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WO1999018200A1 - Proteines ressemblant au facteur h du complement humain et adnc codant ces proteines - Google Patents

Proteines ressemblant au facteur h du complement humain et adnc codant ces proteines Download PDF

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Publication number
WO1999018200A1
WO1999018200A1 PCT/JP1998/004448 JP9804448W WO9918200A1 WO 1999018200 A1 WO1999018200 A1 WO 1999018200A1 JP 9804448 W JP9804448 W JP 9804448W WO 9918200 A1 WO9918200 A1 WO 9918200A1
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WIPO (PCT)
Prior art keywords
protein
cells
proteins
present
cell
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PCT/JP1998/004448
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English (en)
Inventor
Seishi Kato
Shingo Sekine
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Sagami Chemical Research Center
Protegene Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Sagami Chemical Research Center, Protegene Inc. filed Critical Sagami Chemical Research Center
Priority to CA002305386A priority Critical patent/CA2305386A1/fr
Priority to EP98945576A priority patent/EP1021530A1/fr
Priority to AU92819/98A priority patent/AU9281998A/en
Priority to JP2000514998A priority patent/JP2001519152A/ja
Publication of WO1999018200A1 publication Critical patent/WO1999018200A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • the present invention relates to proteins alike to human complement factor H and cDNAs coding for these proteins.
  • the proteins of the present invention can be employed as pharmaceuticals.
  • the human cDNAs of the present invention can be utilized as probes for the gene diagnosis and gene sources for the gene therapy.
  • the cDNAs can be utilized as gene sources for large-scale production of the proteins encoded by said cDNAs .
  • Eucaryotic cells wherein expression vectors of said cDNAs are introduced can be utilized for secretory production of the proteins alike to human complement factor H.
  • Human complement factor H is one of glycoproteins that are abundantly contained in the serum and are involved in the regulation of enzymatic processing of the major complement component C3. Cloning of the cDNA of the human complement factor H, followed by analysis of its amino acid sequence, has revealed that this protein has 20 short consensus repeats (SCR) , each consisting of about 60 amino acid residues [Ripoche, J. et al . , Bioche . J. 29: 593-602 (1988)]. Recently, several cDNAs of proteins, which are related to complement factor H and possess plural SCRs, have been reported and it has been recognized that these proteins are organized as one family [Estaller, C. et al . , J. Immunol.
  • the object of the present invention is to provide novel proteins alike to human complement factor H and cDNAs coding for these proteins.
  • the present inventors have been successful in cloning of human cDNAs coding for proteins alike to human complement factor H, thereby completing the present invention.
  • the present invention provides proteins alike to human complement factor H, namely proteins containing the amino acid sequence represented by Sequence No. 1.
  • the present invention provides cDNAs coding for the above- mentioned proteins and containing the base sequence represented by Sequence No. 2 or Sequence No. 3 as well as transformation eucaryotic cells that are capable of expressing said cDNAs .
  • Figure 1 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP01265.
  • Figure 2 A figure illustrating the results of the dot matrix analysis of the amino acid sequence of the protein encoded by clone HP01265.
  • the proteins of the present invention can be obtained, for example, by a method for isolation from human organs, cell lines, etc., a method for preparation of peptides by the chemical synthesis, or a method for production with the recombinant DNA technology using the DNAs coding for the proteins alike to human complement factor H of the present invention, wherein the method for obtainment by the recombinant DNA technology is employed preferably.
  • in vitro expression can be achieved by preparation of an RNA by the in vitro transcription from a vector having one of cDNAs of the present invention, followed by in vitro translation using this RNA as a template.
  • recombination of the translation region into a suitable expression vector by the method known in the art leads to expression of a large amount of the encoded protein by using prokaryotic cells such as Escheri chia coli , Bacill us subtili s, etc., and eucaryotic cells such as yeasts, insect cells, mammalian cells, etc.
  • prokaryotic cells such as Escheri chia coli , Bacill us subtili s, etc.
  • eucaryotic cells such as yeasts, insect cells, mammalian cells, etc.
  • a recombinant expression vector bearing the translation region in the cDNA of the present invention is constructed in an expression vector having an origin, a promoter, a ribosome-binding site, a cDNA-cloning site, a terminator etc., which can be replicated in the prokaryotic cells, and, after transformation of the host cells with said expression vector, the thus-obtained transformant is incubated, whereby the protein encoded by said cDNA can be produced on a large scale in the prokaryotic cells.
  • a fusion protein with another protein can be expressed.
  • a protein portion coding for said cDNA can be obtained by cleavage of said fusion protein with an appropriate protease.
  • Said fusion protein provided that it possesses the activity alike to human complement factor H, shall come within the scope of the present invention.
  • the protein of the present invention can be produced by extracellular secretion, when the translation region of said cDNA is subjected to recombination to an expression vector for eucaryotic cells that has a promoter, a splicing region, a poly (A) addition site, etc., followed by introduction into the eucaryotic cells.
  • the expression vector is exemplified by pKAl, pED6dpc2, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pYES2, and so on.
  • eucaryotic cells to be used in general include mammalian culture cells such as simian kidney cells COS7, Chinese hamster ovary cells CHO, etc., budding yeasts, fission yeasts, silkworm cells, Xenopus laevi s egg cells, and so on, but any eucaryotic cells may be used, provided that they are capable of effecting secretory expression of the present proteins.
  • the proteins of the present invention include peptide fragments (more than 5 amino acid residues) containing any partial amino acid sequence in the amino acid sequence represented by Sequence No . 1. These peptide fragments can be utilized as antigens for preparation of antibodies.
  • the proteins of the present invention are secreted in an extracellular manner. Since a portion capable of binding sugar chains exists in the amino acid sequence, proteins where sugar chains are added can be obtained by expression in appropriate eucaryotic cells. Accordingly, such proteins or peptides wherein sugar chains are added shall come within the scope of the present invention.
  • the DNAs of the present invention include all DNAs coding for the above-mentioned proteins. Said DNAs can be obtained by using a method by chemical synthesis, a method by cDNA cloning, and so on.
  • the human cDNAs of the present invention can be cloned from cDNA libraries of the human cell origin. These cDNA libraries are constructed by using as templates poly (A) " " RNAs extracted from human cells.
  • the human cells may be cells delivered from the human body, for example, by the operation or may be the culture cells.
  • a poly (A) + RNA isolated from the liver is used in Examples.
  • the cDNAs can be synthesized by using any method selected from the Okayama-Berg method [Okayama, H. and Berg, P., Mol. Cell. Biol.
  • the identification of the cDNAs is carried out by the determination of the whole base sequence by the sequencing, the search of known proteins having sequences analogous to the amino acid sequence predicted from the base sequence, expression of proteins by in vitro translation, and expression by cultured cells .
  • the cDNAs of the present invention are characterized by containing the base sequence represented by Sequence No. 2 or Sequence No. 3.
  • that represented by Sequence No. 3 possesses a 2033-bp base sequence with a 1737-bp open reading frame.
  • This open reading frame codes for a protein consisting of 578 amino acid residues.
  • This protein is partially identical with the protein alike to human complement factor H in the amino acid sequence level.
  • the same clones as the cDNAs of the present invention can be easily obtained by screening of the human cDNA libraries constructed from the human cells by the use of an oligonucleotide probe synthesized on the basis of the cDNA base sequence described in Sequence No. 3.
  • any cDNA that is subjected to insertion or deletion of one or plural nucleotides and/or substitution with other nucleotides in Sequence No. 2 or Sequence No. 3 shall come within the scope of the present invention.
  • any protein that is formed by these modifications comprising insertion or deletion of one or plural amino acids and/or substitution with other amino acids shall come within the scope of the present invention, as far as the protein possesses the activity of the protein alike to human complement factor H .
  • the cDNAs of the present invention include cDNA fragments (more than 10 bp) containing any partial base sequence in the base sequence represented by Sequence No. 3. Also, DNA fragments consisting of a sense chain and an anti-sense chain shall come within this scope . These DNA fragments can be utilized as the probes for the gene diagnosis.
  • polynucleotides and proteins of the present invention may exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA) .
  • the polynucleotides provided by the present invention can be used by the research community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states) ; as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodiesusing DNA immunization
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al . , Cell 75:791-803 (1993) ) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • the proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state) ; and, of course, to isolate correlative receptors or ligands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein 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 peptide or small molecule inhibitors or agonists of the binding interaction. Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
  • Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
  • the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules .
  • the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
  • a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • Many protein factors discovered to date, including all known cytokines have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preBM+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al . , J. Immunol. 137:3494-3500, 1986; Bertagnolli et al . , J. Immunol.
  • Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon ⁇ , Schreiber, R.D. In Current Protocols in Immunology . J.E . e.a. Coligan eds . Vol 1pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al . , Nature 336:690-692, 1988; Greenberger et al . , Proc. Natl . Acad. Sci. U.S.A.
  • Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic 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.
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID) ) , e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SCID severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial orfungal infections, or may result from autoimmune disorders.
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp . and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus- host disease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma
  • T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD) .
  • B lymphocyte antigen functions such as , for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal.
  • a B7 lymphocyte antigen e.g., B7-1, B7-3 or blocking antibody
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant .
  • the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al . , Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
  • murine models of GVHD can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block costimulation of T cells by disrupting receptor : ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856) .
  • Upregulation of an antigen function (preferably a B lymphocyte antigen function) , as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the commoncold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically .
  • anti-viral immune responses maybe enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfeet them with a nucleic acid encoding aprotein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides .
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and/or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class Hoc chain protein and an MHC class H ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I ⁇ chain protein and ⁇ 2 microglobulin protein or an MHC class Hoc chain protein and an MHC class H ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte 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.
  • T-cell-dependent immunoglobulin responses and isotype switching include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Gueryetal., J. Immunol. 134:536-544, 1995; Inaba et al . , Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al . , Journal of Immunology 154:5071-5079, 1995; Porgador et al . , Journal of Experimental Medicine 182:255-260, 1995; Nair et al . , Journal of Virology 67:4062-4069, 1993; Huang et al .
  • lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al. , Cytometry 13 : 795-808, 1992; Gorczyca et al .
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: 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 88:7548-7551, 1991.
  • a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for proliferation and differentiation of various hematopoietic lines are cited above.
  • Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: 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 include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G.
  • a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and m the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and/or bone growth m circumstances where bone is not normally formed, has application m the healing of bone fractures and cartilage damage or defects m humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also m the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful m cosmetic plastic surgery.
  • a protein of this invention may also be used the treatment of periodontal disease, and m other tooth repair processes . Such agents may provide an environment to attract bone-formmg cells, stimulate growth of bone-formmg cells or induce differentiation of progenitors of bone-formmg cells.
  • a protein of the invention may also be useful the treatment of osteoporosis or osteoarth ⁇ tis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
  • a protein of the present invention which induces tendon/ligament-like tissue or other tissue formation circumstances where such tissue is not normally formed, has application m the healing of tendon or ligament tears, deformities and other tendon or ligament defects humans and other animals.
  • Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use m preventing damage to tendon or ligament tissue, as well as use the improved fixation of tendon or ligament to bone or other tissues, and m repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful m cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon or ligament-form g cells, stimulate growth of tendon- or ligament-formmg cells, induce differentiation of progenitors of tendon- or ligament-formmg cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return m vivo to effect tissue repair.
  • the compositions of the invention may also be useful the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
  • Tne compositions may also include an appropriate matrix and/ or sequestering agent as a carrier as is well known the art.
  • Tne protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer ' s, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.
  • diseases of the peripheral nervous system such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer ' s, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.
  • Further conditions which may be treated m accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.
  • Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention. Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, Kidney, skin, endotheliu ) , muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues . Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate.
  • a protein of the invention may also exhibit angiogenic activity.
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury m various tissues, and conditions resulting from systemic cytokine damage.
  • a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
  • the activity of a protein of the invention may, among otner means, be measured by the following methods :
  • Assays for tissue generation activity include, without limitation, those described in: International Patent Publication
  • Assays for wound nealmg activity include, witnout limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.,, Year Book Medical
  • a protein of the present invention may also exhibit activ - or hibm-related activities. Inhibms are characterized oy their ability to inhibit the release of follicle stimulating hormone (FSH), while activms and are characterized by their ability to stimulate the release of follicle stimulating hormone
  • FSH follicle stimulating hormone
  • a protein of the present invention alone cr heterodimers with a member of the inhibin ⁇ family, may be useful as a contraceptive based on the ability of mhibms to decrease fertility m female mammals and decrease spermatogenesis m male mammals. Administration of sufficient amounts of other mhibms can induce infertility these mammals.
  • the protein of the invention as a homodimer or as a heterodimer with other protein subumts of the mhibm- ⁇ group, may be useful as a fertility inducing therapeutic, based upon the ability of activ molecules m stimulating FSH release from cells of the anterior pituitary.
  • a protein of the invention may also be useful for advancement of the onset of fertility sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for activm/mhibm activity include, witnout limitation, those described m: Vale et al . , Endocrino l ogy 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, 1985; Forage et al . , Proc. Natl . Acad. Sci . USA 83: 3091-3095, 1986. Chemotactic/Chemokmeti c. Acr.ivi ty
  • a protein of the present invention may have chemotactic or cnemokinetic activity (e.g., act as a che okme) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosmophils, epithelial and/or endothelial cells.
  • Chemotactic and cnemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or cnemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as m treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result improved immune responses against the tumor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide m any known assay for cell chemotaxis.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
  • Suitable assays for movement and adhesion include, witnout limitation, those described m: Current Protocols m Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub.
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful m treatment of various coagulation disorders (mclud ghereditary disorders, such as hemophilias or to enhance coagulation and other hemostatic events m treating wounds resulting from trauma, surgery or other causes.
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kmases and their ligands, receptor phosphatases and their ligands, receptors involved m cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectms, mtegrms and their ligands) and receptor/ligand pairs involved m antigen presentation, antigen recognition and development of cellular and humoral immune responses) .
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention may themselves be useful as inhibitors of receptor/ligand interactions.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for receptor-ligand activity include without limitation those described m: Current Protocols m Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevacn, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al . , Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al . , J. Exp. Med.168:1145-1156, 1988; Rosenstem et al . , J.Exp.Med.
  • Proteins of the present invention may also exhibit anti- flammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved m the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions) , including without limitation inflammation associated with infection (such as septic snock, sepsis or systemic inflammatory response syndrome
  • SIRS ischemia-reperfusion injury
  • endotoxm lethality arthritis
  • complement-mediated hyperacute rejection nephritis
  • cytokine or chemokme-mduced lung injury inflammatory bowel disease, Crohn's disease or resulting from over production of ytok es such as TNF or IL-1.
  • Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material .
  • a protein of the invention may exhibit other anti-tumor activities.
  • a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC; .
  • a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis) , by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth
  • a protein of the invention may also exhibit one or more of tne following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and otner parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change m bone form or shape) ; effecting biorhythms or ca ⁇ cadic cycles or rhythms; effecting the fertility of male or female subjects; effecting tne metaoolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component (s) ; effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders) , depression (including depressive disorders )
  • the decapped poly (A) RNA and 3 nmol of a chimeric DNA- RNA ol i gonucleotide ( 5' -dG-dG-dG-dG-dA-dA-dT-dT-dC-dG-dA-G-G- A-3') were dissolved m a solution containing 50 mM Tris- hydrochloride buffer solution (pH 7.5), 0.5 mM ATP, 5 mM MgC l 10 mM 2-mercaptoethanol, and 25% polyethylene glycol, whereto was added 50 units of T4RNA ligase and a total 30 ⁇ l volume of the resulting mixture was reacted at 20°C for 12 hours. After the reaction solution was subjected to phenol extraction, fol l owed by ethanol precipitation, the resulting pellet was dissolved m water to obtain a chimeric-oligo-capped poly (A) + RNA.
  • the reaction solution was sub j ected to phenol extraction, followed by ethanol precipitation, the resulting pellet was dissolved in a solution containing 50 mM T ⁇ s- hydrochloride buffer solution (pH 7.5, , 100 mM NaCl, 10 mM MgCl , and 1 mM dithiothreitol. Thereto were added 100 units of EcoRI and a total 20 ⁇ l volume of the resulting mixture was reacted at 37°C for one hour.
  • 50 mM T ⁇ s- hydrochloride buffer solution pH 7.5, 100 mM NaCl, 10 mM MgCl , and 1 mM dithiothreitol.
  • the reaction solution was sub j ected to pnenol extraction, followed by ethanol precipitation, the resulting pellet was dissolved m a solution containing 20 mM T ⁇ s-hydrcchloride buffer solution (pH7.5), lOOmMKCl, 4 MMgCl , 10 mM (NH ), S0 4 , and 50 ⁇ g/ml of tne bovine serum albumin.
  • Tnereto were added 60 units of an Escheri chi a coli DNA ligase and the resulting mixture was reacted at 16°C for 16 hours.
  • the reaction solution was added 2 ⁇ l of 2 mM dNTP, 4 units of Escheri chia coli DNA polymerase I, and 0.1 unit of Escheri chia coli RNase H and the resulting mixture was reacted at 12°C for one hour and then at 22°C for one hour.
  • the cDNA-synthesis reaction solution was used for transformation of Escheri chi a coli DH12S (GIBCO-BRL) .
  • the transformation was carried out by the electroporation method. A portion of the transformant was sprayed on the 2xYT agar culture medium containing 100 ⁇ g/ml ampicillm and the mixture was incubated at 37°C overnight.
  • a colony formed on the agar medium was picked up at random and inoculated on 2 ml of the 2xYT culture medium containing 100 ⁇ g/ml ampicillm. After incubation at 37°C overnight, the culture mixture was cent ⁇ fuged to separate the mycelia, from which a plasmid DNA was prepared by the alkaline lysis method. The plasmid DNA was subjected to double digestion with EcoRI and NotI, followed oy 0.8 agarose gel electrophoresis, to determine the size of the cDNA insert.
  • the sequence reaction was carried out by using an M13 universal primer labeled with a fluorescent dye and a Taq polymerase (a kit of Applied Biosystems) and then the product was examined with a fluorescent DNA sequencer (Applied Biosystems) to determine an about 400-bp base sequence at the 5' -terminus of the cDNA.
  • the sequence data were file d as the homo/protein cDNA bank database.
  • Clone HP01265 was obtained as the result of a large-scale sequencing of cDNA clones selected from the aoove-mentioned cDNA library.
  • the present clone has a structure consisting of a 77-bp 5' -nontranslation region, a 1737-bp open reading frame, and a 219-bp 3' -nontranslation region (Sequence No.3) .
  • the open reading frame codes for a protein consisting of 578 amino acid residues and there existed in the N-terminus a hydrophobic region that is considered to be a secretory signal sequence.
  • Figure 1 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte-Doolittle method, of this protein of the present invention.
  • the dot matrix analysis of the thus-obtained amino acid sequence revealed that two highly analogous domains are connected in a direct manner in the present protein, as shown in Figure 2.
  • Table 1 shows the comparison between the two domain sequences.
  • the marks of -, *, and . represent a gap, an amino acid residue identical with the protein of the present invention, and an amino acid residue analogous to the protein of the present invention, respectively.
  • Vector pHP01265 bearing the cDNA of the present invention was used for m vitro translation with a T T rabbit reticulocyte lysate kit (Promega) .
  • [ S]meth ⁇ onme was added to label the expression product with a radioisotope .
  • Each of the reactions was carried out according to the protocols attached to the kit.
  • Two micrograms of the plasmid pHP01265 was reacted at 30°C for 90 minutes m a total 100 ⁇ l volume of the reaction solution containing 50 ⁇ l of T h T rabbit reticulocyte lysate, 4 ⁇ l of a buffer solution (attached to kit) , 2 ⁇ l of an ammo acid mixture
  • the resulting fragment was inserted between Hindlll and PvuII in pSSD3 (DDBJ/EMBL/GenBank Registration No. AB007632), thereby constructing a vector expressing a fusion protein of the secretory signal sequence of the target cDNA and the urokinase protease domain.
  • Escheri chia coli (host: JM109) bearing the fusion-protein expression vector was incubated at 37°C for 2 hours in 2 ml of the 2xYT culture medium containing 100 ⁇ g/mi of ampicillin, the helper phage M13K07 (50 1) was added and the incubation was continued at 37 °C overnight.
  • a supernatant separated by centrifugation underwent precipitation with polyethylene glycol to obtain single-stranded phage particles. These particles were suspended in 100 ⁇ l of 1 mM Tris-0.1 mM EDTA, pH 8 (TE) .
  • the culture cells originating from the simian kidney, C0S7 were incubated at 37°C in the presence of 5% CO, in the Dulbecco' s modified Eagle's culture medium (DMEM) containing 10% fetal calf albumin.
  • DMEM Dulbecco' s modified Eagle's culture medium
  • the culture medium was removed, the cell surface was washed with a phosphate buffer solution and then washed again with DMEM containing 50 mM Tris-hydrochloric acid (pH 7.5) (TDMEM).
  • each of the samples formed a clear circle to identify that urokinase was secreted in the culture medium.
  • the inserted cDNA fragment codes for the ammo acid sequence that functions as tne secretory signal sequence.
  • Application of the (-3,-1) rule a method for predicting the cleavage site m the secretory signal sequence, allows to expect that the maturation protein starts from glutamme at position 19. (6) Expression by C0S7
  • Escne ⁇ chia coli bearing pHP01265 of the present invention was mfecte ⁇ with helper phage M13K07 and smgle-stranded phage particles were obtained by the above-mentioned procedure.
  • the thus-obtained phage was used for introducing pHP01265 m the culture cells originating from the simian kidney, C0S7. After incubation at 37°C for 2 days m the presence of 5% CO ⁇ , the incubation was continued for one hour m the culture medium containing [ J3 S]cystme.
  • the present invention provides human cDNAs coding for proteins alike to human complement factor H and proteins encoded by these human cDNAs .
  • the human cDNAs of the present invention can be utilized as probes for the gene diagnosis and gene sources for the gene therapy.
  • the cDNAs can be utilized as gene sources for large-scale production of the proteins encoded by said cDNAs .
  • Eucaryotic cells wherein expression vectors of said cDNAs are introduced can be utilized for secretory production of the proteins alike to human complement factor H.
  • Said recombinant proteins can be employed as pharmaceuticals/research reagents, particularly as pharmaceuticals and research reagents for the immunological mechanism involving the complement reaction.
  • the present invention also provides genes corresponding to the polynucleotide sequences disclosed herein.
  • “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with 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 appropriate genomic libraries or other sources of genomic materials.
  • An "isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
  • Organisms that have enhanced, reduced, or modified 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 ribozy es that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al . , 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res . Mol. Biol. 58: 1-39; all of which are incorporated by reference herein) .
  • Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
  • organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s).
  • Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (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 by reference herein) , or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al., 1988, Nature 336: 348-352; U.S. Patent Nos .
  • the present invention also provides for soluble forms of such protein.
  • the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell which it is expressed.
  • the intracellular and transmembrane domains of proteins of the invention can be identified m accordance with known techniques for determination of such domains from sequence information.
  • Proteins and protein fragments of the present invention include proteins with ammo acid sequence lengths that are at least 25% (more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, wnere sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous ammo acids that shares at least 75% sequence identity (more preferably, at least 85 identity; most preferably at least 95- identity) with any such segment of any of the disclosed proteins.
  • Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention.
  • a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide, as determined by those of skill m the art.
  • Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein ano screening a suitable nucleic acid source from the desired species.
  • the invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally- occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous, or related to that encoded by the polynucleotides.
  • the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
  • the present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein.
  • stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
  • the hybrid length is that anticipated for the hybridized reg ⁇ on(s) of the hybridizing polynucleotides.
  • the hybrid length is assumed to be that of the hybridizing polynucleotide.
  • the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
  • SSPE SSPE
  • IxSSC 0.15M NaCl and 15mM sodium citrate
  • T B - T R The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (T m ) of the hybrid where T m is determined according to the following equations.
  • T m (°C) 2(#of A + T bases) + 4(# of G + C bases).
  • N the number of bases m the hybrid
  • [Na + ] is the concentration of sodium ions in the hybridization buffer
  • each such hybridizing polynucleotide has a length that is at least 25% (more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.

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Abstract

La présente invention se rapporte à de l'ADNC codant pour des protéines semblables au facteur H du complément humain, ainsi qu'aux protéines codées par cet ADNC humain. L'ADNC humain de la présente invention peut être utilisé en tant que sonde moléculaire pour le diagnostic génétique et en tant que sources de gènes pour la thérapie génique.
PCT/JP1998/004448 1997-10-06 1998-10-02 Proteines ressemblant au facteur h du complement humain et adnc codant ces proteines WO1999018200A1 (fr)

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CA002305386A CA2305386A1 (fr) 1997-10-06 1998-10-02 Proteines ressemblant au facteur h du complement humain et adnc codant ces proteines
EP98945576A EP1021530A1 (fr) 1997-10-06 1998-10-02 PROTEINES RESSEMBLANT AU FACTEUR H DU COMPLEMENT HUMAIN ET ADNc CODANT CES PROTEINES
AU92819/98A AU9281998A (en) 1997-10-06 1998-10-02 Proteins alike to human complement factor h and cdnas encoding these proteins
JP2000514998A JP2001519152A (ja) 1998-10-02 1998-10-02 ヒト補体因子Ｈ様蛋白質およびそれをコードするｃＤＮＡ

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008032053A1 (fr) * 2006-09-15 2008-03-20 Ares Trading S.A. Variantes d'épissure fhr-4a
KR101623526B1 (ko) 2013-12-24 2016-05-23 재단법인 목암생명공학연구소 인간 보체 인자 h의 생산 방법

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE202016103409U1 (de) 2016-06-28 2017-09-29 Rehau Ag + Co Kantenleiste für Möbelstücke

Non-Patent Citations (4)

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Title
SKERKA C ET AL: "A novel short consensus repeat-containing molecule is related to human complement factor H.", J BIOL CHEM, FEB 5 1993, 268 (4) P2904-8, UNITED STATES, XP002091108 *
SKERKA C ET AL: "Molecular cloning of a human serum protein structurally related to complement factor H.", J BIOL CHEM, JUN 25 1991, 266 (18) P12015-20, UNITED STATES, XP002091109 *
SKERKA C ET AL: "The human factor H-related protein 4 (FHR-4). A novel short consensus repeat-containing protein is associated with human triglyceride-rich lipoproteins.", J BIOL CHEM, FEB 28 1997, 272 (9) P5627-34, UNITED STATES, XP002091107 *
ZIPFEL PF ET AL: "COMPLEMENT FACTOR-H AND RELATED PROTEINS - AN EXPANDING FAMILY OF COMPLEMENT-REGULATORY PROTEINS", IMMUNOLOGY TODAY, 1994, 15, 121-126, XP002091110 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008032053A1 (fr) * 2006-09-15 2008-03-20 Ares Trading S.A. Variantes d'épissure fhr-4a
KR101623526B1 (ko) 2013-12-24 2016-05-23 재단법인 목암생명공학연구소 인간 보체 인자 h의 생산 방법

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