JP2001519151A - CDNA encoding human protein with transmembrane domain - Google Patents

Abstract

  (57) [Summary] The present invention provides a cDNA encoding a human protein having a transmembrane domain, and a eukaryotic cell expressing the cDNA. The cDNA of the present invention can be used as a probe for gene diagnosis or a gene source for gene therapy. Further, by using the cDNA, the protein can be expressed in a large amount. Cells into which these membrane protein genes have been introduced to express membrane proteins in large amounts can be applied to detection of corresponding ligands, screening of new low-molecular-weight drugs, and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD [0001] The present invention relates to a cDNA encoding a human protein having a transmembrane domain, and a eukaryotic cell expressing the cDNA. The human cDNA of the present invention can be used as a probe for gene diagnosis or a gene source for gene therapy. In addition, it can be used as a gene source for mass-producing the protein encoded by the cDNA. Cells expressing the cDNA can be used for detection of a corresponding ligand, screening of a new low-molecular-weight drug, and the like.

(Background Art) [0002] Membrane proteins play important roles as signal receptors, ion channels, transporters, and the like in transporting substances and transmitting information through cell membranes.
For example, receptors for various cytokines, ion channels for sodium ions, potassium ions, chloride ions, and the like, transporters for sugars, amino acids, and the like are known, and many of them have already been cloned.

[0003] These membrane protein abnormalities have been found to be associated with many diseases of unknown origin. For example, a gene for a membrane protein having 12 transmembrane domains has been identified as a causative gene for cystic fibrosis [Rommens, J. et al. M
. et al. , Science 245: 1059-1065 (1989).
)]. In addition, several membrane proteins have been found to function as receptors when viruses infect cells. For example, HIV-1 has been shown to infect cells intracellularly via the membrane protein on the T cell membrane, the CD4 antigen and the membrane protein Fugin, which has seven transmembrane domains [Feng, Y. et al. et al. , Sc
issue 272: 872-877 (1996)]. Therefore, the discovery of a new membrane protein is expected to lead to the elucidation of the causes of many diseases, and the isolation of a new gene encoding a membrane protein is desired.

[0004] Conventionally, membrane proteins have been difficult to purify, and thus many have been isolated by a gene approach. A general method is to introduce a cDNA library into eukaryotic cells, express the cDNA, and then express the cell expressing the target membrane protein on the membrane using an immunological technique using an antibody. This is a so-called expression cloning in which a change in the permeability of a membrane is detected by a physiological method. However, this method can clone only genes of known membrane proteins.

[0005] In general, a membrane protein has a hydrophobic transmembrane domain inside the protein, and after being synthesized by ribosomes, this domain stays in the phospholipid membrane and is trapped in the membrane. Therefore, the entire base sequence of the full-length cDNA is determined, and if a highly hydrophobic transmembrane domain is present in the amino acid sequence of the protein encoded by the cDNA, the cDNA encodes a membrane protein. Conceivable.

(Disclosure of the Invention) It is an object of the present invention to provide a novel human protein having a transmembrane domain, a DNA encoding the protein, and a transformed eukaryotic cell capable of expressing the cDNA.

As a result of intensive studies, the present inventors have cloned a cDNA encoding a protein having a transmembrane domain from a human full-length cDNA bank and completed the present invention.
That is, the present invention provides a cDNA encoding a human protein having a transmembrane domain,
For example, a cDNA containing any of the nucleotide sequences represented by SEQ ID NO: 1 to SEQ ID NO: 2
And a transformed eukaryotic cell capable of expressing the cDNA.

(Best Mode for Carrying Out the Invention) The cDNA of the present invention can be cloned, for example, from a cDNA library derived from human cells. cDNA is synthesized using poly (A) ⁺ RNA extracted from human cells as a mold. The human cells may be cells extracted from a human body by surgery or the like or cultured cells. The cDNA was prepared according to the Okayama-Berg method [Okayama, H.
. and Berg, P.M. , Mol. Cell. Biol. 2: 161-1
70 (1982)], Gubler-Hoffman method [Gubler, U.S.A.
and Hoffman, J .; , Gene 25: 263-269 (1983).
], But in order to obtain a full-length clone efficiently, the capping method [Kato, S. et al. et al
. , Gene 150: 243-250 (1994)].

[0009] The primary selection of cDNA encoding a human protein having a transmembrane domain comprises c
Partial base sequence determination of a cDNA clone arbitrarily selected from a DNA library,
The amino acid sequence encoded by the nucleotide sequence is determined, and the presence or absence of a hydrophobic portion in the obtained N-terminal amino acid sequence region is confirmed. Next, a second selection is performed by determining the entire nucleotide sequence by sequencing and expressing the protein by in vitro translation.

[0010] The cDNA of the present invention is characterized by containing either the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or the nucleotide sequence represented by SEQ ID NO: 3 to SEQ ID NO: 4. The clone numbers (HP numbers), the cells from which the cDNA clones were obtained, the total number of bases of the cDNA, and the number of amino acid residues of the encoded protein are summarized in Table 1.

[0011]

[Table 1]

[0012] A cDNA library prepared from the human cell line or human tissue used in the present invention was prepared using an oligonucleotide probe synthesized based on the nucleotide sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 2. By screening the rally, a clone identical to the cDNA of the present invention can be easily obtained.

In general, polymorphism due to individual differences is frequently observed in human genes. Therefore, SEQ ID NO: 1
To SEQ ID NO: 4, cDNAs in which one or more nucleotides have been added, deleted and / or substituted with other nucleotides are also included in the scope of the present invention.

The cDNA of the present invention includes a cDNA fragment (10 bp or more) containing the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or any partial nucleotide sequence represented by SEQ ID NO: 3 to SEQ ID NO: 4 Is also included. Also, a DNA fragment consisting of a sense strand and an antisense strand is included in this category. These DNA fragments can be used as probes for gene diagnosis.

When the cDNA of the present invention is expressed in eukaryotic cells, the translation region of the cDNA is recombined into an expression vector for eukaryotic cells having a promoter, a splicing region, a poly (A) addition site, and the like. When introduced into eukaryotic cells, the cDNA of the present invention can be expressed on the cell membrane surface as a membrane protein. As an expression vector,
pKA1, pED6pdc2, pCDM8, pSVK3, pMSG, pSVL,
Examples include pBK-CMV, pBK-RSV, EBV vector, pRS, pYES2 and the like. Eukaryotic cells include monkey kidney cells COS7, cultured mammalian cells such as Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells,
Xenopus egg cells and the like are generally used, but any eukaryotic cells may be used as long as they can express the present cDNA on the membrane surface. In order to introduce the expression vector into eukaryotic cells, known methods such as an electroporation method, a calcium phosphate method, a liposome method, and a DEAE dextran method can be used.

In addition to the activities and uses described above, the polynucleotides and proteins of the invention exhibit one or more of the following uses or biological activities, including those associated with the assays cited herein. obtain. The uses or activities described with respect to the proteins of the present invention may be due to the administration or use of such proteins, or the administration or use of polynucleotides encoding such proteins (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 for various purposes by a research population. To express a recombinant protein for analysis, 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 chromosome identification or mapping of relevant gene locations; To identify disorders; as a probe to hybridize to and thus discover novel related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; other novel polynucleotides As a probe to "subtract" a known sequence in the process of discovering; to select and create oligomers for attachment to a "gene chip" or other support (including for testing 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. If the polynucleotide encodes a protein that binds or potentially binds to another protein (e.g., as in a receptor-ligand interaction), the polynucleotide encoding the other protein with which binding occurs can be used. Identify or
Alternatively, the polynucleotide can be used in an interaction trap assay to identify inhibitors of the binding interaction (eg, as described in Gyuris et al., Cell 75: 791-803 (1993)).

The proteins provided by the present invention can be used in assays to measure biological activity (including in large panels of proteins for high-throughput screening); 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 tissue marker (either constitutively or at a particular stage of tissue differentiation or development, or in a disease state); and, of course, to isolate the relevant receptor or ligand It can be used as well. A protein binds or potentially binds to another protein (eg, a receptor-
If so (as in a ligand interaction), the protein can be used to identify other proteins with which binding occurs, or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for binding interaction peptides or small molecule inhibitors or agonists.

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

[0020] 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 eds. 1989, and "Methods in Enzy
mology: Guide to Molecular Cloning Techniques, '' Academic Press, Berger,
Includes SL and AR Kimmel eds. 1987.

Nutritional Uses The polynucleotides and proteins of the present 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, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the protein or polynucleotide of the invention can be added to the diet of the particular organism, or can be 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 a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured.

Cytokines and Cell Proliferation / Differentiation Activity The proteins of the present invention can exhibit cytokine activity, cell proliferation activity (either induction or inhibition) or cell differentiation activity (either induction or inhibition), or a specific activity. It can 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 protein of the present invention can be measured in 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) for any of a number of conventional factor-dependent cell proliferation assays.

The activity of the protein of the invention may be measured, inter alia, by the following method:

Assays for proliferation of T cells or thymocytes include, but are not limited to, Current Protocols in Immunology, JE Coligan, AM Kruisbeek, DH M
argulies, EM Shevach, W Strober, Pub.Green 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.
4 described above.

Assays for cytokine production and / or proliferation of spleen cells, lymph node cells or thymocytes include, but are not limited to, Polyclonal T cell stimulants.
ation, Kruisbeek, AM and Shevac, EM Current Protocols in Immunolo
gy. JEea Coligan 1st volume 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. Ed. JEea Coligan Volume 1 6.8
.1-6.8.8, John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic and lymphocyte producing cells include, but are not limited to, Measurement of Human and Murine Interleukin 2 and In
terleukin 4, Bottomly, K., Davis, LS and Lipsky, PE Current Protoco
ls in Immunology. Ed. JEea Coligan, Vol. 1, pages 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 page 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, Vol. 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, Without limitation, Current Protocols in Immunology, JE Coligan, AM Kruisbeek, DH
Margulies, EM Shevach, W Strober, Pub.Green 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; including those described in 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 described herein for assays. The protein is used in a variety of immunodeficiencies and disorders (severe combined immunodeficiency (SC
ID), for example, in the regulation of T and / or B lymphocyte proliferation (up-regulation or down-regulation)
And may be useful in exerting the cytolytic activity of NK cells and other cell populations. These immunodeficiencies can be genetic or caused by viral (eg, HIV) and bacterial or fungal infections, or can result from autoimmune disorders. More particularly, infectious diseases caused by viruses, bacteria, fungi or other infections (HIV, hepatitis virus, herpes virus, mycobacteria, infection by Leishmania spp., Malaria spp., And various diseases such as candidiasis) (Including fungal infections) may be treatable using the proteins of the present invention. Of course, in this regard, the proteins of the invention may also be useful where a boost to the immune system may generally be desired, ie, in the treatment of cancer.

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

The proteins of the present invention can also be used to enable an immune response in a number of ways. Down-regulation may be in a form that inhibits or blocks an immune response already in progress, or may include preventing the induction of an immune response. The function of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. In general, immunosuppression of a T cell response is an active, non-antigen-specific process that requires continuous exposure of the T cell to a suppressor. Tolerance (including unresponsiveness or induction of anergy in T cells) is generally antigen-specific,
It is distinguished from immunosuppression in that exposure to the tolerizing agent persists after cessation. Operationally, tolerance can be demonstrated by the lack of a T cell response upon re-exposure to a specific antigen in the absence of a tolerizing agent.

[0031] Down-regulation or prevention of one or more antigen functions, including but not limited to B-lymphocyte antigen function (eg, 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 transplantation and in graft versus host disease (GVHD). For example, blocking T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue grafts, graft rejection is initiated through the recognition of the tissue piece as foreign by T cells, followed by an immune response that destroys the graft. Molecules that inhibit or block the interaction of B7 lymphocyte antigen with its natural ligand (s) on immune cells (soluble monomeric form of the peptide alone with B7-2 activity, or another B lymphocyte antigen ( For example, B7-1, B7-
The pre-implantation administration of the 3) active monomeric form of the peptide in combination with a monomeric form of the activity or a blocking antibody), without transmitting the corresponding costimulatory signal, can be carried out without transducing the corresponding ligand (s) on the immune cells. Can lead to the binding of the molecule to Blocking B lymphocyte antigen function in this way prevents cytokine synthesis by immune cells such as T cells and thus acts as an immunosuppressant. In addition, the lack of costimulation may also be sufficient to anerge the T cells and thereby induce tolerance in the subject. Induction of long-term tolerance by B lymphocyte antigen blocking reagents may obviate the need for repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of the B lymphocyte antigen combination.

The efficacy of a particular blocking reagent in preventing organ graft rejection or GVHD can be evaluated using animal models that are predictive of efficacy in humans. Examples of suitable systems that can be used include allogeneic heart grafts in rats and xenogeneic islet cell grafts in mice, see Lenschow et al., Science 257: 789-792.
Natl. Acad. Sci. USA, 89: 11102-11105 (1992). It is used. Furthermore, a mouse model of GVHD (Paul, Ed., Fundamental Immunology, Raven Press, New York, 1989,
Pp. 846-847) using B in vivo against disease progression.
The effect of blocking lymphocyte antigen function can be determined.

[0033] Blocking antigen function may also be therapeutically useful in the treatment of autoimmune diseases. Many autoimmune disorders are reactive to self-tissues and are the result of inappropriate activation of T cells that promote the production of cytokines and autoantibodies involved in the pathology of the disease. Preventing activation of self-reactive T cells may reduce or eliminate the symptoms of the disease. Receptors for B lymphocyte antigens: The use of reagents that block T cell costimulation by disrupting the ligand interaction, using autoantibodies or T antibodies that inhibit T cell activation and may be involved in disease processes 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 a blocking reagent in preventing or ameliorating an autoimmune disorder 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, autoimmune collagen arthritis in mice, diabetes in NOD mice and BB rats, and mouse experimental myasthenia gravis (Paul, Ed., Fundamental Immunology, Raven Pr.
ess, New York, 1989, pp. 840-856).

Up-regulation of antigen function (preferably B-lymphocyte antigen function) as a means of up-regulating the immune response may also be useful in therapy. Up-regulation of the immune response may be in a form that enhances an existing immune response or elicits an initial immune response. For example, enhancing the immune response through stimulation of B lymphocyte antigen function may be useful in case of viral infection. In addition, systemic viral diseases such as influenza, common cold, and encephalitis can be ameliorated by systemic administration of a stimulating form of B lymphocyte antigen.

Alternatively, T cells are removed from the patient and express the peptide of the invention,
Alternatively, costimulate the T cells in vitro with APCs pulsed with viral antigens, either together with a stimulatory form of the soluble peptide of the invention, and reintroduce the T cells activated in vitro into the patient This 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 so that the cells are Bringing all or part of the protein into expression and reintroducing the transfected cells into the patient. Thus, the infected cells can deliver a costimulatory signal to the T cells in vivo, thereby activating the T cells.

In another application, up-regulation 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 B7-2-like activity alone or in combination with a peptide having B7-1-like activity and / or B7-3-like activity is provided. Can be used to transfect ex vivo. The transfected tumor cells are returned to the patient and the peptide is expressed on the surface of the transfected cells. Alternatively, tumor cells can be targeted for transfection in vivo using gene therapy techniques.

The presence of a peptide of the invention having the activity of the B lymphocyte antigen (s) on the surface of the tumor cells is indicative of the side effects required to induce a T cell mediated immune response against the transfected tumor cells. A stimulus signal is provided to the T cells. In addition, tumor cells that lack MHC class I or MHC class II molecules, or that cannot reexpress sufficient amounts of MHC class I or MHC class II molecules,
MHC class Iα chain protein and beta ₂ microglobulin protein or M HC Class IIα chain protein and all or part of the MHC class IIβ chain protein (e.g., the cytoplasmic domain shortened moiety) were transfected with a nucleic acid encoding, Thereby, MHC class I or MHC class II protein can be expressed on the cell surface. B lymphocyte antigen (for example, B7-1
, B7-2, B7-3), the expression of appropriate class I or class II MHC in combination with a peptide having the activity of induces a T cell-mediated immune response against the transfected tumor cells. Optionally, co-transfecting a gene encoding an antisense construct, such as an invariant chain, that blocks the expression of an MHC class II binding protein with a DNA encoding a peptide having B lymphocyte antigen activity Thus, the presentation of tumor-associated antigens can be promoted and tumor-specific immunity can be induced. Thus, induction of a T cell-mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.

[0038] The activity of the protein of the invention may be measured, inter alia, by the following method:

Assays suitable 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.Green 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
79-3092, 1994.

Assays for T cell-dependent immunoglobulin responses and isotype switching, particularly, but not limited to, those that regulate T cell-dependent antibody responses and identify proteins that affect Th1 / Th2 profiles , 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 Volume 1 3.8.1-3.8.16, John Wiley and
Includes the assay described in Sons, Toronto. 1994.

The mixed lymphocyte reaction (MLR) assay, which specifically identifies 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.

[0042] Dendritic cell-dependent assays (particularly identifying proteins expressed by dendritic cells that activate naive T cells) include, but are not limited to, Guery et al.
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 the assays described in Inaba et al., Journal of Experimental Medicine 172: 631-640, 1990.

Assays for lymphocyte survival / apoptosis, specifically identifying proteins that prevent apoptosis after superantigen induction and 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
y1: 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 modulatory activity The proteins of the present invention may be useful in regulating hematopoiesis and, consequently, in treating myeloid or lymphoid cell deficiencies. Even near-limit biological activity supporting colony forming cells or factor-dependent cell lines indicates an involvement in the regulation of hematopoiesis, such as: For example, supporting the proliferation of erythroid progenitor cells alone or in combination with other cytokines (e.g., for treating various anemias, or in combination with radiation therapy / chemotherapy, for example. Or for use in stimulating the production of erythroid cells); eg, granulocytes and monocytes / cells useful in combination with chemotherapy to prevent or treat the resulting myelosuppression. Support the growth of myeloid cells such as macrophages (ie, traditional CSF activity); support the growth of megakaryocytes and consequent platelets, thereby preventing or treating various platelet disorders such as thrombocytopenia And is generally used instead of or in addition to platelet transfusion); and / or Support for the growth of hematopoietic stem cells, which can be any and all hematopoietic cells (and thus various stem cell disorders, including but not limited to aplastic anemias and paroxysms Stem cells compartments after irradiation / chemotherapy, either in vivo or ex vivo (ie, in combination with bone marrow transplantation or peripheral progenitor cell transplantation (allogeneic or xenogeneic)) as normal cells Or show therapeutic utility in genetically manipulating and repopulating for gene therapy).

The activity of the protein of the invention may be measured, inter alia, by the following method:

Assays suitable for the growth and differentiation of various hematopoietic cell lines have been cited above.

Assays for embryonic stem cell differentiation, particularly those that identify 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 modulate lympho-hematopoiesis, include, but are not limited to, Methylcellulose colony
forming assays, Freshney, MG Culture of Hematopoietic Cells.RI Fre
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 Bridell, 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, 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.
l Includes assays described in pages 139-162, Wiley-Liss, Inc., New York, NY. 1994.

Tissue Growth Activity The protein of the present invention is useful in compositions used for the growth or regeneration of bone, cartilage, tendon, ligament and / or nervous tissue, as well as wound healing and tissue repair and replacement. It may also have utility in treating ulcers.

The proteins of the present invention that induce cartilage and / or bone growth in situations where bone is not formed normally 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 present invention may be used in the treatment of periodontal disease and other tooth restoration processes. Such an agent may provide an environment that attracts osteogenic cells, stimulates the proliferation of osteogenic cells, or induces the differentiation of osteogenic cell precursors. The proteins of the present invention may be used, for example, through the stimulation of bone and / or cartilage repair or by blocking the process of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory or inflammatory processes. It may also be useful in treating osteoporosis or osteoarthritis.

Another category of tissue regeneration activity that can be attributed to the proteins of the invention is tendon / ligament formation. Proteins of the invention that induce the formation of tendon / ligament-like tissue or other tissue in situations where such tissue is not formed normally are torn, deformed and tendon- or ligament-like of tendons or ligaments in humans and other animals. It has application in the healing of ligament defects. Such preparations using tendon / ligament-like tissue-inducing proteins are useful for prophylactic use in preventing damage to tendon or ligament tissue, as well as for improving fixation of tendon or ligament to bone or other tissue, and for tendon or ligament tissue May have application in the repair of defects against. The 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 tendon or ligament defects of other origin, It is also useful in cosmetic surgery for attachment or repair. The compositions of the present invention can attract tendon or ligament forming cells, stimulate the growth of tendon or ligament forming cells, induce differentiation of tendon or ligament forming cell precursors, or return to vivo for tissue repair To provide an environment to induce the growth of tendon / ligament cells or progenitors ex vivo.
The compositions of the present invention may also be useful for treating 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 present invention may be involved in the growth of nerve cells and regeneration of nerve and brain tissue, involving degeneration, death or trauma to nerve cells or nerve tissue, ie, central and peripheral nervous system diseases and neuropathies and mechanical And may also be useful in the treatment of traumatic disorders. More specifically, peripheral nervous system disorders such as peripheral nerve injury, peripheral neuropathy and localized neuropathy, and such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Shy-Drager syndrome The proteins may be used for treating central nervous system diseases. Additional conditions that can be treated according to 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 therapy may also be treatable using the proteins of the invention.

The proteins of the present invention can be used to improve or speed up non-healing wounds, including but not limited to pressure ulcers, ulcers associated with vascular dysfunction, wounds due to surgery and trauma, etc. It may also be useful for promoting closure.

The proteins of the present invention can be used in organ (eg, including pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal, or cardiac) and vascular (including vascular endothelium) tissues It is anticipated that they may also exhibit activities for the generation or regeneration of other tissues such as, or for promoting the growth of cells constituting such tissues. Some of the desired effects are obtained by inhibiting or modulating fibrotic scarring, which allows for normal tissue regeneration. The proteins of the present invention may also exhibit angiogenic activity.

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

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

The activity of the protein of the invention may be measured, inter alia, by the following method:

Assays for tissue generating activity include, but are not limited to, WO WO
95/16035 (bones, cartilage, tendons); WO95 / 05846 (nerves, neurons); WO91 / 07471 (skin, endothelium).

Assays for wound healing activity include, but are not limited to, Winter, Epid
ermal Wound Healing, 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 present invention may also exhibit activin or inhibin-related activity. Inhibin is characterized by its ability to inhibit follicle stimulating hormone (FSH) release, and activin is characterized by its ability to stimulate follicle stimulating hormone (FSH) release. Thus, the protein of the invention, alone or in a heterodimer with a member of the inhibin alpha family, reduces fertility in female mammals,
Based on the ability of inhibin to reduce spermatogenesis in male mammals, it may be useful as a contraceptive. Administration of a sufficient amount of another inhibin can induce infertility in these mammals. Alternatively, a protein of the invention may be used as a homodimer or as a heterodimer with other protein subunits of the inhibin-β group, based on the ability of activin molecules to stimulate FSH release from cells of the anterior pituitary gland. It may be useful as a function-inducing therapeutic. See, for example, U.S. Pat. No. 4,798,885. The protein of the present invention enhances the onset of fertility in a sexually immature mammal (so that
To increase the reproductive performance of livestock such as cattle, sheep and pigs during their lifetime).

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

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

The activity of the protein of the invention may be measured, inter alia, by the following method:

Assays for chemotactic activity (identifying proteins that induce or prevent chemotaxis) are based on the ability of a protein to induce the migration of cells across a membrane, and the ability of one cell population to the other Consists of an assay that measures the ability of the protein to induce the attachment of DNA. Assays suitable 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.Green 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 present invention may also exhibit hemostatic or thrombolytic activity. As a result, such proteins are useful in the treatment of various clotting disorders, including hereditary 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 present invention may also be useful in treating and preventing thrombolysis or inhibition of thrombus formation and conditions resulting therefrom, such as myocardial infarction and central nervous system infarction such as stroke.

The activity of the protein of the invention may be measured, inter alia, by the following method:

Assays for hemostatic 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 Proteins of the invention may also exhibit activity as receptors, receptor ligands or inhibitors or agonists of 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 (
Without limitation, cell adhesion molecules (such as selectins, integrins and their ligands) and receptor / ligand pairs involved in antigen presentation, antigen recognition, and the development of cellular and humoral immune responses Is included. Receptors and ligands are also useful for screening for potential peptide or small molecule inhibitors of the relevant receptor / ligand interaction. The proteins of the present invention, including but not limited to receptor and ligand fragments, are
As such, it may be useful as an inhibitor of receptor / ligand interaction.

The activity of the protein of the invention may be measured, inter alia, by the following method:

Assays suitable 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.Green 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 present invention may also exhibit anti-inflammatory activity. Anti-inflammatory activity inhibits or promotes cell-cell interactions (such as cell adhesion) by providing stimulation to cells involved in the inflammatory response, thereby chemotaxis cells involved in the inflammatory process. It can be achieved by inhibiting or promoting sex, by inhibiting or promoting cell extravasation, or by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Using proteins exhibiting such activity, inflammatory conditions (including, but not limited to, chronic or acute conditions), including but not limited to infections (septic shock, sepsis or systemic inflammatory response syndrome (SIRS))
Related to 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 the overproduction of cytokines such as). The proteins of the invention may also be useful for treating anaphylaxis and hypersensitivity to antigenic substances or materials.

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

Other Activities The proteins of the present 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 Height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or the size or form of an organ or body part Effects on body characteristics (including but not limited to breast augmentation or vice versa, changes in bone morphology or shape) (suppression or enhancement); effects on biorhythms or cardiac cycle or rhythm; male or female Influence on the subject's fertility; fed fats, lipids, proteins, carbohydrates, vitamins, minerals, cofactors or other nutritional factors or ingredients (one or more) ) Effects on metabolism, catabolism, assimilation, processing, utilization, storage or elimination; appetite, libido, stress, cognition (including cognitive impairment), depression (including depressive disorder) and violent behavior (including Providing analgesic or other pain-relieving effects; promoting differentiation and proliferation of embryonic stem cells in non-hematopoietic lineages; hormonal or endocrine activity; Correcting deficiencies in and treating deficiency-related diseases; treating hyperproliferative disorders such as psoriasis; immunoglobulin-like activity such as the ability to bind antigen or complement; and vaccine compositions Elicits an immune response to such a protein or another substance or object that is cross-reactive with such a protein Ability.

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

(1) Preparation of Poly (A) ⁺ RNA As human cells for extracting mRNA, histological lymphoma cell line U937 (ATCC CRL 1593) stimulated with phorbol ester and gastric cancer tissue removed by surgery were used. Using. The cell line was cultured according to a conventional method.

After homogenizing about 1 g of a human cell in 20 ml of a 5.5 M guanidium thiocyanate solution, a reference [Okayama, H .; et al. , "Method
s in Enzymology "Vol.164, Academic Pr
ess, 1987]. This was applied to an oligo dT cellulose column washed with 20 mM Tris-HCl buffer (pH 7.6), 0.5 M NaCl, and 1 mM EDTA to obtain poly (A) ⁺ RNA according to the above-mentioned literature.

(2) Preparation of cDNA Library 10 μg of the above poly (A) ⁺ RNA was dissolved in 100 mM Tris-HCl buffer (pH 8), and 1 unit of alkaline phosphatase derived from bacteria not containing RNase was added thereto. Reacted. After the reaction solution was extracted with phenol, ethanol precipitation was performed.
, 0.1% 2-mercaptoethanol, 0.01% Triton X-100 solution. To this, 1 unit of tobacco-derived acid pyrophosphatase (manufactured by Epicenter Technologies) was added and reacted at 37 ° C. for 1 hour in a total volume of 100 μl.
After phenol extraction of the reaction solution, ethanol precipitation was performed, and the pellet was dissolved in water.
An uncapped poly (A) ⁺ RNA solution was obtained.

The uncapped poly (A) ⁺ RNA, DNA-RNA chimeric oligonucleotide (5′-dG-dG-dG-dG-dA-dA-dT-dT-dC-dG
-DA-GG-A-3 ') in 50 mM Tris-HCl buffer (pH 7.
5), dissolved in 0.5 mM ATP, 5 mM MgCl ₂ , 10 mM 2-mercaptoethanol, 25% aqueous polyethylene glycol solution, added with 50 units of T4 RNA ligase, and reacted at 20 ° C. for 12 hours in a total volume of 30 μl. After the reaction solution was extracted with phenol, ethanol precipitation was performed, and the pellet was dissolved in water to obtain chimeric oligo-capped poly (A) ⁺ RNA.

The vector pKA1 (JP-A-4-117292) developed by the present inventors was digested with KpnI, and about 60 dT tails were added with a terminal transferase. This was digested with EcoRV to remove the dT tail on one side, and used as a vector primer.

After annealing 6 μg of the chimeric oligo-capped poly (A) ⁺ RNA prepared above with 1.2 μg of vector primer, 50 mM Tris-HCl buffer (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ Was dissolved in a 10 mM dithiothreyl, 1.25 mM dNTP (dATP + dCTP + dGTP + dTTP) solution, 200 units of reverse transcriptase (manufactured by GIBCO-BRL) was added, and the mixture was reacted at 42 ° C. for 1 hour in a total volume of 20 μl. After the reaction solution was extracted with phenol, ethanol precipitation was performed, and the pellet was treated with 50 mM Tris-HCl buffer (pH 7.5), 100 mM
NaCl, 10 mM MgCl ₂ , dissolved in 1 mM dithiothreitol solution. To this, 100 units of EcoRI was added and reacted at 37 ° C. for 1 hour in a total volume of 20 μl. After phenol extraction of the reaction solution, ethanol precipitation was performed, and the pellet was
M Tris-HCl buffer (pH 7.5), 100 mM KCl, 4 mM MgCl ₂ , 10 mM (NH ₄ ) ₂ SO ₄ , dissolved in 50 μg / ml bovine serum albumin solution. Escherichia coli DNA ligase (60 units) was added thereto and reacted at 16 ° C. for 16 hours. 2 μl of 2 mM dNTP, 4 units of E. coli DNA polymerase I,
Escherichia coli RNase H (0.1 unit) was added, and the mixture was reacted at 12 ° C for 1 hour and then at 22 ° C for 1 hour.

Next, Escherichia coli DH12S (manufactured by GIBCO-BRL) was transformed using the cDNA synthesis reaction solution. Transformation was performed by electroporation. A part of the transformant was inoculated on 2 × YT agar medium containing 100 μg / ml ampicillin and cultured overnight at 37 ° C. Pick up any colonies that formed on the agar and 100μ
2 ml of 2 × YT medium containing g / ml ampicillin was inoculated and cultured at 37 ° C. overnight. The culture solution was centrifuged, and plasmid DNA was prepared from the cells by alkaline lysis. The plasmid DNA was double-digested with EcoRI and NotI, and then subjected to 0.8% agarose gel electrophoresis to determine the size of the cDNA insert. Further, using the obtained plasmid as a template, a sequence reaction was performed using M13 universal primer labeled with a fluorescent dye and Taq polymerase (a kit manufactured by Applied Biosystems), and then subjected to a fluorescent DNA sequencer (Applied Biosystems). The nucleotide sequence of about 400 bp at the 5 'end of the cDNA was determined. Sequence data was filed as a homoprotein cDNA bank database.

(3) Selection of cDNA Encoding a Protein Having a Transmembrane Domain The base sequence registered in the homoprotein cDNA bank is converted into a three-frame amino acid sequence, and the open reading frame (ORF) starting from the start codon is converted.
). Next, those having a signal sequence specific to a secreted protein at the N-terminus of the portion encoded by the ORF were selected. These clones were subjected to sequencing from both the 5 ′ and 3 ′ directions using the deletion method using Exonuclease III, and the entire nucleotide sequence was determined. For the protein encoded by the ORF, the method of Kyte-Doolittle [Kyte, J & Do
ottle, R .; F. , J. et al. Mol. Biol. 157: 105-132
(1982)], the hydrophobicity / hydrophilicity profile was determined, and the presence or absence of a hydrophobic region was examined. The protein was considered to be a membrane protein if the amino acid sequence of the encoded protein contained a hydrophobic region that appeared to be a transmembrane domain.

[0087] (4) using a 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 reticular erythrocyte lysate kit (Promega). At this time, [ ³⁵ S] methionine was added, and the expression product was labeled with a radioisotope. All reactions were performed according to the protocol attached to the kit. Plasmid 2
The [mu] g, T _N T rabbit reticulocyte lysate 12.5 [mu] l, buffer (supplied with the kit) (methionine-free) 0.5 [mu] l, amino acid mixture 2μl, ^[35 S] methionine (Amersham) 2 [mu] l (0. 37 MBq / μl), T7 RNA polymerase 0.5 μl, RNasin 20 U
Reaction was performed at 90 ° C. for 90 minutes. In 3 μl of the reaction solution, add SDS sampling buffer (12
After adding 2 μl of 5 mM Tris-HCl buffer, pH 6.8, 120 mM 2-mercaptoethanol, 2% SDS solution, 0.025% bromophenol blue, 20% glycerol) and heating at 95 ° C. for 3 minutes, SDS-polyacrylamide It was subjected to gel electrophoresis. Autoradiography was performed to determine the molecular weight of the translation product.

(5) Expression by COS7 100 cells of Escherichia coli (host: JM109) having the expression vector of the protein of the present invention
After culturing at 37 ° C. for 2 hours in 2 ml of 2 × YT medium containing μg / ml ampicillin,
Helper phage M13KO7 (50 μl) was added and cultured at 37 ° C. overnight.
Single-stranded phage particles were obtained from the supernatant separated by centrifugation by polyethylene glycol precipitation. This was added to 100 μl of 1 mM Tris-0.1 mM EDTA, p
Suspended in H8 (TE). As a control, pSSD3 and a vector pKA1-UPA containing full-length cDNA of urokinase [Yokoyama-Koba]
yashi, M .; et al. , Gene 163: 193-196 (199).
5)], a single-stranded phage particle suspension prepared in the same manner as in [1] was used.

Monkey kidney-derived cultured cells COS7 were cultured in Dulbecco's modified Eagle (DMEM) medium containing 10% fetal calf serum at 37 ° C. in the presence of 5% CO ₂ . 1 × 10 ⁵ COS7 cells were seeded on a 6-well plate (Nunc, 3 cm in diameter of the hole), and 5% C
The cells were cultured at 37 ° C. for 22 hours in the presence of O ₂ . After removing the medium, the cell surface was washed with a phosphate buffer, and then DMEM (TDME) containing 50 mM Tris-HCl (pH 7.5).
M) and washed again. 1 μl of a single-stranded phage suspension was added to the cells, and DMEM medium 0
. 6 ml of a suspension of 3 μl of TRANSFECTAM ^™ (IBF) was added, and the cells were cultured at 37 ° C. for 3 hours in the presence of 5% CO ₂ . After removing the sample solution, the cell surface was washed with TDMEM, and 2 ml of DMEM containing 10% fetal bovine serum was added per well.
In addition, the cells were cultured at 37 ° C. for 2 days in the presence of 5% CO ₂ . Furthermore, after 1 hour in medium containing ^[35 S] system in or ^[35 S] methionine, were dissolved collect cells, was subjected to SDS-PAGE, bands observed corresponding to the expression product of each protein Was. For example, HP00991 produced an 18 kDa band in the membrane fraction.

(6) Example of clone <HP00567> (SEQ ID NOS: 1 and 3) Human histiocytic lymphoma cell line U937 cDN stimulated with phorbol ester
The entire base sequence of the cDNA insert of clone HP00567 obtained from the A library was determined. The 5 'untranslated region of 119 bp and the OR of 1431 bp were determined.
F had a structure consisting of a 2020 bp 3 ′ untranslated region. ORF is 476
It encodes a protein consisting of amino acid residues and has at least 9 transmembrane domains. FIG. 1 shows the hydrophobicity / hydrophilicity profile of the present protein determined by the method of Kyte-Doolittle. As a result of in vitro translation, a band corresponding to a molecular weight of 52,264 predicted from the ORF was not observed, and a high-molecular-weight smeared translation product was generated.

When a protein database was searched using the amino acid sequence of the present protein,
It was completely identical with the rat protein transport protein Sec61α subunit (SWISS-PROT accession number P38378). When GenBank was searched using the nucleotide sequence of the present cDNA, ESTs having a homology of 90% or more (for example, accession number AA301007) were found.
Since it is a partial sequence, it cannot be determined whether it encodes the same protein as the protein of the present invention.

The rat protein transport protein Sec61α subunit has an important role in protein transport in the endoplasmic reticulum membrane [Gorlich, D. et al. et al. ,
Cell 71: 489-503 (1992)]. Therefore, this protein is a human homolog of the protein transport protein Sec61α subunit. This cDNA
Can be used to diagnose and treat diseases involving ER dysfunction.

<HP00991> (SEQ ID NOS: 2 and 4) cDN of clone HP00991 obtained from human gastric cancer cDNA library
When the entire nucleotide sequence of the A insert was determined, the 5 'untranslated region of 65 bp, 55
It had a structure consisting of an 8 bp ORF and a 196 bp 3 ′ untranslated region. OR
F encodes a protein consisting of 185 amino acid residues and has four transmembrane domains. FIG. 2 shows the hydrophobicity / hydrophilicity profile of the present protein determined by the method of Kyte-Doolittle. As a result of the in vitro translation, a 21 kDa translation product was generated, which substantially matched the molecular weight predicted from the ORF of 21,080.

When a protein database was searched using the amino acid sequence of the present protein,
It had similarity to rat translocon binding protein γ subunit (SWISS-PROT accession number Q08013). Table 2 shows a comparison of the amino acid sequences of the human protein (HP) of the present invention and the rat translocon-binding protein γ subunit (RN). -Indicates a gap, * indicates the same amino acid residue as the protein of the present invention,. Represents an amino acid residue similar to that of the protein of the present invention. Both are 9 in all areas
It had 8.4% homology.

[0095]

[Table 2]

Further, when GenBank was searched using the nucleotide sequence of the present cDNA,
T having a homology of 90% or more (for example, accession number AA3
17439), but it cannot be determined whether or not it encodes the same protein as the protein of the present invention since it is a partial sequence.

The rat translocon binding protein γ subunit is one of the subunits of the complex involved in the localization of endoplasmic reticulum proteins [Hartmann, E. et al. et al
. , Eur. J. Biochem. 214: 375-381 (1993)].
Therefore, it is considered that this protein plays an important role in protein retention and folding in the endoplasmic reticulum. This cDNA can be used for diagnosis and treatment of diseases involving dysfunction of the endoplasmic reticulum.

(Possibility of Industrial Use) The present invention provides a cDNA encoding a human protein having a transmembrane domain, and a eukaryotic cell expressing the cDNA. The cDNA of the present invention can be used as a probe for gene diagnosis or a gene source for gene therapy. In addition, the DN
By using A, the protein can be expressed in a large amount. Cells into which these membrane protein genes have been introduced to express membrane proteins in large amounts can be applied to detection of corresponding ligands, screening of new low-molecular-weight drugs, and the like.

[0099] 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 an mRNA from which the cDNA polynucleotide sequence is derived, and may include contiguous regions of the genome required for regulated expression of such a gene. Therefore, the corresponding gene is
It may include, but is not limited to, coding sequences, 5 'and 3' untranslated regions, alternatively 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 a suitable genomic library or other source of genomic material. An "isolated gene" is a gene that has been separated from flanking coding sequences, if present, present in the genome of the organism from which it was isolated.

[0100] Organisms having enhanced, reduced or altered expression of the gene (s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind to 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.
1, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1-39; all of which are incorporated herein by reference). Transgenic animals having multiple copies of the gene (s) corresponding to the polynucleotide sequences disclosed herein, preferably a cell construct with a genetic construct that is stably maintained in the transformed cells And its progeny are provided. Also provided are transgenic animals having altered gene regulatory regions that increase or decrease gene expression levels or alter the temporal or spatial pattern of gene expression (see EP 0 649 464 B1; Incorporated by reference in the description). In addition, the gene (s) corresponding to the polynucleotide sequences disclosed herein may be modified by insertion of a foreign sequence (s) into the corresponding gene (s), or by inserting the corresponding gene (s) into the corresponding gene (s). ) Is partially or completely inactivated via deletion of all or part of the organism. Partial or complete gene inactivation can be achieved via insertion of a transposable element and preferably 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 of which are incorporated herein by reference), or preferably via homologous recombination detected by a positive / negative gene selection strategy (Mansour et al., 1988). U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614. No. 5,396,491;
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 may be used to develop non-human models for the study of disorders involving the corresponding gene (s) and to interact with the protein product (s) of the corresponding gene (s). Useful for the development of assay systems for the identification of working molecules.

Where the proteins of the invention are membrane-bound (eg, a receptor), the invention also provides soluble forms of such proteins. In such a form, the protein is deleted by deleting some or all of the intracellular and transmembrane domains of the protein,
Ensure complete secretion from the expressing cell. 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 at least 25% (more preferably at least 50%, most preferably at least 75%) of the length of the disclosed proteins, It has at least 60% sequence identity (more preferably at least 75% identity; most preferably at least 90% or 95% identity) with the disclosed proteins. Sequence identity is determined by comparing the amino acid sequences of proteins when aligned to maximize overlap and identity while minimizing sequence gaps. Shares 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 Proteins and protein fragments containing segments comprising (more preferably 20 or more, most preferably 30 or more) contiguous amino acids are also encompassed by the present invention.

[0103] 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 a different origin than the given protein or polynucleotide, but as determined by one of skill in the art, a significant sequence for a given protein or polynucleotide. A protein or polynucleotide having similarity. Species homologs may be isolated and identified by generating appropriate probes or primers from the sequences provided herein and screening for an appropriate nucleic acid source from the desired species.

The present invention relates to allelic variants of the disclosed polynucleotides or proteins,
That is, it also encompasses other naturally occurring forms of the isolated polynucleotide that encode the same, homologous, or related protein to the protein encoded by the polynucleotide.

The present invention also encompasses polynucleotides having sequences that are complementary to the sequences of the polynucleotides disclosed herein.

The present invention hybridizes to polynucleotides described herein under conditions of reduced stringency, more preferably under stringent conditions, and most preferably under highly stringent conditions. The resulting polynucleotide 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 as, for example, conditions GL. Conditions of reduced stringency are at least as stringent as, for example, conditions M-R.

[0107]

[Table 3]

⁽¹⁾ : The 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, the hybrid length is assumed to be the length of the hybridizing polynucleotide. When hybridizing polynucleotides of known sequence, the hybrid length can be determined by juxtaposing the polynucleotide sequences and identifying the region (s) of optimal sequence complementarity. ⁽²⁾ : In hybridization and washing buffer, SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1.25 mM EDTA, pH 7.4) was converted to SSC (1 × SSC Can be replaced by 0.15 M NaCl and 15 mM sodium citrate); after hybridization is complete, washing is performed for 15 minutes. ^* T _B -T _R : The hybridization temperature for hybrids that are expected to be less than 50 base pairs in length should be 5-10 ° C. below the melting temperature (T _m ) of the hybrid. T _m is determined according to the following equation: For hybrids less than 18 base pairs in length, T _m (° C.) = 2 (A + T bases) +4 (G + C bases). For hybrids between 18 and 49 base pairs in length, the Tm
(° C.) = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) − (600 / N), where N is the number of bases in the hybrid and [Na ⁺ ] is the hybridization The sodium ion concentration in the buffer ([Na ⁺ ] for 1 × SSC = 0.165 M).

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 6.3-6.4, which are incorporated herein by reference.

Preferably, each such hybridizing polynucleotide is at least 25% (more preferably at least 50%, most preferably at least 75%) of the length of the polynucleotide of the invention to which it hybridizes. %) With a polynucleotide of the present invention to which it hybridizes.
Has 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.

[0111]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the hydrophobicity / hydrophilicity profile of the protein encoded by clone HP00567.

FIG. 2 shows the hydrophobicity / hydrophilicity of the protein encoded by clone HP00991.

Claims (4)

[Claims] 1. A cD comprising the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
NA. 2. The cDNA according to claim 1, comprising the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. 3. An expression vector capable of in vitro translation or expression in a eukaryotic cell of the DNA according to claim 1 or 2. 4. A transformed eukaryotic cell capable of expressing the DNA of any one of claims 1 to 2 and producing a protein encoded by the DNA.