US20020128459A1 - Physiologically active protein originating in mammals - Google Patents

Physiologically active protein originating in mammals Download PDF

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US20020128459A1
US20020128459A1 US10/139,262 US13926202A US2002128459A1 US 20020128459 A1 US20020128459 A1 US 20020128459A1 US 13926202 A US13926202 A US 13926202A US 2002128459 A1 US2002128459 A1 US 2002128459A1
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protein
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Yusuke Nakamura
Toshihiro Tanaka
Shuichi Tsukuda
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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,.
  • ⁇ 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.
  • PTCA percutaneous transluminal coronary angioplasty
  • 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.
  • 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.
  • differential display method Nucleic Acids Research, Vol.21, No.18, pp.4272-4280 (1993); and Science, Vol.257, pp.967-971 (1992)).
  • 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.
  • 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.
  • the two novel protein-encoding genes of the present invention are expressed specifically after PTCA, and are thought to be genes involved in onset and progress of arteriosclerosis and/or coronary artery restenosis.
  • Clone BA0306 has the following characteristics.
  • 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.
  • clone BA0306 is thought to inhibit active oxygen such as nitrogen monoxide (NO), which is involved in the progress of arteriosclerosis and/or restenosis.
  • active oxygen such as nitrogen monoxide (NO)
  • Clone BA2303 has the following characteristics.
  • 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.
  • 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.
  • G protein GTP binding protein
  • 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.
  • 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.
  • DNAs genes
  • proteins proteins
  • 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.
  • model animals can be produced by disrupting (inactivating) the endogenous gene corresponding to the DNA.
  • 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.
  • model animals that have only the human-derived gene of the present invention can be produced.
  • drugs compounds, antibodies, and so on
  • the therapeutic effect of the drug can be estimated.
  • the present invention provides the DNAS, proteins, expression vectors, transformants, antibodies, pharmaceutical compositions, transgenic mice, and knockout, mentioned below.
  • a DNA comprising a nucleotide sequence corresponding to nucleotide residues 97 to 1419 of the nucleotide sequence of SEQ ID NO: 3.
  • 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.
  • a pharmaceutical composition comprising the protein fragment of (6) or the fusion protein of (7) and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising the antibody or its portion of (10) or (11) and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • a pharmaceutical composition comprising the protein fragment of (19) or the fusion protein of (20) and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising the antibody or its portion of (23) or (24) and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • a transmembrane protein exists as a monomer, homodimer, heterodimer, or oligomer with another chain(s) having the same or different amino acid sequence.
  • extracellular region 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.
  • “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.
  • Alphabetical triplet or single letter codes used to represent amino acids in the present specification or figures mean amino acids as follows.
  • 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).
  • 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).
  • 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 1 and IgA2).
  • 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.
  • the heavy chain of IgG1 is composed of VH, C ⁇ 1 1 domain, hinge region, C ⁇ 1 2 domain, and C ⁇ 1 3 domain in this order from N terminus.
  • the heavy chain of IgG2 is composed of VH, C ⁇ 2 1 domain, hinge region, C ⁇ 2 2 domain, and C ⁇ 2 3 domain in this order from N terminus.
  • the heavy chain of IgG3 is composed of VH, C ⁇ 3 1 domain, hinge region, C ⁇ 3 2 domain, and C ⁇ 3 3 domain in this order from N terminus.
  • the heavy chain of IgG4 is composed of VH, C ⁇ 4 1 domain, hinge region, C ⁇ 4 2 domain, and C ⁇ 4 3 domain in this order from N terminus.
  • the heavy chain of IgA is composed of VH, C ⁇ 1 domain, hinge region, C ⁇ 2 domain, and C ⁇ 3 domain in this order from N terminus.
  • the heavy chain of IgA1 is composed of VH, C ⁇ 1 1 domain, hinge region, C ⁇ 1 2 domain, and C ⁇ 1 3 domain in this order from N terminus.
  • the heavy chain of IgA2 is composed of VH, C ⁇ 2 1 domain, hinge region, C ⁇ 2 2 domain, and C ⁇ 2 3 domain in this order from N terminus.
  • the heavy chain of IgD is composed of VH, C ⁇ 1 domain, hinge region, C ⁇ 2 domain, and C ⁇ 3 domain in this order from N terminus.
  • the heavy chain of IgM is composed of VH, C ⁇ 1 domain, C ⁇ 2 domain, C ⁇ 3 domain, and C ⁇ 4 domain in this order from N terminus and have no hinge region as seen in IgG, IgA, and IgD.
  • the heavy chain of IgE is composed of VH, C ⁇ 1 domain, C ⁇ 2 domain, C ⁇ 3 domain, and C ⁇ 4 domain in this order from N terminus and have no hinge region as seen in IgG, IgA, and IgD.
  • 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).
  • 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.
  • 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.
  • 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.
  • Ig immunoglobulin
  • 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.
  • IgG1 is preferable among IgG.
  • a protein derived from human, mouse, or rat is preferable as the protein of the present invention.
  • 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.
  • Fc constant region
  • 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.
  • 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.
  • 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.
  • the DNA of the present invention comprises either a genomic DNA or cDNA.
  • the DNA includes any DNA composed of any codons encoding the same amino acids.
  • 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.
  • Tm 82.3° C.+0.41 ⁇ (G+C) %—500/n—0.61 ⁇ (formamide) % (n means the number of the nucleotide of probe).
  • Tm varies as (1) and (2) mentioned below.
  • Tm descends by about 1° C. per 1% mismatch.
  • the temperature conditions for the combination of completely complementary strands can be set as follows.
  • the temperature conditions for the combination of incompletely complementary strands can be set as follows.
  • the temperature conditions when a probe with 23 or less nucleotides is used can be 37° C. or can be calculated using the following formula.
  • the DNA of the present invention can be a DNA obtained by any method.
  • 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.
  • cDNA complementary DNA
  • 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.
  • cDNA can be cloned from the mRNA encoding the protein of the present invention by, for example, the method described below.
  • 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.
  • 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.
  • 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, ⁇ gt10, ⁇ gt11, and so on.
  • the vector having a promoter that can express a gene encoding the polypeptide of the present invention in a host is preferably used.
  • 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 ⁇ , MC1061/P3, etc.).
  • 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).
  • 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.
  • 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 32 P-labeled chemically synthesized oligonucleotides as probes, which are corresponding to the amino acid sequence of the polypeptide of the present invention.
  • 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.
  • a cDNA library prepared using a cDNA expression vector for example, ⁇ ZAPII phage vector
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • plasmid DNA and bacteriophage DNA plasmid DNA and bacteriophage DNA
  • 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.
  • phages are a bacteriophage such as ⁇ phage, and an animal or insect virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and nuclear polyhedrosis virus.
  • 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.
  • 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.
  • 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.
  • 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).
  • SD Shine-Dalgarno
  • the host when the host is Escherichia, it preferably comprises Trp promoter, lac promoter, recA promoter, ⁇ 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.
  • examples thereof are SL01 promoter, SP02 promoter, penP promoter and so on.
  • 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.
  • the promoter is not limited to the above examples.
  • an enhancer is effective for expression.
  • a preferable initiation codon is, for example, a methionine codon (ATG).
  • termination codon for example, TAG, TGA, TAA, and so on
  • TAG TAAA
  • TAA TAA
  • 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.
  • a preferable plasmids are pBR322 or its artificial derivatives (DNA fragment obtained by treating pBR322 with appropriate restriction enzymes) for E. coli , yeast 2 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • DHFR dihydrofolate reductase
  • thymidine kinase gene thymidine kinase gene
  • neomycin resistance gene glutamate synthase gene
  • glutamate synthase gene glutamate synthase gene
  • adenosine deaminase gene adenosine deaminase gene
  • ornithine decarboxylase gene hygromycin-B-phophotransferase gene
  • aspartate transcarbamylase gene etc
  • 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.
  • appropriate DNA fragments for example, linkers, restriction sites generated with other restriction enzyme
  • Transformants of the present invention can be prepared by introducing the expression vector mentioned above into host cells.
  • 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.
  • E. coli or animal cells are preferably used. Specific examples are E. coli (DH5 ⁇ , 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.).
  • E. coli DH5 ⁇ , 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. monkey-derived cells
  • human-derived cells Hela
  • An expression vector can be introduced (transformed (transduced)) into host cells by known method.
  • 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.
  • 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.
  • the nutrient media preferably comprise carbon source, inorganic nitrogen source, or organic nitrogen source necessary for the growth of host cells (transformants).
  • the carbon source are glucose, dextran, soluble starch, and sucrose
  • 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.
  • 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.).
  • 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.
  • liquid media comprising the nutrient source mentioned above are appropriate.
  • the media with pH 5 to 8 are preferably used.
  • 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° C. for about 3 to 24 hours with aeration and stirring, if necessary.
  • cultivation can be performed usually at 30 to 40° C. for about 16 to 96 hours with aeration and stirring, if necessary.
  • 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° C. for about 14to 144 hours with aeration and stirring, if necessary.
  • 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° C. for about 15 to 72 hours with aeration and stirring, if necessary.
  • 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° C. for 15 to 100 hours with aeration and stirring, if necessary.
  • 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.
  • 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.
  • 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.
  • 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
  • charges such as ion exchange chromatography and hydroxylapatite chromatography
  • the fungus bodies or cells are harvested by the usual method such as filtration or centrifugation and suspended in appropriate buffer.
  • 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.
  • the polypeptide or the polypeptide fragment is isolated and purified from the crude extract by the usual method as illustrated above.
  • 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.
  • 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)).
  • embryonic stem 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.
  • “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.).
  • the “antibody” of the present invention can be a polyclonal antibody (antiserum) or a monoclonal antibody, and preferably a monoclonal antibody.
  • 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.
  • an immunogen 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.
  • the monoclonal antibody includes those having any one isotype of IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable.
  • 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.
  • 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.
  • 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.
  • 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.
  • a mammal such as a mouse, rat, guinea pig, hamster, rabbit, or human, or more preferably, a mouse, rat, or human.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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 ⁇ SSC.
  • appropriate restriction enzymes for example, EcoRI and HindIII
  • electrophoresed using, for example, 0.7% agarose gel
  • 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
  • the filter After the filter is sufficiently dried, it is baked at 75° C. for3 hours. After baking, the filter is treated with 0.1 ⁇ SSC/0.1% SDS at 65° C. for 30 minutes. Then, it is soaked in 3 ⁇ SSC/0.1% SDS. The filter obtained is treated with prehybridization solution in a plastic bag at 65° C. for 3 to 4 hours.
  • 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, ⁇ EMBL3, ⁇ EMBL4, etc.).
  • E. coli for example, LE392, NM539, etc.
  • the genomic library is screened by plaque hybridization such as Benton-Davis method (Science, Vol.196, pp.
  • human CH gene and human CL gene used for chimerization are isolated.
  • C ⁇ 1 gene as a CH gene and C ⁇ gene as a CL gene, are isolated.
  • These genes can be isolated from human genomic library with mouse C ⁇ 1 gene and mouse C ⁇ gene, corresponding to human C ⁇ 1 gene and human C ⁇ gene, respectively, as probes, taking advantage of high homology between the nucleotide sequences of mouse immunoglobulin gene and that of human immunoglobulin gene.
  • DNA fragments comprising human C ⁇ gene and an enhancer region are isolated from human ⁇ 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.
  • mouse VH gene is inserted downstream mouse VH gene
  • 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.
  • 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.
  • Chimeric gene-inserted expression vector(s) thus prepared are introduced into myelomas that do not produce antibodies, for example, P3X63 ⁇ Ag8 ⁇ 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.
  • Desired chimeric monoclonal antibodies are obtained from the culture supernatant of antibody-producing cells thus screened.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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′) 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)).
  • F(ab′) 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.
  • a protease such as pepsin and papain
  • 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 ⁇ 1 ( ⁇ 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′) 2 .
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition can be administered to an adult in a dose of 10 ⁇ g to 1000 mg (or 10 ⁇ g to 500 mg) per one administration.
  • 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.
  • 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 ⁇ g antibody/ml carrier to 10 mg antibody/ml carrier.
  • a non-toxic, pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection with adjusting a concentration to 0.1 ⁇ 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 ⁇ g to 100 mg/kg body weight, preferably 50 ⁇ g to 50 mg/kg body weight once or more times a day.
  • administration route are medically appropriate administration routes such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection, preferably intravenous injection.
  • 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.
  • a non-aqueous diluent for example, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and alcohol such as ethanol
  • suspension or emulsion.
  • 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.
  • 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.
  • FIG. 1 is a photograph showing an electrophoresis image of rabbit BA2303 cDNA samples obtained by RT-PCR.
  • 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.
  • FIG. 2 is a photograph showing an electrophoresis image of rabbit BA0306 cDNA samples obtained by RT-PCR.
  • 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.
  • FIG. 3 shows a plot of the hydrophobicity and hydrophilicity of the amino acid residues composing rabbit BA2303 protein.
  • FIG. 4 shows a plot of the hydrophobicity and hydrophilicity of the amino acid residues composing human BA0306 protein.
  • FIG. 5 shows a plot of the hydrophobicity and hydrophilicity of the amino acid residues composing human BA2303 protein.
  • FIG. 6 is a photograph showing the result of Northern blot analysis of the expression of human BA2303 mRNA in various human tissues.
  • FIG. 7 is a photograph showing the result of Northern blot analysis of the expression of human BA0306 mRNA in various human tissues.
  • FIG. 8 shows a plot of the hydrophobicity and hydrophilicity of the amino acid residues composing mouse BA2303 protein.
  • FIG. 9 shows the sequence homology at the amino acid level between BA2303 proteins from rabbit, human, and mouse.
  • FIG. 10 shows the sequence homology at the amino acid level between BA0306 proteins from rabbit, human, and mouse.
  • 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.
  • 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.
  • RNA samples each 2 ⁇ l, 1 ⁇ g/ml sampled with the passage of time or mRNA samples (each 2 ⁇ l, 0.5 ⁇ g/ml), which were obtained in Example 2, were dissolved in diethyl pirocarbonate (DEPC)-treated distilledwater(8 ⁇ l).
  • Anchorprimer(GT15MA, 1 ⁇ l, 25 pmol/ ⁇ l) was added to make the total volume 10 ⁇ l, and the mixture was then incubated 5 min at 65° C. The samples were placed on ice immediately after completion of the incubation.
  • 5x first strand buffer (4 ⁇ l, composition: 0.25 M Tris-HCl (pH 7.5), 0.375 M KCl, 0.05 M DTT, 0.015 M MgCl 2 ), 0.1 M DTT (2 ⁇ l), 250 ⁇ M dNTP (1 ⁇ l), distilled water (1 ⁇ l), and reverse transcriptase (Superscript, GIBCO BRL, 1 ⁇ l, 200 U/ ⁇ l) were added to make the total volume 20 ⁇ l.
  • cDNA was synthesized by incubating the reaction mixture for 1 hr at 42° C., and then DEPC-treated water (30 ⁇ l) was added to make the final volume 50 ⁇ l.
  • the template cDNA (each 2 ⁇ l) was mixed with distilled water (10.75 ⁇ l), 10x EX Taq buffer (2 ⁇ l), 25 ⁇ M dNTP (1.5 ⁇ l), arbitrary primer (sequence: GATCAATCGC, 1 ⁇ l, 25 pmol/ ⁇ l), anchor primer (1 ⁇ l, 25 pmol/ ⁇ l), EX Taq DNA polymerase (0.25 ⁇ l), and ⁇ 35S-dATP (1.5 ⁇ l, 10 mCi/ml, Amersham) to make the total volume 20 ⁇ l.
  • PCR was carried out with a cycle of 95° C. for 3 min, 40° C. for 5 min, 72° C. for 5 min; 40 cycles of 95° C. for 30 sec, 40° C. for 2 min, 72° C. for 1 min; and a step of 72° C. for 5 min, and then the samples were kept at 4° C.
  • Each of the resulting PCR products was mixed with stop buffer (5 ⁇ l, composition: formamide (30 ml), xylenecyanol (30 mg), bromophenol blue (10 mg), 0.5 M EDTA (200 ⁇ l, (pH 8.0)), and then, 3.5 ⁇ 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.
  • Each template cDNA (3 ⁇ l) was mixed with 10x Vogelstein buffer (2.5 ⁇ l), 2.5 mM dNTP (1.5 ⁇ l), forward primer (1 ⁇ l, 25 pmol/ ⁇ l), reverse primer (1 ⁇ l, 25 pmol/ ⁇ l), ⁇ -actin primer mix (each 25 pmol/ ⁇ l), and EX Taq DNA polymerase (0.2 ⁇ l), adjusting the total volume to 25 ⁇ l.
  • RT-PCR was carried out with a step of 94° C. for 2 min; 35 cycles of 94° C. for 3 sec, 55° C. for 30 sec, 72° C. for 1 min; and a step of 72° C. for 3 min, and then the samples were kept at 4° C.
  • 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).
  • 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).
  • 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)).
  • human homologues containing the nucleotide sequences described in SEQ ID NO: 3 (BA2303) and in SEQ ID NO: 9 (BA0306) were obtained.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • oligopeptide (Gln-Asp-Ala-Gln-Gly-Gln-Arg-Ile-Gly-His-Phe-Glu-Phe-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 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • MBP maltose binding protein
  • NEB New England Bio Labs.
  • 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.
  • 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.
  • IPTG isopropanol- ⁇ -D-thiogalactopyranoside
  • Amylose resin (15 ml, BIORAD) was packed in a disposable column (2.5 dia. ⁇ 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.
  • 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 ⁇ 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.
  • 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 ⁇ 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 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.
  • 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.
  • the expression plasmid pQE-32 (QIA expression type IV construct, QIAGEN) was digested with BamHI and SalI, and then blunted.
  • 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.
  • 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° C. with shaking, with measuring the OD.
  • IPTG isopropanol- ⁇ -D-thiogalactopyranoside
  • shaking culturing was further performed at 37° 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.
  • 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.
  • 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 ⁇ g/well), and the fluorescence intensity was measured to determine the antibody titers.
  • the antibody against the ecombinant protein was produced.
  • 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.
  • a knockout mouse whose endogenous gene encoding mouse BA2303 protein was inactivated, was generated as follows. (1) Construction of a targeting vector
  • 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.
  • the cDNA encoding mouse BA2303 protein (SEQ ID NO: 5), which was cloned in the previous Example, was labeled with 32 P 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.
  • 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.
  • 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.
  • Tk thymidine kinase gene
  • ES cells 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 ⁇ 10 7 cells/ml.
  • the targeting vector was added to the cells (25 ⁇ g/1 ml cell suspension), and electroporation was performed with a single pulse of 350 V/cm (25 ⁇ F).
  • the ES cells were seeded into 10 cm dishes (1 ⁇ 107 cells/dish), cultured for one day in maintenance medium, and then the medium was replaced with selection medium (containing G418 (250 ⁇ g/ml) and 2 ⁇ M gancyclovir). The culture was continued with replacing the medium every two days.
  • selection medium containing G418 (250 ⁇ g/ml) and 2 ⁇ M gancyclovir. The culture was continued with replacing the medium every two days.
  • 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.
  • 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.
  • 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.
  • 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 32 P 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.
  • 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).
  • NotI-digested pBluescript II SK(-) was ligated with an insert of the above mouse BA0306 genomic DNA (exons I to V).
  • 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.
  • the resulting pBluescript II SK(-) was digested with SacII and linealized to use as a targeting vector.
  • ES cells Mouse embryonic stem cells (ES cells, 1 ⁇ 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 ⁇ 10 7 cells/ml.
  • the targeting vector was added to the (25 ⁇ g/ ml cell suspension), and electroporation was performed with a single pulse of 350 V/cm (25 ⁇ F).
  • the ES cells were seeded into 10 cm dishes (1 ⁇ 10 7 cells/dish), cultured 1 day in maintenance medium, and then the medium was replaced with selection medium (containing G418 (250 ⁇ g/ml) and 2 ⁇ M gancyclovir). The culture was continued with replacing the medium every two days.
  • selection medium containing G418 (250 ⁇ g/ml) and 2 ⁇ M gancyclovir. The culture was continued with replacing the medium every two days.
  • 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.
  • 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.
  • the 5′ and 3′ flanking genes encompassing the integrated neo gene can be detected as 7 kb and 5 kb bands, respectively.
  • 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.
  • 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.
  • active oxygen species such as nitrogen monoxide (NO)
  • GTP binding protein GTP binding protein
  • 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.
  • the DNA itself is very useful as an antisense medicine
  • the extracellular domain fragment of the protein is useful as a soluble receptor medicine
  • the antibody or its part is useful as an antibody medicine.
  • 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.
  • model animals can be generated by disrupting (inactivating) a corresponding endogenous gene.
  • 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.
  • model animals having a human gene of the invention alone by crossing the model animals, whose endogenous gene is disrupted, with the transgenic animals.
  • a drug which targets the introduced human gene compounds, and antibodies, etc.

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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.

Description

    TECHNICAL FIELD
  • 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. [0001]
    • BACKGROUND ART
  • 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. [0002]
  • 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. [0003]
  • 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. [0004]
  • 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. [0005]
  • 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,. [0006]
  • Since particular decisive causes of arteriosclerosis are unclear, only symptomatic treatment by chemotherapy is currently possible. For example, β 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. [0007]
  • 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. [0008]
  • 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. [0009]
  • 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. [0010]
  • 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. [0011]
  • 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. [0012]
  • 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. [0013]
  • 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)). [0014]
  • 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. [0015]
    • DISCLOSURE OF THE INVENTION
  • 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. [0016]
  • 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 ([0017] day 1 to 7) after PTCA and completed the present invention.
  • 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. [0018]
  • Clone BA0306 has the following characteristics. [0019]
  • (1) Its increased expression is observed on [0020] day 1 to 7 after PTCA of coronary artery (the peak is observed on day 4).
  • (2) Northern blotting reveals the expression of the mRNA as about 3.5 k and about 4.4 k bands in various human tissues. [0021]
  • (3) It has ten putative transmembrane regions. [0022]
  • (4) It has amino acid sequence homology with [0023] S. cerevisiae oxidative stress resistance protein, S. cerevisiae zinc/cadmium resistance protein, heavy metal ion resistance protein, and so on.
  • (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. [0024]
  • 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. [0025]
  • Clone BA2303 has the following characteristics. [0026]
  • (1) Its increased expression is observed from [0027] day 1 after PTCA of coronary artery, and the expression continues until day 7 with the maximum expression on day 2 to 4.
  • (2) Northern blotting reveals the expression of the mRNA as about 3.9 k and about 2.1 k bands in various human tissues. [0028]
  • (3) It has seven putative transmembrane regions. [0029]
  • (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. [0030]
  • 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. [0031]
  • 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. [0032]
  • 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. [0033]
  • 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. [0034]
  • 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. [0035]
  • Namely, the present invention provides the DNAS, proteins, expression vectors, transformants, antibodies, pharmaceutical compositions, transgenic mice, and knockout, mentioned below. [0036]
  • (1) A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4. [0037]
  • (2) A DNA encoding a protein fragment comprising the extracellular region of a protein having the amino acid sequence of SEQ ID NO: 4. [0038]
  • (3) A DNA comprising a nucleotide sequence corresponding to nucleotide residues 97 to 1419 of the nucleotide sequence of SEQ ID NO: 3. [0039]
  • (4) A DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 3 under stringent conditions. [0040]
  • (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. [0041]
  • (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. [0042]
  • (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. [0043]
  • (8) An expression vector comprising the DNA of any one of (1) to (4). [0044]
  • (9) A transformant carrying the expression vector of (8). [0045]
  • (10) An antibody or its portion reactive with the protein of (5) or the protein fragment of (6). [0046]
  • (11) The antibody or its portion of (10), wherein the antibody is a monoclonal antibody. [0047]
  • (12) A pharmaceutical composition comprising the protein fragment of (6) or the fusion protein of (7) and a pharmaceutically acceptable carrier. [0048]
  • (13) A pharmaceutical composition comprising the antibody or its portion of (10) or (11) and a pharmaceutically acceptable carrier. [0049]
  • (14) A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 10. [0050]
  • (15) A DNA encoding a protein fragment comprising the extracellular region of a protein having the amino acid sequence of SEQ ID NO: 10. [0051]
  • (16) A DNA having a nucleotide sequence corresponding to nucleotide [0052] 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. [0053]
  • (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. [0054]
  • (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. [0055]
  • (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. [0056]
  • (21) An expression vector comprising the DNA of any one of (14) to (17). [0057]
  • (22) A transformant carrying the expression vector of (21). [0058]
  • (23) An antibody or its portion reactive with the protein of (18) or the protein fragment of (19). [0059]
  • (24) The antibody or its portion of (23), wherein the antibody is a monoclonal antibody. [0060]
  • (25) A pharmaceutical composition comprising the protein fragment of (19) or the fusion protein of (20) and a pharmaceutically acceptable carrier. [0061]
  • (26) A pharmaceutical composition comprising the antibody or its portion of (23) or (24) and a pharmaceutically acceptable carrier. [0062]
  • (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. [0063]
  • (28) A transgenic mouse in which the human-derived DNA comprising a DNA having a nucleotide sequence corresponding to nucleotide [0064] 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. [0065]
  • (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. [0066]
  • 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. [0067]
  • 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. [0068]
  • 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. [0069]
  • 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. [0070]
  • 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. [0071]
  • 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. [0072]
  • 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. [0073]
  • “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). [0074]
  • 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). [0075]
  • 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[0076] 1 and IgA2).
  • 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. [0077]
  • Similarly, the heavy chain of IgG1 is composed of VH, [0078] Cγ 11 domain, hinge region, Cγ 12 domain, and Cγ 13 domain in this order from N terminus. The heavy chain of IgG2 is composed of VH, Cγ 21 domain, hinge region, Cγ 22 domain, and Cγ 23 domain in this order from N terminus. The heavy chain of IgG3 is composed of VH, Cγ 31 domain, hinge region, Cγ 32 domain, and Cγ 33 domain in this order from N terminus. The heavy chain of IgG4 is composed of VH, Cγ 41 domain, hinge region, Cγ 42 domain, and Cγ 43 domain in this order from N terminus.
  • The heavy chain of IgA is composed of VH, Cα1 domain, hinge region, Cα2 domain, and Cα3 domain in this order from N terminus. [0079]
  • Similarly, the heavy chain of IgA1 is composed of VH, [0080] Cα 11 domain, hinge region, Cα 12 domain, and Cα 13 domain in this order from N terminus. The heavy chain of IgA2 is composed of VH, Cα 21 domain, hinge region, Cα 22 domain, and Cα 23 domain in this order from N terminus.
  • The heavy chain of IgD is composed of VH, Cδ1 domain, hinge region, Cδ2 domain, and Cδ3 domain in this order from N terminus. [0081]
  • The heavy chain of IgM is composed of VH, Cμ1 domain, Cμ2 domain, Cμ3 domain, and Cμ4 domain in this order from N terminus and have no hinge region as seen in IgG, IgA, and IgD. [0082]
  • The heavy chain of IgE is composed of VH, Cε1 domain, Cε2 domain, Cε3 domain, and Cε4 domain in this order from N terminus and have no hinge region as seen in IgG, IgA, and IgD. [0083]
  • 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). [0084]
  • 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. [0085]
  • 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. [0086]
  • 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. [0087]
  • 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. [0088]
  • 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. [0089]
  • (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. [0090]
  • (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. [0091]
  • (3) A DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 3 under stringent conditions. [0092]
  • (4) A DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 9 under stringent conditions. [0093]
  • 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 [0094] 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.
  • 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. [0095]
  • 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. [0096]
  • Tm=82.3° C.+0.41×(G+C) %—500/n—0.61×(formamide) % (n means the number of the nucleotide of probe). [0097]
  • Temperature=Tm −25° C. [0098]
  • 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. [0099]
  • (1) Tm descends by about 1° C. per 1% mismatch. [0100]
  • (2) Tm descends by 0.6 to 0.7° C. per 1% formamide. [0101]
  • Accordingly, the temperature conditions for the combination of completely complementary strands can be set as follows. [0102]
  • (A) 65 to 75° C. (formamide not added) [0103]
  • (B) 35 to 45° C. (in the presence of 50% formamide) [0104]
  • The temperature conditions for the combination of incompletely complementary strands can be set as follows. [0105]
  • (A) 45 to 55° C. (formamide not added) [0106]
  • (B) 35 to 42° C. (in the presence of 30% formamide) [0107]
  • The temperature conditions when a probe with 23 or less nucleotides is used can be 37° C. or can be calculated using the following formula. [0108]
  • Temperature=2° C.×(the number of A+T)+4° C.×(the number of C+G) −5° C. [0109]
  • 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. [0110]
  • 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. [0111]
  • (1) cDNA can be cloned from the mRNA encoding the protein of the present invention by, for example, the method described below. [0112]
  • 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. [0113]
  • 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 [0114] E. coli with plasmid vectors, phage vectors, or cosmid vectors having this cDNA or by transfecting E. coli after in vitro packaging.
  • 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, λgt10, λgt11, 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. [0115]
  • 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, [0116] E. coli: HB101, DH5α, MC1061/P3, etc.).
  • 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). [0117]
  • 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. [0118]
  • 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 [0119] 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.
  • When a cDNA library prepared using a cDNA expression vector (for example, λZAPII 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. [0120]
  • 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. [0121]
  • (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. [0122]
  • (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. [0123]
  • 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. [0124]
  • 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 [0125] 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 λ phage, and an animal or insect virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and nuclear polyhedrosis virus.
  • 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. [0126]
  • When bacteria, particularly [0127] 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.
  • 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. [0128]
  • 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, λPL 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. [0129]
  • A preferable initiation codon is, for example, a methionine codon (ATG). [0130]
  • The commonly used termination codon (for example, TAG, TGA, TAA, and so on) is illustrated as a termination codon. [0131]
  • Usually used natural or synthetic terminators are used as a terminator region. [0132]
  • 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 [0133] E. coli, yeast 2 μ 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.
  • 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. [0134]
  • 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. [0135]
  • 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. [0136]
  • 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. [0137]
  • Transformants of the present invention can be prepared by introducing the expression vector mentioned above into host cells. [0138]
  • 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. [0139]
  • [0140] E. coli or animal cells are preferably used. Specific examples are E. coli (DH5α, 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.).
  • An expression vector can be introduced (transformed (transduced)) into host cells by known method. [0141]
  • 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 ([0142] 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.
  • 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. [0143]
  • 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.). [0144]
  • 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. [0145]
  • Specific media and cultivation conditions used depending on host cells are illustrated below, but are not limited thereto. [0146]
  • When the hosts are bacteria, actinomycetes, yeasts, filamentous fungi, liquid media comprising the nutrient source mentioned above are appropriate. The media with [0147] pH 5 to 8 are preferably used.
  • When the host is [0148] 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° C. for about 3 to 24 hours with aeration and stirring, if necessary.
  • When the host is Bacillus, cultivation can be performed usually at 30 to 40° C. for about 16 to 96 hours with aeration and stirring, if necessary. [0149]
  • 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° C. for about 14to 144 hours with aeration and stirring, if necessary. [0150]
  • 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° C. for about 15 to 72 hours with aeration and stirring, if necessary. [0151]
  • 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° C. for 15 to 100 hours with aeration and stirring, if necessary. [0152]
  • 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. [0153]
  • 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. [0154]
  • 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. [0155]
  • 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. [0156]
  • 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. [0157]
  • 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, [0158] Chapter 7, pp.361-408, (1990)).
  • 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. [0159]
  • “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.). [0160]
  • The “antibody” of the present invention can be a polyclonal antibody (antiserum) or a monoclonal antibody, and preferably a monoclonal antibody. [0161]
  • Specifically, it is an antibody reactive to (against, which binds to) the above-mentioned protein or its fragment of the present invention. [0162]
  • 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. [0163]
  • The monoclonal antibody includes those having any one isotype of IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable. [0164]
  • 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. [0165]
  • 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. [0166]
  • 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. [0167]
  • 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. [0168]
  • 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. [0169]
  • 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. [0170]
  • 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. [0171]
  • 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. [0172]
  • 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. [0173]
  • 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. [0174]
  • 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. [0175]
  • 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. [0176]
  • 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. [0177]
  • 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×SSC. After the filter is sufficiently dried, it is baked at 75° C. for3 hours. After baking, the filter is treated with 0.1×SSC/0.1% SDS at 65° C. for 30 minutes. Then, it is soaked in 3×SSC/0.1% SDS. The filter obtained is treated with prehybridization solution in a plastic bag at 65° C. for 3 to 4 hours. [0178]
  • Next, [0179] 32P-labeled probe DNA and hybridization solution are added to the bag and reacted at 65° C. about 12 hours. After hybridization, the filter is washed under appropriate salt concentration, reaction temperature, and time (for example, 2×SSC-0.1% SDS, room temperature, 10 minutes). The filter is put into a plastic bag with a little 2×SSC, and subjected to autoradiography after the bag is sealed.
  • 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, λEMBL3, λEMBL4, etc.). [0180] 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 (κ) 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.
  • Separately, human CH gene and human CL gene used for chimerization are isolated. For example, when a chimeric antibody with human IgG1 is produced, Cγ1 gene as a CH gene, and Cκ gene as a CL gene, are isolated. These genes can be isolated from human genomic library with mouse Cγ1 gene and mouse Cκ gene, corresponding to human Cγ1 gene and human C κ gene, respectively, as probes, taking advantage of high homology between the nucleotide sequences of mouse immunoglobulin gene and that of human immunoglobulin gene. [0181]
  • Specifically, DNA fragments comprising human Cκ gene and an enhancer region are isolated from human λ 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 λ788 and then human Cγ1 gene is isolated with the probes mentioned above. [0182]
  • 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. [0183]
  • Chimeric gene-inserted expression vector(s) thus prepared are introduced into myelomas that do not produce antibodies, for example, P3X63·Ag8·653 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. [0184]
  • Desired chimeric monoclonal antibodies are obtained from the culture supernatant of antibody-producing cells thus screened. [0185]
  • 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. [0186]
  • 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). [0187]
  • 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. [0188]
  • 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. [0189]
  • 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. [0190]
  • 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. [0191]
  • 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. [0192]
  • 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. [0193]
  • 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. [0194]
  • 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′)[0195] 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)).
  • “F(ab′)[0196] 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 CHγ1 (γ1 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′)2.
  • 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. [0197]
  • 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. [0198]
  • 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 μg to 1000 mg (or 10 μg 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. [0199]
  • 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 μg 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 μg to 100 mg/kg body weight, preferably 50 μg 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. [0200]
  • 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. [0201]
  • 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. [0202]
  • 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.[0203]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a photograph showing an electrophoresis image of rabbit BA2303 cDNA samples obtained by RT-PCR. [0204]
  • 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. [0205]
  • FIG. 2 is a photograph showing an electrophoresis image of rabbit BA0306 cDNA samples obtained by RT-PCR. [0206]
  • 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. [0207]
  • FIG. 3 shows a plot of the hydrophobicity and hydrophilicity of the amino acid residues composing rabbit BA2303 protein. [0208]
  • FIG. 4 shows a plot of the hydrophobicity and hydrophilicity of the amino acid residues composing human BA0306 protein. [0209]
  • FIG. 5 shows a plot of the hydrophobicity and hydrophilicity of the amino acid residues composing human BA2303 protein. [0210]
  • FIG. 6 is a photograph showing the result of Northern blot analysis of the expression of human BA2303 mRNA in various human tissues. [0211]
  • FIG. 7 is a photograph showing the result of Northern blot analysis of the expression of human BA0306 mRNA in various human tissues. [0212]
  • FIG. 8 shows a plot of the hydrophobicity and hydrophilicity of the amino acid residues composing mouse BA2303 protein. [0213]
  • FIG. 9 shows the sequence homology at the amino acid level between BA2303 proteins from rabbit, human, and mouse. [0214]
  • FIG. 10 shows the sequence homology at the amino acid level between BA0306 proteins from rabbit, human, and mouse. [0215]
  • 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. [0216]
  • 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.[0217]
    • BEST MODE FOR IMPLEMENTING THE INVENTION
  • The present invention is illustrated in detail below with reference to examples, but is not to be construed as being limited thereto. [0218]
    • EXAMPLE 1
  • Generation of a rabbit model whose aortal endothelium is detached by PTCA [0219]
  • 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 [0220] day 1 to six months after PTCA.
    • EXAMPLE 2
  • Preparation of total RNA from removed aortae [0221]
  • 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). [0222]
  • Also, the aorta was removed from a normal Japanese white rabbit, which was not subjected to PTCA, and total RNA was prepared as described. [0223]
    • EXAMPLE 3
  • cDNA synthesis [0224]
  • Total RNAs (each 2 μl, 1 μg/ml) sampled with the passage of time or mRNA samples (each 2 μl, 0.5 μg/ml), which were obtained in Example 2, were dissolved in diethyl pirocarbonate (DEPC)-treated distilledwater(8 μl). Anchorprimer(GT15MA, 1 μl, 25 pmol/μl) was added to make the [0225] total volume 10 μl, and the mixture was then incubated 5 min at 65° C. The samples were placed on ice immediately after completion of the incubation.
  • Then, 5x first strand buffer (4 μl, composition: 0.25 M Tris-HCl (pH 7.5), 0.375 M KCl, 0.05 M DTT, 0.015 M MgCl[0226] 2), 0.1 M DTT (2 μl), 250 μM dNTP (1 μl), distilled water (1 μl), and reverse transcriptase (Superscript, GIBCO BRL, 1 μl, 200 U/μl) were added to make the total volume 20 μl. cDNA was synthesized by incubating the reaction mixture for 1 hr at 42° C., and then DEPC-treated water (30 μl) was added to make the final volume 50 μl.
    • EXAMPLE 4
  • Analysis of the time course of gene expression [0227]
  • 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)). [0228]
  • 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). [0229]
  • The template cDNA (each 2 μl) was mixed with distilled water (10.75 μl), 10x EX Taq buffer (2 μl), 25 μM dNTP (1.5 μl), arbitrary primer (sequence: GATCAATCGC, 1 μl, 25 pmol/μl), anchor primer (1 μl, 25 pmol/μl), EX Taq DNA polymerase (0.25 μl), and α35S-dATP (1.5 μl, 10 mCi/ml, Amersham) to make the total volume 20 μl. PCR was carried out with a cycle of 95° C. for 3 min, 40° C. for 5 min, 72° C. for 5 min; 40 cycles of 95° C. for 30 sec, 40° C. for 2 min, 72° C. for 1 min; and a step of 72° C. for 5 min, and then the samples were kept at 4° C. [0230]
  • Each of the resulting PCR products was mixed with stop buffer (5 μl, composition: formamide (30 ml), xylenecyanol (30 mg), bromophenol blue (10 mg), 0.5 M EDTA (200 μl, (pH 8.0)), and then, 3.5 μl 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. [0231]
  • 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. [0232]
  • 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. [0233]
  • 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. [0234]
  • Each template cDNA (3 μl) was mixed with 10x Vogelstein buffer (2.5 μl), 2.5 mM dNTP (1.5 μl), forward primer (1 μl, 25 pmol/μl), reverse primer (1 μl, 25 pmol/μl), β-actin primer mix (each 25 pmol/μl), and EX Taq DNA polymerase (0.2 μl), adjusting the total volume to 25 μl. RT-PCR was carried out with a step of 94° C. for 2 min; 35 cycles of 94° C. for 3 sec, 55° C. for 30 sec, 72° C. for 1 min; and a step of 72° C. for 3 min, and then the samples were kept at 4° C. [0235]
  • The obtained PCR products were separated by electrophoresis. The results were shown in FIGS. [0236] 1 (BA2303) and 2 (BA0306).
  • It was confirmed that the expression of BA2303 was increased from [0237] 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
  • Isolation of long strand cDNA [0238]
  • 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)). [0239]
  • The PCR was performed twice using the Marathon cDNA Amplification Kit (CLONTECH) and the cDNA fragments obtained in Example 4 as a template. [0240]
  • 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). [0241]
  • 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). [0242]
  • 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). [0243]
    • EXAMPLE 6
  • Isolation of human counterpart genes [0244]
  • 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. [0245]
  • 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). [0246]
  • 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). [0247]
  • 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). [0248]
  • 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). [0249]
  • Homology search between known proteins indicated that human BA0306 has sequence homology at the amino acid level with [0250] S. cerevisiae oxidative stress resistance protein, S. cerevisiae zinc/cadmium resistance protein, and heavy metal ion resistance protein, etc.
    • EXAMPLE 7
  • Isolation of mouse BA2303 cDNA [0251]
  • 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. [0252]
  • 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). [0253]
  • 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). [0254]
    • EXAMPLE 8
  • Isolation of mouse BA0306 cDNA [0255]
  • 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. [0256]
  • 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). [0257]
    • EXAMPLE 9
  • Preparation of anti-peptide antibody against human BA2303 [0258]
  • An oligopeptide (Gln-Asp-Ala-Gln-Gly-Gln-Arg-Ile-Gly-His-Phe-Glu-Phe-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 μg/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. [0259]
  • 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. [0260]
    • EXAMPLE 10
  • Preparation of recombinant fusion protein with human BA2303 protein [0261]
  • 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. [0262]
  • 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 [0263] 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-β-D-thiogalactopyranoside (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 μl, phenylmethylsulfonyl fluoride) to suppress protease digestion.
  • 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. [0264]
  • Amylose resin (15 ml, BIORAD) was packed in a disposable column (2.5 dia.×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 [0265] flow rate 1 ml/min, and washed with ice-cold column buffer.
  • 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 μl) 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. [0266]
    • EXAMPLE 11
  • Preparation of antibody against human BA2303 protein [0267]
  • 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 μg/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 ([0268] 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
  • Construction of an expression vector for recombinant human BA2303 [0269]
  • 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. [0270]
  • 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. [0271]
    • EXAMPLE 13
  • Preparation of recombinant rabbit BA0306 protein [0272]
  • 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. [0273]
  • The expression plasmid pQE-32 (QIA expression type IV construct, QIAGEN) was digested with BamHI and SalI, and then blunted. [0274]
  • 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. [0275]
  • Next, [0276] 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° C. with shaking, with measuring the OD. Then, IPTG (isopropanol-β-D-thiogalactopyranoside) was added to the culture to the final concentration of 1 mM, and shaking culturing was further performed at 37° 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.
  • 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. [0277]
    • EXAMPLE 14
  • Preparation of antibody against human BA0306 [0278]
  • 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 μg/well), and the fluorescence intensity was measured to determine the antibody titers. The antibody against the ecombinant protein was produced. [0279]
  • 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. [0280]
    • EXAMPLE 15
  • Generation of knockout mice of mouse BA2303 gene [0281]
  • A knockout mouse, whose endogenous gene encoding mouse BA2303 protein was inactivated, was generated as follows. (1) Construction of a targeting vector [0282]
  • 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. [0283]
  • The cDNA encoding mouse BA2303 protein (SEQ ID NO: 5), which was cloned in the previous Example, was labeled with [0284] 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.
  • 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. [0285]
  • (2) Transfection of the targeting vector into ES cells [0286]
  • 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 1×10[0287] 7 cells/ml. The targeting vector was added to the cells (25 μg/1 ml cell suspension), and electroporation was performed with a single pulse of 350 V/cm (25 μF). Then, the ES cells were seeded into 10 cm dishes (1×107 cells/dish), cultured for one day in maintenance medium, and then the medium was replaced with selection medium (containing G418 (250 μg/ml) and 2 μM 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.
  • (3) Screening of knockout ES cells [0288]
  • Each neomycin resistant ES clone was examined by PCR whether its endogenous gene encoding mouse BA2303 protein was inactivated (knocked out) by homologous recombination. [0289]
  • 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. [0290]
  • (4) Generation of knock out mice [0291]
  • 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. [0292]
    • EXAMPLE 16
  • Generation of knockout mice of mouse BA0306 gene [0293]
  • (1) Construction of a targeting vector. [0294]
  • 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 [0295] 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.
  • 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. [0296]
  • (2) Transfection of the targeting vector into ES cells. [0297]
  • Mouse embryonic stem cells (ES cells, 1×10 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 1×10[0298] 7 cells/ml. The targeting vector was added to the (25 μg/ ml cell suspension), and electroporation was performed with a single pulse of 350 V/cm (25 μF). Then, the ES cells were seeded into 10 cm dishes (1×107 cells/dish), cultured 1 day in maintenance medium, and then the medium was replaced with selection medium (containing G418 (250 μg/ml) and 2 μM 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.
  • (3) Screening of knockout ES cells [0299]
  • 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. [0300]
  • 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. [0301]
  • (probe 1) [0302]
  • 5′ flanking DNA which was amplified using two primers described in SEQ ID NO: 39 and SEQ ID NO: 40. [0303]
  • (probe 2) [0304]
  • 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). [0305]
  • 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. [0306]
  • 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. [0307]
  • (4) Generation of knock out mice [0308]
  • 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. [0309]
  • (clone 0-16-9) [0310]
  • Total number of injected cells: 83 [0311]
  • Littermates: 13 [0312]
  • Chimera mice: 7 [0313]
  • Chimera where contribution to hair color is 80% or more: [0314]
  • (clone 0-22-11) [0315]
  • Total number of injected cells: 202 [0316]
  • Littermates: 12 [0317]
  • Chimera mice: 3 [0318]
  • Chimera where contribution to hair color is 80% or more: 3 [0319]
  • (clone 0-22-18) [0320]
  • Total number of injected cells: 148 [0321]
  • Littermates: 9 [0322]
  • Chimera mice: 5 [0323]
  • 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. [0324]
    • INDUSTRIAL APPLICABILITY
  • 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. [0325]
  • [BA0306][0326]
  • 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. [0327]
  • (1) Its expression is increased from [0328] day 1 to day 7 after PTCA of the coronary aorta (peak at day 4).
  • (2) Its mRNA is expressed in various human tissues as detected by Northern blotting as approximately 3.5 kb and 4.4 kb bands. [0329]
  • (3) Its 10 predicted transmembrane regions. [0330]
  • (4) Its sequence homology at the amino acid level with [0331] S. cerevisiae oxidative stress resistance protein, S. cerevisiae zinc/cadmium resistance protein, and heavy metal ion resistance protein, etc.
  • [BA2303][0332]
  • 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. [0333]
  • (1) Its expression is increased [0334] day 1 after PTCA of the coronary aorta, reaches the maximum on day 2 to day 4, and continued until day 7.
  • (2) Its mRNA is expressed in various human tissues as detected by Northern blotting as approximately 3.9 kb and 2.1 kb bands. [0335]
  • (3) having seven predicted transmembrane regions. [0336]
  • 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. [0337]
  • 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. [0338]
  • 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. [0339]
  • 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. [0340]
  • 0
    SEQUENCE LISTING
    <160> NUMBER OF SEQ ID NOS: 42
    <210> SEQ ID NO 1
    <211> LENGTH: 1399
    <212> TYPE: DNA
    <213> ORGANISM: Oryctolagus cuniculus
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)..(1158)
    <400> SEQUENCE: 1
    gtc aga atc aac aac ata gca gta gct gta gga aaa gaa gct aaa ctt 48
    Val Arg Ile Asn Asn Ile Ala Val Ala Val Gly Lys Glu Ala Lys Leu
    1 5 10 15
    tac ctg ttc caa gcc cag gaa tgg ctg aag ctg cag gaa agc agt cat 96
    Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys Leu Gln Glu Ser Ser His
    20 25 30
    gat tac agc tgt cat gaa aaa tta tcc aaa gcc caa ttg aca atg acc 144
    Asp Tyr Ser Cys His Glu Lys Leu Ser Lys Ala Gln Leu Thr Met Thr
    35 40 45
    atg aac cag agt gaa cat aat atg aca gtg tcc cag att cca tct cca 192
    Met Asn Gln Ser Glu His Asn Met Thr Val Ser Gln Ile Pro Ser Pro
    50 55 60
    caa acg tgg cac gtg ttt tat gca gac aag tat aca tgc cga gtt gac 240
    Gln Thr Trp His Val Phe Tyr Ala Asp Lys Tyr Thr Cys Arg Val Asp
    65 70 75 80
    gag gag aat tgg caa gtg gaa gat atc cca ttt gaa atg gtg tta cta 288
    Glu Glu Asn Trp Gln Val Glu Asp Ile Pro Phe Glu Met Val Leu Leu
    85 90 95
    aac cca gat gct gaa gga aat ccg ttt gat cat ttt ggt gct gga gaa 336
    Asn Pro Asp Ala Glu Gly Asn Pro Phe Asp His Phe Gly Ala Gly Glu
    100 105 110
    tct ggg tta cat gag ttc ttt ttc ctc cta gtc cta gtg tac ttt gtg 384
    Ser Gly Leu His Glu Phe Phe Phe Leu Leu Val Leu Val Tyr Phe Val
    115 120 125
    act gct tgc att tat gcg cag tca ttg tgg cag gct ctt aag aaa gga 432
    Thr Ala Cys Ile Tyr Ala Gln Ser Leu Trp Gln Ala Leu Lys Lys Gly
    130 135 140
    ggg ccc atg cac atg att cta aag gtg ctg aca act gca ctg ctg ttg 480
    Gly Pro Met His Met Ile Leu Lys Val Leu Thr Thr Ala Leu Leu Leu
    145 150 155 160
    caa gct ggt tca gct gta gct aat tac atc cat ttc tcc agt tac tcc 528
    Gln Ala Gly Ser Ala Val Ala Asn Tyr Ile His Phe Ser Ser Tyr Ser
    165 170 175
    aaa gat gga atc ggg gta cct ttt atg gga agc ttg gca gaa ttt ttt 576
    Lys Asp Gly Ile Gly Val Pro Phe Met Gly Ser Leu Ala Glu Phe Phe
    180 185 190
    gac atc gct tcc caa att cag atg tta tac ctg ctt ctg agt ctg tgc 624
    Asp Ile Ala Ser Gln Ile Gln Met Leu Tyr Leu Leu Leu Ser Leu Cys
    195 200 205
    atg ggc tgg acc ata gtc agg atg aag aag tct caa agc aga cct ctc 672
    Met Gly Trp Thr Ile Val Arg Met Lys Lys Ser Gln Ser Arg Pro Leu
    210 215 220
    cag tgg gat tcg acc cct gcc tcc act ggc att gcc gtg ttc att gtc 720
    Gln Trp Asp Ser Thr Pro Ala Ser Thr Gly Ile Ala Val Phe Ile Val
    225 230 235 240
    ctg aca cag agt gtt ttg ctg ctt tgg gaa cag ttt gaa gat acc ggt 768
    Leu Thr Gln Ser Val Leu Leu Leu Trp Glu Gln Phe Glu Asp Thr Gly
    245 250 255
    cat cat agc tcc cat tca cac cac aac tta gca ggg atc ctt ctg atc 816
    His His Ser Ser His Ser His His Asn Leu Ala Gly Ile Leu Leu Ile
    260 265 270
    gtt tta aga att tgc ctg gca ttg tca tta ggc tgt gga ctc tat cag 864
    Val Leu Arg Ile Cys Leu Ala Leu Ser Leu Gly Cys Gly Leu Tyr Gln
    275 280 285
    atc atc aca gtg gag agg agc aca ctc aaa agg gag ttc tac atc aca 912
    Ile Ile Thr Val Glu Arg Ser Thr Leu Lys Arg Glu Phe Tyr Ile Thr
    290 295 300
    ttt gcc aaa ggc tgt atc tta tgg ttt ttg tgc cat cca agt ctg gca 960
    Phe Ala Lys Gly Cys Ile Leu Trp Phe Leu Cys His Pro Ser Leu Ala
    305 310 315 320
    tgc att tct gtc att ttt aat gac tac caa aga gat aag gtt att aca 1008
    Cys Ile Ser Val Ile Phe Asn Asp Tyr Gln Arg Asp Lys Val Ile Thr
    325 330 335
    ata ggt gtt atc ctt ggc cag tct gtt gcc atg gtt atc ctc tac aga 1056
    Ile Gly Val Ile Leu Gly Gln Ser Val Ala Met Val Ile Leu Tyr Arg
    340 345 350
    ctc ttt ctc tcc cac agt cta tac tgg gaa gtt tct tcc ctt tcc tca 1104
    Leu Phe Leu Ser His Ser Leu Tyr Trp Glu Val Ser Ser Leu Ser Ser
    355 360 365
    gta aca cta cca ctg acc gta tcg tct gga cac aaa agc cgc cct cat 1152
    Val Thr Leu Pro Leu Thr Val Ser Ser Gly His Lys Ser Arg Pro His
    370 375 380
    ttc tga tacttgattt ctgtggaaaa gaaaagtgaa ggggttaaaa gagtgcaata 1208
    Phe
    385
    aggacccaaa tacagtgact tttttttcat acatttggta tgaaaaatcg aatagcaaaa 1268
    gcagagcatg tttctgtgat aactgcattt aagcagtacc aaaactgaac aaaggtaata 1328
    actgaaatgt tttaaaatac atgtaaacaa taaactttca ggaaattctg ttgttaaaaa 1388
    aaaaaaaaaa c 1399
    <210> SEQ ID NO 2
    <211> LENGTH: 385
    <212> TYPE: PRT
    <213> ORGANISM: Oryctolagus cuniculus
    <400> SEQUENCE: 2
    Val Arg Ile Asn Asn Ile Ala Val Ala Val Gly Lys Glu Ala Lys Leu
    1 5 10 15
    Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys Leu Gln Glu Ser Ser His
    20 25 30
    Asp Tyr Ser Cys His Glu Lys Leu Ser Lys Ala Gln Leu Thr Met Thr
    35 40 45
    Met Asn Gln Ser Glu His Asn Met Thr Val Ser Gln Ile Pro Ser Pro
    50 55 60
    Gln Thr Trp His Val Phe Tyr Ala Asp Lys Tyr Thr Cys Arg Val Asp
    65 70 75 80
    Glu Glu Asn Trp Gln Val Glu Asp Ile Pro Phe Glu Met Val Leu Leu
    85 90 95
    Asn Pro Asp Ala Glu Gly Asn Pro Phe Asp His Phe Gly Ala Gly Glu
    100 105 110
    Ser Gly Leu His Glu Phe Phe Phe Leu Leu Val Leu Val Tyr Phe Val
    115 120 125
    Thr Ala Cys Ile Tyr Ala Gln Ser Leu Trp Gln Ala Leu Lys Lys Gly
    130 135 140
    Gly Pro Met His Met Ile Leu Lys Val Leu Thr Thr Ala Leu Leu Leu
    145 150 155 160
    Gln Ala Gly Ser Ala Val Ala Asn Tyr Ile His Phe Ser Ser Tyr Ser
    165 170 175
    Lys Asp Gly Ile Gly Val Pro Phe Met Gly Ser Leu Ala Glu Phe Phe
    180 185 190
    Asp Ile Ala Ser Gln Ile Gln Met Leu Tyr Leu Leu Leu Ser Leu Cys
    195 200 205
    Met Gly Trp Thr Ile Val Arg Met Lys Lys Ser Gln Ser Arg Pro Leu
    210 215 220
    Gln Trp Asp Ser Thr Pro Ala Ser Thr Gly Ile Ala Val Phe Ile Val
    225 230 235 240
    Leu Thr Gln Ser Val Leu Leu Leu Trp Glu Gln Phe Glu Asp Thr Gly
    245 250 255
    His His Ser Ser His Ser His His Asn Leu Ala Gly Ile Leu Leu Ile
    260 265 270
    Val Leu Arg Ile Cys Leu Ala Leu Ser Leu Gly Cys Gly Leu Tyr Gln
    275 280 285
    Ile Ile Thr Val Glu Arg Ser Thr Leu Lys Arg Glu Phe Tyr Ile Thr
    290 295 300
    Phe Ala Lys Gly Cys Ile Leu Trp Phe Leu Cys His Pro Ser Leu Ala
    305 310 315 320
    Cys Ile Ser Val Ile Phe Asn Asp Tyr Gln Arg Asp Lys Val Ile Thr
    325 330 335
    Ile Gly Val Ile Leu Gly Gln Ser Val Ala Met Val Ile Leu Tyr Arg
    340 345 350
    Leu Phe Leu Ser His Ser Leu Tyr Trp Glu Val Ser Ser Leu Ser Ser
    355 360 365
    Val Thr Leu Pro Leu Thr Val Ser Ser Gly His Lys Ser Arg Pro His
    370 375 380
    Phe
    385
    <210> SEQ ID NO 3
    <211> LENGTH: 2130
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (97)..(1419)
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 2001, 2003, 2019, 2033, 2035, 2053, 2079, 2114, 2116,
    2119, 2129
    <223> OTHER INFORMATION: n is a or g or c or t
    <400> SEQUENCE: 3
    ctccggcgcc caccccgcct cccccagctg ccgacgtggg gcgggcagcc gccggcggct 60
    gggagccgag gcgtcggtgc agacctggag acgggc atg ggg ggg ctg cgg ctg 114
    Met Gly Gly Leu Arg Leu
    1 5
    ctg gct gtg gcc ctc acg tgc tgc tgg tgg ccg cag ggc agc cag ggt 162
    Leu Ala Val Ala Leu Thr Cys Cys Trp Trp Pro Gln Gly Ser Gln Gly
    10 15 20
    aag acc ctg cgg ggc agc ttc agc agc acc gcg gcc cag gac gcc cag 210
    Lys Thr Leu Arg Gly Ser Phe Ser Ser Thr Ala Ala Gln Asp Ala Gln
    25 30 35
    ggc cag cgc atc ggc cac ttc gag ttc cat ggt gac cat gct ctt ctg 258
    Gly Gln Arg Ile Gly His Phe Glu Phe His Gly Asp His Ala Leu Leu
    40 45 50
    tgt gtc aga atc aac aac ata gca gta gct gtt gga aaa gaa gct aaa 306
    Cys Val Arg Ile Asn Asn Ile Ala Val Ala Val Gly Lys Glu Ala Lys
    55 60 65 70
    ctc tac ctg ttc caa gcc cag gaa tgg cta aag cta cag caa agc agt 354
    Leu Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys Leu Gln Gln Ser Ser
    75 80 85
    cat ggt tat agc tgt agt gaa aaa tta tcc aaa gct cag ttg aca atg 402
    His Gly Tyr Ser Cys Ser Glu Lys Leu Ser Lys Ala Gln Leu Thr Met
    90 95 100
    acc atg aac cag acc gaa cat aat ctg aca gtg tcc cag att ccg tct 450
    Thr Met Asn Gln Thr Glu His Asn Leu Thr Val Ser Gln Ile Pro Ser
    105 110 115
    cca caa acg tgg cat gtg ttt tat gca gac aag tat aca tgc caa gat 498
    Pro Gln Thr Trp His Val Phe Tyr Ala Asp Lys Tyr Thr Cys Gln Asp
    120 125 130
    gac aag gag aat tct cag gtg gaa gat atc cca ttt gaa atg gtg tta 546
    Asp Lys Glu Asn Ser Gln Val Glu Asp Ile Pro Phe Glu Met Val Leu
    135 140 145 150
    cta aac cca gat gcc gaa ggg aat cca ttt gat cat ttt agt gct gga 594
    Leu Asn Pro Asp Ala Glu Gly Asn Pro Phe Asp His Phe Ser Ala Gly
    155 160 165
    gaa tct ggg tta cat gag ttc ttt ttc ctc cta gtc cta gtg tac ttt 642
    Glu Ser Gly Leu His Glu Phe Phe Phe Leu Leu Val Leu Val Tyr Phe
    170 175 180
    gtg att gct tgc att tat gct caa tca ttg tgg cag gct att aag aaa 690
    Val Ile Ala Cys Ile Tyr Ala Gln Ser Leu Trp Gln Ala Ile Lys Lys
    185 190 195
    ggc gga ccc atg cac atg att tta aag gtt ctg aca act gca ttg ctg 738
    Gly Gly Pro Met His Met Ile Leu Lys Val Leu Thr Thr Ala Leu Leu
    200 205 210
    tta caa gct ggt tca gct tta gct aat tac att cat ttc tcc agt tac 786
    Leu Gln Ala Gly Ser Ala Leu Ala Asn Tyr Ile His Phe Ser Ser Tyr
    215 220 225 230
    tcc aaa gat gga ata ggg gta cca ttt atg gga agt ttg gca gaa ttt 834
    Ser Lys Asp Gly Ile Gly Val Pro Phe Met Gly Ser Leu Ala Glu Phe
    235 240 245
    ttt gac atc gct tcc caa att cag atg tta tac tta ctt ttg agt cta 882
    Phe Asp Ile Ala Ser Gln Ile Gln Met Leu Tyr Leu Leu Leu Ser Leu
    250 255 260
    tgc atg ggt tgg aca ata gtc aga atg aag aag tct caa agc aga cct 930
    Cys Met Gly Trp Thr Ile Val Arg Met Lys Lys Ser Gln Ser Arg Pro
    265 270 275
    ctc cag tgg gat tct acg cct gca tcc act ggc att gca gta ttc att 978
    Leu Gln Trp Asp Ser Thr Pro Ala Ser Thr Gly Ile Ala Val Phe Ile
    280 285 290
    gtc atg aca cag agt gtt ttg cta ctt tgg gaa cag ttt gaa gat atc 1026
    Val Met Thr Gln Ser Val Leu Leu Leu Trp Glu Gln Phe Glu Asp Ile
    295 300 305 310
    agt cat cat agc tac cat tca cac cac aac tta gca ggg atc ctc cta 1074
    Ser His His Ser Tyr His Ser His His Asn Leu Ala Gly Ile Leu Leu
    315 320 325
    att gtt cta aga att tgc cta gca ttg tca tta ggc tgt gga ctc tat 1122
    Ile Val Leu Arg Ile Cys Leu Ala Leu Ser Leu Gly Cys Gly Leu Tyr
    330 335 340
    cag atc atc aca gtg gag aga agt aca ctc aaa agg gag ttc tac atc 1170
    Gln Ile Ile Thr Val Glu Arg Ser Thr Leu Lys Arg Glu Phe Tyr Ile
    345 350 355
    aca ttt gcc aaa ggc tgt atc ttg tgg ttt tta tgc cat cca gtt ctt 1218
    Thr Phe Ala Lys Gly Cys Ile Leu Trp Phe Leu Cys His Pro Val Leu
    360 365 370
    gca tgc att tct gtc att ttt agc gac tac caa aga gac aag gtt att 1266
    Ala Cys Ile Ser Val Ile Phe Ser Asp Tyr Gln Arg Asp Lys Val Ile
    375 380 385 390
    aca ata ggt gtt atc ctt tgc cag tct gtt tcc atg gtt att ctc tac 1314
    Thr Ile Gly Val Ile Leu Cys Gln Ser Val Ser Met Val Ile Leu Tyr
    395 400 405
    aga ctc ttt ctg tct cac agt cta tac tgg gaa gtt tct tca ctt tct 1362
    Arg Leu Phe Leu Ser His Ser Leu Tyr Trp Glu Val Ser Ser Leu Ser
    410 415 420
    tca gta aca cta cca ctg acc ata tca tct gga cac aaa agt cgc cct 1410
    Ser Val Thr Leu Pro Leu Thr Ile Ser Ser Gly His Lys Ser Arg Pro
    425 430 435
    cat ttc tga tacttgattt ttgttgagag gaaaagtgaa ttggttaaaa 1459
    His Phe
    440
    gagtgcaata aggatccaaa tacagtgact tttttttcat acatttagta tgaaaacttg 1519
    aacagcgaaa gcagagcatg ttatttatat aactgcattt aagcagtacc aagactgaaa 1579
    aaaaaggtaa taaatgaaat gttttgaaat atacttaaac aacaaacttt gaagaaagtg 1639
    ttgttataaa attattgaag cgatttctat gtggaaataa atgtgaaaaa taaaactatg 1699
    atattttggt aaaatattca ccacttataa tgcctcatct taatagctaa ctcasgttta 1759
    atartcttat aaaaagtaat cagttaaatg aatacttgct tataaatatc taaactaatc 1819
    cactttatga aatcagtgtt atacattgaa ttttaaaact gctgcctttt atgcctttaa 1879
    ggaaaatgtt tttccctatt ttgaatttta aaggaattga aattcctccc ggaaattaat 1939
    ataaataggg ttccccgtta aatgaaataa accctggttt aattggtggg gtggaattaa 1999
    tncncccaat tttttcccgn cccttttttg gggncncatt ttccgggttt taanccttga 2059
    ataaaccaaa gggtttttgn aaaaaccctt tttttgaaaa aaaattaaaa ccttnanttn 2119
    cctttacccn g 2130
    <210> SEQ ID NO 4
    <211> LENGTH: 440
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 4
    Met Gly Gly Leu Arg Leu Leu Ala Val Ala Leu Thr Cys Cys Trp Trp
    1 5 10 15
    Pro Gln Gly Ser Gln Gly Lys Thr Leu Arg Gly Ser Phe Ser Ser Thr
    20 25 30
    Ala Ala Gln Asp Ala Gln Gly Gln Arg Ile Gly His Phe Glu Phe His
    35 40 45
    Gly Asp His Ala Leu Leu Cys Val Arg Ile Asn Asn Ile Ala Val Ala
    50 55 60
    Val Gly Lys Glu Ala Lys Leu Tyr Leu Phe Gln Ala Gln Glu Trp Leu
    65 70 75 80
    Lys Leu Gln Gln Ser Ser His Gly Tyr Ser Cys Ser Glu Lys Leu Ser
    85 90 95
    Lys Ala Gln Leu Thr Met Thr Met Asn Gln Thr Glu His Asn Leu Thr
    100 105 110
    Val Ser Gln Ile Pro Ser Pro Gln Thr Trp His Val Phe Tyr Ala Asp
    115 120 125
    Lys Tyr Thr Cys Gln Asp Asp Lys Glu Asn Ser Gln Val Glu Asp Ile
    130 135 140
    Pro Phe Glu Met Val Leu Leu Asn Pro Asp Ala Glu Gly Asn Pro Phe
    145 150 155 160
    Asp His Phe Ser Ala Gly Glu Ser Gly Leu His Glu Phe Phe Phe Leu
    165 170 175
    Leu Val Leu Val Tyr Phe Val Ile Ala Cys Ile Tyr Ala Gln Ser Leu
    180 185 190
    Trp Gln Ala Ile Lys Lys Gly Gly Pro Met His Met Ile Leu Lys Val
    195 200 205
    Leu Thr Thr Ala Leu Leu Leu Gln Ala Gly Ser Ala Leu Ala Asn Tyr
    210 215 220
    Ile His Phe Ser Ser Tyr Ser Lys Asp Gly Ile Gly Val Pro Phe Met
    225 230 235 240
    Gly Ser Leu Ala Glu Phe Phe Asp Ile Ala Ser Gln Ile Gln Met Leu
    245 250 255
    Tyr Leu Leu Leu Ser Leu Cys Met Gly Trp Thr Ile Val Arg Met Lys
    260 265 270
    Lys Ser Gln Ser Arg Pro Leu Gln Trp Asp Ser Thr Pro Ala Ser Thr
    275 280 285
    Gly Ile Ala Val Phe Ile Val Met Thr Gln Ser Val Leu Leu Leu Trp
    290 295 300
    Glu Gln Phe Glu Asp Ile Ser His His Ser Tyr His Ser His His Asn
    305 310 315 320
    Leu Ala Gly Ile Leu Leu Ile Val Leu Arg Ile Cys Leu Ala Leu Ser
    325 330 335
    Leu Gly Cys Gly Leu Tyr Gln Ile Ile Thr Val Glu Arg Ser Thr Leu
    340 345 350
    Lys Arg Glu Phe Tyr Ile Thr Phe Ala Lys Gly Cys Ile Leu Trp Phe
    355 360 365
    Leu Cys His Pro Val Leu Ala Cys Ile Ser Val Ile Phe Ser Asp Tyr
    370 375 380
    Gln Arg Asp Lys Val Ile Thr Ile Gly Val Ile Leu Cys Gln Ser Val
    385 390 395 400
    Ser Met Val Ile Leu Tyr Arg Leu Phe Leu Ser His Ser Leu Tyr Trp
    405 410 415
    Glu Val Ser Ser Leu Ser Ser Val Thr Leu Pro Leu Thr Ile Ser Ser
    420 425 430
    Gly His Lys Ser Arg Pro His Phe
    435 440
    <210> SEQ ID NO 5
    <211> LENGTH: 1918
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (82)..(1383)
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (469)..(471)
    <223> OTHER INFORMATION: n is a or g or c or t
    Xaa is Tyr or His or Gln or Asn or Lys or Asp or Glu
    <400> SEQUENCE: 5
    ggcacgagcc gccctctgct gccgacgtgg gctgcaggcc gcaggcggtt gccgggcgag 60
    caaacggagc gggcggcggg c atg ggc ggc ctg cgg ctg ctg gcg gta gcc 111
    Met Gly Gly Leu Arg Leu Leu Ala Val Ala
    1 5 10
    ctc acg tgc agc tgc tgg tgg ccg cag ggc ggc cag ggc aag acc ctg 159
    Leu Thr Cys Ser Cys Trp Trp Pro Gln Gly Gly Gln Gly Lys Thr Leu
    15 20 25
    cgt ggc agc ttc agc agc gcc gcg gcc cgc gac gcc cag ggc cag agc 207
    Arg Gly Ser Phe Ser Ser Ala Ala Ala Arg Asp Ala Gln Gly Gln Ser
    30 35 40
    atc ggc cat ttc gag ttc cac cga atc aac aac gta gca gtg gct gtt 255
    Ile Gly His Phe Glu Phe His Arg Ile Asn Asn Val Ala Val Ala Val
    45 50 55
    gga aaa gaa gct aaa ctc tac ctg ttc caa gcc cag gaa tgg ctg aag 303
    Gly Lys Glu Ala Lys Leu Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys
    60 65 70
    ctg ctg gag agc agc ccc ggc tac agc tgc agt gag cgg cta gcc cga 351
    Leu Leu Glu Ser Ser Pro Gly Tyr Ser Cys Ser Glu Arg Leu Ala Arg
    75 80 85 90
    gct cag ctg aca gtg aca gtg acc cag acg gag cac aac ctc aca gtg 399
    Ala Gln Leu Thr Val Thr Val Thr Gln Thr Glu His Asn Leu Thr Val
    95 100 105
    tcc cag ctg ccc gct ccc cag aca tgg cga gtg ttc tat gcc gac aag 447
    Ser Gln Leu Pro Ala Pro Gln Thr Trp Arg Val Phe Tyr Ala Asp Lys
    110 115 120
    ttc acc tgc agg gat gac tca nas agc ccc cag ggg gag gag atc ccc 495
    Phe Thr Cys Arg Asp Asp Ser Xaa Ser Pro Gln Gly Glu Glu Ile Pro
    125 130 135
    ttt gaa atg gtg ctc ctc aac ccg gac gcc gag gga aac ccg ctg gat 543
    Phe Glu Met Val Leu Leu Asn Pro Asp Ala Glu Gly Asn Pro Leu Asp
    140 145 150
    cat ttt agc gcc aga gag tcc ggg ctc cac gag ttc ttt ttc ctc ctc 591
    His Phe Ser Ala Arg Glu Ser Gly Leu His Glu Phe Phe Phe Leu Leu
    155 160 165 170
    gtc cta gtg tac ttt gtg act gcg tgc atc tat gcg cag tct ctg tgg 639
    Val Leu Val Tyr Phe Val Thr Ala Cys Ile Tyr Ala Gln Ser Leu Trp
    175 180 185
    cag gct atg aag aag gga gga ccc atg cac acc atc tta aag gtc ctc 687
    Gln Ala Met Lys Lys Gly Gly Pro Met His Thr Ile Leu Lys Val Leu
    190 195 200
    acc act gca ctg ctg ctt caa gct gct tca gcc tta gct aat tac atc 735
    Thr Thr Ala Leu Leu Leu Gln Ala Ala Ser Ala Leu Ala Asn Tyr Ile
    205 210 215
    cac ttg tcc agg tac tcc aga gat ggg cta gga gtg cct ctc ata gga 783
    His Leu Ser Arg Tyr Ser Arg Asp Gly Leu Gly Val Pro Leu Ile Gly
    220 225 230
    agc ctg gca gaa gtt ttt gac att gcc tcc caa att cag atg ctg tac 831
    Ser Leu Ala Glu Val Phe Asp Ile Ala Ser Gln Ile Gln Met Leu Tyr
    235 240 245 250
    ctg ctt ctg agc ctg tgt atg ggc tgg aca ata gtg cgg atg aag aag 879
    Leu Leu Leu Ser Leu Cys Met Gly Trp Thr Ile Val Arg Met Lys Lys
    255 260 265
    tcg cag agc aga ccg ctc cag tgg gac tcg aca ccc gcg tcc acg ggc 927
    Ser Gln Ser Arg Pro Leu Gln Trp Asp Ser Thr Pro Ala Ser Thr Gly
    270 275 280
    atc gca gtt ttc aty gtc atc aca cag agc att ttg cta cty tgg gag 975
    Ile Ala Val Phe Ile Val Ile Thr Gln Ser Ile Leu Leu Leu Trp Glu
    285 290 295
    cag ttt gaa gac acc agt cac cac agc gca cat tca cac cgc agc tta 1023
    Gln Phe Glu Asp Thr Ser His His Ser Ala His Ser His Arg Ser Leu
    300 305 310
    gcc ggg ctc ttg ctg att gtc tta cgg atc tgc ctg gcg ctg tcg ctg 1071
    Ala Gly Leu Leu Leu Ile Val Leu Arg Ile Cys Leu Ala Leu Ser Leu
    315 320 325 330
    ggc tgc gga ctt tac cag gtc atc aca gtg gag agg agc gcg ctc aag 1119
    Gly Cys Gly Leu Tyr Gln Val Ile Thr Val Glu Arg Ser Ala Leu Lys
    335 340 345
    aga gag ttc tac atc acg ttt gcc aag ggc tgc atc ctg tgg ttc ttg 1167
    Arg Glu Phe Tyr Ile Thr Phe Ala Lys Gly Cys Ile Leu Trp Phe Leu
    350 355 360
    tgc cag cca gcg ctc gca tgc att gct gtc gct ttt aat gac tac caa 1215
    Cys Gln Pro Ala Leu Ala Cys Ile Ala Val Ala Phe Asn Asp Tyr Gln
    365 370 375
    aga gat aag ctt atc aca gta ggt gtc atc ctg tgt cag gcc gtg gcc 1263
    Arg Asp Lys Leu Ile Thr Val Gly Val Ile Leu Cys Gln Ala Val Ala
    380 385 390
    atg gtc att ctg tac aga ctt ttc ctg tcc cac agt ctt tac tgg gag 1311
    Met Val Ile Leu Tyr Arg Leu Phe Leu Ser His Ser Leu Tyr Trp Glu
    395 400 405 410
    gtc tcc tcg ctc tcc tca gta acg cta cca ctg acc atc tcg tct gca 1359
    Val Ser Ser Leu Ser Ser Val Thr Leu Pro Leu Thr Ile Ser Ser Ala
    415 420 425
    cac aga ggg cgc cct cat ttc tga tgcttgagtt ttgtggagag aaccagtgaa 1413
    His Arg Gly Arg Pro His Phe
    430
    tggagaagtg caataggatc caacgcagca ccgtcttgct gtgcctttgc gtgacagctg 1473
    agcggtggaa gcagggcgtc ttatttatag aactgaacgt cagcgggctc agcagaaagg 1533
    aatagaagct ccggagtgaa ctcaaacagt gaacttccca gaaagaatgt tgtttcaagg 1593
    tgactgaaac agtttccacg tggaaataaa tgtgaaaagg actgcttaga gtacacgtgg 1653
    gccaggtggt cacacctgcg atgcctcgtc actagcaaac tcaggcctga tagtcctaca 1713
    gtattcacct agacaatact tgcctgtgcg tgcccagctc gcccagttat gaaatcagcg 1773
    ggatgtgctg attttaaaac tacttctttt tatcctttaa agaacgtgca tttcaaatta 1833
    taatttaaag gacttgaaag tgaaattact taggaaataa atagaaaata tgttaacagt 1893
    taaacgaaaa aaaaaaaaaa aaaaa 1918
    <210> SEQ ID NO 6
    <211> LENGTH: 433
    <212> TYPE: PRT
    <213> ORGANISM: Mus musculus
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 130
    <223> OTHER INFORMATION: Xaa is Tyr or His or Gln or Asn or Lys or Asp
    or Glu
    <400> SEQUENCE: 6
    Met Gly Gly Leu Arg Leu Leu Ala Val Ala Leu Thr Cys Ser Cys Trp
    1 5 10 15
    Trp Pro Gln Gly Gly Gln Gly Lys Thr Leu Arg Gly Ser Phe Ser Ser
    20 25 30
    Ala Ala Ala Arg Asp Ala Gln Gly Gln Ser Ile Gly His Phe Glu Phe
    35 40 45
    His Arg Ile Asn Asn Val Ala Val Ala Val Gly Lys Glu Ala Lys Leu
    50 55 60
    Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys Leu Leu Glu Ser Ser Pro
    65 70 75 80
    Gly Tyr Ser Cys Ser Glu Arg Leu Ala Arg Ala Gln Leu Thr Val Thr
    85 90 95
    Val Thr Gln Thr Glu His Asn Leu Thr Val Ser Gln Leu Pro Ala Pro
    100 105 110
    Gln Thr Trp Arg Val Phe Tyr Ala Asp Lys Phe Thr Cys Arg Asp Asp
    115 120 125
    Ser Xaa Ser Pro Gln Gly Glu Glu Ile Pro Phe Glu Met Val Leu Leu
    130 135 140
    Asn Pro Asp Ala Glu Gly Asn Pro Leu Asp His Phe Ser Ala Arg Glu
    145 150 155 160
    Ser Gly Leu His Glu Phe Phe Phe Leu Leu Val Leu Val Tyr Phe Val
    165 170 175
    Thr Ala Cys Ile Tyr Ala Gln Ser Leu Trp Gln Ala Met Lys Lys Gly
    180 185 190
    Gly Pro Met His Thr Ile Leu Lys Val Leu Thr Thr Ala Leu Leu Leu
    195 200 205
    Gln Ala Ala Ser Ala Leu Ala Asn Tyr Ile His Leu Ser Arg Tyr Ser
    210 215 220
    Arg Asp Gly Leu Gly Val Pro Leu Ile Gly Ser Leu Ala Glu Val Phe
    225 230 235 240
    Asp Ile Ala Ser Gln Ile Gln Met Leu Tyr Leu Leu Leu Ser Leu Cys
    245 250 255
    Met Gly Trp Thr Ile Val Arg Met Lys Lys Ser Gln Ser Arg Pro Leu
    260 265 270
    Gln Trp Asp Ser Thr Pro Ala Ser Thr Gly Ile Ala Val Phe Ile Val
    275 280 285
    Ile Thr Gln Ser Ile Leu Leu Leu Trp Glu Gln Phe Glu Asp Thr Ser
    290 295 300
    His His Ser Ala His Ser His Arg Ser Leu Ala Gly Leu Leu Leu Ile
    305 310 315 320
    Val Leu Arg Ile Cys Leu Ala Leu Ser Leu Gly Cys Gly Leu Tyr Gln
    325 330 335
    Val Ile Thr Val Glu Arg Ser Ala Leu Lys Arg Glu Phe Tyr Ile Thr
    340 345 350
    Phe Ala Lys Gly Cys Ile Leu Trp Phe Leu Cys Gln Pro Ala Leu Ala
    355 360 365
    Cys Ile Ala Val Ala Phe Asn Asp Tyr Gln Arg Asp Lys Leu Ile Thr
    370 375 380
    Val Gly Val Ile Leu Cys Gln Ala Val Ala Met Val Ile Leu Tyr Arg
    385 390 395 400
    Leu Phe Leu Ser His Ser Leu Tyr Trp Glu Val Ser Ser Leu Ser Ser
    405 410 415
    Val Thr Leu Pro Leu Thr Ile Ser Ser Ala His Arg Gly Arg Pro His
    420 425 430
    Phe
    <210> SEQ ID NO 7
    <211> LENGTH: 2857
    <212> TYPE: DNA
    <213> ORGANISM: Oryctolagus cuniculus
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (415)..(2199)
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (745)..(747)
    <223> OTHER INFORMATION: Xaa is Met or Leu
    <400> SEQUENCE: 7
    gtgttactgt gtttcactaa atgtttgaag gctgtcggac tttttgaatc atatgatctc 60
    ctgaaagtag ttcacattgt tcagttcgtt tttatattaa aacttgggac tgcatttttt 120
    atggttttgt ttcaaaagcc attttcttct gggaaaacta ttaccaaaca ccagtggatc 180
    acaatattta aacatgcagt tgccgggtgt atcatttcac tcttgtggtt ttttggcctt 240
    accctttgtg gaccactaag gactttgctg ctgtttgaac acagtgaaat tgttgtcatc 300
    tcgctcctca gtgttttgtt caccagttct ggaggaggac cagcaaagac aagaggggct 360
    gcttttttca tcattgctgt gatctgttta ttgctttttg acaatgatga tctc atg 417
    Met
    1
    gct aaa atg gca gaa cac cct gaa gga cat cat gac agt gct cta act 465
    Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser Ala Leu Thr
    5 10 15
    cac atg ctt tac aca gcc att gcc ttc tta ggt gtg gca gat cac aag 513
    His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly Val Ala Asp His Lys
    20 25 30
    ggt gga gta ttg ttg cta gta ctg gct ttg tgt tgt aaa gtt ggt ttt 561
    Gly Gly Val Leu Leu Leu Val Leu Ala Leu Cys Cys Lys Val Gly Phe
    35 40 45
    cac aca gct tcc aga aaa ctc tct ata gat gtt ggg gga gcc aaa cgt 609
    His Thr Ala Ser Arg Lys Leu Ser Ile Asp Val Gly Gly Ala Lys Arg
    50 55 60 65
    ctt caa gct tta tcc cat ctt gtt tct gtg ctt ctc ttg tgc cca tgg 657
    Leu Gln Ala Leu Ser His Leu Val Ser Val Leu Leu Leu Cys Pro Trp
    70 75 80
    gtc att gtt ctt tct atg aca act gag agt aaa gtt gag tct tgg ttt 705
    Val Ile Val Leu Ser Met Thr Thr Glu Ser Lys Val Glu Ser Trp Phe
    85 90 95
    tct ctc att atg cct ttc acg atg gtt att ttt ttt gtc wtg atc ctg 753
    Ser Leu Ile Met Pro Phe Thr Met Val Ile Phe Phe Val Xaa Ile Leu
    100 105 110
    gat ttc tac gtg gat tcc att tgt tca gtc aaa atg gaa gtt tcc aaa 801
    Asp Phe Tyr Val Asp Ser Ile Cys Ser Val Lys Met Glu Val Ser Lys
    115 120 125
    tgt gcc cgc tat gga tcc ttg ccc att ttt att agt gct ctc ctt ttt 849
    Cys Ala Arg Tyr Gly Ser Leu Pro Ile Phe Ile Ser Ala Leu Leu Phe
    130 135 140 145
    gga aat ttc tgg acc cac ccc ata act gac cag ctt cgg gca atg agc 897
    Gly Asn Phe Trp Thr His Pro Ile Thr Asp Gln Leu Arg Ala Met Ser
    150 155 160
    aga gca gca cac cag ggg agc acg gaa cac gtt ctg tct gga gga gtg 945
    Arg Ala Ala His Gln Gly Ser Thr Glu His Val Leu Ser Gly Gly Val
    165 170 175
    gtc gtg agc gca gtg ttc ttc atc ttg tct gcc aac atc ctg tca tct 993
    Val Val Ser Ala Val Phe Phe Ile Leu Ser Ala Asn Ile Leu Ser Ser
    180 185 190
    cct tcg aag agg ggg cag aag ggc acc ctg att gga tac tct cct gaa 1041
    Pro Ser Lys Arg Gly Gln Lys Gly Thr Leu Ile Gly Tyr Ser Pro Glu
    195 200 205
    gga gca cct ctt tac aac ttc atg ggg gat gct ttt cag cac agc tca 1089
    Gly Ala Pro Leu Tyr Asn Phe Met Gly Asp Ala Phe Gln His Ser Ser
    210 215 220 225
    cag tcc gtg cct cgg ttt att aag gaa tcg ctg aaa cag att ctt gag 1137
    Gln Ser Val Pro Arg Phe Ile Lys Glu Ser Leu Lys Gln Ile Leu Glu
    230 235 240
    gag agt gac tct agg cag atc ttt tac ttc ttg tgc ttg aat ctg ctt 1185
    Glu Ser Asp Ser Arg Gln Ile Phe Tyr Phe Leu Cys Leu Asn Leu Leu
    245 250 255
    ttt acc ttt gtg gaa tta ttc tat gga gtg ctg acg aat agt ctg ggt 1233
    Phe Thr Phe Val Glu Leu Phe Tyr Gly Val Leu Thr Asn Ser Leu Gly
    260 265 270
    ctg atc tca gat ggc ttt cac atg ctc ttt gac tgc tct gcc ttg gtc 1281
    Leu Ile Ser Asp Gly Phe His Met Leu Phe Asp Cys Ser Ala Leu Val
    275 280 285
    atg gga ctt ttt gct gcc ctg atg agt aga tgg aaa gca act cgg att 1329
    Met Gly Leu Phe Ala Ala Leu Met Ser Arg Trp Lys Ala Thr Arg Ile
    290 295 300 305
    ttc tcc tac ggg tat ggc cga ata gaa att ctt tct gga ttt att aat 1377
    Phe Ser Tyr Gly Tyr Gly Arg Ile Glu Ile Leu Ser Gly Phe Ile Asn
    310 315 320
    gga ctt ttt cta ata gta ata gct ttt ttt gtg ttt atg gag tca gtt 1425
    Gly Leu Phe Leu Ile Val Ile Ala Phe Phe Val Phe Met Glu Ser Val
    325 330 335
    gcc aga ttg att gat cct ccg gaa tta gac aca aac atg cta aca cca 1473
    Ala Arg Leu Ile Asp Pro Pro Glu Leu Asp Thr Asn Met Leu Thr Pro
    340 345 350
    gtg tca gtt gga ggg ctg ata gta aac ctt att ggt atc tgt gcc ttt 1521
    Val Ser Val Gly Gly Leu Ile Val Asn Leu Ile Gly Ile Cys Ala Phe
    355 360 365
    agc cac gcc cat aat cac acc cat gga tct tcc caa gga agc tgt cac 1569
    Ser His Ala His Asn His Thr His Gly Ser Ser Gln Gly Ser Cys His
    370 375 380 385
    tca tcc gat cac agc cat tca cac cac atg cat gga cac agt gac cat 1617
    Ser Ser Asp His Ser His Ser His His Met His Gly His Ser Asp His
    390 395 400
    gga cat ggt cac agc cat gga tcc cca ggc ggc ggc atg aat gct aac 1665
    Gly His Gly His Ser His Gly Ser Pro Gly Gly Gly Met Asn Ala Asn
    405 410 415
    atg agg ggt gtg ttt ttc cat gtt ttg gca gac acg ctt ggc agt att 1713
    Met Arg Gly Val Phe Phe His Val Leu Ala Asp Thr Leu Gly Ser Ile
    420 425 430
    ggt gtg att gta ttt aca gtt ttt ata gag cag ttt ggg tgg ttc att 1761
    Gly Val Ile Val Phe Thr Val Phe Ile Glu Gln Phe Gly Trp Phe Ile
    435 440 445
    gcg gat ccc ctc tgt tct ctc ttt att gct gta tta ata ttt ctc agt 1809
    Ala Asp Pro Leu Cys Ser Leu Phe Ile Ala Val Leu Ile Phe Leu Ser
    450 455 460 465
    gtt gtc cca ctg atc aaa gat gcc tgt cag gtt cta ctt ttg aga ctg 1857
    Val Val Pro Leu Ile Lys Asp Ala Cys Gln Val Leu Leu Leu Arg Leu
    470 475 480
    cca cca gag tat gaa aaa gaa cta cat att gct tta gaa aag ata caa 1905
    Pro Pro Glu Tyr Glu Lys Glu Leu His Ile Ala Leu Glu Lys Ile Gln
    485 490 495
    aaa att gar gga tta ata tca tac cga gat cct cat ttc tgg cgc cat 1953
    Lys Ile Glu Gly Leu Ile Ser Tyr Arg Asp Pro His Phe Trp Arg His
    500 505 510
    tct gcc agt att gtg gca gga aca att cat ata caa gtg aca tct gat 2001
    Ser Ala Ser Ile Val Ala Gly Thr Ile His Ile Gln Val Thr Ser Asp
    515 520 525
    gtg cta gaa caa aga ata gta cag cag gtt aca gga ata ctt aaa gat 2049
    Val Leu Glu Gln Arg Ile Val Gln Gln Val Thr Gly Ile Leu Lys Asp
    530 535 540 545
    gca gga gta aac aat tta aca att caa gta gaa aaa gaa gca tac ttt 2097
    Ala Gly Val Asn Asn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr Phe
    550 555 560
    caa cat atg tct ggc cta agt act gga ttt cat gat gtt ctg gct atg 2145
    Gln His Met Ser Gly Leu Ser Thr Gly Phe His Asp Val Leu Ala Met
    565 570 575
    aca aaa caa atg gag tcc atg aaa tac tgc aag gat ggc act tac att 2193
    Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys Asp Gly Thr Tyr Ile
    580 585 590
    atg tga gagaactcac agattacccc cgatgtgagc agtgaagatt cagtgactca 2249
    Met
    595
    gtgttgtaac attgccagca ggacagaaac tgcgtgtaat tgtacagaga ttttaaagct 2309
    ccctattctt ggatcaagga ctctttccta aaggaaattt aaatattgat tgaaacattg 2369
    atcacacagt aaaatagtga tttgagttat gtattttaaa tgactcttac aatttgaaca 2429
    taatgtgtct catcatcttc agaaatggac acaatgatgg attctaatga agaccaaaag 2489
    tacttctgtg tttcctttct gtcagaaagc atctccattg taaatatgta tttacatgtt 2549
    tattacaaag atccaaatga aaaattttta gtccattttt tgcatagcct aaagataaaa 2609
    taggaataaa agttctatat ttatgaattt tctgtacata aaactggttt ctaattataa 2669
    ctgaagtcca ctgggtaaaa tctgtattgc caccttaaat gtaaactaaa ttatttgaga 2729
    gaaacttcaa ccactgatat gacataagca gtgagaacag ggagtctata acattacagt 2789
    tttggatgtt accaaaacca accactctgt aaaataaatt ttttactttt gtcaaaaaaa 2849
    aaaaaaaa 2857
    <210> SEQ ID NO 8
    <211> LENGTH: 594
    <212> TYPE: PRT
    <213> ORGANISM: Oryctolagus cuniculus
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 111
    <223> OTHER INFORMATION: Xaa is Met or Leu
    <400> SEQUENCE: 8
    Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser Ala Leu
    1 5 10 15
    Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly Val Ala Asp His
    20 25 30
    Lys Gly Gly Val Leu Leu Leu Val Leu Ala Leu Cys Cys Lys Val Gly
    35 40 45
    Phe His Thr Ala Ser Arg Lys Leu Ser Ile Asp Val Gly Gly Ala Lys
    50 55 60
    Arg Leu Gln Ala Leu Ser His Leu Val Ser Val Leu Leu Leu Cys Pro
    65 70 75 80
    Trp Val Ile Val Leu Ser Met Thr Thr Glu Ser Lys Val Glu Ser Trp
    85 90 95
    Phe Ser Leu Ile Met Pro Phe Thr Met Val Ile Phe Phe Val Xaa Ile
    100 105 110
    Leu Asp Phe Tyr Val Asp Ser Ile Cys Ser Val Lys Met Glu Val Ser
    115 120 125
    Lys Cys Ala Arg Tyr Gly Ser Leu Pro Ile Phe Ile Ser Ala Leu Leu
    130 135 140
    Phe Gly Asn Phe Trp Thr His Pro Ile Thr Asp Gln Leu Arg Ala Met
    145 150 155 160
    Ser Arg Ala Ala His Gln Gly Ser Thr Glu His Val Leu Ser Gly Gly
    165 170 175
    Val Val Val Ser Ala Val Phe Phe Ile Leu Ser Ala Asn Ile Leu Ser
    180 185 190
    Ser Pro Ser Lys Arg Gly Gln Lys Gly Thr Leu Ile Gly Tyr Ser Pro
    195 200 205
    Glu Gly Ala Pro Leu Tyr Asn Phe Met Gly Asp Ala Phe Gln His Ser
    210 215 220
    Ser Gln Ser Val Pro Arg Phe Ile Lys Glu Ser Leu Lys Gln Ile Leu
    225 230 235 240
    Glu Glu Ser Asp Ser Arg Gln Ile Phe Tyr Phe Leu Cys Leu Asn Leu
    245 250 255
    Leu Phe Thr Phe Val Glu Leu Phe Tyr Gly Val Leu Thr Asn Ser Leu
    260 265 270
    Gly Leu Ile Ser Asp Gly Phe His Met Leu Phe Asp Cys Ser Ala Leu
    275 280 285
    Val Met Gly Leu Phe Ala Ala Leu Met Ser Arg Trp Lys Ala Thr Arg
    290 295 300
    Ile Phe Ser Tyr Gly Tyr Gly Arg Ile Glu Ile Leu Ser Gly Phe Ile
    305 310 315 320
    Asn Gly Leu Phe Leu Ile Val Ile Ala Phe Phe Val Phe Met Glu Ser
    325 330 335
    Val Ala Arg Leu Ile Asp Pro Pro Glu Leu Asp Thr Asn Met Leu Thr
    340 345 350
    Pro Val Ser Val Gly Gly Leu Ile Val Asn Leu Ile Gly Ile Cys Ala
    355 360 365
    Phe Ser His Ala His Asn His Thr His Gly Ser Ser Gln Gly Ser Cys
    370 375 380
    His Ser Ser Asp His Ser His Ser His His Met His Gly His Ser Asp
    385 390 395 400
    His Gly His Gly His Ser His Gly Ser Pro Gly Gly Gly Met Asn Ala
    405 410 415
    Asn Met Arg Gly Val Phe Phe His Val Leu Ala Asp Thr Leu Gly Ser
    420 425 430
    Ile Gly Val Ile Val Phe Thr Val Phe Ile Glu Gln Phe Gly Trp Phe
    435 440 445
    Ile Ala Asp Pro Leu Cys Ser Leu Phe Ile Ala Val Leu Ile Phe Leu
    450 455 460
    Ser Val Val Pro Leu Ile Lys Asp Ala Cys Gln Val Leu Leu Leu Arg
    465 470 475 480
    Leu Pro Pro Glu Tyr Glu Lys Glu Leu His Ile Ala Leu Glu Lys Ile
    485 490 495
    Gln Lys Ile Glu Gly Leu Ile Ser Tyr Arg Asp Pro His Phe Trp Arg
    500 505 510
    His Ser Ala Ser Ile Val Ala Gly Thr Ile His Ile Gln Val Thr Ser
    515 520 525
    Asp Val Leu Glu Gln Arg Ile Val Gln Gln Val Thr Gly Ile Leu Lys
    530 535 540
    Asp Ala Gly Val Asn Asn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr
    545 550 555 560
    Phe Gln His Met Ser Gly Leu Ser Thr Gly Phe His Asp Val Leu Ala
    565 570 575
    Met Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys Asp Gly Thr Tyr
    580 585 590
    Ile Met
    <210> SEQ ID NO 9
    <211> LENGTH: 2519
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)..(1785)
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (289)..(291)
    <223> OTHER INFORMATION: Xaa is Phe or Leu
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1015)..(1017)
    <223> OTHER INFORMATION: Xaa is Lys or Arg
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1492)..(1494)
    <223> OTHER INFORMATION: n is a or g or c or t
    Xaa is Gln or Lys or Glu
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1540)..(1542), (1582)..(1584), (1693)..(1695)
    <223> OTHER INFORMATION: Xaa is Phe or Ser
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1609)..(1611)
    <223> OTHER INFORMATION: Xaa is Gln or Arg
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 2493, 2516
    <223> OTHER INFORMATION: n is a or g or c or t
    <400> SEQUENCE: 9
    atg gct aaa atg gct gaa cac cct gaa gga cat cat gac agt gct cta 48
    Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser Ala Leu
    1 5 10 15
    act cat atg ctt tac aca gcc att gcc ttc tta ggt gtg gca gat cac 96
    Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly Val Ala Asp His
    20 25 30
    aag ggt gga gta tta ttg cta gta ctg gct ttg tgt tgt aaa gtt ggt 144
    Lys Gly Gly Val Leu Leu Leu Val Leu Ala Leu Cys Cys Lys Val Gly
    35 40 45
    ttt cat aca gct tcc aga aag ctc tct gtc gac gtt ggt gga gct aaa 192
    Phe His Thr Ala Ser Arg Lys Leu Ser Val Asp Val Gly Gly Ala Lys
    50 55 60
    cgt ctt caa gct tta tct cat ctt gtt tct gtg ctt ctc ttg tgc cca 240
    Arg Leu Gln Ala Leu Ser His Leu Val Ser Val Leu Leu Leu Cys Pro
    65 70 75 80
    tgg gtc att gtt ctt tct gtg aca act gag agt aaa gtg gag tct tgg 288
    Trp Val Ile Val Leu Ser Val Thr Thr Glu Ser Lys Val Glu Ser Trp
    85 90 95
    ytt tct ctc att atg cct ttt gca acg gtt atc ttt ttt gtc atg atc 336
    Xaa Ser Leu Ile Met Pro Phe Ala Thr Val Ile Phe Phe Val Met Ile
    100 105 110
    ctg gat ttc tac gtg gat tcc att tgt tca gtc aaa atg gaa gtt tcc 384
    Leu Asp Phe Tyr Val Asp Ser Ile Cys Ser Val Lys Met Glu Val Ser
    115 120 125
    aaa tgt gct cgt tat gga tcc ttt ccc att ttt att agt gct ctc ctt 432
    Lys Cys Ala Arg Tyr Gly Ser Phe Pro Ile Phe Ile Ser Ala Leu Leu
    130 135 140
    ttt gga aat ttt tgg aca cat cca ata aca gac cag ctt cgg gct atg 480
    Phe Gly Asn Phe Trp Thr His Pro Ile Thr Asp Gln Leu Arg Ala Met
    145 150 155 160
    aac aaa gca gca cac cag gag agc act gaa cac gtc ctg tct gga gga 528
    Asn Lys Ala Ala His Gln Glu Ser Thr Glu His Val Leu Ser Gly Gly
    165 170 175
    gtg gta gtg agt gct ata ttc ttc att ttg tct gcc aat atc tta tca 576
    Val Val Val Ser Ala Ile Phe Phe Ile Leu Ser Ala Asn Ile Leu Ser
    180 185 190
    tct ccc tct aag aga gga caa aaa ggt acc ctt att gga tat tct cct 624
    Ser Pro Ser Lys Arg Gly Gln Lys Gly Thr Leu Ile Gly Tyr Ser Pro
    195 200 205
    gaa gga aca cct ctt tat aac ttc atg ggt gat gct ttt cag cat agc 672
    Glu Gly Thr Pro Leu Tyr Asn Phe Met Gly Asp Ala Phe Gln His Ser
    210 215 220
    tct caa tcg atc cct agg ttt att aag gaa tca cta aaa caa att ctt 720
    Ser Gln Ser Ile Pro Arg Phe Ile Lys Glu Ser Leu Lys Gln Ile Leu
    225 230 235 240
    gag gag agt gac tct agg cag atc ttt tac ttc ttg tgc ttg aat ctg 768
    Glu Glu Ser Asp Ser Arg Gln Ile Phe Tyr Phe Leu Cys Leu Asn Leu
    245 250 255
    ctt ttt acc ttt gtg gaa tta ttc tat ggc gtg ctg acc aat agt ctg 816
    Leu Phe Thr Phe Val Glu Leu Phe Tyr Gly Val Leu Thr Asn Ser Leu
    260 265 270
    ggc ctg atc tcg gat gga ttc cac atg ctt ttt gac tgc tct gct tta 864
    Gly Leu Ile Ser Asp Gly Phe His Met Leu Phe Asp Cys Ser Ala Leu
    275 280 285
    gtc atg gga ctt ttt gct gcc ctg atg agt agg tgg aaa gcc act cgg 912
    Val Met Gly Leu Phe Ala Ala Leu Met Ser Arg Trp Lys Ala Thr Arg
    290 295 300
    att ttc tcc tat ggg tac ggc cga ata gaa att ctg tct gga ttt att 960
    Ile Phe Ser Tyr Gly Tyr Gly Arg Ile Glu Ile Leu Ser Gly Phe Ile
    305 310 315 320
    aat gga ctt ttt cta ata gta ata gcg ttt ttt gtg ttt atg gag tca 1008
    Asn Gly Leu Phe Leu Ile Val Ile Ala Phe Phe Val Phe Met Glu Ser
    325 330 335
    gtg gct ara ttg att gat cct cca gaa tta gac act cac atg tta aca 1056
    Val Ala Xaa Leu Ile Asp Pro Pro Glu Leu Asp Thr His Met Leu Thr
    340 345 350
    cca gty tca gtt gga ggg ctg ata gta aac ctt att ggt atc tgt gcc 1104
    Pro Val Ser Val Gly Gly Leu Ile Val Asn Leu Ile Gly Ile Cys Ala
    355 360 365
    ttt agc cat gcc cat agc cat gcc cat gga gct tct caa gga agc tgt 1152
    Phe Ser His Ala His Ser His Ala His Gly Ala Ser Gln Gly Ser Cys
    370 375 380
    cac tca tct gat cac agc cat tca cay cat atg cat gga cac agt gac 1200
    His Ser Ser Asp His Ser His Ser His His Met His Gly His Ser Asp
    385 390 395 400
    cat ggg cat ggt cac agc cac gga tct gcg ggt gga ggc atg aat gct 1248
    His Gly His Gly His Ser His Gly Ser Ala Gly Gly Gly Met Asn Ala
    405 410 415
    aac atg agg ggt gta ttt yta cat gtt ttg gca gat acw ctt ggc agc 1296
    Asn Met Arg Gly Val Phe Leu His Val Leu Ala Asp Thr Leu Gly Ser
    420 425 430
    att ggt gtg att gta tcc aca gtt ttt ata gag cag ttt gga tgg ttc 1344
    Ile Gly Val Ile Val Ser Thr Val Phe Ile Glu Gln Phe Gly Trp Phe
    435 440 445
    atc gct gac cca ctc tgt tct ctt ttt att gct ata tta ata ttt ctc 1392
    Ile Ala Asp Pro Leu Cys Ser Leu Phe Ile Ala Ile Leu Ile Phe Leu
    450 455 460
    agt gtt gtt cca ctg att aaa gat gcc tgc cag gtt tta ctc ctg aga 1440
    Ser Val Val Pro Leu Ile Lys Asp Ala Cys Gln Val Leu Leu Leu Arg
    465 470 475 480
    ttg cca cca gaa tat gga aaa gaa cta cat att gct tta gaa aag ata 1488
    Leu Pro Pro Glu Tyr Gly Lys Glu Leu His Ile Ala Leu Glu Lys Ile
    485 490 495
    cag naa att gaa gga tta ata tca tac cga gac cct cat ttt tgg cgt 1536
    Gln Xaa Ile Glu Gly Leu Ile Ser Tyr Arg Asp Pro His Phe Trp Arg
    500 505 510
    cat tyt gct agt att gtg gca gga aca att cat ata cag gtg aca tyt 1584
    His Xaa Ala Ser Ile Val Ala Gly Thr Ile His Ile Gln Val Thr Xaa
    515 520 525
    gat gtg cta gaa caa aga ata gta crg cag gtt aca gga ata ctt aaa 1632
    Asp Val Leu Glu Gln Arg Ile Val Xaa Gln Val Thr Gly Ile Leu Lys
    530 535 540
    gat gct gga gta aac aat tta aca att caa gtg gaa aag gag gca tac 1680
    Asp Ala Gly Val Asn Asn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr
    545 550 555 560
    ttt caa cat atg tyt ggc cta agt act gga ttt cat gat gtt ctg gct 1728
    Phe Gln His Met Xaa Gly Leu Ser Thr Gly Phe His Asp Val Leu Ala
    565 570 575
    atg aca aaa caa atg gaa tcc atg aaa tac tgc aaa gat ggt act tac 1776
    Met Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys Asp Gly Thr Tyr
    580 585 590
    atc atg tga gataactcaa gaattacccc tggagaataa acaatgaaga 1825
    Ile Met
    595
    ttaaatgact cagtatttgt aatattgcca gaaggataaa aattacacat taactgtaca 1885
    gaaacagagt tccctactac tggatcaagg aatctttctt gaaggaaatt taaatacaga 1945
    atgaaacatt aatggtaaaa gtggagtaat tatttaaatt atgtgtataa aaggaatcaa 2005
    attttgagta aacatgatgt attacatcat cttcaaaaat agatatgatg gattctagtg 2065
    aagaccaaaa ttacttctgt ttactttcta tcaggaagca tctccattgt aaatatgtat 2125
    ttacatgttt attacaaaga cccaaatgaa aaatttttag tccatttttt gcatagccta 2185
    aagataaaat aggaataaaa gttctatatt tatgggattt tctgtatata aaactggttt 2245
    ctaattataa cttaagtcca ttaagtaaaa tctgtattgc cactttaaat gtaaactaaa 2305
    ttatttggga gaaacttcaa ccactgatat gagataagca atgagaatag ggaagtgtat 2365
    aacatcacag tttttgatgt attacaaaaa tcaaccactt tataaaataa atttttttta 2425
    cttttggtaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaag cggccgctga 2485
    attctagnta gaattcagcg gccgctgaat ncta 2519
    <210> SEQ ID NO 10
    <211> LENGTH: 594
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 97
    <223> OTHER INFORMATION: Xaa is Phe or Leu
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 339
    <223> OTHER INFORMATION: Xaa is Lys or Arg
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 498
    <223> OTHER INFORMATION: Xaa is Gln or Lys or Glu
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 514, 528, 565
    <223> OTHER INFORMATION: Xaa is Phe or Ser
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 537
    <223> OTHER INFORMATION: Xaa is Gln or Arg
    <400> SEQUENCE: 10
    Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser Ala Leu
    1 5 10 15
    Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly Val Ala Asp His
    20 25 30
    Lys Gly Gly Val Leu Leu Leu Val Leu Ala Leu Cys Cys Lys Val Gly
    35 40 45
    Phe His Thr Ala Ser Arg Lys Leu Ser Val Asp Val Gly Gly Ala Lys
    50 55 60
    Arg Leu Gln Ala Leu Ser His Leu Val Ser Val Leu Leu Leu Cys Pro
    65 70 75 80
    Trp Val Ile Val Leu Ser Val Thr Thr Glu Ser Lys Val Glu Ser Trp
    85 90 95
    Xaa Ser Leu Ile Met Pro Phe Ala Thr Val Ile Phe Phe Val Met Ile
    100 105 110
    Leu Asp Phe Tyr Val Asp Ser Ile Cys Ser Val Lys Met Glu Val Ser
    115 120 125
    Lys Cys Ala Arg Tyr Gly Ser Phe Pro Ile Phe Ile Ser Ala Leu Leu
    130 135 140
    Phe Gly Asn Phe Trp Thr His Pro Ile Thr Asp Gln Leu Arg Ala Met
    145 150 155 160
    Asn Lys Ala Ala His Gln Glu Ser Thr Glu His Val Leu Ser Gly Gly
    165 170 175
    Val Val Val Ser Ala Ile Phe Phe Ile Leu Ser Ala Asn Ile Leu Ser
    180 185 190
    Ser Pro Ser Lys Arg Gly Gln Lys Gly Thr Leu Ile Gly Tyr Ser Pro
    195 200 205
    Glu Gly Thr Pro Leu Tyr Asn Phe Met Gly Asp Ala Phe Gln His Ser
    210 215 220
    Ser Gln Ser Ile Pro Arg Phe Ile Lys Glu Ser Leu Lys Gln Ile Leu
    225 230 235 240
    Glu Glu Ser Asp Ser Arg Gln Ile Phe Tyr Phe Leu Cys Leu Asn Leu
    245 250 255
    Leu Phe Thr Phe Val Glu Leu Phe Tyr Gly Val Leu Thr Asn Ser Leu
    260 265 270
    Gly Leu Ile Ser Asp Gly Phe His Met Leu Phe Asp Cys Ser Ala Leu
    275 280 285
    Val Met Gly Leu Phe Ala Ala Leu Met Ser Arg Trp Lys Ala Thr Arg
    290 295 300
    Ile Phe Ser Tyr Gly Tyr Gly Arg Ile Glu Ile Leu Ser Gly Phe Ile
    305 310 315 320
    Asn Gly Leu Phe Leu Ile Val Ile Ala Phe Phe Val Phe Met Glu Ser
    325 330 335
    Val Ala Xaa Leu Ile Asp Pro Pro Glu Leu Asp Thr His Met Leu Thr
    340 345 350
    Pro Val Ser Val Gly Gly Leu Ile Val Asn Leu Ile Gly Ile Cys Ala
    355 360 365
    Phe Ser His Ala His Ser His Ala His Gly Ala Ser Gln Gly Ser Cys
    370 375 380
    His Ser Ser Asp His Ser His Ser His His Met His Gly His Ser Asp
    385 390 395 400
    His Gly His Gly His Ser His Gly Ser Ala Gly Gly Gly Met Asn Ala
    405 410 415
    Asn Met Arg Gly Val Phe Leu His Val Leu Ala Asp Thr Leu Gly Ser
    420 425 430
    Ile Gly Val Ile Val Ser Thr Val Phe Ile Glu Gln Phe Gly Trp Phe
    435 440 445
    Ile Ala Asp Pro Leu Cys Ser Leu Phe Ile Ala Ile Leu Ile Phe Leu
    450 455 460
    Ser Val Val Pro Leu Ile Lys Asp Ala Cys Gln Val Leu Leu Leu Arg
    465 470 475 480
    Leu Pro Pro Glu Tyr Gly Lys Glu Leu His Ile Ala Leu Glu Lys Ile
    485 490 495
    Gln Xaa Ile Glu Gly Leu Ile Ser Tyr Arg Asp Pro His Phe Trp Arg
    500 505 510
    His Xaa Ala Ser Ile Val Ala Gly Thr Ile His Ile Gln Val Thr Xaa
    515 520 525
    Asp Val Leu Glu Gln Arg Ile Val Xaa Gln Val Thr Gly Ile Leu Lys
    530 535 540
    Asp Ala Gly Val Asn Asn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr
    545 550 555 560
    Phe Gln His Met Xaa Gly Leu Ser Thr Gly Phe His Asp Val Leu Ala
    565 570 575
    Met Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys Asp Gly Thr Tyr
    580 585 590
    Ile Met
    <210> SEQ ID NO 11
    <211> LENGTH: 178
    <212> TYPE: DNA
    <213> ORGANISM: Oryctolagus cuniculus
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: 54, 68, 97
    <223> OTHER INFORMATION: n is a or g or c or t
    <400> SEQUENCE: 11
    gttttttttt tttttcatac atttggtatg aaacatcgaa tagcaaaagc agancatgtt 60
    tctgtatnac tgcatttaag cagtaccaaa actgaanaaa ggtaataact gaaatgtttt 120
    aaaatacatg taaacaataa actttcagga aattctgttg ttaaaaaaaa aaaaaaac 178
    <210> SEQ ID NO 12
    <211> LENGTH: 167
    <212> TYPE: DNA
    <213> ORGANISM: Oryctolagus cuniculus
    <400> SEQUENCE: 12
    cttgattgcc accttaaatg taaactaaat tatttgagag aaacttcaac cactgatatg 60
    acataagcag tgagaacagg gagtctataa cattacagtt ttggatgtta ccaaaaccaa 120
    ccactctgta aaataaattt tttacttttg taaaaaaaaa aaaaaac 167
    <210> SEQ ID NO 13
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(24)
    <400> SEQUENCE: 13
    tcatacattt ggtatgaaac atcg 24
    <210> SEQ ID NO 14
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(24)
    <400> SEQUENCE: 14
    tttttttaac aacagaattt cctg 24
    <210> SEQ ID NO 15
    <211> LENGTH: 27
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(27)
    <400> SEQUENCE: 15
    aatttcctga aagtttattg tttacat 27
    <210> SEQ ID NO 16
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(25)
    <400> SEQUENCE: 16
    gaaacatgct ctgcttttgc tattc 25
    <210> SEQ ID NO 17
    <211> LENGTH: 27
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(27)
    <400> SEQUENCE: 17
    atccctgcta agttgtggtg tgaatgg 27
    <210> SEQ ID NO 18
    <211> LENGTH: 27
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(27)
    <400> SEQUENCE: 18
    tctgctttga gacttcttca tcctgac 27
    <210> SEQ ID NO 19
    <211> LENGTH: 27
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(27)
    <400> SEQUENCE: 19
    ccatcctaat acgactcact atagggc 27
    <210> SEQ ID NO 20
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(23)
    <400> SEQUENCE: 20
    actcactata gggctcgagc ggc 23
    <210> SEQ ID NO 21
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(22)
    <400> SEQUENCE: 21
    ttgattgcca ccttaaatgt aa 22
    <210> SEQ ID NO 22
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(22)
    <400> SEQUENCE: 22
    gtggttggtt ttggtaacat cc 22
    <210> SEQ ID NO 23
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(25)
    <400> SEQUENCE: 23
    gttatagact ccctgttctc actgc 25
    <210> SEQ ID NO 24
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(25)
    <400> SEQUENCE: 24
    agactccctg ttctcactgc ttatg 25
    <210> SEQ ID NO 25
    <211> LENGTH: 27
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(27)
    <400> SEQUENCE: 25
    atcttcactg ctcacatcgg gggtaat 27
    <210> SEQ ID NO 26
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(23)
    <400> SEQUENCE: 26
    tcactgctca catcgggggt aat 23
    <210> SEQ ID NO 27
    <211> LENGTH: 1703
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <220> FEATURE:
    <221> NAME/KEY: CDS
    LOCATION: (10)..(1701)
    <400> SEQUENCE: 27
    gatgatctc atg gca aag atg gct gaa cac ccg gaa gga cat cat gat agt 51
    Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser
    1 5 10
    gct cta act cac atg ctc tat aca gcc att gcc ttt tta ggg gtg gca 99
    Ala Leu Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly Val Ala
    15 20 25 30
    gat cac aag ggt gga gta ctc ttg ctg gtg ctg gct tta tgt tgt aaa 147
    Asp His Lys Gly Gly Val Leu Leu Leu Val Leu Ala Leu Cys Cys Lys
    35 40 45
    gtt ggt ttt cat acg gct tcc aga aag ctc tct ata gat gtt ggt gga 195
    Val Gly Phe His Thr Ala Ser Arg Lys Leu Ser Ile Asp Val Gly Gly
    50 55 60
    gct aag cgc ctt cag gcc tta tct cag ctt gtt tct gtg ttt ctc ctg 243
    Ala Lys Arg Leu Gln Ala Leu Ser Gln Leu Val Ser Val Phe Leu Leu
    65 70 75
    tgc cca tgg gtg att gtc ctt tct gtg aca act gaa agt aag gtt gag 291
    Cys Pro Trp Val Ile Val Leu Ser Val Thr Thr Glu Ser Lys Val Glu
    80 85 90
    tct tgg ttc tct ctc atc atg cct ttc acc aca gtc atc ttt ttt gtc 339
    Ser Trp Phe Ser Leu Ile Met Pro Phe Thr Thr Val Ile Phe Phe Val
    95 100 105 110
    atg atc ctg gat ttc tat atg gat tct gtt tgt tca gtc aaa atg gac 387
    Met Ile Leu Asp Phe Tyr Met Asp Ser Val Cys Ser Val Lys Met Asp
    115 120 125
    gtg tcc aaa tgt gcc cgc tat ggg tcc ttt ccc att ttt att agt gcc 435
    Val Ser Lys Cys Ala Arg Tyr Gly Ser Phe Pro Ile Phe Ile Ser Ala
    130 135 140
    ctc ctg ttt cga aat ttc tgg aca cac cca ata aca gac caa ctc cgg 483
    Leu Leu Phe Arg Asn Phe Trp Thr His Pro Ile Thr Asp Gln Leu Arg
    145 150 155
    gct atg aac aga gca gca cac cag gag agc aca gaa cac gtc ctg tct 531
    Ala Met Asn Arg Ala Ala His Gln Glu Ser Thr Glu His Val Leu Ser
    160 165 170
    gga gga gtg gta gtg agc gct gtg ttc ttc att ttg tcg gcc aac att 579
    Gly Gly Val Val Val Ser Ala Val Phe Phe Ile Leu Ser Ala Asn Ile
    175 180 185 190
    cta tca tct ccc tct aag aga gga cag aaa ggt acc ctt att gga tat 627
    Leu Ser Ser Pro Ser Lys Arg Gly Gln Lys Gly Thr Leu Ile Gly Tyr
    195 200 205
    tct cct gaa gga aca cca ctc tat cac ttc atg ggg gac gct ttt cag 675
    Ser Pro Glu Gly Thr Pro Leu Tyr His Phe Met Gly Asp Ala Phe Gln
    210 215 220
    cac agc tct cag tcg gtg cct agg ttc att aag gac tca cta aag cag 723
    His Ser Ser Gln Ser Val Pro Arg Phe Ile Lys Asp Ser Leu Lys Gln
    225 230 235
    gtt ctc gag gag agc gac tct agg cag atc ttt tac ttc ttg tgc ttg 771
    Val Leu Glu Glu Ser Asp Ser Arg Gln Ile Phe Tyr Phe Leu Cys Leu
    240 245 250
    aat ctg ctt ttt acc ttt gtg gag ttg ttc tat ggc gtg cta acc aac 819
    Asn Leu Leu Phe Thr Phe Val Glu Leu Phe Tyr Gly Val Leu Thr Asn
    255 260 265 270
    agt cta ggc ctg atc tca gat gga ttt cac atg ctc ttt gac tgc tcg 867
    Ser Leu Gly Leu Ile Ser Asp Gly Phe His Met Leu Phe Asp Cys Ser
    275 280 285
    gct ttg gtc atg gga ctg ttt gct gcc ctg atg agc cgc tgg aaa gcc 915
    Ala Leu Val Met Gly Leu Phe Ala Ala Leu Met Ser Arg Trp Lys Ala
    290 295 300
    acc cgg att ttc tcc tat ggg tat ggc cga ata gag att ctc tct ggc 963
    Thr Arg Ile Phe Ser Tyr Gly Tyr Gly Arg Ile Glu Ile Leu Ser Gly
    305 310 315
    ttt att aat ggg ctt ttt ctg atc gtg ata gca ttt ttt gtg ttt atg 1011
    Phe Ile Asn Gly Leu Phe Leu Ile Val Ile Ala Phe Phe Val Phe Met
    320 325 330
    gaa tca gtg gct aga ctg atc gat cct ccg gaa cta gac aca aac atg 1059
    Glu Ser Val Ala Arg Leu Ile Asp Pro Pro Glu Leu Asp Thr Asn Met
    335 340 345 350
    ctg aca cca gtt tcc gtc gca ggg ctg ata gta aac ctt att ggt atc 1107
    Leu Thr Pro Val Ser Val Ala Gly Leu Ile Val Asn Leu Ile Gly Ile
    355 360 365
    tgt gcc ttc agc cac gcc cac agc cat ggc cat ggc gct tct caa gga 1155
    Cys Ala Phe Ser His Ala His Ser His Gly His Gly Ala Ser Gln Gly
    370 375 380
    aac tgc cac tct gat cac ggc cat tca cac cat gca cat gga cac ggc 1203
    Asn Cys His Ser Asp His Gly His Ser His His Ala His Gly His Gly
    385 390 395
    cat gat cac ggt cac agc cac ggc ttc acg ggt gga ggc atg aat gcg 1251
    His Asp His Gly His Ser His Gly Phe Thr Gly Gly Gly Met Asn Ala
    400 405 410
    aac atg agg ggt gta ttt ctc cat gtg ttg gca gac aca ctg ggc agc 1299
    Asn Met Arg Gly Val Phe Leu His Val Leu Ala Asp Thr Leu Gly Ser
    415 420 425 430
    atc ggc gtg att gtg tcc aca gtt ctc ata gag cag ttt gga tgg ttc 1347
    Ile Gly Val Ile Val Ser Thr Val Leu Ile Glu Gln Phe Gly Trp Phe
    435 440 445
    att gct gat ccc ctg tgt tct ctt ttt att gcc gtg ttg ata ttt ctc 1395
    Ile Ala Asp Pro Leu Cys Ser Leu Phe Ile Ala Val Leu Ile Phe Leu
    450 455 460
    agt gtg atc cca ctg att aaa gat gcc tgt caa gtt cta ctt ctg aga 1443
    Ser Val Ile Pro Leu Ile Lys Asp Ala Cys Gln Val Leu Leu Leu Arg
    465 470 475
    cta cca cct gac cat gaa aaa gaa ctg cat att gct tta gaa aag ata 1491
    Leu Pro Pro Asp His Glu Lys Glu Leu His Ile Ala Leu Glu Lys Ile
    480 485 490
    cag aaa att gag gga tta ata tca tac cga gac cct cat ttt tgg cgc 1539
    Gln Lys Ile Glu Gly Leu Ile Ser Tyr Arg Asp Pro His Phe Trp Arg
    495 500 505 510
    cat tct gcc agt att gta gcg gga aca att cat ata caa gtg aca tct 1587
    His Ser Ala Ser Ile Val Ala Gly Thr Ile His Ile Gln Val Thr Ser
    515 520 525
    gag gtg ctg gag cag aga att gta cag cag gtt aca ggg ata ctt aaa 1635
    Glu Val Leu Glu Gln Arg Ile Val Gln Gln Val Thr Gly Ile Leu Lys
    530 535 540
    gat gca gga gta aac aac cta acc ata caa gtg gaa aag gag gca tac 1683
    Asp Ala Gly Val Asn Asn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr
    545 550 555
    ttt cag cat atg tcc ggc ct 1703
    Phe Gln His Met Ser Gly
    560
    <210> SEQ ID NO 28
    <211> LENGTH: 564
    <212> TYPE: PRT
    <213> ORGANISM: Mus musculus
    <400> SEQUENCE: 28
    Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser Ala Leu
    1 5 10 15
    Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly Val Ala Asp His
    20 25 30
    Lys Gly Gly Val Leu Leu Leu Val Leu Ala Leu Cys Cys Lys Val Gly
    35 40 45
    Phe His Thr Ala Ser Arg Lys Leu Ser Ile Asp Val Gly Gly Ala Lys
    50 55 60
    Arg Leu Gln Ala Leu Ser Gln Leu Val Ser Val Phe Leu Leu Cys Pro
    65 70 75 80
    Trp Val Ile Val Leu Ser Val Thr Thr Glu Ser Lys Val Glu Ser Trp
    85 90 95
    Phe Ser Leu Ile Met Pro Phe Thr Thr Val Ile Phe Phe Val Met Ile
    100 105 110
    Leu Asp Phe Tyr Met Asp Ser Val Cys Ser Val Lys Met Asp Val Ser
    115 120 125
    Lys Cys Ala Arg Tyr Gly Ser Phe Pro Ile Phe Ile Ser Ala Leu Leu
    130 135 140
    Phe Arg Asn Phe Trp Thr His Pro Ile Thr Asp Gln Leu Arg Ala Met
    145 150 155 160
    Asn Arg Ala Ala His Gln Glu Ser Thr Glu His Val Leu Ser Gly Gly
    165 170 175
    Val Val Val Ser Ala Val Phe Phe Ile Leu Ser Ala Asn Ile Leu Ser
    180 185 190
    Ser Pro Ser Lys Arg Gly Gln Lys Gly Thr Leu Ile Gly Tyr Ser Pro
    195 200 205
    Glu Gly Thr Pro Leu Tyr His Phe Met Gly Asp Ala Phe Gln His Ser
    210 215 220
    Ser Gln Ser Val Pro Arg Phe Ile Lys Asp Ser Leu Lys Gln Val Leu
    225 230 235 240
    Glu Glu Ser Asp Ser Arg Gln Ile Phe Tyr Phe Leu Cys Leu Asn Leu
    245 250 255
    Leu Phe Thr Phe Val Glu Leu Phe Tyr Gly Val Leu Thr Asn Ser Leu
    260 265 270
    Gly Leu Ile Ser Asp Gly Phe His Met Leu Phe Asp Cys Ser Ala Leu
    275 280 285
    Val Met Gly Leu Phe Ala Ala Leu Met Ser Arg Trp Lys Ala Thr Arg
    290 295 300
    Ile Phe Ser Tyr Gly Tyr Gly Arg Ile Glu Ile Leu Ser Gly Phe Ile
    305 310 315 320
    Asn Gly Leu Phe Leu Ile Val Ile Ala Phe Phe Val Phe Met Glu Ser
    325 330 335
    Val Ala Arg Leu Ile Asp Pro Pro Glu Leu Asp Thr Asn Met Leu Thr
    340 345 350
    Pro Val Ser Val Ala Gly Leu Ile Val Asn Leu Ile Gly Ile Cys Ala
    355 360 365
    Phe Ser His Ala His Ser His Gly His Gly Ala Ser Gln Gly Asn Cys
    370 375 380
    His Ser Asp His Gly His Ser His His Ala His Gly His Gly His Asp
    385 390 395 400
    His Gly His Ser His Gly Phe Thr Gly Gly Gly Met Asn Ala Asn Met
    405 410 415
    Arg Gly Val Phe Leu His Val Leu Ala Asp Thr Leu Gly Ser Ile Gly
    420 425 430
    Val Ile Val Ser Thr Val Leu Ile Glu Gln Phe Gly Trp Phe Ile Ala
    435 440 445
    Asp Pro Leu Cys Ser Leu Phe Ile Ala Val Leu Ile Phe Leu Ser Val
    450 455 460
    Ile Pro Leu Ile Lys Asp Ala Cys Gln Val Leu Leu Leu Arg Leu Pro
    465 470 475 480
    Pro Asp His Glu Lys Glu Leu His Ile Ala Leu Glu Lys Ile Gln Lys
    485 490 495
    Ile Glu Gly Leu Ile Ser Tyr Arg Asp Pro His Phe Trp Arg His Ser
    500 505 510
    Ala Ser Ile Val Ala Gly Thr Ile His Ile Gln Val Thr Ser Glu Val
    515 520 525
    Leu Glu Gln Arg Ile Val Gln Gln Val Thr Gly Ile Leu Lys Asp Ala
    530 535 540
    Gly Val Asn Asn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr Phe Gln
    545 550 555 560
    His Met Ser Gly
    <210> SEQ ID NO 29
    <211> LENGTH: 29
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(29)
    <400> SEQUENCE: 29
    gggaattcat gggtaagacc ctgcggggc 29
    <210> SEQ ID NO 30
    <211> LENGTH: 35
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(35)
    <400> SEQUENCE: 30
    ggaagctttc actcatgtaa cccagattct ccagc 35
    <210> SEQ ID NO 31
    <211> LENGTH: 35
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(35)
    <400> SEQUENCE: 31
    ggttttctag agctcgtgca gacctggaga cgggc 35
    <210> SEQ ID NO 32
    <211> LENGTH: 39
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(39)
    <400> SEQUENCE: 32
    gggttttcta gatcttcaga aatgagggcg acttttgtg 39
    <210> SEQ ID NO 33
    <211> LENGTH: 29
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(29)
    <400> SEQUENCE: 33
    gggatccgca tagtacagca ggttacagg 29
    <210> SEQ ID NO 34
    <211> LENGTH: 29
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(29)
    <400> SEQUENCE: 34
    ggtcgaccct ttaaaataca taactcaaa 29
    <210> SEQ ID NO 35
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(30)
    <400> SEQUENCE: 35
    aatgtatgca gtatggaaat ggaaagttgc 30
    <210> SEQ ID NO 36
    <211> LENGTH: 28
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(28)
    <400> SEQUENCE: 36
    cgaatgggct gaccgcttcc tcgtgctt 28
    <210> SEQ ID NO 37
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(30)
    <400> SEQUENCE: 37
    cggcaggagc aaggtgagat gacaggagat 30
    <210> SEQ ID NO 38
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(30)
    <400> SEQUENCE: 38
    ttacagtgtc aggaataaag gctatgcttc 30
    <210> SEQ ID NO 39
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(20)
    <400> SEQUENCE: 39
    gactcgatct catggcaaag 20
    <210> SEQ ID NO 40
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(21)
    <400> SEQUENCE: 40
    tagagcatgt gagttagagc a 21
    <210> SEQ ID NO 41
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(21)
    <400> SEQUENCE: 41
    ccaacattct atcatctccc t 21
    <210> SEQ ID NO 42
    <211> LENGTH: 19
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence:an
    artificially synthesized primer sequence
    <220> FEATURE:
    <221> NAME/KEY: primer_bind
    <222> LOCATION: (1)..(19)
    <400> SEQUENCE: 42
    agcggctcat cagggcagc 19

Claims (30)

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.
US10/139,262 1997-02-28 2002-05-07 Physiologically active protein originating in mammals Abandoned US20020128459A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/139,262 US20020128459A1 (en) 1997-02-28 2002-05-07 Physiologically active protein originating in mammals

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP9/62259 1997-02-28
JP6225997 1997-02-28
JP10062263A JPH10295388A (en) 1997-02-28 1998-02-25 Mammalian-derived, physiologically active protein
JP10/62263 1998-02-25
US09/380,287 US6710171B1 (en) 1997-02-28 1998-02-27 Physiologically active protein originating in mammals
US10/139,262 US20020128459A1 (en) 1997-02-28 2002-05-07 Physiologically active protein originating in mammals

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
PCT/JP1998/000835 Continuation WO1998038305A1 (en) 1997-02-28 1998-02-27 Physiologically active protein originating in mammals
US09/380,287 Continuation US6710171B1 (en) 1997-02-28 1998-02-27 Physiologically active protein originating in mammals

Publications (1)

Publication Number Publication Date
US20020128459A1 true US20020128459A1 (en) 2002-09-12

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Family Applications (3)

Application Number Title Priority Date Filing Date
US09/380,287 Expired - Fee Related US6710171B1 (en) 1997-02-28 1998-02-27 Physiologically active protein originating in mammals
US10/139,262 Abandoned US20020128459A1 (en) 1997-02-28 2002-05-07 Physiologically active protein originating in mammals
US10/255,969 Abandoned US20030083486A1 (en) 1997-02-28 2002-09-27 Physiologically active protein originating in mammals

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US09/380,287 Expired - Fee Related US6710171B1 (en) 1997-02-28 1998-02-27 Physiologically active protein originating in mammals

Family Applications After (1)

Application Number Title Priority Date Filing Date
US10/255,969 Abandoned US20030083486A1 (en) 1997-02-28 2002-09-27 Physiologically active protein originating in mammals

Country Status (6)

Country Link
US (3) US6710171B1 (en)
EP (1) EP0974651A4 (en)
JP (1) JPH10295388A (en)
AU (1) AU6118398A (en)
CA (1) CA2282557A1 (en)
WO (1) WO1998038305A1 (en)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020137890A1 (en) * 1997-03-31 2002-09-26 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP1661997A1 (en) * 1999-12-01 2006-05-31 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP1275717A4 (en) * 2000-03-29 2004-10-06 Kyowa Hakko Kogyo Kk Proliferative glomerular nephritis-associated gene
KR100986225B1 (en) * 2001-02-16 2010-10-07 아스비오파마 가부시키가이샤 Methods of treating diseases in association with decrease in the expression of aop-1 gene or aop-1 and remedies for the diseases
WO2005113604A2 (en) * 2004-05-14 2005-12-01 Hematech, Llc Methods for immunoglobulin purification
RU2453605C2 (en) * 2007-09-14 2012-06-20 Игорь Эдуардович Грановский Differentiating and specific oligonucleotides for identification of dna sequences of transgene plants in foodstuff, method for identifying transgene products, biochip, combination of oligonucleotides (versions) and kit for implementing such method

Also Published As

Publication number Publication date
WO1998038305A1 (en) 1998-09-03
US20030083486A1 (en) 2003-05-01
EP0974651A4 (en) 2003-05-07
EP0974651A1 (en) 2000-01-26
AU6118398A (en) 1998-09-18
CA2282557A1 (en) 1998-09-03
JPH10295388A (en) 1998-11-10
US6710171B1 (en) 2004-03-23

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