WO1998042836A1 - Recepteur de cytokine mammifere - Google Patents

Recepteur de cytokine mammifere Download PDF

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Publication number
WO1998042836A1
WO1998042836A1 PCT/US1998/005254 US9805254W WO9842836A1 WO 1998042836 A1 WO1998042836 A1 WO 1998042836A1 US 9805254 W US9805254 W US 9805254W WO 9842836 A1 WO9842836 A1 WO 9842836A1
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leu
glu
val
gly
ser
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PCT/US1998/005254
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English (en)
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Si Lok
Robyn L. Adams
Tracey A. Pownder
Choon J. Kho
Theodore Whitmore
Theresa M. Farrah
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Zymogenetics, Inc.
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Priority to AU67623/98A priority Critical patent/AU6762398A/en
Publication of WO1998042836A1 publication Critical patent/WO1998042836A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

  • hormones and polypeptide growth factors are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue.
  • hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF) , epidermal growth factor (EGF) , granulocyte-macrophage colony stimulating factor (GM-CSF) , erythropoietin (EPO) and calcitonin.
  • Proteins may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of proteins are soluble molecules, such as the transcription factors.
  • cytokines molecules that promote the proliferation and/or differentiation of cells.
  • examples of cytokines include erythropoietin (EPO) , which stimulates the development of red blood cells; thro bopoietin (TPO) , which stimulates development of cells of the megakaryocyte lineage; and granulocyte- colony stimulating factor (G-CSF) , which stimulates development of neutrophils.
  • EPO erythropoietin
  • TPO thro bopoietin
  • G-CSF granulocyte- colony stimulating factor
  • the present invention addresses this need by providing a novel polypeptide and related compositions and methods.
  • the present invention provides an isolated polynucleotide encoding a mammalian cytokine receptor termed Zcytor4.
  • Zcytor4 polypeptides are comprised of a sequence of amino acids 438 amino acids long with the initial Met as shown in SEQ ID NO : 1 and SEQ ID NO : 2 for the human and SEQ ID NO: 15 and SEQ ID NO: 16 for the mouse.
  • the polypeptide further comprises an affinity tag.
  • the polynucleotide is DNA.
  • an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding Zcytor4 polypeptide, and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked.
  • a cultured eukaryotic cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a protein polypeptide encoded by the DNA segment .
  • a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond.
  • the first portion of the chimeric polypeptide consists essentially of (a) a Zcytor4 polypeptide as shown in either SEQ ID NO: 2 or SEQ ID NO: 4; (b) allelic variants of SEQ ID NO : 2 ; and (c) protein polypeptides that are at least 80% identical to (a) or (b) .
  • the second portion of the chimeric polypeptide consists essentially of another polypeptide such as an affinity tag.
  • the affinity tag is an immunoglobulin F c polypeptide.
  • the invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
  • an antibody that specifically binds to a Zcytor4 polypeptide as disclosed above, and also an anti- idiotypic antibody which neutralizes the antibody to a Zcytor4 polypeptide.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene .
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems .
  • “Operably linked” when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e . g . transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • polynucleotide is a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules.
  • promoter is used herein for its art- recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a "soluble protein” is a protein polypeptide that is not bound to a cell membrane.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO : 1 and SEQ ID NO : 4 , or a sequence complementary thereto, under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Typical stringent conditions are those in which the salt concentration is about 0.02 M or less at pH 7 and the temperature is at least about 60°C.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • RNA DNA and DNA are well known in the art. It is generally preferred to isolate RNA from testis, including whole testis tissue extracts or testicular cells, such as Sertoli cells, Leydig cells, spermatogonia, or epididymis, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA.
  • Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al . , Biochemistry 28:52-94 (1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Na tl . Acad . Sci . USA £9:1408-1412 (1972).
  • Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. Polynucleotides encoding Zcytor4 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • the polynucleotides of the present invention can be synthesized using DNA synthesizers.
  • the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short DNA fragments (60 to 80 bp) can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer DNA molecules the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • synthetic genes double- stranded are assembled in modular form from single- stranded fragments that are from 20 to 100 nucleotides in length.
  • One method for building a synthetic gene requires the initial production of a set of overlapping, complementary oligonucleotides, each of which is between 20 to 60 nucleotides long.
  • the sequences of the strands are planned so that, after annealing, the two end segments of the gene are aligned to give blunt ends.
  • Each internal section of the gene has complementary 3 ' and 5 ' terminal extensions that are designed to base pair precisely with an adjacent section.
  • synthetic genes can be designed with terminal sequences that facilitate insertion into a restriction endonuclease sites of a cloning vector and other sequences should also be added that contain signals for the proper initiation and termination of transcription and translation. See Glick, Bernard R. and Jack J. Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994), Itakura, K. et al . Synthesis and use of synthetic oligonucleotides . Annu . Rev. Biochem . 53 : 323-356 (1984), and Climie, S. et al . Chemical synthesis of the thymidylate synthase gene. Proc . Natl . Acad . Sci . USA 87 :633-637 (1990) .
  • sequences disclosed in SEQ ID ⁇ 0S:1, 2 represent a single allele of the human and SEQ ID NOS : 3 and 4 represent a single allele of the mouse Zcytor4. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures .
  • the present invention further provides counterpart proteins and polynucleotides from other species ("species orthologs”) .
  • species orthologs are Zcytor4 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primates.
  • Species orthologs of the human and mouse Zcytor4 protein can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a protein- encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human or mouse cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
  • a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the sequences disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the protein. Similar techniques can also be applied to the isolation of genomic clones.
  • the present invention also provides isolated protein polypeptides that are substantially homologous to the protein polypeptides of SEQ ID NO: 2 or SEQ ID NO : 4 and their species orthologs.
  • isolated is meant a protein or polypeptide that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • substantially homologous is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO : 2 , or 4 , or their species orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO : 2 , or 4 or their species orthologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull . Ma th . Bio . 48 : 603-616 (1986) and Henikoff and Henikoff, Proc . Natl . Acad . Sci . USA 89:10915-10919 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blossom 62" scoring matrix of
  • the percent identity is then calculated as: Total number of identical matches x 100
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .
  • Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 3) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl -terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag) , such as a poly-histidine tract, protein A, Nilsson et al . , EMBO J.
  • an affinity tag such as a poly-histidine tract, protein A, Nilsson et al . , EMBO J.
  • Acidic glutamic acid aspartic acid
  • Polar glutamine asparagine
  • Hydrophobic leucine isoleucine
  • Table 3 continued valine
  • Aromatic phenylalanine tryptophan tyrosine
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244 : 1081-1085 (1989); Bass et al . , Proc . Natl . Acad . Sci . USA 88:4498-4502 (1991) .
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., ligand binding and signal transduction) to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of ligand-protein interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al . , Science 255:306-312 (1992); Smith et al . , J. Mol . Biol . 224:899-904 (1992); Wlodaver et al . , FEBS Lett . 309:59-64 (1992) . The identities of essential amino acids can also be inferred from analysis of homologies with related proteins.
  • Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned, mutagenized proteins in host cells.
  • Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below.
  • Mutagenized DNA molecules that encode active proteins or portions thereof e.g., ligand-binding fragments
  • polypeptides that are substantially homologous to residues SEQ ID NO: 2 or SEQ ID NO : 4 or allelic variants thereof and retain the properties of the wild-type protein.
  • the protein polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al . , Molecular Cloning: A Laboratory Manual , 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and Ausubel et al . , ibid .
  • a DNA sequence encoding a Zcytor4 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of the protein, or may be derived from another secreted protein (e.g., t- PA) or synthesized e novo .
  • the secretory signal sequence is joined to the Zcytor4 DNA sequence in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al . , U.S. Patent No. 5,037,743; Holland et al . , U.S. Patent No. 5,143,830).
  • Cultured mammalian cells are preferred hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell 24:725 (1978); Corsaro and Pearson, Soma tic Cell Genetics 7:603 (1981) : Graham and Van der Eb, Virology 52:456 (1973), electroporation ⁇ Neumann et al . , EMBO J. 2:841-845
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293, ATCC No. CRL 1573; Graham et al . , J. Gen . Virol . 36:59-72 (1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus .
  • promoters include those from metallothionein genes (U.S. Patent Nos . 4,579,821 and 4,601,978 and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants” . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants . " A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as
  • Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate .
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al . , U.S. Patent No. 5,162,222; Bang et al . , U.S. Patent No. 4,775,624; and WIPO publication WO 94/06463.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J " . Biosci . (Bangalore) 22:47-58 (1987) .
  • Fungal cells including yeast cells, and particularly cells of the genus Saccharomyces , can also be used within the present invention, such as for producing protein fragments or polypeptide fusions.
  • Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S. Patent No. 4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) .
  • a preferred vector system for use in yeast is the POT2 vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
  • a novel protein is produced by a cultured cell, and the cell is used to screen for ligands for the protein, including the natural ligand, as well as agonists and antagonists of the natural ligand.
  • ligands for the protein including the natural ligand, as well as agonists and antagonists of the natural ligand.
  • a cDNA or gene encoding the protein is combined with other genetic elements required for its expression (e.g., a transcription promoter) , and the resulting expression vector is inserted into a host cell.
  • Cells that express the DNA and produce functional protein are selected and used within a variety of screening systems.
  • PROTEIN ISOLATION Expressed recombinant polypeptides (or chimeric polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography . Suitable anion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide , specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ) being particularly preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl , butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryvilie, PA) , Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art . Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affini ty
  • the polypeptides of the present invention can be isolated by exploitation of their properties.
  • immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins. Briefly, a gel is first charged with divalent metal ions to form a chelate, E. Sulkowski, Trends in Biochem. 3:1-7 (1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • Zcytor4 useful to promote adhesion of tissue following an operation. Also the Zcytor4 polypeptide of the present invention would be useful to coat microtiter plates to promote adhesion of cells of interest on the plates.
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the Zcytor4 gene has been mapped on chromosome
  • a Zcytor4 nucleic acid probe could to used to check for abnormalities in chromosome 12. If the probe does not hybridize to chromosome 12 or if it only binds to one chromosome, this would indicate an abnormality in
  • a Northern blot containing poly A RNA from various human leukemia lines was also probed with the 285 bp cDNA probe and the 3.1 kb transcript was found to be expressed in Burkitt's lymphoma, T-cell leukemia lineages and myeloid leukemia lineages.
  • an antibody to zcytor4 or nucleic acid probe could by used as a diagnostic tool in detecting these diseases.
  • the present invention also provides reagents with significant therapeutic value.
  • the Zcytor4 polypeptide naturally occurring or recombinant
  • fragments thereof, antibodies and anti-idiotypic antibodies thereto should be useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions.
  • abnormal proliferation e.g., cancerous conditions, or degenerative conditions.
  • the Zcytor4 gene is highly expressed in thymus, lymph node, spleen, peripheral blood leukocytes (PBLs) and bone marrow, treatment and modulation of development of lymphoid cells is likely. Abnormal proliferation, regeneration, degeneration, and atrophy may be modulated by appropriate therapeutic treatment using the compositions provided herein.
  • a disease or disorder associated with abnormal expression or abnormal signaling by a Zcytor4 polypeptide should be a likely target for an agonist or antagonist of the Zcytor4 polypeptide.
  • the Zcytor4 polypeptide may play a role in regulation or development of hematopoietic cells, e.g., lymphoid cells, which affect immunological responses, e . g . , lymphoid cells which affect immunological responses, e.g., autoimmune disorders.
  • Antibodies to the Zcytor4 can be purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in pharmaceutically acceptable carriers or diluents along with physiologically innocuous stabilizers and excipients. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates use of antibodies, binding fragments thereof or single- chain antibodies of the antibodies including forms which are not complement binding.
  • the quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medications administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vi tro may provide useful guidance in the amounts useful for in vivo administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage.
  • Methods for administration include oral, intravenous, peritoneal, intramuscular, or transdermal administration.
  • Pharmaceutically acceptable carriers will include water, saline, buffers to name just a few. Dosage ranges would ordinarily be expected from l ⁇ g to lOOO ⁇ g per kilogram of body weight per day. However, the doses by be higher or lower as can be determined by a medical doctor with ordinary skill in the art. For a complete discussion of drug formulations and dosage ranges see Remington ' s Pharmaceutical Sciences, 19 Ed., (Mack Publishing Co.,
  • a gene encoding a Zcytor4 polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV) , papillomavirus, Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell.
  • defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt et al . , Mol ec . Cell . Neurosci . , 2 : 320-330 (1991) ] , an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al . , J. Clin . Invest . , 90 : 626-630 (1992), and a defective adeno- associated virus vector [Samulski et al . , J. Virol . , 62:3096-3101 (1987); Samulski et al . J. Virol . , 63:3822- 3828 (1989) ] .
  • HSV1 herpes virus 1
  • the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al . , U.S. Patent No. 5,399,346; Mann et al . , Cell , 33:153 (1983); Temin et al . , U.S. Patent No. 4,650,764; Temin et al . , U.S. Patent No. 4,980,289; Markowitz et al . , J. Virol . , 62:1120 (1988); Temin et al . , U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al . ; and Blood, 82:845 (1993).
  • a retroviral vector e.g., as described in Anderson et al . , U.S. Patent No. 5,399,346; Mann et al . , Cell , 33
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner et al . , Proc . Na tl . Acad . Sci . USA, 84:7413-7417 (1987); see Mackey et al . , Proc . Na tl . Acad . Sci . USA, 85:8027-8031 (1988) ] .
  • the sue of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit.
  • directing transfection to particular cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides, e . g. , hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • DNA vector for gene therapy can be introduced into the desired host cells by methods known in the art, e . g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter [see, e . g . , Wu et al . , J. Biol . Chem . , 267:963-967 (1992); Wu et al . , J. Biol . Chem . , 263:14621- 14624 (1988) ] .
  • Zcytor4 polypeptides can also be used to prepare antibodies that specifically bind to Zcytor4 epitopes, peptides or polypeptides.
  • the Zcytor4 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • Suitable antigens would be the Zcytor4 polypeptide or a contiguous 9 amino acid or larger fragment thereof.
  • Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al . (eds.),
  • polyclonal antibodies can be generated by inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a Zcytor4 polypeptide or a fragment thereof.
  • the antibodies produced by the inoculated animal are then isolated either from the blood serum, polyclonal antibodies, or from antibody-producing spleen cells which are fused with a myeloma cell to produce a hybridoma which secretes monoclonal antibodies.
  • the immunogenicity of a Zcytor4 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zcytor4 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof.
  • polypeptide portion is "hapten-like"
  • such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen- binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting non- human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to Zcytor4 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zcytor4 protein or peptide) .
  • Genes encoding polypeptides having potential Zcytor4 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli .
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , US
  • Patent NO. 5,223,409 Ladner et al . , US Patent NO. 4,946,778; Ladner et al . , US Patent NO. 5,403,484 and
  • Random peptide display libraries can be screened using the Zcytor4 sequences disclosed herein to identify proteins which bind to Zcytor .
  • binding proteins which interact with Zcytor4 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as Zcytor4
  • anti-Zcytor4 binding proteins would be useful in the purification of Zcytor4.
  • Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules.
  • antibodies herein specifically bind if they bind to a Zcytor4 polypeptide, peptide or epitope with a binding affinity -i —j -
  • O _1 more preferably 10 M or greater, and most preferably 10 9 M-1 or greater.
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis, Scatchard, G.,
  • antibodies are determined to specifically bind if they do not significantly cross-react with related polypeptides.
  • Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zcytor4 but not known related polypeptides using a standard Western blot analysis (Ausubel et al . , ibid. ) .
  • Examples of known related polypeptides are orthologs, proteins from the same species that are members of a protein family (e.g. IL-16) , Zcytor4 polypeptides, and non-human Zcytor4.
  • antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds to the inventive polypeptides.
  • antibodies raised to Zcytor4 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to Zcytor4 will flow through the matrix under the proper buffer conditions.
  • Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.) (Raven Press, 1993); Getzoff et al .
  • assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to Zcytor4 proteins or peptides. Exemplary assays are described in detail in Antibodies : A Labora tory Manual , Harlow and Lane (Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant Zcytor4 protein or polypeptide .
  • Antibodies to Zcytor4 may be used for tagging cells that express Zcytor4; for isolating Zcytor4 by affinity purification; for diagnostic assays for determining circulating levels of Zcytor4 polypeptides; for detecting or quantitating soluble Zcytor4 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block Zcytor4 in vi tro and in vivo .
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to Zcytor4 or fragments thereof may be used in vi tro to detect denatured Zcytor4 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • An expressed sequence tag SEQ ID NO: 5 was identified and a cDNA containing 2130 base pairs obtained from a kidney cDNA library.
  • the DNA was transfected into E . coli strain DHlOb.
  • the plasmid was designated pSL6907.
  • a nested 5' RACE reaction was used to extend the open reading frame.
  • the reaction was used to extend the open reading frame.
  • the reaction was carried out on kidney MARATHON-READYTM cDNA (Clontech Laboratories, Inc., Palo
  • Alto, CA Alto, CA template in accordance with the manufacturer' s protocol.
  • the outer nest PCR reaction employed primers SEQ ID NO: 1
  • reaction products were diluted 1:500 with water, and 1 ⁇ l of the solution was used as a template for PCR employing the inner nested primers SEQ ID NO: 8 (obtained from Clontech Laboratories) and SEQ ID NO: 9. Reaction conditions were the same as disclosed above.
  • the resulting product was purified on a low-melt agarose gel and was cloned using a PCR-SCRIPTTM kit (Stratagene Cloning Systems, La Jolla, CA) to produce the plasmid pSLR4105-2. Sequence analysis of pSLR4105-2 revealed the 5' RACE product extended the open reading frame of pSL6907 by an additional 43 codons .
  • reaction mixture was incubated at 94°C for 1 minute, followed by 30 cycles at 95°C, 15 seconds;
  • the labeled probe was purified on a push
  • the first strand cDNA reaction contained 10 ⁇ l of human fetal kidney twice poly d (T) -selected poly (A) mRNA (Clontech Laboratories) at a concentration of 1.0 ⁇ g/ ⁇ l, and 4 ⁇ l of 20 pmole/ ⁇ l first strand primer ZC6171 (SEQ ID NO: 11) containing an Xho I restriction site. The mixture was heated at 70°C for 3 minutes and cooled by chilling on ice.
  • First strand cDNA synthesis was initiated by the addition of 8 ⁇ l of first strand buffer (5X SUPERSCRIPTTM buffer; Life Technologies, Gaithersburg, MD) , 4 ⁇ l of 100 mM dithiothreitol, and 3 ⁇ l of a deoxynucleotide triphosphate solution containing 10 mM each dTTP, dATP, and 5-methyl-CTP (Pharmacia LKB Biotechnology, Piscataway, NJ) to the RNA-primer mixture. The reaction mixture was incubated at 37°C for 2 minutes, followed by the addition of 10 ⁇ l of 200 U/ ⁇ l RNase H- reverse transcriptase
  • the unincorporated nucleotides and primers in the unlabeled first strand were also removed by chromatography on 400 pore size gel filtration column (Clontech Laboratories). The length of labeled first strand cDNA was determined by agarose gel electrophoresis.
  • the second strand reaction contained 135 ⁇ l of the unlabeled first stand cDNA, 40 ⁇ l of 5x polymerase I buffer (125 mM TRIS: HC1, pH 7.5, 500 mM KC1, 25 mM MgCl 2 , 50 mM (NH 4 ) 2 S0 4 )), 2.5 ⁇ l of 100 mM dithiothreitol, 5 ⁇ l of a solution containing 10 mM of each deoxynucleotide triphosphate, 7 ⁇ l of 5 mM ⁇ -NAD, 2.5 ⁇ l of3 U/ ⁇ l E. coli DNA ligase (New England Biolabs, Beverly, MA) , 7 ⁇ l of 10 U/ ⁇ l E.
  • 5x polymerase I buffer 125 mM TRIS: HC1, pH 7.5, 500 mM KC1, 25 mM MgCl 2 , 50 mM (NH 4 ) 2 S0 4
  • Unincorporated 32 P- ⁇ dCTP in the labeled reaction was removed by chromatography through a 400 pore size gel filtration column (Clontech Laboratories) before analysis by agarose gel electrophoresis.
  • the reaction was terminated by the addition of 20 ⁇ l 0.5 mM EDTA and extraction with phenol/chloroform and chloroform followed by ethanol precipitation in the presence of 2.5 M ammonium acetate.
  • the yield of cDNA was estimated to be approximately 2 ⁇ g from starting mRNA template of 10 ⁇ g.
  • Eco RI adapters were ligated onto the 5' ends of the cDNA described above to enable cloning into an expression vector.
  • a 12 ⁇ l aliquot of cDNA (approximately 2 ⁇ g) and 4 ⁇ l of 65 pmole/ ⁇ l of Eco RI adapter (Pharmacia LKB) were ligated onto the 5' ends of the cDNA described above to enable cloning into an expression vector.
  • the cDNA was digested with Xho I, resulting in a cDNA having a 5' Eco RI cohesive end and a 3' Xho I cohesive end.
  • the Xho I restriction site at the 3' end of the cDNA had been previously introduced using primer SEQ ID NO: 11.
  • Restriction enzyme digestion was carried out in a reaction mixture containing 25 ⁇ l of cDNA as described above, 10 ⁇ l of lOx H Buffer (Boehringer Mannheim), 64 ⁇ l H 2 0, and 1.0 ⁇ l of 40 U/ ⁇ l Xho I (Boehringer Mannheim) . Digestion was carried out at 37°C for 40 minutes. The reaction was terminated by incubation at 70°C for 20 minutes and chromatography through a 400 pore size gel filtration column (Clontech Laboratories) .
  • the cDNA was ethanol precipitated, washed with 70% ethanol, air dried and resuspended in 13.5 ⁇ l of water.
  • the cDNA was phosphorylated by the addition of 2 ⁇ l of lOx kinase buffer (660 mM TRIS, pH 7.5, 100 mM MgCl 2 ) , 3 ⁇ l of 10 mM ATP, 0.5 of 100 mM DTT, and 1 ⁇ l of polynucleotide kinase (10 U/ ⁇ l; obtained from Life Technologies, Inc.) .
  • the cDNA was precipitated in the presence of ⁇ volume of 7.5 M ammonium acetate and 2.5 volume of ethanol, and resuspended in 20 ⁇ l of lx gel loading buffer (10 mM TRIS-HC1, pH 8.0, 1 mM EDTA, 5% glycerol, and 0.125% bromphenol blue).
  • the resuspended cDNA was heated to 65°C for 5 minutes, cooled on ice and electrophoresed on a 0.8% low melt agarose gel.
  • the contaminating adapters and cDNA below 3.5 kb in length were excised from the gel.
  • the electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane origin.
  • the area of the gel containing the concentrated cDNA was excised and placed in a microfuge tube, and the approximate volume of the gel slice was determined.
  • An aliquot of water approximately three times the volume of the gel slice (300 ⁇ l) and 35 ⁇ l lOx ⁇ - agarose I buffer (New England Biolabs) was added to the tube, and the agarose was melted by heating to 65°C for 15 minutes.
  • 3 ⁇ l of 1 U/ ⁇ l ⁇ -agarose I (New England Biolabs) was added, and the mixture was incubated for 60 minutes at 45°C to digest the agarose.
  • the resulting cDNA was cloned into the lambda phage vector ⁇ ZAP® ii (Stratagene Cloning Systems) that was predigested with Eco RI and Xho I and dephosphorylated.
  • Ligation of the cDNA to the ⁇ ZAP II vector was carried out in a reaction mixture containing 1.0 ⁇ l of prepared vector, 1.0 ⁇ l of rat testis cDNA, 1.0 ⁇ l 10X Ligase Buffer (Promega Corp.), 1.0 ⁇ l of 10 mM ATP, 5 ⁇ l water, and 1.0 ⁇ l of T4 DNA Ligase at 15 U/ml (Promega Corp.).
  • the ligation mixture was incubated at 5°C-15°C overnight in a temperature gradient. After incubation, the ligation mixture was packaged into phage using an in vi tro
  • the human fetal kidney ⁇ ZAP II library was used to infect E. coli host cells (XLl-Blue MRF' strain;
  • the filters were prehybridized in hybridization solution (5X SSC, 5X Denhardt's solution,
  • a probe was prepared as a PCR product by using oligonucleotide primers designed to amplify the Zcytor4 cDNA coding region.
  • a PCR reaction mixture containing 2 ⁇ l of ZC10,941 (SEQ ID NO: 6) and 2 ⁇ l of ZC12,014 (SEQ ID NO: 10), 1 ⁇ l ( approximately 100 femtogram) pSLR4105-2, I ⁇ l of 10 M dNTP, 10 ⁇ l of 10X KlenTaq buffer (Clontech Laboratories), 82 ⁇ l water, and 2 ⁇ l KlenTaq DNA polymerase (Clontech Laboratories).
  • the PCR reaction was run as follows: 1 cycle at 94°C for 1 minute; 30 cycles at 95°C for 15 seconds, 56°C for 20 seconds, 68° for 3 minutes; then held at 68°C for 5 minutes.
  • the PCR product was gel purified on a 1.5% low melt agarose gel.
  • P-dCTP by random priming using the MEGAPRIMETM DNA Labeling System (Amersham) , according to the manufacturer's specifications.
  • the prehybridization solution was replaced with fresh hybridization solution containing 1.4 x 10 cpm/ml labeled probe and allowed to hybridize for 64 hours at 60°C.
  • the hybridization solution was removed and the filters were rinsed in a wash solution containing 0.25 X SSC, 0.25% SDS and 1 mM EDTA at 55°C.
  • the filters were placed on autoradiograph film and exposed at 70°C with intensifying screens for 96 hours.
  • the plates were incubated overnight at 37°C, and filter lifts were prepared, prehybridized, hybridized, washed and autoradiographed as described above. Examination of the resulting autoradiographs revealed positive signals on 5 filter lifts. The phage plaques were subjected to an additional round of plaque purification.
  • the plasmids were excised using an ExASSIST/SOLRTM system (Stratagene Cloning Systems), according to the manufacturer's specification. These plasmids were amplified by PCR for size determination and sequencing.
  • a 285 bp double stranded DNA probe for Northern analysis was prepared from pSL6907 by PCR using oligonucleotide primers SEQ ID NO: 12 and SEQ ID NO: 13.
  • the 285 bp PCR fragment was gel-purified using a QIAQUICKTM purification kit (Qiagen Inc., Chatsworth, CA) and random- primer labeled using a MULTIPRIMETM kit (Amersham Corp.). Labeled cDNA was purified from free counts using a Stratagene push column.
  • Human multiple tissue Northern blots (MTN, MTN II and MTN III from Clontech Laboratories) were pre-hybridized for three hours at 68°C using Clontech
  • the P-labeled cDNA probe was then added to 10 ml of fresh hybridization solution at 10 cpm/ml overnight at 68°C.
  • the blots were washed several times at room temperature in wash solution containing 2X SSC, 0.05% sodium dodecyl sulfate (SDS) at 50°C for 40 minutes with one change of wash solution.
  • SDS sodium dodecyl sulfate
  • a single transcript of about 3.1 kb was detected after exposure to film.
  • the transcript was present in highest abundance in spleen, thymus, lymph nodes and bone marrow followed by testis and peripheral blood lymphocytes.
  • the 3.1 kb transcript was also apparent in the fetal tissue blot with highest abundance in the liver, then kidney with minimal expression in lung and brain.
  • the 285 bp probe was also used to probe a human RNA master dot blot (Clontech Laboratories) pre-hybridized in EXPRESSHYBTM hybridization solution for 3 hours at 65°C and hybridized with 10 cpm/ml of labeled probe overnight at 65°C. The blots were then washed in 0. IX SSC, 0.05% SDS and agitated for 40 minutes at 65°C. The blots were then washed in 0.1X SSC, 0.1% SDS at 50°C for 40 minutes with one change of solution.
  • RNA samples which have been normalized to the mRNA expression levels of eight different housekeeping genes, was highest in kidney followed by whole brain, pituitary gland, fetal brain, various areas of the brain occipital lobe, putamen, cerebellum, cerebellum hippocampus), stomach and testis.
  • the MTNs were repeated with a different set of Northern blots. The result of this probing was the same as seen previously.
  • a Northern blot containing poly A RNA from various human leukemia lines was also probed with the 285 bp cDNA probe and the 3.1 kb transcript was found to be expressed in Burkitt's lymphoma, T-cell leukemia lineages and myeloid leukemia lineages.
  • the GeneBridge 4 Radiation Hybrid Panel contains DNAs on which PCR on be used from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient) .
  • WWW World Wide Web
  • PCR reactions in the 95 wells consisted of 2.5 ⁇ l lOx KLENTAQTM PCR reaction buffer (Clontech Laboratories Inc., Palo Alto, CA) , 2 ⁇ l dNTPs mix 2.5 mM each of dATP, dGTP, dCTP and dTTP, Perkin-Elmer, Foster City, CA) , 1.25 ⁇ l sense primer, CTG CGC CCG AGG AGA TGG CCG ACA GGA (SEQ ID NO: 17), 1.25 ⁇ l antisense SEQ ID 13, 2.5 ⁇ l "REDILOADTM" (Research Genetics, Inc., Huntsville, AL) , 0.5 ⁇ l 50X ADVANTAGE KLENTAQTM Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and water is added to bring the total volume to 25 ⁇ l .
  • CCTCCTGCCT CAGCCTCCTG AGTAGCTGGG ATTACAGGTG TCGGCCACCA CACCTGGCTA 2069
  • ATGTGAATTA ⁇ TTTTTCAT ⁇ TTACCAGT AGTAAAAAAG TAGAAAAGCA AGTATGATAT 2669
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • GAC ⁇ C ACA GCC CAA GAT TAC AGG CTT CAG TTC CGC AAA TGT ACT GCA 722 Asp Phe Thr Ala Gin Asp Tyr Arg Leu Gin Phe Arg Lys Cys Thr Ala 225 230 235 AAT CAT TTT GAA GAC GTG TAT GTA GGT TCT GAA ACG GAG TTC ATA GTG 770 Asn His Phe Glu Asp Val Tyr Val Gly Ser Glu Thr Glu Phe He Val 240 245 250
  • GGA GAC GGC AGG CAG GAA TGG AGT CCT TGG AGT GTT CCC CAG ACA GGC 866 Gly Asp Gly Arg Gin Glu Trp Ser Pro Trp Ser Val Pro Gin Thr Gly 270 275 280
  • AAA GAA ATG ACC AAT CAG ⁇ G CCT GCA GTT ACC TCT GGC TCC ACT GTC 1202 Lys Glu Met Thr Asn Gin Leu Pro Ala Val Thr Ser Gly Ser Thr Val 385 390 395
  • AAATCGTGGT CTTGTTATAC CAGCATACAT ATAAT ⁇ TTA ATGTGAATTT TTGTTTGTTT 2177 ⁇ TATCAATA GTAAAAAAAA AAAGWRRAAA AGCAMGTGTG GTAT iTGT AAGATAA ⁇ T 2237
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • AAA AAG GCC TCA CAC ATT CAG CTG GAC AGC TTA CCA GAA GTG CCC ⁇ G 432 Lys Lys Ala Ser His He Gin Leu Asp Ser Leu Pro Glu Val Pro Leu 130 135 140
  • GGT TCT GAA ACG GAG TTC ATA GTG TTG CAC ATA GAC CCC AAT GTA GAT 720 Gly Ser Glu Thr Glu Phe He Val Leu His He Asp Pro Asn Val Asp 225 230 235 240
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal

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Abstract

L'invention concerne de nouveaux polypeptides protéiniques récepteurs de cytokine mammifères, les polynucléotides codant ces polypeptides, ainsi que les compositions et les procédés relatifs à ces polypeptides.
PCT/US1998/005254 1997-03-21 1998-03-18 Recepteur de cytokine mammifere WO1998042836A1 (fr)

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AU67623/98A AU6762398A (en) 1997-03-21 1998-03-18 Mammalian cytokine receptor

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US60/041,570 1997-03-21

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001083536A1 (fr) * 2000-04-27 2001-11-08 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, recepteur 15 du facteur cellulaire, et polynucleotide codant pour ce polypeptide

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0490410A1 (fr) * 1990-12-14 1992-06-17 Shionogi Seiyaku Kabushiki Kaisha Récepteur du TXA2 et gène codant pour celui-ci
WO1996040923A1 (fr) * 1995-06-07 1996-12-19 Icos Corporation Chemokine et analogues de chemokine derives de macrophages

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0490410A1 (fr) * 1990-12-14 1992-06-17 Shionogi Seiyaku Kabushiki Kaisha Récepteur du TXA2 et gène codant pour celui-ci
WO1996040923A1 (fr) * 1995-06-07 1996-12-19 Icos Corporation Chemokine et analogues de chemokine derives de macrophages

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001083536A1 (fr) * 2000-04-27 2001-11-08 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, recepteur 15 du facteur cellulaire, et polynucleotide codant pour ce polypeptide

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