WO1996001843A1 - Polypeptide produced by stromal cell - Google Patents

Abstract

A polypeptide produced by mouse marrow stromal cells and composed of 211 amino acid residues, and a human homolog thereof; a process for producing the polypeptide; a DNA coding for the polypeptide; a fragment hybridizing selectively with the DNA sequence; a replication or expression vector comprising the DNA; a host cell transformed by the vector; an antibody against the polypeptide; and a pharmaceutical composition containing the polypeptide or the antibody. The polypeptide is usable for the prevention or treatment of the hypoplasia or abnormal growth of hematopoietic cells, the hyperergasia or hypergasia of the nervous system, diseases related to the hyperergasia or hypergasia of the immune system, such as inflammatory diseases, the decrease of hematopoietic cells after bone marrow transplantation, the decrease of leucocytes, platelets, B cells or T cells after radiation therapy, anemia and infectious diseases; the prevention or treatment of cancer, leukemia and AIDS; the prevention or treatment of various degenerative diseases and nerve injury; the prevention of treatment of metabolic disorder of the bone; and tissue repair.

Description

 Description Polypeptide produced by stromal cells

 The present invention relates to a novel polypeptide produced by a stromal cell, a method for producing the same, a DNA encoding the polypeptide, a vector comprising the DNA, a host cell transformed with the vector, The present invention relates to an antibody of a polypeptide, and a pharmaceutical composition containing the peptide or antibody. More specifically, a novel polypeptide produced by a certain mouse stromal cell line, a human homolog of the polypeptide, a method for producing the polypeptide, a DNA encoding the polypeptide, and a vector comprising the DNA The present invention relates to host cells transformed with the vectors, antibodies of the polypeptides, and pharmaceutical compositions containing the polypeptides or the antibodies. Background art

Bone marrow stromal cells form the bone marrow microenvironment such as the immune system and hematopoietic system, and are factors that are indispensable for inducing the proliferation and differentiation of their stem cells, such as IL-17, SCF, IL-11, M—CSF, G— It is known that it produces and secretes factors such as CSF, GM—CSF, IL-16, TGF— / 3, and LIF. It has also been shown that some bone marrow stromal cells are involved in bone metabolism. However, only the group of factors isolated so far cannot completely fulfill the role of stromal cells. This suggests that some factors have not yet been isolated. Disclosure of the invention

 The present inventors have paid attention to this point, and made intensive studies to find out a novel factor (polypeptide) produced by a certain type of stromal cells, particularly a secretory protein.

 Conventionally, when attempting to obtain a specific polypeptide or a DNA encoding the same, the desired biological activity is confirmed in a tissue or cell culture, and then the polypeptide is isolated and purified, followed by gene expression. Or a method of expressing and cloning a gene using its biological activity as an index has been generally used.

 However, biologically active polypeptides in vivo often have various biological activities, and as a result of cloning a gene using a certain activity as an index, it is the same as a known polypeptide. More and more cases are found later. In addition, most of the factors produced by bone marrow stromal cells produce only trace amounts, which makes isolation, purification and confirmation of biological activity difficult.

 In recent years, the production and sequence technologies of cDNA have been rapidly developed, and a large amount of cDNA can be sequenced quickly. Therefore, using these techniques, cDNA libraries are produced from various cells and tissues, the cDNA is randomly cloned, the nucleotide sequence is determined, and the corresponding polypeptide is expressed. The method of analyzing physiological functions is being developed. This method has the characteristic that the gene can be cloned without any biochemical or genetic analysis and information on its nucleotide sequence can be obtained. Has a large accidental factor.

The present inventors have studied the cloning of genes for growth and differentiation factors that work in the hematopoietic and immune systems. And growth differentiation factors (eg, various Most of secreted proteins such as cytokins and membrane proteins such as their receptors (hereinafter collectively referred to as secreted proteins, etc.) have a sequence called signal peptide at the N-terminus. Focusing on the fact that the method of the present invention has been carried out, the present inventors have conducted intensive studies on a method for efficiently and selectively closing a gene encoding a signal peptide. As a result, we found a method to efficiently amplify the N-terminal fragment and to easily search for the presence or absence of a signal peptide (see Japanese Patent Publication No. Hei 6-315380 or European Patent Application Publication No. 0607054A2). The inventors have succeeded in finding a novel factor (peptide) produced by bone marrow stromal cells and a DNA encoding the same, and have completed the present invention.

 The amino acid sequence of a known polypeptide registered in Swiss Prot Release 26 was investigated, but none of the polypeptides of the present invention had a sequence identical or highly homologous to that of the polypeptide of the present invention. Nothing. Furthermore, the nucleotide sequence registered in DDBB (DNA Data Bank of Japan Release 17) was also examined, but no sequence identical or highly homologous to cDNA encoding the polypeptide of the present invention was found. Therefore, it was confirmed that the polypeptide of the present invention was completely novel.

 That is, the present invention

1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5 in a substantially pure form, a homolog thereof, a fragment thereof, or a homolog of the fragment thereof;

 2) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in a substantially pure form, a homolog thereof, a fragment thereof, or a homolog of the fragment thereof;

3) The polymer according to the above 2, which comprises the amino acid sequence represented by SEQ ID NO: 1. Pseudo,

 4) DNA encoding the polypeptide described in 3 above,

 5) The DNA according to 4 above, which has the nucleotide sequence of SEQ ID NO: 2, or a fragment that selectively hybridizes to the sequence,

6) The DNA according to 4 above, which has the nucleotide sequence of SEQ ID NO: 3, or a fragment that selectively hybridizes to the sequence,

 7) A replication or expression vector comprising the DNA according to any one of the above items 4 to 6,

 8) a host cell transformed with the replication or expression vector according to 7 above; 9) culturing the host cell according to 8 above under conditions for expressing the polypeptide described in 2 or 3 above. A method for producing the polypeptide comprising:

 10) a monoclonal or polyclonal antibody of the polypeptide described in 2 or 3 above;

1 1) a pharmaceutical composition comprising the polypeptide according to 2 or 3 or the antibody according to 10 and a pharmaceutically acceptable excipient and Z or a carrier;

 12) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 in a substantially pure form, a homolog thereof, a fragment thereof, or a homolog of the fragment thereof;

 13) The polypeptide according to the above 12, comprising the amino acid sequence represented by SEQ ID NO: 5,

 14) DNA encoding the polypeptide described in 13 above,

 15) The DNA according to 14 above, which has the nucleotide sequence of SEQ ID NO: 6, or a fragment that selectively hybridizes to the sequence,

16) The DNA according to the above 14, which has a base sequence represented by SEQ ID NO: 7, Or a fragment that selectively hybridizes to that sequence,

 17) a replication or expression vector comprising the DNA according to any one of the above items 14 to 16,

 18) a host cell transformed with the replication or expression vector according to 17 above,

 19) A method for producing the polypeptide according to the above 12 or 13, comprising culturing the host cell according to the above 18 under conditions for expressing the polypeptide.

 20) a monoclonal or polyclonal antibody of the polypeptide described in 12 or 13 above, and

 21) A pharmaceutical composition comprising the polypeptide according to 12 or 13 or the antibody according to 20 and a pharmaceutically acceptable excipient and / or carrier. is there. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a conceptual diagram of a method for preparing a cDNA library according to the present invention. FIG. 2 is a construction diagram of the plasmid vector pUCSRaML2. Figure 3 shows the construction of ML2-hT ac with pUCSR.

 FIG. 4 shows the hydrophobicity profile of the polypeptide of the present invention (mouse, part).

 FIG. 5 shows the hydrophobic profile of the polypeptide of the present invention (human and partial).

詳細 Detailed description of the invention

 The present invention

(1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, (2) DNA encoding the polypeptide described in (1) above,

(3) a DNA having the nucleotide sequence of SEQ ID NO: 2,

 (4) a DNA having the nucleotide sequence of SEQ ID NO: 3,

 (5) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5, (6) a DNA encoding the polypeptide described in (5),

(7) a DNA having the nucleotide sequence of SEQ ID NO: 6 and

 (8) DNA having the nucleotide sequence of SEQ ID NO: 7

About.

 In particular, the present invention relates to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5 in a substantially pure form, a homolog thereof, a fragment of the sequence and a homolog thereof.

 The invention further relates to DNAs encoding those polypeptides. More specifically, a DNA having the nucleotide sequence of SEQ ID NO: 2, 3, 6, or 7 and a fragment that selectively hybridizes to the nucleotide sequence of SEQ ID NO: 2, 3, 6, or 7 Having DNA. A polypeptide having the amino acid sequence of SEQ ID NO: 1 or 5 in substantially pure form is generally defined as at least 90%, for example, 95, 98 or 99%, of the polypeptide at the time of production. This means that the polypeptide has the amino acid sequence shown in SEQ ID NO: 1.

 A homologue of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5 generally means at least 20, preferably at least 30, for example, 40, 60 or 100 contiguous amino acid regions. It is at least 70%, preferably at least 80 or 90%, more preferably 95% or more homologous, and such homologs are hereinafter described as polypeptides of the present invention.

Further, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 5 A fragment of a peptide, or a fragment of a homologue thereof, means a portion of at least 10 amino acids, preferably at least 15 amino acids, for example a 20, 25, 30, 40, 50 or 60 amino acid portion. DNAs that selectively hybridize to DNA having the nucleotide sequence shown in SEQ ID NO: 2, 3, 6, or 7 are generally at least 20, preferably at least 30, for example, 40, 60 or It is at least 70%, preferably at least 80 or 90%, more preferably 95% or more homologous in 100 contiguous nucleotide sequence regions. Described as DNA.

 The fragment of DNA having the nucleotide sequence represented by SEQ ID NO: 2, 3, 6, or 7 means at least 10 bases, preferably at least 15 bases, for example, 20, 25, 30 or 40 bases, and Such a fragment is also included in the DNA of the present invention.

 Further, the present invention includes a replication or expression vector comprising the DNA of the present invention. Examples of the vector include a plasmid, a virus or a phage vector comprising an ori region and, if necessary, a promoter for expression of the above DNA, a promoter and a control element, and the like. The vector may contain one or more selectable marker genes, for example, an ampicillin resistance gene. The vector can be used in vitro, for example, for the production of RNA corresponding to DNA and for the transformation of host cells.

Further, the present invention provides a vector for replicating or expressing the DNA of the present invention, which comprises the nucleotide sequence represented by SEQ ID NO: 2, 3, 6, or 7, or a DNA having an open reading frame thereof. Also included are host cells transformed with. Cells include, for example, bacteria, yeast, insect cells or mammalian cells. Furthermore, the present invention also includes a method for producing the polypeptide of the present invention, which comprises culturing the host cell of the present invention under conditions for expressing the polypeptide of the present invention. The cultivation is preferably performed under conditions in which the polypeptide of the present invention is expressed and produced from host cells.

 An antisense RNA can also be produced by inserting the DNA of the present invention into the antisense region of a vector as described above. Such antisense RNA can also be used to control the level of the polypeptide of the present invention in cells.

 The present invention also includes the monoclonal or polyclonal antibodies of the polypeptide of the present invention. Furthermore, the present invention includes a method for producing a monoclonal or polyclonal antibody of the polypeptide of the present invention. Monoclonal antibodies can be produced by ordinary hybridoma technology using the peptides of the present invention or fragments thereof as antigens. The polyclonal antibody can be produced by an ordinary method in which a host animal (for example, rat egret) is inoculated with the polypeptide of the present invention and immune serum is collected. The present invention also includes a pharmaceutical composition comprising the polypeptide of the present invention, its antibody, and a pharmaceutically acceptable excipient and / or carrier.

 Examples of the polypeptide of the present invention of (1) or (5) include those having a part of the amino acid sequence shown in SEQ ID NO: 1 or 5 and a part of which is deleted (for example, SEQ ID NO: 1). In 1 or 5, a polypeptide consisting of only a part essential for the expression of biological activity and a soluble polypeptide lacking the cytoplasmic domain and transmembrane domain, etc.), some of which are combined with other amino acids Substitutions (for example, substitutions with amino acids having similar properties), and those in which other amino acids are added or inserted into a part thereof are also included.

As is well known, one codon codes for one amino acid. ~ 6 types (for example, 1 type for Met and 6 types for Leu) are known. Therefore, the base sequence of DNA can be changed without changing the amino acid sequence of the polypeptide.

 The DNA of the present invention specified by (2) or (6) includes all base sequence groups encoding the polypeptide represented by SEQ ID NO: 1 of (1) or SEQ ID NO: 5 of (5). Is included. By changing the nucleotide sequence, the productivity of the polypeptide may be improved.

 The DNA specified by (3) or (7) is one embodiment of the DNA represented by (2) or (6), and represents a natural sequence.

 The DNA shown in (4) or (8) represents the sequence obtained by adding the natural untranslated portion to the DNA specified in (3) or (7), respectively.

 Preparation of DNA having the base sequence of SEQ ID NO: 3 is performed according to the following method.

Preparation of cDNA library of signal peptide

 (1) After synthesizing a single-stranded DNA against mRNA isolated from the target cell using random primers, add oligo dG to the 3 ′ end of the obtained single-stranded DNA,

 (2) The double-stranded DNA for the single-stranded DNA obtained in (1) is synthesized using a poly C oligomer to which a specific restriction enzyme (enzyme I) site is linked as a primer,

 (3) Divide the double-stranded DNA obtained in (2), fractionate by fragment size, ligate a linker containing a specific restriction enzyme (enzyme Π) site different from enzyme I, and re- Fractionate,

(4) The amplified cDNA was subjected to polymerase chain reaction (hereinafter abbreviated as PCR) using a primer containing the enzyme I site and a primer containing the enzyme II site, and the amplified cDNA I was added to the enzyme I-enzyme II. After digestion with 1 42

(5) a step of ligating the obtained cDNA fragment upstream of a gene for a known secretory protein or the like from which a signal peptide has been deleted, incorporating the fragment into a eukaryotic cell expression plasmid, and then transforming.

 To explain each step in detail, in step (1), after stimulating the target cells with an appropriate stimulant if necessary, a method such as Okayama H. (Method in Enzymology, 154, _3 ( 1987)). The target cell may be any cell that may be producing a secretory protein or the like. For example, neural cells and hematopoietic cells can be mentioned. The synthesis of single-stranded cDNA using a random primer is performed by a known method. Commercially available random primers are used. Subsequently, oligo dT is added to the 3 'end of the single-stranded cDNA by terminal hydroxytransferase.

 The synthesis of the double-stranded DNA in step (2) is also performed by a known method. The restriction enzyme (enzyme I) site linked to the poly-G oligomer serving as a primer and the restriction enzyme (enzyme II) site used in the next step (3) can be any one as long as they are different from each other. Is also good. Preferably, Sa1I is used as enzyme I and SacI is used as enzyme II.

 In step (3), the cDNA is fragmented by sonication so that the cDNA length becomes 500 bp on average, fractionated into 400-800 bp cDNA by agarose electrophoresis (AGE), and then T4 DNA polymerase This is carried out by blunting the ends with ligation, ligating an enzyme II adapter, and fractionating again into a 400-800 bp DNA by agarose electrophoresis. Enzyme II can be anything different from enzyme I as described above. By this step, the possibility that the cDNA fragment containing the fernal peptide is present between the enzyme I and the enzyme II is increased.

In step (4), enzyme I, which may contain a signal peptide, and enzyme To further increase the probability of the existence of the part sandwiched by II, it is attached to PCR. PCR is a well-known method, and automated devices for it are commercially available. Twenty-five to thirty amplifications are sufficient. The amplified cDNA is digested with Enzyme I-Enzyme II and fractionated into 400-800 bp cDNA by agarose gel electrophoresis.

 In step (5), a known secretory protein or other gene (called a reporter gene) obtained by removing a signal peptide from a plasmid vector for eukaryotic cell expression, and the cDN obtained in (4) upstream thereof This is a step of transforming by incorporating the A fragment. Here, various plasmid vectors for eukaryotic cell expression are known. For example, pcDL-SR chick pcEV-4, which functions in Escherichia coli, is used.

 As the reporter gene, genes of all kinds of soluble secreted proteins and mature proteins of membrane proteins are used. Furthermore, the expression of these reporter genes must be confirmed by any method such as an antibody method. Preferably, the human IL-2 receptor α gene is used. Many Escherichia coli strains for transformation are already known, and any of them may be used, but a DH5 competent cell (described in Gene, 96, 23 (1990)) is preferred. The transformant is cultured by a conventional method to obtain the cDNA library of the present invention (a conceptual diagram is shown in FIG. 1).

According to the method for preparing a cDNA library of the present invention, it is highly possible that a library contains a gene fragment encoding a signal peptide, but not all clones contain the fragment. Not all gene fragments encode an unknown (new) signal peptide. Then, it is necessary to screen a gene fragment encoding an unknown signal peptide from the library. That is, the cDNA library is subdivided into pools of an appropriate size and incorporated into an expression system. Expression systems for producing polypeptides include mammalian cells (eg, monkey COS-7 cells, Chinese hamster CHO cells, mouse L cells, etc.). Transfection is performed by known methods, for example the DEAE-Dextran method. After culture, the presence or absence of reporter gene expression is determined.

 The reporter gene is known to be expressed even if the signal peptide is specific to other secretory proteins. That is, expression of the reporter gene indicates that a signal peptide of some secretory protein was incorporated into the library. Positive pools are further subdivided, and expression and determination are repeated until a single clone is obtained. The expression of the reporter gene is determined depending on the type of the reporter gene, but is determined by a fluorescent-labeled antibody method, an enzyme-labeled antibody method (ELISA), a radiolabeled antibody method (RIA), or the like.

 Next, the nucleotide sequence of the isolated positive clone was determined, and for cDNA that was found to encode an unknown protein, a full-length clone was isolated using it as a probe, and the full-length nucleotide sequence was sequenced. You can decide. These operations are performed by methods known to those skilled in the art. For example, the nucleotide sequence is determined by the Maxam-Gilbert method and the dideoxy terminator method. The full-length sequence is performed according to the method described in Molecular Cloning (published by Cold Spring Harbor Laboratory Press, 1989, by Sambrook, J., Fritsch, EF, and Maniatis, T.).

 When the nucleotide sequences of SEQ ID NOs: 2 and 3 are partially, preferably completely, determined, the DNA encoding the protein of the present invention present in mammals is also included.

2 one Alternatively, a DNA encoding a homolog and a subset of the protein of the present invention can be obtained. Oligonucleotides having the appropriate mouse nucleotide sequence are synthesized and used to hybridize from a mammalian cDNA library or mRNA by the PCR method, or using a fragment of the appropriate mouse nucleotide sequence as a probe. By doing so, a mammalian type DNA encoding the protein can be obtained from one of the mammalian cDNA libraries or the genomic library.

 In more detail, for example,

 (i) In order to isolate cDNA encoding the human peptide of the present invention, cells producing the peptide can be selected by Northern blot analysis or PCR.

 (ii) It has been confirmed that the gene for the human homolog of the mouse is expressed by Northern blot or the like.For example, mRNA was isolated from a cell line or tissue,

(iii) First strand (single-stranded cDNA) and then second strand (double-stranded cDNA) are synthesized from the mRNA (synthesis of cDNA), and (iv) the cDNA is appropriately synthesized. Built into the phage vector,

(V) Infecting host cells with the obtained recombinant phage (preparation of cDNA library),

(vi) Screening by plaque hybridization using a cDNA fragment encoding the peptide obtained by labeling the obtained cDNA library as a probe,

 (vii) A phage DNA was prepared from the obtained positive clone, and the excised cDNA was subcloned into a plasmid vector.

(viii) It can be created by sequencing the entire length. More specifically, in step (i), a human glioblastoma cell line is applied. Stimulation with an appropriate stimulant (eg, IL-1 or the like) or no stimulation is performed according to the method of Okayama (H.) (described in Method in Enzymology, 154, 3 (1987)). It is. As a cell producing the polypeptide, preferably, a human glioblastoma cell line T98G (ATCC strain number, CRL-1690) can be mentioned, but any cell producing the peptide can be used. Any cell may be used.

 The step (ii) is performed according to the method of Okayama (H) or the like (see Method in Enzymology, 154, 3 (1987)).

 Steps (iii), (iv) and (V) are steps for preparing a cDNA library and are performed according to the modified Gubler & Hoffman method (described in Gene, 25, 263 (1983)). .

 Examples of the plasmid vector used in step (iv) include plasmid vector Yuichi (eg, pBR322, pBluescript II, etc.) and phage vectors (eg, λ £ £ 10, λDASΗII, etc.) Many phage vectors are known, but preferably a phage vector λgt22A (42.73 kbp sold by BRL) is used.

 As the host cell used in the step (v), Escherichia coli Y 1090 (r-1) (sold by BEL) is preferably used.

 Steps (vi) and (vii) are performed according to the method described in Molecular Cloning (Sambrook, J, Frits, E, F, and Maniatis, T, Cold Spring Harbor Laboratory Press (1989)).

 The sequencing in step (viii) is performed by the Maxam-Gilbert method, the didexy, and the Yuichi Minei Yuichi method.

It is necessary to confirm that the cDNA thus obtained is full-length or almost full-length. This confirmation is performed by Northern analysis using the cDNA as a probe (Molecu / 0183

lar Cloning). The size of the mRNA obtained from the hybridized band and the size of the cDNA are compared, and if they are almost the same, the cDNA is considered to be almost full length.

 Once the nucleotide sequences shown in SEQ ID NOs: 2, 3, 6, and 7 are determined, the present invention is carried out by chemical synthesis or by chemically synthesizing a fragment of the nucleotide sequence and hybridizing this as a probe. DNA can be obtained. Furthermore, the required amount of the desired DNA can be obtained by introducing the vector containing the present DNA into an appropriate host and growing it.

 As a method for obtaining the polypeptide of the present invention,

 (1) a method of purification and isolation from living organisms or cultured cells,

 (2) a method for peptide synthesis, or

 (3) a method of producing using genetically modified technology,

Etc., but the method described in (3) is industrially preferable. An expression system for producing polypeptides using genetic engineering technology

Examples of (host-vector system) include bacterial, yeast, insect cell, and mammalian cell expression systems.

 For example, when expressing in E. coli, an initiation codon (ATG) is added to the 5 'end of the DNA encoding the mature protein portion, and the resulting DNA is ligated to an appropriate promoter (eg, trp promoter, 1 ac promoter overnight;; iPL promoter, T7 promoter, etc.) and inserted into a vector that functions in E. coli (eg, pBR322, pUC18, pUC19, etc.) to insert the expression vector. Make it.

Next, Escherichia coli (eg, E. Coli DH1, E. Coli J Ml09, E. Coli HB101, etc.) transformed with this expression vector is cultured in a suitable medium, and the cells are used for the purpose. Can be obtained. In addition, if a bacterial signal peptide (for example, the signal peptide of pe1B) is used, the target polypeptide can be secreted into periplasm. In addition, it can produce fusion proteins with other polypeptides.

 When expressed in mammalian cells, for example,

The DNA encoding the nucleotide sequence represented by 7 is inserted into an appropriate vector (for example, a retrovirus vector, a herpes pilomavirus vector, a vaccinia virus vector, an SV40 vector vector, etc.). An expression vector is prepared by inserting it downstream of a suitable promoter (eg, the SV40 promoter, LTR promoter, meta-mouth thionein promoter, etc.). Next, appropriate mammalian cells (eg, monkey COS-7 cells, Chinese hamster CHO cells, mouse L cells, etc.) are transformed with the obtained expression vector, and the transformants are cultured in an appropriate medium. As a result, the desired polypeptide is secreted into the culture solution. The polypeptide obtained as described above can be isolated and purified by a general biochemical method. Industrial applicability

 Since the polypeptide of the present invention is produced and secreted by a stromal cell line, biological activity related to the differentiation, proliferation, growth or maintenance of hematopoietic cells, biological activity related to the function of the immune system, It is thought to have biological activities related to proliferation, growth or inflammation, and also to bone metabolism.

For example, proliferation of B cells, T cells, mast cells, class-specific induction by promoting immunoglobulin class switch, differentiation of B cells into antibody-producing cells, proliferation or differentiation of granulocyte precursor cells, monocyte / macrophage precursor Proliferation or differentiation of cells, proliferation of neutrophils, monocytes, macrophages, eosinophils, basophils Proliferation or hyperactivity, megakaryocyte progenitor cell proliferation, neutrophil progenitor cell proliferation or differentiation, B or T progenitor cell proliferation or differentiation, erythrocyte production promotion, erythrocyte, neutrophil, eosinophil, eosinophil Supports the growth of basophils, monocytes, macrophages, mast cells, megakaryocyte progenitors, promotes the migration of neutrophils, monocytes, macrophages, B cells or T cells, proliferates thymocytes, suppresses the differentiation of fat cells, Proliferation of natural killer cells, proliferation of hematopoietic stem cells, suppression of proliferation of stem cells and various hematopoietic progenitor cells, or promotion of bone resorption by promoting differentiation of monocytes into osteoclasts, differentiation of mesenchymal stem cells into osteoblasts It may have the effect of promoting or proliferating or activating osteoclasts by the polypeptide alone or by acting synergistically with other cytokines.

 In addition to these, any factor that acts on the immune system is also expected to act on the nervous system. Promoting proliferation of neurites, extending neurites, maintaining survival of ganglion cells, promoting proliferation or differentiation of astrocytosis, proliferation or maintaining survival of peripheral nerves, proliferation of Schwann cells, growth or survival of motor nerves. There may be.

 In addition, during the early embryonic development, the polypeptide is used to induce the formation of epidermis, brain, spine, and nerves by ectoderm-inducing action, dorsal cord connective tissue (bone, muscle, tendon), blood cells, It promotes or inhibits the formation of heart, kidney, gonad organs, or the formation of digestive organs (stomach, intestine, liver, kidney) and respiratory system (lung, trachea) by endoderm induction In addition to its potential, it may also have the effect of inhibiting the growth or growth of the above organs in living organisms.

Therefore, the polypeptide of the present invention itself is a disease relating to hypoplasia or abnormal proliferation of hematopoietic cells, a decrease or increase in the function of the immune or nervous system or bone metabolism, such as inflammatory diseases (rheumatism, ulcers). Ulcerative colitis ), Hematopoietic stem cell depletion after bone marrow transplantation, Leukocyte, platelet, B cell or T cell depletion after radiation or chemotherapy for cancer, leukemia, anemia, infectious disease, cancer, leukemia, AIDS, It is expected to be used as a therapeutic agent for various degenerative diseases (Alheimer's disease, multiple sclerosis, etc.), nerve damage, and bone metabolism disorder (osteoporosis, etc.).

 In addition, since the polypeptide may have an action of differentiating or proliferating ectoderm, mesoderm, or endoderm-derived organs, the polypeptide (epidermal, bone, muscle, tendon, heart, kidney, stomach, intestine, liver, It is also expected to be used as a tissue repair agent for the viscera, lung, trachea, etc.).

 In addition, the polypeptide can be quantified in a living body using a polypeptide monoclonal antibody or a monoclonal antibody of the polypeptide, whereby the polypeptide can be used for studying the relationship between the polypeptide and a disease or diagnosing a disease. Can be Polyclonal and monoclonal antibodies can be prepared by a conventional method using the polypeptide or a fragment thereof as an antigen.

The DNA of the present invention is not only an important and essential type II for producing the polypeptide of the present invention, which is expected to have tremendous utility, but also the diagnosis and treatment of genetic diseases (treatment of gene deficiency or It can be used for antisense DNA (RNA) to stop the expression of polypeptides and other treatments. In addition, genomic DNA can be separated using the DNA of the present invention as a probe. Similarly, it is also possible to isolate a gene for a polypeptide having high homology to the DNA of the present invention and a gene for a polypeptide having high homology to the polypeptide of the present invention in an organism other than human. It is possible. The polypeptides of the present invention may be used for diseases related to hypoplasia or abnormal growth of hematopoietic cells, abnormal or increased nervous system function, or increased or decreased immune system function, such as inflammatory diseases (rheumatism, ulcerative colitis, etc.), bone marrow Decrease in hematopoietic stem cells after transplantation Disease, leukocyte, platelet, B-cell or T-cell depletion after radiation therapy or chemotherapy, anemia, infectious disease, cancer, leukemia, AIDS, various degenerative diseases (Alzheimer's disease, multiple sclerosis) ) Or for preventing or treating nerve damage, for preventing or treating bone metabolic disorders (such as osteoporosis), or for repairing tissues, etc., usually systemically or locally, generally orally or parenterally. It is administered in the form of Preferred are oral administration, intravenous administration and intraventricular administration.

 Dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually in the range of 1 to 10 mg per adult, once to several times a day. It is administered orally or parenterally, once to several times a day, in the range of 10 tg to 100 mg / adult per adult.

 Of course, as described above, the dose varies depending on various conditions, so that a dose smaller than the above dose may be sufficient, or may be required beyond the range. When administering the compound of the present invention, it is used as a solid composition, a liquid composition and other compositions for oral administration, an injection, an external preparation, a suppository and the like for parenteral administration.

 Solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like. Capsules include soft capsules and hard carbs.

In such a solid composition, the one or more active substances include at least one inert diluent (e.g., lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch). , Polyvinylpyrrolidone, magnesium aluminate metasilicate, etc.). The composition is prepared according to a conventional method, using additives other than inert diluents, such as lubricants (magnesium stearate, etc.), disintegrants (cellulose It may contain a calcium cholate, etc.), a stabilizer (human serum albumin, lactose, etc.) and a solubilizing agent (arginine, aspartic acid, etc.).

 Tablets or pills may be coated with gastric or enteric film such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate if necessary, or two or more layers. May be applied. Also included are capsules of absorbable materials, such as gelatin.

 Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and commonly used inert diluents (eg, purified water, Ethanol etc.). Such compositions may contain, in addition to the inert diluent, adjuvants such as wetting agents and suspending agents, sweetening agents, flavoring agents, flavoring agents and preservatives.

 Other compositions for oral administration include sprays which contain one or more active substances and are formulated in a manner known per se. This composition contains, in addition to the inert diluent, a stabilizer such as sodium bisulfite to provide isotonicity, an isotonic agent such as sodium chloride, sodium citrate or citric acid. It may be. The preparation of sprays is described in detail, for example, in U.S. Pat. Nos. 2,868,691 and 3,095,355.

Injections for parenteral administration according to the present invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. For aqueous or non-aqueous solutions and suspensions, one or more active substances are mixed with at least one inert diluent. Aqueous diluents include, for example, distilled water for injection and physiological saline. Non-aqueous diluents include, for example, propylene glycol, polyethylene glycol, and olive oil. Vegetable oils, alcohols such as ethanol, polysorbate 80 (registered trademark), and the like.

 Such compositions may further include preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg, human serum albumin, lactose, etc.), solubilizing agents (eg, arginine, aspartic acid, etc.) ) May be included.

 These are sterilized by filtration through a pateria retaining filter, blending of a bactericide or irradiation. They can also be used to produce a sterile solid composition (for example, by a freeze-drying method) and to dissolve in sterile distilled water for injection or other solvents before use.

 Other compositions for parenteral administration include, for example, topical solutions, softeners, liniments, suppositories and pessaries for rectal administration, which contain one or more active substances and are formulated in a conventional manner. included. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described more specifically with reference to Examples and Reference Examples, but these do not limit the scope of the present invention.

Example 1: Construction of a plasmid for producing an expression vector

 The P c D_SR 296 vector (described in Mol. Cell. Biol., 8, 966 (1988)) prepared by Takebe et al.

It is an excellent mammalian cell expression vector having a promoter system (SR) composed of the R region of I LTR and part of the U5 sequence. However, there are drawbacks such as (1) one cloning site for the insert, EcoRI, and (2) the amount of vector recovered from Escherichia coli is low because the pBR322 vector is used as the backbone of the vector region. Therefore, the pUC19 vector, which has a high yield from large intestine orchids, is A modified pcD-SRa296 vector having a multicloning site was prepared by the following method.

 pc D—SRa 296 vector (provided by Takebe, National Institute of Health) was digested with Sa1I and the 1.7 kb P digestion containing the SRa promoter was separated using agarose gel electrophoresis. After recovery, the fragment was treated with Klenow to obtain blunt ends.

 After cutting the pUC19 vector with NdeI and HindIII, a 2.4 kbp fragment containing the AmpR and pUCori regions was separated and recovered using agarose electrophoresis, and treated with Klenow to make blunt ends. Thereafter, the cells were further treated with BAP (bacterial alkaline phosphatase) to remove the phosphate group at the 5 'end.

The 1.7 kbp fragment containing the SRa promoter and the 2.4 kbp fragment containing pUC ori thus obtained were circularized by ligation to construct a new vector. The PstI-KpnI fragment was removed from the obtained vector, and the following synthetic polylinker having the following T7 and sp6 promoters was synthesized.

Pst l T7 promoter Sac Sal Sma

, EcoRI ^Nde

 CO CO ACTAGTCTATAGTGTCACCTAAATCGTGGTACC

Spe SP 6 promoter Kpn

Was replaced. The plasmid vector thus constructed (about 3.9 kbp, shown in Fig. 2) was named pUCSRaML2.

 pUCSRML2 has the following features as a multipurpose plasmid vector.

1. Hajime Okayama Berg method and Gubl err & Hoffman method can be applied.

 2. High plasmid yield per culture.

 3. Single-stranded DNA can also be prepared.

 4. Easy to cut out cDNA inserts.

 5. It is easy to make a 'deletion mutant' for the sequence.

 6. Transcription is possible in vitro.

 7. Has a promoter that is expressed in mammalian cells. Example 2: cDNA library with selectivity for signal peptides —Construction of construction vector

 Next, a plasmid was constructed in which the cDNA encoding hT ac (used as a reporter gene, such as human IL-2 receptor) was transcribed into pUCSR ML2 as described above. And named pUC SRa-hT ac. Incorporates cDNA into the vector downstream of the hTac gene, downstream of the SRα promoter of the vector, and forms a fusion protein with hTac by translation of the protein from the cDNA having a signal sequence. Then, the fusion protein is expressed on the membrane.

(1) That is, hT a was added to the HindIII site of pBluescript SK (+) (sold by Stratagene, pBS). of. After digestion of pBS-hTac into which DNA has been incorporated, digestion with KpnI, blunting with T4 DNA polymerase, digestion with SacI, removal of leader sequence, The I-Eco RI adapter was ligated to obtain an Eco RI-blunt end fragment. This fragment was incorporated into the EcoRI-Smal site of pUCSRaML2, which had broken the SacI site, to obtain pUCSRML2-hTac (shown in FIG. 3). Example 3: Generation of a cDNA library with selectivity for signal peptide

 From the mouse stromal cell line ST2 (a cell that supports the survival and proliferation of hematopoietic stem cells and the proliferation and differentiation of B cells and myeloid cell lines and is described in EMBO J., _7, 1337 (1988)). The total RNA was extracted by the AGPC (acid guanidine-funool-clo mouth form) method (described in detail in the Cell Engineering Experiment Protocol (published by Shujunsha), pages 28-31). Poly A-RNA was purified using oligo (dT) -latex (Oligotex-dT30 (trade name, sold by Takara Shuzo Co., Ltd.)). Using a random hexamer as a primer, single-stranded cDNA was synthesized by reverse transcriptase, and dC was added to its 3 ′ end by terminal deoxytransferase. DC of 17 m er connecting restriction enzyme sites including S a1

5 'GATGCGGCCGCGTCGACGAATTC (d C) 17 3'

 Not I Sac I Eco RI

The primers were used as primers to synthesize double-stranded cDNA. Fragmented cDNA by sonication to an average length of 500 bp Thereafter, cDNA of 400 to 800 bp was fractionated by agarose electrophoresis. After blunting the ends with T4 DNA polymerase, the mouth containing the Sac I site

5 'GAGGTACAAGCTTGATATCGAGCTCGCGG 3' 3 'CATGTTCGAACTATAGCTCGAGCGCC 5'

 Hind ΠΙ Eco RV Sac I

(Refer to Nucreic Acids Res., 18, 4293 (1990)), and the cDNA of 400 to 800 bp was fractionated again by agarose electrophoresis. Primer (NLC) containing Sa1I site,

5 'GATGCGGCCGCGTCGACGAATTC 3'

Primers (L L H E S) containing

Using 5′3 ′, 25 cycles of PCR were performed at 94 ° C. for 1 minute, 50 ° C. for 2 minutes, and 72 ° C. for 2 minutes. The amplified cDNA was digested with SacI and EcoRI, and a 400-800 bp cDNA was fractionated by agarose gel electrophoresis. This cDNA and plasmid obtained by digesting pUCSR and ML2-hTac (produced in Example 2) with SacI and Sa1I were ligated with T4 DNA ligase, and Escherichia coli DH was ligated. The 5α strain was transformed to obtain a library of the C D Ν あ る that was selective for the signal peptide. 01843

Example 4: Signal peptide coding cDNA screening and analysis

 The colonies obtained from the library prepared in Example 3 were subdivided into pools (about 50 colonies / pool). For each pool, plasmid was isolated by the miniprep method and transfected into COS-7 cells by the DAE-dextran method (Current Protocol in Molecular Biology, § 9.2.1). After 48 hours, the cells were detached from the dish, and incubated with mouse anti-Tac IgG antibody for 20 minutes on ice. After removing the free antibodies, the goat anti-mouse IgG antibody labeled with FITC (fluorescein isothiosinate) was incubated for 20 minutes on ice, and the free antibodies were removed again. Then, fluorescent staining for Tac on the cell surface was performed. Then, a positive pool was selected using a fluorescence microscope. One pool was further subdivided into colonies, and the same method was repeated until a single clone was obtained, thereby obtaining one positive clone (pBS-ST-T1). Next, pUCSR <ML2-hTac vector Two types of synthetic primers specific to one

5 'GCCTGTACGGAAGTGTTACTTCTGC 3' (12 base upstream from PstI cloning site, for sense) and

 5 'CCATGGCTTTGAATGTGGCG 3'

 (2 Obase downstream from SaGI cloning site, for antisense)

Was used to determine the base sequence of the ST—T1 insert. D NA's sequencing was done by Sidde (Sanger, F) 's dideoxy * termine.

- twenty one - The assay was performed by a cycle sequence method using an ABI (Applied Biosystems Inc.) fluorescent dye overnight, one minute, and one day based on the evening method. ABI DNA Sequencer (Model 373A) was used for reading the sequence. In addition, an open reading frame following the in frame (in-frame) was searched for T acc DNA from which the leader sequence had been deleted from the obtained DNA sequence, and it was converted to the expected amino acid sequence. By drawing a sex profile, it was confirmed that the signal peptide had a characteristic N-terminal hydrophobic region (shown in Fig. 4). Furthermore, a homologous search with the database at the DNA and amino acid levels revealed that ST-T1 encodes an unknown protein. Example 5: Screening of full-length cDNA and determination of nucleotide sequence

The cDNA library was prepared from mRNA derived from the mouse stromal cell line ST-2 using SuperScript (registered trademark) lambda system (sold by BRL). This cDNA was ligated to Sgt22A (available from BRL) having a phosphatase-treated Sa1I, N0tI arm. In vitro packaging, carried out in accordance with professional tocol of in vitro-Nono ⁰ Kkei Managing Kit Lambda Yi down (in vitro Packaging Kit LAMDA INN) ( Nippon Gene), the recombinant phage host E. coli Y 1 090 (r —) (Sold by BBL). The result was a cDNA library of 1 million plaques. Next, pBS-ST-T1 was digested with Sail and Notl, and an ST-T1 fragment was prepared by agarose electrophoresis. Using the oligo-labeled ST-Tlc DNA fragment as a probe, the library was screened to obtain a number of positive clones. The insert is about 1.0k 96/01 43

For the bp clone, the Sa1I-NotI fragment cut out from the igt22A vector was subcloned into pUCS RaML2 to obtain plasmid pUCSRML2-ST-T1. Using the T7 primer, the base sequence of 300 bp on the 5 'side of the STT1 cDNA was determined, and the sequence matching the ST-T1 of the probe was the most 5% of the ST-T1 from the phage library. 'I first confirmed that it was on the side. Example 6: Determining the full-length sequence and open reading frame of cDNA

 c Full length sequence of DNA is by Molecular Cloning (Sambrook,

J., Fritsch, EF, and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) according to the method described in Random Sequence.

 That is, plasmid was recovered from the ST—T1 clone, and the cDNA insert was separated and purified. This was ligated and fragmented, the end of the DNA fragment was blunt-ended with T4 polymerase, and the DNA fragment having a length of around 400 bp was recovered. The obtained DNA fragment was cloned into the SmaI site of a plasmid vector, BLUESCRIPT II (sold by Stratagene), and then transformed into E. coli. After randomly picking 20 colonies and preparing plasmid DNA, the DNA sequence of these 20 plasmids (all of which have a fragment of ST-T1 cDNA as an insert). Quensing was performed. Sequencing of DNA and reading of the sequence were performed by the method described in Example 4.

 The sequence data for the ST—T1 cDNA fragment is the DNA fragment of the DNAS

Use a DNA sequence ligation program to create a continuous sequence After editing, the base sequence shown in SEQ ID NO: 3 was obtained. An open reading frame was determined from the cDNA full-length sequence data, and was further translated into an amino acid sequence to obtain the sequence shown in SEQ ID NO: 1. Example Ί: Confirmation of the source of cDNA encoding human ST-T1 peptide

The human glioblastoma cell line T98G (ATCC strain number, CRL-1690) was homogenized and incubated with oligo dT-cellulose. After washing unnecessary substances, polyA-RNA was eluted and recovered (see Venn storm, B. et. Al., Cell, 28, 135 (1982)). The RNA 2 / g and after 1.0% Agarosu electrophoresis, block tee Ngushi to nitrocellulose main Npuren, ^{32 P-labeled} mouse ST- T lc DNA is High Priestess soybean as a probe permitted the expression of mRNA A of 1.4kb Was. Example 8: Separation and purification of human mRNA

 After stimulating a human glioblastoma cell line T98G3x10 'with 100 units of Zm1 human IL-11 ^ for 4 hours, the method of Okayama, H. et al. in Enzymology, 154, 3 (1987)).

 That is, 5.5M GT C solution (5.5 metaguanidine thiosinate,

After solubilizing the cells with 25 mM sodium citrate and 0.5% sodium lauryl sarcosine, cell lysate was dissolved in cesium trifluoroacetate (CsTFA) with a density of 1.51 and cesium trifluoroacetate (CsTFA). The solution was placed on a cushion and subjected to ultracentrifugation (120,000 xg, 20 hours) to recover 1.26 mg of total RNA during precipitation. This was passed twice through an oligo dT cellulose column to recover 46 / zg po1y (A) ^T RNA. Example 9: Preparation of human cDNA library

The cDNA library was prepared by a modification of the Gubler & Hoffmann method (see Gene, 25, 263 (1983)). From p 0 1 y prepared in Example ^{8 (A) τ RN A (} 5 g), using an oligo dT primer with No t I site, by reverse transcriptase to synthesize first strand. Subsequently, a second strand was synthesized, subjected to Sail adapter ligation and N0tI digestion, and then subjected to gel filtration column chromatography (Sephacryl S-500HR (Pharmacia)). Excluding the adapter and primers, 820 ng of the cDNA fraction was recovered. The above cDNA synthesis step was performed using a kit of Super Script Lambda System (Super Script System, sold by BRL). Example 10: Screening of human ST-Tlc DNA by cross-hybridization from a human cDNA library

As described in Example 5, a recombinant phage was prepared to obtain a cDNA library consisting of 1,000,000 plaques. Illustrated 1,000,000 plaques obtained on LB plates Purotti Ngushi to nitrocellulose main Npuren, with ³² P-labeled mouse ST- T lc DNA (Example 5 SEQ ID NO: 3 of the same fragments as prepared in Were used as probes, and 40 positive clones were obtained. Example 11: Isolation of positive clones of human ST-Tlc DNA Phage DNA was prepared from 6 of the clones by a conventional method (see Cell Engineering Laboratory Protocol, Shujunsha, page 8). , S a 1 I, N After digestion with 0 tI, the length of the insert cDNA was determined by agarose electrophoresis, and 5 clones were found to be 1.4 kb. From the results of Northern analysis, this 1.4 kb clone was considered to be almost the full length of human ST-T1.

 Then, for one of the five 1.4kb clones, S a l

1. After agarose electrophoresis, the cDNA digested with N0tI was cut out and subcloned into the Sa1I and NotI sites of the ML2 plasmid pUCSR. Example 12: 8 human-shaped 1 0 ⁷ Eight Sequencing

 From this full-length cDNA sequence data, an open reading frame (shown as SEQ ID NO: 6) was determined, and further translated into an amino acid sequence to obtain a sequence shown as SEQ ID NO: 5. The hydrophobic profile of the obtained amino acid sequence was drawn, and the hydrophobic region at the N-terminal portion characteristic of the signal peptide was confirmed (shown in FIG. 5). By comparing the 30 to 40 amino acids on the N-terminal side of this peptide with known signal peptides, the signal peptide portion of this peptide was estimated (Von Heuane, G. Nucleic Acids Res. 14, 4683 (1986)), and the sequence shown in SEQ ID NO: 8 was obtained.

 The nucleotide sequence of DNA was determined by a cycle sequence method using a fluorescent dye terminator (available from Applied Biosystems Inc.). The sequence was read using a DNA sequencer (Model 373A, available from Applied Biosystems Inc.).

The nucleotide sequence of the obtained human ST-T1 cDNA and the predicted amino acid sequence were compared with those of mouse ST-T1, and found to be 92.4% in the amino acid sequence and 92.4% in the DNA sequence. Has 76.9% homology Arrangement table

SEQ ID NO: 1

Array Length: 2 1 1

Sequence type: amino acid

 Topology: linear

Sequence type: protein

Array

 Met Ala Val Leu Ser Leu Leu Leu Leu Gly Gly Leu Trp Ser Ala Val 1 5 10 15 Gly Ala Ser Asn Met Ala Val Val Tyr Cys Gly Ser Val Val Lys Leu

 20 25 30

 Leu Asn Tyr Arg His Asn Val Arg Leu His Ser His Asp Val Arg Tyr

 35 40 45

 Gly Ser Gly Ser Gly Gin Gin Ser Val Tyr Gly Val Tyr Ser Val Asp 50 55 60

 Asp Ser Asn Ser Tyr Trp Arg lie Arg Gly Lys Tyr Ala Tyr Val Cys 65 70 75 80

Glu Arg Gly Tyr Pro ly Lys Cys Gly Gin Pro lie Arg Leu Tyr His

 85 90 95 lie Asn Tyr Gly Arg Asn Leu His Ser His His Phe Tyr Ser Pro Leu

 100 105 110

 Ser Gly Asn Gin Glu Val Ser Ser Phe Gly Glu Glu Gly Glu Gly Asp

 115 120 125

Tyr Leu Asp Asp Trp Tyr Val Leu Cys lie Gly Pro Tyr Trp Val Arg 130 135 140 Asp Gly Glu Val Arg Phe Lys His Ser Ser Tyr Asp Val Leu Leu Ser 145 150 155 160

Val Tyr Gly Glu Gin Tyr Gly Arg Pro lie Ser Gly Gin Gin Glu Val

 165 170 175

His Gly Met Ala Gin Pro Ser Gin Asn Asn Tyr Trp Lys Ala Met Glu

 180 185 190

 Gly lie Phe Met Lys Pro Ser Glu Leu Leu Arg Ala Glu Val His His

 195 200 205

 Ala Glu Leu *

 210 SEQ ID NO: 2

Array length: 6 3 6

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: cDNA to mRNA

Array

 ATGGCTGTCT TGTCGCTTCT GTTGTTGGGT GGTTTGTGGA GCGCTGTGGG AGCGTCCAAC 60 ATGGCTGTCG TTACTTGCGG CTCCGTGGTG AAGCTACTCA ATACACGCCA CAATGTCCGA 120 CTGCATTCTC ACGACGTGCG CTATGGGTCA GGTAGTGGGC AGCAGTCAGT GACAGGTGTG 180

ACCTCTGTGG ATGACAGCAA CAGTTACTGG AGGATACGGG GGAAGACAGC CACAGTGTGT 240

GAAAGGGGAA CCCCCATCAA ATGCGGGCAG CCCATCCGTC TGACACACAT CAACACAGGT 300

CGAAACCTGC ACAGCCACCA TTTCACTTCA CCTCTCTCTG GAAACCAGGA AGTGAGTTCC 360 TTTGGCGAAG AAGGTGAAGG CGACTATCTG GATGACTGGA CAGTGCTCTG TATTGGACCT 420

TACTGGGTTA GAGATGGTGA GGTGAGGTTC AAACATTCTT CCACTGACGT ACTGCTGTCT 480 GTCACAGGAG AACAGTACGG ACGACCCATA AGTGGACAAC AAGAGGTACA TGGTATGGCC 540

CAGCCAAGTC AGAACAACTA CTGGAAGGCC ATGGAAGGCA TCTTCATGAA GCCCAGTGAG 600

TTGCTGAGGG CAGAGGTCCA TCATGCAGAG CTGTGA 636 SEQ ID NO: 3

Array length: 1 0 2 0

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: cDNA to mRNA

Array

 GGGGCAGTTT CTCGGATCTA GGATGGCTGT CTTGTCGCTCTT CTGTTGTTGG GTGGTTTGTG 60 GAGCGCTGTG GGAGCGTCCA ACATGGCTGT CGTTACTTGC GGCTCCGTGG TGAAGCTACT 120 CAATACACGC CACAATGTCC GACTGCATTC TCACGACGTG CGCTGATC GCGTCAGGTCGATCCAGGTCGATCGATCCATC GTC ATC

GGGGAAGACA GCCACAGTGT GTGAAAGGGG AACCCCCATC AAATGCGGGC AGCCCATCCG 300

TCTGACACAC ATCAACACAG GTCGAAACCT GCACAGCCAC CATTTCACTT CACCTCTCTC 360

TGGAAACCAG GAAGTGAGTT CCTTTGGCGA AGAAGGTGAA GGCGACTATC TGGATGACTG 420

GACAGTGCTC TGTATTGGAC CTTACTGGGT TAGAGATGGT GAGGTGAGGT TCAAACATTC 480 TTCCACTGAC GTACTGCTGT CTGTCACAGG AGAACAGTAC GGACGACCCA TAAGTGGACA 540

ACAAGAGGTA CATGGTATGG CCCAGCCAAG TCAGAACAAC TACTGGAAGG CCATGGAAGG 600

CATCTTCATG AAGCCCAGTG AGTTGCTGAG GGCAGAGGTC CATCATGCAG AGCTGTGACT 660

CTAAATACTC TGAGCCACTG TCGCCACACA GTGTTTGTAG ACATCTGCTG CTGCTTTACT 720

GCGGGATCCC TGCTCCAGGT CCTCTGAGCG TGCTGCCTTG GGACCTGCAG CCCTTCACAC 780 TTGAATTGGT TGCTCTCCCA GGCTTAGTCA GTTCTCTCGG GAAAGGACTC ACTCGTCTGT 840

GAAGGTGGAG ATAGCTTTCT TCATGCTGGT AAAAGAAGTT GAGGAGCTTG CCTGTCTTAG 900 CTGGGAAGTG TAACTCCGCA GGAACTGGGC ATCATGGATT CTTAGTTTGT GATTTCTCCT 960 GTTTTGTCTT TCTCCAAAAT AATGAATAAA TAAATAAATA AAAAGAAAAA AAAAAAAAAA 1020

SEQ ID NO: 4

Array length: 1 0 2 0

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: cDNA to mRNA

Origin

 Organism Name: Mouse

 Cell line: S T 2

Array features

 Characteristic symbol: CDS

 Location: 23. .658

 Method used to determine characteristics: P Symbol representing characteristics: sig peptide

 Location: 23.. 76

 Method used to determine features: S Symbol representing feature: mat peptide

 Location: 77. .658

How the features were determined: S Array

 GGGGCAGTTT CTCGGATCTA GG ATG GCT GTC TTG TCG CTT CTG TTG TTG GGT 52

 Met Ala Val Leu Ser Leu Leu Leu Leu Gly

 -15-10

GGT TTG TGG AGC GCT GTG GGA GCG TCC AAC ATG GCT GTC GTT ACT TGC 100

Gly Leu Trp Ser Ala Val Gly Ala Ser Asn Met Ala Val Val Tyr Cys

 -5 1 5

 GGC TCC GTG GTG AAG CTA CTC AAT ACA CGC CAC AAT GTC CGA CTG CAT 148 Gly Ser Val Val Lys Leu Leu Asn Tyr Arg His Asn Val Arg Leu His

 10 15 20

 TCT CAC GAC GTG CGC TAT GGG TCA GGT AGT GGG CAG CAG TCA GTG ACA 196 Ser His Asp Val Arg Tyr Gly Ser Gly Ser Gly Gin Gin Ser Val Tyr

 25 30 35 40

 GGT GTG ACC TCT GTG GAT GAC AGC AAC AGT TAC TGG AGG ATA CGG GGG 244 Gly Val Tyr Ser Val Asp Asp Ser Asn Ser Tyr Trp Arg lie Arg Gly

 45 50 55

 AAG ACA GCC ACA GTG TGT GAA AGG GGA ACC CCC ATC AAA TGC GGG CAG 292 Lys Tyr Ala Tyr Val Cys Glu Arg Gly Tyr Pro lie Lys Cys Gly Gin

 60 65 70

CCC ATC CGT CTG ACA CAC ATC AAC ACA GGT CGA AAC CTG CAC AGC CAC 340

Pro lie Arg Leu Tyr His lie Asn Tyr Gly Arg Asn Leu His Ser His

 75 80 85

 CAT TTC ACT TCA CCT CTC TCT GGA AAC CAG GAA GTG AGT TCC TTT GGC 388 His Phe Tyr Ser Pro Leu Ser Gly Asn Gin Glu Val Ser Ser Phe Gly

90 95 100 GAA GAA GGT GAA GGC GAC TAT CTG GAT GAC TGG ACA GTG CTC TGT ATT 436 Glu Glu Gly Glu Gly Asp Tyr Leu Asp Asp Trp Tyr Val Leu Cys lie 105 110 115 120

GGA CCT TAC TGG GTT AGA GAT GGT GAG GTG AGG TTC AAA CAT TCT TCC 484 Gly Pro Tyr Trp Val Arg Asp Gly Glu Val Arg Phe Lys His Ser Ser

 125 130 135

 ACT GAC GTA CTG CTG TCT GTC ACA GGA GAA CAG TAC GGA CGA CCC ATA 532 Tyr Asp Val Leu Leu Ser Val Tyr Gly Glu Gin Tyr Gly Arg Pro lie

 140 145 150

 AGT GGA CAA CAA GAG GTA CAT GGT ATG GCC CAG CCA AGT CAG AAC AAC 580 Ser Gly Gin Gin Glu Val His Gly Met Ala Gin Pro Ser Gin Asn Asn

 155 160 165

 TAC TGG AAG GCC ATG GAA GGC ATC TTC ATG AAG CCC AGT GAG TTG CTG 628 Tyr Trp Lys Ala Met Glu Gly lie Phe Met Lys Pro Ser Glu Leu Leu

 170 175 180

 AGG GCA GAG GTC CAT CAT GCA GAG CTG TGA CTCTAAATAC TCTGAGCCAC 678 Arg Ala Glu Val His His Ala Glu Leu *

185 190

TGTCGCCACA CAGTGTTTGT AGACATCTGC TGCTGCTTTA CTGCGGGATC CCTGCTCCAG 738 GTCCTCTGAG CGTGCTGCCT TGGGACCTGC AGCCCTTCAC ACTTGAATTG GTTGCTCTCC 798 CAGGCTTAGT CAGTTCTCTC GGGAAAGGAC TCACTCGTCT GTGAAGGTGG AGATAGCTTT 858 CTTCATGCTG GTAAAAGAAG TTGAGGAGCT TGCCTGTCTT AGCTGGGAAG TGTAACTCCG 918 CAGGAACTGG GCATCATGGA TTCTTAGTTT GTGATTTCTC CTGTTTTGTC TTTCTCCAAA 978 ATAATGAATA AATAAATAAA TAAAAAGAAA AAAAAAAAAA AA 1020 SEQ ID NO: 5 One 6 ε —

09Τ 99ΐ 09ΐ 9Η 52

• I3S nai ng Λ J¾ JGS S S H sAq aqj 3JV I¾ nio dsy

 OH gsi οετ

 S-ry A dJX JA OJJ A \ usy SA ngq J¾ dJX dsy dsy ns αΑ

 9Ζΐ OZT 9ΐΐ

dsv ^ ΙΟ "10 ^Λ Ι3" 19 "ΐθ ^ Ιΰ ⁹ ¾ί ^B IV ΙΗ u \ usy J9S 'OZ

 0Π 90T 001

^{Π37 OJJ J9S J¾ aqj STH STH J3} S S TH Η9 Ί US V 3J V ^ 19 usy I ^ A

 96 06 S8

 STH -im ngq Sjy an OJJ u SAQ sAq 9Π OJJ Λιο Sjy

 08 9A Oi S9 5ΐ

SXQ jq BIV J3S sAq Sjy Bjy ^ ll usy jas dsy

 09 S9 09

 dsv s i ^ io Ϊ ^ Λ o ^ io s JQS

 Ot ^ 98

J ^ 3JV ΐ ^ Λ dsv STH J3S STH ^η3 l ^e A ^US V ^S TH V-"ίΧ usy naq 01

 OS 02

 "31 sA iBA A -I3S f) s 八 ΐ Α ^ Α ^ Ι ^ ΐν ^

 9ΐ Οΐ 5 I

Ι ^ Α ^ ιν -I3S d nai 3 ^ ig naq na naq na old l¾ BIV 9 «

 Garden 9 geese, Ba ^: bacteria germ

Leakage: — α ^ Μ

Zf £ lOIS6dr / JDd £ 10/96 OAV Val Thr Gly Glu Gin Tyr Gly Arg Pro lie Ser Gly Gin Lys Glu Val

165 170 175

 His Gly Met Ala Gin Pro Ser Gin Asn Asn Tyr Trp Lys Ala Met Glu

 180 185 190

Gly He Phe Met Lys Pro Ser Glu Leu Leu Lys Ala Glu Ala His His

 195 200 205

 Ala Glu Leu *

 210 SEQ ID NO: 6

Array length: 6 3 6

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: cDNA to mRNA

Array

 ATGGCTGTAG TACCTCTGCT GTTGTTGGGG GGTTTGTGGA GCGCTGTGGG AGCGTCCAGC 60 CTGGGTGTCG TTACTTGCGG CTCCGTGGTG AAGCTACTCA ATACGCGCGCCA CAACGTCCGA 120 CTGCACTCAC ACGACGTGCG CTATGGGTCA AGTAGTGGGC AGCAGTCAGT GACAGGAG AGCGAGTGACTGAGTGAGCAGGATCAGGTCAGCGAGTCAGCGTCAGCGTCAGCGTCAGCGTCAGCGTCAGCGTCAGCGAGTCAGCGTCAGCGTCAGCGTCAGCGTCAGTGAGTCAGCGTCAGCGTCAGCGTCAGTCAGTCAGCGTCAGCGTCAGTGAGTCAGCGTCAGCGTCAGTGAGTCAGCGTCAGCGTCAGTGAGTCAGCGTCAGCGTCAGCGTCAGTGAGTCAGCGTCAGTGAGTCAGCGTCAGCGTGA

GAGAGGGGAA CCCCCATCAA GTGTGGCCAG CCCATCCGGC TGACACATGT CAACACTGGC 300

CGAAACCTCC ATAGTCACCA CTTCACTTCA CCTCTTTCTG GAAACCAGGA AGTGACTGCT 360

TTTGGTGAAG AAGGTGAAGG TGATTATCTG GATGACTGGA CAGTGCTCTG TAATGGACCC 420

TACTGGGTGA GAGATGGTGA GGTGCGGTTC AAACACTCTT CCACTGAGGT ACTGCTGTCT 480 GTCACAGGAG AACAATATGG TCGACCTATC AGTGGGCAAA AAGAGGTGCA TGGCATGGCC 540

CAGCCAAGTC AGAACAACTA CTGGAAAGCC ATGGAAGGCA TCTTCATGAA GCCCAGTGAG 600 TTGTTGAAGG CAGAAGCCCA CCATGCAGAG CTGTGA 636 SEQ ID NO: 7

Array length: 1 1 0 0

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: cDNA to niRNA

Array

GTTTTCTTCG AAGATTTGGG GCTCCGCGAT ACAGTTAGGA TGGCTGTAGT ACCTCTGCTG 60 TTGTTGGGGG GTTTGTGGAG CGCTGTGGGA GCGTCCAGCC TGGGTGTCGT TACTTGCGGC 120 TCCGTGGTGA AGCTACTCAA TACGCGCCAC AACGTCCGAC TGCACTCACACGGACGCG

TATGGGTCAA GTAGTGGGCA GCAGTCAGTG ACAGGTGTAA CCTCTGTGGA TGACAGCAAC 240

AGTTACTGGA GGATACGGCG GAAGAGTGCC ACAGTGTGTG AGAGGGGAAC CCCCATCAAG 300 TGTGGCCAGC CCATCCGGCT GACACATGTC AACACTGGCC GAAACCTCCA TAGTCACCAC 360

TTCACTTCAC CTCTTTCTGG AAACCAGGAA GTGACTGCTT TTGGTGAAGA AGGTGAAGGT 420

GATTATCTGG ATGACTGGAC AGTGCTCTGT AATGGACCCT ACTGGGTGAG AGATGGTGAG 480

GTGCGGTTCA AACACTCTTC CACTGAGGTA CTGCTGTCTG TCACAGGAGA ACAATATGGT 540

CGACCTATCA GTGGGCAAAA AGAGGTGCAT GGCATGGCCC AGCCAAGTCA GAACAACTAC 600 TGGAAAGCCA TGGAAGGCAT CTTCATGAAG CCCAGTGAGT TGTTGAAGGC AGAAGCCCAC 660

CATGCAGAGC TGTGAATCTT GAGGCTCTGA GGCACTGTTA ACGCACAATG TTCACAGACA 720

TCTGTTGCTG CCTCACCTTG GGATCCCTGC CACAAGTTCC TTGGGCAGTG GCCATGTCAC 780

CATTGAGATG AAGATATACA ACAGAGAAAT AGTGGCTGTG TTTGGGAAGC TTCAGCCCTG 840

CACATTTTGA ACTAGTCACT CTCCCAGACT TGGCGGTGGG TCAGTTCTTT CCTGAGTAGA 900 GGACTTGCTG GTAAAAGGGG CAGATGCTTT TTATTAGTAC TGATTAAACC ACACTGAGGG 960

AAACATCCCT CTTAGCTGGG AAACTGTTTA CTCTTCAGGA GCTTGGCATC ATGGACTGTT 1020 AATGTATGTG ATTTTCCCCC TATTTTCTCT CCCCCACAAT GATAAAAACA ATAATTTTAT 1080

TATGAAAAAA AAAAAAAAAA 1100 SEQ ID NO: 8

Array length: 1 1 0 0

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: cDNA to mRNA

Origin

 Organism name: Homo Sapiens

 Cell line: T98G

Array features

 Characteristic symbol: CDS

 Location: 40. .675

 Method used to determine characteristics: P Symbol representing characteristics: sig peptide

 Location: 40..94

 Method used to determine features: S Symbol representing feature: mat peptide

 Location: 95. .675

How the features were determined: S Array

 GTTTTCTTCG AAGATTTGGG GCTCCGCGAT ACAGTTAGG ATG GCT GTA GTA CCT 54

 Met Ala Val Val Pro

 -15

CTG CTG TTG TTG GGG GGT TTG TGG AGC GCT GTG GGA GCG TCC AGC CTG 102

Leu Leu Leu Leu Gly Gly Leu Trp Ser Ala Val Gly Ala Ser Ser Leu

 -10 -5 1

 GGT GTC GTT ACT TGC GGC TCC GTG GTG AAG CTA CTC AAT ACG CGC CAC 150 Gly Val Val Thr Cys Gly Ser Val Val Lys Leu Leu Asn Thr Arg His

 5 10 15

 AAC GTC CGA CTG CAC TCA CAC GAC GTG CGC TAT GGG TCA AGT AGT GGG 198 Asn Val Arg Leu His Ser His Asp Val Arg Tyr Gly Ser Ser Ser Gly

 20 25 30 35

 CAG CAG TCA GTG ACA GGT GTA ACC TCT GTG GAT GAC AGC AAC AGT TAC 246 Gin Gin Ser Val Thr Gly Val Thr Ser Val Asp Asp Ser Asn Ser Tyr

 40 45 50

 TGG AGG ATA CGG CGG AAG AGT GCC ACA GTG TGT GAG AGG GGA ACC CCC 294 Trp Arg lie Arg Arg Lys Ser Ala Thr Val Cys Glu Arg Gly Thr Pro

 55 60 65

ATC AAG TGT GGC CAG CCC ATC CGG CTG ACA CAT GTC AAC ACT GGC CGA 342 lie Lys Cys Gly Gin Pro lie Arg Leu Thr His Val Asn Thr Gly Arg

 70 75 80

 AAC CTC CAT AGT CAC CAC TTC ACT TCA CCT CTT TCT GGA AAC CAG GAA 390 Asn Leu His Ser His His Phe Thr Ser Pro Leu Ser Gly Asn Gin Glu

85 90 95 1 2

GTG ACT GCT TTT GGT GAA GAA GGT GAA GGT GAT TAT CTG GAT GAC TGG 438 Val Thr Ala Phe Gly Glu Glu Gly Glu Gly Asp Tyr Leu Asp Asp Trp 100 105 110 115

ACA GTG CTC TGT AAT GGA CCC TAC TGG GTG AGA GAT GGT GAG GTG CGG 486 Thr Val Leu Cys Asn Gly Pro Tyr Trp Val Arg Asp Gly Glu Val Arg

 120 125 130

 TTC AAA CAC TCT TCC ACT GAG GTA CTG CTG TCT GTC ACA GGA GAA CAA 534 Phe Lys His Ser Ser Thr Glu Val Leu Leu Ser Val Thr Gly Glu Gin

 135 140 145

TAT GGT CGA CCT ATC AGT GGG CAA AAA GAG GTG CAT GGC ATG GCC CAG 582

Tyr Gly Arg Pro lie Ser Gly Gin Lys Glu Val His Gly Met Ala Gin

 150 155 160

 CCA AGT CAG AAC AAC TAC TGG AAA GCC ATG GAA GGC ATC TTC ATG AAG 630 Pro Ser Gin Asn Asn Tyr Trp Lys Ala Met Glu Gly lie Phe Met Lys

 165 170 175

 CCC AGT GAG TTG TTG AAG GCA GAA GCC CAC CAT GCA GAG CTG TGA 675 Pro Ser Glu Leu Leu Lys Ala Glu Ala His His Ala Glu Leu *

180 185 190

 ATCTTGAGGC TCTGAGGCAC TGTTAACGCA CAATGTTCAC AGACATCTGT TGCTGCCTCA 735 CCTTGGGATC CCTGCCACAA GTTCCTTGGG CAGTGGCCAT GTCACCATTG AGATGAAGAT 795

ATACAACAGA GAAATAGTGG CTGTGTTTGG GAAGCTTCAG CCCTGCACAT TTTGAACTAG 855 TCACTCTCCC AGACTTGGCG GTGGGTCAGT TCTTTCCTGA GTAGAGGACT TGCTGGTAAA 915 AGGGGCAGAT GCTTTTTATT AGTACTGATT AAACCACACT GAGGGAAACA TCCCTCTTAG 975 CTGGGAAACT GTTTACTCTT CAGGAGCTTG GCATCATGGA CTGTTAATGT ATGTGATTTT 1035 CCCCCTATTT TCTCTCCCCC ACAATGATAA AAACAATAAT TTTATTATGA AAAAAAAAAA 1095

AAAAA 1100

Claims

 The scope of the claims

1) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5 in a substantially pure form, a homolog thereof, a fragment thereof, or a homolog of the fragment thereof.

 2) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in a substantially pure form, a homolog thereof, a fragment thereof, or a homolog of the fragment thereof.

 3) The polypeptide according to claim 2, consisting of the amino acid sequence represented by SEQ ID NO: 1.

 4) A DNA encoding the polypeptide described in claim 3.

5) The DNA of claim 4, which has the nucleotide sequence of SEQ ID NO: 2, or a fragment that selectively hybridizes to the sequence.

6) The DNA according to claim 4, which has the nucleotide sequence represented by SEQ ID NO: 3, or a fragment that selectively hybridizes to the sequence.

7) A replication or expression vector comprising the DNA according to any one of claims 4 to 6.

 8) A host cell transformed with the replication or expression vector according to claim 7.

9) A method for producing a polypeptide according to claim 8, comprising culturing the host cell according to claim 8 under conditions for expressing the polypeptide according to claim 2 or 3.

 10) A monoclonal or polyclonal antibody of the polypeptide according to claim 2 or 3.

11) The polypeptide described in claim 2 or 3 or the antibody described in claim 10 and a pharmaceutically acceptable excipient and And / or a carrier.

 12) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 in a substantially pure form, a homolog thereof, a fragment thereof or a homolog of the fragment thereof.

 13) The polypeptide according to claim 12, comprising an amino acid sequence represented by SEQ ID NO: 5.

 14) A DNA encoding the polypeptide described in claim 13.

15) The DNA according to claim 14 having the nucleotide sequence of SEQ ID NO: 6, or a fragment _c that selectively hybridizes to the sequence.

 16) The DNA according to claim 14, which has the nucleotide sequence of SEQ ID NO: 7, or a fragment that selectively hybridizes to the DNA h0.

17 7) A replication or expression vector comprising the DNA according to any one of claims 14 to 16.

 18) A host cell transformed with the replication or expression vector according to claim 17.

 19) The polypeptide comprising culturing the host cell according to claim 18 under conditions for expressing the polypeptide according to claim 12 or 13. Manufacturing method of peptide.

 20) A monoclonal or polyclonal antibody of the polypeptide according to claim 12 or 13.

 21 1) containing the polypeptide described in claim 12 or 13 or the antibody described in claim 20 and a pharmaceutically acceptable excipient and / or carrier A pharmaceutical composition, comprising: