MXPA02000138A

MXPA02000138A - Helical polypeptide zalpha29.

Info

Publication number: MXPA02000138A
Application number: MXPA02000138A
Authority: MX
Inventors: Darrell C Conklin
Original assignee: Zymogenetics Inc
Priority date: 1999-06-28
Filing date: 2000-06-16
Publication date: 2002-07-02
Also published as: CA2377580A1; WO2001000831A1; JP2003503056A; EP1192254A1; AU5621000A

Abstract

Novel cytokine polypeptides, materials and methods for making them, and method of use are disclosed. The polypeptides comprise at least 15 contiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO:4, and may be prepared as polypeptide fusions comprise heterologous sequences, such as affinity tags. The polypeptides and polynucleotides encoding them may be used within a variety of therapeutic, diagnostic, and research applications.

Description

ZALPHA HELICOIDAL POLYPEPTIDE29 BACKGROUND OF INVENTION Cytokines are polypeptide hormones that are produced by a cell and affect the growth or metabolism of this cell or another cell. In multicellular animals, cytokines control growth, migration, differentiation, and cell maturation. Cytokines play a role in both the development and normal pathogenesis, including the development of solid tumors. Cytokines are physicochemically diverse, ranging in size from 5 kDa (TGF-a) to 140 kDa (Mullerian inhibitory substance). They include chains of a single peptide, as well as homodimers and heterodimers linked with disulfide. Cytokines have an influence on cellular events by agglutination to surface-cell receptors. Agglutination initiates a chain of signaling events within the cell, which ultimately leads to phenotypic changes such as cell division, protease production, cellular migration, protein expression of the surface of the cell. cell, and the production of additional growth factors. Cell differentiation and maturation are also under the control of cytokines. For example, hematopoietic factors, erythropoietin, thrombopoietin, and G-CSF stimulate the production of erythrocytes, platelets, and neutrophils, respectively, from precursor cells in the bone marrow. The development of mature cells from pluripotent progenitors may require the presence of a plurality of factors. The role of cytokines in the control of cellular processes makes them candidates and probable targets for therapeutic intervention; Actually, several cytokines have been approved for clinical use. Interferon-alpha (IFN-a), for example, is used in the treatment of hair cell leukemia, chronic myeloid leukemia, Kaposi's sarcoma, condilomata acuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal and Puri, " Common and Uncommon Features of Cytokines and Cytokine Receptors: An Overview, "in Aggarwal and Puri, eds., Human Cytokines: Their Role in Disease and Therapy, Blackwell Science, Cambridge, MA, 1995, 3-24). Platelet derived growth factor (PDGF) has been approved in the United States and other countries for the treatment of skin ulcers in diabetic patients. Erythropoietin of the hematopoietic cytokine has been developed for the treatment of anemias (eg, EP 613,683). G-CSF, GM-CSF, IFN-β, IFN- ?, and IL-2 have also been approved for use in humans (Aggarwal and Puri, ibid.). Experimental evidence supports additional therapeutic uses of cytokines and their inhibitors. Inhibition of PDGF receptor activity has been shown to reduce intimal hyperplasia in the arteries of injured mandrels (Giese et al., Restenosis Summit VIII, Poster Session # 23, 1996, U.S. Patent No. 5,620,687). Vascular endothelial growth factors (VEGFs) have been shown to promote the growth of blood vessels in ischemic limbs (Isner et al., The Lancet 348: 370-374, 1996), and have been proposed for use as agents for the healing of wounds, for the treatment of periodontal disease, to promote endothelialization in vascular graft surgery, and to promote collateral circulation following myocardial infarction (WIPO Publication No. WO 95/24473; No. 5,219,739). A soluble VEGF receptor (soluble flt-1) has been found to block agglutination of VEGF to receptors on the cell surface and inhibit vascular tissue growth in vitro (Biotechnology News 1-5 (17): 5- 6, 1996). An experimental evidence suggests that the inhibition of angiogenesis can be used to block the development of the tumor. { Biotechnology News, November 13, 1997) and that angiogenesis is an initial indicator of cervical cancer (Br. J. Cancer 76: 1410-1415, 1997). More recently, thrombopoietin has been shown to stimulate platelet production in vivo (Kaushansky et al., Nature 369: 568-571, 1994) and has been the subject of several clinical trials (reviewed by von dem Borne et al. , Bailliere's Clinic Haematol, l: 1: 427-445, 1998). In view of the proven clinical usefulness of cytokines, there is a need in the art for such additional molecules to be used both as therapeutic agents and as research tools and reagents. Cytokines are used in the laboratory to study development processes, and in the laboratory and industry facilities as components of cell culture media.

BRIEF DESCRIPTION OF THE INVENTION Within an aspect of the invention there is provided an isolated polypeptide comprising an amino acid residue sequence selected from the group consisting of residues 48-62 of SEQ ID NO: 2, residues 47- 61 of SEQ ID NO: 4, residues 63-104 of SEQ ID NO: 2, residues 62-103 of SEQ ID NO: 4, residues 105-119 of SEQ ID NO: 2, waste 104-118 of SEQ ID NO: 4, residues 120-137 of SEQ ID NO: 2, residues 119-136 of SEQ ID NO: 4, residues 138-152 of SEQ ID NO: 2, residues 137-151 of SEQ ID NO: 4, residues 153-170 of SEQ ID NO: 2, residues 152-169 of SEQ ID NO: 4, residues 171-185 of SEQ ID NO: 2, and residues 170-184 of SEQ ID NO: 4. Within one embodiment, the isolated polypeptide has from 15 to 1500 amino acid residues. Within a related embodiment, the sequence of the amino acid residues is operably linked via a peptide linker or polypeptide linker to a second polypeptide selected from the group consisting of the maltose agglutination protein, an immunoglobulin constant region , a polyhistidine tag, and a peptide as shown in SEQ ID NO: 5. Within another embodiment, the isolated polypeptide comprises at least 30 contiguous residues of SEQ ID NO: 2 or SEQ ID NO: 4. Within of other embodiments, the isolated polypeptide comprises residues 48-185 or residues 27-190 of SEQ ID NO: 6. Within the additional embodiments, the isolated polypeptide comprises residues 48-185 of SEQ ID NO: 2, residues 47-184 of SEQ ID NO: 4, residues 27-190 of SEQ ID NO: 2, or residues 26-188 of SEQ ID NO: 4.

Within a second aspect of the invention there is provided an expression vector comprising the following operatively linked elements: a transcription promoter; a segment of DNA encoding a polypeptide as described above; and a transcription terminator. Within one embodiment, the DNA segment comprises nucleotides 79 to 570 of SEQ ID NO: 7. Within another embodiment, the expression vector further comprises a sequence of the secretory signal operably linked to the DNA segment. Within a third aspect the invention provides a cultured cell into which an expression vector has been introduced as described above, wherein the cell expresses the DNA segment. Within one embodiment, the expression vector further comprises a sequence of the secretory signal operably linked to the DNA segment, and the polypeptide is secreted by the cell. Within a fourth aspect the invention provides a method of making a protein comprising culturing a cell into which an expression vector has been introduced as described above under conditions whereby the DNA segment is expressed and the polypeptide is produced, and recover the protein. When the expression vector further comprises a sequence of the secretory signal operably linked to the DNA segment, the polypeptide is secreted by the cell and recovered from a medium in which the cell is cultured. Within a fifth aspect the invention provides a protein produced by the method described above. Within an sixth aspect of the invention there is provided an antibody that binds specifically to the protein described above. Within a seventh aspect of the invention there is provided a method of detecting, in a test sample, the presence of an antagonist of zalpha29 activity. The method comprises the steps of: (a) culturing a cell that functions in response to zalpha29; (b) exposing the cell to a zalpha29 polypeptide in the presence and absence of a test sample; (c) comparing the levels of response to zalpha29 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and (d) determining from the comparison the presence of an antagonist of zalpha29 activity in the test sample.

These and other aspects of the invention will become apparent during reference to the following detailed description of the invention and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1D are a Hopp / Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO: 2. The profile is based on a window of six sliding residues. The buried or masked G, S, and T residues and the exposed H, Y, and W residues were ignored. The waste is indicated in the Figure by lowercase letters. Figure 2 is an alignment of the amino acid sequences of human zalpha29 (SEQ ID NO: 2) and mouse (SEQ ID NO: 4) representative.

DETAILED DESCRIPTION OF THE INVENTION Prior to the description of the invention in detail, the definition of the following terms may be useful for understanding it. The term "affinity tag" is used herein to denote a segment of the polypeptide that can be attached to a second polypeptide to provide for the purification or detection of the second polypeptide or to provide sites for the attachment of the second polypeptide to a substrate. In the beginning, any peptide or protein for which an antibody or other specific agglutination agent is available, such as an affinity tag, can be used. Affinity tags include a polyhistidine tract, protein A (Nilsson et al., EMBO J. 4: 1075, 1985, Nilsson et al., Methods Enzymol 198: 3 1991), glutathione S transferase (Smith and Johnson, Gene _6_7: 31, 1988), the maltose agglutination protein (Kellerman and Ferenci, Methods Enzymol, 90: 459-463, 1982, Guan et al., Gene 67: 21-30, 1987), affinity tag of Glu-Glu (Grussenmeyer et al., Proc. Nati, Acad. Sci. USA 82: 7954-4, 1985, see SEQ ID NO: 5, substance P, peptide Flag ™ (Hopp et al., Biotechnology 6: 1204-10, 1988), streptavidin binding peptide, thioredoxin, ubiquitin, cellulose agglutination proteins, T7 polymerase, or other antigenic epitope or agglutination domain. See, in general Ford et al., Protein Expression and Purification 2: 95-107, 1991. The DNAs encoding affinity tags are available from commercial providers (for example, Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA; and Eastman Kodak, New Haven, CT). The term "allelic variant" as used herein, denotes any of two or more alternative forms of a gene that occupies the same chromosomal site. Allelic variation arises naturally through mutation, and can lead to phenotypic polymorphism within populations. Genetic mutations can be inactive (without change in the encoded polypeptide) or can encode polypeptides having the altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote the positions within the polypeptides. Wherever the context permits, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain carboxyl-terminal placed sequence with respect to a reference sequence within a polypeptide is located near the carboxyl terminus of the reference sequence, but not necessarily at the carboxyl terminus of the complete polypeptide. "Angiogenic" denotes the ability of a compound to stimulate the formation of new blood vessels from existing vessels, acting alone or in a coordinated manner with one or more additional compounds. Angiogenic activity can be measured as the activation of endothelial cells, the stimulation of protease secretion by endothelial cells, the migration of endothelial cells, the formation of capillary buds, and the proliferation of endothelial cells. Angiogenesis can also be measured using any of several of the in vivo assays as described herein. A "complement" of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and the reverse orientation when compared to a reference sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary with 5 'CCCGTGCAT 3'. The term "corresponding to", when applied to the positions of the amino acid residues in the sequences, means the corresponding positions in a plurality of sequences when the sequences are optimally aligned. The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons (when compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of the nucleotides, but they encode the same amino acid residue (ie, the GAU and GAC triplets each encode Asp). The term "expression vector" is used to denote a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to the additional segments that provide for its transcription. Such additional segments include the 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 the plasmid or viral DNA, or may contain elements of both. The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic environment and therefore is free of other foreign or undesirable coding sequences, and is in a form suitable for use within the genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include the cDNA and the genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include the 5 'and 3' untranslated regions that are naturally present, such as promoters and terminators. The identification of the associated regions will be evident to a person with ordinary experience in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985). An "isolated" polypeptide or protein is a protein or polypeptide that is found in a condition other than its natural environment, such as away from the blood and tissue of the animal. The isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. The polypeptides can be prepared in a highly purified form, i.e. with a purity greater than 95%, more preferably of a purity greater than 99%. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as trimers or alternatively derived or glycosylated forms. "Operationally linked" means that two or more entities are joined together in such a way that they function in a coordinated manner for their intended purposes. When referring to DNA segments, the phrase indicates, for example, that the coding sequences are linked in the correct reading structure, and the transcription starts at the promoter and proceeds through the segment (s) of coding to the terminator. When referring to the polypeptides, "operably linked" includes sequences linked either covalently (e.g., by disulfide linkage) or non-covalently (e.g., by hydrogen bonding), hydrophobic interactions, or by interactions with a salt bridge), where the desired function (s) of the sequences are retained. The term "ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The sequence differences between orthologs are the result of speciation. A "polynucleotide" is a single-stranded or double-stranded polymer of the deoxyribonucleotide or ribonucleotide bases read from the 5f to 3 'end. The polynucleotides include RNA and AD ?, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The sizes of the polynucleotides are expressed as the base pairs ("abbreviated" pb "), the nucleotides (" nt "), or kilobases (" kb "). Where the context allows, these last two terms can describe the polynucleotides that are single-stranded or double-stranded When the term is applied to double-stranded molecules, it is used to denote the total length and it will be understood that it will be equivalent to the term "base pairs". by those skilled in the art, that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; therefore all nucleotides within a double-stranded polynucleotide molecule are not necessarily grouped by pairs. Such extremes that are not even in general will not exceed 20 nt in length. A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, produced either naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides". The term "promoter" is used herein for its recognized meaning in the art to denote a portion of a gene that contains the DNA sequences that are provided for the binding of RNA polymerase and the initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'non-coding regions of the genes.

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other nonpeptide substituents can be added to a protein by the cell in which the protein is produced, and will vary with the type of the cell. Proteins are defined here in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may nevertheless be present. A "secretory signal sequence" is a DNA sequence that encodes. a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. A "segment" is a portion of a larger molecule (e.g., a polynucleotide or polypeptide) that has specified attributes. For example, a segment of DNA encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5 'to 3' direction, encodes the sequence of amino acids of the specified polypeptide. The weights and molecular lengths of the polymers determined by imprecise analytical methods (for example, gel electrophoresis) will be understood to be approximate values. When such value is expressed as "almost" X or "approximately" X, the established value of X will be understood to be accurate to + 10%. All references cited here are incorporated for reference in their entirety. The present invention provides novel cytokine proteins and polypeptides. This novel cytokine, called "zalpha29", was identified by the presence of remarkable characteristics of polypeptides and polynucleotides of bundle cytokines or packets of four helices (e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin, and growth hormone). Analysis of the amino acid sequence of human zalpha29 shown in SEQ ID NO: 2 indicates the presence of four amphipathic alpha-helical regions. These regions include at least 48 to 62 (propeller A), 105 to 119 (propeller B), 138 to 152 (propeller C), and 171 to 185 (helix D) amino acid residues. Within these helical regions, the residues that are expected to rest within the core of the four-helix package are presented at positions 48, 51, 52, 55, 58, 59, 62, 105, 108, 109, 112, 115, 116, 119, 138, 141, 142, 145, 148, 149, 152, 171, 174, 175, 178, 181, 182, and 185 of SEQ ID NO: 2. The residues 49, 50, 53, 54, 56, 57, 60, 61, 106, 107, 110, 111, 113, 114, 117, 118, 139, 140, 143, 144, 146, 147, 150, 151, 172, 173, 176, 177, 179, 180, 183, and 184 are expected to rest on the exposed surface of the package. The interleave loops comprise approximately residues 63-104 (AB loops), 120-137 (BC loops), and 153-170 (CD loops). The human zalpha29 cDNA (SEQ ID NO: 1) encodes a polypeptide of 190 amino acid residues. Although not wished to be limited by theory, this sequence is predicted to include a 26-residue secretory peptide. Segmentation after residue 26 will lead to a mature polypeptide (residues 27-190 of SEQ ID NO: 2) having a calculated molecular weight (exclusive of glycosylation) of 18,558 Da. Those skilled in the art will recognize, however, that some cytokines (e.g., endothelial cell growth factor, basic FGF, and IL-lß) do not comprise conventional secretory peptides and are secreted by a mechanism that does not It is understood. There is a single consensus N-linked glycosylation site in SEQ ID NO: 2 in residues 111-113. The cDNA also includes a clear polyadenylation signal. The mouse zalpha29 polypeptide (SEQ ID NO: 4) is predicted to include helices and loops at analogous positions, including helices at residues 47-61, 104-118, 137-151, and 170-184; and the loops in waste 62-103, 119-136, and 152-169. See Figure 2. Those skilled in the art will recognize that the boundaries of the predicted domain are somewhat imprecise and can vary by up to + 5 amino acid residues. The polypeptides of the present invention comprise at least 15 contiguous amino acid residues of SEQ ID NO: 2. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of the SEC ID NO: 2, up to the full predicted mature polypeptide (residues 27 to 190 of SEQ ID NO: 2) or the primary translation product (residues 1 to 190 of SEQ ID NO: 2). The corresponding zalpha29 mouse polypeptides (see SEQ ID NO:) are also provided by the invention. As will be described in more detail below, these polypeptides may comprise additional polypeptide sequence (s), other than zalpha29.

Within the polypeptides of the present invention are polypeptides comprising a portion carrying an epitope of a protein as shown in SEQ ID NO: 2 or SEQ ID NO: 4. An "epitope" is a region of a protein to which an antibody can be agglutinated. See, for example, Geysen et al., Proc. Nati Acad. Sci. USA 81: 3998-4002, 1984. The epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope during the folding or unfolding of the protein. Linear epitopes are generally at least 6 amino acid residues in length. Relatively short synthetic peptides that mimic a portion of a protein sequence are routinely capable of producing an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219: 660-666, 1983. Antibodies that recognize linear, short epitopes are particularly useful in analytical and diagnostic applications that employ a denatured protein, such as the Western blotting assay (Tobin, Proc. Nati, Acad. Sci. USA 76: 4350-4356, 1979), or in the analysis of fixed cells or tissue samples. Antibodies to linear epitopes are also useful for detecting zalpha29 fragments, such as could occur in body fluids or cell culture media. The antigenic epitope-bearing polypeptides of the present invention are useful for enhancing or elevating antibodies, including monoclonal antibodies, which specifically agglutinate a zalpha29 protein. The antigenic epitope-bearing polypeptides contain a sequence of at least six, often at least nine, commonly from 15 to about 30 amino acid residues of a zalpha29 protein (eg, SEQ ID NO: 2). Polypeptides comprising a larger portion of a zalpha29 protein, ie from 30 to 50 residues up to the complete sequence, are included. It is preferred that the amino acid sequence of the epitope-bearing polypeptide be selected to provide substantial solubility in the aqueous solvents, i.e. that the sequence includes relatively hydrophilic residues, and the hydrophobic residues are substantially avoided. Such regions include the interdomain loops of zalpha29 and the fragments thereof, in particular the B-C loops (residues 120-137 of SEQ ID NO: 2), which are markedly hydrophilic (see Figure 1C). Polypeptides in this regard include those comprising residues 99-104, 129-134, and 162-167 of SEQ ID NO: 2. Of particular interest within the present invention are polypeptides comprising the complete four-helices package of the zalpha29 polypeptide (eg, residues 48-185 of SEQ ID NO: 2). Such polypeptides may further comprise all or part of one or both amino-terminal regions (residues 27-47 of SEQ ID NO: 2) and carboxyl-terminus (residues 186-190 of SEQ ID NO: 2) of zalpha29, natural, as well as amino acid residues other than zalpha29 or the polypeptide sequences that are described in greater detail below. The polypeptides of the present invention can be prepared with one or more amino acid substitutions, deletions or additions, when compared to SEQ ID NO: 2. These changes will usually be minor in nature, ie conservative amino acid substitutions and other changes which do not significantly affect the cleavage or activity of the protein or polypeptide, and include amino- or carboxyl-terminal extensions, such as the amino-terminal methionine residue, a carboxyl-terminal cysteine residue to facilitate subsequent binding to a marine lame hemocyanin activated with maleimide, a small linker peptide of up to about 20-25 residues, or an extension that facilitates purification (an affinity tag) as described above. Two or more affinity tags can be used in combination. Polypeptides comprising affinity tags may further comprise a polypeptide linker and / or a proteolytic cleavage site between the zalpha29 polypeptide and the affinity tag. Exemplary cleavage sites include thrombin cleavage sites and factor Xa cleavage sites. The present invention also provides a variety of other polypeptide fusions. For example, a zalpha29 polypeptide can be prepared as a fusion to a dimerization protein as described in U.S. Pat. Nos. 5,155,027 and 5,567,584. Suitable dimerization proteins in this regard include the constant region domains of the immunoglobulin. Zalpha29-immunoglobulin polypeptide fusions can be expressed in cells genetically engineered to produce a variety of multimeric zalpha29 analogues. In addition, a zalpha29 polypeptide can be linked to another bioactive molecule, such as a cytokine, to provide a multifunctional molecule. One or more helices of a zalpha29 polypeptide can be linked to another cytokine to improve or otherwise modify its biological properties. Auxiliary domains can be fused to zalpha29 polypeptides to target them with respect to cells, tissues, or specific macromolecules (eg, collagen). For example, a zalpha29 polypeptide or protein can be targeted to a predetermined cell type by fusing a zalpha29 polypeptide to a ligand that binds specifically to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for diagnostic or therapeutic purposes. A zalpha29 polypeptide can be fused to two or more portions, such as an affinity tag for purification and a targeting domain as target. Polypeptide fusions may also comprise one or more cleavage sites, particularly between the domains. See, Tuan et al., Connective Tissue Research 34: 1-9, 1996. Polypeptide fusions of the present invention will generally contain no more than about 1,500 amino acid residues, frequently no more than about 1,200 residues, usually not more than about 1, 000 waste, and will in many cases be considerably smaller. For example, a zalpha29 polypeptide of 164 residues (residues 27-190 of SEQ ID NO: 2) can be fused to the E. coli β-galactosidase (1.021 residues, see Casadaban et al., J. Bacteriol. 143: 971-980, 1980), a spacer of 10 residues, and a factor Xa cleavage site of 4 residues to give a polypeptide of 1,199 residues. In a second example, residues 27-190 of SEQ ID NO: 2 can be fused to the maltose agglutination protein (approximately 370 residues), a 4-residue segmentation site, and a 6-residue polyhistidine label. As described above, the polypeptides of the present invention comprise at least 15 contiguous residues of SEQ ID NO: 2 or SEQ ID NO: 4. These polypeptides may further comprise additional residues as shown in SEQ ID NO: 2, a variant of SEQ ID NO: 2, or another protein as described herein. "Variants of SEQ ID NO: 2" include polypeptides that are at least 85%, at least 90%, or at least 95% identical with the corresponding region of SEQ ID NO: 2. The identity of the percentage sequence it is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986, and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA J39: 10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment marks or values using a gap opening penalty of 10, a gap extension penalty of 1, and the evaluation matrix " BLOSUM62"by Henikoff and Henikoff (ibid.) As shown in Table 1 (amino acids are indicated by standard letter codes). The percentage identity is then calculated as: Total number of identical matches x 100 [length of the longest sequence plus the number of holes introduced in the longest sequences to align the two sequences] • ^ 2 CM oo i LO CM CM 1 I CO rH 00 CM CM «H ITO rH O rH cn CM CM I I < 3 < CM 00 rH or cn CM vH or 00 I 1 1 1 1 oo pn HNH CM rH CM CM CM oo lllll 1 1 1 1 1 o * CM «= 3 * ^ CM cn cn CM or CM CM 00 00 lilil í 1 1 1 1 1? The CM or OO OO IH CM OO rH or rH 00 CM CM IIIII 1 1 1 1 1 sm NNO (. NHO Cl rH or rH CM rH CM lll I 1 1 1 1 1 or C? P. ^ m ro HH n H iM 00 rH rH CM CM rH lllllllll 1 1 1 1 1 1 or wr.oc. H Ht. 'rh o rh o rh 00 oo IIIIIIII 1 1 1 1 rh oooo rp ooo cM o CM rH O «3 < CM 00 iiiii 1 1 1 1 -l ON n HONO i? CM N ri o. CM rH rH cn CM 00 IIIIIII 1 1 1 1 1 1 < f HN CM OHHOM ri HHH (M or cn CM olll II lilil í 1 1 cq SQ? Sw? M r! _ ^ S fe r ü. EH S: H> I LO 8 »The level of identity between the amino acid sequences can be determined by the similarity search algorithm" FASTA "described by Pearson and Lipman (Proc. Nati, Acad. Sci. USA 85_: 2444, 1988) and by Pearson, (Meth. Enzymol, 183: 63, 1990.) Briefly, FASTA first characterizes the sequence similarity by identifying the shared sequences. by the interrogation sequence (e.g., SEQ ID NO: 2) and a test sequence q that has either the highest density of the identities (if the variable ktup is 1) or the pairs of identities (if ktup = 2), without considering substitutions, insertions, or deletions of conservative amino acids. The ten regions with the highest density of identities are then re-evaluated by comparing the similarity of all amino acids formed in pairs using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest evaluation. If there are several regions with evaluations larger than the "cut" value (calculated by a predetermined formula based on the length of the sequence and the value of ktup), then the trimmed initial regions are examined to determine if the regions can be joined to form a close alignment with the gaps. Finally, the highest evaluation regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 444, 1970; Sellers, SIAM J. Appl. Math ^ 26: 787, 1974), which allows the insertions and deletions of amino acids. The preferred parameters for the FASTA analysis are: ktup = l, gap opening penalty = 10, gap extension penalty = l, and substitution matrix = BLOSUM62. These parameters can be introduced in a FASTA program by modifying the file of the evaluation matrix ("SMATRIX"), as explained in Appendix 2 of Pearson, 1990 (ibid.) .- FASTA can also be used to determine the identity of the sequence of the nucleic acid molecules using a relationship such as that described above. For comparisons of nucleotide sequences, the value of ktup can vary from one to six, preferably three to six, more preferably three, with other parameters set as failure characteristics. The present invention includes polypeptides having one or more conservative amino acid changes when compared to the amino acid sequence of SEQ ID NO: 2. The BLOSUM62 matrix (Table 1) is an amino acid substitution matrix derived from approximately 2,000 multiple alignments local segments of the protein sequence, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc Nati, Acad Sci USA 89: 10915, 1992). Accordingly, substitution frequencies of BLOSUM62 can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. When used here, the term "substitution of conservative amino acids" preferably refers to a substitution represented by a BLOSUM62 value greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. The preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2 or 3), although substitutions of the most preferred conservative amino acids are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3). The proteins of the present invention can also comprise the amino acid residues that are not naturally present. Amino acids that are not naturally present include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allorthonine, methyltreonine. , hydroxyethyl-cysteine, hydroxy-ethyl-homocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Various methods are known in the art to incorporate amino acid residues that are not naturally present in proteins. For example, an in vitro system can be used wherein the antisense mutations are suppressed using the chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are already known in the art. The transcription and translation of the plasmids containing the antisense mutations is typically carried out in a cell-free system comprising an extract of E. coli S30 and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991, Ellman et al., Methods Enzymol. 202: 301, 1991, Chung et al., Science 259: 806-809, 1993, and Chung et al., Proc. Na ti. Acad. Sci. USA 90: 10145-10149, 1993. In a second method, translation is carried out in Xenopus oocytes by microinjection of the mutated mRNA and the chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271: 19991-19998, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid to be replaced (eg, with phenylalanine) and in the presence of the amino acid (s) that are not present naturally (for example, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The amino acid that is not naturally present is incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem. 33: 7470-7476, 1994. Amino acid residues that are naturally present can be converted to species that are not naturally present by chemical modification in vitro. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993). Changes of the amino acid sequence are made in zalpha29 polypeptides to minimize alteration of the higher order structure essential for biological activity. For example, changes in amino acid residues will be made so as not to alter the characteristic of the four-helical bundles of the protein family. The effects of amino acid sequence changes can be predicted by computer modeling as described above or determined by analysis of the crystal structure (see, for example, Lapthorn et al., Ibid.). A hydrophilicity profile of SEQ ID NO: 2 is shown in Figures 1A-1D. Those skilled in the art will recognize that this hydrophilicity will be taken into account when designing alterations in the amino acid sequence of a zalpha polypeptide., so as not to alter the total profile. The residues within the core of the four-helix package can be replaced with other residues as shown in SEQ ID NO: 6. The residues that are predicted to be on the exposed surface of the four-helix package will be relatively intolerant to a substitution Other substitutions of candidate amino acids within human zalpha29 are suggested by the alignment of the human (SEQ ID NO: 2) and mouse (SEQ ID: O: 4) sequences as shown in Figure 2, such sequences are approximately 85% in their entirety. The cysteine residue at position 160 of SEQ ID NO: 2 (position 159 of SEQ ID NO: 4) lies in the C-D loops suggesting its participation in an interchain disulfide bond. This residue is expected to be relatively intolerant of a substitution. One skilled in the art can employ many well-known techniques, independently or in combination, to analyze and compare structural features that affect the cleavage of a variable protein or polypeptide to a standard molecule to determine whether such modifications could be significant. An accepted and well-known method for measuring the unfolding or folding is the circular dichroism (CD). The measurement and comparison of CD spectra generated by a modified molecule and the standard molecule are routine in the art (Johnson, Proteins _7: 205-214, 2,990). Crystallography is another well-known and accepted method for analyzing splitting and structure. Nuclear magnetic resonance (NMR), mapping of digestive peptides and epitope mapping are other known methods for analyzing the cleavage and structural similarities between proteins and polypeptides (Schaanan et al., Science 257: 961-964, 1992). The essential amino acids in the polypeptides of the present invention can be identified according to methods 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. Nati, Acad. Sci. USA 88: 4498-4502, 1991). In this last technique, single alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested to verify biological activity as described below to identify the amino acid residues that are critical for the activity of the molecule . Multiple amino acid substitutions can be made and tested using the known methods of mutagenesis and selection, such as those described by Reidhaar-Olson and Sauer (Science 241: 53-57, 1988) or Bowie and Sauer (Proc. Nati. Acad. Sci. USA 86: 2152-2156, 1989). Briefly, these authors describe methods for simultaneously randomly placing two or more positions on a polypeptide, selecting a functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that may be used include phage display (eg, Lowman et al., Biochem. ^: 10832-10837, 1991).; Ladner et al., U.S. Patent No. 5,223,409; Huse, Publication WIPO WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986, Ner et al., DNA 1 _.- 121, 1988). The variants of the polypeptide and zalpha29 DNA sequences described can be generated by means of DNA splicing as described by Stemmer, Nature 370: 389-391, 1994 and Stemmer, Proc. Nati Acad. Sci. USA 91: 10747-10751, 1994. Briefly, variant genes are generated by in vitro homologous recombination by the random fragmentation of a paternal gene followed by re-coupling using PCR, leading to randomly introduced point mutations. This technique can be modified using a family of parental genes, such as allelic variants or genes from different species, to introduce additional variability in the process. The selection or choice of the desired activity, followed by further iterations of the mutagenesis and the assay, provide the rapid "evolution" of the sequences by the selection of the desirable mutations while simultaneously being selected against the deleterious changes. In many cases, the structure of the final polypeptide product will result from the processing of the incipient polypeptide chain by the host cell, therefore the final sequence of a zalpha29 polypeptide produced by a host cell does not always correspond to the total sequence encoded by the host. expressed polynucleotide. For example, expression of the complete zalpha29 sequence in a cultured mammalian cell is expected to lead to the removal of at least the secretory peptide, although the same polypeptide produced in a prokaryotic host could be expected to not be cleaved. The differential processing of the individual chains can lead to the heterogeneity of the expressed polypeptides. The zalpha29 proteins of the present invention are characterized by their activity, ie the modulation of the proliferation, differentiation, migration, adhesion, or metabolism of the cell types responsible. The biological activity of zalpha29 proteins is evaluated using in vitro or in vivo assays designed to detect the proliferation, differentiation, migration or adhesion of cells; or changes in cellular metabolism (eg, the production of other growth factors or other macromolecules). Many suitable assays are known in the art, and representative assays are described herein. Trials using cultured cells are more convenient for selection, such as to determine the effects of substitutions, deletions, or amino acid insertions. However, in view of the developmental processes (eg, angiogenesis, wound healing), in vivo assays will generally be employed to further confirm and characterize the biological activity. Certain in vitro models, such as the model of the three-dimensional collagen gel matrix of Pepper et al., (Biochem Biophys, Res. Comm. 189: 824-831, 1992), are sufficiently complex to evaluate the histological effects. The assays can be carried out using the exogenously produced proteins, or they can be carried out in vivo or in vitro using the cells expressing the polypeptides of interest. Assays can be carried out using the zalpha29 proteins alone or in combination with other growth factors, such as members of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, the the cells of the stem). The representative tests are described later. Mutagenesis methods as described above can be combined with high throughput or high volume screening methods to detect the biological activity of zalpha29 variable polypeptides. Trials that can be scaled up for high performance include mitogenesis assays, which in turn can be carried out in a 96-cavity format. The mutagenized DNA molecules encoding the active zalpha29 polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. Using the methods described above, a person of ordinary skill in the art can prepare a variety of fragments or variants of polypeptides from the SEQ ID NO: 2 or SEQ ID NO: 4 that retain zalpha29 activity of the wild type. The present invention also provides polynucleotide molecules, including DNA and RNA molecules, which encode zalpha29 polypeptides described above. A representative DNA sequence encoding the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 1, and a representative DNA sequence encoding the amino acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 3. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation between these polynucleotide molecules is possible. SEQ ID NO: 7 is a degenerate DNA sequence encompassing all of the DNAs encoding the zalpha29 polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 7 also provides all RNA sequences encoding SEQ ID NO: 2 substituting U for T. Accordingly, the polynucleotides encoding the zalpha29 polypeptide comprising nucleotides 1-534 or nucleotides 52-534 of SEQ ID NO: 7, and their RNA equivalents, are contemplated by the present invention, because they are the segments of SEQ ID NO: 7 that encode other polypeptides of zalpha29 described therein. Table 2 describes the one-letter codes used within SEQ ID NO: 7 to denote the positions of the degenerate nucleotides. The "resolutions" are in the nucleotides denoted by a letter of the code. "Complement" indicates the code for the complementary nucleotide (s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A is complementary to T, and G is complementary to C.

TABLE 2 Nucleotide Resolution Complement Resolution A A T T C C G G G C C T T A A R A | G Y C | T Y C | T R A | G M A | C K G | T K G | T M A | C S C | G S C | G w A | T W AIT H A | C | T D A | G | T B C | G | T V A | C | G V A | C | G B CIGIT D A | G | T H A | C | T N AICIGIT N A | C | G | T The degenerate codons used in SEQ ID NO: 7, which encompass all possible codons for a given amino acid, are described in Table 3, which is given below.

TABLE 3 Amino Code - One Codon Codes Acid Degenerate Letter Cys C TGC TGT TGY Ser S AGC AGT TCA TCG CT WSN Thr T ACA ACC ACG ACT CAN Pro P CCA CCC CCG CCT CCN Wing A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R - AGA AGG CGA CGC CGG MGT Lys K AAA AAG AAR Met M ATG ATG lie I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY T and T T T T T T Trp W TG TG TG T TA T T T T TR TR Asp | Asp B RAY GluIG In Z SAR Any X NNN Hollow - A person with ordinary experience in the art will appreciate that some ambiguity is introduced in the determination of a degenerate codon, representative of all possible codons that encode each amino acid. For example, the degenerate codon for serine (WSN), in some circumstances, can encode arginine (ARG), and the degenerate codon for arginine (MGN), in some circumstances, can encode serine (AGY). There is a similar relationship between the codons that encode phenylalanine and leucine. Accordingly, some polynucleotides encompassed by the degenerate sequence can encode the variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2. Variant sequences can be easily tested to verify functionality as described here. A person with ordinary experience in the art will also appreciate that different species may exhibit a use of the preferential codon. In general, see, Grantham, et al., Nuc. Acids Res. 8_: 1893-912, 1980, Haas, et al. Curr. Biol. 6: 315-24, 1996; Wain-Hobson, et al., Gene 13: 355-64, 1981; Grosjean and Fiers, Gene 1_8: 199-209, 1982; Holm, Nuc. Acíds Res. 14: 3075-87, 1986; Ikemura, J. Mol. Biol. 158: 573-97, 1982. When used here. The introduction of the preferential codon sequences into the recombinant DNA, for example, can improve the production of the protein making the translation of the protein more efficient within a particular type or species of cell. Therefore, the sequence of the degenerate codon described in SEQ ID NO: 7 serves as a template for the optimization of the expression of polynucleotides in various types and species of cells commonly used in the art and described herein. Within certain embodiments of the invention the isolated polynucleotides will hybridize to regions of similar size of SEQ ID NO: 1, or to a sequence complementary thereto, under severe conditions. In general, severe conditions are selected to be about 5 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined pH and ionic strength conditions) at which 50% of the target sequence is hybridized to a perfectly matched or matched probe. Typical severe conditions are those in which the concentration of the salt is up to about 0.03 M at pH 7 and the temperature is at least about 60 ° C. As previously noted, isolated polynucleotides of the present invention include DNA and RNA. The methods to prepare the AD? and the AR? They are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zalpha29 RNA. Zalpha29 transcripts have also been detected in numerous tissues as described below. Total RNA can be prepared using guanine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry, 18: 52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc Nati Acad Sci USA 69: 1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. In the alternative, genomic DNA can be isolated. The polynucleotides encoding zalpha29 polypeptides are then identified and isolated, for example, by hybridization or PCR. The full-length clones encoding zalpha29 can be obtained by conventional cloning procedures. Complementary DNA clones (cDNAs) are commonly used within protein production systems, although for some applications (for example, in expression in transgenic animals) it may be preferable to use a genomic clone, or modify a cDNA clone to include at least one genomic intron. A sequence of partial human genomic zalpha29 is shown in SEQ ID NO: 14. This sequence comprises an exon from nucleotide 1885 to nucleotide 2112 (corresponding to nucleotides 483-710 of SEQ ID NO: 1). The partial mouse genomic sequences are shown in SEQ ID NO: 15 and SEQ ID NO: 16. Within SEQ ID NO: 15, nucleotides 6-165 are an exon corresponding to nucleotides 40-199 of SEQ ID NO: 3. Within SEQ ID NO: 16, nucleotides 175-295 are an exon corresponding to nucleotides 200-320 of SEQ ID NO: 3. Methods for preparing cDNA and clones Genomics are well known and are within the level of ordinary skill in the art, and include the use of the sequence described herein, or parts thereof, to probe or to prepare a library. Expression libraries can be probed with antibodies to zalpha29, receptor fragments, or other specific agglutination partners. The zalpha29 polynucleotide sequences described herein can also be used as probes or primers to clone the 5 'non-coding regions of a zalpha29 gene. Promoter elements of a zalpha29 gene can be used to direct the expression of heterologous genes in, for example, animals or transgenic patients treated with gene therapy. Cloning flanking sequences 5 * also facilitates the production of zalpha29 proteins by "gene activation" as described in U.S. Pat. No. 5,641,670. Briefly, the expression of an endogenous zalpha29 gene in a cell is altered by the introduction into the zalpha29 site of a DNA construct comprising at least one targeting sequence, a regulatory sequence, an exon, and a donor site. for the splice, not grouped in pairs. The targeting sequence as target is a 5 'non-coding sequence of zalpha29 that allows for homologous recombination of the construct with the endogenous zalpha29 site, whereby the sequences within the construct become operatively linked to the coding sequence of endogenous zalpha29. In this manner, an endogenous zalpha29 promoter can be replaced or supplemented with other regulatory sequences to provide improved expression, tissue-specific, or otherwise regulated. Those experts in the. The art will recognize that the sequences described in SEQ ID NOS: 1-2 and 3-4 represent a single allele of human and mouse zalpha29, respectively. Allelic variants of these sequences can be cloned by probing the cDNA or the genomic libraries of different individuals according to standard procedures.

The present invention also provides counterpart polypeptides and polynucleotides from other species ("orthologs"). Of particular interest are zalpha29 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Human zalpha29 orthologs can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a type of tissue or cell that expresses zalpha29 as described above. A library is then prepared from the mRNA of a cell line or positive tissue. A cDNA encoding zalpha29 can then be isolated by a variety of methods, such as probing with a partial or complete human cDNA or with one or more sets of degenerate probes based on the described sequence. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using the primers designed from the representative human and mouse zalpha29 sequences, described herein. Within a further method, the cDNA library can be used to transform or transfect the host cells, and expression of the cDNA of interest can be detected with an antibody to a zalpha29 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. For any zalpha29 polypeptide, including variants and fusion proteins, a person of ordinary skill in the art can easily generate a degenerate polynucleotide sequence encoding this polypeptide using the information described in Tables 3 and 4, above. In addition, those skilled in the art can use the standard program to contemplate zalpha29 variants based on the amino acid and polynucleotide sequences described herein. The present invention thus provides a means that can be read by the computer encoded with a data structure that provides at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEC ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and portions thereof. Suitable forms of media that can be read by computers include magnetic media and media that can be read optically. Examples of the magnetic means include a fixed or hard device, a random access memory (RAM) microcircuit, a flexible magnetic disk, a digital linear tape (DLT), a temporary disk memory, and a ZIP disk. Media that can be read optically are exemplified by compact discs (for example, memory only for CD reading (ROM), video discs that can be written to CD (RW), and those that can be recorded on CD) , and digital video / versatile (DVD) discs (for example, DVD-ROM, DVD-RAM, and DVD + RW). The zalpha29 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced according to conventional techniques using cells into which an expression vector has been introduced. which encodes the polypeptide. When used herein, a "cell into which an expression vector has been introduced" includes both cells that have been manipulated directly by the introduction of the exogenous DNA molecules and the progeny thereof containing the introduced DNA. Suitable host cells are those types of cells that can be transformed or transfected with the exogenous DNA and grown in a culture, and include bacteria, fungal cells, and the highest eukaryotic cells cultured. Techniques for manipulating the cloned DNA molecules and introducing the exogenous DNA into a variety of host cells are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 / a. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. In general, a sequence of DNA encoding a zalpha29 polypeptide is operably linked to other genetic elements required for its expression, generally including a promoter and transcription 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 the selectable markers can be provided on separate vectors, and the replication of the exogenous DNA can be provided by the integration into the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary experience in art. Many such elements are described in the literature and are available through commercial providers. To direct a zalpha29 polypeptide within the secretory pathway of a host cell, a sequence of the secretory signal (also known as a leader sequence, prepro sequence or pre-sequence) is provided in the expression vector. The sequence of the secretory signal may be that of zalpha29, or it may be derived from another secreted protein (e.g., t-PA; see, U.S. Pat. No. 5,641,655) or synized de novo. sequence of secretory signal is operably linked to zalpha29 DNA sequence, ie, two sequences are linked in correct reading frame and positioned to direct newly synized polypeptide into secretory pathway of host cell. sequences of secretory signal are commonly placed 5 'with respect to sequence of DNA encoding polypeptide of interest, although certain sequences of signal can be placed elsewhere in DNA sequence of interest (see, for example, Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). expression of zalpha29 polypeptides by means of a secretory pathway of huospedera cell is expected to lead to production of multimeric proteins. Such multimers include both homomultimers and hetero-ultimers, latter including proteins comprising only zalpha29 polypeptides and proteins that include zalpha29 and heterologous polypeptides (eg, a second four-helical package cytokine polypeptide). If a mixture of proteins results from expression, individual species are isolated by conventional methods. higher order monomers, dimers, and multimers are separated, for example, by size exclusion chromatography. heteromultimers can be separated from homomultimers by immunoaffinity chromatography using antibodies specific for individual dimers or by sequential immunoaffinity steps using antibodies specific for polypeptides of individual components. See, generally, U.S. Pat. No. 5,094,941. multimers may also be joined or coupled in vitro during incubation of component polypeptides under suitable conditions. In general, in vitro coupling will include incubation of protein mixture under denaturing and reducing conditions followed by retraction and reoxidation of polypeptides from homodimers and heterodimers. recovery and coupling of proteins expressed in bacterial cells is described later. Cultured mammalian cells can be used as hosts within present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate mediated transfection (Wigler et al., Cell 1: 725, 1978, Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981, Graham et al. Van der Eb, Virology _52: 456, 1973), electroporation (Neu ann et al., EMBO J. 1: 841-845, 1982), transfection mediated by DEAE-dextran (Ausubel et al., Ibid.), And transfection mediated by liposomes (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). production of recombinant polypeptides in cultured mammalian cells is described, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, .. Patente U.S. No. 4,656,134. Suitable cultured mammalian cells include cell lines 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 ovary of Chinese hamster (for example CHO-Kl; ATCC No. CCL 61 and DG44 CHO, Chasin et al., Som. Cell, Molec. Genet, 1: 2: 555-666, 1986). Additional suitable cell lines are already known in art and are available from public depositaries such as American Type Culture Collection, Manassas, VA. Promoters for use in cultured mammalian cells include SV-40 or cytomegalovirus promoters, (see, for example, U.S. Patent No. 4,956,288, promoters of metallothionein genes (U.S. Patent Nos. 4, 579,821 and 4,601,978) and the major final promoter of the adenovirus. Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited in the American Type Culture Collection, Manassas, VA USA under accession numbers 98669 and 98668, respectively, and derived from them. The selection of the drug is generally used to select the cultured mammalian cells within which the 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 capable of passing the gene of interest to their progeny are referred to as "stable transfectants". A preferred selectable marker is a gene that encodes antibiotic neomycin resistance. The selection is carried out in the presence of a drug of the neomycin type, such as G-418 or the like. The selection systems can also be used to increase the level of expression of the gene of interest, a process referred to as "amplification". The amplification is carried out by culturing the transfectants in the presence of a low level of the selective agent and then increasing the amount of the selective agent to select the cells that produce high levels of the products of the introduced genes. A selectable, amplifiable, preferred marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, resistance to multiple drugs, puromycin acetyltransferase), may also be used. The adenovirus system (described in more detail below) can also be used for the production of the protein in vitro. By culturing cells other than 293 infected with the adenovirus under conditions where the cells are not dividing rapidly, cells can produce proteins for prolonged periods of time. For example, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows the infected cells to survive for several weeks without significant cell division. In an alternative method, 293 cells infected with the adenovirus vector can be grown as adherent cells or in a suspension culture at a relatively high cell density to produce significant amounts of the protein (See Garnier et al., Cytotechnol 15: 145-55, 1994). With any protocol, a secreted heterologous protein, expressed, can be repeatedly isolated from the cell culture supernatant, the lysate, or the membrane fractions depending on the arrangement of the protein expressed in the cell. Within the production protocol of infected 293 cells, non-secreted proteins can also be obtained effectively. Insect cells can be infected with the recombinant baculovirus, commonly derived from Autographa calif omica nuclear polyhedrosis virus (AcNPV) according to methods known in the art. Within one method, the recombinant baculovirus is produced through the use of a transposon-based system described by Luckow et al. (J. Virol. 67: 4566-4579, 1993). This system, which uses the transfer vectors, is commercially available in the form of a set (Bac-to-Bac ™ set); Life Technologies, Rockville, MD). The transfer vector (e.g., pFastBacl ™, Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest in a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid". See, Hill-Perkins and Possee, J. Gen. Virol. 71: 971-976, 1990; Bonning et al., J. Gen. Virol. 7 ^: 1551-1556, 1994; and Chazenblak and Rapoport, J. Biol. Chem. 270: 1543-1549, 1995. In addition, the transfer vectors can include a fusion in the structure with the DNA encoding an extension of the affinity polypeptide or tag as described above. Using techniques known in the art, a transfer vector containing a sequence encoding zalpha29 is transformed into the host cells of E. coli, and the cells are selected for the bacmides which contain an interrupted lacZ gene, indicative of baculovirus recombinant. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. The recombinant virus expressing the zalpha29 protein is subsequently produced. Viral, recombinant storage materials are made by the methods commonly used in the art. For the production of the protein, the recombinant virus is used to infect the host cells, typically a cell line derived from autumn borer worm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g. High Five ™ cells; Invitrogen, Carisbad, CA). See, for example, U.S. Pat. No. 5,300,435. The serum free media is used to grow and maintain the cells. Formulations of suitable media are already known in the art and can be obtained from commercial suppliers. The cells are grown from an inoculation density of about 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells, at which time a material in recombinant viral storage is added to a multiplicity of infections (MOI) of 0.1 to 10, more typically about 3. The methods used are already generally known in the art. Other higher eukaryotic cells can also be used as hosts, including plant cells and bird cells. The use of Agrobacterium rhizogenes as a vector for the expression of genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 1: 1: 47-58, 1987. Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces. cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with the exogenous DNA and producing the recombinant polypeptides therefrom are described, for example, by Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. 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. Pat. No. 4,845,075. Transformed cells are selected by the phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). An exemplary vector system for use in Saccharomyces cerevisiae is the POTl vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows the transformed cells to be selected for growth in the glucose-containing medium. Promoters and terminators suitable for use in yeast include those of the glycolytic enzyme genes (see, for example, Kawasaki, US Patent No. 4,599,311, Kingsman et al., US Patent No. 4,615,974, and Bitter, US Pat. No. 4,977,092) and the alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltose are already known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-65, 1986; Cregg, U.S. Patent No. 4,882,279 and Raymond et al., Yeast 14_, 11-23, 1998. Aspergillus cells can be used according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are described by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are described by Lambowitz, U.S. Pat. No. 4,486,533. The production of the recombinant proteins in Pichia methanolica are described in U.S. Pat. Nos. 5,716,808, 5,376,383, 5,854, and 5,888,768. Prokaryotic host cells, including strains of the bacteria of Escherichia coli, Bacillus and other genera, are also useful host cells within the present invention. Techniques for the transformation of these hosts and the expression of the foreign DNA sequences cloned there are well known in the art (see, for example, Sambrook et al., Ibid.). When a polypeptide of zalpha29 is expressed in bacteria such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed to the periplasmic space by a sequence of bacterial secretion. In the first case, the cells are used, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be re-folded and dimerized by dilution with the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by altering or breaking the cells (for example, by the application of sound or by osmotic shock) to release the contents of the periplasmic space and recover the protein, whereby the need for denaturation and re-folding is eliminated. The transformed or transfected host cells are cultured according to the conventional procedures in a culture medium containing the nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a source of carbon, a source of nitrogen, essential amino acids, vitamins and minerals. The media may also contain components such as growth factors or serum, when required. The growth medium will generally be selected for cells containing the exogenously added DNA, for example, by drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or cotransfected in the host cell. Liquid cultures are provided with sufficient ventilation by conventional means, such as stirring small containers or spraying fermenters. Depending on the proposed use, the polypeptides and proteins of the present invention can be purified up to > 80% purity, > 90% purity, > 95% purity, or even a pharmaceutically pure state, which is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. A purified protein or polypeptide can be prepared substantially free of other proteins or polypeptides, particularly those of animal origin. The recombinant zalpha29 proteins expressed (including chimeric polypeptides and multimeric proteins) are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principies & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically approximately 6 histidine residues) are purified by affinity chromatography on a chelate resin. of nickel. See, for example, Houchuli et al., Bio / Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., Ibid. The fusions of agglutination proteins to maltose are purified on an amylose column according to methods known in the art. The zalpha29 polypeptides can also be prepared by chemical synthesis in accordance with methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85: 2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co. , Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3: 3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides. Using the methods known in the art, zalpha29 proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated, treated with PEG or not treated with PEG; and may or may not include an initial methionine amino acid residue. Target cells for use in assays for zalpha29 activity include, without limitation, vascular cells (especially endothelial cells and smooth muscle cells), hematopoietic cells (myeloid and lymphoid), liver cells (including hepatocytes, fenestrated endothelial cells, Kupffer cells, and Ito cells), fibroblasts (including human dermal fibroblasts and lung fibroblasts), fetal lung cells, articular synoviocytes, pericytes, chondrocytes , osteoblasts, and epithelial cells of the prostate. Endothelial cells and hematopoietic cells are derived from a common ancestral cell, the hemangioblast (Choi et al., Development 125: 725-732, 1998).

The activity of zalpha29 proteins can be • measured in vitro using the cultured cells or in vivo by the. administration of the molecules of the claimed invention to an appropriate animal model. Tests that measure cell proliferation or differentiation are well known in the art. For example, assays that measure proliferation include assays such as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs _8: 347-354, 1989), incorporation of radiolabeled nucleotides (as described, for example. , by Raines and Ross, Methods Enzymol, 109: 749-773, 1985, Wahl et al., Mol Cell Biol. 8: 5016-5025, 1988, and Cook et al., Analytical Biochem. 119: 1-7, 1990), the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of proliferating cells (Porstmann et al., J. Immunol, Methods J32: 169-179, 1985), and the use of salts of tetrazolium (Mosmann, J. Immunol., Methods 65: 55-63, 1983; Alley et al., Cancer Res. 48: 589-601, 1988; Marshall et al., Growth Reg. 5: 69-84, 1995; Scudiero et al., Cancer Res. 8 ^: 4827-4833, 1988). Differentiation can be tested using suitable precursor cells that can be induced to differentiate into a more mature phenotype. Tests that measure differentiation include, for example, the measurement of surface-cell markers associated with tissue-specific expression, enzymatic activity, functional activity, or morphological changes (Watt, FASEB, 5: 281- 284, 1991; Francis, Differentiation 57: 63-75, 1994; Raes, Adv. Anirm. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated aguí for reference. The activity of zalpha29 can also be detected using assays designed to measure the production induced by zalpha29 of one or more additional growth factors or other macromolecules. Such assays include those for determining the presence of hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFa), interleukin-6 (IL-6), VEGF, the growth factor of the acid fibroblasts (aFGF), angiogenin, and other macromolecules produced by the liver. Suitable assays include mitogenesis assays that utilize target cells in response to the macromolecules of interest, receptor agglutination assays, agglutination assays for competition, immunological assays (eg, ELISA), and other formats known in art. The secretion of metalloprotease is measured from the treated primary human dermal fibroblasts, synoviocytes and chondrocytes. The relative levels of collagenase, gelatinase and stromelysin produced in response to culture in the presence of a zalpha29 protein are measured using the zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1: 96-106, 1990). The synthesis of pre-collagen / collagen by fibroblasts and dermal chondrocytes in response to a test protein is measured using the incorporation of 3H-proline into the secreted secreted collagen. Collagen labeled with 3H is visualized by SDS-PAGE followed by autoradiography (Unemori and Amento, J. Biol. Chem. 265: 10681-10685, 1990). The secretion of glycosaminoglycan (GAG) from fibroblasts and dermal chondrocytes is measured using a 1, 9-dimethylmethylene blue dye agglutination assay (Farndale et al., Biochim Biophys. Acta 883: 173-177, 1986). Collagen and GAG assays are also carried out in the presence of IL-lß or TGF-β to examine the ability of the zalpha29 protein to modify the established responses to these cytokines. Activation assays of monocytes are carried out (1) to investigate the ability of zalpha29 proteins to further stimulate monocyte activation, and (2) to examine the ability of zalpha29 proteins to modulate activation of monocytes. monocytes induced by binding or induced by endotoxin (Fuhlbrigge et al., J. Immunol. 138: 3799-3802, 1987). The levels of IL-lß and TNFα produced in response to activation are measured by ELISA (Biosource, Inc. Camarillo, CA). Monocyte / macrophage cells, by virtue of CD14 (LPS receptor), are exquisitely sensitive to endotoxin, and proteins with moderate levels of endotoxin-like activity will activate these cells. The hematopoietic activity of the zalpha29 proteins can be evaluated on several hematopoietic cells in the culture. Suitable assays include assays of the major bone marrow colonies and assays of colonies restricted by the lineage of the final stage, which are already known in the art (eg, Holly et al., Publication WIPO 95 / 21920). Bone marrow cells placed on a suitable semi-solid medium (eg, 50% methylcellulose containing 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% antibiotic PSN mixture) they are incubated in the presence of the test polypeptide, then examined microscopically to verify the formation of colonies. The known hematopoietic factors with used as controls.- The mitogenic activity of zalpha29 polypeptides on hematopoietic cell lines can be measured as described above.

Cell migration is assayed essentially as described by Kahler et al. (Arteriosclerosis, Thrombosis, and Vascular Biology 11_: 932-939, 1997). A protein is considered to be chemotactic if it induces the migration of cells from an area of low concentration of proteins to an area of high protein concentration. A typical test is carried out using the Boyden chambers modified with a polystyrene membrane that separates the two chambers (for example, Transwell®, Corning Costar Corp.). The test sample, diluted in a medium containing 1% BSA, is added to the lower chamber of a 24-well plate containing Transwells. The cells are then placed on the Transwell insert which has been pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of incubation at 37 ° C. The non-migrating cells are removed by rubbing the upper part of the Transwell membrane, and the cells attached to the underside of the membrane are fixed and stained with 0.1% crystalline violet. The stained cells are then extracted with 10% acetic acid and the absorbance at 600 nm is measured. The migration is then calculated from a standard calibration curve. Cell migration can also be measured using the matrigel meter of Grant et al. ("Angiogenesis as a component of epithelial-mesenchymal interactions" in Goldberg and Rosen, Epithelial -Mesenchymal Interaction in Cancer, Birkhauser Verlag, 1995, 235-248, Baatout, Anticancer Research 3/7: 451-456, 1997). The activity of cell adhesion is essentially tested as described by LaFleur et al. (J. Biol. Chem. 272: 32798-32803, 1997). Briefly, the microtiter plates are coated with the test protein, the non-specific sites are blocked with BSA, and the cells (such as the smooth le cells, leukocytes, or endothelial cells) are plated at a density of about 104-105 cells / well. The cells are incubated at 37 ° C (typically for about 60 minutes), then the non-adherent cells are removed by gentle washing. The adhered cells are quantified by conventional methods (for example, by staining with crystalline violet, the lysate of the cells, and the determination of the optical density of the lysate). The control cavities are coated with a known adhesive protein, such as fibronectin or vitronectin. The activity of zalpha29 proteins can be measured with a silicon-based biosensor microphysiometer that measures the rate of extracellular acidification or proton excretion associated with receptor agglutination and subsequent physiological cellular responses. An exemplary device thereof is the Cytosensor ™ Microfiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulator and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science 257: 1906-1912, 1992; Pitchford et al., Meth. Enzymol. 228: 84-108, 1997; Arimilli et al, J. Immunol. Meth. 212: 49-59, 1998; and Van Liefde et al., Eur. J. Pharmacol. 346: 87-95, 1998. The microphysiometer can be used to test adherent or non-adherent eukaryotic or prokaryotic cells. By measuring the changes of extracellular acidification in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zalpha29 proteins, their agonists, and antagonists. The microphysiometer can be used to measure the responses of a eukaryotic cell responsive to zalpha29, compared to a eukaryotic cell that does not respond to zalpha29 polypeptide. Eukaryotic cells responsive to zalpha29 comprise cells in which a receptor for zalpha29 has been transfected creating a cell that functions in response to zalpha29., as well as the cells that respond naturally to zalpha29. The differences, measured by a change in extracellular acidification, in the response of cells exposed to the polypeptide of zalpha29 in relation to a control not exposed to zalpha29, are a direct measurement of cell responses modulated by zalpha29. In addition, such responses modulated by zalpha29 can be evaluated under a variety of stimuli. The present invention thus provides methods for identifying agonists and antagonists of zalpha29 proteins, which comprise providing the response cells to a zalpha29 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detect a change in a cellular response of the second portion of the cells when compared to the first portion of the cells. The change in cellular response is shown as a measurable change in the rate of extracellular acidification. Culturing a third portion of the cells in the presence of a zalpha29 protein and the absence of a test compound provides a positive control for the zalpha29 response cells and a control to compare the agonist activity of a test compound with that of zalpha29 polypeptide. Antagonists of zalpha29 can be identified by exposing the cells to the zalpha29 protein in the presence or absence of the test compound, whereby a reduction in activity stimulated by zalpha29 is indicative of the antagonist activity in the test compound. The expression of zalpha29 polynucleotides in animals provides models for further study of the biological effects of overproduction or inhibition of protein activity in vivo. The polynucleotides encoding the zalpha29 and the antisense polynucleotides can be introduced into the test animals, such as • the mice, using viral vectors or the naked or pure DNA, or transgenic animals can be produced. An in vivo method for testing the proteins of the present invention utilizes the viral delivery systems. Exemplary viruses for this purpose include adenoviruses, herpesviruses, retroviruses, vaccinia viruses, and adeno-associated viruses (AAV). Adenovirus, a double-stranded DNA virus, is usually the best-studied gene transfer vector for the delivery of heterologous nucleic acids. For a review, see Becker et al., Meth Cell Biol. 43: 161-189, 1994, and Douglas and Curiel, Science & amp; amp;; Medicine 4: 44-53, 1997. The adenovirus system offers several advantages. The adenovirus (i) accommodates or adapts relatively large DNA inserts; (ii) makes them grow at a high concentration, (iii) infect a wide range of mammalian cell types, and (iv) be used with many different promoters including the appropriate, tissue-specific, and regulatable promoters. Because the adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of the heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by ligation or direct binding or by a homologous recombination with a cotransfected plasmid. In an exemplary system, the essential gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (eg, the human 293 cell line). When administered intravenously to intact animals, the adenovirus primarily targets the liver. If the adenoviral delivery system has a deletion of the El gene, the virus can not replicate in the host cells. However, host tissue (eg, the liver) will express and process (and, if a signal sequence is present, secrete) the heterologous protein. The secreted proteins will enter the circulation in the highly vascularized liver, and the effects on the infected animal can be determined. An alternative method of gene delivery involves removing the cells from the body and introducing a vector into the cells as a naked or pure DNA plasmid. The transformed cells are then re-implanted in the body. Vectors of pure or naked DNA are introduced into the host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, the use of a gene gun , or the use of a DNA vector transporter. See, Wu et al., J. Biol. Chem. 263: 14621-14624, 1988; Wu et al., J. Biol. Chem. 267: 963-967, 1992; and Johnston and Tang, Meth. Cell Biol. 43: 353-365, 1994. Transgenic mice, designed to express a zalpha29 gene and mice exhibiting a complete absence of zalpha29 gene function, referred to as "knockout mice" (Snouwaert et al., Science 257: 1083, 1992), can also be generated (Lowell et al., Nature 366: 740-742, 1993). These mice can be used to study the zalpha29 gene and the protein encoded by it in an in vivo system. Transgenic mice are particularly useful for investigating the role of zalpha29 proteins in initial development because they allow the identification of developmental abnormalities or blocks resulting from the over or under expression of a specific factor. See also, Maisonpierre et al., Science, 277: 55-60, 1997 and Hanahan, Science 277: 48-50, 1997. Promoters for transgenic expression include the promoters of the metallothionein and albumin genes. The antisense methodology can be used to inhibit the transcription of the zalpha29 gene to examine the effects of such inhibition in vivo. Polynucleotides that are complementary to a segment of a polynucleotide encoding zalpha29 (eg, a polynucleotide as described in SEQ ID NO: 1) are designed to bind to the mRNA encoding zalpha29 and to inhibit the translation of such mRNA. . Such antisense oligonucleotides can also be used to inhibit the expression of the genes encoding zalpha29 polypeptide in cell culture. Most four-helical package cytokines as well as other proteins produced by activated lymphocytes play an important biological role in differentiation, activation, recruitment and cellular homeostasis of cells throughout the body. Zalpha29 and inhibitors of zalpha29 activity are expected to have a variety of therapeutic applications. These therapeutic applications include the treatment of diseases which require immune regulation, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, erythematosis due to systemic lupus, and diabetes. Zalpha29 may be important in the regulation of inflammation, and therefore could be useful in the treatment of rheumatoid arthritis, asthma and sepsis. There may be a role for zalpha29 in the mediation of tumorigenesis, whereby a zalpha29 antagonist could be useful in the treatment of cancer. Zalpha29 may be useful in the modulation of the immune system, whereby zalpha29 and zalpha29 antagonists can be used to reduce rejection of the grafts, preventing host disease against the graft, strengthening immunity against infectious diseases, the treatment of immunocompromised patients (for example, patients with HIV +), or in the improvement of vaccines. The zalpha29 polypeptides can be administered alone or in combination with other vasculogenic or angiogenic agents, including VEGF. When zalpha29 is used in combination with an additional agent, the two compounds can be administered simultaneously or 'sequentially when appropriate for the specific condition being treated. For pharmaceutical use, the zalpha29 proteins are formulated for topical or parenteral delivery, particularly intravenous or subcutaneous, according to conventional methods. In general, the pharmaceutical formulations will include a polypeptide of zalpha29 in combination with a pharmaceutically acceptable carrier, such as a salted solution, a buffered saline solution, 5% dextrose in water, or the like. The formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss of protein on the surfaces of the small vial, etc. Methods of the formulation are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19 / a. ed., 1995. Zalpha29 will be used preferably at a concentration of approximately 10 to 100 μg / ml of total volume, although concentrations in the range of 1 ng / ml to - 1000 μg / ml can be used. For topical application, such as for the promotion of wound healing, the protein will be applied - in the range of 0.1-10 μg / cm2 of the area of the wound, with the exact dose determined by the doctor according to the standards accepted, taking into account the nature and severity of the condition that is going to be treated, the characteristics of the patient, etc. The determination of the dose is within the level of ordinary experience in the art. The dosage is given daily or intermittently during the treatment period. Intravenous administration will be by injection into the bolus or infusion for a typical period of one to several hours. Sustained-release formulations can also be employed. In general, a The therapeutically effective amount of zalpha29 is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant change in hematopoietic or immune function, a significant reduction in morbidity, or a significantly increased histological evaluation. The proteins, agonists, and antagonists of zalpha29 are useful for modulating the expansion, proliferation, activation, differentiation, migration, or metabolism of the types of response cells, which include both primary cells and cultured cell lines. Of particular interest in this regard are hematopoietic cells (including stem cells and mature myeloid and lymphoid cells), endothelial cells, smooth muscle cells, fibroblasts, and hepatocytes. The zalpha29 polypeptides are added to the tissue culture medium for these cell types at a concentration of about 10 pg / ml to about 100 ng / ml. Those skilled in the art will recognize that zalpha29 proteins can be advantageously combined with other growth factors in the culture medium. Within the field of laboratory research, zalpha29 proteins can also be used as molecular weight standards or as reagents in assays to determine levels of protein circulation, such as in the diagnosis of disorders characterized by over or underproduction. of the zalpha29 protein or in the analysis of the cellular phenotype. Zalpha29 proteins can also be used to identify inhibitors of their activity. The test compounds are added to the assays described above to identify compounds that inhibit the activity of zalpha29 protein. In addition to those assays described above, samples can be tested to verify the inhibition of zalpha29 activity within a variety of assays designed to measure receptor agglutination or stimulation / inhibition of zalpha-dependent cellular responses29. For example, zalpha29 response cell lines can be transfected with a reporter gene construct that functions in response to a cell path stimulated by zalpha29. Constructs of the reporter gene of this type are already known in the art, and will generally comprise a response element of activated serum with zalpha29 (SER) operably linked to a gene encoding a testable protein, such as luciferase. The candidate compounds, solutions, mixtures or extracts are tested to verify their ability to inhibit the activity of zalpha29 on target cells as evidenced by a reduction in zalpha29 stimulation of reporter gene expression. Tests of this type will detect compounds that directly block the agglutination of zalpha29 to surface-cell receptors, as well as compounds that block processes in the cell pathway subsequent to agglutination of the receptor-ligand. In the alternative, Compounds or other samples can be tested for direct blocking agglutination zalpha29 receptor zalpha29 using labeled with a detectable label (e.g., 125I, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the agglutination of zalpha29 labeled to the receptor is indicative of inhibitory activity, which can be confirmed by secondary assays. The receptors used within the agglutination assays can be cellular receptors or immobilized receptors, isolated. When used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, fragments agglutination antigen thereof such as fragments F (ab ') 2 and Fab, antibodies, single chain, and the like , including genetically engineered antibodies. Non-human antibodies can be humanized by grafting non-human CDRs onto the human skeleton and constant regions, or by incorporating the entire non-human variable domains (optionally "covering" them with a human-like surface by replacing the exposed waste, where the result is a "coated" antibody). In some cases, humanized antibodies can retain non-human residues within the skeletal domains of the human variable region to improve the appropriate agglutination characteristics. By means of humanizing antibodies, the biological half-life can be increased, and the potential for adverse immune reactions during administration to humans is reduced. A person skilled in the art can generate humanized antibodies with specific and different domains, constants (ie, different subclasses of Ig) to facilitate or inhibit various immune functions associated with the constant domains of the particular antibody. Antibodies are defined to be specific agglutination if they agglutinate to a zalpha29 polypeptide or protein with an affinity at least 10 times greater than the agglutination affinity for controlling the polypeptide or protein (other than zalpha29). The affinity of a monoclonal antibody can easily be determined by a person of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949). Methods for the preparation of polyclonal and monoclonal antibodies are well known in the art (see for example, Hurell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982, which is incorporated herein for reference). As would be apparent to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. . The immunogenicity of a zalpha29 polypeptide can be increased by the use of an auxiliary such as . alum (aluminum hydroxide) or Freund's complete or incomplete auxiliary. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a zalpha29 polypeptide or a portion thereof with an immunoglobulin polypeptide or with a maltose agglutination protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide portion is "similar to the hapten", such portion can be advantageously linked or linked to a macromolecular carrier (such as the marine limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization. Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to zalpha29 polypeptides, and selection of antibody display libraries on phage vectors or the like (eg, through the use of the polypeptide of zalpha.29 immobilized or labeled). Human antibodies can be produced in non-human, transgenic animals, which have been designed to contain the human immunoglobulin genes as described in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination. A variety of assays known to those skilled in the art can be used to detect antibodies that bind specifically to zalpha29 polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such tests include: "Concurrent immunoelectrophoresis, radioimmunoassays, radioimmunoprecipitations, enzyme linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays. antibodies for zalpha29 can be used for affinity purification of the protein, within diagnostic assays to determine circulating levels of the protein; for detecting or quantifying the soluble zalpha29 polypeptide as a marker of the implied disease or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; and as antagonists to block the activity of the protein in vitro and in vivo. Antibodies to zalpha29 can also be used to label cells expressing zalpha29; for affinity purification of the zalpha29 polypeptides and proteins; in the analytical methods that use FACS; for the selection of expression libraries; and for the generation of anti-idiotypic antibodies. The antibodies can be linked to other compounds, including diagnostic and therapeutic agents, using known methods to provide the targeting of these compounds to cells expressing the receptors for zalpha29. For certain applications, including diagnostic uses in vitro and in vivo, it is advantageous to use labeled antibodies. Appropriate labels or direct labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect tags or labels can characterize the use of biotin-avidin or other complementary / anti-complement pairs as intermediates. The antibodies of the present invention can also be conjugated directly or indirectly, with drugs, toxins, radionuclides and the like, and these conjugates used for therapeutic or in vivo diagnostic applications (eg, inhibition of cell proliferation). See, in general, Ra akrishnan et al., Cancer Res. 56: 1324-1330, 1996. The polypeptides and proteins of the present invention can be used to identify and isolate the receptors. The zalpha29 receptors may be involved in the regulation of growth in the liver, the formation of blood vessels, and other developmental processes. For example, zalpha29 polypeptides and proteins can be immobilized on a column, and membrane preparations run on the column (as is generally described in Immobilized Affinity Ligand Techniques, Hermanson et al., Eds., Academic Press, San. Diego, CA, 1992, pp. 195-202). The proteins and polypeptides can also be radiolabelled (Methods Enzymol., Vol.182, "Guide to Protein Purification", M. Deutscher, ed., Academic Press, San Diego, 1990, 721-737) or labeled by photoaffinity (Brunner et al. al., Ann. Rev. Biochem., 62: 483-514, 1993 and Fedan et al., Biochem Pharmacol 33: 1167-1180, 1984) and used to label the surface-specific proteins for the label. In a similar manner, the radiolabeled zalpha29 proteins and polypeptides can be used to clone the analogous receptor in the agglutination assays using cells transfected with a cDNA library of the expression.

The present invention also provides reagents for use in diagnostic applications. For example, the zalpha2 gene, a probe comprising zalpha29 DNA or RNA, or a subsequence thereof, can be used to determine the presence of mutations at or near the zalpha29 site on chromosome 2pl5. Chromosomal aberrations detectable at the zalpha29 gene site include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes, and rearrangements. These aberrations can occur within the coding sequence, within the introns, or within the flanking sequences, including the upstream promoter and regulatory regions, and can be manifested as physical alterations within a coding sequence or changes in the level of expression of the gene. The analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) may be used. PCR primers are at least 5 nucleotides in length, frequently 15 or more nt, and often 20-30 nt. Short polynucleotides can be used when a small region of the gene is targeted for analysis. For the total analysis of the genes, a polynucleotide probe can comprise a complete exon or more. The probes will generally comprise a polynucleotide linked to a signal generating portion such as a radionucleotide. In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as described above, under conditions wherein the polynucleotide will hybridize to the complementary polynucleotide sequence, to produce a first product of the reaction; and (c) comparing the first reaction product with a product of the control reaction. A difference between the first product of the reaction and the product of the control reaction is indicative of a genetic abnormality in the patient. Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be of AR? or AD ?, and will comprise a portion of the SEC ID? O: l, the complement of the SEC ID? O: l, or an equivalent of AR? of the same. Suitable assay methods in this regard include molecular genetic techniques known to those skilled in the art, such as restriction fragment length polymorphism (RFLP) analysis, short series repeat (STR) analysis that employs PCT techniques, linkage or binding chain reaction (Barany, PCR Methods and Applications 1 ^ 5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art. art (Sambrook et al., ibid., Ausubel et al., ibid., AJ Marian, Chest 108: 255-65, 1995). The ribonuclease protection assays (see, for example, Ausubel et al., Ibid., Chapter 4) comprise the hybridization of an RNA probe to a sample of the patient's RNA, after which the product of the reaction ( hybrid of AR? -AR?) is exposed to the R? asa. The hybridized regions of the AR? they are protected from dissolution. Within PCR assays, a genetic sample from the patient is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in the size or quantity of the product recovered are indicative of mutations in the patient. Another PCR-based technique that can be used is the analysis of single-strand conformational polymorphism (SSCP) (Hayashi, PCR Methods and Applications 1: 34-38, 1991). The polypeptides, nucleic acids and / or antibodies of the present invention can be used in the diagnosis or treatment of disorders associated with cell loss or abnormal cell proliferation. (including cancer). The labeled zalpha29 polypeptides can be used to image tumors or other sites of abnormal cell proliferation. Inhibitors of the activity of zalpha29 (zalpha29 antagonists) include anti-zalpha29 antibodies and soluble zalpha29 receptors, as well as other peptidic and non-peptidic agents (including ribozymes). Such antagonists can be used to block the effects of zalpha29 on cells or tissues. Of particular interest is the use of antagonists of zalpha29 activity in cancer therapy. Like the • Initial detection methods improve, it may be possible to intervene in earlier times in the development of tumors, making it feasible to use inhibitors of growth factors to block cell proliferation, angiogenesis, and other events that lead to development and metastasis of the tumor. The inhibitors are also expected to be useful in auxiliary therapy after surgery to prevent the growth of residual cancer cells. The inhibitors can also be used in combination with other cancer therapeutic agents. In addition to the antibodies, the zalpha29 inhibitors include the small molecule inhibitors and the inactive receptor agglutination fragments of zalpha29 polypeptides. The inhibitors are formulated for pharmaceutical use as described above in a general manner, taking into account the precise chemical and physical nature of the inhibitor and the condition to be treated. The relevant determinations are within the level of ordinary experience in the art of formulation. The polynucleotides encoding the zalpha29 polypeptides are useful within the applications of gene therapy where it is desired to increase or inhibit the activity of zalpha29. If a mammal has a zalpha29 gene mutated or absent, a zalpha29 gene can be introduced into the mammalian cells. In one embodiment, a gene encoding a zalpha29 polypeptide is introduced in vivo into the viral vector. Such vectors include a defective or attenuated DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and similar. Defective viruses, which completely or almost completely lack viral genes, are preferred. A defective virus is not infectious after introduction into a cell. The use of defective viral vectors allows administration to cells in a specific, localized area, regardless of whether the vector can infect other cells. Examples of the particular vectors include, but are not limited to, a defective herpes simplex virus 1 vector (HSV1) (Kaplitt et al., Molec. 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., 61: 3096-3101, 1987; Samulski et al., J. Virol. 63: 3822-3888, 1989). Within another embodiment, a zalpha29 gene can be introduced into a retroviral vector as described, for example, by Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell 33: 153, 1983; Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 62: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; Dougherty et al., WIPO Publication WO 95/07358; and Kuo et al., Blood 82: 845, 1993. Alternatively, the vector can be introduced by liposome-mediated transfection ("lipofection"). Synthetic cationic lipids can be used to prepare the liposomes for in vivo transfection of a gene encoding a marker (Felgner et al., Proc Nati Acad Sci USA 84: 7413-7417, 1987; Mackey et al., Proc. Nati Acad. Sci. USA 85: 8027-8031, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages, including the location as molecular targets of liposomes for specific cells. Directing transfection to particular cell types is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. The lipids can be attached or chemically coupled to other molecules for the purpose of location as a target. Peptide and non-peptide molecules can be chemically linked or attached to the liposomes. Within another embodiment, the cells are removed from the body, a vector is introduced into the cells as a plasmid of pure or naked DNA, and the transformed cells are re-planted in the body as described above. The antisense methodology can be used to inhibit the transcription of the zalpha29 gene in a patient as described above in a general manner. Zalpha29 polypeptides and anti-zalpha29 antibodies can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for diagnostic or therapeutic in vivo applications. For example, the polypeptides or antibodies of the present invention can be used to identify or treat tissues and organs that express the corresponding anti-complementary molecule (receptor or antigen, respectively, for example). More specifically, zalpha29 polypeptides or anti-zalpha29 antibodies, or bioactive fragments or portions thereof, can be linked to detectable or cytotoxic molecules and delivered to a mammal that has the cells, tissues, or organs that express the molecule anti-complementary. Suitable detectable molecules can be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles, and the like. Suitable cytotoxic molecules can be attached directly or indirectly to the polypeptide or antibody, and include bacterial or plant toxins (e.g., diphtheria toxin, exotoxin, ricin, abrin, saporin, Preudomonas, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90. These may be either fixed directly to the polypeptide or antibody, or indirectly fixed according to known methods, such as by means of a chelating moiety. The polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with an element of a complementary / anti-complementary pair, wherein the other element is linked to the polypeptide or portion of the antibody. For these purposes, biotin / streptavidin is an exemplary complementary / anti-complementary pair. The toxin-polypeptide fusion proteins or the toxin-fragment / antibody fusion proteins can be used for ablation or inhibition of the targeted cell or tissue, such as in cancer therapy. Of particular interest in this regard are conjugates of a zalpha29 polypeptide and a cytotoxin, which can be used to target the cytotoxin to a tumor or other tissue that is undergoing undesirable angiogenesis or neovascularization. Target cells (ie, those that exhibit the zalpha29 receptor) bind to the zalpha29-toxin conjugate, which is then internalized, killing the cells. The effects of killing the recipient-specific cells (target ablation) are revealed by changes in total animal physiology or by means of histological examination. A) Yes, receptor-directed cytotoxicity, dependent on the ligand, can be used to improve the understanding of the physiological significance of a protein ligand. One such toxin is saporin. Mammalian cells do not have receptors for saporin, which is non-toxic when it remains extracellular. In another embodiment, zalpha29-cytokine fusion proteins or antibody / fragment-cytokine fusion proteins can be used to improve in vitro cytotoxicity (for example, that mediated by monoclonal antibodies against tumor targets). and to improve the in vivo killing of target tissues (e.g., blood cancers and • the bone marrow). See, in general, Hornick et al., Blood 8_9: 4437-4447, 1997). In general, cytokines are toxic if they are administered systemically. The fusion proteins described make it possible to target a cytokine for a desired site of action, such as a cell having binding sites for zalpha29, whereby a high local concentration of cytokine is provided. Cytokines suitable for this purpose include, for example, the stimulating factor of the macrophage colonies of the granulocytes and the interleukin-2 (GM-CSF). Such fusion proteins can be used to elicit the cytokine-induced killing of tumors and other tissues suffering from angiogenesis and neovascularization.

The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or can be introduced locally at the proposed site of action. The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1 Northern blot analyzes for zalpha29 were carried out using "blots" or commercially prepared samples of human RNA (Northern Blots I, II, and III of Human Multiple Tissues, Northern Blot II of Human Fetal Multiple Tissues, and Master Blot of Human RNA; Clontech Laboratories, Inc., Palo Alto, CA). The zalpha29 hybridization probe was generated as a PCR amplification product purified with a gel. The amplification product was made using the oligonucleotides ZC21,720 (SEQ ID NO: 8) and ZC21,721 (SEQ ID NO: 9) as the PCR primers and a cloned zalpha29 cDNA (see SEQ ID NO: 1) like a mold. The PCR amplification was carried out as follows: 1 μl of the zalpha29 cDNA (~ 2 ng) and 40 pmol of each of the oligonucleotide primers ZC21,720 and ZC21,721 were added to a reaction mixture containing the reagents commercially available (KlenTaq Polymerase Set from Advantage ™, Clontech Laboratories, Inc.) following the protocol recommended by the manufacturer. The reaction was carried out as follows: 94 ° C for 30 seconds, 25 cycles of 94 ° C for 5 seconds, 55 ° C for 5 seconds, and 68 ° C for 1 minute, followed by 68 ° C for 3 minutes and a retention at 4 ° C. The 422 bp PCR-amplified fragment was gel-purified and recovered using silica gel particles (QIAEX® II gel extraction set, Qiagen, Valencia, CA) according to the recommended protocol The probe was radioactively labeled using a commercially available pool (primary-random labeling system from Rediprime ™ II, Amersham Corp., Arlington, Heights, IL) according to the manufacturer's protocol. commercially available batch column (column NucTrap®; Stratagene, La Jolla, CA; see U.S. Pat. No. 5,336,412). A hybridization solution (ExpressHyb ™ Hybridization Solution, Clontech Laboratories, Inc.) was used for the prehybridization and hybridization solutions for the "Northern blots" assays. Hybridization was carried out overnight at 65 ° C. Following hybridization, the "blots" or samples were washed in 2X SSC, 0.1% SDS at room temperature, followed by a wash in 0.1X SSC and 0.1%. SDS at 50 ° C. The "blots" or samples were exposed to the film (BIOMAX, Eastman Kodak, New Haven, CT). An all-night exposure showed a band of approximately 870 bases in each strip on all of the "blots" or samples. Each RNA sample on the RNA Master Blot was positive while negative controls were negative. Positive tissues on Northerns included those of the heart, ovaries, fetal lung, brain, small intestine, fetal liver, placenta, colon (mucosal lining), fetal kidney, lung, peripheral blood leukocytes, liver, stomach, musculoskeletal, thyroid, kidney, spinal cord, pancreas, lymph node, spleen, trachea, thymus, adrenal gland, prostate, bone marrow, testes, fetal brain. The positive tissues on the RNA Master Blot that were not on the Northerns also included those of the tonsils, aorta, caudate nucleus, bladder, cerebellum, uterus, cerebral cortex, pituitary gland, frontal lobe, salivary gland, hippocampus, mammary gland, medulla oglongata, appendix, occipital lobe, trachea, putamen, fetal heart, substantia nigra, fetal spleen, thalamus, fetal thymus, and subthalamic nucleus. The Northern blots assays were retested for the human transferrin receptor. The resulting signal generated from the transferrin receptor probe was used to normalize the zalpha29 signal. The tissues with the largest ratio of the zalpha29 signal to the transferrin receptor signal were the heart, liver, and testes.

Example 2 Zalpha29 was mapped or mapped with respect to chromosome 2 using the commercially available version of the Stanford G3 Radiation Hybrid Mapping Panel (Research Genetics, Inc., Huntsville, AL). This panel contains the DNAs that can be treated by PCR of each of the 83 clones of the radiation hybrid of the complete human genome, plus two control DNAs (the MR donor and the A3 receiver). A publicly available WWW server (http: // shgc-www-stanford.edu) allows the chromosomal localization of the markers. For the mapping of zalpha29 with the Stanford G3 RH Panel, the 20 μl reaction mixtures were placed in a 96-well microtiter plate that can be treated with PCR (Strategene, La Jolla, CA) and used in a thermal recycling machine (RoboCycler® • Gradient 96; Strategene). Each of the 85 reactions of PCR consisted of 2 μl of the buffer (KlenTaq 10X PCR reaction buffer (Clontech Laboratories, Inc., Palo Alto, CA), 1.6 μl of the dNTPs mixture (2.5 mM each, Perkin-Elmer, Foster City, CA) ), 1 μl of the sense primer, ZC 22,737, (SEQ ID NO: 10), 1 μl of the antisense primer, ZC 22,738 (SEQ ID NO: 11), 2 μl of an agent for increasing density and dyeing of screening (RediLoad Research Genetics, Inc., Huntsville, AL), 0.4 μl of a commercially available DNA polymerase / antibody mixture (50X Advantage ™ KlenTaq Polymerase Mix, Clontech Laboratories, Inc.), 25 ng of a hybrid clone DNA individual or control and x μl of ddH20 for a total volume of 20 μl The reactions were superimposed with an equal amount of mineral oil and sealed.The conditions of the recycling machine for the PCR were as follows: an initial denaturation of 5 minutes of at 94 ° C, 35 cycles of denaturation from 45 seconds to 94 ° C, 45 seconds of annealing at 64 ° C and 75 seconds of extension at 72 ° C, followed by a final extension of 7 minutes at 72 ° C. The reactions were separated by electrophoresis on a 2% agarose gel (obtained from Life Technologies, Gaithersburg, MD). The results showed the binding of zalpha29 to the SHGC-30949 marker of the skeleton of chromosome 2 with an LOD evaluation of > 11 and at a distance of 0 cR_10000 from the marker. The use of surrounding genes that have been physically mapped places zalpha29 in the 2pl6-pl5 region on chromosome 2.

EXAMPLE 3 The coding region of the mouse zalpha29 protein was amplified by PCR using the primers that added the Fsel and AscI restriction sites at the 5"and 3T terminations respectively, the PCR primers ZC23019 (SEQ ID NO: 12) and ZC23018 (SEQ ID NO: 13) were used with a template plasmid (pT7T3D-Pac) containing the full-length murine zalpha29 cDNA in a PCR reaction as follows: a cycle at 95 ° C for 5 minutes; followed by 15 cycles at 95 ° C for 0.5 minutes, 58 ° C for 0.5 minutes, and 72 ° C for 0.5 minutes, followed by 72 ° C for 7 minutes, followed by soaking at 4 ° C. of the reaction by PCR was loaded onto a 1.2% agarose gel (low melt) (SeaPlaque® GTG; FMC Corp., Rockland, ME) in the TAE buffer (0.04M Tris-acetate, 0.001M EDTA). PCR product of zalpha29 was excised from the gel.The gel slices were melted at 65 °, and the DNA was extracted two times s with phenol and precipitated with ethanol. The PCR product was then solubilized with Fsel + AscI, extracted with phenol / chloroform, precipitated with EtOH, and rehydrated in 20 μl of TE (Tris / EDTA pH 8). The 567 bp zalpha29 fragment was ligated or ligated into the Fsel-Ascl sites of a modified CMV pAdTrack (He et al., Proc. Nati, Acad. Sci. USA 95: 2509-2514, 1998). This construct also contained the marker gene of the green fluorescent protein (GFP) The expression of GFP that activates the CMV promoter was replaced with the SV40 promoter and the SV40 polyadenylation signal was replaced with the polyadenylation signal of human growth hormone. In addition, the natural polylinker was replaced with the Fsel sites, EcoRV, and Ascl. This modified form of the pAdTrack CMV was called pZyTrack. The ligation or union was carried out using a set of linkage and DNA selection (Fast-Link ™, Epicenter Technologies, Madison, Wl). Clones containing zalpha29 cDNA were identified by standard mini-prep procedures. To linearize the plasmid, approximately 5 μg of plasmid pZyTrack zalpha29 were solubilized with Pmel. Approximately 1 μg of the linearized plasmid was cotransformed with 200 ng of the pAdEasy superarolate (He et al., Ibid.) In BJ5183 cells. Cotransformation was done using an electroporator (Gene Pulser®, Bio-Rad Laboratories, Inc., Hercules, CA) at 2.5 kV, 200 ohms, and 25 μFa. The complete cotransformation mixture was plated onto 4 LB plates containing 25 μg / ml kanamycin. Smaller colonies were absorbed and expanded in LB / kanamycin, and recombinant adenovirus DNA was identified by standard DNA miniprep procedures. Digestion of the recombinant adenovirus DNA with Fsel + Asci confirmed the presence of the zalpha29 sequence. DNA from the recombinant adenovirus miniprep was transformed into the E. coli host cells (DH10B ™, Life Technologies, Gaithersburg, MD), and the DNA was prepared using a commercially available plasmid isolation kit (QIAGEN® Plasmid Maxi Kit; Qiagen, Inc., Valencia, CA) according to the supplier's instructions. Approximately 5 μg of the recombinant adenoviral DNA were digested with the Pací enzyme (New England Biolabs) for 3 hours at 37 ° C in a reaction volume of 100 μl containing 20-30U of Pací. The digested DNA was extracted twice with an equal volume of phenol / chloroform and precipitated with ethanol. The DNA microspheres were resuspended in 5 μl of distilled water. A T25 vessel of the QBI-293A cells (Quantum Biotechnologies, Inc., Montreal, Canada), inoculated the day before and grown to 60-70% of the confluence, were transfected with the PacI digested DNA. The DNA digested with Paci was diluted to a total volume of 50 μl with sterile HBS (150 mM NaCl, 20 mM HEPES). In a separate tube, 25 μl of 1 mg / ml of the N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethyl-ammonium (DOTAP) salts (Boehringer Mannheim, Indianapolis, IN ) were diluted to a total volume of 100 μl with HBS. The DNA was added to DOTAP, mixed gently from top to bottom by means of a pipette, and left at room temperature for 15 minutes. The media were removed from the 293A cells, and the cells were washed with 5 ml of serum free MEMalpha containing 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, and 25 mM HEPES buffer (the components of the medium obtained from Life Technologies, Gaithersburg, MD). 5 ml of the serum free MEM were added to the 293A cells and maintained at 37 ° C. The DNA / lipid mixture was added dropwise to the T25 vessel of the 293A cells, mixed gently and incubated at 37 ° C for 4 hours. After 4 hours the medium containing the DNA / lipid mixture was removed by aspiration and replaced with 5 ml of the complete MEM containing 5% fetal bovine serum. - The transfected cells were verified by the expression of GFP and. the formation of the foci (viral plaques). Seven days after the transfection of the 293A cells with the recombinant adenoviral DNA, the cells expressed the GFP and began to form foci. The crude viral lysate was collected with a scraper from the cells and transferred to a 50 ml conical tube. To release most of the virus particles from the cells, three freeze / thaw cycles were carried out in a dry ice / ethanol bath and a 37 ° water bath. The crude lysate was amplified (primary amplification (1 °)) to obtain a "storage material" for the lysate work of the zalpha29 recombinant adenovirus (rAdV). Ten 10 cm plates of almost confluent 293A cells (80-90%) were established 20 hours previously. 200 ml of the crude rAdV lysate were added to each 10 cm plate, and the plates were checked for 48 to 72 hours to verify the CPE (cytopathic effect) under the white light microscope and the GFP expression under the fluorescent microscope. When all of the 293A cells showed CPE, the lysate of the 1 st storage material was collected, and the freeze / thaw cycles were performed as above. For the secondary amplification (2 °), 20 15 cm tissue culture discs of the 293A cells were prepared so that the cells had a confluence of 80-90%. Almost 20 ml of the 5% MEM medium were removed, and each disc was inoculated with 300-500 ml of the amplified rAdv lysate of 1 °. After 48 hours the cells were used for virus production. This lysate was collected in 250 ml polypropylene centrifuge bottles. To purify the rAdV, the NP-40 detergent was added to a final concentration of 0.5% to the bottles of the crude lysate to lyse all the cells. The bottles were placed on a rotating platform for 10 minutes, shaking as fast as possible without the bottles falling off. The wastes were converted into microspheres by centrifugation at 20,000 X G for 15 minutes. The supernatants were transferred to 250 ml polycarbonate centrifuge bottles, and 0.5 volumes of the PEG-8000 / 2M NaCl solution were added. The bottles were stirred overnight on ice. The bottles were centrifuged at 20,000 x G for 15 minutes, and the supernatants were discarded in a bleaching solution.

Using a scraper from the sterile cells, the precipitate from the 2 bottles was resuspended in 2.5 ml of PBS. The virus solution was placed in 2 ml microcentrifuge tubes and centrifuged at 14,000 X G for 10 minutes to remove any additional cellular debris. The supernatant of the 2 ml microcentrifuge tubes was transferred to a tube with 15 ml polypropylene cap and adjusted to a density of 1.34 g / ml with CsCl. The volume of the virus solution was estimated, and 0.55 g / ml of CsCl was added. The CsCl was dissolved, and 1 ml of this -solution weighed 1.34 g. The solution was transferred to the polycarbonate thin-walled centrifuge tubes (3.2 ml; Beckman # 362305) and spun at 348,000 XG for 3-4 hours at 25 ° C in a Bexman Optimal TLX microcentrifuge with a TLA-100.4 rotor. The virus formed a white band. Using tips from the wide-bore pipette, the virus band was collected. The salt was removed by filtration in a gel using the commercially available columns and the crosslinked dextran medium (columns PD-10 pre-packed with Sephadex® G-25M; Pharmacia, Piscataway, NJ). The column was equilibrated with 20 ml of PBS.

The virus was loaded and allowed to move to the column. 5 ml of PBS was added to the column, and fractions of 8-10 drops were collected. The optical densities of the 1:50 dilutions of each fraction were determined at 260 nm on a spectrophotometer. A peak or peak of clear absorbency was present between fractions 7-12. These fractions were pooled, and the optical density (OD) of a 1:25 dilution was determined. The concentration of the virus was determined by the formula: (OD at 260 nm) (25) (l.l x 1012) = virions / ml. The OD of a 1:25 dilution of rAdV of zalpha29 was 0.059, giving a virus concentration of 3.3 X 10 12 virions / ml. To store the virus, glycerol was added to the purified virus at a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80 ° C. A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Canada) was followed to measure the infectivity of the recombinant virus. Briefly, two 96-well tissue culture plates were seeded with 1 X 104 293A cells per well in MEM containing 2% fetal bovine serum for each recombinant virus. which is going to be rehearsed. After 24 hours, 10-fold dilutions of each virus from 1X10-2 to 1X10"4 were made in MEM containing 2% fetal bovine serum, 100 μl of each dilution were placed in each of the 20 After 5 days at 37 ° C, the cavities were read either positive or negative for the CPE, and a value for the plaque forming units / ml (PFU) was calculated.The TCID50 formulation used was as for Quantum Biotechnologies, Inc., previous. The concentration (T) was determined from a plate in which the virus used was diluted from 10"2 to 10 ~ 14, and read 5 days after infection.In each dilution a ratio (R) of positive cavities for the CPE for a total number of cavities was determined To calculate the concentration of the undiluted virus sample, the factor, "F" = l + d (S-0.5), where "S" is the sum of the relationships (R); and "d" is LoglO of the dilution series, for example, "d" is equal to 1 for a 10-fold dilution series.The concentration of the undiluted sample is T = 10 (1 + F ) = TCID50 / ml To convert TCID50 / ml to pfu / ml, 0.7 is subtracted from the exponent in the calculation for the concentration (T) .The adenovirus zalpha29 had a concentration of 1. 3 X 1010 pfu / ml. From the foregoing, it will be appreciated that, although the specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited, except for the appended claims.

LIST OF THE SEQUENCES • < 110 > ZymoGenetics, Inc .. < 120 > ZALPHA HELICOIDAL POLYPEPTIDE29 < 130 > 99-28PC < 150 > US 09/343, 163 < 151 > 1999-06-28 < 160 > 16 < 170 > FastSEQ for Windows Version 3. 0 < 210 > 1 < 211 > 813 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (twenty-one) . ... (593) < 400 > 1 ggctcgagcc ttcgcagagc atg gcg gcg ggc gag ctt gag ggt ggc aaa ccc 53 Met Ala Ala Gly Glu Leu Glu Gly Gly Lys Pro 1 5 10 ctg age ggg ctg ctg aat gcg ctg gcc cag gac act ttc cae ggg tac 101 Leu Ser Gly Leu Leu Asn Ala Leu Ala Gln Asp Thr Phe His Gly Tyr 15 20 25 ccc ggc ate aca gag gag ctg cta cgg age cag cta tat cea gag gtg. 149 Pro Gly Lie Thr Glu Glu Leu Leu Arg Ser Gln Leu Tyr Pro Glu Val 30 - 35 40 cea ccc gag gag ttc cgc ccc ttt ctg gca aag atg agg ggg att ctt 197 Pro Pro Glu Glu Phe Arg Pro Phe Leu Wing Lys Met Arg Gly He Leu 45 50 55 aag tet att gcg tet gca gac atg gat ttc aac cag ctg gag gca ttc 245 Lys Ser Wing Wing Wing Asp Met Asp Phe Asn Gln Leu Glu Wing Phe 60 65 70 75 ttg act gct caa acc aaaaag caa ggt ggg ate aca tet gac caa gct 293 Leu Thr Ala Gln Thr Lys Lys Gln Gly Gly He Thr Ser Asp Gln Wing 80 85 90 gct gtc att tec aaa ttc tgg aag age falls aag aca aaa ate cgt gag 341 Wing Val lie Ser Lys Phe Tf Lys Ser His Lys Thr Lys He Arg Glu 95 100 105 age ctc atg aac cag age cgc tgg aat age ggg ctt cgg cgc ggc ctg age 389 Ser Leu Met Asn Gln Ser Arg Trp Asn Ser Gly Leu Arg Gly Leu Ser 110 115 120 tgg aga gtt gat ggc aag tet cag tca agg falls tca gct caa ata falls 437 Trp Arg Val Asp Gly Lys Ser Gln Ser Arg His Be Wing Gln He His 125 130 135 aca cct gtt gcc att ata gag ctg gaa tta ggc aaa tat gga cag gaa 485 Thr Pro Val Ala He lie Glu Leu Glu Leu Gly Lys Tyr Gly Gln Glu 140 145 150 155 tet gaa ttt ctg tgt ttg gaa ttt gat gag gtc aaa gtc aac caa att 533 Ser Glu Phe Leu Cys Leu Glu Phe Asp Glu Val Lys Val Asn Gln lie 160 165 170 ctg aag acg ctg tca gag gta gaa agt ate age aca ctg ate age 581 Leu Lys Thr Leu Ser Glu Val Glu Glu Ser He Thr Leu He Ser 175 180 185 cag cct aac tga agatgatgta tgaaggagtt ggagttgttg aaaccaaggt 633 Gln Pro Asn * 190 gtccatgatc cctccccact gaccttttct aagaaaattc ttgtgcccgc attggtatta 693 aatcctcgca ttcagteaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 753 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 813 < 210 > 2 < 211 > 190 < 212 > PRT < 213 > Homo sapiens 400 > 2 Met Wing Wing Gly Glu Leu Glu Gly Gly Lys Pro Leu Ser Gly Leu Leu 1 5 10 15 Asn Ala Leu Ala Gln Asp Thr Phe His Gly Tyr Pro Gly He Thr Glu 20 25 30 Glu Leu Leu Arg Ser Gln Leu Tyr Pro Glu Val Pro Pro Glu Glu Phe 35 40 45 Arg Pro Phe Leu Wing Lys Met Arg Gly He Leu Lys Ser Wing Ser 50 55 60 Wing Asp Met Asp Phe Asn Gln Leu Glu Wing Phe Leu Thr Wing Gln Thr 65 70 75 80 Lys Lys Gln Gly Gly He Thr Ser Asp Gln Wing Wing Val He Ser Lys 85 90 95 Phe Trp Lys Ser His Lys Thr L? S He Arg Glu Ser Leu Met Asn Gln 100 105 110 Ser Arg Trp Asn Ser Gly Leu Arg Gly Leu Ser Trp Arg Val Asp Gly 115 120 125 Lys Ser Gln Ser Arg His Ser Wing Gln He His Thr Pro Val Wing 130 135 140 lie Glu Leu Glu Leu Gly Lys Tyr Gly Gln Glu Ser Glu Phe Leu Cys 145 150 155 160 Leu Glu Phe Asp Glu Val Lys Val Asn Gln He Leu Lys Thr Leu Ser 165 170 175 Glu Val Glu Glu Ser He Thr Leu He Ser Gln Pro Asn 180 185 190 < 210 > 3 < 211 > 805 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > CDS < 222 > (2. 3) . . . (589) < 400 > 3 ggatcttggg ccctccttag cc atg gcg ggc gat ctg gag ggt ggc aag tec 52 Met Wing Gly Asp Leu Glu Gly Gly Lys Ser - 1 5 10 ctg age ggg ctg ctg age ggc cta gcg cag aac gcc ttt drops gga drops 100 Leu Ser Gly Leu Leu Be Gly Leu Wing Gln Asn Wing Phe His Gly His 15 20 25 tcg ggt gtc acg gag gag ctg ctg fall age caa ctc tat ccg gaa gtg 148 Ser Gly Val Thr Glu Glu Leu Leu His Ser Gln Leu Tyr Pro Glu Val 30 35 40 cea ccg gag gag ttc cgc ccc ttc ctg gcg aag atg aga gga ctt ctc 196 Pro Pro Glu Glu Phe Arg Pro Phe Leu Ala Lys Met Arg Gly Leu Leu 45 50 55 aag tet att gca tet gca gac atg gat ttc aac cag tta gag gca ttc 244 Lys Ser Wing Wing Wing Asp Met Asp Phe Asn Gln Leu Glu Wing Phe 60 65 70 ctg act gct caa acc aaaaag aa ggt ggc ate aac agt gag caa gct 292 Leu Thr Ala Gln Thr Lys Lys Gln Gly Gly He Thr Ser Glu Gln Ala 75 80 85 90 gca gtc ate tec aag ttt tag aag age falls aag ata aaa ate cga gag 340 Wing Val He Ser Lys Phe Trp Lys Ser His Lys He Lys He Arg Glu 95 100 105 agt ctc atg aag cag age cgc tgg gac aac ggc ctt cgg ggc ctg age 388 Ser Leu Met Lys Gln Ser Arg Trp Asp Asn Gly Leu Arg Gly Leu Ser 110 115 120 tgg aga gtc gat ggc aag tet cag tca cgg falls tca act cag ata falls 436 Trp Arg Val Asp Gly Lys Ser Gln Ser Arg His Ser Thr Gln He His 125 130 135 age cct gtt gcc ata ata gag ctg gaa ttt gga aaa aat gga cag gaa 484 Ser Pro Val Wing He He Glu Glu Leu Glu Lys Asn Gly Gln Glu 140 145 150 tet gaa ttt ttg tgt ctg gaa ttt gat gaa gtt aaa gtc aag caa ate 532 Ser Glu Phe Leu Cys Leu Glu Phe Asp Glu Val Lys Val Lys Gln He 155 160 165 170 ctg aag aag ctg tca gag gta gag agt ate aac agg ctg atg cag 580 Leu Lys Lys Leu Ser Glu Val Glu Glu Ser lie Asn Arg Leu Met Gln 175 180 185 gca gcc 'taa ctgaagagag tatcaatagg ctgatgcagg cagcctaact 629 Ala Ala * gaaggctgga ggaaggggcg tttgaagtga agctgctcac agactttctc cactgaccct 689 ttgaaagtcc tgtttgccca ctggtgttac caaaagacat tgtatacatg catgaaagtc 749 ttcaagaata aataaaaata tattttaaaa agtgggtaaa aaagagaaac ctctca 805 < 210 > 4 < 211 > 188 < 212 > PRT < 213 > Mus musculus < 400 > 4 Met Wing Gly Asp Leu Glu Gly Gly Lys Ser Leu Ser Gly Leu Leu Ser 1 5 10 '15 Gly Leu Wing Gln Asn Wing Phe His Gly His Ser Gly Val Thr Glu Glu 20 25 30 Leu Leu His Ser Gln Leu Tyr Pro Glu Val Pro Pro Glu Glu Phe Arg 35 40 45 Pro Phe Leu Wing Lys Met Arg Gly Leu Leu Lys Ser lie Wing Ser Wing 50 55 60 Asp Met Asp Phe Asn Gln Leu Glu Wing Phe Leu Thr Wing Gln Thr Lys 65 70 75 80 Lys Gln Gly Gly He Thr Ser Glu Gln Wing Ala Val He Ser Lys Phe 85 90 95 Trp Lys Ser His Lys He Lys He Arg Glu Be Leu Met Lys Gln Ser 100 105 110 Arg Trp Asp Asn Gly Leu Arg Gl? Leu Ser Trp Arg Val Asp Gly Lys 115 120 125 Ser Gln Ser Arg His Ser Thr Gln He His Pro Val Wing He He 130 135 140 Glu Leu Glu Phe Gly Lys Asn Gly Gln Glu Ser Glu Phe Leu Cys Leu 145 150 155 160 Glu Phe Asp Glu Val Lys Val Lys Gln He Leu Lys Lys Leu Ser Glu 165 170 175 Val Glu Glu Ser He Asn Arg Leu Met Gln Wing Ala 180 185 < 210 > 5 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > peptide tag < 400 > 5 Glu Tyr Met Pro Met Glu 1 5 < 210 > 6 < 211 > 190 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Variable polypeptides < 221 > VARIANT < 222 > (48) ... (105) < 223 > Xaa is Leu, Lie, Val, Met, Phe, Trp, Gly or Ala < 221 > VARIANT < 222 > (108) ... (108) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly, Ala or Ser < 221 > VARIANT < 222 > (109) ... (109) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly or Ala < 221 > VARIANT < 222 > (112) ... (112) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly, Ala or Gln < 221 > VARIANT < 222 > (115) ... (115) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly or Ala < 221 > VARIANT < 222 > (116) ... (116) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly, Ala or Asp < 221 > VARIANT < 222 > (119) ... (138) - < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly or Ala < 221 > VARIANT < 222 > (141) ... (141) < 223 > Xaa is .Leu, He, Val, Met, Phe, Trp, Gly, Ala or Pro < 221 > VARIANT < 222 > (142) ... (145) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly or Ala < 221 > VARIANT < 222 > (148) ... (148) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly, Ala or Glu < 221 > VARIANT < 222 > (149) ... (149) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly or Ala < 221 > VARIANT < 222 > (152) ... (152) < 223 > Xaa- is Leu, He, Val, Met, Phe, Trp, Gly, Ala, Asn or Tyr < 221 > VARIANT < 222 > (171) ... (171) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly or Ala < 221 > VARIANT < 222 > (174) ... (174) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly, Ala, Lys or Thr < 221 > VARIANT < 222 > (175) ... (178) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly or Ala < 221 > VARIANT < 222 > (181) ... (181) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly, Ala or Ser < 221 > . VARIANT < 222 > (182) ... (185) < 223 > Xaa is Leu, He, Val, Met, Phe, Trp, Gly or Ala 400 > 6 Met Wing Wing Gly Glu Leu Glu Gly Gly Lys Pro Leu Ser Gly Leu Leu 1 5 10 15 Asn Ala Leu Ala Gln Asp thr Phe His Gly Tyr Pro Gly He Thr Glu 20 25 30 Glu Leu Leu Arg Ser Gln Leu Tyr Pro Glu Val Pro Pro Glu Glu Xaa 35 40 45 Arg Pro Xaa Xaa Ala Lys Xaa Arg Gly Xaa Xaa Lys Ser Xaa Ala Ser 50 - 55 60 Wing Asp Met Asp Phe Asn Gln Leu Glu Wing Phe Leu Thr Wing Gln Thr 65 70 75 80 Lys Lys Gln Gly Gly He Thr Ser Asp Gln Ala Wing Val He Ser Lys 85 90 95 Phe Trp Lys Ser His Lys Thr Lys Xaa Arg Glu Xaa Xaa Met Asn Xaa 100 105 110 Ser Arg Xaa Xaa Ser Gly Xaa Arg Gly Leu Ser Trp Arg Val Asp Gly 115 120 125 K Lys Ser Gln Ser Arg His Ser Ala Gln Xaa His Thr Xaa Xaa Ala He _ > 130 135 140 Xaa Glu Leu Xaa Xaa Gly Lys Xaa Gly Gln Glu Ser Glu Phe Leu Cys 145 150 155 160 Leu Glu Phe Asp Glu Val Lys Val Asn Gln Xaa Leu Lys Xaa Xaa Ser 165 170 175 Glu Xaa Glu Glu Xaa Xaa Ser Thr Xaa He Ser Gln Pro Asn 180 185 190 < 210 > 7 < 211 > 570 < 212 > DNA < 213 > Artificial Sequence 40 < 220 > < 223 > degenerate sequence < 221 > mise feature '< 222 > (1) . . . (570) 45 < 223 > n = A, T, C or G < 400 > 7 atggcngcng gngarytnga rggnggpaar ccnytnwsng gnytnytnaa ygcnytngcn 60 cargayacnt tycayggnta yccnggnath acngargary tnytnmgnws ncarytntay 120 cnccngarga ccngargtnc rttymgnccn ttyytngcna aratgmgngg nathytnaar 180 wsnathgcnw sngcngayat ggayttyaay carytngarg cnttyytnac ngencaraen 240 aaraarcarg gnggnathac nwsngaycar gengengtna thwsnaartt ytggaarwsn 300 cayaaracna arathmgnga rsnytnatg aaycarwsnm gntggaayws nggnytnmgn 360 ggmgngtnga ggnytnwsnt yggnaarwsn carwsnmgnc aywsngcnca ratheayaen 420 ccngtngcna thathgaryt ngarytnggn aartayggnc argarwsnga rttyytntgy 480 ytngarttyg aygargtnaa rgtnaayear athytnaara cnytnwsnga rgtngargar 540 wsnathwsna cnytnathws nearecnaay 570 < 210 > < 211 > 18 < 212 > DNA '< 213 > Artificial Sequence '< 220 > < 223 > oligonucleotide primer ZC21.720 < 400 > 8 ggtaccccgg catcacag 18 < 210 > 9 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide primer ZC21.721 < 400 > 9 gacctcatca aattccaaac here 23 < 210 > 10 < 211 > 18 '< 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide primer ZC22.737 < 400 > 10 gctggcccag gacacttt 18 < 210 > 11 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucl primer < 400 > 11 gaatccccct catctttg 18 < 210 > 12 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide primer ZC23019 < 400 > 12 cacacaggcc ggccaccatg gcgggcgatc tggagggtgg 40 < 210 > 13 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide primer ZC23018 < 400 > 13 cacacaggcg cgcctttagg ctgcctgcat cagcctgttg 40 < 210 > 14 < 211 > 6644 < 212 > DNA < 213 > Homo sapiens < 400 > 14 tcaactttgc gctttagggc ttacgcacgg acgccagggt gcctgggggg tgagatttga 60 caaaatcgta ttgaactcct ggcttcaagt gatcctcctg cgtcagcctt ccaaaatgct 120 ggggttacag gcatgagcca ccatgcctgg cctggcaggt ctcatttttt agaagtttct 180 gctcttgttc agataatgta aactecaaga cttatttttc tatctgttga gtctcaattg 240 ccttcagcac aaagtaacct ataggccaaa gtggcaaagt tggggctggt atattctgat 300 acttttcagt agttaggaaa atacagagaa aagaacattg acttagtgac tgtcatcaac 360 agagggacat taattageca catcatttaa catgcctggg gcttaattta attatetaga 420 aaacaaggca atgctctgta attggattat ctgctctaaa aggattcata ttacttgatt 480 aggattttga attgttctga atgtagttgc tectagatac ataaaattta tccttgtctc 540 tcaagataat gcagatgtag aatttgtcta gtgtgcatga aattcctgct gccataactc 600 tgactgggca agagatctta acatactgat cctcccccta tacaaaaaaa cccccatctc 660 tttttttttt aaaaaaaatt agttaaccgg ggcagggttg gcaaccctgt agtecaagea 720 actcaggagg ctgaggtggg aggatcactt tagcccagga gtttgaggct acagtgagct 780 atgatcatgc cactgcactc cagcctggaa acagaggggg aaaaaaatct tattttttga 840 tatctaccat ctacctaacc taggattgat tgaccaatcc taacagtgat tgaagggaac 900 gtatttaagg gagctgtgag gagggttgct tgcagcaagt ggagggaccc cagaaatgtt 960 tgttgttatt tcaagatctg gttatctttc agttatctgg ctcatgtttc ccaagcaact 1020 tgtcacaatt tctggcataa ccactaaatc caagttaget cacttcccag actaaetcag 1080 agtccatcag agtcagtcaa aattctcctt tcatttttgt aatccaaggt gctgtggaga 1140 ttcggcgtca cagtatggac tecagagetg agagttecca cagtctgatt ctgcctttat 1200 atggtattta tttttgatac aggttaacct ctttgtgcct cagggtcctc atttttaaaa 1260 cagcactact caagttetta ccttaaaaat tattagagga tecaatgaga tgacaaagag 1320 gaagagcttc teatatgect acatctgaca cctagtgaga tttgtacgcc tettatttat 1380 caccatcata caatatgaaa aaagcacttt tttttttttt tgagatggac tctcactctc 1440 tcccccaggc tggagtgcag tggcccgatc tcggctcact gcaagctccg actttcgggt 1500 tcacgccatt ctcctgcctc agcctcctga gtagctggga ctcacaggtg cccgcaactg 1560 attttttata cgcctggcta tttttagtag agacggggtt tcacccgtgt tagccaggat 1620 ggtctcgatc tcctgacctt gtgatctgcc cgcctcggcc tcccaaagtg ctgggattac 1680 aggtgtgagc cactgcgcct ggcataaaag cacttttatt tagggcatta tgtggatatg 1740 ctttgctggg taaaatatca ettegatatt aaggtctgag ctgggcttgc tggattggga 1800 ccctgggtat tttgagtttg gtcatgccag atggccttgg ctatcttgtg gtttccctct 1860 aaaatatcct tttatgtttt ccaggaatct gaatttctgt gtttggaatt tgatgaggtc 1920 aaattctgaa aaagtcaacc gacgctgtca gaggtagaag aaagtatcag cacactgatc 1980 agccagccta actgaagatg atgtatgaag gagttggagt tgttgaaacc aaggtgtcca 2040 tgatccctcc ccactgacct tttctaagaa aattcttgtg cccgcattgg tattaaatec 2100 tegeatteag tcttcctgcc tctacttgct cagatttett tttttctagc ttteatttag 2160 tettacattt gttccagtgc agaggttctc acccttcagt gtgcataaat gttataaggg 2220 gtacttgtaa aagcattcac ttttttgttg ttattattaa attcggagtg ttgctctgtt 2280 gcccaggctg gagtgeagtg gtgeagtcat ggctcactgc agcctcaagc tcctggactc 2340 aagcgagcct cccacctcag cctctcaagt agctgggact acaggtgcat gccaccacac 2400 tcaggtaatt tttgtatttt ttgtagagat ggggtttcac catgttgccc aggctggtct 2460 ggaaatcctg ggctcaagtg atcctcccac cctggcttcc caaagcccaa agtgctggga 2520 gagaaaagca ttacaggcgt tttacattta aaaaaaaaaa aaaaaaaaaa aagtaggctt 2580 ccagggctct atccccagag acttggattc aataggatta gggtgagaga gatcagcaat 2640 ggaaatcctt gatatagtgg tttgtcctgg gtggggtttt aagaatttat acatgataaa 2700 attattttta tcatgtagga aagtagaaaa aaaagctttt atcatgcaaa tatagggctg 2760 accaaagtgc tatgtactta gctgaggcat aggagcacct acctaaccta gaaaagatgt 2820 acctgaccct agttaaaacc tgagctcttt ctgaaactga tttgggcatt tggattagtt 2880 ctgcttaaat ctggggcatc tggtttgatc tgaactactg agagaetcag gcttttctgg 2940 aacctagaac taaattggcc tcacaacaaa gggactccct tcacttgcct caagtcagga 3000 tcatgggaag gggcagatgt ctgctgagac tgatgtgagg tcttttacct cagaaaattt 3060 tacctgagtc attaaaataa aacccctttc aaaaaatttc tttaagaaaa actagtgtaa 3120 taaaaagtag gtcctattag agaaettace aaaeaccaag aacattctaa cggcggaggc 3180 tgtcaactag catattgagg ctatggtcct tgttgaacag gttttgtcat ctgatactga 3240 aaatetaaga taatatttag tgcctttgga gtataatatg ttcaaaaaat gtggttatct 3300 tggte tgtga ttacageata tgtccatgct aaggagtttg tttcaggaca ggaataagtc 3360 ctcttctgtt aagcagtttc tectaaatca gtttggagac atttcaggag cttttctaac 3420 acccaagctg aaattattgg cttcttctct gattaaaacc atcccagcag ttagcaaaca 3480 ataaccagaa ggttttcaat gtagcccctg tgcacccttc agaaaacatc ttgaaacagt 3540 gattcaagaa actgtaaata aggaatgtgg tttggaaaaa aaaaaaacta ttttaaactt 3600 gccttctgtt cccagggctg ctgtcatgta attttgataa atctaggaga ggtctcctgc 3660 agcaatagaa tgtaaaatgg gataatcgga tatetcatca ttccagatgt ccttggaagg 3720 aataactaga gctatcacct tagtattgac teatatatec catggaagtc tgtggaagtg 3780 gcacatgggc tgaaggaaca cacaaggaga ggaaatcatt gtcatagtct gaaactctgt 3840 ttaggtcatc ccatgaaagt aatagetaca tgggctttta agagtggcca aaattgtatt 3900 cccaaaccct aggtcattgg aagactacag ttaatgtata ctgagttttc aagaattaaa 3960 aagaaaacca aaaactggtt gttgcaggtg gtccccacat ttaacactaa gcacttctga 4020 atgcaagttg tttctaacag ggtatatttt atatttactg atgattttta attttttatt 4080 tatatgttta atcaaaggta ttatagtcta ttatgaaaat aeagaaacat actaagaaca 4140 gttaatgacc catcaatcta atgtacagaa agaaggctag aactaggaaa agagttgact 4200 ttccttgaat aaattcccag aagtggagta cacagtttta tttatttatt tagagacaga 4260 gtttcactct gtcacccatg ctggaatgca gtggcgcaat ctcagctcac tgcaacctcc 4320 gcctcccggg ttcaagtgat tctcctgtct cagcctcctg agtagctggg actacaggca 4380 cgtccggcta cctgccacca attttttttt gtatttttag tagagacagg gtttcactat 4440 gttggccagg ctggtctcga actcctgacc tccagtgatt gacccgcctc agcctcccaa 4500 agtgctggga ttacaggcgt gagccactgc acccggccta gtacacagtt tttaactttg 4560 ataaacattg ccaaattcct ctccaggaag gctgtattaa tttgtattcc ctctgagaaa 4620 gtataagact aaattacccc ctctcttgcc taattggcta tcatcatttt ttgtattttc 4680 tgggagtaag ttcttagaaa gttttgtaag ggacaettac attaaaccag gacatctccc 4740 tggtaacaat aaaageatgg agaaaggacc agggaaggag aaaacaggta taaagttccc 4800 agagacccca ctaggttttc tacctgtgcg atectagatt aaaaccactt gttttgattt 4860 caggaaatta gggacaaaat aaaaatctca gcctgaactg gaccttgtag aaattatece 4920 tgcttgagca ataageaetc taaattcagt ctgtttagaa agattcctgc ccgttagcca 4980 ggtgtggtag cacaggcctc aagtcc AAGC tgctcaggag gctgaggaag gaggatgcct 5040 tgagcccagg agtttggggc ttcaggcaac aacagcaaga gcccatctct aaaaaagaaa 5100 gagagagaga gaaaaggaga gagagagaga gatgagagag agagaaagat gagagaagaa 5160 aagaaaaaaa cagtccagcc aagctaaaag ttagctttca gaataaagtc agaaaataac 5220 tccagatttt ggtagcgttg tgttgatacg aagcaaaaga tttggcctta ttcttaggtc 5280 aggctttcct tggaagctct agttcttctc agctgtaaca gcaaaagcct aaattecatt 5340 atagactctt tatttccttt atataacctc tcttccccca gtcttatttt aataatgatt 5400 tccagcatta caaaaagagt aaaaaaagta gtttaactct tcacccccaa atgcaagaag 5460 gtggtgaaaa gcagaggatg atgttgagta tcttaaatag ctgacatcat gtcaaactat 5520 taattgttga agttattttt ttacacctga gtgaacattt agaaaataat ataaatagaa 5580 attaaaggga aataaatgct aaaccgatgt tagaaaatac tgttttctga agtgtacagt 5640 aagtatcttt ttgtatgttt ttttttcttt ttaatttatt tattgaaatg gagtctcact 5700 ctgtcaccca ggctggagtg cagtggcgcg atcttggctc actgcaacct ccgccctttg 5760 agttcaagcg attctcctgc ctcagcctcc tgagtacctg ggatcacagg cacctgccac 5820 cgcacccagc taattttttt tttactttta gtagagacgg agtttcacca tcttggccag 5880 gctagtcttg aactcctgac ctcatgatcc atccgcctcg gcctcccaaa gtgctgggat 5940 tacaggtgtg agccaccatg cccagccttt tatttattta tttatttttg agaaggagtc 6000 tcactctgtc gcccagggtg gagtgeagtg gtgcaatctc tgctcactgc aacctctgcc 6060 tcccaggttc aagcgattct cctgtgtcag cctcccgagt agctgggatt acaggcatgc 6120 gccaccgcac ccagctaatt tttatatttt tagtagagac gtggtttcac catgttggcc 6180 aggctggtct caaactcctg accttcggtg atccacccac ctcggcctcc caaagtgctg 6240 ggatgacagg catgagccgc tgcacccagc ctcaaagtgt atagtaaata tctaaacaaa 6300 tgaaagggac aagatataga aggaatctta ggatcagctg agagataatt gaatactttc 6360 ctaaaagaac acaatactgg aagggatggg gctttgtggg acaattgeta ttttgaattc 6420 ttaggtgtcc aactttacaa ccaaggttta caaatatttt aaatggtgat ttagtcagca 6480 ctcaaataga gaagggáaga acataattag ettaagetta cctctagttg tagagtatac 6540 aggttttgac ctcaaaattt gaaaaatcgc aatttttatc taagtgcaat caagttttcc 6600 atggccataa ttatttgggg ttgtctctca tggcatcttt GTAA 6644 < 210 > 15 < 211 > 560 < 212 > DNA < 213 > Mus musculus < 400 > fifteen accatgggtg gcaagtccct gagcgggctg ctgagcggcc tagcgcagaa cgcctttcac. 60 ggacactcgg gtgtcacgga ggagctgctg cacagccaac tctatccgga agtgccaccg 120 gaggagttcc gccccttcct ggcgaagatg agaggacttc tcaaggtacg gtggttccgc 180 cgagcagccc tgccctctcg cagcctcagg cccgccccag cctcgggtgc tgctgtcttt 240 gggcgctcag ggacccttct gagccgtgga ggtcggtctg ttgcggcctt gttttaggga 300 cacataaegg tgaaaacatt ggattttttt ttctctccct tgtgtctgta caagaettte 360 gtatagataa gtttcgagtt tttttcgcct cggactttga tgttgcaccg ggcgttgtag 420 tgcactcctt taatctgtgc acttggagag gcagaggctg gcagagagtt gtgtgagttc 480 gaggccagcc tgttgcacag agttccgggg cagtcagggc aatgtggtga gacccttgtt 540 taaagagagc gagagcgtgc 560 < 210 > 16 < 211 > 295 < 212 > DNA < 213 > Mus musculus < 400 > 16 ggtcctacag acccacagct tccaggatct ccatgacaca gggcaacagc aggctatccg 60 agaggagccc tggtgaaact aagttcaatc aanatatgtt ctgtagctag gcagctagct 120 ttgtctagtt atctaccaag ttcaaatata ttgctttttc ttttatcttt atagtctatt 180 gcatctgcag acatggattt caaccagtta gaggcattcc tgactgctca aaccaaaaag 240 tcaccagtga caaggtggca gcaagctgca gtcatctcca agttttggaa gagcc 295 It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims

CLAIMS Having been described. The invention as claimed above is claimed as property contained in the following claims: 1. An isolated polypeptide, characterized in that it comprises a sequence of amino acid residues selected from the group consisting of residues 48-62 of SEQ ID NO: 2, residues 47-61 of SEQ ID NO: 4, residues 63-104 of SEQ ID NO: 2, residues 62-103 of SEQ ID NO: 4, residues 105-119 of SEQ ID NO: 2 , the • residues 104-118 of SEQ ID NO: 4, residues 120-137 of SEQ ID NO: 2, residues 119-136 of SEQ ID NO: 4, residues 138-152 of SEQ ID NO: 2, residues 137-151 of SEQ ID NO: 4, residues 153-170 of SEQ ID NO: 2, residues 152-169 of SEQ ID NO: 4, residues 171-185 of SEQ ID NO: 2, and residues 170-184 of SEQ ID NO: 4.
2. The isolated polypeptide according to claim 1, characterized in that it is from 15 to 1500 amino acid residues in length.
3. The isolated polypeptide according to claim 2, characterized in that the amino acid residue sequence is operably linked by means of a peptide linker or polypeptide linker to a second polypeptide selected from the group consisting of the agglutination protein of the maltose, a constant region of immunoglobulin, a polyhistidine tag, and a peptide as shown in SEQ ID NO: 5.
4. The isolated polypeptide according to claim 1, characterized in that it comprises at least 30 contiguous residues of the SEC ID NO: 2 or SEQ ID NO: 4. The isolated polypeptide according to claim 1, characterized in that it comprises residues 48-185 of SEQ ID NO: 6 or residues 27-190 of SEQ ID NO: 6. NO: 6. The polypeptide isolated according to claim 1, characterized in that it comprises residues 48-185 of SEQ ID NO: 2, residues 47-184 of SEC XD NO: 4, the residues s 27-190 of SEQ ID NO: 2, or residues 26-188 of SEQ ID NO:. 7. An expression vector, characterized in that it comprises the following operatively linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising an amino acid residue sequence selected from the group consisting of residues 48-62 of SEQ ID NO: 2, residues 47-61 of SEQ ID NO: 4, the residues 63-104 of SEQ ID NO: 2, residues 62-103 of SEQ ID NO: 4, residues 105-119 of SEQ ID NO: 2, residues 104-118 of SEQ ID NO: 4, residues 120-137 of SEQ ID NO: 2, residues 119-136 of SEQ ID NO: 4, residues 138-152 of SEQ ID NO: 2, residues 137-151 of SEQ ID NO: 4, residues 153-170 of SEQ ID NO: 2, residues 152-169 of SEQ ID NO: 4, residues 171-185 of SEQ ID NO: 2, and residues 170-184 of SEC ID NO: 4; and a transcription terminator. 8. The expression vector according to claim 7, characterized in that the DNA segment comprises nucleotides 79 to 570 of SEQ ID NO: 7. 9. The expression vector according to claim 7, characterized in that the polypeptide comprises residues 48-185 of SEQ ID NO: 6 or residues 27-190 of SEQ ID NO: 6. 10. The expression vector according to claim 7, characterized in that the polypeptide comprises residues 48- 185 of SEQ ID NO: 2, residues 47-184 of SEQ ID NO: 4, residues 27-190 of SEQ ID NO: 2, or residues 26-188 of SEQ ID NO: 4. 11 The expression vector according to claim 7, characterized in that it further comprises a sequence of the secretory signal operably linked to the DNA segment. 12. A cultured cell into which the expression vector has been introduced according to any of claims 7-11, characterized in that the cell expresses the DNA segment. A method of manufacturing a polypeptide, characterized in that it comprises: culturing the cell of claim 12 under conditions whereby the DNA segment is expressed and the polypeptide is produced; and recovering the polypeptide. 14. A polypeptide, characterized in that it is produced by the method according to claim 13. 15. An antibody that binds specifically to the polypeptide according to claim 14. 16. A detection method, in a test sample, of the presence of an antagonist of zalpha29 activity, characterized in that it comprises: culturing a cell that functions in response to zalpha29; expose the cell to a zalpha29 polypeptide in the presence? absence of a test sample; compare the response levels for the zalpha29 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of an antagonist of zalpha29 activity in the test sample. 'HELICOIDAL POLIPEPTIDO ZALPHA29 SUMMARY OF THE INVENTION The present invention relates to novel cytokine polypeptides, materials and methods for making them, and to a method of use. The polypeptides comprise at least 15 contiguous amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 4, and can be prepared as polypeptide fusions comprising heterologous sequences, such as affinity tags. The polypeptides and polynucleotides that encode them can be used within a variety of therapeutic, diagnostic, and research applications.