressing polypeptides of the present
invention cultured on a 12-well plate for tissue culture are washed
twice with assay buffer (1 ml) and then assay buffer is added (490
.mu.l per well).
[0243] (b) Labeled subjects (5 .mu.l) are added and the solution is
reacted at room temperature for 1 hour. Non-labeled subjects (5
.mu.l) are added to measure the quantity of non-specific
binding.
[0244] (c) The reaction solution is removed and the remaining cells
are washed with a washing solution (1 ml) 3 times. Labeled subjects
binding to cells are dissolved with 0.2M NaOH (included 1% SDS) and
the solution is mixed with liquid scintillator A (4 ml) (Wako Pure
Chemical Industries, Ltd.).
[0245] (d) The radioactivity is measured by liquid scintillation
counter (Beckman Coulter).
[0246] (15) A Screening Method of Expression Regulatory
Substances
[0247] Polynucleotide A, polypeptide B or antibodies against the
polypeptides are useful as a reagent to search or screen expression
regulatory substances to markers of the present invention.
[0248] Namely, a screening method of expression regulatory
substances of the present invention is, for example, a screening
method of expression regulatory substances of markers of the
present invention by measuring quantity of mRNAs or proteins of
KIAA1036 polypeptides or the partial peptides included in (i) blood
of a non-human mammal, a specific organ, a tissue or cells isolated
from the organ or (ii) transformants and so on.
[0249] (16) A Kit for Screening of Expression Regulatory
Substances
[0250] A kit for screening of expression regulatory substances of
KIAA1036 polypeptides or its salt is a kit comprising cells
expressing polypeptide A or polypeptide B or cell culture solutions
including the polypeptides. The Example of a kit for screening of
the present invention is the following.
[0251] (17) A Kit for Screening on the Basis of the Immunologic
Assay
[0252] An assay of KIAA1036 polypeptides is, for example, sandwich
ELISA using two kinds of monoclonal antibodies that their epitopes
are different in antibodies reacting with the polypeptide in the
liquid phase, or a radioimmunoassay using KIAA1036 polypeptides
labeled with a radioisotope such as .sup.126I and antibodies
specifically recognizing it. Therefore, in a case of diagnosis with
antibodies, KIAA1036 polypeptide is included as standard antigens
in addition to antibodies of the present invention. Furthermore,
standard curve can be included in a kit.
[0253] (18) A Kit for Screening on the Basis of a
Molecular-Biological Assay
[0254] The expression level of DNA encoding KIAA1036 polypeptide
can be quantitated in mRNA level by Northern hybridization or PCR
using oligonucleotides prepared from KIAA1036 polynucleotides.
[0255] For example, (i) an agent, for example, an anticancer agent
is given into a normal or a disease model non-human mammal (e.g.,
mouse, rat, rabbit, sheep, pig, cattle, cat, dog or monkey) and
after a fixed time, blood, a specific organ, for example, brain,
kidney, colon, ovary, tissues or cells isolated from an organ are
obtained. mRNAs of a polypeptide of the present invention or the
partial peptide included in the obtained cells can be for example,
extracted by the extraction method widely known in this field,
quantitated by the method such as TaqManPCR or analyzed by Northern
blot widely known, or (ii) transformants expressing KIAA1036
polypeptides or the partial peptides are produced by the above
mentioned method and mRNAs of KIAA1036 polypeptides or the partial
peptides included in the transformant can be
quantitated/analyzed.
[0256] And in a case of diagnosis with polynucleotides, labeled
KIAA1036 polynucleotides are included in a kit.
[0257] (19) A Pharmaceutical Composition
[0258] A pharmaceutical composition comprising at least one
selected from substances such as polynucleotide A, a complementary
polynucleotide to the polynucleotide, polypeptide B, its antibody
or a gene therapeutic vector having polynucleotide A can be given
independently as a therapeutic agent. But preferred is usually that
a pharmaceutical composition is mixed with one or more
pharmaceutically acceptable carrier(s) and provided as a
pharmaceutical preparation produced by the any method widely known
in the technical field of galenical pharmacy. Because KIAA1036
inhibit DNA synthesis in vascular endothelial cells and cell
migrating activity, DNA synthesis inhibitors and cell migrating
activity inhibitors are included. And substances obtained by a
screening method or a kit for screening of the present invention
and their salts are included. The substance is, for example, (a) a
substance enhancing or reducing cell stimulation activity through
binding between binding substances and KIAA1036 polypeptides, for
example, DNA synthesis activity, migrating activity, network
formation activity of vascular endothelial cells, (b) a substance
enhancing or reducing a binding activity between a binding
substance and KIAA1036 polypeptide or (c) a substance enhancing or
reducing an expression level of KIAA1036 polypeptide. The substance
is, for example, a low-molecular weight compound, a peptide, a
protein, a nonpeptide substance, a synthetic substance or a ferment
product. These substances can be new or well-known substances and
natural or synthetic substances. Because agonists against KIAA1036
polypeptides have the same activity as bioactivation of binding
substances against KIAA1036 polypeptides, they are useful as safe
and low-toxic drugs for the binding substance activity. Because
antagonists against KIAA1036 polypeptides can inhibit bioactivation
of binding substances against KIAA1036 polypeptides, they are
useful as safe and low-toxic drugs, which inhibit the binding
substance activity. Substances enhancing binding activity between
binding substances and KIAA1036 polypeptides are useful as safe and
low-toxic drugs to enhance bioactivation of binding substances
against KIAA1036 polypeptides. Substances reducing binding activity
between binding substances and KIAA1036 polypeptides are useful as
safe and low-toxic drugs to reduce bioactivation of binding
substances against KIAA1036 polypeptides. And gene therapeutic
expression vectors expressing KIAA1036 are included. Because
KIAA1036 can be a marker for neovascularization, vascular disease,
inflammatory disease, entoptic neovascular disease, reproductive
system disease, central nervous system disease or cancer, it is
suggested that KIAA1036 relates to those diseases and they are
useful for gene therapies of these diseases. The gene therapeutic
expression vectors are expression vectors having a part or all of a
polynucleotide of KIAA1036 and when they are introduced into cells
or tissues, they can normalize disorders which cause some natural
or acquired disease at a gene level. In other words, the vectors
can compensate for cells with normal genes, repair/modify defects
of genes and regulate expression of genes.
[0259] (20) A Therapeutic Agent
[0260] A pharmaceutical composition comprising polypeptide B, an
antibody against the polypeptide, polynucleotide A, complementary
polynucleotide to the polynucleotide, a binding substance, a bond
regulatory substance, an expression regulatory substance, a DNA
synthesis inhibitor, a cell migrating activity inhibitor or a gene
therapeutic vector can be given independently as a therapeutic
agent. But preferred is usually that a pharmaceutical composition
is mixed with one or more pharmaceutically acceptable carrier(s)
and provided as a pharmaceutical preparation produced by the any
method widely known in the technique field of galenical
pharmacy.
[0261] The most effective route of administration in treats is
desirable as a route of administration, for example, an oral
administration or a non-oral administration such as intraoral,
tracheobronchial, intrarectal, subcutaneous, intramuscular or
intravenous. A form of administration is, for example, spray,
capsule, tablet, granule, syrup, emulsion, suppository, injection,
ointment or tapes.
[0262] An appropriate pharmaceutical preparation for an oral
administration is, for example, emulsion, syrup, capsule, tablet,
powder or granule. For example, a liquid preparation such as
emulsion or syrup can be produced by using water, saccharide such
as sucrose, sorbitol or fructose, glycol such as polyethylene
glycol or propylene glycol, oil such as sesame oil, olive oil or
soybean oil, antiseptic such as p-hydroxy ester benzoate or flavor
such as strawberry or peppermint as a excipient. Capsule, tablet,
powder or granule can be produced by using a vehicle such as
lactose, dextrose, sucrose or mannitol, a disintegrating agent such
as starch or sodium alginate, a lubricant such as magnesium
stearate or talc, a binding agent such as polyvinyl alcohol,
hydroxypropylcellulose or gelatin, a surface active agent such as
fatty acid ester or a plasticizer such as glycerol as a
excipient.
[0263] An appropriate pharmaceutical preparation for non-oral
administration is, for example, injection, suppository or spray.
For example, an injection is prepared by using a carrier comprising
salt solution, dextrose solution or their mixture. A suppository is
prepared by using a carrier such as cacao butter, fat hydride or
carboxylic acid. And a spray is prepared by using the substance or
a carrier, which does not stimulate oral cavity and airway mucous
membrane of a recipient and spread the substance as microparticles
to become easy to absorb. The carrier includes, for example,
lactose or glycerol. A pharmaceutical preparation such as aerosol
or dry powder is possible if a nature of the substance and that of
the used carrier are appropriate for it. And in these non-oral
agents, a component used as an excipient in oral agents can be
added.
[0264] A substance obtained by using a screening method or a kit
for screening of the present invention or the salt is a binding
substance, a binding activity regulatory substance, an expression
regulatory substance, a DNA synthesis inhibitor or a cell migrating
activity inhibitor, for example, (a) a substance enhancing or
reducing cell stimulation activity through bonds between binding
substances and polypeptide B, for example, an activity to promote
or inhibit DNA synthesis, which can be monitored by BrdU
(bromodeoxyuridine) uptake, or an activity to promote or inhibit of
cell migrating activity, (b) a substance enhancing or reducing a
binding activity between binding substances and polypeptide B or
(c) a substance enhancing or reducing an expression level of a
marker of the present invention.
[0265] The substance is a low-molecular weight compound, peptide,
protein, non-peptide substance, a synthetic substance or a ferment
product. These substances can be new or well-known substances and
natural or synthetic substances. Because agonists against KIAA1036
polypeptides have the same activity as bioactivation of binding
substances against KIAA1036 polypeptides, they are useful as safe
and low-toxic drugs for the binding substance activity. Because
antagonists against KIAA1036 polypeptides can inhibit bioactivation
of binding substances against KIAA1036 polypeptide or something
like this, they are useful as safe and low-toxic drugs, which
inhibit the binding substance activity. Substances enhancing
binding activity between binding substances and KIAA1036
polypeptides are useful as safe and low-toxic drugs to enhance
bioactivation of binding substances against KIAA1036 polypeptides.
Substances reducing binding activity between binding substances and
KIAA1036 polypeptides are useful as safe and low-toxic drugs to
reduce bioactivation of binding substances against KIAA1036
polypeptides. Substances reducing binding activity between binding
substances and KIAA1036 polypeptides are useful as safe and
low-toxic drugs to reduce bioactivation of binding substances
against KIAA1036 polypeptides. And therapeutic agent KIAA1036
polypeptides for any kinds of diseases comprising substances
changing the expression level of KIAA1036 polypeptides seem to have
any important role, for example, a central nervous system as
described above. Therefore an expression regulatory substance
changing the expression level of KIAA1036 polypeptides or the
partial peptide can be used as a therapeutic agent for a disease
related insufficiency of KIAA1036 polypeptides. When the substance
is used as a therapeutic agent for a disease relating insufficiency
of KIAA1036 polypeptide, it can be turned a pharmaceutical
preparation by the stereotyped method. For example, the substance
can be used orally as a tablet with sugar-coating, a capsule, an
elixir or a microcapsule as occasion demandsor parentally as a
sterile solution with water or the other pharmaceutically
acceptable solution or an injection of suspension. For example, the
substances can be produced by mixing as the unitary dose form
required to accomplish popularly acceptable pharmaceutical
preparation with physiologically acceptable well-known carrier,
spice, vehicle, antiseptic, stabilizer or binding agent. The amount
of active principle in these pharmaceutical preparations is decided
to be able to obtain an appropriate capacity in a directed
range.
[0266] An excipient, which can mix with tablet or capsule, is, for
example, a binding agent such as gelatin, corn starch, tragacanth
or Arabian gum, a vehicle such as crystal cellulose, a swelling
agent such as corn starch, gelatin or alginic acid, a lubricant
agent such as magnesium stearate, a sweetening agent such as
sucrose, lactose or saccharin or a spice such as peppermint or
cherry. In the case where the dosage form is a capsule, the above
type of ingredients and a liquid carrier such as fat and oil can be
included. An aseptic composition for the injection can be
prescribed by the usual pharmaceutical preparation, for example, by
dissolving or suspending an activity substance in vehicle such as
water for the injection or natural plant oil such as sesame oil or
coconut oil. An aqueous solution for the injection is, for example,
an isotonic solution such as D-sorbitol, D-mannitol or sodium
chloride including physiological salt solution, dextrose or
adjuvant. It can be used with appropriate solubilizing agent, for
example, alcohol such as ethanol, propylene glycol or polyethylene
glycol, a nonionic surface-active agent such as polysorbate 80 or
HCO-50. Oil solution is, for example, sesame oil or soybean oil and
it can be used with a solubilizing agent such as benzyl benzoate or
benzyl alcohol.
[0267] And the above preventive/therapeutic agent can be mixed
with, for example, a buffer such as phosphate buffer or sodium
acetate buffer, a soothing agent such as benzalkonium chloride or
procaine hydrochloride, a stabilizer such as human serum albumin or
polyethylene glycol, a preservatives such as benzyl alcohol or
phenol or an antioxidant. A prepared injection solution is usually
filled up into an appropriate ampoule.
[0268] (21) A Method of Treating
[0269] Because the above-mentioned pharmaceutical composition can
be a therapeutic agent for vascular disease, inflammatory disease,
entoptic neovascular disease, reproductive system disease, central
nervous system disease or cancer, a method of treating disease such
as vascular disease, inflammatory disease, entoptic neovascular
disease, reproductive system disease, central nervous system
disease or cancer is provided by giving that pharmaceutical
composition by an appropriate method. It is desirable that the
above-mentioned pharmaceutical composition is used in the most
effectively medication method in treats. And the method is, for
example, an oral administration or a non-oral administration such
as intraoral, tracheobronchial, intrarectal, subcutaneous,
intramuscular or intravenous.
[0270] A therapeutic agent as described above is safe and low-toxic
and can be given to, for example, a mammal such as human, rat,
mouse, rabbit, sheep, pig, cattle, cat, dog or monkey. A dosage of
a preventive or therapeutic agent per 1 time varies depending on
medication subject, an object organ, a condition of a disease or a
medication method. But in the case of a oral administration, for
example, for a patient with hypertonia whose body weight is 60 kg,
usually approximately 0.1 to 100 mg per a day, preferably
approximately 1.0 to 50 mg, more preferably approximately 1.0 to 20
mg are given. For example, in the case of an injection of
parentally giving, for example, for a patient with hypertonia whose
body weight is 60 kg, usually approximately 0.01 to 30 mg per a
day, preferably approximately 0.1 to 20 mg, more preferably
approximately 0.1 to 10 mg are given by an intravenous injection.
In the case of the other animal, the amount converted into the
amount per kg of body weight can be given. The dosage or times of a
dosage varies depending on treating effect targeted, medication
method, treating period, age or body weight, but usually it is 10
.mu.g to 8 mg/kg per a day for an adult.
EXAMPLES
[0271] The following examples are provided to exemplify and do not
restrict the present invention. As a genetic engineering method, if
there is not a special provision, the method described in Molecular
Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor
Laboratory), is used.
Example 1
[0272] An Identification of Gene Whose Expression in Human
Umbilical Vein Endothelial Cells (HUVEC) is Enhanced by the
Stimulation of Vascular Endothelial Cell Propagation Factors
(VEGF)
[0273] Three Endocell HUVEC (KURABO) which is a human umbilical
vein vascular endothelial cell were thawed and then inoculated on
16 culture plates coating with collagen (IWAKI) poured EBM medium
including 10% fetal calf serum (FCS) (Sanko Junyaku). After
culturing at 37.degree. C. for 3 days, the medium was changed with
the same medium and cultured for 2 days. After the cells reached to
confluence, the medium was changed with M199 medium including 1%
FCS (Nissui) and cells were cultured for 24 hours. For a culture
with VEGF treatment, the medium was changed to M199 medium
including 1% FCS and 1 nM human VEGF.sub.165 (R&D Systems). For
a culture without VEGF (a control), medium was changed with the
same medium not including human VEGF.sub.165. Cells were cultured
at 37.degree. C. and collected after 0 hour, 0.5 hours, 2 hours, 6
hours, 12 hours and 24 hours. Collected cells were washed with PBS
twice and total RNA were prepared by ISOGEN (Nippon Gene) and mRNAs
were prepared by oligo dT cellulose column. These mRNAs were
analyzed by LifeArrayhumanUniGENE (Incyte Genomics) through Gene
Expression Microarray analysis on assignment of Incyte
Genomics.
[0274] As a result, for 6393 human genes, gene expression level was
calculated as a ratio of a expression level in VEGF-treated cells
compared to one in untreated cells in each time point. Among these
genes, the expression level of a gene GenBank ID: AF055021, whose
function was unknown, was increased by treatment with VEGF reaching
the expression level of was 3.8 fold after 24 hours. Because a
protein translated region was unclear from the information of
sequence of AF055021 gene in a database of NCBI (National Center
for Biotechnology Information), a base sequence of the same gene
was searched with AssEst (maze) database and a consensus sequence
of EST cluster was obtained. Based on this consensus sequence,
homology search using Blastx database in AssEst revealed that it
encoded KIAA1036 polypeptide (SEQ ID NO: 2) whose function was
unknown. Additionally, it had 58% homology with a polypeptide of
AK022567 whose function was unknown (FIG. 1). KIAA1036 gene and
AK022567 gene on NCBI database have open reading frames
corresponding to 365 and 290 amino acid residues respectively,
which are initiated from translation initiation codons and they are
seem to be almost full length cDNA. A polypeptide of AK022567 was
already published internationally as a secretory protein
(WO99/3881). And KIAA1036 is predicted as a secretory protein
because the homology of their amino acid sequences is 58%.
Example 2
[0275] Expression of KIAA1036 in Human Normal Tissues
[0276] Cloning of KIAA1036 gene from human umbilical vein vascular
endothelial cell was tried. Two kinds of oligonucleotides were
synthesized as primers for PCR on the basis of a nucleic acid
sequence of 3' untranslated region of KIAA1036 gene from NCBI
database.
1 Primer1: 5'-GTTCAGGACTGTCTTTCAGC-3' (SEQ ID NO: 4) Primer2:
5'-GTCAATACTGATGGACTTGC-3' (SEQ ID NO: 5)
[0277] Total RNA was prepared according to an attached manual with
RNeasy Midi (QIAGEN) from HUVEC which are human umbilical vein
vascular endothelial cells. Single strand cDNA fragments were
prepared from the obtained total RNA (10 .mu.g) with SuperscriptII
(Invitrogen). The obtained single strand cDNAs were used as
templates and gene fragments were amplified with Primer1 (SEQ ID
NO: 4) and Primer2 (SEQ ID NO: 5) by PCR.
[0278] To one fiftieth of single strand cDNAs, was added TaKaRa Ex
Taq (TaKaRa) (1.25 unit), Primer1 and Primer2 (1 .mu.M each), a
buffer (Mg.sup.2+ plus) for PCR reaction attached TaKaRa Ex Taq (5
.mu.l) and dNTP Mixture whose final concentration was 200 .mu.M.
Distilled water was added to the mixture to be 50 .mu.l in total.
This was how samples for PCR were prepared. PCR reaction was held
under the condition that is 94.degree. C. for 3 minutes was once
and the cycle (94.degree. C. for 1 minute, 50.degree. C. for 1
minute and 72.degree. C. for 1 minute) was 30 times and then
72.degree. C. for 5 minutes was once. The amplified cDNA fragment
by the PCR reaction was separated by an agarose electrophoresis and
750 bp cDNA fragment (SEQ ID NO: 6) was collected. The obtained
cDNA fragment was digested with restriction enzymes, ApaI, HincII
and PstI and the lengths of cDNA fragments separated by an agarose
gel electrophoresis were measured. And the collected cDNA fragment
was confirmed as a target. The cDNA fragment (50 ng) amplified with
Primer1 and Primer2 was labeled with .sup.32P using
[.alpha.-.sup.32P] dCTP (3000 Ci/m mol, 10 mCi/ml) (Amersham
Pharmacia Bioteque) by an attached manual with Rediprime II DNA
Labeling System (Amersham Pharmacia Bioteque). Northern blot was
held with these cDNA fragments labeled with .sup.32P as a
probe.
[0279] Nylon membrane of Multiple Tissue Northern (MTN.TM.) Blots
Human 12-Lane MTN Blot (Clontech) was immersed with GMC buffer
(including 0.5M phosphoric acid buffer, pH 7.2, 1% cattle serum
albumin, 1 mM EDTA and 7% SDS) and prehybridization was held at
65.degree. C. for 1 hour. After that, the nylon membrane was
immersed with GMC buffer containing probes labeled with .sup.32P
and hybridization was held at 65.degree. C. for 16 hours. After
hybridization, the nylon membrane was washed with 2.times.SSC
(1.times.SSC was 15 mM sodium citrate, 150 mM NaCl, pH 7.0) at a
room temperature for 5 minutes, 1.times.SSC (including 0.2% SDS) at
50.degree. C. for 30 minutes and 0.5.times.SSC (including 0.2% SDS)
at 50.degree. C. for 30 minutes one after another and dried with
air. Subsequently, this nylon membrane was put on an imaging plate
cassette (FUJI FHOTO FILM CO., LTD.) with an imaging plate (FUJI
FHOTO FILM CO., LTD.). Image analysis was held with image analyzer
FLA3000 (FUJI FHOTO FILM CO., LTD.).
[0280] As a result, signals which showed about 6 kb length were
detected from brain, heart, skeletal muscle, spleen, kidney, liver,
small intestine and placenta and the strong expression was
selectively observed in brain and placenta.
Example 3
[0281] Expression of KIAA1036 in Human Umbilical Vein Endothelial
Cells
[0282] As the VEGF-induced expression of KIAA1036 was originally
found by Gene Expression Microarray Analysis using DNA chip as
described in Example 1, the result was confirmed by Northern blot
with KIAA1036 probe and total RNA prepared from VEGF-treated HUVEC.
Endocell HUVEC (KURABO) which are human umbilical vein endothelial
cells were cultured in M199 medium containing 1 nM human
VEGF.sub.165 (R&D Systems) and 1% FCS as described in Example
1. After 0, 0.5, 1, 2, 6, 12 and 24 hours of culture, total RNA was
prepared from these cells according to an attached manual with
RNeasy Midi (QIAGEN).
[0283] After electrophoresis of each RNA sample (5 .mu.g), the
samples were transferred to a nylon membrane, Hybond-XL (Amersham
Pharmacia Bioteque). That nylon membrane was immersed with GMC
buffer and prehybridization was held at 65.degree. C. for 1 hour.
After prehybridization, the nylon membrane was immersed with GMC
buffer containing probes labeled with .sup.32P as Example 2 and
hybridization was held at 65.degree. C. for 16 hours. After
hybridization, the nylon membrane was washed with 2.times.SSC at a
room temperature for 5 minutes, 1.times.SSC (including 0.2% SDS) at
50.degree. C. for 30 minutes and 0.5.times.SSC (including 0.2% SDS)
at 50.degree. C. for 30 minutes one after another and dried with
air. Subsequently, this nylon membrane was put on an imaging plate
cassette (FUJI FHOTO FILM CO., LTD.) with an imaging plate (FUJI
FHOTO FILM CO., LTD.). Image analysis was held with image analyzer
FLA3000 (FUJI FHOTO FILM CO., LTD.).
[0284] Next, probes were dehybridized by immersing the same nylon
membrane in a bath with boiling water and Northern blot was held
with human .beta.-actin cDNA fragments labeled with .sup.32P as
Example 2 as probes. Signal intensities of KIAA1036 gene were
normalized with that of .beta.-actin gene at each time point. And
then a ratio of the expression level of KIAA1036 in each time point
to 0 hour was calculated.
[0285] As a result, the expression was inhibited at 0.5 to 2 hours
after adding VEGF but a high gene expression, which was 3.2 fold,
was induced after 24 hours. This result was same as that obtained
by DNA chip (FIG. 2).
Example 4
[0286] Expression of KIAA1036 in Human Cancer Tissues
[0287] The difference of expression levels of the genes in a normal
tissue and a cancer tissue of all kinds of organ from human was
analyzed.
[0288] Matched Tumor/Normal Expression Array (Clontech), which is a
nylon membrane with cDNAs synthesized from a cancer tissue and a
normal tissue of chest, uterus, colon, stomach, ovary, lung,
kidney, rectum, cervix, small intestine or prostate of the same
patient, was immersed with GMC buffer containing probes labeled
with .sup.32P as Example 2 and hybridization was held at 65.degree.
C. for 16 hours. After hybridization, the nylon membrane was washed
with 2.times.SSC at a room temperature for 5 minutes, 1.times.SSC
(including 0.2% SDS) at 50.degree. C. for 30 minutes and
0.5.times.SSC (including 0.2% SDS) at 50.degree. C. for 30 minutes
one after another and dried with air. Subsequently, this nylon
membrane was put on an imaging plate cassette (FUJI FHOTO FILM CO.,
LTD.) with an imaging plate (FUJI FHOTO FILM CO., LTD.). Image
analysis was held with image analyzer FLA3000 (FUJI FHOTO FILM CO.,
LTD.).
[0289] After hybridization of the nylon membrane, signal
intensities were measured with .beta.-actin genes labeled with
.sup.32P as probes as described in Example 3. Signal intensities of
KIAA1036 was normalize with signal intensities by .beta.-actin
probe in each organ, and a ratio of cancer tissues to normal
tissues in the same patient was calculated. Then a change of an
expression level of the genes in all kinds of cancer tissue was
analyzed.
[0290] As a result, expression of KIAA1036 genes were increased in
cancer tissues of all 4 patients with ovarian cancer and 11
patients with colon cancer patient (FIG. 3).
Example 5
[0291] Expression of KIAA1036 in COS Cells
[0292] cDNA encoding the polypeptide that 3.times.FLAG sequence was
added to a carboxy-terminal of KIAA1036 amino acid sequence as set
forth in Met of 1-Val of 365 of SEQ ID NO: 2 was inserted to a
expression vector in animal cells and expressed in COS cells.
[0293] A base sequence as set forth in A of 386-C of 1480 of SEQ ID
NO: 1 encoding KIAA1036 was inserted in the NotI/XbaI sites of an
expression vector in animal cells, p3.times.FLAG-CMV-13 and
p3.times.FLAG-CMV-14(SIG- MA-Aldrich) and plasmids, pFLAG13-1036
and pFLAG14-1036 which express polypeptides that 3.times.FLAG was
added to carboxy- or amino-terminals of KIAA1036, respectively,
were constructed.
[0294] COS-7 cells (African green monkey kidney cells
(SV40-transformed cells)) were used as host of pFLAG13-1036 and
pFLAG14-1036 and transformation was operated by an attached manual
with FuGENE6 reagent (Roche Diagnostics).
[0295] COS-7 cells (5.times.10.sup.5 cell/well in 60 mm dishes)
were cultured in D-MEM medium containing 10% FCS at 37.degree. C.
for 16 hours. After medium was changed, pFLAG13-1036 (2 .mu.g) and
FuGENE6 (10 .mu.l) were added to the cells and the cells were
cultured for 48 hours. pFLAG14-1036 and p3.times.FLAG-CMV-14
without KIAA1036 cDNA as a control were also used in a same method
to make transfectants and the transfectants were cultured.
[0296] Expression of KIAA1036 polypeptide fused with FLAG was
analyzed by Western blotting, a method of Laemli.
[0297] The culture supernatants (10 .mu.l) of all kinds of COS-7
transfectants were subjected to SDS-PAGE and electrically
transfered to a nitrocellulose membrane in a nitrocellulose
membrane kit (TEFCO). The nitrocellulose membrane was immersed with
a blocking reagent (TBS, pH 7.6; including 5%(w/v) skim milk) and
shook at a room temperature for 1 hour. And the membrane was
immersed in TBS reagent (pH7.6; including 5%(w/v) skim milk)
containing anti-FLAG M2 antibody labeled with peroxidase (HRP)
(SIGMA-Aldrich), put static at a room temperature for 1 hour. Then
the nitrocellulose membrane was washed with a washing reagent (TBS,
pH7.6; including 0.05%(v/v) TritonX-100) for 10 minutes 3 times.
The nitrocellulose membrane and ECL Plus Western Blotting Detection
Reagents (Amersham Pharmacia Bioteque) were reacted at a room
temperature. The nitrocellulose membrane and X-ray film X-OMAT AR
(Kodak) were put on X-OMAT cassette (Kodak) immediately and exposed
for a few minutes.
[0298] As a result, expected bands, which show about 50 kDa, from
the culture supernatant of COS cells transfected with pFLAG13-1036
and pFLAG14-1036 were detected but the band was not detected in a
control. Therefore, it was confirmed that KIAA1036 gene encodes a
KIAA1036 protein, which is able to express, and that KIAA1036 was a
secretory protein in animal cells.
Example 6
[0299] Expression of KIAA1036 Protein in a Baculovirus System
[0300] Expressions in a baculovirus system were operated with
Bac-to-Bac Baculovirus Expression System (Invitrogen) according to
an attached manual.
[0301] cDNA fragments encoding KIAA1036 with 3.times.FLAG at the
carboxy-terminal described in Example 5 was inserted in NotI/XhoI
sites of a plasmid pFastBac1, an expression plasmid for insect
cells, to make pFASTBac1036. The recombinant plasmid was introduced
in Max Efficiency DH10 Bac Competent Cells and cDNA was transferred
into baculovirus genome (bacmid DNA) to give rise to a recombinant
bacmid DNA. The recombinant bacmid DNA was introduced in Sf9 (ATCC:
CRL-1711, ovary cell) cell with CellFECTIN Reagent (Invitrogen) to
obtain a recombinant baculovirus. A culture supernatant (0.5 ml) of
Sf9 transfectants was added into a culture medium (50 ml) of Sf9
cells with vascular disease, inflammatory disease, entoptic
neovascular disease, reproductive system disease, central nervous
system disease or malignant neoplasma (The concentration was
1.5.times.10.sup.6 cells/ml) and cultured at 28.degree. C. for 96
hours. The transformed cells were collected by centrifugation.
Expression of the FLAG-tagged KIAA1036 was confirmed by Western
blotting as described in Example 5.
[0302] The collected transformants were washed once with PBS (5 ml,
pH7.4). After washing, the cells were suspended in Lysis reagent (5
ml) (including 50 mM Tris-HCl, pH7.4; 0.15M NaCl, 0.1 mM EDTA/2Na,
0.1 mM EGTA, 1 mM DTT, 0.1 mM Amidinophenyl methansulfonyl fluride
hydrochloride or 0.1% NP-40), treated with ultrasonic waves on ice
for 15 seconds for 4 times by MICROCON (HEART SYSTEMS) and
separated by centrifugation at 14,500.times.g for 20 minutes. The
same amount of Lysis reagent was added to the supernatant to make
it a crude protein solution.
[0303] After a crude protein solution (10 .mu.l) was subjected to
SDS-PAGE, it was electrically transferred on a nitrocellulose
membrane in nitrocellulose membrane kit (TEFCO). The nitrocellulose
membrane was immersed in a blocking reagent (TBS, pH7.6; including
5%(w/v) skim milk) and shook at a room temperature for 1 hour.
After that, it was immersed in a TBS reagent (pH7.6; including
5%(w/v) skim milk) containing anti-FLAG M2 antibody labeled with
peroxidase (HRP) (SIGMA-Aldrich), put static at a room temperature
for 1 hour. And then the nitrocellulose membrane was washed with
the washing reagent (TBS, pH7.4; including 0.05%(v/v) TritonX-100)
for 10 minutes 3 times. The nylon membrane and ECL Plus Western
Blotting Detection Reagents (Amersham Pharmacia Bioteque) were
reacted at a room temperature. The nitrocellulose membrane and
X-ray hyper film ECL (Amersham Pharmacia Bioteque) were put on
X-OMAT cassette Kodak) immediately and exposed for a few
minutes.
[0304] KIAA1036 FLAG proteins were purified by an affinity
chromatography with anti-FLAG antibodies.
[0305] After an ANTI-FLAG M2-Agarose (SIGMA-Aldrich) affinity
column was equilibrated with TBS, pH7.4 (including 0.1% NP-40), a
crude protein solution was added and FLAG proteins were separated.
The columns were then washed with TBS (pH7.4; including 0.1% NP-40)
and the protein was eluted with Gly-HCl (pH3.5; including 0.1%
NP-40) to be 1 ml/fraction. Each fraction was neutralized with 1M
Tris-HCl (pH8.0; 20 .mu.l) to make it an affinity purification
fraction.
[0306] A crude protein fraction (10 .mu.l) and an affinity purified
fraction (10 .mu.l) were subjected to SDS-PAGE and the gel was
stained by Bio-Safe Coomassie (BIO-RAD).
[0307] As a result, an expected band with about 50 kDa was
detected.
[0308] Furthermore, after SDS-PAGE, the purified fraction was
electrically transferred to PVDF membrane (BIO-RAD) and a band
stained with Bio-Safe Coomassie (BIO-RAD) was cut off, and then a
sequence of an amino terminal was analyzed by an amino acid
sequencer, Procise Model 491 (Applied Biosystems).
[0309] As a result, an amino acid sequence as set forth in Pro of
2-Gly of 10 of SEQ ID NO: 2 was detected and it was confirmed that
the expressed protein was KIAA1036.
Example 7
[0310] A Blocking Effect of DNA Synthesis by KIAA1036 in Vascular
Endothelial Cells
[0311] To examine an effect of KIAA1036 protein to the
proliferation of vascular endothelial cells, BrdU
(bromodeoxyuridine) incorporation was measured in HUVEC cells
treated with VEGF in the presence (a treated group) or the absence
(a control group) of KIAA1036. A KIAA1036 protein, which was
expressed by a baculovirus expression system in Example 6 and
purified by an affinity chromatography, was used. Incorporation of
BrdU into cells was measured with cell propagation ELISA and a BrdU
chemiluminescence kit according to an attached manual.
[0312] Endocell HUVEC (KURABO) which are human vascular endothelial
cells (3000 cells/plate) were cultured on collagen-coated plates
(IWAKI) with M199 medium (Nissui) containing 5% fetal calf serum
(FCS) at 37.degree. C. for 24 hours. For the VEGF-treated group,
medium was changed to M199 medium including 1% FCS, 1 nM human
VEGF.sub.165 (R&D Systems) and 0, 1, 10 nM KIAA1036 protein.
For the VEGF-untreated group (control group) medium was changed to
the same mediums without human VEGF.sub.165 containing 0, 1 or 10
nM KIAA1036 protein. A solution labeled with BrdU (0.02 ml/well)
was added and the cells were cultured at 37.degree. C. for 12
hours. The cultured cells were put static at a room temperature for
1 hour to immobilize and an anti-BrdU antibody solution labeled
with POD (0.1 ml/well) was added and the cells were put static at a
room temperature for 1 hour. The cells were washed with a washing
buffer for 5 minutes 3 times and a luminecsence substrate (0.1
ml/well) was added. After 3 minutes of shaking, the instantaneous
fluorescence for 5 seconds was measured by Luminescencer JNR (ATTO)
within 10 minutes.
[0313] As a result, VEGF promoted DNA synthesis of HUVEC, but
KIAA1036 protein doses-dependently inhibited it (FIG. 4).
Example 8
[0314] A Blocking Effect to the Migration of Vascular Endothelial
Cells by KIAA1036 Protein
[0315] An effect to the migration of vascular endothelial cells of
KIAA1036 protein was examined by a modified Boyden chamber
method.
[0316] To the lower chambers of Transwell (Corning coaster), M199
medium (600 .mu.l /well) containing 0.25 nM human VEGF.sub.165
(R&D Systems) and 1 or 10 nM KIAA1036 protein was added for
samples treated with VEGF (test group), and M199 medium (600
.mu.l/well) containing 1 or 10 nM KIAA1036 protein without VEGF was
added for a control group. Endocell HUVEC (KURABO)
(4.times.10.sup.4 cells/well) were cultured in M199 medium
containing 5% FCS for 16 hours and then seeded in the upper
chambers and cultured at 37.degree. C. for 4 hours. Filters of
Transwell were immobilized with Diffquick fix reagent (Sysmex) and
then stained with Diffquick stain (Sysmex). After wiping the side
of the upper chamber of filter, the cell migration ability toward
VEGF was measured by counting the number of the cells migrating to
the lower part.
[0317] As a result, VEGF promoted migration of HUVEC, but KIAA1036
protein significantly inhibited it (FIG. 5).
Example 9
[0318] A Blocking Effect to Network Formation of Vascular
Endothelial Cells by KIAA1036 Protein
[0319] To examine an effect to network formation of vascular
endothelial cells by KIAA1036 protein, network formation of HUVEC
in the presence (treated group) or the absence (control group) of
KIAA1036 protein was observed.
[0320] Matrigel (Becton Dickinson Biosciences) (1 ml/well) was
divided in 6-well tissue culture plates chilled on ice and put
static at 37.degree. C. for 1 hour to be gelatinized. Endocell
HUVEC (KURABO) were suspended in EBM medium containing 10% FCS and
poured onto the matrigel at 5.times.10.sup.4 cells/well with a
final concentration of KIAA1036 protein at 1 or 10 nM (test group)
or without KIAA1036 protein. After 9 hours of incubation at
37.degree. C., the network formations of HUVEC in test divisions
and control divisions were observed with an inverted
microscope.
[0321] As a result, it was confirmed that KIAA1036 protein
obviously inhibits the network formation.
Industrial Applicability
[0322] The present invention provides with a detection method of
mRNA of KIAA1036, a detection method of a polypeptide of KIAA1036,
a marker for neovascularization, vascular disease, inflammatory
disease, entoptic neovascular disease, reproductive system disease,
central nervous system disease or malignant neoplasm, a diagnostic
kit and a therapeutic agent.
Sequence CWU 0
0
* * * * *