U.S. patent application number 10/139262 was filed with the patent office on 2002-09-12 for physiologically active protein originating in mammals.
Invention is credited to Nakamura, Yusuke, Tanaka, Toshihiro, Tsukuda, Shuichi.
Application Number | 20020128459 10/139262 |
Document ID | / |
Family ID | 26403327 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020128459 |
Kind Code |
A1 |
Nakamura, Yusuke ; et
al. |
September 12, 2002 |
Physiologically active protein originating in mammals
Abstract
The present invention provides novel physiologically active
protein molecules originating in mammals, which are specifically
expressed in arteriosclerosis and/or coronary restenosis, and are
predicted to relate closely to the onset and progress of these
diseases; DNAs encoding the protein molecules; antibodies reactive
with the molecules; and pharmaceutical compositions comprising the
above protein molecule or the antibody. The protein molecules,
DNAs, and antibodies are useful for treating and preventing
arteriosclerosis.
Inventors: |
Nakamura, Yusuke;
(Yokohama-shi, JP) ; Tanaka, Toshihiro;
(Minato-ku, JP) ; Tsukuda, Shuichi; (Yokohama-shi,
JP) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
26403327 |
Appl. No.: |
10/139262 |
Filed: |
May 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10139262 |
May 7, 2002 |
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09380287 |
Nov 16, 1999 |
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09380287 |
Nov 16, 1999 |
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PCT/JP98/00835 |
Feb 27, 1998 |
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Current U.S.
Class: |
536/23.2 ;
424/146.1; 424/94.63; 435/226; 435/320.1; 435/325; 530/388.26 |
Current CPC
Class: |
A01K 2217/075 20130101;
C07K 2319/00 20130101; A01K 2217/05 20130101; A61K 38/00 20130101;
C07K 14/705 20130101; A61P 9/10 20180101; C07K 14/4703 20130101;
C07K 14/82 20130101 |
Class at
Publication: |
536/23.2 ;
530/388.26; 435/320.1; 435/325; 424/146.1; 424/94.63; 435/226 |
International
Class: |
A61K 038/48; C07H
021/04; A61K 039/395; C12N 009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 1997 |
JP |
9/62259 |
Feb 25, 1998 |
JP |
10/62263 |
Claims
1. A DNA encoding a protein having the amino acid sequence of SEQ
ID NO: 4.
2. A DNA encoding a protein fragment comprising the extracellular
region of a protein having the amino acid sequence of SEQ ID NO:
4.
3. A DNA comprising a nucleotide sequence corresponding to
nucleotide residues 97 to 1419 of the nucleotide sequence of SeQ ID
NO: 3.
4. A DNA hybridizing with a DNA having the nucleotide sequence of
SEQ ID NO: 3 under stringent conditions.
5. A protein having the amino acid sequence of SEQ ID NO: 4 or an
amino acid sequence substantially the same as said amino acid
sequence.
6. A protein fragment comprising the extracellular region of a
protein having the amino acid sequence of SEQ ID NO: 4 or an amino
acid sequence substantially the same as said amino acid
sequence.
7. A fusion protein between the extracellular region of the protein
of claim 5 and the constant region of the heavy chain of human
immunoglobulin (Ig) or a portion of the constant region.
8. An expression vector comprising the DNA of any one of claims 1
to 4.
9. A transformant carrying the expression vector of claim 8.
10. An antibody or its portion reactive with the protein of claim 5
or the protein fragment of claim 6.
11. The antibody or its portion of claim 10, wherein the antibody
is a monoclonal antibody.
12. A pharmaceutical composition comprising the protein fragment of
claim 6 or the fusion protein of claim 7 and a pharmaceutically
acceptable carrier.
13. A pharmaceutical composition comprising the antibody or its
portion of claim 10 or 11 and a pharmaceutically acceptable
carrier.
14. A DNA encoding a protein having the amino acid sequence of SEQ
ID NO: 10.
15. A DNA encoding a protein fragment comprising the extracellular
region of a protein having the amino acid sequence of SEQ ID NO:
10.
16. A DNA having a nucleotide sequence corresponding to nucleotide
residues 1 to 1785 of the nucleotide sequence of SEQ ID NO: 9.
17. A DNA hybridizing with a DNA having the nucleotide sequence of
SEQ ID NO: 9 under stringent conditions.
18. A protein having the amino acid sequence of SEQ ID NO: 10 or an
amino acid sequence substantially the same as said amino acid
sequence.
19. A protein fragment comprising the extracellular region of a
protein having the amino acid sequence of SEQ ID NO: 10 or an amino
acid sequence substantially the same as said amino acid
sequence.
20. A fusion protein comprising the extracellular region of the
protein of claim 18 and the constant region of the heavy chain of
human immunoglobulin (Ig) or a portion of the constant region.
21. An expression vector comprising the DNA of any one of claims 14
to 17.
22. A transformant carrying the expression vector of claim 21.
23. An antibody or its portion reactive with the protein of claim
18 or the protein fragment of claim 19.
24. The antibody or its portion of claim 23, wherein the antibody
is a monoclonal antibody.
25. A pharmaceutical composition comprising the protein fragment of
claim 19 or the fusion protein of claim 20 and a pharmaceutically
acceptable carrier.
26. A pharmaceutical composition comprising the antibody or its
portion of claim 23 or 24 and a pharmaceutically acceptable
carrier.
27. A transgenic mouse in which the human-derived DNA comprising a
DNA having a nucleotide sequence corresponding to nucleotide
residues 97 to 1419 of the nucleotide sequence of SEQ ID NO: 3 is
integrated into an endogenous gene of said mouse.
28. A transgenic mouse in which the human-derived DNA comprising a
DNA having a nucleotide sequence corresponding to nucleotide
residues 1 to 1785 of the nucleotide sequence of SEQ ID NO: 9 is
integrated into an endogenous gene of said mouse.
29. A knockout mouse whose endogenous gene encoding a mouse-derived
protein having the amino acid sequence of SEQ ID NO: 6 is
inactivated so that said protein is not produced.
30. A knockout mouse whose endogenous gene encoding a mouse-derived
protein comprising the amino acid sequence of SEQ ID NO: 28 is
inactivated so that said protein is not produced.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel physiologically
active protein originating in mammals, a DNA encoding said protein,
and an antibody reactive with said protein.
BACKGROUND ART
[0002] A so-called geriatric disease, which is regarded as a
current disease in high living standard society, includes
arteriosclerosis as well as hypertension and diabetes. Important
measures for preventing these diseases are not only development of
therapeutic methods but also daily life control.
[0003] Arteriosclerosis begins with pathological changes (for
example, (1) invasive growth of smooth muscle cells into inner
membrane, (2) qualitative and quantitative changes of collagen,
elastin, and acidic mucopolysaccharides, and (3) cell foaming by
lipid accumulation in the cytoplasm of grown smooth muscle cells
and macrophages implanting tissues) occurring in inner membrane of
artery. As the result of such pathological changes, (1) foam cells
found in the inner membrane produces fat spots on the surface of
the inner membrane, (2) lipid accumulates between tissues (deep
part of midmembrane) and the inner membrane surface is covered with
thick glass-like membrane, accompanied by fibrous growth and
calcification, and (3) bleeding and necrosis occur in tissues to
cause combined pathological changes involving thrombogenesis,
calcification, and deposition of lipid crystals. Such pathological
changes, in time, distribute in artery of a whole body and narrow
the cavity of the artery. In addition, the site of pathological
changes becomes bursal and the vascular wall loses elasticity,
thereby hardening blood vessels. The vessels then wind, and normal
blood flow is inhibited.
[0004] Epidemiological studies so far have illustrated age (about
thirties or more), hypercholesterolemia, hypo-HDL-cholesterolemia,
systolic hypertension, obesity, hemoglobin high value, and diabetes
as risk factors of the onset of arteriosclerosis. Dynamics of in
vivo factors inducing the onset include secretion of adrenalin,
increase of thromboxane A2, decrease of prostacyclin, increase of
serum peroxylipid, increase of free fatty acid, increase of
platelet, increase of fibrinogen, increase of blood coagulation
factors (XII and XIII), decrease of tissue plasmin, increase of
prostaglandin, decrease of antithrombin III, increase of serum LDL,
decrease of serum HDL, increase of insulin, and increase of
renin.
[0005] Studies so far have revealed only that multiple conditions,
for example, physical conditions such as age and obesity,
complication with other diseases, and abnormalities of the dynamics
of many in vivo factors complicatedly are related to each other to
cause arteriosclerosis.
[0006] Treatments of arteriosclerosis are divided with their
purpose into (1) preventive treatments to retract arteriosclerosis
and to prevent the onset of arteriosclerosis by correcting
lifestyle and physical abnormalities such as obesity (for example,
diet therapy and therapeutic exercise) and (2) chemotherapy or
surgical therapy to remove vessel occlusion symptoms occurring with
the progress of arteriosclerosis or to prevent the onset of vessel
cavity occlusion symptoms by thrombus or embolus,.
[0007] Since particular decisive causes of arteriosclerosis are
unclear, only symptomatic treatment by chemotherapy is currently
possible. For example, .beta. blocker is applied when the
enhancement of a catecholamine derivative such as adrenalin is
suspected as the cause, eicosapentaenoic acid is applied for a
prostaglandin derivative, vitamin E is applied for peroxylipid, and
urokinase is applied for thrombus. No effective pharmaceuticals for
treating the arteriosclerosis have been provided yet.
[0008] In the surgical therapy for arterial occulsion, percutaneous
transluminal coronary angioplasty (PTCA) based on the observation
by angiography prevails clinically as an effective means to enlarge
vessel cavity. PTCA has remarkably progressed and prevailed since
it was clinically applied by Gruntzig for the first time in 1977,
and the number of the operation has rapidly increased in Japan.
[0009] PTCA is the method in which the occlusion (constriction)
site is enlarged by inserting a thin catheter with a balloon at the
tip in a thick catheter into the coronary artery occlusion site and
by expanding the balloon.
[0010] However, in cavity enlargement by PTCA, restenosis occurs at
the operation site of the artery in about 30 to 50% of the cases
within a few months after the operation, and this restenosis is a
major drawback of PTCA.
[0011] The restenosis has been thought to occur by the
amplification of neonatal inner membrane proliferation based on the
repair reaction of the injury site of the vascular wall, which has
been inevitably caused by the enlargement of the occlusion site by
PTCA. Although chemotherapy has been tried for preventing this
restenosis, almost no effective drugs have been reported so
far.
[0012] As mentioned above, at present, a method for the complete
treatment and prevention of arteriosclerosis comprising the
prevention of the recurrence of arteriosclerosis and the occurrence
of restenosis has not established. It is thus desired to clarify
the cause of the onset and progress of arteriosclerosis and to
develop a method for the effective treatment and prevention
thereof, and therapeutic and preventive drugs.
[0013] Coronary artery restenosis occurring after PTCA is regarded
as a clinical model of arteriosclerosis from pathological
viewpoints such as neonatal inner membrane proliferation or intimal
thickening. Therefore, to diagnose the tissue characteristics of
the vascular wall at the restenosis site after PTCA and to
elucidate the difference between the characteristics and those of
normal vascular wall by comparing them pathologically and at the
gene level are effective to identify the cause and factors of
restenosis, and further, arteriosclerosis.
[0014] In such comparative studies, a useful method for comparison
and examination at the gene level using the genetic engineering
technique is called differential display method (Nucleic Acids
Research, Vol.21, No.18, pp.4272-4280 (1993); and Science, Vol.257,
pp.967-971 (1992)).
[0015] Specifically, PCTA is applied to the coronary artery of a
large mammal such as a rabbit, the expression patterns of genes in
the inner membrane tissue at the PTCA site are examined by
differential display method, and they are compared with the gene
expression patterns in the inner membrane tissue without PCTA, to
thereby identify genes specifically or increasingly expressed after
PTCA.
DISCLOSURE OF THE INVENTION
[0016] Genes that express specifically or increasingly after PTCA
and proteins derived from said genes may be closely related to
arteriosclerosis and restenosis. The present invention provides
pharmaceuticals and methods for preventing and treating
arteriosclerosis and restenosis by identifying genes and proteins
expressing specifically in arteriosclerosis and coronary artery
restenosis.
[0017] As the result of studies on the analyses of genes specific
to arteriosclerosis and/or coronary artery restenosis, the present
inventors have discovered genes encoding two novel proteins (clone
BA0306 and BA2303) that express increasingly at the comparatively
early stage (day 1 to 7) after PTCA and completed the present
invention.
[0018] The two novel protein-encoding genes of the present
invention, whose characteristics are mentioned below, are expressed
specifically after PTCA, and are thought to be genes involved in
onset and progress of arteriosclerosis and/or coronary artery
restenosis.
[0019] Clone BA0306 has the following characteristics.
[0020] (1) Its increased expression is observed on day 1 to 7 after
PTCA of coronary artery (the peak is observed on day 4).
[0021] (2) Northern blotting reveals the expression of the mRNA as
about 3.5 k and about 4.4 k bands in various human tissues.
[0022] (3) It has ten putative transmembrane regions.
[0023] (4) It has amino acid sequence homology with S. cerevisiae
oxidative stress resistance protein, S. cerevisiae zinc/cadmium
resistance protein, heavy metal ion resistance protein, and so
on.
[0024] (5) The molecules derived from humans and rabbits have the
amino acid sequences of SEQ ID NO: 10 and 8, respectively. The
molecule derived from mice has the amino acid sequence of SEQ ID
NO: 28.
[0025] Judging from these characteristics, clone BA0306 is thought
to inhibit active oxygen such as nitrogen monoxide (NO), which is
involved in the progress of arteriosclerosis and/or restenosis.
[0026] Clone BA2303 has the following characteristics.
[0027] (1) Its increased expression is observed from day 1 after
PTCA of coronary artery, and the expression continues until day 7
with the maximum expression on day 2 to 4.
[0028] (2) Northern blotting reveals the expression of the mRNA as
about 3.9 k and about 2.1 k bands in various human tissues.
[0029] (3) It has seven putative transmembrane regions.
[0030] (4) The molecules derived from humans and mice have the
amino acid sequences of SEQ ID NO: 4 and 6, respectively. The
molecule derived from rabbits has the amino acid sequence of SEQ ID
NO: 2.
[0031] Judging from these characteristics, clone BA2303 is thought
to be a GTP binding protein (G protein)-coupled receptor that
transmits a specific signal through intracellular G protein to an
effector on the plasma membrane or the surface of the cytoplasm by
binding to an in vivo ligand involved in the onset or progress of
arteriosclerosis and/or restenosis.
[0032] Therefore, the genes (DNAs), proteins, or their fragments of
the present invention and antibodies or a portion of them reactive
with the proteins of the present invention are extremely useful for
developing drugs for treatment and prevention of arteriosclerosis
and for treatment and prevention of restenosis after PTCA for
artery occlusion symptom and so on, targeting said genes or protein
molecules. In addition, the DNAs of the present invention
themselves are useful as antisense pharmaceuticals, extracellular
region fragments of said proteins, for example, as soluble receptor
pharmaceuticals, and said antibodies and a portion of them as
antibody pharmaceuticals.
[0033] Genes (DNAs), proteins, and antibodies of the present
invention are useful as reagents for searching proteins (ligands)
interacting with the proteins of the present invention, thereby
elucidating the function of said ligands, and developing
therapeutic drugs targeting said ligands.
[0034] Based on the genetic information of the rabbit- or
mouse-derived DNA, one embodiment of DNAs of the present invention,
model animals (knockout animals) can be produced by disrupting
(inactivating) the endogenous gene corresponding to the DNA.
Similarly, transgenic animals can be produced as model animals by
introducing the human-derived DNA, one embodiment of DNAs of the
present invention, into nonhuman mammals such as mice. Function of
genes and proteins of the present invention can be elucidated by
analyzing the physical, biological, pathologic, and genetic
characteristics of these model animals.
[0035] Moreover, by mating the model animals whose endogenous gene
is thus disrupted with the transgenic animals, model animals that
have only the human-derived gene of the present invention can be
produced. By administering drugs (compounds, antibodies, and so on)
targeting the introduced human gene to these model animals, the
therapeutic effect of the drug can be estimated.
[0036] Namely, the present invention provides the DNAS, proteins,
expression vectors, transformants, antibodies, pharmaceutical
compositions, transgenic mice, and knockout, mentioned below.
[0037] (1) A DNA encoding a protein having the amino acid sequence
of SEQ ID NO: 4.
[0038] (2) A DNA encoding a protein fragment comprising the
extracellular region of a protein having the amino acid sequence of
SEQ ID NO: 4.
[0039] (3) A DNA comprising a nucleotide sequence corresponding to
nucleotide residues 97 to 1419 of the nucleotide sequence of SEQ ID
NO: 3.
[0040] (4) A DNA hybridizing with a DNA having the nucleotide
sequence of SEQ ID NO: 3 under stringent conditions.
[0041] (5) A protein having the amino acid sequence of SEQ ID NO: 4
or an amino acid sequence substantially the same as said amino acid
sequence.
[0042] (6) A protein fragment comprising the extracellular region
of a protein having the amino acid sequence of SEQ ID NO: 4 or an
amino acid sequence substantially the same as said amino acid
sequence.
[0043] (7) A fusion protein between the extracellular region of the
protein of (5) and the constant region of the heavy chain of human
immunoglobulin (Ig) or a portion of the constant region.
[0044] (8) An expression vector comprising the DNA of any one of
(1) to (4).
[0045] (9) A transformant carrying the expression vector of
(8).
[0046] (10) An antibody or its portion reactive with the protein of
(5) or the protein fragment of (6).
[0047] (11) The antibody or its portion of (10), wherein the
antibody is a monoclonal antibody.
[0048] (12) A pharmaceutical composition comprising the protein
fragment of (6) or the fusion protein of (7) and a pharmaceutically
acceptable carrier.
[0049] (13) A pharmaceutical composition comprising the antibody or
its portion of (10) or (11) and a pharmaceutically acceptable
carrier.
[0050] (14) A DNA encoding a protein having the amino acid sequence
of SEQ ID NO: 10.
[0051] (15) A DNA encoding a protein fragment comprising the
extracellular region of a protein having the amino acid sequence of
SEQ ID NO: 10.
[0052] (16) A DNA having a nucleotide sequence corresponding to
nucleotide residues 1 to 1785 of the nucleotide sequence of SEQ ID
NO: 9.
[0053] (17) A DNA hybridizing with a DNA having the nucleotide
sequence of SEQ ID NO: 9 under stringent conditions.
[0054] (18) A protein having the amino acid sequence of SEQ ID NO:
10 or an amino acid sequence substantially the same as said amino
acid sequence.
[0055] (19) A protein fragment comprising the extracellular region
of a protein having the amino acid sequence of SEQ ID NO: 10 or an
amino acid sequence substantially the same as said amino acid
sequence.
[0056] (20) A fusion protein comprising the extracellular region of
the protein of (18) and the constant region of the heavy chain of
human immunoglobulin (Ig) or a portion of the constant region.
[0057] (21) An expression vector comprising the DNA of any one of
(14) to (17).
[0058] (22) A transformant carrying the expression vector of
(21).
[0059] (23) An antibody or its portion reactive with the protein of
(18) or the protein fragment of (19).
[0060] (24) The antibody or its portion of (23), wherein the
antibody is a monoclonal antibody.
[0061] (25) A pharmaceutical composition comprising the protein
fragment of (19) or the fusion protein of (20) and a
pharmaceutically acceptable carrier.
[0062] (26) A pharmaceutical composition comprising the antibody or
its portion of (23) or (24) and a pharmaceutically acceptable
carrier.
[0063] (27) A transgenic mouse in which the human-derived DNA
comprising a DNA having a nucleotide sequence corresponding to
nucleotide residues 97 to 1419 of the nucleotide sequence of SEQ ID
NO: 3 is integrated into an endogenous gene of said mouse.
[0064] (28) A transgenic mouse in which the human-derived DNA
comprising a DNA having a nucleotide sequence corresponding to
nucleotide residues 1 to 1785 of the nucleotide sequence of SEQ ID
NO: 9 is integrated into an endogenous gene of said mouse.
[0065] (29) A knockout mouse whose endogenous gene encoding a
mouse-derived protein having the amino acid sequence of SEQ ID NO:
6 is inactivated so that said protein is not produced.
[0066] (30) A knockout mouse whose endogenous gene encoding a
mouse-derived protein comprising the amino acid sequence of SEQ ID
NO: 28 is inactivated so that said protein is not produced.
[0067] In the following, the present invention is explained in
detail by clarifying the meanings of terms used in the present
application and the general production methods of proteins, protein
fragments, fusion proteins, DNAs, antibodies, transgenic mice, and
knockout mice of the present invention.
[0068] A "protein" of the present invention means a protein and its
fragment derived from mammals such as humans, rabbits, and mice,
and preferably, a human-derived protein and its fragment.
[0069] Particularly preferable examples are (1) a protein having
the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence
substantially the same as said amino acid sequence, (2) a protein
fragment comprising the extracellular region of a protein having
the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence
substantially the same as said amino acid sequence, (3) a protein
having the amino acid sequence of SEQ ID NO: 10 or an amino acid
sequence substantially the same as said amino acid sequence, and
(4) a protein fragment comprising the extracellular region of a
protein having the amino acid sequence of SEQ ID NO: 10 or an amino
acid sequence substantially the same as said amino acid
sequence.
[0070] The term "extracellular region" used herein is explained
below. A transmembrane protein such as a G protein-coupled
receptors or cell surface molecule connects with the membrane
through the hydrophobic peptide region penetrating the lipid
bilayer of the membrane once or several times and has structure
composed of three main regions, that is, extracellular region,
transmembrane region, and cytoplasmic region. Such a transmembrane
protein exists as a monomer, homodimer, heterodimer, or oligomer
with another chain(s) having the same or different amino acid
sequence.
[0071] The term "extracellular region" used herein means the
partial structure (partial sequence) existing outside of the
membrane that holds the transmembrane protein as mentioned above
among the whole structure of said membrane protein. In other words,
it corresponds to the region excluding the region incorporated into
the membrane (transmembrane region) and the region existing in the
cytoplasm following the transmembrane region (cytoplasmic region).
If desired, one to five amino acids derived from the amino acids
constituting the transmembrane and/or cytoplasmic region can be
added to the N- terminus and/or C- terminus of the extracellular
region in the present invention.
[0072] Here, "having substantially the same amino acid sequence"
means to include a protein having an amino acid sequence where
multiple amino acids, preferably 1 to 10 amino acids, particularly
preferably 1 to 5 amino acids, in the amino acid sequence shown in
SEQ ID NO: 4 or 10, are substituted, deleted, and/or modified, and
a protein having an amino acid sequence where multiple amino acids,
preferably 1 to 10 amino acids, particularly preferably 1 to 5
amino acids, are added to said amino acid sequence, as far as the
protein has substantially the same biological properties as the
protein having said amino acid sequence.
[0073] Alphabetical triplet or single letter codes used to
represent amino acids in the present specification or figures mean
amino acids as follows. (Gly/G) glycine, (Ala/A) alanine, (Val/V)
valine, (Leu/L) leucine, (Ile/I) isoleucine, (Ser/S) serine,
(Thr/T) threonine, (Asp/D) aspartic acid, (Glu/E) glutamic acid,
(Asn/N) asparagine, (Gln/Q) glutamine, (Lys/K) lysine, (Arg/R)
arginine, (Cys/C) cysteine, (Met/M) methionine, (Phe/F)
phenylalanine, (Tyr/Y) tyrosine, (Trp/W) tryptophane, (His/H)
histidine, (Pro/P) proline.
[0074] "The constant region or a portion of the constant region of
human immunoglobulin (Ig) heavy chain" used herein means the
constant region or the Fc region of human-derived immunoglobulin
heavy chain (H chain) as described, or a portion of them. The
immunoglobulin can be any immunoglobulin belonging to any class and
any subclass. Specifically, examples of the immunoglobulin are IgG
(IgG1, IgG2, IgG3, and IgG4), IgM, IgA (IgA1 and IgA2), IgD, and
IgE. Preferably, the immunoglobulin is IgG (IgG1, IgG2, IgG3, or
IgG4), or IgM. Examples of particularly preferable immunoglobulin
of the present invention are those belonging to human-derived IgG
(IgG1, IgG2, IgG3, or IgG4).
[0075] Immunoglobulin has a Y-shaped structural unit in which four
chains composed of two homologous light chains (L chains) and two
homologous heavy chains (H chains) are connected through disulfide
bonds (S-S bonds). The light chain is composed of the light chain
variable region (VL) and the light chain constant region (CL). The
heavy chain is composed of the heavy chain variable region (VH) and
the heavy chain constant region (CH).
[0076] The heavy chain constant region is composed of some domains
having the amino acid sequences inherent in each class (IgG, IgM,
IgA, IgD, and IgE) and each subclass (IgG1, IgG2, IgG3, and IgG4,
IgAl.sub.1 and IgA2).
[0077] The heavy chain of IgG (IgG1, IgG2, IgG3, and IgG4) is
composed of VH, CH1 domain, hinge region, CH2 domain, and CH3
domain in this order from N terminus.
[0078] Similarly, the heavy chain of IgG1 is composed of VH,
C.gamma..sub.11 domain, hinge region, C.gamma..sub.12 domain, and
C.gamma..sub.13 domain in this order from N terminus. The heavy
chain of IgG2 is composed of VH, C.gamma..sub.21 domain, hinge
region, C.gamma..sub.22 domain, and C.gamma..sub.23 domain in this
order from N terminus. The heavy chain of IgG3 is composed of VH,
C.gamma..sub.31 domain, hinge region, C.gamma..sub.32 domain, and
C.gamma..sub.33 domain in this order from N terminus. The heavy
chain of IgG4 is composed of VH, C.gamma..sub.41 domain, hinge
region, C.gamma..sub.42 domain, and C.gamma..sub.43 domain in this
order from N terminus.
[0079] The heavy chain of IgA is composed of VH, C.alpha.1 domain,
hinge region, C.alpha.2 domain, and C.alpha.3 domain in this order
from N terminus.
[0080] Similarly, the heavy chain of IgA1 is composed of VH,
C.alpha..sub.11 domain, hinge region, C.alpha..sub.12 domain, and
C.alpha..sub.13 domain in this order from N terminus. The heavy
chain of IgA2 is composed of VH, C.alpha..sub.21 domain, hinge
region, C.alpha..sub.22 domain, and C.alpha..sub.23 domain in this
order from N terminus.
[0081] The heavy chain of IgD is composed of VH, C.delta.1 domain,
hinge region, C.delta.2 domain, and C.delta.3 domain in this order
from N terminus.
[0082] The heavy chain of IgM is composed of VH, C.mu.1 domain,
C.mu.2 domain, C.mu.3 domain, and C.mu.4 domain in this order from
N terminus and have no hinge region as seen in IgG, IgA, and
IgD.
[0083] The heavy chain of IgE is composed of VH, C.epsilon.1
domain, C.epsilon.2 domain, C.epsilon.3 domain, and C.epsilon.4
domain in this order from N terminus and have no hinge region as
seen in IgG, IgA, and IgD.
[0084] If, for example, IgG is treated with papain, it is cleaved
at the slightly N terminal side beyond the disulfide bonds existing
in the hinge region where the disulfide bonds connect the two heavy
chains to generate two homologous Fab, in which a heavy chain
fragment composed of VH and CH1 is connected with one light chain
through a disulfide bond, and one Fc, in which two homologous heavy
chain fragments composed of the hinge region, CH2 domain, and CH3
domain are connected through disulfide bonds (See "Immunology
Illustrated", original 2nd ed., Nankodo, pp.65-75 (1992); and
"Focus of Newest Medical Science `Recognition Mechanism of Immune
System`", Nankodo, pp.4-7 (1991); and so on).
[0085] Namely, "a portion of a constant region of immunoglobulin
heavy chain" of the present invention means a portion of a constant
region of an immunoglobulin heavy chain having the structural
characteristics as mentioned above, and preferably, is the constant
region without C1 domain, or the Fc region. Specifically, examples
thereof are the region composed of hinge region, C2 domain, and C3
domain from each of IgG, IgA, and IgD, and are the region composed
of C2 domain, C3 domain, and C4 domain from each of IgM and IgE. A
particularly preferable example thereof is the Fc region of
human-derived IgG1.
[0086] The "fusion protein" of the present invention is that
composed of the above-described extracellular region of the protein
of the present invention and a constant region or a portion of a
constant region of human immunoglobulin (Ig) heavy chain.
Preferably, it is a fusion polypeptide composed of an extracellular
region of a protein of the present invention and a portion of a
constant region of human IgG heavy chain, and particularly
preferably, it is a fusion polypeptide composed of an extracellular
region of a protein of the present invention and the region (Fc)
composed of a hinge region, CH2 domain, and CH3 domain of human IgG
heavy chain. Moreover, IgG1 is preferable among IgG. In addition, a
protein derived from human, mouse, or rat (preferably, human) is
preferable as the protein of the present invention.
[0087] The fusion protein of the present invention has the
advantage that the fusion polypeptide can be purified extremely
easily by using affinity column chromatography using the property
of protein A, which binds specifically to the immunoglobulin
fragment because the fusion polypeptide of the present invention
has a portion of a constant region (for example Fc) of an
immunoglobulin such as IgG as mentioned above as a fusion partner.
Moreover, since various antibodies against the Fc of various
immunoglobulin are available, an immunoassay for the fusion
polypeptides can be easily performed with antibodies against the
Fc.
[0088] The protein, protein fragment, and fusion protein of the
present invention can be produced not only by recombinant DNA
technology as mentioned below but also by a method well known in
the art such as a chemical synthetic method and a cell culture
method, or a modified method thereof.
[0089] The DNA of the present invention encodes the above-mentioned
protein of the present invention, and includes any nucleotide
sequence that can encode the protein of the present invention. The
DNA preferably encodes a human-derived protein of the present
invention. Specific examples of the DNA are described below.
[0090] (1) A DNA encoding a protein having the amino acid sequence
of SEQ ID NO: 4, a protein fragment composed of the extracellular
region of said protein, or a biological analog obtained by
substituting, deleting, and/or modifying multiple amino acids,
preferably 1 to 10 amino acids, particularly preferably 1 to 5
amino acids in the amino acid sequence of said protein or fragment,
or by inserting multiple amino acids, preferably 1 to 10 amino
acids, particularly preferably 1 to 5 amino acids, in said amino
acid sequence.
[0091] (2) A DNA encoding a protein having the amino acid sequence
of SEQ ID NO: 10, a protein fragment composed of the extracellular
region of said protein, or a biological analog obtained by
substituting, deleting, and/or modifying multiple amino acids,
preferably 1 to 10 amino acids, particularly preferably 1 to 5
amino acids, in the amino acid sequence of said protein or
fragment, or by inserting multiple amino acids, preferably 1 to 10
amino acids, particularly preferably 1 to 5 amino acids, in said
amino acid sequence.
[0092] (3) A DNA hybridizing with a DNA having the nucleotide
sequence of SEQ ID NO: 3 under stringent conditions.
[0093] (4) A DNA hybridizing with a DNA having the nucleotide
sequence of SEQ ID NO: 9 under stringent conditions.
[0094] Specific examples thereof are (1) a DNA having a nucleotide
sequence corresponding to nucleotide residues 97 to 1419 of the
nucleotide sequence of SEQ ID NO: 3, (2) a DNA comprising a
nucleotide sequence corresponding to nucleotide residues 1 to 1419
of the nucleotide sequence of SEQ ID NO: 3, (3) a DNA having a
nucleotide sequence corresponding to nucleotide residues 1 to 1785
of the nucleotide sequence of SEQ ID NO: 9, and (4) a DNA
comprising a nucleotide sequence corresponding to nucleotide
residues 1 to 1785 of the nucleotide sequence of SEQ ID NO: 9.
[0095] The DNA of the present invention comprises either a genomic
DNA or cDNA. In addition, the DNA includes any DNA composed of any
codons encoding the same amino acids.
[0096] Examples of "stringent conditions" are as follows. When a
probe with 50 or more nucleotides is used and hybridization is
performed in 0.9% NaCl, the standard of temperature where 50%
dissociation occurs (Tm) is calculated using the following formula
and the temperature for hybridization can be determined according
to the following formula.
[0097] Tm=82.3.degree. C.+0.41.times.(G+C)
%--500/n--0.61.times.(formamide- ) % (n means the number of the
nucleotide of probe).
[0098] Temperature=Tm -25.degree. C.
[0099] In addition, when a probe with 100 or more nucleotides
(G+C=40 to 50%) is used, it should be considered that Tm varies as
(1) and (2) mentioned below.
[0100] (1) Tm descends by about 1.degree. C. per 1% mismatch.
[0101] (2) Tm descends by 0.6 to 0.7.degree. C. per 1%
formamide.
[0102] Accordingly, the temperature conditions for the combination
of completely complementary strands can be set as follows.
[0103] (A) 65 to 75.degree. C. (formamide not added)
[0104] (B) 35 to 45.degree. C. (in the presence of 50%
formamide)
[0105] The temperature conditions for the combination of
incompletely complementary strands can be set as follows.
[0106] (A) 45 to 55.degree. C. (formamide not added)
[0107] (B) 35 to 42.degree. C. (in the presence of 30%
formamide)
[0108] The temperature conditions when a probe with 23 or less
nucleotides is used can be 37.degree. C. or can be calculated using
the following formula.
[0109] Temperature=2.degree. C..times.(the number of A+T)+4.degree.
C..times.(the number of C+G) -5.degree. C.
[0110] The DNA of the present invention can be a DNA obtained by
any method. For example, the DNA includes complementary DNA (cDNA)
prepared from mRNA, DNA prepared from genomic DNA, DNA prepared by
chemical synthesis, DNA obtained by PCR amplification with RNA or
DNA as a template, and DNA constructed by appropriately combining
these methods.
[0111] The DNA encoding the protein of the present invention can be
obtained by the usual method such as a method to clone cDNA from
mRNA encoding the protein of the present invention, a method to
isolate genomic DNA and then splice them, chemical synthesis and so
on.
[0112] (1) cDNA can be cloned from the mRNA encoding the protein of
the present invention by, for example, the method described
below.
[0113] First, the mRNA encoding the protein of the present
invention is prepared from the above-described tissues or cells
expressing and producing a cell surface molecule (polypeptide) of
the present invention. mRNA can be prepared isolating total RNA by
a known method such as quanidine-thiocyanate method (Chirgwin et
al., Biochemistry, Vol.18, p5294, 1979), hot phenol method, or AGPC
method, and subjecting it to affinity chromatography using oligo-dT
cellulose or poly-U Sepharose.
[0114] Then, with the mRNA obtained as a template, cDNA is
synthesized, for example, by a well-known method using reverse
transcriptase such as the method of Okayama et al. (Mol. Cell.
Biol. Vol.2, p.161 (1982); ibid. Vol.3, p.280 (1983)) or the method
of Hoffman et al. (Gene Vol.25, p.263 (1983)), and converted into
double-stranded cDNA. A cDNA library is prepared by transforming E.
coli with plasmid vectors, phage vectors, or cosmid vectors having
this cDNA or by transfecting E. coli after in vitro packaging.
[0115] The plasmid vectors used in this invention are not limited
as long as they are replicated and maintained in hosts. Any phage
vectors that can be replicated in hosts can also be used. Examples
of usually used cloning vectors are pUC19, .lambda.gt10,
.lambda.gt11, and so on. When the vector is applied to
immunological screening as mentioned below, the vector having a
promoter that can express a gene encoding the polypeptide of the
present invention in a host is preferably used.
[0116] cDNA can be inserted into a plasmid by, for example, the
method of Maniatis et al. (Molecular Cloning, A Laboratory Manual,
second edition, Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA
can be inserted into a phage vector by, for example, the method of
Hyunh et al. (DNA cloning, a practical approach, Vol.1, p.49
(1985)). These methods can be simply performed by using a
commercially available cloning kit (for example, a product from
Takara Shuzo). The recombinant plasmid or phage vector thus
obtained is introduced into appropriate host cells such as a
prokaryote (for example, E. coli: HB101, DH5.alpha., MC1061/P3,
etc.).
[0117] Examples of a method for introducing a plasmid into a host
are calcium chloride method, calcium chloride/rubidium chloride
method described in Molecular Cloning, A Laboratory Manual (second
edition, Cold Spring Harbor Laboratory, p.1.74 (1989)), and
electroporation method. Phage vectors can be introduced into host
cells by, for example, a method in which the phage DNAs are
introduced into grown hosts after in vitro packaging. In vitro
packaging can be easily performed with a commercially available in
vitro packaging kit (for example, a product from Stratagene or
Amersham).
[0118] The cDNA encoding the protein of the present invention can
be isolated from the cDNA library so prepared according to the
method mentioned above by combining general cDNA screening
methods.
[0119] For example, a clone comprising the desired cDNA can be
screened by a known colony hybridization method (Crunstein et al.
Proc. Natl. Acad. Sci. USA, Vol.72, p.3961 (1975)) or plaque
hybridization method (Molecular Cloning, A Laboratory Manual,
second edition, Cold Spring Harbor Laboratory, p.2.108 (1989))
using .sup.32P-labeled chemically synthesized oligonucleotides as
probes, which are corresponding to the amino acid sequence of the
polypeptide of the present invention. Alternatively, a clone having
a DNA fragment encoding a specific region within the polypeptide of
the present invention can be screened by amplifying the region by
PCR with synthetic PCR primers.
[0120] When a cDNA library prepared using a cDNA expression vector
(for example, .lambda.ZAPII phage vector) is used, the desired
clone can be screened by the antigen-antibody reaction using an
antibody against the polypeptide of the present invention. A
screening method using PCR method is preferably used when many
clones are subjected to screening.
[0121] The nucleotide sequence of the DNA thus obtained can be
determined by Maxam-Gilbert method (Maxam et al. Proc. Natl. Acad.
Sci. USA, Vol.74, p.560 (1977)) or the dideoxynucleotide synthetic
chain termination method using phage M13 (Sanger et al. Proc. Natl.
Acad. Sci. USA, Vol.74, pp.5463-5467 (1977)). The whole or a
portion of the gene encoding the polypeptide of the present
invention can be obtained by excising the clone obtained as
mentioned above with restriction enzymes and so on.
[0122] (2) The DNA encoding the polypeptide of the present
invention can be isolated from the genomic DNA derived from the
cells expressing the polypeptide of the present invention as
mentioned above by the following methods. Such cells are
solubilized preferably by SDS or proteinase K, and the DNAs are
deproteinized by repeating phenol extraction. RNAs are digested
preferably with ribonuclease. The DNAs obtained are partially
digested with appropriate restriction enzymes, and the DNA
fragments obtained are amplified with appropriate phage or cosmid
to generate a library. Then, clones having the desired sequence are
detected, for example, by using radioactively labeled DNA probes,
and the whole or a portion of the gene encoding the protein of the
present invention is obtained from the clones by excision with
restriction enzyme and so on.
[0123] (3) The DNA of the present invention can also be chemically
synthesized by the usual method, based on the nucleotide sequence
of SEQ ID NO: 1, 3, 5, 7, 9, or 27.
[0124] The present invention also relates to a recombinant vector
comprising the DNA encoding the protein of the present invention.
The recombinant vector of the present invention is not limited as
long as it can be replicated and maintained or can autonomously
replicate in various prokaryotic and/or eukaryotic hosts. The
vector of the present invention includes plasmid vectors and phage
vectors.
[0125] The recombinant vector can easily be prepared by ligating
the DNA encoding the protein of the present invention with a vector
for recombination available in the art (plasmid DNA and
bacteriophage DNA) by the usual method. Specific examples of the
vectors for recombination used are E. coli-derived plasmids such as
pBR322, pBR325, pUC12, pUC13, and pUC19, yeast-derived plasmids
such as pSH19 and pSH15, and Bacillus subtilis-derived plasmids
such as pUB110, pTP5, and pC194. Examples of phages are a
bacteriophage such as .lambda. phage, and an animal or insect virus
(pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and
nuclear polyhedrosis virus.
[0126] An expression vector is useful for expressing the DNA
encoding the protein of the present invention and for producing the
polypeptide of the present invention. The expression vector is not
limited as long as it expresses the gene encoding the polypeptide
of the present invention in various prokaryotic and/or eukaryotic
host cells and produces this protein. Examples thereof are pMAL C2,
pEF-BOS (Nucleic Acids Res. Vol.18, p.5322 (1990)), pME18S
(Experimental Medicine: SUPPLEMENT, "Handbook of Genetic
Engineering" (1992)), and so on.
[0127] When bacteria, particularly E. coli are used as host cells,
an expression vector is generally comprised of, at least, a
promoter/operator region, an initiation codon, the DNA encoding the
protein of the present invention, termination codon, terminator
region, and replicon.
[0128] When yeast, animal cells, or insect cells are used as hosts,
an expression vector is preferably comprised of, at least, a
promoter, an initiation codon, the DNA encoding the protein of the
present invention, and a termination codon. It may also comprise
the DNA encoding a signal peptide, enhancer sequence, 5'- and
3'-untranslated region of the gene encoding the protein of the
present invention, splicing junctions, polyadenylation site,
selectable marker region, and replicon. The expression vector may
also contain, if required, a gene for gene amplification (marker)
that is usually used.
[0129] A promoter/operator region to express the polypeptide of the
present invention in bacteria comprises a promoter, an operator,
and a Shine-Dalgarno (SD) sequence (for example, AAGG). For
example, when the host is Escherichia, it preferably comprises Trp
promoter, lac promoter, recA promoter, .lambda.PL promoter, lpp
promoter, tac promoter, or the like. Examples of a promoter to
express the polypeptide of the present invention in yeast are PH05
promoter, PGK promoter, GAP promoter, ADH promoter, and so on. When
the host is Bacillus, examples thereof are SL01 promoter, SP02
promoter, penP promoter and so on. When the host is a eukaryotic
cell such as a mammalian cell, examples thereof are SV40-derived
promoter, retrovirus promoter, heat shock promoter, and so on, and
preferably SV-40 and retrovirus-derived one. As a matter of course,
the promoter is not limited to the above examples. In addition, to
use an enhancer is effective for expression.
[0130] A preferable initiation codon is, for example, a methionine
codon (ATG).
[0131] The commonly used termination codon (for example, TAG, TGA,
TAA, and so on) is illustrated as a termination codon.
[0132] Usually used natural or synthetic terminators are used as a
terminator region.
[0133] A replicon means a DNA capable of replicating the whole DNA
sequence in host cells, and includes a natural plasmid, an
artificially modified plasmid (DNA fragment prepared from a natural
plasmid), a synthetic plasmid, and so on. Examples of a preferable
plasmids are pBR322 or its artificial derivatives (DNA fragment
obtained by treating pBR322 with appropriate restriction enzymes)
for E. coli, yeast 2 .mu. plasmid or yeast chromosomal DNA for
yeast, and pRSVneo ATCC 37198, pSV2dhfr ATCC 37145, pdBPV-MMTneo
ATCC 37224, pSV2neo ATCC 37149, etc. for mammalian cells.
[0134] An enhancer sequence, polyadenylation site, and splicing
junction that are usually used in the art, such as those derived
from SV40 can be also used.
[0135] A selectable marker usually used can be used according to
the usual method. Examples thereof are resistance genes for
antibiotics, such as tetracycline, neomycin, ampicillin, or
kanamycin, and thymidine kinase gene.
[0136] Examples of a gene for gene amplification are dihydrofolate
reductase (DHFR) gene, thymidine kinase gene, neomycin resistance
gene, glutamate synthase gene, adenosine deaminase gene, ornithine
decarboxylase gene, hygromycin-B-phophotransferase gene, aspartate
transcarbamylase gene, etc.
[0137] The expression vector of the present invention can be
prepared by continuously and circularly linking at least the
above-mentioned promoter, initiation codon, DNA (gene) encoding the
polypeptide of the present invention, termination codon, and
terminator region, to an appropriate replicon. If desired,
appropriate DNA fragments (for example, linkers, restriction sites
generated with other restriction enzyme), can be used by the usual
method such as digestion with a restriction enzyme or ligation
using T4 DNA ligase.
[0138] Transformants of the present invention can be prepared by
introducing the expression vector mentioned above into host
cells.
[0139] Host cells used in the present invention are not limited as
long as they are compatible with an expression vector mentioned
above and can be transformed. Examples thereof are various cells
such as natural cells or artificially established recombinant cells
usually used in technical field of the present invention (for
example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces,
Pichia, etc.), animal cells, or insect cells.
[0140] E. coli or animal cells are preferably used. Specific
examples are E. coli (DH5.alpha., TB1, HB101, etc.), mouse-derived
cells (COP, L, C127, Sp2/0, NS-1, NIH 3T3, etc.), rat-derived
cells, hamster-derived cells (BHK, CHO, etc.), monkey-derived cells
(COS1, COS3, COS7, CV1, Velo, etc.), and human-derived cells (Hela,
diploid fibroblast-derived cells, HEK293, myeloma, Namalwa,
etc.).
[0141] An expression vector can be introduced (transformed
(transduced)) into host cells by known method.
[0142] Transformation can be performed, for example, according to
the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, Vol.69,
p.2110 (1972)), protoplast method (Mol. Gen. Genet., Vol.168, p.111
(1979)), or competent method (J. Mol. Biol., Vol.56, p.209 (1971))
when the hosts are bacteria (E. col, Bacillus subtilis, etc.), the
method of Hinnen et al. (Proc. Natl. Acad. Sci. USA, Vol.75, p.1927
(1978)), or lithium method (J. Bacteriol., Vol.153, p.163 (1983))
when the host is Saccharomyces cerevisiae, the method of Graham
(Virology, Vol.52, p.456 (1973)) when the hosts are animal cells,
and the method of Summers et al. (Mol. Cell. Biol., Vol.3,
pp.2156-2165 (1983)) when the hosts are insect cells.
[0143] The protein of the present invention can be produced by
cultivating transformants (in the following this term includes
transductants) comprising an expression vector prepared as
mentioned above in nutrient media.
[0144] The nutrient media preferably comprise carbon source,
inorganic nitrogen source, or organic nitrogen source necessary for
the growth of host cells (transformants). Examples of the carbon
source are glucose, dextran, soluble starch, and sucrose, and
examples of the inorganic or organic nitrogen source are ammonium
salts, nitrates, amino acids, corn steep liquor, peptone, casein,
meet extract, soy bean cake, and potato extract. If desired, they
may comprise other nutrients (for example, an inorganic salt (for
example, calcium chloride, sodium dihydrogenphosphate, and
magnesium chloride), vitamins, antibiotics (for example,
tetracycline, neomycin, ampicillin, kanamycin, etc.).
[0145] Cultivation is performed by a method known in the art.
Cultivation conditions such as temperature, pH of the media, and
cultivation time are selected appropriately so that the protein of
the present invention is overproduced.
[0146] Specific media and cultivation conditions used depending on
host cells are illustrated below, but are not limited thereto.
[0147] When the hosts are bacteria, actinomycetes, yeasts,
filamentous fungi, liquid media comprising the nutrient source
mentioned above are appropriate. The media with pH 5 to 8 are
preferably used.
[0148] When the host is E. coli, examples of preferable media are
LB media, and M9 media (Miller et al. Exp. Mol. Genet., Cold Spring
Harbor Laboratory, p.431 (1972)). Using these media, cultivation
can be performed usually at 14 to 43.degree. C. for about 3 to 24
hours with aeration and stirring, if necessary.
[0149] When the host is Bacillus, cultivation can be performed
usually at 30 to 40.degree. C. for about 16 to 96 hours with
aeration and stirring, if necessary.
[0150] When the host is yeast, examples of media are Burkholder
minimal media (Bostian, Proc. Natl. Acad. Sci. USA, Vol.77, p.4505
(1980)). The pH of the media is preferably 5 to 8. Cultivation can
be performed usually at 20 to 35.degree. C. for about 14to 144
hours with aeration and stirring, if necessary.
[0151] When the host is an animal cell, examples of media are MEM
media containing about 5 to 20% fetal bovine serum (Science,
Vol.122, p.501 (1952)), DMEM media (Virology, Vol.8, p.396 (1959)),
RPMI1640 media (J. Am. Med. Assoc., Vol.199, p.519 (1967)), and 199
media (Proc. Soc. Exp. Biol. Med., Vol.73, p.1 (1950)). The pH of
the media is preferably about 6 to 8. Cultivation can be performed
usually at about 30 to 40.degree. C. for about 15 to 72 hours with
aeration and stirring, if necessary.
[0152] When the host is an insect cell, an example of media is
Grace's media containing fetal bovine serum (Proc. Natl. Acad. Sci.
USA, Vol.82, p.8404 (1985)). The pH thereof is preferably about 5to
8. Cultivation can be performed usually at about 20 to 40.degree.
C. for 15 to 100 hours with aeration and stirring, if
necessary.
[0153] The protein of the present invention can be produced as a
transmembrane protein by cultivating transformants as mentioned
above, in particular animal cells to overexpress the protein of the
present invention on the surface of the cells. The protein of the
present invention can be produced as a soluble protein fragment
such as an extracellular region protein fragment by preparing the
transformants as mentioned above using the DNA encoding the
extracellular region and by cultivating the transformants to allow
them to secrete the soluble polypeptide into the culture
supernatant.
[0154] Namely, a culture filtrate (supernatant) is obtained by the
method such as filtration or centrifugation of the obtained
culture, and the protein of the present invention is purified and
isolated from the culture filtrate by the usual method commonly
used in order to purify and isolate a natural or synthetic
protein.
[0155] Examples of the isolation and purification method are a
method utilizing solubility, such as salting out and solvent
precipitation method, a method utilizing the difference in
molecular weight, such as dialysis, ultrafiltration, gel
filtration, and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, a method utilizing charges, such as ion exchange
chromatography and hydroxylapatite chromatography, a method
utilizing specific affinity, such as affinity chromatography, a
method utilizing the difference in hydrophobicity, such as reverse
phase high performance liquid chromatography, and a method
utilizing the difference in isoelectric point, such as isoelectric
focusing.
[0156] When the protein of the present invention exists in the
periplasm or cytoplasm of cultured transformants, first, the fungus
bodies or cells are harvested by the usual method such as
filtration or centrifugation and suspended in appropriate buffer.
After the cell wall and/or cell membrane of the cells and so on are
disrupted by the method such as lysis with sonication, lysozyme,
and freeze-thawing, the membrane fraction comprising the protein of
the present invention is obtained by the method such as
centrifugation or filtration. The membrane fraction is solubilized
with a detergent such as Triton-X100 to obtain the crude extract.
Finally, the polypeptide or the polypeptide fragment is isolated
and purified from the crude extract by the usual method as
illustrated above.
[0157] The "transgenic mouse" of the present invention is a
transgenic mouse wherein the DNA (cDNA or genomic DNA) prepared as
mentioned above encoding the protein of the present invention
derived from animals except mice (non-self protein) have been
integrated into its endogenous locus of the mouse. The transgenic
mouse expresses the non-self protein and secretes the protein into
its body.
[0158] The transgenic mouse can be prepared according to the method
as usually used for producing a transgenic animal (for example, see
"Newest Manual of Animal Cell Experiment", LIC press, Chapter 7,
pp.361-408, (1990)).
[0159] Specifically, for example, embryonic stem cells (ES cells)
obtained from normal mouse blastocysts are transformed with an
expression vector in which the gene encoding human-derived
polypeptide of the present invention (i.e. "uman JTT-1 antigen")
has been operably inserted. ES cells in which the gene encoding the
human-derived polypeptide of the present invention has been
integrated into the endogenous gene are screened by the usual
method. Then, the ES cells screened are microinjected into a
fertilized egg obtained from another normal mouse (blastocyst)
(Proc. Natl. Acad. Sci. USA, Vol.77, No.12, pp.7380-7384 (1980);
U.S. Pat. No. 4,873,191). The blastocyst is transplanted into the
uterus of another normal mouse as the foster mother. Then, founder
mice (progeny mice) are born from the foster mother mouse. By
mating the founder mice with normal mice, heterogeneic transgenic
mice are obtained. By mating the heterogeneic transgenic mice with
each other, homogeneic transgenic mice are obtained according to
Mendel's laws.
[0160] "Knockout mouse" of the present invention is a mouse wherein
the endogenous gene encoding the mouse-derived protein of the
present invention has been knocked out (inactivated). It can be
prepared, for example, by positive-negative selection method in
which homologous recombination is applied (U.S. Pat. No. 5,464,764;
No. 5,487,992; No. 5,627,059; Proc. Natl. Acad. Sci. USA, Vol.86,
pp.8932-8935 (1989); Nature, Vol.342, pp.435-438 (1989); etc.).
[0161] The "antibody" of the present invention can be a polyclonal
antibody (antiserum) or a monoclonal antibody, and preferably a
monoclonal antibody.
[0162] Specifically, it is an antibody reactive to (against, which
binds to) the above-mentioned protein or its fragment of the
present invention.
[0163] The antibody of the present invention can be natural
antibodies obtained by immunizing mammals such as mice, rats,
hamsters, guinea pigs, and rabbits with an immunogen (antigen),
such as the protein of the present invention (natural, recombinant,
or synthetic ones), cells expressing the protein of the present
invention, or transformants overexpressing the designed protein on
the surface thereof prepared using recombinant DNA technology as
described above on the cell surface. The antibody of the present
invention also includes chimeric antibodies and humanized
antibodies (CDR-grafted antibodies) that can be produced by
recombinant DNA technology, and human antibodies that can be
produced using human antibody-producing transgenic animals.
[0164] The monoclonal antibody includes those having any one
isotype of IgG, IgM, IgA, IgD, or IgE. IgG or IgM is
preferable.
[0165] The polyclonal antibody (antisera) or monoclonal antibody of
the present invention can be produced by the known methods. Namely,
a mammal, preferably, a mouse, rat, hamster, guinea pig, rabbit,
cat, dog, pig, goat, horse, or cattle, or more preferably, a mouse,
rat, hamster, guinea pig, or rabbit is immunized, for example, with
an immunogen (antigen) mentioned above with Freund's adjuvant, if
necessary. The polyclonal antibody can be obtained from the
antiserum obtained from the animal so immunized. In addition, the
monoclonal antibodies are produced as follows. Hybridomas are
prepared from the antibody-producing cells obtaind from the animal
so immunized and myeloma cells that are not capable of producing
autoantibodies. The hybridomas are cloned, and clones producing the
monoclonal antibodies showing the specific affinity to the antigen
used for immunizing the mammal are screened.
[0166] Specifically, the monoclonal antibody can be produced as
follows. Immunizations are performed by injecting or implanting
once or several times the protein of the present invention, cells
expressing the protein and so on as mentioned above as an
immunogen, if necessary, with Freund's adjuvant, subcutaneously,
intramuscularly, intravenously, through the footpad, or
intraperitoneally into a mouse, rat, hamster, guinea pig, or
rabbit, preferably a mouse, rat, or hamster (including a transgenic
animal generated so as to produce antibodies derived from another
animal such as the transgenic mouse producing human antibody).
Usually, immunizations are performed once to four times every one
to fourteen days after the first immunization. Antibody-producing
cells are obtained from the mammal so immunized in about one to
five days after the last immunization.
[0167] Hybridomas that secrete a monoclonal antibody can be
prepared by the method of Kbhler and Milstein (Nature, Vol.256,
pp.495-497 (1975)) and by its modified method. Namely, hybridomas
are prepared by fusing antibody-producing cells contained in a
spleen, lymph node, bone marrow, or tonsil obtained from the mammal
immunized as mentioned above, preferably a spleen, with myelomas
without autoantibody-producing ability, which are derived from,
preferably, a mammal such as a mouse, rat, guinea pig, hamster,
rabbit, or human, or more preferably, a mouse, rat, or human.
[0168] For example, mouse-derived myeloma P3/X63-AG8.653 (653),
P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0,
Sp2), PAI, F0, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., or
human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR,
D1R11, or CEM-T15 can be used as a myeloma used for the cell
fusion.
[0169] Hybridoma clones producing monoclonal antibodies can be
screened by cultivating hybridomas, for example, in microtiter
plates and by measuring the reactivity of the culture supernatant
in the well in which hybridoma growth is observed, to the immunogen
used for the immunization mentioned above, for example, by enzyme
immunoassay such as RIA and ELISA.
[0170] The monoclonal antibodies can be produced from hybridomas by
cultivating the hybridomas in vitro or in vivo such as in the
ascites fluid of amouse, rat, guinea pig, hamster, or rabbit,
preferably a mouse or rat, more preferably mouse and isolating the
antibodies from the resulting the culture supernatant or ascites
fluid of a mammal.
[0171] Cultivating hybridomas in vitro can be performed depending
on the property of cells to be cultured, on the object of a test
study, and on the various conditions of a cultivating method, by
using known nutrient media or any nutrient media derived from known
basal media for growing, maintaining, and storing the hybridomas to
produce monoclonal antibodies in culture supernatant.
[0172] Examples of basal media are low calcium concentration media
such as Ham'F12 medium, MCDB153 medium, or low calcium
concentration MEM medium, and high -calcium concentration media
such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium,
ASF104 medium, or RD medium. The basal media can contain, for
example, sera, hormones, cytokines, and/or various inorganic or
organic substances depending on the objective.
[0173] Monoclonal antibodies can be isolated and purified from the
culture supernatant or ascites fluid mentioned above by saturated
ammonium sulfate precipitation, euglobulin precipitation method,
caproic acid method, caprylic acid method, ion exchange
chromatography (DEAE or DE52), affinity chromatography using
anti-immunoglobulin column or protein A column.
[0174] The "chimeric antibody" of the present invention is a
monoclonal antibody prepared by genetic engineering, and
specifically means a chimeric antibody such as mouse/human chimeric
monoclonal antibody whose variable regions or the other regions are
derived from mouse immunoglobulin and whose constant regions are
derived from human immunoglobulin.
[0175] The constant region derived from human immunoglobulin has
the amino acid sequence inherent in each isotype such as IgG, IgM,
IgA, IgD, and IgE. The constant region of the recombinant chimeric
monoclonal antibody of the present invention can be that of human
immunoglobulin belonging to any isotype. Preferably, it is the
constant region of human IgG.
[0176] The chimeric monoclonal antibody of the present invention
can be produced, for example, as follows. Needless to say, the
production method is not limited thereto.
[0177] A mouse/human chimeric monoclonal antibody can be prepared,
referring to Experimental Medicine: SUPPLEMENT, Vol.1.6, No.10
(1988); and examined published Japanese patent application (JP-B)
No. Hei3-73280. Namely, it can be prepared by operably inserting CH
gene (C gene encoding the constant region of H chain) obtained from
the DNA encoding human immunoglobulin downstream of active VH genes
(rearranged VDJ gene encoding the variable region of H chain)
obtained from the DNA encoding a mouse monoclonal antibody isolated
from the hybridoma producing the mouse monoclonal antibody, and CL
gene (C gene encoding the constant region of L chain) obtained from
the DNA encoding human immunoglobulin downstream of active VL genes
(rearranged VJ gene encoding the variable region of L chain)
obtained from the DNA encoding the mouse monoclonal antibody
isolated from the hybridoma, into the same or different vectors so
as for them to be expressed, following by transforming host cells
with the expression vector, and then by cultivating the
transformants.
[0178] Specifically, DNAs are first extracted from mouse monoclonal
antibody-producing hybridomas by the usual method, digested with
appropriate restriction enzymes (for example, EcoRI and HindIII),
electrophoresed (using, for example, 0.7% agarose gel), and
analyzed by Southern blotting. After an electrophoresed gel is
stained, for example, with ethidium bromide and photographed, the
gel is given with marker positions, washed twice with water, and
soaked in 0.25 M HCl for 15 minutes. Then, the gel is soaked in 0.4
N NaOH solution for 10 minutes with gently stirring. The DNAs are
transferred to a filter for 4 hours by the usual method. The filter
is recovered and washed twice with 2.times.SSC. After the filter is
sufficiently dried, it is baked at 75.degree. C. for3 hours. After
baking, the filter is treated with 0.1.times.SSC/0.1% SDS at
65.degree. C. for 30 minutes. Then, it is soaked in
3.times.SSC/0.1% SDS. The filter obtained is treated with
prehybridization solution in a plastic bag at 65.degree. C. for 3
to 4 hours.
[0179] Next, .sup.32P-labeled probe DNA and hybridization solution
are added to the bag and reacted at 65.degree. C. about 12 hours.
After hybridization, the filter is washed under appropriate salt
concentration, reaction temperature, and time (for example,
2.times.SSC-0.1% SDS, room temperature, 10 minutes). The filter is
put into a plastic bag with a little 2.times.SSC, and subjected to
autoradiography after the bag is sealed.
[0180] Rearranged VDJ gene and VJ gene encoding H chain and L chain
of a mouse monoclonal antibody are identified by Southern blotting
mentioned above. The region comprising the identified DNA fragment
is fractioned by sucrose density gradient centrifugation and
inserted into a phage vector (for example, Charon 4A, Charon 28,
.lambda.EMBL3, .lambda.EMBL4, etc.). E. coli (for example, LE392,
NM539, etc.) is transformed with the phage vector to generate a
genomic library. The genomic library is screened by plaque
hybridization such as Benton-Davis method (Science, Vol.196, pp.
180-182 (1977)) using appropriate probes (H chain J gene, L chain
(.kappa.) J gene, etc.) to obtain positive clones comprising
rearranged VDJ gene or VJ gene. By making the restriction map and
determining the nucleotide sequence of the clones obtained, it is
confirmed that genes comprising the desired, rearranged VH (VDJ)
gene or VL (VJ) gene are obtained.
[0181] Separately, human CH gene and human CL gene used for
chimerization are isolated. For example, when a chimeric antibody
with human IgG1 is produced, C.gamma.1 gene as a CH gene, and
C.kappa. gene as a CL gene, are isolated. These genes can be
isolated from human genomic library with mouse C.gamma.1 gene and
mouse C.kappa. gene, corresponding to human C.gamma.1 gene and
human C .kappa. gene, respectively, as probes, taking advantage of
high homology between the nucleotide sequences of mouse
immunoglobulin gene and that of human immunoglobulin gene.
[0182] Specifically, DNA fragments comprising human C.kappa. gene
and an enhancer region are isolated from human .lambda. Charon 4A
HaeIII-AluI genomic library (Cell, Vol.15, pp.1157-1174 (1978)),
for example, with a 3 kb HindIII-BamHI fragment of clone Igl46
(Proc. Natl. Acad. Sci. USA, Vol.75, pp.4709-4713 (1978)) and a 6.8
kb EcoRI fragment of clone MEP10 (Proc. Natl. Acad. Sci. USA,
Vol.78, pp.474-478 (1981)) as probes. In addition, for example,
after human fetal hepatocyte DNA is digested with HindIII and
fractioned by agarose gel electrophoresis, a 5.9 kb fragment is
inserted into .lambda.788 and then human C.gamma.1 gene is isolated
with the probes mentioned above.
[0183] Using mouse VH gene, mouse VL gene, human CH gene, and human
CL gene so obtained, and taking promoter region and enhancer region
into consideration, human CH gene is inserted downstream mouse VH
gene and human CL gene is inserted downstream mouse VL gene into an
expression vector such as pSv2gpt or pSV2neo with appropriate
restriction enzymes and DNA ligase by the usual method. In this
case, chimeric genes of mouse VH gene/human CH gene and mouse VL
gene/human CL gene can be respectively inserted in the same
expression vector or in different expression vectors.
[0184] Chimeric gene-inserted expression vector(s) thus prepared
are introduced into myelomas that do not produce antibodies, for
example, P3X63.multidot.Ag8.multidot.653 cells or SP210 cells by
protoplast fusion method, DEAE-dextran method, calcium phosphate
method, or electroporation method. The transformants are screened
by cultivating in media containing a drug corresponding to the drug
resistance gene inserted into the expression vector and, then,
cells producing desired chimeric monoclonal antibodies are
obtained.
[0185] Desired chimeric monoclonal antibodies are obtained from the
culture supernatant of antibody-producing cells thus screened.
[0186] The "humanized antibody (CDR-grafted antibody)" of the
present invention is a monoclonal antibody prepared by genetic
engineering and specifically means a humanized monoclonal antibody
wherein a portion or the whole of the complementarity determining
regions of the hypervariable region are derived from the
complementarity determining regions of the hypervariable region
from a mouse monoclonal antibody, the framework regions of the
variable region are derived from the framework regions of the
variable region from human immunoglobulin, and the constant region
is derived from human a constant region from immunoglobulin.
[0187] The complementarity determining regions of the hypervariable
region exists in the hypervariable region in the variable region of
an antibody and means three regions which directly and
complementary binds to an antigen (complementarity-determining
residues, CDR1, CDR2, and CDR3). The framework regions of the
variable region means four comparatively conserved regions lying
upstream, downstream or between the three complementarity
determining regions (framework region, FR1, FR2, FR3, and FR4).
[0188] In other words, a humanized monoclonal antibody means that
in which the whole region except a portion or the whole of the
complementarity determining regions of the hypervariable region of
a nonhuman mammal-derived monoclonal antibody have been replaced
with their corresponding regions derived from human
immunoglobulin.
[0189] The constant region derived from human immunoglobulin has
the amino acid sequence inherent in each isotype such as IgG (IgG1,
IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of a
humanized monoclonal antibody in the present invention can be that
from human immunoglobulin belonging to any isotype. Preferably, it
is the constant region of human IgG. The framework regions of the
constant region derived from human immunoglobulin are not
particularly limited.
[0190] The humanized monoclonal antibody of the present invention
can be produced, for example, as follows. Needless to say, the
production method is not limited thereto.
[0191] For example, a recombinant humanized monoclonal antibody
derived from mouse monoclonal antibody can be prepared by genetic
engineering, referring to unexamined Japanese patent publication
(JP-WA) No. Hei 4-506458 and unexamined Japanese patent publication
(JP-A) No. Sho 62-296890. Namely, at least one mouse H chain CDR
gene and at least one mouse L chain CDR gene corresponding to the
mouse H chain CDR gene are isolated from hybridomas producing mouse
monoclonal antibody, and human H chain gene encoding the whole
regions except human H chain CDR corresponding to mouse H chain CDR
mentioned above and human L chain gene encoding the whole region
except human L chain CDR correspond to mouse L chain CDR mentioned
above are isolated from human immunoglobulin genes.
[0192] The mouse H chain CDR gene(s) and the human H chain gene(s)
so isolated are operably inserted into an appropriate vector so
that they can be expressed. Similarly, the mouse L chain CDR
gene(s) and the human L chain gene(s) are operably inserted into
another appropriate vector so that they can be expressed.
Alternatively, the mouse H chain CDR gene(s)/human H chain gene(s)
and mouse L chain CDR gene(s)/human L chain gene(s) can be operably
inserted into the same expression vector so that they can be
expressed. Host cells are transformed with the expression vector
thus prepared to obtain transformants producing humanized
monoclonal antibody. By cultivating the transformants, desired
humanized monoclonal antibody is obtained from culture
supernatant.
[0193] The "human monoclonal antibody" of the present invention is
immunoglobulin in which the entire regions comprising the variable
and constant region of H chain, and the variable and constant
region of L chain constituting immunoglobulin are derived from the
gene encoding human immunoglobulin.
[0194] The human antibody can be produced in the same way as the
production method of polyclonal or monoclonal antibodies mentioned
above by immunizing, with an antigen, a transgenic animal which for
example, at least human immunoglobulin gene(s) have been integrated
into the locus of a non-human mammal such as a mouse by the usual
method. For example, a transgenic mouse producing human antibodies
is prepared by the methods described in Nature Genetics, Vol.15,
pp.146-156(1997); Nature Genetics, Vol. 7, pp. 13-21 (1994); JP-WA
Nos. Hei 4-504365, International patent publication No. WO94/25585;
Nikkei Science, No.6, pp.40-50 (1995); Nature, Vol.368, pp.856-859
(1994); and JP-WA No. Hei 6-500233.
[0195] The "portion of an antibody" used in the present invention
means a partial region of the antibody, preferably monoclonal
antibody of the present invention as mentioned above, and
specifically, means F(ab').sub.2, Fab', Fab, Fv (variable fragment
of antbody), sFv, dsFv (disulfide stabilized Fv), or dAb (single
domain antibody) (Exp. Opin. Ther. Patents, Vol.6, No.5, pp.441-456
(1996)).
[0196] "F(ab').sub.2" and "Fab'" can be produced by treating
immunoglobulin (monoclonal antibody) with a protease such as pepsin
and papain, and means an antibody fragment generated by digesting
immunoglobulin near the disulfide bonds existing between the hinge
regions in each of the two H chains. For example, papain cleaves
IgG upstream of the disulfide bonds existing between the hinge
regions in each of the two H chains to generate two homologous
antibody fragments in which an L chain composed of VL (L chain
variable region) and CL (L chain constant region), and an H chain
fragment composed of VH (H chain variable region) and CH.gamma.1
(.gamma.1 region in the constant region of H chain) are connected
at their C terminal regions through a disulfide bond. Each of such
two homologous antibody fragments is called Fab'. Pepsin also
cleaves IgG downstream of the disulfide bonds existing between the
hinge regions in each of the two H chains to generate an antibody
fragment slightly larger than the fragment in which the two
above-mentioned Fab' are connected at the hinge region. This
antibody fragment is called F(ab').sub.2.
[0197] The "pharmaceutical composition" of the present invention
comprises any one of the protein, protein fragment, fusion protein
antibody, or portion of an antibody of the present invention as
defined above; and a pharmaceutically acceptable carrier.
[0198] The "pharmaceutically acceptable carrier" includes a
excipieut, a diluent, an expander, a decomposition agent, a
stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a
colorant, a sweetener, a viscosity increasing agent, a flavor, a
solubility increasing agent, or other additives. Using one or more
of such carriers, a pharmaceutical composition can be fomulated
into tablets, pills, powders, granules, injections, solutions,
capsules, troches, elixirs, suspensions, emulsions, or syrups. The
pharmaceutical composition can be administered orally or
parenterally. Other forms for parenteral administration include a
solution for external application, suppository for rectal
administration, and pessary, prescribed by the usual method, which
comprises one or more active ingredient.
[0199] The dosage can vary depending on the age, sex, weight, and
symptom of a patient, effect of treatment, administration route,
period of treatment, or the kind of active ingredient (polypeptide
or antibody mentioned above) contained in the pharmaceutical
composition. Usually, the pharmaceutical composition can be
administered to an adult in a dose of 10 .mu.g to 1000 mg (or 10
.mu.g to 500 mg) per one administration. Depending on various
conditions, the dosage less than that mentioned above may be
sufficient in some cases, and the dosage more than that mentioned
above may be necessary in other cases.
[0200] In particular, the injection can be produced by dissolving
or suspending the antibody in a non-toxic, pharmaceutically
acceptable carrier such as physiological saline or commercially
available distilled water for injection with adjusting a
concentration to 0.1 .mu.g antibody/ml carrier to 10 mg antibody/ml
carrier. The injection thus produced can be administered to a human
patient in need of treatment in a dose of 1 .mu.g to 100 mg/kg body
weight, preferably 50 .mu.g to 50 mg/kg body weight once or more
times a day. Examples of administration route are medically
appropriate administration routes such as intravenous injection,
subcutaneous injection, intradermal injection, intramuscular
injection, or intraperitoneal injection, preferably intravenous
injection.
[0201] The injection can also be prepared into a non-aqueous
diluent (for example, propylene glycol, polyethylene glycol,
vegetable oil such as olive oil, and alcohol such as ethanol),
suspension, or emulsion.
[0202] The injection can be sterilized by filtration with a
bacteria-non-penetrated filter, by mixing bacteriocide, or by
irradiation. The injection can be produced in the form that is
prepared upon use. Namely, it is freeze-dried to be a sterile solid
composition, and can be dissolved in sterile distilled water for
injection or another solvent before use.
[0203] The pharmaceutical composition of the present invention can
be used to treat or prevent arteriosclerosis and restenosis after
the treatment of artery occlusion, such as PTCA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0204] FIG. 1 is a photograph showing an electrophoresis image of
rabbit BA2303 cDNA samples obtained by RT-PCR.
[0205] The numerals indicate days from the exfoliation of the
artery endothelium using a balloon catheter to the removal of the
artery; thus, the figure shows the time course of the cDNA
expression.
[0206] FIG. 2 is a photograph showing an electrophoresis image of
rabbit BA0306 cDNA samples obtained by RT-PCR.
[0207] The numerals indicate days from the exfoliation of the
artery endothelium using a balloon catheter to the removal of the
artery; thus, the figure shows the time course of the cDNA
expression.
[0208] FIG. 3 shows a plot of the hydrophobicity and hydrophilicity
of the amino acid residues composing rabbit BA2303 protein.
[0209] FIG. 4 shows a plot of the hydrophobicity and hydrophilicity
of the amino acid residues composing human BA0306 protein.
[0210] FIG. 5 shows a plot of the hydrophobicity and hydrophilicity
of the amino acid residues composing human BA2303 protein.
[0211] FIG. 6 is a photograph showing the result of Northern blot
analysis of the expression of human BA2303 mRNA in various human
tissues.
[0212] FIG. 7 is a photograph showing the result of Northern blot
analysis of the expression of human BA0306 mRNA in various human
tissues.
[0213] FIG. 8 shows a plot of the hydrophobicity and hydrophilicity
of the amino acid residues composing mouse BA2303 protein.
[0214] FIG. 9 shows the sequence homology at the amino acid level
between BA2303 proteins from rabbit, human, and mouse.
[0215] FIG. 10 shows the sequence homology at the amino acid level
between BA0306 proteins from rabbit, human, and mouse.
[0216] FIG. 11 schematically shows the structures of mouse genomic
DNA containing exons that encode mouse BA2303 protein, and of the
targeting vector for knockout mice generation.
[0217] FIG. 12 schematically shows the structures of mouse genomic
DNA containing exons that encode mouse BA0306 protein, and of the
targeting vector for knockout mice generation.
BEST MODE FOR IMPLEMENTING THE INVENTION
[0218] The present invention is illustrated in detail below with
reference to examples, but is not to be construed as being limited
thereto.
EXAMPLE 1
[0219] Generation of a rabbit model whose aortal endothelium is
detached by PTCA
[0220] According to the method described in "Protocols in
Circulation Research" (Jikken-Igaku Zoukan (1996) Vol.14 (12), 87),
a balloon catheter was inserted into the thoracic artery of
Japanese white rabbits by surgical operation and was inflated to
perform PTCA. The artery including the operation site was removed
at certain periods from day 1 to six months after PTCA.
EXAMPLE 2
[0221] Preparation of total RNA from removed aortae
[0222] The aorta was removed at 1, 2, 4, 7, 14, 23, 30, 54, 112,
and 137 days after PTCA, and total RNA was prepared from the aortae
by the standard method using the TRIZOL reagent (GIBCO BRL).
[0223] Also, the aorta was removed from a normal Japanese white
rabbit, which was not subjected to PTCA, and total RNA was prepared
as described.
EXAMPLE 3
[0224] cDNA synthesis
[0225] Total RNAs (each 2 .mu.l, 1 .mu.g/ml) sampled with the
passage of time or mRNA samples (each 2 .mu.l, 0.5 .mu.g/ml), which
were obtained in Example 2, were dissolved in diethyl pirocarbonate
(DEPC)-treated distilledwater(8 .mu.l). Anchorprimer(GT15MA, 1
.mu.l, 25 pmol/.mu.l) was added to make the total volume 10 .mu.l,
and the mixture was then incubated 5 min at 65.degree. C. The
samples were placed on ice immediately after completion of the
incubation.
[0226] Then, 5x first strand buffer (4 .mu.l, composition: 0.25 M
Tris-HCl (pH 7.5), 0.375 M KCl, 0.05 M DTT, 0.015 M MgCl.sub.2),
0.1 M DTT (2 .mu.l), 250 .mu.M dNTP (1 .mu.l), distilled water (1
.mu.l), and reverse transcriptase (Superscript, GIBCO BRL, 1 .mu.l,
200 U/.mu.l) were added to make the total volume 20 .mu.l. cDNA was
synthesized by incubating the reaction mixture for 1 hr at
42.degree. C., and then DEPC-treated water (30 .mu.l) was added to
make the final volume 50 .mu.l.
EXAMPLE 4
[0227] Analysis of the time course of gene expression
[0228] The time course of gene expression after PTCA was analyzed
by the standard method using differential display (Nucleic Acid
Research (1993) Vol. 21(18), 4272-4280; Science (1992) Vol. 257,
967-971), and RT-PCR (reverse transcription-polymerase chain
reaction; "PCR and its Application" (Jikken-Igaku Zoukan (1990)
Vol. 8(9); "Gene Amplification PCR Method/Principles and Novel
Applications" Kyoritsu-Syuppan (1992)).
[0229] One hundred-fold dilution of the cDNA samples (each time
point) which were prepared in Example 3 was used as a template for
PCR in differential display. Fifty fold dilution was used for cDNA
samples that were synthesized from mRNA (each time point).
[0230] The template cDNA (each 2 .mu.l) was mixed with distilled
water (10.75 .mu.l), 10x EX Taq buffer (2 .mu.l), 25 .mu.M dNTP
(1.5 .mu.l), arbitrary primer (sequence: GATCAATCGC, 1 .mu.l, 25
pmol/.mu.l), anchor primer (1 .mu.l, 25 pmol/.mu.l), EX Taq DNA
polymerase (0.25 .mu.l), and .alpha.35S-dATP (1.5 .mu.l, 10 mCi/ml,
Amersham) to make the total volume 20 .mu.l. PCR was carried out
with a cycle of 95.degree. C. for 3 min, 40.degree. C. for 5 min,
72.degree. C. for 5 min; 40 cycles of 95.degree. C. for 30 sec,
40.degree. C. for 2 min, 72.degree. C. for 1 min; and a step of
72.degree. C. for 5 min, and then the samples were kept at
4.degree. C.
[0231] Each of the resulting PCR products was mixed with stop
buffer (5 .mu.l, composition: formamide (30 ml), xylenecyanol (30
mg), bromophenol blue (10 mg), 0.5 M EDTA (200 .mu.l, (pH 8.0)),
and then, 3.5 .mu.l of each resulting mixture was subjected to
sequence gel electrophoresis on a 6% acrylamide gel (composition
(in 500 ml total): urea (240 g), 10x TBE (50 ml), 40% acrylamide
(75 ml, a mixture of 38% monoacrylamide and 2% bisacrylamide)). The
result showed that there were two bands whose expression was
changed in the time course.
[0232] Both bands were excised from the gel, and two DNA fragments
containing the nucleotide sequences described in SEQ ID NO: 11 (178
bp) and SEQ ID NO: 12 (167 bp) were isolated according to the
standard method ("Gene Engineering Handbook"Jikken-Igaku, Yodosya
(1992)). The fragments were named as BA2303 (SEQ ID NO: 11), and
BA0306 (SEQ ID NO: 12), respectively. To confirm the expression of
the DNAs containing the two fragments in the time course, RT-PCR
was performed using cDNA samples obtained in Example 3 (each time
point) as a template.
[0233] For amplification of BA2303, synthetic DNA fragments
described in SEQ ID NO: 13 and SEQ ID NO: 14 were used as forward
and reverse primers, respectively.
[0234] For amplification of BA0306, synthetic DNA fragments
described in SEQ ID NO: 21 and SEQ ID NO: 22 were used as forward
and reverse primers, respectively.
[0235] Each template cDNA (3 .mu.l) was mixed with 10x Vogelstein
buffer (2.5 .mu.l), 2.5 mM dNTP (1.5 .mu.l), forward primer (1
.mu.l, 25 pmol/.mu.l), reverse primer (1 .mu.l, 25 pmol/.mu.l),
.beta.-actin primer mix (each 25 pmol/.mu.l), and EX Taq DNA
polymerase (0.2 .mu.l), adjusting the total volume to 25 .mu.l.
RT-PCR was carried out with a step of 94.degree. C. for 2 min; 35
cycles of 94.degree. C. for 3 sec, 55.degree. C. for 30 sec,
72.degree. C. for 1 min; and a step of 72.degree. C. for 3 min, and
then the samples were kept at 4.degree. C.
[0236] The obtained PCR products were separated by electrophoresis.
The results were shown in FIGS. 1 (BA2303) and 2 (BA0306).
[0237] It was confirmed that the expression of BA2303 was increased
from day 1 after the vascular endothelium was detached by PTCA,
reached the maximal level from about day 2 to day 4, and continued
until about day 7. The expression of BA0306 was detected over a
period from day 1 to day 7 after PTCA, with peak expression at day
4.
EXAMPLE 5
[0238] Isolation of long strand cDNA
[0239] To isolate long strand cDNAs containing the two cDNA
fragments (BA2303 andBA0306) obtained in Example 4, RACE (rapid
amplification ends)-PCR was performed (Proc. Natl. Acad. Sci. USA
(1988) Vol. 85, 8998-9002; "PCR Method for Gene
Amplification/Principles and Novel Applications" Kyoritsu-Syuppan
(1992)).
[0240] The PCR was performed twice using the Marathon cDNA
Amplification Kit (CLONTECH) and the cDNA fragments obtained in
Example 4 as a template.
[0241] BA2303 was amplified by PCR using synthetic DNA primers
described in SEQ ID NO: 15 and SEQ ID NO: 19 (1), andwithprimers
described in SEQ ID NO: 16 and SEQ ID NO: 20 (2), and subsequently
using synthetic DNA primers described in SEQ ID NO: 17 and SEQ ID
NO: 19 (3), and with primers described in SEQ ID NO: 18 and SEQ ID
NO: 20 (4).
[0242] BA0306 was amplified by PCR using synthetic DNA primers
described in SEQ ID NO: 23 and SEQ ID NO: 19 (1), and with primers
described in SEQ ID NO: 24 and SEQ ID NO: 20 (2), and subsequently
using synthetic DNA primers described in SEQ ID NO: 25 and SEQ ID
NO: 19 (3), and with primers described in SEQ ID NO: 26 and SEQ ID
NO: 20 (4). The above PCR produced DNAs described in SEQ ID NO: 1
(BA2303) and in SEQ ID NO: 7 (BA0306).
[0243] Analysis of the deduced amino acid sequence by plotting the
hydrophilicity and hydrophobicity and by PSORT program suggested
that BA2303 is a protein having seven transmembrane regions (FIG.
3).
EXAMPLE 6
[0244] Isolation of human counterpart genes
[0245] The rabbit cDNAs (BA2303 and BA0306) obtained in Example 5
were used as a probe to screen a human cDNA library (Fetal Brain,
STRATAGENE, code:937-227) by colony hybridization according to the
standard method ("Gene Engineering HandBook" Jikken-Igaku Zokan,
Yodosya, (1992)). Thus, human homologues containing the nucleotide
sequences described in SEQ ID NO: 3 (BA2303) and in SEQ ID NO: 9
(BA0306) were obtained.
[0246] Analysis of the deduced amino acid sequence of BA0306
protein by plotting the hydrophilicity and hydrophobicity and by
PSORT program suggested that the protein has 10 transmembrane
regions (FIG. 4). It is also suggested that human BA2303 is a
protein having seven transmembrane regions as is the rabbit one
obtained in Example 5 (FIG. 5).
[0247] Using the respective human DNA as a probe, the expression of
mRNA of the two genes in various human tissues was examined using
the Human Multiple Tissue Northern Blot (CLONTECH, code: #7760-1,
#7759-1).
[0248] BA2303 mRNA was expressed in various human tissues as
evident as two bands of about 3.9 kb and about 2.1 kb (FIG. 6).
[0249] BA0306 mRNA was also expressed in various human tissues as
detected as two bands of about 3.5 kb and about 4.4 kb (FIG.
7).
[0250] Homology search between known proteins indicated that human
BA0306 has sequence homology at the amino acid level with S.
cerevisiae oxidative stress resistance protein, S. cerevisiae
zinc/cadmium resistance protein, and heavy metal ion resistance
protein, etc.
EXAMPLE 7
[0251] Isolation of mouse BA2303 cDNA
[0252] As was described in Example 6, rabbit BA2303 gene was used
as a probe for screening a mouse cDNA library (STRATAGENE, code:
936-309), and the mouse homologue containing the nucleotide
sequence described in SEQ ID NO: 5 was isolated. The deduced amino
acid sequence of the coding region was described in SEQ ID NO:
6.
[0253] Analysis of the deduced amino acid sequence by plotting the
hydrophilicity and hydrophobicity and by PSORT program suggested
that mouse BA2303 protein has seven transmembrane regions as do
rabbit and human BA2303 (FIG. 8).
[0254] BA2303 proteins of the present invention, from rabbit, human
and mouse, have a high sequence homology at the amino acid level
between each other (FIG. 9).
EXAMPLE 8
[0255] Isolation of mouse BA0306 cDNA
[0256] As was described in Example 6, rabbit BA0306 gene was used
as a probe for screening a mouse cDNA library (STRATAGENE, code:
936-309), and the mouse homologue containing the nucleotide
sequence described in SEQ ID NO: 27 was isolated. The deduced amino
acid sequence of the coding region was described in SEQ ID NO:
28.
[0257] BA0306 proteins of the present invention, from rabbit, human
and mouse, have a high sequence homology at the amino acid level
with each other (FIG. 10).
EXAMPLE 9
[0258] Preparation of anti-peptide antibody against human
BA2303
[0259] An oligopeptide
(Gln-Asp-Ala-Gln-Gly-Gln-Arg-Ile-Gly-His-Phe-Glu-Ph- e-His-Gly)
containing amino acid residues from 35 to 49 in the sequence
described in SEQ ID NO: 4 was synthesized. Two rabbits were
immunized three times with peptide and Freund's complete adjuvant.
The rabbit sera obtained after each immunization were subjected to
ELISA using horse radish peroxidase-conjugated goat anti-rabbit IgG
and microplates having wells coated with the peptide (1
.mu.g/well), and the fluorescence intensity was measured at 492 nm
to determine the antibody titers. Titers were determined as
dilution of sera to obtain a fluorescence intensity at 492 nm not
more than 0.2. The result showed that the titers of antisera taken
from a rabbit A were 50-fold or less before immunization (3 to 5
ml), 30,600-fold after the first immunization (16 ml), 40,900-fold
after the second immunization (25 ml), and 41,100-fold after the
third immunization (23 ml), indicating that the titer was increased
with the number of immunization. The titers of antisera from the
other rabbit B were not more than 50-fold before immunization (3 to
5 ml), 149,200- fold after the first immunization (25 ml),
327,500-fold after the second immunization (25 ml), and
500,000-fold or more after the third immunization (25 ml),
indicating that the titer was increased and that antibody against
the peptide was produced.
[0260] Next, the forth immunization was performed on both rabbits A
and B. The titers after the forth immunization were 46,500-fold in
rabbit A, and 500,000-fold or more in rabbit B as was after the
third immunization. Then, the antisera taken from rabbit A after
the forth immunization were purified by affinity chromatography
using a column absorbed with the peptide that had been used as an
antigen. The titer of the sera from rabbit A after purification was
69,800-fold.
EXAMPLE 10
[0261] Preparation of recombinant fusion protein with human BA2303
protein
[0262] Fusion proteins of the present invention were prepared as a
fusion protein with maltose binding protein (MBP) using the
expression plasmid pMAL-C2 (New England Bio Labs. (NEB)), which
contains a DNA encoding MBP. Experimental procedures were performed
according to the manufacturer's instructions (Catalogue number:
#800, `Protein Fusion & Purification System` Ver. 3.03, 12/1994
revised) and by the standard method of recombinant DNA
technology.
[0263] Using a template of the DNA encoding human BA2303 (SEQ ID
NO: 3), which was cloned in the previous Example, a DNA containing
the nucleotide sequence corresponding to the N-terminal amino acids
(residues 22 (Gly) to 171 (His)), having EcoRI and HindIII
restriction sites at 5' and 3' termini, respectively, was amplified
by PCR according to the standard method. Oligonucleotides described
in SEQ ID NO: 29 and SEQ ID NO: 30 were used as 5' and 3' primers,
respectively. The above pMAL-C2 expression plasmid (NEB, inserted
with a DNA encoding MBP) was digested with EcoRI and HindIII, and
the resulting fragments were recovered. Using a commercially
available DNA ligation kit, the above PCR products of human BA2303
were ligated into the pMAL-C2, and the resulting plasmid was used
to transfected E. coli TB1 cells. The bacterial expression plasmid
was prepared in a large quantitiy from the transformed colony. A
culture of the transformed colony (1/100 volume) was inoculated
into 1 liter of LB broth containing ampicillin and glucose, and
incubated with shaking until the OD value became up to 0.5. Then,
isopropanol-.beta.-D-thiogalac- topyranoside (IPTG) was added to
the culture to the final concentration of 0.3 mM, and shaking
culture was performed further (3 hr). The culture was then
centrifuged to remove the supernatant, and the precipitated
bacteria was resuspended in cold column buffer (50 ml, composition:
20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, and 10 mM mercaptoethanol),
which was supplemented with 0.1 M PMSF (50 .mu.l,
phenylmethylsulfonyl fluoride) to suppress protease digestion.
[0264] The following procedures were carried out on ice unless
otherwise noted. The obtained bacteria suspension was sonicated on
ice to disrupt cells. Then, the suspension was centrifuged (9000
rpm, 15 to 30 min) to recover soluble fraction. The soluble
fraction was diluted with ice-cold column buffer to load on a
column.
[0265] Amylose resin (15 ml, BIORAD) was packed in a disposable
column (2.5 dia..times.10 cm), washed, and equilibrated with 8
volumes of ice-cold column buffer. The sample was loaded onto the
column using a pump to keep the flow rate 1 ml/min, and washed with
ice-cold column buffer.
[0266] The fusion protein was eluted and fractionated with ice-cold
column buffer containing 10 mM maltose. Each fraction was separated
by SDS-PAGE, and analyzed by western blotting using antisera
against MBP (NEB). Fractions producing a band detected by western
blotting at the position approximately corresponding to that of the
full-length fusion protein were determined to be positive. Next,
the positive fractions were further purified. MBP/BA2303 fusion
protein can be digested by adding 1 mg/ml factor Xa (5 .mu.l) to
the solution containing the fusion protein and incubating it for 24
hr. Digestion of the fusion protein can be determined by SDS-PAGE
followed by western blotting using antisera against MBP.
EXAMPLE 11
[0267] Preparation of antibody against human BA2303 protein
[0268] Recombinant protein prepared in Example 10, containing
approximately 150 N-terminal amino acids of human BA2303 protein
(residues 22 (Gly) to 171 (His)), was used as an immunogen. Two
rabbits were immunized with the recombinant protein and Freund's
complete adjuvant. The rabbit sera was subjected to ELISA using
horse radish peroxidase-conjugated goat anti-rabbit IgG and
microplates having wells coated with the peptide (1 .mu.g/well),
and the fluorescence intensity was measured at 492 nm to determine
the antibody titers. The titer was determined as dilution of serum
to obtain a fluorescence intensity at 492 nm not more than 0.2. The
result showed that the titer of sera taken from a rabbit was
50-fold or less before immunization (3to 5 ml), and 316,900- fold
after immunization (18 ml), indicating that the titer was
increased. The titer of the sera from the other rabbit was less
than 50-fold before immunization (3 to 5 ml), and increased to
312,300-fold after immunization (23 ml), indicating that antibody
against the recombinant protein was produced.
EXAMPLE 12
[0269] Construction of an expression vector for recombinant human
BA2303
[0270] Using a template of the DNA encoding human BA2303 (SEQ ID
NO: 3), which was cloned in the previous Example, a DNA containing
the nucleotide residues 77 to 1419 (containing the entire open
reading frame (ORF)), having XbaI restriction sites at both 5' and
3' termini, was amplified by PCR according to the standard method.
Oligonucleotides described in SEQ ID NO: 31 and in SEQ ID NO: 32
were used as 5' and 3' primers, respectively.
[0271] The resulting PCR products were ligated into the XbaI site
of the pcDNA expression plasmid (Invitrogen) using a commercially
available DNA ligation kit to construct an expression vector for
recombinant human BA2303. Higher eukaryotic host cells such as COS
cells can be transfected with the vector, and the resulting
colonies are selected to obtain transfected cells. Human BA2303
proteins can be expressed abundantly on the cell surface of the
transfected cells by incubating the them in appropriate medium such
as DMEM containing 10% FCS.
EXAMPLE 13
[0272] Preparation of recombinant rabbit BA0306 protein
[0273] Using a template of the DNA encoding rabbit BA0306 (SEQ ID
NO: 7), which was cloned in the previous Example, a DNA containing
the nucleotide residues 2017 (Ile) to 2196 (Met), having BamHI and
SalI restriction sites at 5' and 3' termini, respectively, was
amplified by PCR according to the standard method. In the amino
acid sequence (60 residues) encoded by the rabbit nucleotide
sequence (nucleotides 2017 (Ile) to 2196 (Met)), 58 residues are
the same as those in the corresponding human BA0306 sequence
(residues 535 to 594 in SEQ ID NO: 10). Oligonucleotides described
in SEQ ID NO: 33 and in SEQ ID NO: 34 were used as 5' and 3'
primers, respectively.
[0274] The expression plasmid pQE-32 (QIA expression type IV
construct, QIAGEN) was digested with BamHI and SalI, and then
blunted.
[0275] According to the instruction manual for handling pQE-32, the
obtained PCR products were ligated into the blunted ends of pQE-32
digested with BamHI-SalI using a commercially available DNA
ligation kit. The resulting expression vector for recombinant human
BA0306 was named as pQE-32R7-15.
[0276] Next, E. coli cells (XL-1 blue MRF') were transformed with
the pQE-32R7-15 according to the standard method, and the
transformed colonies were selected ("Gene Engineering Handbook"
Jikken-Igaku Bessatsu, Yodosha (1992) 46-51). A culture of the
transformed cells was inoculated into LB broth containing
ampicillin and glucose, and incubated at 37.degree. C. with
shaking, with measuring the OD. Then, IPTG
(isopropanol-.beta.-D-thiogalactopyranoside) was added to the
culture to the final concentration of 1 mM, and shaking culturing
was further performed at 37.degree. C. for 4 hrs. The culture was
centrifuged to remove the supernatant, and the precipitated
bacteria was resuspended in column buffer. The suspension was
sonicated on ice to disrupt cells, then centrifuged, and soluble
fraction was recovered. The soluble fraction was diluted with
ice-cold column buffer to load on a column.
[0277] A column was packed with Ni-NTA resin, washed, and
equilibrated with column buffer. The samples were applied on the
column and washed with column buffer. The eluted fractions were
collected, and thus recombinant rabbit BA0306 protein was
obtained.
EXAMPLE 14
[0278] Preparation of antibody against human BA0306
[0279] Recombinant rabbit BA0306 protein prepared in Example 13 was
used as an immunogen. The protein and Freund's complete adjuvant
were used to immunize chickens. The chicken sera were subjected to
ELISA using horse radish peroxidase-conjugated anti-chicken IgG and
microplates having wells coated with the recombinant protein (1
.mu.g/well), and the fluorescence intensity was measured to
determine the antibody titers. The antibody against the ecombinant
protein was produced.
[0280] Furthermore, rabbit BA0306 protein fragment, which was used
as an immunogen in this example, and the above recombinant human
BA0306 protein were detected by western blotting using the chicken
antisera, indicating that the antisera had a cross reactivity with
human BA0306 protein.
EXAMPLE 15
[0281] Generation of knockout mice of mouse BA2303 gene
[0282] A knockout mouse, whose endogenous gene encoding mouse
BA2303 protein was inactivated, was generated as follows. (1)
Construction of a targeting vector
[0283] A targeting vector for generation of a knockout mouse, in
which an endogenous gene encoding mouse BA2303 protein was
inactivated (knocked out) by homologous recombination
(Nikkei-Science (1994) May, 52-62), was constructed as follows.
[0284] The cDNA encoding mouse BA2303 protein (SEQ ID NO: 5), which
was cloned in the previous Example, was labeled with .sup.32P by
the standard method to obtain a probe used in hybridization. The
probe was used to screen a cosmid mouse genomic DNA library
("Labomanual Human Genome Mapping" Hori M., and Nakamura Y. edit.,
Maruzen Syuppan), and thus, a mouse genomic DNA clone containing
exons (E1, E2, and E3) which encode mouse BA2303 protein was
isolated. The structure of the genomic DNA was schematically shown
in FIG. 11. The genomic DNA was subcloned into a plasmid, and
digested with SacII to remove the region of 124 bp encompassing E1
and the intron between E1 and E2, and then ligated with an insert
of a neomycin resistance gene of 1143 bp (neo, as a positive
selection marker), which had been digested with restriction enzymes
and blunted.
[0285] The plasmid pBluescript II SK(-) was digested with SacII,
and ligated with an insert of a thymidine kinase gene (Tk, as a
negative selection marker). Then, the resulting pBluescript II
SK(-) was digested with XbaI, and ligated with an insert of the
above mouse BA2303 genomic DNA having a neo gene insertion.
[0286] (2) Transfection of the targeting vector into ES cells
[0287] Mouse embryonic stem cells (ES cells) (Nature (1993) 362,
255-258; Nature (1987) 326, 292-295), which were cultured in DMEM
containing 15% fetal bovine serum, were trypsinized to obtain
single isolated cells, washed three times in phosphate buffer, and
prepared as a cell suspension of 1.times.10.sup.7 cells/ml. The
targeting vector was added to the cells (25 .mu.g/1 ml cell
suspension), and electroporation was performed with a single pulse
of 350 V/cm (25 .mu.F). Then, the ES cells were seeded into 10 cm
dishes (1.times.107 cells/dish), cultured for one day in
maintenance medium, and then the medium was replaced with selection
medium (containing G418 (250 .mu.g/ml) and 2 .mu.M gancyclovir).
The culture was continued with replacing the medium every two days.
On the tenth day after transfection of targeting vector, 540
neomycin resistant ES clones were isolated using a micropipet under
microscopic observation. The clones were cultured separately in 24
well plates layered with feeder cells, and replica of 540 neomycin
resistant ES cells were obtained.
[0288] (3) Screening of knockout ES cells
[0289] Each neomycin resistant ES clone was examined by PCR whether
its endogenous gene encoding mouse BA2303 protein was inactivated
(knocked out) by homologous recombination.
[0290] PCR was performed using genomic DNA extracted from each
neomycin resistant ES clone as a template, with two primers
designed based on the sequence of the neo gene (SEQ ID NO: 36 and
SEQ ID NO: 37) (1) and on the mouse BA2303 genomic DNA sequence
which locates on the flanking region of the BA2303 DNA which was
inserted in the targeting vector (SEQ ID NO: 35 and SEQ ID NO: 38)
(2). DNA was purified using an automated DNA purification robot
(Kubota). The result showed that desired PCR products were obtained
in several clones among the ES clones examined. Further selection
of these clones can be performed by genomic Southern blotting.
Genomic DNA was extracted from each clone, digested with
restriction enzymes, and separated by electrophoresis on an agarose
gel. Then, the DNA was transferred onto a nylon membrane, and
subjected to hybridization using a probe designed based on the
genomic sequence of mouse BA2303. The probe was designed based on
the sequence which locates in the flanking region of the site of
homologous recombination, and thus enabled to distinguish mutated
genome from normal one by size. The knockout ES clone selected in
this way was used for generation of knockout mice as described
below.
[0291] (4) Generation of knock out mice
[0292] The above obtained ES cells, having inactivation in the
endogenous gene encoding mouse BA2303 protein as a result of
homologous recombination, were injected into blastocysts obtained
by crossing C57BL6 mice (Japan Charles River) (15 cells/embryo,
microinjection). Immediately after microinjection, the blastocysts
were implanted into uterines of ICR mice (Clea Japan), which had
undergone pseudopregnancy treatment two days and half before (10
blastocysts/one side of the uterine). Thus, desired chimera mice
were obtained. The chimera were crossed with normal C57BL6 mice to
obtain agouti mice, whose color is attributed to a gene determining
hair color, originating from ES cells.
EXAMPLE 16
[0293] Generation of knockout mice of mouse BA0306 gene
[0294] (1) Construction of a targeting vector.
[0295] A targeting vector for generation of a knockout mouse, in
which the endogenous gene encoding mouse BA0306 protein was
inactivated (knocked out) by homologous recombination
(Nikkei-Science (1994) May, 52-62), was constructed as follows. The
cDNA encoding mouse BA0306 protein (SEQ ID NO: 27), which was
cloned in the previous Example, was labeled with .sup.32P by the
standard method to obtain a probe used in hybridization. The probe
was used to screen a 129SVJ mouse genomic DNA library (STRATAGENE),
and a mouse genomic DNA clone containing exons (exon I, II, III,
IV, and V) that encode mouse BA0306 protein was isolated.
[0296] The plasmid pBluescript II SK(-) was digested with XhoI and
HindIII, and ligated with XhoI-HindIII-digested thymidine kinase
gene (TK, as a negative selection marker). Next, NotI-digested
pBluescript II SK(-) was ligated with an insert of the above mouse
BA0306 genomic DNA (exons I to V). Then, the neomycin resistance
gene (neo, as a positive selection marker) was digested with BamHI
and XhoI, blunted, and ligated into the Aor51HI site of the exon V
in the mouse BA0306 genomic DNA. Finally, the resulting pBluescript
II SK(-) was digested with SacII and linealized to use as a
targeting vector.
[0297] (2) Transfection of the targeting vector into ES cells.
[0298] Mouse embryonic stem cells (ES cells, 1.times.10 cells)
(Nature (1993) 362, 255-258; Nature (1987) 326, 292-295), which
were cultured in DMEM containing 15% fetal bovine serum, were
trypsinized to obtain single isolated cells, washed three times in
phosphate buffer, and then prepared as a cell suspension of
1.times.10.sup.7 cells/ml. The targeting vector was added to the
(25 .mu.g/ ml cell suspension), and electroporation was performed
with a single pulse of 350 V/cm (25 .mu.F). Then, the ES cells were
seeded into 10 cm dishes (1.times.10.sup.7 cells/dish), cultured 1
day in maintenance medium, and then the medium was replaced with
selection medium (containing G418 (250 .mu.g/ml) and 2 .mu.M
gancyclovir). The culture was continued with replacing the medium
every two days. On the tenth day after transfection, 573 neomycin
resistant ES clones were isolated using a micropipet under
microscopic observation. The clones were cultured separately in 24
well plates layered with feeder cells, and replica of 573 neomycin
resistant ES cells were obtained.
[0299] (3) Screening of knockout ES cells
[0300] Each neomycin resistant ES clone was examined by genomic
Southern blotting whether its endogenous gene encoding mouse BA0306
protein was inactivated (knocked out) through homologous
recombination.
[0301] Genomic DNA was extracted from each neomycin resistant ES
clone, and genomic Southern blotting was performed on EcoRI
digested genomic DNA fragments according to the standard method
using the following probes.
[0302] (probe 1)
[0303] 5' flanking DNA which was amplified using two primers
described in SEQ ID NO: 39 and SEQ ID NO: 40.
[0304] (probe 2)
[0305] 3' flanking DNA which was amplified using two primers
described in SEQ ID NO: 41 and SEQ ID NO: 42. DNA was purified
using an automated DNA purification robot (Kubota).
[0306] If the endogenous gene encoding BA0306 is normally targeted
by the targeting vector, the 5' and 3' flanking genes encompassing
the integrated neo gene can be detected as 7 kb and 5 kb bands,
respectively.
[0307] The result showed that desired knockout of the gene was
occurred in three ES clones (named as 0-16-9, 0-22-11, and
0-22-18), which were used for generation of knockout mice as
described below.
[0308] (4) Generation of knock out mice
[0309] The ES clones obtained above, having inactivation in the
endogenous gene encoding mouse BA0306 protein as a result of
homologous recombination, were microinjected into blastocysts
obtained by crossing C57BL6 mice (Japan Charles River) (15
cells/embryo). Immediately after microinjection, the blastocysts
were transferred to uterines of ICR mice (Clea Japan) (10
blastocysts/one side of the uterine), which had undergone
pseudopregnancy treatment two days and half before. As a result,
desired knockout chimera mice were obtained from each ES clone as
followings.
[0310] (clone 0-16-9)
[0311] Total number of injected cells: 83
[0312] Littermates: 13
[0313] Chimera mice: 7
[0314] Chimera where contribution to hair color is 80% or more:
[0315] (clone 0-22-11)
[0316] Total number of injected cells: 202
[0317] Littermates: 12
[0318] Chimera mice: 3
[0319] Chimera where contribution to hair color is 80% or more:
3
[0320] (clone 0-22-18)
[0321] Total number of injected cells: 148
[0322] Littermates: 9
[0323] Chimera mice: 5
[0324] The chimera were crossed with normal C57BL6 mice to obtain
agouti mice whose color is attributed to a gene determining hair
color, originating from ES cells.
INDUSTRIAL APPLICABILITY
[0325] The present invention provides two novel physiologically
active protein molecules (BA0306, and BA2303) having
characteristics described below, which are specifically expressed
in arteriosclerosis or coronary restenosis, and are predicted to
relate closely to the onset and progress of these diseases; their
fragments; a gene (DNA) encoding the protein molecules; an antibody
reactive with the molecule, and its fragment; and pharmaceutical
compositions comprising the above protein molecule or the
antibody.
[0326] [BA0306]
[0327] A molecule having the following characteristics, and
presumed to have inhibitory effects on active oxygen species such
as nitrogen monoxide (NO), which has been identified to be involved
in the progress of arteriosclerosis and restenosis.
[0328] (1) Its expression is increased from day 1 to day 7 after
PTCA of the coronary aorta (peak at day 4).
[0329] (2) Its mRNA is expressed in various human tissues as
detected by Northern blotting as approximately 3.5 kb and 4.4 kb
bands.
[0330] (3) Its 10 predicted transmembrane regions.
[0331] (4) Its sequence homology at the amino acid level with S.
cerevisiae oxidative stress resistance protein, S. cerevisiae
zinc/cadmium resistance protein, and heavy metal ion resistance
protein, etc.
[0332] [BA2303]
[0333] A molecule having the following characteristics, and
presumed to be a G protein(GTP binding protein)-coupled receptor
that transduces a specific signal through intracellular G protein
to an effector on the plasma membrane or in the cytoplasm by
binding to an in vivo ligand which is involved in the onset and
progress of arteriosclerosis and restenosis.
[0334] (1) Its expression is increased day 1 after PTCA of the
coronary aorta, reaches the maximum on day 2 to day 4, and
continued until day 7.
[0335] (2) Its mRNA is expressed in various human tissues as
detected by Northern blotting as approximately 3.9 kb and 2.1 kb
bands.
[0336] (3) having seven predicted transmembrane regions.
[0337] Therefore, a gene (DNA) or protein of the present invention
or its part, and an antibody reactive with the protein, or a part
of the antibody are extremely useful in developing the drugs
targeting the gene or the protein molecule for treatment and
prevention of arteriosclerosis as well as restenosis after PTCA of
arterial embolism. Also, the DNA itself is very useful as an
antisense medicine, the extracellular domain fragment of the
protein is useful as a soluble receptor medicine, and the antibody
or its part is useful as an antibody medicine.
[0338] Furthermore, the gene (DNA), protein, and antibody of the
present invention are useful as a reagent for screening a protein
(ligand) interacting with the protein of the invention,
identification of the function of the ligand, and developing a drug
which targets the ligand.
[0339] In addition, based on the nucleotide sequence originating
from rabbit or mouse, as an embodiment of the DNA of the present
invention, model animals (knockout animals) can be generated by
disrupting (inactivating) a corresponding endogenous gene.
Similarly, trasngenic animals can be generated as a model animal by
introducing human DNA, as an embodiment of the DNA of the present
invention, into mammals such as mice except human. It is possible
to identify the functions of the gene and protein of the invention
by analyzing the physical, biological, pathological, and genetical
characteristics of the model animals.
[0340] Moreover, it is possible to generate model animals having a
human gene of the invention alone by crossing the model animals,
whose endogenous gene is disrupted, with the transgenic animals.
Thus, it is possible to estimate the therapeutic effects of a drug
which targets the introduced human gene (compounds, and antibodies,
etc.) by administrating the drug into the model animals.
Sequence CWU 0
0
* * * * *