JP2003504021A - Human protein having hydrophobic domain and DNA encoding the same - Google Patents

Abstract

  (57) [Summary] The present invention provides a human protein having a hydrophobic domain, a DNA encoding the same, an expression vector for the DNA, a transformed eukaryotic cell expressing the DNA, and an antibody against the protein.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a human protein having a hydrophobic domain and a DNA encoding the same.
, An expression vector of this DNA, a eukaryotic cell expressing this DNA, and an antibody against this protein. The protein of the present invention can be used as a medicine or as an antigen for producing an antibody against this protein. The human cDNA of the present invention can be used as a gene diagnostic probe or a gene source for gene therapy. Further, it can be used as a gene source for mass-producing the protein encoded by this cDNA. Cells in which these genes have been introduced to express a large amount of secretory proteins or membrane proteins can be used for detection of corresponding receptors and ligands, screening of new small molecule drugs, and the like. The antibody of the present invention can be used for detection, quantification, purification, etc. of the protein of the present invention.

BACKGROUND ART Cells secrete many proteins extracellularly. These secreted proteins play important roles in cell growth control, differentiation induction, substance transport, biological defense and the like. Since secreted proteins act extracellularly, unlike intracellular proteins, they can be administered to the body by injection or drip, and have potential as medicines. In fact, many human secretory proteins such as interferons, interleukins, erythropoietins and thrombolytic agents are currently used as medicines. In addition, clinical tests are underway for secreted proteins other than these, and application development aiming at pharmaceuticals is being made. It is considered that human cells still produce many unknown secretory proteins, and if these secretory proteins and the gene encoding them are available, new drug development using them can be expected.

On the other hand, the membrane protein plays an important role in the substance transport and signal transduction through the cell membrane as a signal receptor, an ion channel, a transporter and the like. For example, receptors for various cytokines, ion channels for sodium ions, potassium ions, chlorine ions, etc., transporters for sugars, amino acids, etc. are known, and most of them have already been cloned. Abnormalities in these membrane proteins have been found to be associated with many previously unknown diseases. Therefore, if a new membrane protein can be found, it is expected to lead to the elucidation of the causes of many diseases, and the isolation of a new gene encoding the membrane protein is desired.

Conventionally, it has been difficult to purify these secretory proteins and membrane proteins from human cells, and thus many of them have been isolated by the approach from the gene side. A general method is to introduce a cDNA library into a eukaryotic cell, express the cDNA, and then screen cells that secrete or express a protein having a desired activity on the membrane surface. This is an expression cloning method. However, this method can clone only the genes of proteins whose functions are known.

Generally, secretory proteins and membrane proteins have at least one hydrophobic domain inside the protein, and after being synthesized by ribosome, this domain functions as a secretory signal or remains in the phospholipid membrane and remains in the membrane. To be trapped. Therefore, full length cDNA
If the entire nucleotide sequence of A is determined and a highly hydrophobic region exists in the amino acid sequence of the protein encoded by the cDNA, the cDNA is considered to encode a secretory protein or a membrane protein.

(Object of the Invention) The main object of the present invention is to provide a novel human protein having a hydrophobic domain, a DNA encoding this protein, an expression vector for this DNA, and a transformed eukaryotic cell capable of expressing this DNA. , And to provide antibodies against this protein. These and other objects and advantages of the invention will be apparent to those skilled in the art from the following description with reference to the accompanying drawings.

(Summary of the Invention) As a result of intensive studies, the present inventors have completed the present invention by cloning a cDNA encoding a protein having a hydrophobic domain from a human full-length cDNA bank.
That is, the present invention provides a human protein having a hydrophobic domain, the protein including any of the amino acid sequences represented by SEQ ID NOs: 1 to 10. The present invention also provides DNAs encoding the above proteins, such as SEQ ID NO: 11 to SEQ ID NO: 30.
And an expression vector capable of in vitro translation or expression of this DNA in a eukaryotic cell, a transformed eukaryotic cell capable of expressing this DNA and producing the above protein, and Provide an antibody against the protein.

(Detailed Description of the Invention) The protein of the present invention is isolated from human organs, cell lines, etc., a peptide is prepared by chemical synthesis based on the amino acid sequence of the present invention, or a hydrophobic protein of the present invention is used. It can be obtained by a method of producing by recombinant DNA technology using a DNA encoding a sex domain, but a method of obtaining by recombinant DNA technology is preferably used. For example, a protein can be expressed in vitro by preparing RNA from the vector having the cDNA of the present invention by in vitro transcription and performing in vitro translation using this as a template. When the translation region is recombined into an appropriate expression vector by a known method, the protein encoded by prokaryotic cells such as Escherichia coli and Bacillus subtilis and eukaryotic cells such as yeast, insect cells and mammalian cells can be obtained. It can be expressed in large quantities.

When the protein of the present invention is produced by expressing DNA by in vitro translation, the translation region of this cDNA is recombined into a vector having an RNA polymerase promoter to contain an RNA polymerase corresponding to the promoter. When added to an in vitro translation system such as reticulocyte lysate or wheat germ extract, the protein of the present invention can be produced in vitro. Examples of RNA polymerase promoters include T7, T3 and SP6. Vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T7
18, pT7 / 3 19, pBluescript II and the like can be exemplified.
Also, by adding canine pancreatic microsomes or the like to the reaction system, the protein of the present invention can be expressed in a secretory form or in a form incorporated into a microsomal membrane.

When the protein of the present invention is produced by expressing DNA in a microorganism such as Escherichia coli, an origin capable of replicating in the microorganism, a promoter, a ribosome binding site, c
An expression vector in which the translation region of the cDNA of the present invention is recombined into an expression vector having a DNA cloning site, a terminator, etc. is prepared, and a host cell is transformed with this expression vector, and then the obtained transformant is cultured. If this cDNA
It is possible to mass-produce the protein encoded by P. At this time, if a start codon and a stop codon are added before and after an arbitrary translation region for expression, a protein fragment containing the arbitrary region can be obtained. Alternatively, it can be expressed as a fusion protein with another protein. It is also possible to obtain only the protein portion encoded by this cDNA by cleaving this fusion protein with an appropriate protease. Expression vectors for E. coli include pUC system, pBluescript
II, pET expression system, pGEX expression system and the like can be exemplified.

When the protein of the present invention is produced by expressing DNA in eukaryotic cells, the expression region for eukaryotic cells having a promoter, splicing region, poly (A) addition site, etc. in the translation region of this cDNA. If it is recombined into a vector and introduced into a eukaryotic cell,
The protein of the present invention can be secreted or produced as a membrane protein on the surface of a cell membrane. Expression vectors include pKA1, pED6dpc2, pCDM8,
Examples include pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS and pYES2. Examples of eukaryotic cells include cultured mammalian cells such as monkey kidney cell COS7 and Chinese hamster ovary cell CHO,
Saccharomyces cerevisiae, fission yeast, silkworm cells, Xenopus egg cells and the like are generally used, but any eukaryotic cells can be used as long as they can express the present protein. To introduce the expression vector into eukaryotic cells, known methods such as electroporation, calcium phosphate method, liposome method, and DEAE dextran method can be used.

In order to isolate and purify the target protein from the culture after expressing the protein of the present invention in prokaryotic cells or eukaryotic cells, known separation operations can be combined.
For example, treatment with a denaturant such as urea or a surfactant, ultrasonic treatment, enzymatic digestion, salting out or solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.

The protein of the present invention also includes a peptide fragment (5 amino acid residues or more) containing any partial amino acid sequence of the amino acid sequences represented by SEQ ID NOs: 1 to 10. These peptide fragments can be used as an antigen for producing an antibody. Further, among the proteins of the present invention, those having a signal sequence are secreted in the form of a mature protein after the signal sequence is removed. Therefore, these mature proteins fall within the scope of the proteins of the present invention. The N-terminal amino acid sequence of the mature protein can be easily obtained by using the signal sequence cleavage site determination method [JP-A-8-187100]. Also, some membrane proteins become secreted by being processed on the cell surface. Such secretory proteins or peptides also fall within the scope of the protein of the present invention. If a sugar chain binding site is present in the amino acid sequence, a protein with a sugar chain added can be obtained by expressing it in an appropriate eukaryotic cell. Therefore, a protein or peptide to which such a sugar chain is added falls within the category of the protein of the present invention.

The DNA of the present invention includes all DNAs encoding the above proteins. This DNA can be obtained using a method by chemical synthesis, a method by cDNA cloning, or the like.

The cDNA of the present invention can be cloned from, for example, a human cell-derived cDNA library. cDNA is synthesized using poly (A) ⁺ RNA extracted from human cells as a template. The human cells may be cells that have been extracted from the human body by surgery or the like, or cultured cells. The cDNA is the Okayama-Berg method [Okayama, H
． and Berg, P.M. , Mol. Cell. Biol. 2: 161-1
70 (1982)], the Gubler-Hoffman method [Gubler, U .;
and Hoffman, J .; , Gene 25: 263-269 (1983).
, Etc., but in order to efficiently obtain a full-length clone, the capping method as described in the Examples [Kato, S. et al. et al
． , Gene 150: 243-250 (1994)]. A commercially available human cDNA library can also be used. To clone the cDNA of the present invention from a cDNA library, an oligonucleotide is synthesized based on the nucleotide sequence of an arbitrary part of the cDNA of the present invention, and this is used as a probe to colony or plaque high by a known method. Screening by hybridization may be performed. In addition, by synthesizing an oligonucleotide that hybridizes to both ends of the target cDNA fragment, and using this as a primer, the cDNA of the present invention can be prepared from mRNA isolated from human cells by RT-PCR.
Fragments can also be prepared.

The cDNA of the present invention is characterized by containing either the base sequence represented by SEQ ID NO: 11 to SEQ ID NO: 20 or the base sequence represented by SEQ ID NO: 21 to SEQ ID NO: 30. Table 1 shows the respective clone numbers (HP numbers), the cells from which the cDNA clones were obtained, the total number of bases in the cDNA, and the number of amino acid residues in the encoded protein.

[0017]

[Table 1]

A cDNA live prepared from the human cell line or human tissue used in the present invention is prepared by using an oligonucleotide probe synthesized based on the nucleotide sequence of the cDNA described in any of SEQ ID NOs: 11 to 30. By screening a rally, a clone identical to the cDNA of the present invention can be easily obtained.

In general, human genes are frequently found to have polymorphisms due to individual differences. Therefore, SEQ ID NO: 1
A cDNA in which 1 to SEQ ID NO: 30 has one or more nucleotides added, deleted and / or replaced with another nucleotide is also included in the scope of the present invention.

Similarly, a protein having one or more amino acids added, deleted, and / or substituted with another amino acid, which is generated by these changes, also includes the amino acids represented by SEQ ID NOs: 1 to 10. As long as it has the activity of each protein having a sequence, it falls within the scope of the present invention.

The cDNA of the present invention includes a cDNA fragment (10 bp or more) containing any partial base sequence of the nucleotide sequences represented by SEQ ID NO: 11 to SEQ ID NO: 20 or the nucleotide sequences represented by SEQ ID NO: 21 to SEQ ID NO: 30. Is also included. In addition, a DNA fragment consisting of a sense strand and an antisense strand also falls into this category. These DNA fragments can be used as a probe for gene diagnosis.

The antibody of the present invention can be obtained from serum after immunizing an animal using the protein of the present invention as an antigen. As the antigen, a peptide chemically synthesized based on the amino acid sequence of the present invention or a protein expressed in eukaryotic cells or prokaryotic cells can be used.
Alternatively, it can be prepared by introducing the above expression vector for eukaryotic cells into the muscle or skin of an animal by injection or gene gun, and then collecting serum [JP-A-7-313187]. As animals, mice, rats, rabbits, goats, chickens and the like are used. Monoclonal antibodies against the protein of the present invention can be produced by fusing B cells collected from the spleen of an immunized animal with myeloma to prepare a hybridoma.

In addition to the above activities and uses, the polynucleotides and proteins of the present invention exhibit one or more of the following uses or biological activities, including those associated with the assays cited herein. obtain. The use or activity described for a protein of the invention is due to the administration or use of such a protein, or the administration or use of a polynucleotide encoding such a protein (eg,
(Such as in a vector suitable for gene therapy or introduction of DNA).

Research Uses and Uses The polynucleotides provided by the present invention can be used by the research population for a variety of purposes. To express a recombinant protein for analytical, characterization or therapeutic use; the corresponding protein is preferentially expressed (constitutively or at a particular stage of tissue differentiation or development, or in a disease state). either)
As a marker for tissue; as a molecular weight marker for Southern gels; as a chromosomal marker or tag (if labeled) for identification of chromosomes or mapping of related gene locations; potential genetic relative to endogenous DNA sequences in a patient To identify a disorder; as a probe to hybridize to and thus discover a novel related DNA sequence; as an information source to derive PCR primers for genetic fingerprinting; other novel polynucleotides As a probe to "subtract" known sequences in the process of discovering; to select and generate oligomers for attachment to "gene chips" or other supports, including for inspection of expression patterns; DNA To raise anti-protein antibodies using immunization techniques; To as an antigen to induce either, or another immune response eliciting an anti-DNA antibody, it is possible to use a polynucleotide. Where the polynucleotide encodes a protein that binds or potentially binds (eg, as in a receptor-ligand interaction) to another protein, the polynucleotide encoding another protein with which binding occurs. Identify or
Alternatively, the polynucleotides can be used in an interaction trap assay to identify inhibitors of binding interactions (eg, as described in Gyuris et al., Cell 75: 791-803 (1993)).

The proteins provided by the invention may be used in assays to measure biological activity (including in a panel of multiple proteins for high throughput screening); eliciting antibodies or alternative immunity. To elicit a response; as a reagent (including a labeling reagent) in an assay designed to quantitatively measure the level of a protein (or its receptor) in a biological fluid; the corresponding protein is preferred As a marker of tissue expressed either constitutively, or at a particular stage of tissue differentiation or development, or in a disease state; and, of course, for isolating the relevant receptor or ligand It can be used as well. A protein binds or potentially binds to another protein (eg, receptor-
Proteins (as in ligand interactions), the proteins can be used to identify other proteins with which binding occurs, or to identify inhibitors of binding interactions. The proteins involved in these binding interactions can also be used to screen peptides or small molecule inhibitors or agonists of binding interactions.

Any or all of these research applications can be developed in reagent grade or kit format for commercialization as research products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include, but are not limited to, "Molecular Cloning: A
Laboratory Manual ", 2nd Edition, Cold Spring Harbor Laboratory Press, Sambr
ook, J., EF Fritsch and T. Maniatis, 1989, and Methods in Enzy.
mology: Guide to Molecular Cloning Techniques '', Academic Press, Berger,
Includes SL and AR Kimmel ed. 1987.

Nutritional Use The polynucleotides and proteins of the invention can also be used as a nutritional source or supplement. Such uses include, but are not limited to, use as a protein or amino acid supplement, as a carbon source, as a nitrogen source and as a carbohydrate source. In such cases, the protein or polynucleotide of the invention may be added to the diet of a particular organism, or a separate solid or liquid preparation such as in the form of a powder, pill, solution, suspension or capsule. It can be administered as a product. In the case of microorganisms, the protein or polynucleotide of the present invention can be added to the medium in which the microorganisms are cultured.

Cytokines and Cell Proliferation / Differentiation Activity The proteins of the invention may exhibit cytokine activity, cell proliferation activity (either induction or inhibition) or cell differentiation activity (either induction or inhibition), or It may induce the production of other cytokines in the cell population. Many proteinaceous factors found to date, including all known cytokines, have shown activity in one or more factor-dependent cell proliferation assays, thus making the assay convenient for cytokine activity. Acts as a confirmation. The activity of the proteins of the present invention is not limited to cell lines (including, but not limited to 32D, DA2, DA
1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (preB M
+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, T
(Including F-1, Mo7e and CMK) is confirmed by any of a number of conventional factor dependent cell proliferation assays.

[0030]   The activity of the proteins of the invention may be measured, inter alia, by the following method:

Assays for T cell or thymocyte proliferation include, but are not limited to, Current Protocols in Immunology, JE Coligan, AM Kruisbeek, DH M.
argulies, EM Shevach, W Strober, Pub. Greene Publishing Associates a
nd Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Func
tion 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immun.
ol. 137: 34943500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990;
Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli et al., J.
Immunol. 149: 3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 199.
Including those described in 4.

Assays for spleen cell, lymph node cell or thymocyte cytokine production and / or proliferation include, but are not limited to, Polyclonal T cell stimul.
ation, Kruisbeek, AM and Shevach, EM Current Protocols in Immunolo
gy. JEea Coligan, Volume 1, 3.12.1-3.12.14, John Wiley and Sons, Tor
onto.1994; and Measurement of mouse and human Interferon g, Schrei
ber, RD Current Protocols in Immunology. JEea Coligan, Volume 1, 6.8
.1-6.8.8, including those described by John Wiley and Sons, Toronto. 1994.

Assays for hematopoietic and lymphopoietic cell proliferation and differentiation include, but are not limited to, the Measurement of Human and Murine Interleukin 2 and In
terleukin 4, Bottomly, K., Davis, LS and Lipsky, PE Current Protoco
ls in Immunology. JEea Coligan, Vol. 1, 6.3.1-6.3.12, John Wiley a
nd Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173: 1205-1211, 1991; Mor.
eau et al., Nature 336: 690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.
.SA 80: 2931-2938, 1983; Measurement of mouse and human interleukin 6-
Nordan, R. Current Protocols in Immunology. JEea Coligan, Vol. 1, 6
.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. A
cad. Sci. USA 83: 1857-1861, 1986; Measurement of human Interleukin 11
-Bennett, F., Giannotti, J., Clark, SC and Turner, KJ Current Pr
otocols in Immunology. JEea Coligan, Vol. 1, 6.15.1 John Wiley and
Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9-Cia
rletta, A., Giannotti, J., Clark, SC and Turner, KJ Current Protoco
ls in Immunology. JEea Coligan, Volume 1, 6.13.1, John Wiley and Son
s, Toronto. 1991.

Assays for T cell clonal responses to antigens (specifically identifying proteins that affect APC-T cell interactions and direct T cell effects by measuring proliferation and cytokine production) include: Without limitation, Current Protocols in Immunology, JE Coligan, AM Kruisbeek, DH
Margulies, EM Shevach, W Strober, Pub. Greene Publishing Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Fu
nction; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunolo
gic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77: 6091.
-6095, 1980; Weinberger et al., Eur. J. Immun. 11: 405-411, 1981; Takai et al., J.
Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988.

Immunostimulatory or Suppressive Activity The proteins of the present invention may also exhibit immunostimulatory or immunosuppressive activity, including, but not limited to, the activities for which assays are described herein. Proteins are associated with various immunodeficiencies and disorders (severe combined immunodeficiency (SC
ID)), for example, in the regulation of proliferation of T and / or B lymphocytes (upregulation or downregulation),
And in exerting the cytolytic activity of NK cells and other cell populations. These immunodeficiencies may be genetic or may be caused by viral (eg HIV) and bacterial or fungal infections, or may result from autoimmune disorders. More particularly, infectious diseases caused by viruses, bacteria, fungi or other infections (HIV, hepatitis virus, herpes virus, mycobacteria, leishmania spp., Infections by malaria spp. (Including fungal infections) may be treatable using the proteins of the invention. Of course, in this regard, the proteins of the invention may also be useful if boosts to the immune system may be generally desired, ie, in the treatment of cancer.

Autoimmune disorders that can be treated using the proteins of the invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pneumonia, Guean-Barre syndrome, autoimmunity. Thyroiditis, insulin-dependent diabetes mellitus,
Includes myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such proteins of the invention may also be useful in the treatment of allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions for which immunosuppression is desired, including organ transplantation, may also be treatable using the proteins of the invention.

The proteins of the invention can also be used to enable an immune response in a number of ways. Down-regulation may be in the form of blocking or blocking an already ongoing immune response, or may involve preventing induction of an immune response. The function of activated T cells may be inhibited by suppressing the T cell response, or by inducing specific tolerance in the T cells, or both. In general, immunosuppression of T cell responses is an active, non-antigen specific process that requires continuous exposure of T cells to suppressors. Tolerance, including non-responsiveness or induction of anergy in T cells, is generally antigen-specific,
It is distinguished from immunosuppression in that it persists after termination of exposure to tolerizing factors. Operationally, tolerance can be demonstrated by a lack of T cell response upon re-exposure to a specific antigen in the absence of tolerizing factor.

Down-regulation or prevention of one or more antigenic functions, including but not limited to B lymphocyte antigenic functions (such as B7), eg high levels of lymphokines by activated T cells. Prevention of synthesis is useful in the context of tissue, skin and organ transplants and in graft versus host disease (GVHD). For example, blocking T cell function should result in reduced tissue destruction in tissue transplants. Typically, in tissue grafts, graft rejection is initiated through recognition by the T cells as foreign of the tissue, followed by an immune reaction that destroys the graft. Molecules that inhibit or block the interaction of the B7 lymphocyte antigen with its natural ligand (s) on immune cells (a soluble, monomeric peptide alone with B7-2 activity, or another B lymphocyte antigen ( For example, B7-1, B7-
Pre-implantation administration of 3) in combination with a peptide in active monomeric form, or as a blocking antibody), does not transduce the corresponding costimulatory signal, without the natural ligand (s) on immune cells. Can lead to the binding of molecules to. Thus, blocking B lymphocyte antigen function prevents cytokine synthesis by immune cells such as T cells and thus acts as an immunosuppressant. Moreover, lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in the subject. Induction of long-term tolerance by B lymphocyte antigen blocking reagents may avoid the need for repeated administration of these blocking reagents. It may also be necessary to block the function of the B lymphocyte antigen combination in order to achieve sufficient immunosuppression or tolerance in the subject.

The efficacy of certain blocking reagents in preventing organ transplant rejection or GVHD can be evaluated using animal models that predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart transplants in rats and xenogeneic islet cell transplants in mice, see Lenschow et al., Science 257: 789-792.
(1992) and Turka et al., Proc. Natl. Acad. It is used. Furthermore, a mouse model of GVHD (Paul, Fundamental Immunology, Raven Press, New York, 1989,
846-847) in vivo against disease progression.
The effect of blocking lymphocyte antigen function can be determined.

Blocking antigen function may also be therapeutically useful in the treatment of autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive to self tissues and promote the production of cytokines and autoantibodies involved in the pathology of the disease. By preventing activation of autoreactive T cells, symptoms of disease may be reduced or eliminated. Administration of a reagent that blocks T cell costimulation by disrupting the B lymphocyte antigen receptor: ligand interaction is used to inhibit T cell activation and participate in autologous antibodies or T Production of cell-derived cytokines can be prevented. In addition, blocking reagents can induce antigen-specific tolerance of autoreactive T cells that can lead to long-term relief from disease. The efficacy of blocking reagents in preventing or ameliorating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune disease. Examples include mouse experimental autoimmune encephalitis, MRL / lpr / lpr mice or NZ.
Systemic lupus erythematosus in B hybrid mice, mouse autoimmune collagen arthritis, diabetes in NOD mice and BB rats, and mouse experimental myasthenia gravis (Paul, Ed., Fundamental Immunology, Raven Pr.
ess, New York, 1989, 840-856).

Upregulation of antigen function (preferably B lymphocyte antigen function) as a means of upregulating the immune response may also be useful in therapy. Upregulation of an immune response may be in the form of enhancing an existing immune response or inducing an initial immune response. For example, enhancement of immune response via stimulation of B lymphocyte antigen function may be useful in the case of viral infection. In addition, systemic viral diseases such as influenza, common cold, and encephalitis may be ameliorated by systemic administration of the stimulatory form of B lymphocyte antigen.

Alternatively, is the T cell removed from the patient and expressing a peptide of the invention,
The T cells are co-stimulated in vitro with APCs pulsed with a viral antigen, either with stimulatory forms of the soluble peptides of the invention, and the in vitro activated T cells are reintroduced into the patient. May enhance the antiviral immune response in infected patients. Another method of enhancing an anti-viral immune response is to isolate infected cells from a patient and transfect them with a nucleic acid encoding a protein of the invention described herein such that the cells are on their surface. To express all or part of the protein and reintroduce the transfected cells into the patient. Thus, infected cells can deliver costimulatory signals to T cells in vivo, thereby activating T cells.

In another application, upregulation or enhancement of antigen function, preferably B lymphocyte antigen function, may be useful in inducing tumor immunity. Tumor cells transfected with a nucleic acid encoding at least one of the peptides of the invention (eg sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma)
Can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, tumor cells can be transfected to express the peptide combination. For example, an expression vector that directs the expression of tumor cells obtained from a patient with a peptide having a B7-2 like activity alone or in combination with a peptide having a B7-1 like activity and / or a B7-3 like activity. It can be used to transfect ex vivo. The transfected tumor cells are returned to the patient to express the peptide on the surface of the transfected cells. Alternatively, gene therapy techniques can be used to target tumor cells for transfection in vivo.

The presence of the peptides of the invention having the activity of B lymphocyte antigen (s) on the surface of tumor cells is dependent on the sub-requisites to induce a T cell-mediated immune response against the transfected tumor cells. Provides a stimulatory signal to T cells. Further, tumor cells lacking MHC class I or MHC class II molecules, or tumor cells that are unable to re-express a sufficient amount of MHC class I or MHC class II molecules,
MHC class I α chain protein and β ₂ microglobulin protein or M
Transfecting with a nucleic acid encoding all or part of an HC class II α chain protein and an MHC class II β chain protein (eg, a cytoplasmic domain truncation moiety), thereby causing the MHC class I or MHC class II protein to be expressed on the cell surface. Can be expressed on. B lymphocyte antigen (eg, B7-1
, B7-2, B7-3), expression of the appropriate class I or class II MHC in combination with a peptide having activity, induces a T cell mediated immune response against the transfected tumor cells. Optionally, a gene encoding an antisense construct that blocks the expression of MHC class II binding proteins, such as an invariant chain, is co-transfected with DNA encoding a peptide having the activity of a B lymphocyte antigen. Then, the presentation of tumor-associated antigens can be promoted to induce tumor-specific immunity. Therefore, induction of a T cell-mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.

[0045]   The activity of the proteins of the invention may be measured, in particular, by the following method:

Suitable assays for thymocyte or spleen cell cytotoxicity include, but are not limited to, Current Protocols in Immunology, JE Coligan, AM Kruisbeek,
DH Margulies, EM Shevach, W Strober, Pub. Greene Publishing Associ
ates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyt
e Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al.,
Proc. Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Immunol.
128: 1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai.
Et al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512,
1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrm
ann et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-.
1572, 1985; Takai et al., J. Immunol. 137: 3494-3500, 1986; Bowman et al., J. Viro.
logy 61: 1992-1998; Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli.
Et al., Cellular Immunology 133: 327-341, 1991; Brown et al., J. Immunol. 153: 30.
Includes the assays described in 79-3092, 1994.

Assays for T cell-dependent immunoglobulin responses and isotype switching, in particular, identifying proteins that regulate T cell-dependent antibody responses and influence Th1 / Th2 profiles, are not limited. , Maliszewski,
J. Immunol. 144: 3028-3033, 1990; and Assays for B cell function: In vi.
tro antibody production, Mond, JJ and Brunswick, M. Current Protocols
in Immunology JEea Coligan, Vol. 1, 3.8.1-3.8.16, John Wiley and
Includes the assay described in Sons, Toronto. 1994.

The mixed lymphocyte reaction (MLR) assay, particularly identifying proteins that primarily produce Th1 and CTL responses, includes, but is not limited to, Current Pr.
otocols in Immunology, JE Coligan, AM Kruisbeek, DH Margulies, E.
M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Int
erscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.1
9; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 349.
4-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., J.
Immunol. 149: 3778-3783, 1992.

Dendritic cell-dependent assays, particularly those that identify proteins expressed by dendritic cells that activate naive T cells, include, but are not limited to, Guery et al., J.
Immunol. 134: 536-544, 1995; Inaba et al., Journal of Experimental Medicine 1
73: 549-559, 1991; Macatonia et al., Journal of Immunology 154: 5071-5079, 19
95; Porgador et al., Journal of Experimental Medicine 182: 255-260, 1995; Nai.
r et al., Journal of Virology 67: 4062-4069, 1993; Huang et al., Science 264: 961.
-965, 1994; Macatonia et al., Journal of Experimental Medicine 169: 1255-1264.
, 1989; Bhardwaj et al., Journal of Clinical Investigation 94: 797-807, 1994;
And Inaba et al., Journal of Experimental Medicine 172: 631-640, 1990.

Assays for lymphocyte survival / apoptosis, particularly identifying proteins that prevent apoptosis after superantigen induction, and proteins that regulate lymphocyte homeostasis, include, but are not limited to, Darzynkiewicz et al. ,
Cytometry 13: 795-808, 1992; Gorczyca et al., Leukemia 7: 659-670, 1993; Gor
czyca et al., Cancer Research 53: 1945-1951, 1993; Itoh et al., Cell 66: 233-243,
1991; Zacharchuk, Journal of Immunology 145: 4037-4045, 1990; Zamai et al., C.
ytometry 14: 891-897, 1993; Gorczyca et al., International Journal of Oncolog.
y 1: Includes the assay described in 639-648, 1992.

Assays for proteins that affect T cell commitment and early stages of development include, but are not limited to, Antica et al., Blood.
84: 111-117, 1994; Fine et al., Cellular Immunology 155: 111-122, 1994; Galy
Et al., Blood 85: 2770-2778, 1995; Toki et al., Proc. Nat. Acad. Sci. USA 88: 754.
8-7551, 1991.

Hematopoietic regulatory activity The proteins of the invention may be useful in the regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even near-biological activity in favor of colony-forming cells or factor-dependent cell lines is implicated in the regulation of hematopoiesis as follows. For example, supporting the proliferation of erythroid progenitor cells alone or in combination with other cytokines (thereby, for example, treatment of various anemias, or in combination with radiation / chemotherapy, erythroid progenitor cells and / or Or usefulness in use in stimulating the production of erythroid cells); eg granulocytes and monocytes / which are useful for preventing or treating the resulting myelosuppression in combination with chemotherapy Supporting proliferation of myeloid cells such as macrophages (ie, traditional CSF activity); Supporting proliferation of megakaryocytes and consequent platelets, thereby preventing or treating various platelet disorders such as thrombocytopenia. And is generally used instead of or complementary to platelet transfusion); and / or mature to Of the proliferation of hematopoietic stem cells that can become any and all hematopoietic cells (hence various stem cell disorders, usually disorders treated by transplantation including, but not limited to, aplastic anemia and paroxysmal). Stem cell compartments including nocturnal hemoglobinuria, as well as post-irradiation / chemotherapy, as normal cells, either in vivo or ex vivo (ie, combined with bone marrow transplant or peripheral progenitor cell transplant (allogeneic or xenogeneic)) Or show therapeutic utility in genetically manipulating and repopulating for gene therapy).

[0053]   The activity of the proteins of the invention may be measured, in particular, by the following method:

Suitable assays for proliferation and differentiation of various hematopoietic cell lines have been cited above.

Assays for embryonic stem cell differentiation, particularly identifying proteins that affect embryonic hematopoiesis, include, but are not limited to, Johansson et al Cellular Biology.
15: 141-151, 1995; Keller et al., Molecular and Cellular Biology 13: 473-486.
, 1993; McClanahan et al., Blood 81: 2903-2915, 1993.

Assays for stem cell survival and differentiation, particularly identifying proteins that regulate lympho-hematopoiesis, include but are not limited to Methylcellulose colony.
forming assays, Freshney, MG Culture of Hematopoietic Cells. RI Fre
Shney et al. Vol 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama
Et al., Proc. Natl. Acad. Sci. USA 89: 5907-5911, 1992; Primitive hematopoie.
tic colony forming cells with high proliferative potential, McNiece, IK
And Briddell, RA Culture of Hematopoietic Cells. RI Freshney et al.
Vol 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental.
Hematology 22: 353-359, 1994; Cobblestone area forming cell assay, Ploe
macher, RE Culture of Hematopoietic Cells. RI Freshney et al. Vol. 1-21
Page, Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow cultures
in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen,
T. Culture of Hematopoietic Cells. RI Freshney et al. Vol. 163-179, Wile
y-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assa
y, Sutherland, HJ Culture of Hematopoietic Cells. RI Freshney et al. Vo
l 139-162, Wiley-Liss, Inc., New York, NY. 1994.

[0057] Protein tissue growth activity present invention, bone, cartilage, tendon, growth or regeneration of ligaments and / or nerve tissue, and in the compositions used in the repair and replacement of wound healing and tissue, further burns, lacerations and It may also have utility in treating ulcers.

The proteins of the invention that induce cartilage and / or bone growth in situations where bone is not normally formed have applications in the healing of fractures and cartilage damage or defects in humans and other animals. Such preparations using the proteins of the invention may have prophylactic use in reducing closed and open fractures and improving fixation of artificial joints. De novo bone formation induced by osteogenic factors contributes to the repair of congenital, trauma-induced, or oncological resection-induced craniofacial defects and is also useful in cosmetic surgery. is there.

The proteins of the invention may be used in the treatment of periodontal disease and other tooth repair processes. Such agents may provide an environment that attracts osteogenic cells, stimulates proliferation of osteogenic cells, or induces differentiation of osteogenic cell precursors. The proteins of the invention are, for example, by blocking the processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by stimulation of bone and / or cartilage repair or mediated by inflammation or inflammatory processes, It may also be useful in the treatment of osteoporosis or osteoarthritis.

Another category of tissue regeneration activity that can be attributed to the proteins of the present invention is tendon / ligament formation. The proteins of the present invention that induce the formation of tendon / ligament-like tissue or other tissue in a situation where it does not normally form are found in humans and other animals for tendon or ligament lacerations, deformities and other tendon or It has applications in the healing of ligament defects. Such preparations using tendon / ligament-like tissue-derived proteins are for prophylactic use in preventing damage to tendon or ligament tissue, as well as improving fixation of the tendon or ligament to bone or other tissue, and tendon or ligament tissue. May have application in repairing defects. De novo tendon / ligament-like tissue formation induced by the compositions of the present invention contributes to the repair of congenital, trauma-induced, or other origin tendon or ligament defects, It is also useful in cosmetic surgery for attachment or repair. The composition of the present invention attracts tendon or ligament forming cells, stimulates proliferation of tendon or ligament forming cells, induces differentiation of precursors of tendon or ligament forming cells, or returns to in vivo for tissue repair. It may thus provide an environment ex vivo to induce proliferation of tendon / ligament cells or precursors.
The compositions of the present invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The composition may include a suitable matrix and / or sequestering agent as a carrier, as is well known in the art.

The proteins of the invention are involved in the proliferation of nerve cells and the regeneration of nerve and brain tissue involved in degeneration, death or trauma to nerve cells or tissue, ie central and peripheral nervous system diseases and neuropathy and mechanical. And may also be useful in treating traumatic disorders. More particularly, peripheral nervous system disorders such as peripheral nerve injury, peripheral neuropathy and localized neuropathy, as well as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Shy-Drager syndrome. The protein may be used in the treatment of central nervous system disorders. Additional conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders such as spinal cord disorders, head trauma, and cerebrovascular disease (such as stroke). Peripheral neuropathy resulting from chemotherapy or other medical therapies may also be treatable using the proteins of the invention.

The proteins of the present invention are better or more rapid in non-healing wounds including, but not limited to, pressure ulcers, vascular dysfunction-related ulcers, surgical and traumatic wounds, and the like. It can also be useful in facilitating closure.

The protein of the present invention can be used for organ (including pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or myocardium), and blood vessel (including vascular endothelium) tissues. It is expected that they may also exhibit activity for the production or regeneration of other tissues such as, or for promoting the growth of the cells that make up such tissues. Part of the desired effect may be due to the inhibition or modulation of fibrotic scars that allow the regeneration of normal tissue. The proteins of the invention may also exhibit angiogenic activity.

The proteins of the invention may also be useful in the protection or regeneration of the intestine and the treatment of conditions resulting from lung or liver fibrosis, reperfusion injury in various tissues, and systemic cytokine damage.

The proteins of the invention may also be useful in promoting or suppressing differentiation of the tissue from precursor tissues or cells; or inhibiting growth of the tissue.

[0066]   The activity of the proteins of the invention may be measured, inter alia, by the following method:

Assays for tissue-generating activity include, but are not limited to, International Publication WO
95/16035 (bone, cartilage, tendon); WO 95/05846 (nerve, neuron); WO 91/07491 (skin, endothelium).

Assays for wound healing activity include, but are not limited to, Winter, Epid
ermal Wound Healing, pp. 71-112 (Maibach, HI and Rovee, DT), Year Book
Medical Publishers, Inc., Chicago (Eaglstein and Mertz, J. Invest. Derm
atol 71: 382-84 (modified by 1978)).

Activin / Inhibin Activity The proteins of the invention may also exhibit activin or inhibin related activity. Inhibin is characterized by its ability to inhibit the release of follicle stimulating hormone (FSH) and activin is characterized by its ability to stimulate the release of follicle stimulating hormone (FSH). Thus, the protein of the invention, alone or in a heterodimer with a member of the inhibin α family, reduces fertility in female mammals,
Based on inhibin's ability to reduce spermatogenesis in male mammals, it may be useful as a contraceptive. Administration of a sufficient amount of other inhibins can induce infertility in these mammals. Alternatively, the proteins of the present invention, based on the ability of the activin molecule to stimulate FSH release from cells of the anterior pituitary, as a homodimer or as a heterodimer with other protein subunits of the inhibin-β group, conception. It may be useful as a therapeutic agent for induction of activity. See, eg, US Pat. No. 4,798,885. The proteins of the present invention improve the onset of fertility in sexually immature mammals (resulting in
It may also be useful for increasing the reproductive performance of livestock such as cattle, sheep and pigs during their lifetime).

[0070]   The activity of the proteins of the invention may be measured, inter alia, by the following method:

Assays for activin / inhibin activity include, but are not limited to, Vale et al., Endocrinology 91: 562-572, 1972; Ling et al., Nature 321: 779-782,
1986; Vale et al., Nature 321: 776-779, 1986; Mason et al., Nature 318: 659-663, 1
985; Forage et al., Proc. Natl. Acad. Sci. USA 83: 3091-3095, 1986.

Chemotaxis / Chemokinesis Activity The proteins of the present invention can be used in mammalian cells (eg, monocytes, fibroblasts, neutrophils,
It may have chemotactic or chemokinetic activity of T cells, including mast cells, eosinophils, epithelial cells and / or endothelial cells (eg may act as chemokines). Chemotaxis and chemokinesis proteins can be used to recruit or attract a desired cell population to a desired site of action. Chemotaxis or chemokinesis proteins are particularly advantageous for the treatment of wounds and other trauma to tissues as well as the treatment of localized infections. For example, the attraction of lymphocytes, monocytes or neutrophils to the tumor or site of infection may improve the immune response to the tumor or infectious agent.

A protein or peptide has chemotactic activity for a particular population of cells if it can directly or indirectly stimulate the directed orientation or movement of that population of cells. Preferably, the protein or peptide has the ability to directly stimulate the directed movement of cells. Whether a particular protein has chemotactic activity for a cell population can be readily determined by using such protein or peptide in any known assay for cell chemotaxis.

[0074]   The activity of the proteins of the invention may be measured, inter alia, by the following method:

An assay for chemotaxis activity (identifying proteins that induce or prevent chemotaxis) is based on the ability of the protein to induce migration of cells across the membrane, and the ability of one cell population to another. It consists of an assay that measures the ability of a protein to induce the attachment of Suitable assays for movement and attachment include, but are not limited to,
Current Protocols in Immunology, JE Coligan, AM Kruisbeek, DH Marg
ulies, EM Shevach, W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6
.12.1-6.12.28; Taub et al J. Clin. Invest. 95: 1370-1376, 1995; Lind et al APMIS
103: 140-146, 1995; Muller et al. Eur. J. Immunol. 25: 1744-1748; Gruber et al. J.
. of Immunol. 152: 5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-.
1768, 1994.

Hemostatic and thrombolytic activity The proteins of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such proteins are useful in the treatment of a variety of coagulation disorders, including inherited disorders such as hemophilia, or in the treatment of wounds resulting from trauma, surgery or other causes. And is expected to enhance other hemostatic events. The proteins of the invention may also be useful in the treatment and prevention of thrombolysis or inhibition of thrombus formation and the conditions resulting therefrom, such as myocardial infarction and central nervous system vascular infarction (eg stroke).

[0077]   The activity of the proteins of the invention may be measured, inter alia, by the following method:

Assays for hemostasis and thrombolytic activity include, but are not limited to, Li
net et al., J. Clin. Pharmacol. 26: 131-140, 1986; Burdick et al., Thrombosis Res.
45: 413-419, 1987; Humphrey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Pr
ostaglandins 35: 467-474, 1988.

Receptor / Ligand Activity The proteins of the invention may also exhibit activity as inhibitors, agonists of receptors, receptor ligands or receptor / ligand interactions. Examples of such receptors and ligands include, but are not limited to, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and Its ligand (
Cell adhesion molecules (including but not limited to selectins, integrins and their ligands) and receptor / ligand pairs involved in antigen presentation, antigen recognition, and development of cellular and humoral immune responses) Includes. Receptors and ligands are also useful in screening potential peptides or small molecule inhibitors of related receptor / ligand interactions. The proteins of the invention, including but not limited to fragments of the receptor and ligand,
As such, it may be useful as an inhibitor of receptor / ligand interactions.

[0080]   The activity of the proteins of the invention may be measured, inter alia, by the following method:

Suitable assays for receptor-ligand activity include, but are not limited to, Curren
t Protocols in Immunology, JE Coligan, AM Kruisbeek, DH Marguli
es, EM Shevach, W. Strober, Pub. Greene Publishing Associates and W
iley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under stat
ic conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84: 6
864-6868, 1987; Bierer et al., J. Exp. Med. 168: 1145-1156, 1988; Rosenstein.
Et al., J. Exp. Med. 169: 149-160 1989; Stoltenborg et al., J. Immunol. Methods
175: 59-68, 1994; Stitt et al., Cell 80: 661-670, 1995.

Anti-inflammatory activity The proteins of the invention may also exhibit anti-inflammatory activity. Anti-inflammatory activity is the chemotaxis of cells involved in the inflammatory process by providing stimuli to cells involved in the inflammatory response, thereby inhibiting or promoting cell-cell interactions (such as cell adhesion). It can be achieved by inhibiting or promoting sex, by inhibiting or promoting cellular exudation, or by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Inflammatory conditions (including chronic or acute conditions), including but not limited to infections (septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), using proteins exhibiting such activity.
Associated with ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease. Inflammation or TNF or IL-1
Including inflammation resulting from overproduction of cytokines such as The proteins of the invention may also be useful in the treatment of anaphylaxis and hypersensitivity to antigenic substances or materials.

Tumor Inhibitory Activity In addition to the activity described above for immunological treatment or prevention of tumors, the proteins of the invention may exhibit other antitumor activity. The protein may directly or indirectly inhibit tumor growth (eg, via ADCC). A protein inhibits tumor growth by acting on tumor tissue or tumor precursor tissue, thereby inhibiting the formation of tissue necessary to support tumor growth (eg, by inhibiting angiogenesis) By causing the production of other factors, agents or cell types that
Alternatively, it may exhibit tumor inhibitory activity by suppressing, eliminating or inhibiting factors, agents or cell types that promote tumor growth.

Other Activities The proteins of the invention may also exhibit one or more of the following additional activities or effects: Growth of infectious agents, including but not limited to bacteria, viruses, fungi and other parasites. , Inhibition of infection or function or its killing; height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or size or morphology of organs or body parts Effects on body characteristics including (but not limited to) breast augmentation or vice versa, changes in bone morphology or shape; inhibition or enhancement; effects on biorhythm or cardiac cycle or rhythm; male or female Effect on subject's fertility; fed fats, lipids, proteins, carbohydrates, vitamins, minerals, cofactors or other nutritional factors or ingredients (single or multiple) ) Metabolism, catabolism, assimilation, processing, utilization, storage or elimination; appetite, libido, stress, cognition (including cognitive impairment), depression (including depressive disorder) and violent behavior (to these Effects on behavioral characteristics, including (but not limited to); providing analgesic or other pain-relieving effects; promoting differentiation and proliferation of embryonic stem cells in non-hematopoietic lineages; hormones or endocrine activities; Of deficiency and treatment of deficiency-related diseases; treatment of hyperproliferative disorders (such as psoriasis); immunoglobulin-like activity (such as ability to bind antigen or complement); and vaccine compositions Acts as an antigen to elicit an immune response against such a protein or another substance or entity that is cross-reactive with such a protein ability.

Examples Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. The basic manipulations and enzymatic reactions for recombination of DNA are described in the literature ["Molecular Cloning. A Laboratory Man.
ual ", Cold Spring Harbor Laboratory, 1
989]. Unless otherwise specified, the restriction enzymes and various modifying enzymes used were those manufactured by Takara Shuzo. The buffer composition of each enzyme reaction and the reaction conditions were in accordance with the attached instructions. cDNA synthesis is described in the literature [Kato, S. et al. et al. , Gene 15
0: 243-250 (1994)].

(1) Selection of cDNA Encoding Protein Having Hydrophobic Domain As a cDNA library, fibrosarcoma cell line HT-1080cD
The NA library (WO98 / 11217) was used. Also, human thymus mRNA
(Clontech), human kidney mRNA (Clontech), and human umbilical cord blood mRNA were used as cDNA libraries.

A full-length cDNA clone was selected from each library, the entire base sequence was determined, and a homoprotein cDNA bank consisting of the full-length cDNA clone was constructed. Regarding the protein encoded by the full-length cDNA clone registered in the homoprotein cDNA bank, the method of Kyte-Doolittle [K
yte, J & Doolittle, R.Y. F. J. Mol. Biol. 1
57: 105-132 (1982)], the hydrophobic / hydrophilic profile was determined, and the presence or absence of the hydrophobic domain was examined. Clones having a secretory signal or a hydrophobic region that is considered to be a transmembrane domain in the amino acid sequence of the encoded protein were selected as candidate clones.

[0088] (2) using the plasmid vector containing the cDNA of protein synthesis present invention by in vitro translation was carried out in vitro transcription / translation by T N _T rabbit reticulocyte lysate kit (Promega). At this time, [ ³⁵ S] methionine was added, and the expression product was labeled with a radioisotope. Both reactions were performed according to the protocol attached to the kit. Plasmid 2μg, _{T N T} rabbit reticulocyte lysate 12.5 [mu] l, (supplied with the kit) buffer (without methionine) 0.5 [mu] l, amino acid mixture ^{2μl, [35} S] methionine (Amersham) 2 [mu] l (0 .37 MBq / μl), 0.5 μl of T7 RNA polymerase, and 20 U of RNasin in a total reaction volume of 25 μl
The reaction was carried out at 90 ° C for 90 minutes. The experiment in the presence of the membrane system was carried out by adding 2.5 μl of canine pancreatic microsome fraction (Promega) to this reaction system. S to 3 μl of reaction solution
DS sampling buffer (125 mM Tris-HCl buffer, pH 6.8, 12
2 μl of 0 mM 2-mercaptoethanol, 2% SDS solution, 0.025% bromophenol blue, 20% glycerol was added, and the mixture was heated at 95 ° C. for 3 minutes, and then subjected to SDS-polyacrylamide gel electrophoresis. Autoradiography was performed to determine the molecular weight of the translation product.

(3) Expression by COS7 Escherichia coli having the expression vector of the protein of the present invention was cultured in 2 ml of 2 × YT medium containing 100 μg / ml ampicillin at 37 ° C. for 2 hours, and then helper phage M1
3KO7 (50 μl) was added, and the mixture was cultured at 37 ° C. overnight. Single-stranded phage particles were obtained by polyethylene glycol precipitation from the supernatant separated by centrifugation. This was suspended in 100 μl of 1 mM Tris-0.1 mM EDTA, pH 8 (TE).

[0090]   Cultured cells COS7 derived from monkey kidney were modified with Dulbecco's containing 10% fetal bovine serum.
5% CO in Eagle (DMEM) medium_TwoCultured in the presence at 37 ° C. 1x10 ⁵ COS7 cells were seeded in a 6-well plate (Nunc, hole diameter 3 cm) and 5%
CO_TwoIn the presence, the cells were cultured at 37 ° C for 22 hours. After removing the medium, use phosphate buffer to display the cell surface.
The surface was washed, and DMEM (TDM) containing 50 mM Tris-HCl (pH 7.5) was further added.
It was washed again with EM). To this cell, 1 μl of single-stranded phage suspension, DMEM medium
0.6 ml, TRANSFECTAM^TM(IBF Co., Ltd.)
Add 5% CO_TwoIn the presence, it was cultured at 37 ° C for 3 hours. After removing the sample solution,
The cell surface was washed with DMEM, and DMEM containing 10% fetal bovine serum was added to each well 2 times.
Add 5 ml CO_TwoIn the presence, it was cultured at 37 ° C for 2 days. Replace the medium [³⁵S]
Stain or [³⁵After changing to a medium containing [S] methionine, culturing for 1 hour
It was After separating the medium and cells by centrifugation, the proteins in the medium fraction and the cell membrane fraction are separated.
The quality was subjected to SDS-PAGE.

(4) Preparation of Antibody The plasmid vector having the cDNA of the present invention was adjusted to 2 μg / μl with a phosphate buffer (PBS: 145 mM NaCl, 2.68 mM KCl, 8.09 mMN).
a ₂ HPO ₄ , ₂ mM KH ₂ PO ₄ , pH 7.2). Mouse (ICR
Strain) 25 μ of PBS solution of the previously prepared plasmid in the left and right quadriceps femoris
A total of 50 μl each was injected using a 26 gauge needle. The same injection was continued every other week for one month, and then blood was collected. The collected blood is stored at 4 ° C overnight,
After the blood was coagulated, it was centrifuged at 8,000 xg for 5 minutes and the supernatant was taken. NaN ₃ was added to this supernatant so as to be 0.01%, and the mixture was stored at 4 ° C. Antibody production
It was confirmed by immunostaining of COS7 cells into which the corresponding vector was introduced, or by Western blotting using cell lysate or secretory product.

(5) Clone Example <HP03372> (SEQ ID NOS: 1, 11, 21) cDN of clone HP03372 obtained from human thymus cDNA library
When the entire nucleotide sequence of the A insert was determined, a 75 bp 5'untranslated region, 70
It had a structure consisting of a 2 bp ORF and a 531 bp 3'untranslated region. OR
F codes for a protein consisting of 233 amino acid residues, and a putative secretory signal was present at the N-terminus. FIG. 1 shows the hydrophobicity / hydrophilicity profile of the present protein obtained by the method of Kyte-Doolittle. As a result of in vitro translation, a translation product of 25 kDa, which is almost the same as the molecular weight 26,281 predicted from the ORF, was produced. At this time, when microsomes were added, a product of 30 kDa, which is considered to have a sugar chain added, was produced. In the amino acid sequence of this protein, there are two sites where N-glycosylation may occur (34th Asn-Ile-Ser, 9
9th Asn-Asn-Ser) exists. When the (-3, -1) rule, which is a method for predicting cleavage site of secretory signal sequence, is applied, the mature protein is predicted to start from the 20th tyrosine.

When a protein database was searched using the amino acid sequence of the present protein,
It was similar to human gastric lipase (accession number NP_004181). Table 2 shows a comparison of the amino acid sequences of the human protein (HP) of the present invention and human gastric lipase (LP). − Indicates a gap, * indicates the same amino acid residue as the protein of the present invention,
． Represents a protein of the present invention and a similar amino acid residue, respectively. 65 in all areas
． It had 0% homology.

[0094] Table 2 HP MWQLLAAACWMLLLGSMYGYDKKGNNANPEANMNISQIISYWGYPYEEYDVTTKDGYILG    ** **. *... **.. * *. .. ** .. *****. *. ***** ***. *. *. *****. LP MWLLLTMASLISVLGTTHGLFGKLHPGSPEVTMNISQMITYWGYPNEEYEVVTEDGYILE HP IYRIPHGRGCPGRTAPKPAVYLQHGLIASASNWICNLPNNSLAFLLADSGYDVWLGNSRG    . ***. *.. *. * ... *. *. *****. ***. ***. *********. ***. **** ******* LP VNRIPYGKKNSGNTGQRPVVFLQHGLLASATNWISNLPNNSLAFILADAGYDVWLGNSRG HP NTWSRKHLKLSPKSPEYWAFSLDEMAKYDLPATINFIIEKTGQKRLYYVGHSQGTTIAFI    ***. * .. * **. * *. ****. ************. ** .. *****. *. ****** ****. ** LP NTWARRNLYYSPDSVEFWAFSFDEMAKYDLPATIDFIVKKTGQKQLHYVGHSQGTTIGFI HP AFSTNPELAKKIKIFFALAPVVTVKYTQSPMKKLTTLSRRVVKVCDFPSFNLK    ******. ***. **. *. *****. *****. * .. ** ..... * LP AFSTNPSLAKRIKTFYALAPVATVKYTKSLINKLRFVPQSLFKFIFGDKIFYPHNFFDQF

<HP03375> (SEQ ID NO: 2, 12, 22) cDNA of clone HP03375 obtained from human kidney cDNA library
When the entire nucleotide sequence of the A insert was determined, a 59 bp 5'untranslated region, 82
It had a structure consisting of a 2 bp ORF and a 391 bp 3'untranslated region. OR
F codes for a protein consisting of 273 amino acid residues, and a putative secretory signal was present at the N-terminus. FIG. 2 shows the hydrophobicity / hydrophilicity profile of the present protein determined by the Kyte-Doolittle method. As a result of in vitro translation, a translation product of 30 kDa, which is almost the same as the molecular weight 29,598 predicted from the ORF, was produced. At this time, the addition of microsomes produced a 29 kDa product. When the (-3, -1) rule, which is a method for predicting the cleavage site of a secretory signal sequence, is applied, the mature protein is predicted to start at the 23rd alanine.

When a protein database was searched using the amino acid sequence of this protein,
It was similar to the human hypothetical protein (accession number AAB47494).
Table 3 shows a comparison of the amino acid sequences of the human protein (HP) of the present invention and the human virtual protein (HS). -Is a gap, * is the same amino acid residue as the protein of the present invention ,.
Represents a protein of the present invention and a similar amino acid residue, respectively. 35.
It had a homology of 5%.

[0097] Table 3 HP MRGSQEVLLMWLLVLAVGGTEHAYRPGRRVCAVRAHGDPV--SESFVQRVYQP                   .. **.. * .... * .. **. .. *.. **. *. **. * HS MGSRAELCTLLGGFSFLLLLIPGEGAKGGSLRESQGVCSKQTLVVPLHYNESYSQPVYKP HP FLTTCDGHRACSTYRTIYRTAYRRSPGLAPARPRYACCPGWKRTSGLPGACGAAICQPPC    . ** *. *. * ******. **.. *. ...... **. ***... *. ***. ** HS YLTLCAGRRICSTYRTMYRVM-WREVRREVQQTHAVCCQGWKKRHPGALTC-EAICAKPC HP RNGGSCVQPGRCRCPAGWRGDTCQSDVDECSARRGGCPQRCVNTAGSYWCQCWEGHSLSA     *** **. * .. * * .. ** *. *. ***** .... * ... * *****. * * .. * .. HS LNGGVCVRPDQCECAPGWGGKHCHVDVDECRTSITLCSHHCFNTAGSFTCGCPHDLVLGV HP DGTLCVPKGGPPRVAPNPTGVDSAMKEEVQR-LQSRVDLLEEKLQLVLAPLHSLASQALE    ** *.. * ...... *. *.. * *. .. * .. *. * ... * .. .. HS DGRTCMEGSPEPPTSASILSVAVREAEKDERALKQEIHELRGRLE-RLEQWAGQAGAWVR HP HGLP-DPGSLLVHSFQQL ---- GRIDSLSEQISFLEEQLGSCSCKKDS      ** * .. * ... *. **. ***. *.. ***. **. *** ... * HS AVLPVPPEELQPEQVAELWGRGDRIESLSDQVLLLEERLGACSCEDNSLGLGVNHR

In addition, when GenBank was searched using the nucleotide sequence of this cDNA, ES
Those having a homology of 90% or more in T (for example, accession number AA4
48958) was registered, but since it is a partial sequence, it cannot be determined whether it encodes the same protein as the protein of the present invention.

<HP03376> (SEQ ID NOS: 3, 13, 23) When the entire nucleotide sequence of the cDNA insert of clone HP03376 obtained from the human fibrosarcoma cell line HT-1080 cDNA library was determined, 187 bp of 5 ′ untranslated was obtained. The region had a structure consisting of an 849 bp ORF and a 1047 bp 3'untranslated region. The ORF encodes a protein consisting of 282 amino acid residues, and a putative secretory signal was present at the N-terminus and one putative transmembrane domain was present at the C-terminus. FIG. 3 shows the hydrophobicity / hydrophilicity profile of the present protein obtained by the method of Kyte-Doolittle.

When a protein database was searched using the amino acid sequence of this protein,
It was similar to human glycoprotein gp55 (accession number CAA67711). Table 4 shows a comparison of the amino acid sequences of the human protein (HP) of the present invention and the human glycoprotein gp55 (GP). -Is a gap, * is the same amino acid residue as the protein of the present invention ,. Represents a protein of the present invention and a similar amino acid residue, respectively. There was 94.3% homology in all regions.

[0101] Table 4 HP MSGSSLPGALALSLLLVSGSLLPGPGAAQNEPRIVTSEEVIIRESLLPVTLQCNLTSSSH    *******************************************. ****** ********** GP MSGSSLPGALALSLLLVSGSLLPGPGAAQNEPRIVTSEEVIIRDSLLPVTLQCNLTSSSH HP TLMYSYWTRNGVELTATRKNASNMEYRINKPRAEDSGEYHCVYHFVSAPKANATIEVKAA    ********. ********************************************* ********** GP TLMYSYWTKNGVELTATRKNASNMEYRINKPRAEDSGEYHCVYHFVSAPKANATIEVKAA HP PDITGHKRSENKNEGQDAMMYCKSVGYPHPEWIWRKKENGVFEEISNSSGRFFITNKENY    ********************************. ********************* ****. ***** GP PDITGHKRSENKNEGQDAMMYCKSVGYPHPEWMWRKKENGVFEEISNSSGRFFIINKENY HP TELSIVNLQITEDPGEYECNATNSIGSASVSTVLRVRSHLAPLWPFLGILAEIIILVVII    ***. ********************************************** ********** GP TELNIVNLQITEDPGEYECNATNSIGSASVSTVLRVRSHLAPLWPFLGILAEIIILVVII HP VVYEKRKRPDEVPDDDEPAGPMKTNSTNNPKDKNLRQRNTN    *****************************. *********** GP VVYEKRKRPDEVPDDDEPAGPMKTNSTNNHKDKNLRQRNTN

In addition, when GenBank was searched using the nucleotide sequence of this cDNA, ES
Those having a homology of 90% or more in T (for example, accession number AA2
No. 06770) was registered, but since it is a partial sequence, it cannot be determined whether it encodes the same protein as the protein of the present invention.

<HP03377> (SEQ ID NOS: 4, 14, 24) [0103] When the entire nucleotide sequence of the cDNA insert of clone HP03377 obtained from the human fibrosarcoma cell line HT-1080 cDNA library was determined, it was 146 bp of 5'untranslated. The region had a structure consisting of a 717 bp ORF and a 397 bp 3'untranslated region. The ORF encodes a protein consisting of 238 amino acid residues and had three transmembrane domains. Figure 4 shows Kyte-Doo
The hydrophobic / hydrophilic profile of this protein calculated by the method of Little is shown. As a result of the in vitro translation, the molecular weights 26 and 120 expected from the ORF were almost the same 27
A translation product of kDa was produced.

When a protein database was searched using the amino acid sequence of the present protein,
It was similar to the nematode virtual protein 32 kDa (accession number Q09232). Table 5 shows the human protein (HP) of the present invention and the nematode virtual protein 32 kDa (CE
) Shows a comparison of amino acid sequences. -Is a gap, * is the same amino acid residue as the protein of the present invention ,. Represents a protein of the present invention and a similar amino acid residue, respectively. The homology was 48.6% in all regions except the C-terminal side.

[0105] Table 5 HP MSLNEHSMQALSWRKLY-LSRAKLKASS                                    .. * ..... **. * ****. ***** CE PSTAGGGSRNGVGSKEGSVTSLRMPLKKAGDDVDLGHRGELDLSEKYNYDLSRAQLKASS HP RTSALLSGFAMVAMVEVQLDADHDYPPGLLIAFSACTTVLVAVHLFALMISTCILPNIEA    ******. *****. ** *. * .. * *** .... * .. **. ***. ***. ******. * * CE RTSALLAGFAMVCLVE--LQYDQSTPKPLLIVLGVVTSLLVSVHLLALMMSTCILPYMEA HP VSNVHNLNSVKESPHERMHRHIELAWAFSTVIGTLLFLAEVVLLCWVKFLPLKKQPGQPR    .. ... *** .... *. *. * *** ** ****. *. ... *** .. CE TGCTQ ------ DSPHIKLKFYIDLSWLFSTCIGLLLFLVEIGVIFYVKFTAVGYPTAGYI HP PTSKPPASGAAANVSTSGITPGQAAAIASTTIMVPFGLIFIVFAVHFYRSLVSHKTDRQF                                                                  CE TTAMLVPVGVVFVVFSYLIHKNRVSHSLGRFKHKVDTMKQFLDVEANLQKSTLAPSTIRD HP QELNELAEFARLQDQLDHRGDHPLTPGSHYA                                    CE I

In addition, when GenBank was searched using the nucleotide sequence of this cDNA, ES
Those having 90% or more homology in T (for example, accession number W25
Although 208) was registered, it cannot be determined whether it encodes the same protein as the protein of the present invention because it is a partial sequence.

<HP03378> (SEQ ID NOS: 5, 15, 25) cD of clone HP03378 obtained from a human cord blood cDNA library.
When the entire nucleotide sequence of the NA insert was determined, a 281 bp 5'untranslated region,
It had a structure consisting of an ORF of 1119 bp and a 3'untranslated region of 320 bp. The ORF encodes a protein consisting of 372 amino acid residues, and there were 7 putative transmembrane domains. FIG. 5 shows the hydrophobicity / hydrophilicity profile of the present protein obtained by the method of Kyte-Doolittle. In vitro translation resulted in the production of high molecular weight translation products.

When the protein database was searched using the amino acid sequence of this protein,
It had similarities to the human P2Y5-like receptor (accession number AAB66322). Table 6 shows the human protein (HP) of the present invention and human P2Y5-like receptor (
A comparison of the amino acid sequences of P2) is shown. -Is a gap, * is the same amino acid residue as the protein of the present invention ,. Represents a protein of the present invention and a similar amino acid residue, respectively. There was 33.9% homology in all regions.

[0109] Table 6 HP MLANSSSTNSSVLPCPDYRPTHRLHLVVYSLVLAAGLPLN                         *. * .... * .... ... .. *.. ***. *. ** * P2 MGDRRFIDFQFQDSNSSLRPRLGNATANNTCIVD-DSFK--YNLNGAVYSVVFILGLITN HP ALALWVFLRALRVHSVVSVYMCNLAASDLLFTLSLPVRLSYYALHHWPFPDLLCQTTGAI    ... *. ** .... * ..... ***. *****.. ** .. *. **** * ** ... *. P2 SVSLFVFCFRMKMRSETAIFITNLAVSDLLFVCTLPFKIFYNFNRHWPFGDTLCKISGTA HP FQMNMYGSCIFLMLINVDRYAAIVHPLRLRHLRRPRVARLLCLGVWALILVFAVPAARVH    * *. ***. ** *. ***. ***. *. * *. *. *. .. * *** *. * ... *. .. P2 FLTNIYGSMLFLTCISVDRFLAIVYPFRSRTIRTRRNSAIVCAGVWILVLSGGISAS-LF HP RPSRCRYRDLEVRLCFESFSDELWKGRLLPLVLLAEALGFLLPLAAVVYSSGRVFWTLAR    .... ... *** .. *.. **. * .... * .. ** .. ** *. *. * .. **. P2 STTN --- VNNATTTCFEGLSKRVWKTYLSKITIFIEVVGFIIPLILNVSCSSVVLRTLRK HP PDA--TQSQRRRKTVRLLLANLVIFLLCFVPYNSTLAVYGLLRSKLVAASVPARDRVRGV    * .. .... * ..... ..... * .. *******. *. *. *. **. ..... *. *. P2 PATLSQIGTNKKKVLKMITVHMAVFVVCFVPYNSVLFLYALVRSQAITNCF--LERFAKI HP LMVMVL-LAGANCVLDPLVYYFSAEGFRNTLRGLGTPHRARTSATNGTRAALAQSERSAV    ... * **. **. ** .. ***. *. * .... P2 MYPITLCLATLNCCFDPFIYYFTLESFQKSFYINAHIRMESLFKTETPLTTKPSLPAIQE HP TTDATRPDAASQGLLRPSDSHSLSSFTQCPQDSAL                                            P2 EVSDQTTNNGGELMLESTF

In addition, when GenBank was searched using the nucleotide sequence of this cDNA, ES
Those having a homology of 90% or more in T (for example, accession number AA9
Although 93247) was registered, it cannot be determined whether it encodes the same protein as the protein of the present invention because it is a partial sequence.

<HP03379> (SEQ ID NO: 6, 16, 26) cD of clone HP03379 obtained from human cord blood cDNA library
When the entire nucleotide sequence of the NA insert was determined, the 24 bp 5'untranslated region, 4
It had a structure consisting of a 41 bp ORF and a 1772 bp 3'untranslated region.
The ORF encodes a protein consisting of 146 amino acid residues, and there were 9 putative transmembrane domains. FIG. 6 shows the hydrophobicity / hydrophilicity profile of the present protein obtained by the method of Kyte-Doolittle. As a result of in vitro translation, a translation product of 18 kDa, which was slightly larger than the molecular weight of 16,062 predicted from the ORF, was produced.

In addition, when GenBank was searched using the nucleotide sequence of this cDNA, ES
Those having a homology of 90% or more in T (for example, accession number AA6
63042) was registered, but since it is a partial sequence, it cannot be determined whether it encodes the same protein as the protein of the present invention.

<HP03380> (SEQ ID NOs: 7, 17, 27) cD of clone HP03380 obtained from a human cord blood cDNA library.
When the entire nucleotide sequence of the NA insert was determined, a 267 bp 5'untranslated region,
It had a structure consisting of a 909 bp ORF and a 511 bp 3'untranslated region.
The ORF encodes a protein consisting of 302 amino acid residues, and there was a putative transmembrane domain at the N-terminus. FIG. 7 shows the hydrophobicity / hydrophilicity profile of the present protein obtained by the Kyte-Doolittle method. As a result of in vitro translation, a translation product with a molecular weight of 34,178, which is expected from the ORF, and a slightly larger size of 36 kDa was produced.

When a protein database was searched using the amino acid sequence of this protein,
Mouse putative sialyltransferase (accession number CAA07446)
Had similarities with. Table 7 shows a comparison of the amino acid sequences of the human protein (HP) of the present invention and the mouse putative sialyltransferase (MM). -Is a gap, * is the same amino acid residue as the protein of the present invention ,. Represents a protein of the present invention and a similar amino acid residue, respectively. The homology was 89.4% in all regions.

[0115] Table 7 HP MKAPGRLVLIILCSVVFSAVYILLCCWAGLPLCLATCLDHHFPTGSRPTVPGPLHFSGYS    *******. * .. ** .. **** ... ***** **********. *. * ... *. **** ******** MM MKAPGRLLLLTLCILTFSAVCVFLCCWACLPLCLATCLDRHLPAAPRSTVPGPLHFSGYS HP SVPDGKPLVREPCRSCAVVSSSGQMLGSGLGAEIDSAECVFRMNQAPTVGFEADVGQRST    ********. ** *. ******************. **. ****. ********** *. ******* MM SVPDGKPLIRELCHSCAVVSSSGQMLGSGLGAQIDGAECVLRMNQAPTVGFEEDVGQRST HP LRVVSHTSVPLLLRNYSHYFQKARDTLYMVWGQGRHMDRVLGGRTYRTLLQLTRMYPGLQ    ***. *****************. ******. ********************* ********** MM LRVISHTSVPLLLRNYSHYFQHARDTLYVVWGQGRHMDRVLGGRTYRTLLQLTRMYPGLQ HP VYTFTERMMAYCDQIFQDETGKNRRQSGSFLSTGWFTMILALELCEEIVVYGMVSDSYCR    ************************************************** *********. MM VYTFTERMMAYCDQIFQDETGKNRRQSGSFLSTGWFTMILALELCEEIVVYGMVSDSYCS HP EKSHPSVPYHYFEKGRLDECQMYLAHEQAPRSAHRFITEKAVFSRWAKKRPIVFAHPSWR    *** .. ****************** *************************** ******** MM EKSPRSVPYHYFEKGRLDECQMYRLHEQAPRSAHRFITEKAVFSRWAKKRPIVFAHPSWR HP TE MM AK

In addition, when GenBank was searched using the nucleotide sequence of this cDNA, ES
Those having a homology of 90% or more in T (for example, accession number H50
Although 479) was registered, it cannot be determined whether it encodes the same protein as the protein of the present invention because it is a partial sequence.

<HP03396> (SEQ ID NOs: 8, 18, 28) cDNA of clone HP03396 obtained from human kidney cDNA library
When the entire nucleotide sequence of the A insert was determined, the 245 bp 5'untranslated region, 5
It had a structure consisting of an 85 bp ORF and a 133 bp 3'untranslated region. O
RF codes for a protein consisting of 194 amino acid residues, and a putative secretory signal was present at the N-terminus. FIG. 8 shows the hydrophobicity / hydrophilicity profile of the present protein obtained by the method of Kyte-Doolittle. As a result of in vitro translation, a translation product of 23 kDa, which was slightly larger than the molecular weight of 21,417 predicted from the ORF, was produced. At this time, the addition of microsomes produced a 22 kDa product. When the (-3, -1) rule, which is a method for predicting cleavage site of secretory signal sequence, is applied, the mature protein is predicted to start from the 20th serine.

When a protein database was searched using the amino acid sequence of the present protein,
It had similarity with ostrich lysozyme G (accession number P00719). Table 8 shows a comparison of the amino acid sequences of the human protein (HP) of the present invention and ostrich lysozyme G (SC). -Is a gap, * is the same amino acid residue as the protein of the present invention ,. Represents a protein of the present invention and a similar amino acid residue, respectively. There was 42.6% homology in the entire region.

[0119] Table 8 HP MSALWLLLGLLALMDLSESSNWGCYGNIQSLDTPGASCGIGRRHGLNYCGVRASERLAEI                          **** ..... **. **** .... ****** ** .. ** SC RTGCYGDVNRVDTTGASCKSAKPEKLNYCGVAASRKIAER HP DMPYLLKYQPMMQTIGQKYCMDPAVIAGVLSRKSPGDKIL ---- VNMGDRTSMVQ-DPGS    * .. .. * ....... *** *. ******* .. **. * ... * *. *. ... * *. * SC DLQSMDRYKALIKKVGQKLCVDPAVIAGIISRESHAGKALRNGWGDNGNGFGLMQVDRRS HP QAPTS-WISESQVSQTTEVLTTRIKEIQRRFPTWTPDQYLRGGLCAYSGGAG ---- YVRS    . * .. *. * ... *. **. * ... **. ** .. ** **. * *. ** .. ** .. *. * * * SC HKPVGEWNGERHLMQGTEILISMIKAIQKKFPRWTKEQQLKGGISAYNAGPGNVRSYERM HP SQDLSC-DFCNDVLARAKYLKRHGF    ... *. ***. ***. * *. **. SC DIGTTHDDYANDVVARAQYYKQHGY

In addition, when GenBank was searched using the nucleotide sequence of this cDNA, ES
Those having a homology of 90% or more in T (for example, accession number AA4
53324) was registered, but since it is a partial sequence, it cannot be determined whether it encodes the same protein as the protein of the present invention.

<HP10678> (SEQ ID NOs: 9, 19, 29) [0121] When the entire nucleotide sequence of the cDNA insert of clone HP10678 obtained from the human fibrosarcoma cell line HT-1080 cDNA library was determined, it was 228 bp of 5'non-translated. The region had a structure consisting of a 1629 bp ORF and an 810 bp 3'untranslated region. The ORF encodes a protein consisting of 542 amino acid residues, and there were 7 putative transmembrane domains. Kyte in Figure 9
-Shows the hydrophobic / hydrophilic profile of the protein as determined by the method of Doolittle. In vitro translation resulted in the production of high molecular weight translation products.

When the protein database was searched using the amino acid sequence of this protein,
It was similar to the human hypothetical protein KIAA0758 (accession number BAA34478). Table 9 shows the human protein (HP) of the present invention and the human hypothetical protein KIA.
A comparison of the amino acid sequences of A0758 (KI) is shown. -Is a gap, * is the same amino acid residue as the protein of the present invention ,. Represents a protein of the present invention and a similar amino acid residue, respectively. There was 37.9% homology in all regions.

[0123] Table 9 HP MKMKSQATMICCLVFFL                                                              . * KI ISAPINSLLQMAKALIKSPSQDEMLPTYLKDLSISIDKAEHEISSSPGSLGAIINILDLL HP STECSHYRSKIHLKSYSEVANHILDTAAISNWAFIPNK--NASSDLLQSVNLFARQLHIH    ** ... * ... *. * * ** ....... * .... *. **. **. **. * ... *. KI STVPTQVNSEMMTHVLSTV-NVILGKPVLNTWKVLQQQWTNQSSQLLHSVERFSQALQSG HP NNSENIVNELFIQTKGFHINHNTSEKSLNFSMSMNNTTEDILGMVQIPRQELRKLWPNAS    ... ... * .. *... * .. ... *. * * *. *. * ... * KI DSPPLSFSQTNVQMSSTVIKSSHPE --- TYQQRFVFPYFDLWGNVVIDKSYLENL-QSDS HP QAISIAFPTLGAILREAHLQNVSLPRQVNGLVLSVVLPERLQEIILTFEKINKTRNARAQ      ... ***** *** ... *. *. .. * .... *. ** * * ..... .. KI SIVTMAFPTLQAILAQDIQENNFAESLVMTTTVSHNTTMPFR-ISMTF-KNNSPSGGETK HP CVGWHSK ---- KRRWDEKACQMMLDIRNEVKCRCNY--TSVVMSFSILMSSKSMTDKVLD    ** *. .. ** ... * .. .. *. * * .. .. .. ... *... ** KI CVFWNFRLANNTGGWDSSGCYVEEGDGDNVTCICDHLTSFSILMSPDSPDPSSLLGILLD HP YITCIGLSVSILSLVLCLIIEATVWSRVVVTEISYMRHVCIVNIAVSLLTANVWFIIGSH     * ... * .. *****. ** .. **. ** .. *. .. *****. ******. ***. **. ** * .. KI IISYVGVGFSILSLAACLVVEAVVWKSVTKNRTSYMRHTCIVNIAASLLVANTWFIVVA- HP FNIKAQDYNMC ---- VAVTFFSHFFYLSLFFWMLFKALLIIYGILVIFRRMMKSRMMVIG     . * ... *. * **. *** ******. *****. * ... * .. * ... * .. *. KI -AIQDNRYILCKTACVAATFFIHFFYLSVFFWMLTLGLMLFYRLVFILHETSRSTQKAIA HP FAIGYGCPLIIAVTTVAITEPENGYMRPEACWLNWDNTKALLAFAIPAFVIVAVNLIVVL    *. ****** *. *. * .. *. *. * * .. ***** .. *********** .. **. ** .. ... KI FCLGYGCPLAISVITLGATQPREVYTRKNVCWLNWEDTKALLAFAIPALIIVVVNITITI HP VVAVNTQRPSIGSSK-SQDVVIIMRISKNVAILTPLLGLTWGFGIATLIEGTSLTFHIIF    ** ... ***** ... *. ... *** .... ************ .. * .. **. *. *** ** KI VVITKILRPSIGDKPCKQEKSSLFQISKSIGVLTPLLGLTWGFGLTTVFPGTNLVFHIIF HP ALLNAFQGFFILLFGTIMDHKIRDALRMRMSSLKGKSRAAENASLGPTNGSKLMNRQG    *. **. ***. ******. * * ... ** .. *.. *. .... *** .. KI AILNVFQGLFILLFGCLWDLKVQEALLNKFSLSRWSSQHSKSTSLGSSTPVFSMSSPISR

In addition, when GenBank was searched using the nucleotide sequence of this cDNA, ES
Those having a homology of 90% or more in T (for example, accession number AA0
35425) was registered, but since it is a partial sequence, it cannot be determined whether it encodes the same protein as the protein of the present invention.

<HP10688> (SEQ ID NOS: 10, 20, 30) cDNA of clone HP10688 obtained from a human thoracic cDNA library
When the entire nucleotide sequence of the A insert was determined, 173 bp of 5'untranslated region, 8
It had a structure consisting of a 31 bp ORF and a 474 bp 3'untranslated region. O
RF codes for a protein consisting of 276 amino acid residues, and a putative secretory signal was present at the N-terminus and one transmembrane domain was present at the C-terminus. Kyte-
The hydrophobicity / hydrophilicity profile of this protein determined by the method of Doolittle is shown. As a result of in vitro translation, a translation product of 33 kDa, which was slightly larger than the molecular weight of 29,703 predicted from the ORF, was produced.

When a protein database was searched using the amino acid sequence of this protein,
It had similarity with human uroplakin III (accession number AAC34888). Table 10 shows the human protein (HP) of the present invention and human uroplakin III (U).
A comparison of the amino acid sequences of R) is shown. -Is a gap, * is the same amino acid residue as the protein of the present invention ,. Represents a protein of the present invention and a similar amino acid residue, respectively. The homology was 34.3% in all regions except the C-terminal side.

[0127] Table 10 HP MGLPWGQPHLGLQMLLLALNCLRPSLSLELVPYTPQITAWDLEGKVTATTFSLEQPRCVF                   ****. *** .... * * .. * ... * **. **. * *. * UR MPPLWALLALGCLRFGSAVNLQPQLASVT--FATNNPTLTTVALEKPLCMF HP DGLAS--ASDTVWLVVAFSNASRGFQNPETLADIPASPQLL ----- TDGHY--MTLPLSP    *. .. .... *. * * .. * ... .. ... * ... *. *. * ... * * UR DSKEALTGTHEVYLYVLVDSAISRNASVQDSTNTPLGSTFLQTEGGRTGPYKAVAFDLIP HP -DQLPCGDPMAG-SGGAPVL ----- RVGHDHGCHQQP ---- FCNAPLPGPGPYRVKFLLM     .. **. * .... * .... * ***.. *. *. ***** .... ** * .. *. UR CSDLPSLDAIGDVSKASQILNAYLVRVGANGTCLWDPNFQGLCNAPLSAATEYRFKYVLV HP D-TRGSPRAETKWSDPITLHQGKTPGSIDTWPGRRSGSMIVITSILSSLAGLLLLAFLAA    .. * .. * *****. * .. .. **********. ********. **.. ** .. *. * UR NMSTGLVEDQTLWSDPIRTNQLTPYSTIDTWPGRRSGGMIVITSILGSLPFFLLVGFAGA HP STMRFSSLWWPEEAPEQLRIGSFMGKRYMTHHIPPSEAATLPVGCKPGLDPLPSLSP     .... .. UR IALSLVDMGSSDGETTHDSQITQEAVPKSLGASESSYTSVNRGPPLDRAEVYSSKLQD

In addition, when GenBank was searched using the nucleotide sequence of this cDNA, ES
Those having a homology of 90% or more in T (for example, accession number AA4
64826) was registered, but since it is a partial sequence, it cannot be determined whether it encodes the same protein as the protein of the present invention.

INDUSTRIAL APPLICABILITY The present invention relates to a human protein having a hydrophobic domain, DNA encoding the same,
An expression vector for this DNA and a eukaryotic cell expressing this DNA are provided. Since the proteins of the present invention are either secreted or present in the cell membrane, they are considered to be proteins that control cell growth and differentiation. Therefore, the protein of the present invention can be used as a drug such as a carcinostatic agent that is involved in the control of cell growth or differentiation, or as an antigen for producing an antibody against this protein. The DNA of the present invention can be used as a gene diagnostic probe or a gene source for gene therapy. Further, by using this DNA, a large amount of this protein can be expressed. Cells in which these genes have been introduced to express this protein can be used for detection of corresponding receptors and ligands, screening for new low-molecular drugs, and the like. The antibody of the present invention can be used for detection, quantification, purification, etc. of the protein of the present invention.

The present invention also provides genes corresponding to the polynucleotide sequences disclosed herein. A "corresponding gene" is a region of the genome that is transcribed to produce mRNA from which the cDNA polynucleotide sequence is derived, and may include contiguous regions of the genome required for regulated expression of such gene. Therefore, the corresponding gene is
It may include, but is not limited to, coding sequences, 5'and 3'untranslated regions, alternative spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding gene can be isolated according to known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and / or amplification of genes in suitable genomic libraries or other sources of genomic material. An "isolated gene" is a gene that is separated from the flanking coding sequences (if any) that are present in the genome of the organism in which it was isolated.

Organisms having enhanced, reduced or altered expression of the gene (s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired changes in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and / or cleave mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. . Sci. 15 (7): 2
50-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62 (1): 11-22; and Hampe.
l, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1-39; all of which are incorporated herein by reference). Transgenic animals carrying multiple copies of the gene (s) corresponding to the polynucleotide sequences disclosed herein (preferably traits of cells with a genetic construct that is stably maintained in the transformed cells). Produced by transformation) and its progeny. Transgenic animals with altered gene control regions that increase or decrease gene expression levels or alter temporal or spatial patterns of gene expression are also provided (see EP 0 649 464 B1; present). Incorporated by reference in the description). Furthermore, the gene (s) corresponding to the polynucleotide sequences disclosed herein may be mediated by insertion of a foreign sequence into the corresponding gene (s) or to the corresponding gene (s). An organism that is partially or completely inactivated through the deletion of all or part of Partial or complete gene inactivation is mediated by the insertion of a transposable element and preferably by the subsequent incorrect excision (Plasterk, 1992, Bioessays 14 (9): 629-633; Zwaal et al., 1993, Proc.
Natl. Acad. Sci. USA 90 (16): 7431-7435; Clark et al., 1994, Proc. Natl. Acad.
Sci. USA 91 (2): 719-722; all incorporated herein by reference), or preferably via homologous recombination detected by a positive / negative gene selection strategy (Mansour et al., 1988). , Nature 336: 348-352; US Patent No. 5,
Nos. 4,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614,396; 5,616,491.
No. 5,679,523; all of which are incorporated herein by reference). These organisms with altered gene expression are preferably eukaryotes, more preferably mammals. Such organisms interact with the protein product (s) of the corresponding gene (s) for the development of non-human models for the study of disorders involving the corresponding gene (s). It is useful for the development of assay systems for the identification of working molecules. Where the proteins of the invention are membrane bound (eg, receptors), the invention also provides soluble forms of such proteins. In such forms, some or all of the intracellular and transmembrane domains of the protein are deleted so that the protein is completely secreted from the cell in which it is expressed. The intracellular and transmembrane domains of the proteins of the invention can be identified according to known techniques for determining such domains from sequence information.

The proteins and protein fragments of the invention include proteins having an amino acid sequence that is at least 25% (more preferably at least 50%, most preferably at least 75%) the length of the disclosed proteins, It has at least 60% sequence identity with the disclosed protein (more preferably at least 75% identity; most preferably at least 90% or 95% identity). Sequence identity is determined by comparing the amino acid sequences of proteins when aligned to maximize overlap and identity while minimizing sequence gaps. Share at least 75% sequence identity (more preferably at least 85% identity; most preferably at least 95% identity) with any segment of any of the disclosed proteins, preferably 8 or more Also included in the invention are proteins and protein fragments that contain segments containing (more preferably 20 or more, most preferably 30 or more) contiguous amino acids.

Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. As used herein, a "species homolog" is a species of origin different from that of a given protein or polynucleotide, but having significant sequence for the given protein or polynucleotide as determined by one of skill in the art. A protein or polynucleotide having similarities. Species homologs may be isolated and identified by making appropriate probes or primers from the sequences provided herein and screening for an appropriate nucleic acid source from the desired species.

The present invention provides allelic variants of the disclosed polynucleotides or proteins,
That is, it also encompasses the naturally occurring alternative forms of the isolated polynucleotide which encodes a protein which is identical, homologous or related to the protein encoded by the polynucleotide.

The present invention also includes polynucleotides having sequences complementary to the sequences of the polynucleotides disclosed herein.

The present invention hybridizes to a polynucleotide described herein under conditions of reduced stringency, more preferably under stringent conditions, most preferably under highly stringent conditions. The polynucleotide obtained is also included. Examples of stringency conditions are shown in the table below: Highly stringent conditions are at least as stringent as, for example, conditions AF; stringent conditions are at least, for example, conditions GL. As stringent; conditions of reduced stringency are at least as stringent as conditions MR, for example.

[0137]

[Table 2]

⁽¹⁾ : Hybrid length is the expected length for the hybridizing region (s) of the hybridizing polynucleotide. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence,
Hybrid length is assumed to be the length of the hybridizing polynucleotide. When hybridizing a polynucleotide of known sequence, the hybrid length can be determined by aligning the sequences of the polynucleotide and identifying the region (s) of optimal sequence complementarity. ⁽²⁾ : SSPE (1 x S in hybridization and wash buffer ⁾
SPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1.25 mM
EDTA, pH 7.4) can be replaced with SSC (1 × SSC is 0.15M NaCl and 15 mM sodium citrate); after hybridization is complete, a wash is performed for 15 minutes. ^* T _B ~T _R: The hybridization temperature for hybrids length is expected to less than 50 base pairs in 5 than the hybrid melting temperature _{(T m)}
Should be 10 ° C lower. T _m is determined according to the following equation: 18 in length
For hybrids with less than base pairs, T _m (° C.) = 2 (A + T bases) +4 (
G + C base number). For hybrids of 18-49 base pairs in length, T
m (° C.) = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C
)-(600 / N), N is the number of bases in the hybrid and [Na ⁺ ] is the sodium ion concentration in the hybridization buffer ([Na ⁺ ] for 1 × SSC = 0.165M. ).

Further examples of stringency conditions for polynucleotide hybridization are described in Sambrook, J., EF Fritsch, and T. Maniatis, 1989, Mol.
ecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press
, Cold Spring Harbor, NY, Chapters 9 and 11, and Current Protocols in Mol
ecular Biology, 1995, edited by FM Ausubel et al., John Wiley & Sons, Inc., 2.10 and Sections 6.3-6.4, incorporated herein by reference.

Preferably, each such hybridizing polynucleotide has at least 25% (more preferably at least 50%, most preferably at least 75% of the length of the polynucleotide of the invention with which it hybridizes. %) And at least 6 with the polynucleotide of the invention with which it hybridizes.
Have 0% sequence identity (more preferably at least 75% identity; most preferably at least 90% or 95% identity). Sequence identity is determined by comparing the sequences of polynucleotides that hybridize when aligned to maximize overlap and identity while minimizing sequence gaps.

[Sequence list]

[Brief description of drawings]

1 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP03372.

FIG. 2 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP03375.

FIG. 3 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP03376.

FIG. 4 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP03377.

FIG. 5 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP03378.

FIG. 6 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP03379.

FIG. 7 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP03380.

FIG. 8 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP03396.

FIG. 9 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP10678.

FIG. 10 shows the hydrophobic / hydrophilic profile of the protein encoded by clone HP10688.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12N 5/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR) , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, E, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF F term (reference) 4B024 AA01 AA03 AA11 BA80 CA02 CA04 DA02 EA03 EA04 GA11 GA13 GA30 HA20 4B065 AA58X AA72X AAX AA93Y AB01 AC14 BA02 BB01 CA24 CA44 CA46 4H045 AA10 AA11 AA20 BA10 CA40 DA75 EA20 EA50 FA71 FA74

Claims (7)

[Claims] 1. A protein containing any of the amino acid sequences represented by SEQ ID NOs: 1 to 10. 2. An isolated D encoding any of the proteins of claim 1.
NA. 3. An isolated cDNA containing any of the nucleotide sequences represented by SEQ ID NOs: 11 to 20. 4. The cDNA according to claim 3, which comprises any of the nucleotide sequences represented by SEQ ID NOs: 21 to 30. 5. An expression vector capable of in vitro translation or expression in eukaryotic cells of the DNA according to any one of claims 2 to 4. 6. Expressing the DNA according to any one of claims 2 to 4,
A transformed eukaryotic cell capable of producing the protein according to claim 1. 7. An antibody against the protein according to claim 1.