MXPA01002823A - Stomach polypeptide zsig28 - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zsig28, a novel member of the RPV.1 family of proteins. The polynucleotides encoding zsig28 can be used to identify a region of the genome associated with human disease states. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

Description

ZOMIG28 STOMACH POLYPIPTIDE BACKGROUND OF THE INVENTION Appropriate control of the processes of opposition of cell proliferation against terminal differentiation and programmed apoptotic cell death is an important aspect of normal development and homeostasis (Raff, MC, Cell 86: 173-175, 1996), and it has been found to be altered in many human conditions. See, for example, Sa yers, C. L. et al., Cell 64: 337-350, 1991; Meyaard, L. et al., Science 257: 217-129; 1992; Guo, Q et al., Nature Med. 4_: 957-962, 1998; Barinaga, M., Science, 273: 735-737, 1996; Solary, E., et al., Eur. Respir. J., 9: 1293-1305, 1996; Hamet, P. et al., J. Hypertension, _1_4_: S 65-S70, 1996; Roy, N. et al., Cell, 80: 167-178, 1995; and Ambrosini, G., Nature Med., 8_: 917-921, 1997. Much progress has been made towards understanding the regulation of this balance. For example, the signaling cascades through which the extracellular stimulus, such as growth factors, peptide hormones, and cell-cell interactions, control the REF.128168 confinement of the precursor cells to specific cell lineages and their subsequent proliferative expansion (Morrison, S. J. et al., Cel 1 88: 287-298, 1997). In addition, it has been found that cell cycle output and terminal differentiation are coupled in most cell types. See for example, Coppola, J. A. et al., Nature 320: 760-763, 1986; Freytag, S.O., Mol. Cell. Biol. 8_: 1614-1624, 1988; Lee, E. Y. et al., Genes. Dev. 8_: 2008-2021, 1994; Morgenbesser, S. D. et al., Nature 371: 72-74, 1994; Casaccia-Bonnefil, P. et al., Genes Dev. HL_: 2335-2346, 1996; Zacksenhaus, E. et al., Deves Genes. 10: 3051-3064, 1996; and Zhang, P. et al., Nature 387: 151-158, 1997. Apoptosis also plays an important role in many processes of development and homeostasis (Raff, MC, Nature 365: 397-400, 1992; Raff, M. C, supra.), And is often, coordinately regulated with terminal differentiation (Jacobsen, KA et al., Blood 8: 278-2794; Morgenbesser et al., Supra.; Yan, Y. et al., Genes Dev. 11: 973-983, 1997; Zacksenhaus et al., Supra.). Therefore, it seems that the development of individual lines, tissues, organs or even complete multicellular organisms, is the result of a finely synchronized equilibrium between increased cell production due to proliferation and the diminished numbers of cells that result from terminal differentiation and apoptosis. This balance is more likely to be regulated in a coordinated way by the convergence of the multiple regulatory trajectories. The identification of new elements of such systems can provide important insight into both normal cellular processes, as well as, in the etiology and treatment of human disease states. Thus, there is a continuing need to discover new proteins that regulate proliferation, differentiation, and apoptotic trajectories. The activities of both inducers and inhibitors of these trajectories illustrate the enormous clinical potential of, and necessary for, new proliferation, differentiation and apoptotic proteins, their agonists and antagonists. The present invention addresses this need by providing such polypeptides for these and other uses that should be apparent to those skilled in the art of the teachings herein.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention provides an isolated polypeptide that encodes a polypeptide comprising a sequence of amino acid residues that are at least 90% identical to an amino acid sequence selected from the group consisting of: ) the sequence as shown in SEQ ID NO: 2, from amino acid number 24 (Ala), to amino acid number 261 (Val); and (b) the amino acid sequence as shown in SEQ ID NO: 2, from amino acid number 1 (Met) to amino acid number 261 (Val), where the percentage of amino acid identity is determined using a FASTA program with ktup = l, missing by interval opening = 10, lack of interval extension = 1, and substitution matrix = BLOSUM62, with other parameters exposed as automatic allocation . Within one embodiment, the isolated polynucleotide described above, is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO: 1, from nucleotide 139 to nucleotide 853; and (b) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 70 to nucleotide 853. Within another embodiment, the isolated polynucleotide described above comprises nucleotide 1 through nucleotide 783 of SEQ ID NO: 10 . Within another embodiment, the isolated polynucleotide described above, comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2, of amino acid number 24 (Ala) , to amino acid number 261 (Val); and (b) the amino acid sequence as shown in SEQ ID NO: 2, from amino acid number 1 (Met) to amino acid number 261 (Val). Within a second aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a zsig28 polypeptide as shown in SEQ ID NO: 2, from amino acid number 24 (Ala), to amino acid number 261 (Val); and a transcriptional terminator, wherein the promoter is operably linked to the DNA segment, and the DNA segment is operably linked to the transcriptional terminator. Within another embodiment, the expression vector described above, further comprises a secretory signal sequence operably linked to said DNA segment. Within a third aspect, the present invention provides a cultured cell comprising an expression vector as described above, wherein the cell expresses the polypeptide encoded by the DNA segment. Within another aspect, the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first segment of DNA encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of of: (a) the amino acid sequence of SEQ ID NO: 2, of amino acid number 1 (Met), to amino acid number 23 (Ala); (b) the amino acid sequence of SEQ ID NO: 2 of amino acid number 24 (Ala) to amino acid number 82 (Leu); (c) the amino acid sequence of SEQ ID NO: 2 of amino acid number 101 (Leu) to amino acid number 122 (Gly); (d) the amino acid sequence of SEQ ID NO: 2 of amino acid number 141 (Asn) to amino acid number 174 (Ala); (e) the amino acid sequence of SEQ ID NO: 2 of amino acid number 193 (Cys) to amino acid number 261 (Val); and (f) the amino acid sequence of SEQ ID NO: 2 of amino acid number 24 (Ala), to amino acid number 261 (Val); and at least one other segment of DNA encoding an additional polypeptide, wherein the first and the other DNA segments are connected in structure; and wherein the first and the other DNA segments encode the fusion protein. Within another aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA construct encoding a fusion protein as described above; and a transcriptional terminator, wherein the promoter is operably linked to the DNA construct, and the DNA construct is operably linked to the transcriptional terminator. Within another aspect, the present invention provides a cultured cell comprising an expression vector as described above, wherein the cell expresses a polypeptide encoded by the DNA construct. Within another aspect, the present invention provides a method of producing a fusion protein comprising: culturing a cell as described above; and isolation of the polypeptide produced by the cell. Within another aspect, the present invention provides an isolated polypeptide comprising a sequence of amino acid residues that are at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the sequence as shown in SEQ ID NO: 2, from amino acid number 24 (Ala), to amino acid number 261 (Val); and (b) the amino acid sequence as shown in SEQ ID NO: 2, from amino acid number 1 (Met) to amino acid number 261 (Val), wherein the percentage of amino acid identity is determined using a FASTA program with ktup = l, missing by interval opening = 10, missing by extension of interval = 1, and matrix of substitution = BLOSUM62, with other parameters exposed as out of place. Within another embodiment, the isolated polypeptide described above, further contains spaced apart 1 to 4 portions of the N-terminus to the C-terminus in a configuration selected from the group consisting of: (a) Met-. { 47 -50} -Ml-. { 21-22} -M2-. { 73-92} -M3; and (b) Met-. { 47-50} -Ml-. { 21-22} -M2-. { 73-92} -M3-. { 3 } -M4, where M1 is the "portion 1", an amino acid sequence as shown in amino acids 48 to 54 of SEQ ID NO: 2, M2 is "portion 2", an amino acid sequence as shown in amino acids 77 to 82 of SEQ ID NO: 2, M3 is "portion 3", an amino acid sequence as shown at amino acids 174 to 180 of SEQ ID NO: 2, M4 is "portion 4" a amino acid sequence as shown in amino acids 184 to 189 of SEQ ID NO: 2, and. { #} denotes the number of amino acids among the motifs. Within another embodiment, the asylated polypeptide described above, comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 of amino acid number 24 (Ala), to amino acid number 261 (Val); and (b) the amino acid sequence as shown in SEQ ID NO: 2, from amino acid number 1 (Met) to amino acid number 261 (Val). Within another aspect, the present invention provides a method of producing zsig28 polypeptides comprising: culturing a cell as described above; and isolating the zsig28 polypeptide produced by the cell. Within another aspect, the present invention provides a method for producing an antibody to zsig28 polypeptide comprising: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 9 to 238 amino acids, wherein the polypeptide is identical to a contiguous amino acid sequence in SEQ ID NO: 2 of amino acid number 24 (Ala), to amino acid number 261 (Val); (b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 of amino acid number 24 (Ala), to amino acid number 82 (Leu); (c) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 of amino acid number 101 (Leu) to amino acid number 122 (Gly); (d) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 of amino acid number 141 (Asn) to amino acid number 174 (Ala); (e) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 of amino acid number 193 (Cys) to amino acid number 261 (Val); (f) a polypeptide as described above; (g) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 of amino acid number 245 (Ala) to amino acid number 259 (Glu); (h) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 of amino acid number 234 (Asn) to amino acid number 239 (Lys); (i) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 of amino acid number 202 (Glu) to amino acid number 207 (Lys); (j) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 of amino acid number 254 (Lys) to amino acid number 250 (Asp); and (k) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 of amino acid number 110 (Glu) to amino acid number 115 (Ala); and wherein the polypeptide has an immune response in the animal to produce the antibody; and isolation of the animal's antibody. Within another aspect, the present invention provides an antibody produced by the method described above, which is linked to the zsig28 polypeptide. Within another embodiment, the antibody described above is a monoclonal antibody. Within another aspect, the present invention provides an antibody, which is. binds specifically to a polypeptide described above. Within another aspect, the present invention provides a method of detecting, in a test sample, the presence of a modulator of the activity of the zsig28 protein, which comprises: culturing a cell into which an expression vector has been introduced as described above, wherein the cell expresses the zsig28 protein encoded by the DNA segment in the presence and absence of a test sample; and comparing the activity levels of zsig28 in the presence and absence of a test sample, by a biological or biochemical assay; and determination from the comparison, of the presence of the modulator of the zsgi28 activity in the test sample. These and other aspects of the invention will become apparent upon reference to the following detailed description of the invention and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an alignment of zsig2 (SEQ ID NO: 2); murine claudin 1 (CLAUD1) (SEQ ID NO: 3) (Furuse, M. "et al., J. Cell. Biol. 141: 1539-1550, 1998); Genbank Access No. AF072127); Murine CPE receptor (AB007 ) (SEQ ID NO: 4) (Genbank Access No. AB00713) protein similar to the human oligodendrocyte specific protein (OSP) as the protein (HSU899) (SEQ ID NO: 5) (Genbank Access No. U89916); of the human transmembrane suppressed in the Velo-Cardio-Facial Syndrome (AF009) (SEQ ID NO: 6) (Genbank Access No. AF000959); Human OSP (AF0688) (SEQ ID NO: 7) (Genbank Access No. AF068863); murine claudin 2 (AF0721) (SEQ ID NO: 8) (Furuse, M. et al., Supra., Genbank Access No. AF072128); and RVP.l protein (PIR_A3) removal of rat androgen (SEQ ID NO: 9) (Briehl, M.M. et al., Mol.Endocrinol., 5: 1381-1388, 1991). Figure 2 is a hydrophobicity plot of the zsig28 polypeptide.

DETAILED DESCRIPTION OF THE INVENTION Prior to the disclosure of the invention in detail, it may be beneficial for the understanding thereof to define the following terms: The term "affinity tag" is used herein to denote a polypeptide segment that may be attached to a second polypeptide to provide purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principle, any polypeptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine 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 67:31, 1988), Glu-Glu affinity tag, (Grussenmeyer et al., Proc. Nati, Acad. Sci. USA 82: 7952-, 1985), substance P, Flag ™ peptide (Hopp et al. al., Biotechnology 6: 1204-10, 1988), is a trept avidin binding peptide, or other antigenic epitope or binding domain. See generally, Ford et al., Protein Expression and Purification 2: 95-107, 1991. The affinity tags encoding the DNAs are available from commercial suppliers (eg, Pharmacia Biotech, Piscata ay, NJ). The term "allelic variant" is used herein to denote any one of two or more alternative forms of a gene that occupies the same loci or chromosomal sites. Allelic variation is naturally achieved through mutation, and can result in phenotypic polymorphism within populations. Mutations of the gene can be either silent (without change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequences. 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 positions within the polypeptides. Where 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 carboxy 1 -terminal sequence placed to a reference sequence within a polypeptide is located close to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide. The term "complement pair / anticomplement" denotes non-identical portions that form a stable pair, associated non-covalently, under appropriate conditions. For example, biotin and avidin (or streptavidin) are phenotypic elements of a complement / anticomplement pair. Other exemplary complement / anti-complement pairs include receptor / ligand pairs, anti-antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, and the like. Where the subsequent dissociation of the complement / anticomplement pair is desirable, the complement / anticomplement pair preferably has a binding affinity of <; 109 M "1. The term" complements of a polynucleotide molecule "denotes a polynucleotide molecule having a complementary base sequence and reverse orientation compared to a reference sequence, eg, the 5 'sequence ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3' The term "contig" (or contiguous) denotes a polynucleotide having a contiguous elongation of sequence identical or complementary to another polynucleotide The contiguous sequences are said to "overlap" a given elongation or prolongation of polynucleotide sequence. either in its entirety or only a partial elongation of the polynucleotide For example, the contigs (or contiguous) representative of the polynucleotide sequence 5 '-ATGGAGCTT-3' are 5 '-AGCTTgagt-3' and 3 '-tcgacTACC-5 The term "degenerate" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a peptide). The degenerate codons contain different triplets of nucleotides, but they encode the same amino acid residue (ie, the triplets GAU and GAC each encode Asp). A "DNA segment" is a portion of a large DNA molecule that has specified attributes. For example, a segment of DNA encoding a specified polypeptide is a portion of a large DNA molecule, such as a plasmid or a plasmid fragment, which, when read from the 5 'direction to the 3' direction encodes the sequence of amino acids of the specified polypeptide.

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 additional segments that are provided for transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from 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 is also free of other unwanted or foreign coding sequences, and is in a form suitable for use with production systems of the genetically engineered protein. Such isolated molecules are those that are separated from their natural environment and include genomic clones and cDNA. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 'and 3' untranslated regions, such as promoters and terminators. The identification of the associated regions will be apparent to an ordinarily skilled artisan (see for example, Dynan and Tijan, Nature 316: 774-78, 1985). An "isolated" polypeptide or protein is a polypeptide or protein that is in a condition different from its native environment, such as apart from the blood of animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, ie, greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms. The term "operably linked" or "operably linked", when referring to the DNA segments, indicates that the segments are placed in such a way that they function according to their intended purposes, for example, they initiate transcription in the promoter and proceed through the encoding segment to the terminator.

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 the orthologs are the result of the evolution of the species. "Pairs" are different, but structurally related proteins made by an organism. The paralogs are believed to be achieved through the duplication of the gene. For example, α-globin, β-globin, and myoglobin, are paralogs with each other. A "polynucleotide" is a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'end to the 3' end. The polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vi t ro, or prepared from a combination of synthetic or natural molecules. The polynucleotide sizes are expressed as base pairs (abbreviated "bp"), nucelotides ("nt"), or kilobases ("kb"). When the context permits, the last two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules, it is used to denote the full length and will be understood as equivalent to the term "base pairs". It will be recognized 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.; thus, all nucleotides within a double-stranded pollen-lane molecule can not be paired. A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, somehow produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides". The term "promoter" is used herein by its recognized meaning in the art to denote a portion of a gene that contains DNA sequences that are provided for the binding of the 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 can also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents can be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined here in terms of their structures of amino acid skeletons; substituents such as carbohydrate groups are not generally specified, but may nevertheless be present. The term "receptor" denotes a protein associated with the cell that binds to a bioactive molecule (i.e., a ligand), and mediates the effect of the ligand on the cell. Receptors that bind to the membrane are characterized by a structure of multiple domains comprising an extracellular domain that binds to the ligand and an intracellular effector domain that is typically involved in signal transduction. The binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, phosphorylation, increased cyclic production of AMP, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of lipids of inositol and hydrolysis of phospholipids. In general, receptors can be linked to the membrane, cytosolic or nuclear; monomeric (eg, thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (eg, PDFG receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, receptor of eri t ropoiet ina and receptor IL-6). The term "secretory signal sequence" denotes a DNA sequence encoding a polypeptide (a "secretory sequence") that, as a component of a longer polypeptide, directs the longer polypeptide through a secretory path of a cell in a which is synthesized. The longer polypeptide is commonly unfolded to remove the secretory peptide during transit through the secretory pathway. The term "splicing variant" is used herein to denote alternative forms of RNA transcribed from a gene. The variation of binding is achieved naturally through the use of alternative binding sites with a transcribed RNA molecule, or less commonly between the separately transcribed RNA molecules, and can result in several mRNAs transcribed from the same genes. Binding variants can encode polypeptides having an altered amino acid sequence. The term "binding variant" is also used herein to denote a protein encoded by a binding variant of an mRNA transcribed from a gene. The weights and lengths of the polymers determined by imprecise analytical methods (for example, gel electrophoresis), will be understood as approximate values. When such value is expressed as "approximate" X or "approximately" X, the declared value of X will be understood as accurate up to + _ 10%. All references cited herein are incorporated by reference in their entirety. The present invention is based in part on the discovery of a new DNA sequence encoding a polypeptide having homology to a diverse family of receptor proteins, which contain proteins such as human claudin 1 and 2 (Furuse, M., et al. al., J. Cell Biol. 141: 1539-1550, 1998), human and murine oligodendrocyte-specific protein (OSP), (Bronstein, JM, et al., Neurology 4 1_: 772-778, 1996), RVP protein .1 of apoptosis withdrawn from rat androgen (Briehl, MM, Miesfeld, RL, Mol.Endocrinol. 5_: 1381-1388, 1991), and others. The analysis of the tissue distribution of the mRNA corresponding to this new DNA showed high expression in the stomach, and low expression in the lung. The polypeptide has been designated zsig28. The new zisg28 polypeptides of the present invention were initially identified by interrogation of an EST database for proteins homologous to proteins having a secretory signal sequence. These proteins are characterized by an upstream methionine start site (in the 5 direction) and a hydrophobic region of approximately 13 amino acids, followed by a peptidase signal peptidase cleavage site. An EST database is interrogated for new DNA sequences whose translations could fill this search criteria. An EST was found and its corresponding cDNA was sequenced. The new polypeptide encoded by the cDNA showed homology with rat RVP.l. Based on this homology, the nucleotide sequence zsig28 encodes the complete coding sequence of the predicted protein. Zsig28 may be a new protein involved in an apoptotic cell pathway, cell-cell signaling molecule, growth factor receptor, or protein associated with the extracellular matrix with the activity of the growth factor hormone, or the like, and is a new element of the claudin / OSP protein family. The zsig28 polypeptide sequence was obtained from a single clone containing its corresponding polynucleotide sequence. "The clone was obtained from a lung library." Other libraries that could also be searched for such sequences include, stomach, fetal lung, epithelial tissue, and the like The nucleotide sequence of a DNA encoding a representative zsig28 is dibed in FIG. SEQ ID NO: 1, and its 261 deduced amino acid sequence is dibed in SEQ ID NO: 2. In its entirety, the zsig28 polypeptide (SEQ ID NO: 28) represents a full-length polypeptide segment (residue 1 ( Met) to residue 261 (Val) of SEQ ID NO: 2) The domains and structural features of zsig28 are further dibed below The analysis of the zsig28 polypeptide encoded by the DNA sequence of SEQ ID NO: 1, revealed an open reading frame encoding 261 amino acids (SEQ ID NO: 2) comprising a predicted secretory signal peptide of 23 amino acid residues (residue 1 (Met) to residue 23 (Ala) of SEQ ID NO: 2), and a mature polypeptide of 238 a inoperates (residue 24 (Ala) to residue 261 (Val) of SEQ ID NO: 2). The zsig28 polypeptide contains three transmembrane domains. (1) the first transmembrane domain of amino acid 83 (Met) to amino acid 100 (Ala) of SEQ ID NO: 2; (2) the second transmembrane domain of amino acid 123 (lie) to amino acid 140 (Ala) of SEQ ID NO: 2; and (3) the third transmembrane domain is from amino acid 175 (Leu) to amino acid 192 (Met) of SEQ ID NO: 2.

These transmembrane domains are corroborated by the zsig28 hydrophobicity plot (see Figure 2). Between and flanking these domains of the transmembrane leads to regions of the zsig28 molecule that can confer linkage to a ligand, cell-cell interactions, cell signaling functions, and the like. However, such regions and extensions of the hydrophilic amino acids within, could serve as antigenic epitopes suitable for the production of antibodies, as discussed herein. These regions include: (1) "region 1", the amino-terminal region, amino acid 24 (Ala) to amino acid 82 (Leu); (2) "region 2", amino acid 101 (Leu) to amino acid 122 (Gly); (3) "region 3" amino acid 141 (Asn) to amino acid 174 (Ala); and (4) "region 4", the carboxy terminal hydrophilic region, amino acid 193 (Cys) to amino acid 261 (Val). Within zsig28 are the various conserved amino acid motifs, based on the comparison between family members (see Figure 1). However, several regions of low variance are also present within the zsig28 polypeptide. For the determination of low variance regions, see Sheppard, P. et al., Gene 150: 163-167, 1994. Examining a multiple alignment of several known family elements (for example, see figure 1), they revealed the following reasons that are both conserved and have low degeneracy: 1) "portion 1" (a consensus portion pattern encompassing the information in Figure 1 corresponding to amino acids 48 to 54 of SEQ ID NO: 2); f 2) "portion 2" (a consensus portion pattern encompassing the information in Figure 1 corresponding to amino acids 77 to 82 of SEQ ID NO: 2); 3) "portion 3" (a consensus portion pattern encompassing the information in Figure 1 which corresponds to amino acids 174 to 180 of SEQ ID NO: 2); Portions 1 through 3 are separated from the N-terminal to the C-terminal in a configuration represented by the following: Met-. { 47-50} -Ml-. { 21-22} -M2-. { 73-92} -M3, where Met is the initiating methionine residue M # denotes the specific portion described above (for example, M1 is portion 1, etc.) and. { #} denotes the number of amino acids between the portions. In addition, other conserved portions in the third transmembrane domain of zsig28 are evident: 4) "portion 4" (a consensus portion pattern encompassing the information in Figure 1 which corresponds to amino acids 184 to 189 of SEQ ID NO. : 2); Portions 1 through 4 are separated from the N-terminus to the C-terminus in a configuration represented by the following: Met-. { 47-50} -Ml-. { 21-22} -M2-. { 73-92} -M3-. { 3} -M4, where Met is the initiating methionine residue M # denotes the specific portion described above (for example, M4 is the portion 4, etc. •) and. { #} denotes the number of amino acids between the portions. The presence of the transmembrane and conserved regions, and the low variance portions, generally correlate with or define important structural regions in the proteins. Regions of low variability (eg, hydrophobic boxes) are generally present in regions of structural importance (Sheppard, P. et al., Supra). Such regions of low variance often contain rare or infrequent amino acids, such as tryptophan. The flanking regions and between such low variance and conserved portions may be more variable, but are often functionally significant because they may refer to or define important structures and activities such as binding domains, biological and enzymatic activity, transduction signal, cell-cell interaction, tissue localization domains and the like. For example, regions 1 to 4 described above may be functionally different. In addition, there are several individual amino acids conserved through the zsig28 polypeptide, located in SEQ ID NO: 2 at the following amino acid numbers: 30 (Trp), 48 (Gly), 49 (Leu), 50 (Trp), 53 (Cys), 59 (Gly), 63 (Cys), 72 (Leu), 103 (Cys), and 114 (Lys). The regions of amino acid residues conserved in zsig28 described above can be used as tools to identify a new family of elements. For example, the reverse transcriptional polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved regions of RNA obtained from a variety of tissue sources or cell lines. In particular, the highly designed primers of the zsig28 sequences are used for this purpose. The design and use of such degenerate primers can easily be done by one skilled in the art. The corresponding polynucleotides encoding the zsig28 polypeptide regions, domain, portions, residues and sequences described above, are also shown in SEQ ID NO: 1. The present invention also provides polynucleotide molecules, which include the DNA and RNA molecules. , which encode the zsig28 polypeptides described herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible between these polynucleotide molecules. SEQ ID NO: 10 is a degenerate DNA sequence encompassing all of the DNAs encoding the zsig28 polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 10 also provides all RNA sequences encoding SEQ ID NO: 2 by substituting U for T. Thus, polynucleotides encoding zsig28 polypeptide, comprise nucleotide 1 through nucleotide 783 of SEQ ID NO: 10 and their RNA equivalents they are contemplated by the present invention. Table 1 sets forth the one-letter codes used with SEQ ID NO: 10 to denote degenerate nucleotide positions.

"Resolutions" are the nucleotides denoted by a one-letter 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 1 Nucleotide Resolutions Commento 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 A | T W A | T H A | C | T D A | G | T B C | G | T V A | C | G V A | C | G B C | G | T D A | G | T H A | C | T N A | C | G | T N A | C | G | T The degenerate codons used in SEQ ID NO: 10, encompassing all possible codons for a given amino acid, are set forth in Table 2.

Table 2 Code Aminode Code Codons acid letter Degenerate Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T AC AC ACC ACN 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 MGN iys K AAA AAG AAR Met M ATG ATG He I ATTA ATT ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F pc TTT TTY Tyr and TAC TAT TAY Trp W TGG TGG Ter TAA TAG TGA TRR Asn | Asp B RAY Glu | Gln Z SAR any X NNN A ordinary skilled 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 (SN) can in some circumstances, encode arginine (ARG), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). There is a similar relationship between the codons that encode phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence can encode variant amino acid sequences, but one ordinarily skilled in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2. The variant sequences can be easily tested for their functionality as described herein. One of ordinary skill in the art will also appreciate that different species may have "preferential codon use". In general, see Grantham et al., Nuc. Acids Res. , 8_: 1893-912, 1980; Haas, et al., Curr. Biol. , _6: 315-24, 1996; Ain-Hobson, et al., Gene, 13: 355-64, 1981; Grosjean and Fiers, Gene, 1_8_: 199-209, 1982; Holm, Nuc. Acids Res. , I: 3075-87, 1986; Ikemura, J. Mol. Biol. , 158: 573-97, 1982. As used herein, the term "differential codon usage" or "preferential codons" is a technical term that refers to the translation codons of the protein that are most frequently used in the cells of certain species, thus favoring one or a few representative of the possible codons that encode each amino acid (See Table 2). For example, the amino acid threonine (Thr) can be encoded by AC, ACC, ACG, or ACT, but in mammalian cells, the ACC is the most commonly used codon; in other species, for example, different Thr codons may be preferential from insect, yeast, virus or bacterial cells. The preferential codons for particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferential codon sequences in the recombinant DNA can, for example, increase the production of the protein by making the translation of the most efficient protein within a particular cell type or species. Therefore, the degenerate codon sequence, described in SEQ ID NO: 10, serves as a standard for optimizing the expression of polynucleotides in various types and cellular species commonly used in the art and described herein. The sequences that contain the preferential codons can be tested and optimized for expression in several species, and tested for their functionality as described here. Within the preferred embodiments of the invention, the isolated polynucleotides will hybridize to regions of similar sizes of SEQ ID NO: 1, or a sequence complementary thereto, under stringent conditions. In general, stringent 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 ionic strength and defined pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Numerous equations for the calculation of Tm are known in the art, and are specific for DNA, RNA, and DNA-RNA hybrids and variant length polynucleotide probe sequences (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al. , (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 2_6: 227 (1990)). - The sequence analysis software, such as OLIGO 6-0 (LRS, Long Lake, MN) and Primer Prem i er 4. 0 (Premier Biosoft International, Palo Alto, CA), as well as sites on the Internet, are tools available to analyze a given sequence and calculate the Tm based on the user's defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of the longer polynucleotide sequences, (e.g.,> 50 base pairs), is performed at temperatures of about 20-25 ° C below the calculated Tm. For smaller probes, (eg <50 base pairs), hybridization is typically carried out at Tm or below 5-10 ° C. This allows the maximum hybridization ratio for the DNA-DNA and DNA-RNA hybrids. Higher degrees of stringency or severity at lower temperatures can be achieved with the addition of formamide which reduces the Tm of the hybrid by approximately 1 ° C per 1% formamide in the buffer solution. Suitable stringent hydrization conditions are equivalent to about 5 hours to overnight incubation at about 42 ° C in a solution comprising: about 40-50% formamide, up to about 6X SSC, about 5X Denhardt solution, zero to about 10% dextran sulfate, and about 10-20 μg / ml denatured commercially available DNA carrier. In general, such stringent conditions include temperatures of 20-70 ° C and a hybridization buffer containing up to 6x SSC and 0-50% formamide; the hybridization is then followed by washing the filters at up to about 2X SSC. For example, a wash of severity or adequate stringency is equivalent to O.lx SSC up to 2X SSC, 0.1% SDS, at 55 ° C up to 65 ° C. Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence. Typically, washings after hybridization are performed at increased degrees of stringency to remove probes from unhybridized polynucleotides from hybridized complexes. The stringent washing and hybridization conditions depend on the length of the probe, reflected in the Tm, hybridization and washing solutions are used, and are routinely determined empirically by one of skill in the art. As previously noted, isolated polynucleotides of the present invention include DNA and RNA. Methods for the preparation of DNA and RNA are well known in the art. In general, RNA is isolated from a cell or tissue that produces large amounts of zsig28 RNA. Such tissues and cells are identified by Northern blotches (Thomas, Proc. Nati, Acad. Sci. USA 77_: 5201, 1980), and include stomach and lung. Total RNA can be prepared using the extraction of guanidinium isothiocyanate, followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry l_8_: 52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Nati, Acad. Sci. USA 69: 1408-12, 1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. In the alternative, genomic DNA can be isolated. The polynucleotides encoding zsig-28 polypeptides are then identified and isolated by, for example, hybridization or polymerase chain reaction (PCR) (Mullis, U.S. Patent No. 4,683,202). A full-length clone encoding zsig28 can be obtained by conventional cloning procedures. Complementary DNA clones (cDNA) are preferred, although for some applications (eg, expression in transgenic animals) it may be preferable, due to the use of a genomic clone, or to modify a cDNA clone to include at least one genomic intron from the same or different gene. Methods for the preparation of cDNAs and genomic clones are well known and are within the level of ordinary ordinarily skilled in the art, and include the use of the sequence described herein, or parts thereof, for subjecting or priming a library. Expression libraries can be probed with antibodies to zsig28 polypeptides, receptor fragments, or other specific binding patterns. The polynucleotides of the present invention can also be synthesized using DNA synthesis machines. Currently, the method of selection is the phosphoramidite method. If the chemically synthesized double-stranded DNA is required for an application such as the synthesis of a gene or gene fragment, then each complementary strand is processed separately. The production of short polynucleotides (60 to 80 bp) is technically direct and can be accompanied by the synthesis of the complementary strands and then the alignment. However, to produce longer polynucleotides (>300 .bp), special strategies are usually employed, due to the coupling efficiency of each cycle during chemical DNA synthesis are rarely 100%. To overcome this problem, the synthetic genes (double strand) are assembled in modular form from fragments of single strands that are from 20 to 100 nucleotides in length. A method for constructing a synthetic gene requires the initial production of a series of complementary, overlapping oligonucleotides, each of which is within 20 to 60 nucleotides in length. Each internal section of the gene has complementary terminal extensions 3 'and 5' designated for the base pairs with precisely an adjacent section. Thus, after the gene is assembled, the process is completed by the seal of the cuts along with the structures of the two strands with the T4 DNA ligase. In addition to the sequence encoded by the protein, synthetic genes can be designed with terminal sequences that facilitate insertion into a restriction endonuclease site of a cloning vector. However, other sequences must be added, containing signals for the appropriate initiation and termination of transcription and translation. An alternative way to prepare a full-length gene is to synthesize a specified series of overlapping oligonucleotides (40 to 100 nucleotides). Then the short 3 'and 5' overlapping complementary regions (6 to 10 nucleotides) are subjected to heating and cooling, the large intervals still remain, but the short paired base regions are both long and sufficient and sufficiently stable to maintain the structure together. There are full gaps and double DNA is completed via the synthesis of enzymatic DNA by E DNA polymerase I. col i. After the enzymatic synthesis is completed, the sections are sealed with the T4 DNA ligature. The double-stranded constructs are sequentially linked to one of the others to form the sequence of the total gene, which is verified by the analysis of the DNA sequence. See Glick and Pasternak, Molecular Biotechnology, Principies & Applications of Recombinant DNA, (ASM, Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 5_3: 323-56, 1984 and Cumie et al., Proc. Nati Acad. Sci. USA 87: 633-7, 1990. The zsig28 polynucleotide sequences described herein can also be used as probes or primers to clone the 5 'non-coding regions of a zsig28 gene. In view of the tissue-specific expression observed for zsig28 by Northern blotting this region of the gene is expected to provide specific expression to the stomach. The promoter elements of a zsig28 gene could thus be used to direct the tissue-specific expression of the heterologous genes in e.g., transgenic animals or patients treated with the gene therapy. The cloning of the 5 'flanking sequences also facilitates the production of the zsig28 proteins by "gene activation" as described in U.S. Patent No. 5,641,670. Briefly, the expression of an endogenous zsig28 gene in a cell is altered by introducing into the locus or zsig28 site a DNA construct comprising at least one target sequence, a regulatory sequence, an exon and a non-binding donor site. paired The target sequence is a 5 'non-coding sequence of zsig28 that allows homologous recombination of the construct with the locus or endogenous zsig28 site, thereby, the sequences within the construct become operably linked with the endogenous zsig28 coding sequence. In this way, an endogenous zsig28 promoter can be replaced or supplemented with other regulatory sequences to provide tissue specificity, increased or otherwise, regulated expression. The present invention also provides counterparts of polypeptides and polynucleotides of other species (orthologs). These species include, but are not limited to mammals, birds, amphibians, reptiles, fish, insects and others, vertebrate and invertebrate species. Of particular interest are the zsig28 polypeptides of other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine and other primate polypeptides. Human zsig-28 orthologs can be cloned using 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 tissue type or cell expressing zsig28 as described herein. Suitable sources of mRNA can be identified by subjecting Northern spotted probes to probes designated from the sequences described herein. A library is then prepared from mRNA of a positive cell or tissue line. A cDNA encoding zsig28 can then be isolated by a variety of methods, such as by probing with a partial or complete human cDNA or with one or more series of degenerate probes based on the described sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, supra). using designated primers of the representative human zsig28 sequence described herein. Within a further method, the cDNA library can be used to transform or transfect the host cells, and the expression of the cDNA of interest can be detected with an antibody to the zsig28 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequence described in SEQ ID NO: 1 represents a single allele of human zsig28 and such allelic variation and alternative linkages are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals in accordance with standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 2, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are the proteins which are allelic variants of SEQ ID NO: 2. The cDNA degenerated from the alternatively linked mRNAs, which retain the properties of the zsig28 polypeptide, are included within the scope of the present invention, as are the polypeptides that qualify for such cDNAs and mRNAs. The allelic variants and binding variants of these sequences can be cloned by probing cDNAs or libraries of different individuals or tissues in accordance with standard procedures known in the art. The present invention also provides isolated zsig28 polypeptides that are substantially homologous to the polypeptides of SEQ ID NO: 2 and their orthologs. The term "substantially similar" is used herein to denote polypeptides having at least 70%, more preferably at least 80% identity to the sequences shown in SEQ ID NO: 2 or their orthologs. Such polypeptides will more preferably be at least 90% identical and more preferably 95% or more identical to SEQ ID NO: 2 or their orthologs. The percent identity of the sequence is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Biol. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 89: 10915-9, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment records using a gap opening gap of 10, a gap extension gap of 1, and the registration matrix "blosum 62"by Henikoff and Henikoff (ibid.), as shown in Table 3 (amino acids are indicated by standard one-letter codes). The identity percentage is then calculated as: Total number of identical pairs x 100 [length of the longest sequence plus the number of intervals entered in the longest sequence to align the two sequences] ** < t- »H 1 3- H CN ro H 1 E-»? n CN CN or 1 1 C? • < # H ro CN CN 1 1 1 F * H H ro CN 1 1 1 1 1 faith or • «* CN CN H ro H "* CN CN or CN CN H CN H H 1 1 1 1 1 1« i1 CN m H o ro CN H ro H ro 1 1 1 00 rn co H CN CN CN CN CN CN 1 1 1 1 1 1 1 1 1 or vo CN • < * CN m ro CN o CN CN ro ro 1 1 1 1 1 1 1 1 1 1 1 m LD CN sn co H CN co HOH ro CN CN 1 1 1 1 1 1 1 1 1 1 aw N CN o ro CN H or co H or H CN H CN 1 1 1 1 1 1 1 1 1 us? in m n H H rn H CN ro H H CN CN H 1 1 1 1 1 1 1 1 1 1 1 1 1 Q KD with CN H H m H m rn H o H ro ro 1 1 1 1 1 1 1 1 1 1 55 I? ti co o O o H co ro o CN n CN tr o r C o r 1 1 1 1 1 1 1 UO or CN co r-í O N CN CN CN H H CN CN HN CN CN 1 1 1 1 1 1 1 1 1 1 1 • * CN CM or HH or CN HHH CN HHO ro CN o 1 1 1 1 1 1 1 1 1 1 1 1 * S¡ Oí &5 QO a WO a HA «S fe ft CQ E? ÍS > * > The identity sequence of the polynucleotide molecules is determined by similar methods using a ratio as described above. Those skilled in the art will appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method to examine the level of identity carried by an amino acid sequence described herein and the amino acid sequence of a putative variant of zsig28. The FASTA algorithm is described by Pearson and Lipman, Proc. Na t. Aca d. Sci. USA 85: 2 4 4 4, (1988), and by Pea rson, Me th. In zym ol. 1 83: 63, 1990. Briefly, the FASTA first characterizes the similarity of the sequence by identifying the regions carried by the interrogation sequence (e.g., SEQ ID NO: 2), and a test sequence having either the highest density of identities (if the variable ktup is 1) or pairs of identities (if ktup = 2), without considering the substitutions, insertions or deletions of conservative amino acids. The ten regions with the highest density of identities are then registered by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "ordered" to include only those residues that contribute to the highest record. If there are several regions with records larger than the "cut" value (calculated by a predetermined formula based on the length of the sequence and the ktup value), then the initial ordered regions are examined to determine if the regions can be joined to form an approximate alignment with intervals or grooves. Finally, the highest registration regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Bi ol. 48: 4 4 4, 1970; Sellers, SIAM, J. App. Ma th. 2 6: 18 1, 1974), which allows the insertions and deletions of amino acids. The illustrative parameters for the FASTA analysis are: ktup = l, missing by interval opening = 10, missing by interval extension = 1, and substitution matrix = BLOSUM62. These parameters can be entered into a FASTA program by modifying the record matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. In zymol 1 83: 63, 1990. The FAST can also be used to determine the identity of the sequence of the nucleic acid molecules using a ratio as described above. For comparisons of the nucleotide sequence, the ktup value may vary between one to six, preferably three to six, more preferably three, with other parameters as misplaced. Table BLOSUM62 (Table 3) is an amino acid substitution matrix derived from approximately 2, 000 multiple local alignments of the 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 8_9: 10915, 1992). Accordingly, BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. Although it is possible to designate amino acid substitutions based only on the chemical properties (as discussed above), the language "conservative amino acid substitution" 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. According to this system, conservative preferred amino acid substitutions are characterized by a BLOSUM62 value of at least 1 ( for example 1, 2 or 3), while the most preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (for example 2 or 3). Variants of zsig28 polypeptides or substantially homologous zsig28 polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, which are conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the cleavage or activity of the polypeptide.; minor deletions, typically from one to about 30 amino acids; and minor amino or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes polypeptides from about 208 to about 291 amino acid residues comprising a sequence that is at least 80%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of SEQ ID NO. :2. Polypeptides comprising affinity tags that may further comprise a site of proteolytic cleavage between the zsig28 polypeptide and the affinity tag. Preferred sites include thrombin cleavage sites and factor Xa cleavage sites.

Table 4 Conserved basic amino acid substitutions: Arginine Lysine Histidine Acidic: Glutamic acid Aspartic acid Polar: Glutamine Asparagine Hydrophobic: Leucine Isoleucine Valine Aromatic: Phenylalanine Tryptophan Tyrosine Small: Glycine Alanine Serine Threonine Methionine The present invention further provides a variety of other mergers of polypeptides and related multimeric proteins comprising one or more polypeptide fusions. For example, a zsig28 polypeptide can be prepared as a fusion to a dimerizing protein as described in U.S. Patent Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this sense include immunoglobulin constant region domains. The immunoslobulin zsig28 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of mutant zsig28 analogues. Auxiliary domains can be fused to the zsig28 polypeptides to be targeted to specific cells, tissues, or macrogotubules (e.g., collagen). For example, a zsig28 polypeptide or protein could be targeted to a predetermined cell type by fusing a zsig28 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this form, polypeptides and proteins can be targets for therapeutic or diagnostic purposes. A zsig28 polypeptide can be fused to two or more portions, such as an affinity tag for purification and a target domain or dimerization. Fusions of the polypeptides may also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34: 1-9, 1996. Similarly, such fusions can be constructed to allow secretion of regions 1, 2, 3 or 4 of the zsig28 polypeptide described herein, or fragments smaller ones within such regions. The soluble zsig28 regions 1, 2, 3 or 4, bound to the dimerizing proteins may have served as antagonists of the natural ligand for the zsig28 polypeptide, or the polypeptide itself zsig28, for example, by preventing dimerization or multimerization. Such antagonists contain soluble zsig28 regions 1, 2, 3 or 4, can be tested for their functionality as described herein. The proteins of the present invention can also comprise amino acid residues that do not originate naturally. Amino acids that do not originate naturally include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, tetrahydroxyproline, N-methyl-glycine, threonine, methyl threonine, hydroxyethyl cysteine, hydroxyethylhomocysteine, nitro-glutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenyl -alanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for the incorporation of amino acid residues that do not naturally originate in proteins. For example, an in vi t ro system may be employed when nonsense mutations are suppressed using chemically aminoacylated suppression tRNAs. Methods for synthesizing aminoacilant and amino acid tRNAs are known in the art. The transcription and translation of the plasmids containing nonsense mutations are carried out in a cell-free system comprising an E extract. col i S30 and commercially available systems 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: 806-9, 1993; and Chung et al., Proc. Nati Acad. Sci. USA 9_0_: 10145-9, 1993). In a second method, translation is carried out in Xen opus oocytes by mutated mRNA microinj ects and chemically aminoacylated suppressors tRNAs (Turcatti et al., J. Biol. Chem. 271: 1999-1-8, 1996). Within a third method, E cells. col i are cultured in the absence of a natural amino acid that is replaced (eg, phenylalanine) and in the presence of desired amino acid (s) that do not originate naturally (eg, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine). The amino acid that does not originate naturally is incorporated into the protein instead of its natural counterpart. See Koide et al., Bichem. _33_: 7470-6, 1994. Amino acid residues that originate naturally can be converted to species that do not originate naturally by chemical modification in vi t ro. 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). A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, amino acids that do not naturally originate, and unnatural amino acids can be substituted for the amino acid residues zsig28.

The essential amino acids in the polypeptides of the present invention can be identified in accordance with methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 2_44: 1081-5, 1989). Bass et al., Proc. Nati, Acad. Sci. USA 8_8_: 4498-502, 1991). In the latter technique, unique mutations of alanine are introduced to all residues in the molecule, and the resulting mutant molecules are tested for their biological activity as described below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-708, 1996. The sites of receptor-ligand interaction can also be determined by physical structure analyzes, as determined by such techniques as magnetic resonance imaging. nuclear, crystallography, electron diffraction, or photon affinity labeling, in conjunction with the mutation of the putative contact site amino acids. See, for example, de Vos et al., Science 255: 306-12, 1992; Smith et al., J. Mol. Biol. 244: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992.

The identities of the essential amino acids can also be inferred from analyzes of homologies with related proteins, such as claudin (SEQ ID NO: 3), protein such as human OSP (SEQ ID NO: 5), and the like. Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and selection, such as those described by Reidhaar-Olson and Sauer (Science 241: 53-7, 1988) or Bowiw and Sauer (Proc. Nati. Acad. Sci. USA 8_6: 2152-6, 1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selected by the functional polypeptide and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem 30: 10832-7, 1991, Ladner et al., U.S. Patent No. 5,223,409, Huse, WIPO publication WO 92/06204), and mutagenesis. directed to the region (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988). The zsig28 DNA variants described and the polypeptide sequences can be generated through DNA shuffling as described by Stemmer, Nature 370: 389-91, 1994 and Stemmer, Proc. Nati Acad. Sci. USA 91: 10747-51, 1994, and WIPO Publication WO 97/20078. Briefly, the DNA variants are generated by homologous recombination in vi t ro by random fragmentation of an original DNA, followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified using a family of an original DNA, such as allelic variants or genes from different species, to introduce additional variability into the process. The selection or projection for the desired activity, followed by the additional interactions of mutagenesis and assays, provide rapid "evolution" of the sequences by the selection of desirable mutations, while simultaneously being selected against the deleterious changes. Mutagenesis methods as described herein can be combined with automated, high-output screening methods to detect the activity of the mutagenized polypeptides, cloned in the host cells.

Mutagenized DNA molecules that encode active polypeptides (e.g., signal transduction, or linkage activities), 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 structures. Using the methods discussed herein, one ordinarily skilled in the art can identify and / or prepare a variety of fragments or variants of polypeptides of SEQ ID NO: 2 or that retain, for example, linkage, cell-cell communication, or activity Signal transduction of the wild-type zsig28 protein. For example, using the methods described above, one could identify a ligand binding domain in zsgi28; the heterodimeric and homodimeric link domains; other structural or functional domains; or other important domains for protein-protein interactions, cell-cell interactions, or signal transduction. Such polypeptides may also include additional polypeptide segments, such as affinity tags, as generally described herein. For any zsig28 polypeptide, including variants and fusion proteins, one ordinarily skilled in the art can easily generate a completely degenerate polynucleotide sequence encoding such a variant using the information set forth in Tables 1 and 2 above. The zsig28 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells in accordance with conventional techniques. Suitable host cells are those types of cells that can be transformed or transfected with exogenous DNA and grown in cultures, and include bacteria, fungal cells and cultured higher eukaryotic cells. Eukaryotic cells are preferred, particularly cultured cells of multicellular organisms. Techniques for the manipulation of cloned DNA molecules and the introduction of exogenous DNA into a variety of host cells are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY 1987. In general, a DNA sequence. which encodes a zsig28 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. Generally, the promoter is operably linked to the sequence or segment of DNA, and the DNA segment is operably linked to the transcriptional terminator. 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 with certain selectable marker systems they can be provided in separate vectors, and the replication of the exogenous DNA can be provided by the integration of the genome in the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a routine subject designated with the level of one skilled in the art. Many elements are described in the literature and are available through commercial providers. To direct a zsig28 polypeptide in the secretory path of a host cell, a secretory signal sequence (also known as a leader sequence, signal sequence, prepro sequence or pre sequence) is provided in the expression vector. The sequence of the secretory signal may be that of zsig28 or it may be derived from another protein secreted (eg, t-PA) or synthesized from n ovo. The secretory signal sequence is operably linked to the zsig28 DNA sequence, ie, the two sequences are linked in the correct reading structure and positioned to direct the newly synthesized polypeptide into the secretory path of the host cell. The secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences can be placed in any way in the 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). Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides in the secretory pathway. The present invention is provided for such fusion polypeptides. A fusion polypeptide signal can be made where a secretory signal sequence derived from an amino acid residue 1 (Met) to residue 23 (Ala) of SEQ ID NO: 2 is operably linked to a DNA sequence encoding a another polypeptide, using methods known in the art and described herein. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably amino-terminally fused to an additional peptide to di the additional peptide in the secretory pathway. Such constructs have numerous applications known in the art. For example, these new fusion constructs of the secretory signal sequence can di the secretion of an active component of a normally non-secreted protein. Such fusions can be used vi ng or in vi t to di the peptides through the secretory trajectory.

Cultured cells of mammals are suitable hosts within the present invention. Methods for introducing exogenous DNA into host mammalian cells include transfection mediated calcium phosphate (Wigler et al, Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:. 603, 1981: Graham and van der Eb, Virology 5_2: 456, 1973), electroporation (Neumann et al, EMBO J. ". 1: 841-5, 1982), transfection, DEAE dextran (Ausubel et al, ibid).. and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus _15_: 80, 1993; and viral vectors (Miller and Rosamn, BioTechniques 7: 980-90, 1989; .. Wang and Finer, Nature Med 2_: 714-6, 1996) the production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al, U.S. Patent No. 4,713,339; Hagen et al.,. U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include 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. 3_6: 59-72, 1977) and Chinese hamster ovary cell lines (e.g. CH0-K1; ATCC No. CCL 61). Additional suitable cell lines are known in the art and are available from public depositories such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters, such as SV40 or cytomegalovirus promoters, are preferred. See, for example, U.S. Patent No. 4,956,288. Other suitable promoters include those of the metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter. In general, drug selection is used to select the cultured cells of mammals in 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 that 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 resistance to the neomycin antibiotic. The selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. 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 transfectants in the presence of a low level 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 preferred amplifiable selectable marker is dihydrofolate reductases, which confers resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used. Alternate markers that introduce an altered phenotype, such as the green fluorescent protein, or proteins from cell surfaces such as CD4, CD8, MHC Class I, placental alkaline phosphatase, can be used to classify the transfected cells of the cells not transfected by means such as FACS sorting or magnetic bead separation technology. Other higher eukaryotic cells can also be used as hosts or hosts, which include plant cells, insect cells and bird cells. The use of Agroba ct eri um rhi zogen is as a vector for the expression of genes in plant cells have been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11: 47-58, 1987. The transfection of insect cells and the production of foreign polypeptides therein is described by Guarino et al., US Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculoviruses, which are commonly derived from the polypeptide virus in the aquatic cell (AcNPV). See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman &; Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed. , Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. The second method for manufacturing recombinant zsig28 baculoviruses utilizes a transposon-based system described by Luckow (Luckow, VA, et al., J Virol 67_: 4566-79, 19 ~ 93). This system, which uses transfer vectors, is sold in the Bac-to-Bac ™ kit or equipment (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl ™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zsig28 polypeptide to a baculovirus genome maintained in E. coli as a large plasmid called "bacmido". The pFastBacl ™ transfer vector uses the AcNPV polyhedrin promoter to drive expression of the gene of interest, in this case zsig28. However, pFastBacl ™ can be modified to a considerable degree. The polyhedrin promoter can be removed and replaced with the baculovirus basic protein (also known as the Peor promoter, p6.9 or MP) which is expressed early in baculovirus infection and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J. Gen Virol. 7JL_: 971-6, 1990; Bonning, B.C. et al., J. Gen. Virol. 75: 1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, B., J. Biol. Chem. 270: 1543-9, 1995. In such constructs of the transfer vector, a short or long version of the basic protein promoter can be used. In addition, transfer vectors can be constructed which replaces the native zsig28 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from the Glucosiltrans ferase Ecdiester? Ide (EGT), honey bees Melittina (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the secretory signal sequence zsig28 native. In addition, the transfer vectors can include a fusion in the structure with the DNA encoding an epitope tag at the C- or N-terminus of the expressed zsig28 polypeptide, for example, a tag of the Glu-Glu epitope (Grussenmeyer, T. et al., Proc. Nati, Acad. Sci. 8_2: 7952-4, 1985). Using a technique known in the art, a transfer vector containing the zsig28 is transformed into E. coli, and it is separated for the bacmides containing an interrupted lacZ gene indicative of the recombinant baculovirus. The bacmid DNA containing the genome of the recombinant baculovirus is isolated, using common techniques, and used to transfect the Spodoptera frugiperda cells, for example Sf9 cells. The recombinant virus expressing zsig28 occurs subsequently. The recombinant viral material is manufactured by methods commonly used in the art.

The recombinant virus is used to infect the host cells, typically a cell line derived from the autumn soldier worm, Spodop t was frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, Press, Washington, DC, 1994. Another suitable cell line is the High FiveO ™ cell line (Invitrosen) derived from Tri ch opl us iani (Patent North American No. 5,300,435). The commercially available serum free medium is used for the growth and maintenance of the cells. Suitable media are Sf900 II ™ (Life Technologies) or ESF 921 ™ (Expression Systems) for Sf9 cells; and ExcellO405 ™ (JRH Biosciences, Lenexa, KS) or Express FiveO ™ (Life Technologies) for T cells. n i. 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 recombinant viral material is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically about 3. The procedures are generally described in the laboratory manuals (King, LA and Possee, RD, ibid., O'Reilly, DR et al., Ibid., Richardson, CD, ibid. .). Subsequent purification of the zsig28 polypeptide from the supernatant can be achieved using the methods described herein. Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in relation to this include Sa ccha romyces cerevi sia e, Pi chi a pa s tori s, and Pi chia me thanol i ca. The methods to transform S cells. cerevi sia with exogenous DNA and to produce the recombinant polypeptides thereof are described by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373, Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., US Patent 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). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in a medium containing glucose. Promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, for example, Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al., U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092; ) and the alcohol dehydrogenase genes. See also U.S. Patent 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 known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells can be used in accordance with the methods of McKnight et al., US Patent No. 4,935,349. Methods for transforming Acremoium chrysogenum are described by Sumino et al., US Patent No. 5,162,228. Methods for transforming Neurospora are described by Lambowitz, U.S. Patent No. 4,486,533. The use of Pichia methanolica as host or host for the production of recombinant proteins is described in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules to be used in the transformation of P. methanolica will be prepared in a common way as circular plasmids, double-stranded, which are preferred to liberalize before the transformation. For the production of the polypeptide in P. methanolica, it is preferred that the promoter and the terminator in the plasmid be that of a P. methanolica gene, such as an alcohol utilization gene of P. methanolica (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. to facilitate integration of the DNA into the host chromosome, it is preferred to have the complete expression segment of the plasmid flanked at both ends by the host DNA sequences. A preferred selectable marker for use in Pichia methanolica is an ADE2 P. methanolica gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC).; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale industrial processes, where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both the methanol utilization (AUG1 or AUG2) genes are removed. For the production of the secreted proteins, the host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing a DNA encoding a polypeptide of interest in the P cells. That's me. It is preferred to transform the P cells. me than ol i ca by electroporation using a pulse electric field, with exponential decay having a field strength of 2.5 to 4.5 kV / cm, preferably about 3.75 kV / cm and a time constant (t) of 1 to 40 milliseconds, preferably approximately 20 milliseconds. Prokaryotic host cells that include strains of the bacterium Es ch eri chi a col i, Ba ci l l us and other genera are also useful as host cells within the present invention. Techniques for transforming these hosts and expressing the cloned DNA sequences there are well known in the art (see, for example, Sambrook et al., Ibid.). when a zsig28 polypeptide is expressed in bacteria such as E. col i, the polypeptide is 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 above case, the cells are used, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can be redoubled or re-folded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of oxidized and reduced 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 breaking the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recover the protein, thus obviating the need for denaturing and re-folding. Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing 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 medium may also contain such components such as growth factors or serum, as required. The growth medium will generally select cells containing the exogenously added DNA by, for example, the selection of the drug or deficiency in an essential nutrient which is complemented by the selectable marker carried in the expression vector or co-transfected into the host cells. The cells of P. me thanol i ca are grown in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25 ° C to 35 ° C. Liquid cultures are provided with sufficient aeration by conventional means, such as agitation of small flasks or spraying of fermenters. A preferred culture medium for P. I am more than YEPD (2% D-glucose, Peptone Bacto ™ (Difco Laboratories, Detroit, MI), 1% Bacto ™ yeast extract (Difco Laboratories), 0.004% adenine and L-leucine 0.006%). It is preferred to purify the polypeptides of the present invention to a purity > 80%, more preferably up to a purity >; 90 purity even more preferably > 95% purity, and a pharmaceutically pure state, which is greater than 99.9% pure with respect to the contaminating macromolecules, particularly other proteins and nucleic acids, and free of infections and pyrogenic agents is particularly preferred. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. The expressed recombinant zsig28 polypeptides (or chimeric zsig28 polypeptides) can be purified using conventional fractionation and / or purification methods. Precipitation with ammonium sulfate and extraction of chaotrope or acid can be used for the fractionation of samples, exemplary purification steps may include hydroxyapatite, size exclusion, FPLC reverse phase high resolution liquid chromatography. Suitable chromatographic media include the dextrans derivatives, agarose, cellulose, polyacrylamide, special silicas, and the like, PEI, DEAE, QAE and Q. Exemplary chromatographic media include those media derived with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like, or polyacryl resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solids include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, resins crosslinked polyacrylamide salts and the like which are insoluble under the conditions in which they are to be used. These supports can be modified with the reactive groups that allow the binding of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or portions or carbohydrate radicals. Examples of coupling chemistries include activation by cyanogen bromide, activation by hydroxysuccinimide, activation by epoxide, activation by sulfhydryl, activation by hydrazide and coupling chemistries of carboxyl and amino derivatives for carbodiimide. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding the receptor polypeptides to the support medium are well known in the art. The selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principies & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988. The polypeptides of the present invention can be isolated by examination of their biochemical, structural and biological properties. For example, chromatography by adsorption of immobilized metal ions (IMAC) can be used to purify histidine rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem.3: 1-7, 1985). Histidine-rich proteins will be absorbed into this matrix with affinities that differ, depending on the ion used, and will be eluted by competitive elution, lowering pH, or using strong chelating agents. Other purification methods include the purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp. 529-39). Within the additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag or label (e.g., maltose binding protein, an immunoglobulin domain) can be constructed to facilitate purification. In addition, using the methods described in the art, polypeptide fusions, or zsig28 hybrid proteins, are constructed using the regions or domains of the inventive zsig28 in combination with the paralogs (e.g., protein similar to human OSP), orthologs ( for example, OSP or murine RVP.l) or heterologous proteins (Sambrook et al., ibid.; Altschul et al., Ibid.; Picard, Cur. Opin. Biology, _5: 511-5, 1994, and references thereof). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids can alter reaction kinetics, binding, constriction or expansion of substrate specificity, or alter the tissue and cellular location of a polypeptide, and can be applied to polypeptides of unknown structure. The fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide having both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, the part or all of the domain (s) conferring a biological function can be exchanged between the zsig28 of the present invention with the functionally equivalent domains of another member of the family. Such domains include, but are not limited to, the secretory signal sequence, the transmembrane domains, regions 1 through 4, and portions 1 through 4, as described herein. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to the polypeptides of the present invention or proteins to which they are fused, depending on the constructed fusion. In addition, such fusion proteins can exhibit other properties as described herein. Cloning and standard molecular biology techniques can be used to exchange the desired domains between the zsig28 polypeptide and those to which they are fused. In general, a DNA segment encoding a domain of interest, e.g., a domain described above, is operably linked to the structure, and inserted into an appropriate expression vector, as described herein. Such fusion proteins can be expressed, isolated, and assayed for their activity as described herein. The zsig28 polypeptides or fragments thereof can also be prepared through chemical synthesis, the zsig28 polypeptides can be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue. The polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, classical solution synthesis or fragment condensation or 'for the classical solution.

Methods for the synthesis of polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 8_5: 2149, 1963; Kaiser et al., Anal. Biochem. 34: 595, 1970. After complete synthesis of the desired peptide on a solid support, the peptide resin is with a reagent that cleaves the polypeptide from the resin and removes most of the side chain protectants. Such methods are well established in the art. The activity of the molecules of the present invention can be measured using a variety of assays that measure the proliferation and / or differentiation of specific cell types, chemotaxis, adhesion, changes in the ion influx channels, the pH flow, regulation of the levels of the second messenger and the release of the neurotransmitter, the motility of the cells, the binding of the protein, apoptosis, or the like. Such assays are well known in the art. See, for example, in "Basic &Clinical Endocrinology Ser., Vol. 3", Cytochemical Bioassays: Techniques & Applications, Chayen; Chayen, Bitensky, eds., Dekker, New York, 1983. The activity of the molecules of the present invention can be measured using a variety of assays that measure the stimulation of gastrointestinal cell contractibility, peristalsis, secretion modulation and / or Absorption of nutrients from digestive enzymes. Of particular interest are changes in the contractility of smooth muscle cells. For example, the contractile response of the mammalian duodenum segments other gastrointestinal smooth muscle tissues (Depoortere et al., J. Gastrointestinal Motility _! _: 150-159, incorporated herein for reference). An exemplary test uses an ultrasonic micrometer to measure dimensional changes radially between the commissures and longitudinally for the plane of the valve base (Hansen et al., Society of Thoracic Surgeons _60_: S384-390, 1995). Gastric motility is measured in the clinical prescription generally according to the time required for gastric emptying and the subsequent transit time through the gastrointestinal tract. The exploration of gastric emptying is well known to those skilled in the art, and briefly, comprises the use of an oral contrast agent, such as barium, or a radiolabelled food. Solids and liquids can be measured independently. A food or test liquid is radiolabelled with an isotope (for example 99mTc), and after ingestion or administration, the transit time through the gastrointestinal tract and gastric emptying are measured by visualization using gamma cameras (Meyer et al. ., Am. J. Dig. Dis. 2JL_: 296, 1976; Collins et al., Gut 24_: 1117, 1983; Maughan et al., Diabet. Med. 13 9 Supp. _5: S6-10, 1996 and Horowitz et al., Arch. Intern. Med. 145: 1467-1472, 1985). These studies can be performed using before and after the administration of a promoter agent to quantify the efficacy of the drug. In addition, these assays can be used to test the zsig28 agonists and antagonists, discussed below. High expression of the zsig28 polypeptide in the stomach suggests that modulators of zsig28 activity could be therapeutically useful. Modular such may be agonists or antagonists that respectively stimulate or inhibit the activity of the zsig28 polypeptide. The modulating effects of zsig28 activity can be tested by methods well known in the art. The zsig28 agonists or antagonists thereof can be therapeutically useful to promote wound healing. For example, in the stomach. To verify the presence of modulators of zsig28 polypeptides with these capabilities, the agonists or antagonists of the present invention are evaluated for their ability to facilitate wound healing according to procedures known in the art. If desired, the efficacy of the zsig28 polypeptide in relation to this can be compared to the growth factor receptors, such as those for EGF, NGF, TGF-α, TGF-β, insulin, IGF-I, IGF-II, factor growth of fibroblasts (FGF) and the like. In addition, zsig28 polypeptide agonists or antagonists can be evaluated in combination with one or more growth factors to identify the synergistic effects on zsig28 activity. In addition, zsig28 agonists or antagonists may be therapeutically useful for anti-microbial applications. To verify the presence of modulators of zsig28 polypeptides with these capabilities, the agonists or antagonists of the present invention are evaluated for their antimicrobial properties according to procedures known in the art. See for example, Barsum et al., Eur. Breathe J. 8 (5): 709-14, 1995; Sandovsky-Losica et al., J. Med. Vet. Mycol. (England) 28 (4): 279-87, 1990; Mehentee et al., J. Gen. Microbiol (England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage, J. Med. Vet. Mycol. 24: 477-479, 1986 and the like. In addition, zsig28 agonists or antagonists thereof can be evaluated in combination with one or more antimicrobial agents to identify the synergistic effects and modulation of zsig28 polypeptide. The zsig28 polypeptide or fragments thereof can act as antimicrobial agents to block a pathogenic organism to adhere to the zsig28 receptor or as a bowl of bacterial toxin. Such antimicrobial agents operate via the association of the membrane or in the pore-forming mechanisms of binding action directly to the invading microbe. The antimicrobial agents can also act via an enzymatic mechanism, breaking the protective substances of the microbes or the walls / membrane of the cell thereof. Antimicrobial agents capable of inhibiting the proliferation of the microorganism or action or of disruption of the integration of the microorganism by means of either the mechanisms established above, are useful in methods for preventing contamination in cell culture by microbes susceptible to that antimicrobial activity. Such techniques involve culturing cells in the presence of an effective amount of said zsig28 polypeptide, the fragment of the polypeptide, or an agonist or antagonist thereof. Alternatively, such antimicrobial agents could directly bind to a toxin from the invading bacterium, described below, and effectively inactivate its toxicity. The zsig28 polypeptides of the present invention may play a role in the pathogenesis in the human stomach and in intestinal infections. Zsig28 shares homology with the enterotoxin receptor of Cl or tri dium perfringens (CPE) and related proteins that bind to CPE (Katahira, J. et al., J. Cell Biol. 136: 1239-1247, 1997; and, Katahira, J. et al., J. Biol. Chem. 272: 26652-26658, 1997). Similarly, zsig28 can be linked to CPE or other bacterial enterotoxins or exotoxins, collectively described herein as "bacterial toxins". For example, such bacterial toxins are produced by the bacterium that causes the conditions such as food poisoning, botulism, severe diarrhea, inflammation, cramping, or the like (eg, s taphyl oco ccus, E col i enterotoxigenic, campyl oba ct er , Cl tri di um bo t ul in um and the like). In addition, as a receptor, zsig28 can be a site for colonization and the pathogenic effects resulting from pathogenic bacteria in the stomach. For example, Hel i coba cter pyl ori, a causative agent in gastric ulcers, can exert its effects by binding to zsig28 and inducing cell lysis through apoptosis or other mechanisms. Such a paper for zsig28 can be elucidated by one skilled in the art. For example, mammalian cells that are normally insensitive to bacterial toxin can be transfected with zsig28 and can be tested for susceptibility to bacterial toxin (See Katahira, J. et al., Supra.; And, Katahira, J. . et al., supra.). When compared in parallel with non-transfected cells, zsig28 expression cells can be measured by morphological changes associated with death such as (formation of small ampules), lysis, and cell number elimination when compared to the control not transfected. Alternatively, assays that measure the direct linkage of a fluorescent labeled or radiolabelled toxin can be used to measure the direct link to cells expressing zsig28. Similarly, using these assays described above, whole bacterial cells can be tested for their ability to bind and / or cell lysis expressing the zsig28 polypeptide. A) Yes, the zsig28 polypeptides or agonists thereof can be used in cell culture reagents in in vitro studies of the infection of exogenous microorganisms, such as bacterial, viral or fungal infection. Such portions can be used in animal models without infection. Also, the adhesion properties to the microorganisms of the zsig28 polypeptides or agonists thereof can be studied under a variety of conditions in binding assays and the like. As a receptor, the activity of the zsig28 polypeptide can be measured by a silicon-based biosensor microphysiometer, which measures the ratio of extracellular acidification or proton excretion associated with receptor binding and subsequent physiological cellular responses. An exemplary device 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, receptor and regulatory activation, and the like, can be measured by this method. See, for example, McConnell, H.M.- et al., Science 257: 1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228: 84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212: 9-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346: 87-95, 1998. The microphysiometer can be used to test eukaryotic, prokaryotic, adherent or non-adherent cells. By changes in extracellular acidification in cellular media over time, the microphysiometer directly measures cellular responses to various stimuli, including agonists, ligands, or antagonists of the zsig28 polypeptide. Preferably, the microphysiometer is used to measure the responses of a eukaryotic cell expressing zsig28, compared to a control eukaryotic cell that does not express the zsig28 polypeptide. Eukaryotic cells expressing zsig28 comprise cells in which zsig28 has been transfected, as described herein, creating a cell that is responsible for the zsig28 modulator stimulus, or cells that naturally express zsig28, such as cells that express the zsig28 derived from the stomach tissue. The differences, measured by an increase or decrease in extracellular acidification, in the response of cells expressing zsig28, in relation to a control, is a direct measure of the modulated cellular responses of zsig28. In addition, such responses modulated by zsig28 can be tested under a variety of stimuli. Also, using the microphysiometer, a method is provided for identifying agonists and antagonists of the zsig28 polypeptide, which comprises providing cells comprising a zsig28 polypeptide, by culturing a first portion of the cells in the absence of a test compound, by culturing a second portion of cells in the presence of a test compound, and detect an increase or decrease in a second portion of the cells compared to the first portion of the cells. Antagonists and agonists that include the natural ligand for the zsig28 polypeptide can be quickly identified using this method. An in vivo approach to testing the proteins of the present invention involves the viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpes virus, vaccinia virus, retrovirus, and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best-studied gene transfer vector for the delivery of heterologous nucleic acid (for review, see TC Becker et al., Meth Cell Biol. _43_: 161-89, 1994 and JT Douglas and DT Curiel, Science &Medicine 4_: 44-53, 1997). The adenovirus system offers several advantages: (i) the adenovirus can accommodate relatively large DNA inserts; (ii) it can be grown up to a high degree; (iii) infect a wide range of mammalian cell types; and (iv) can be used with many different promoters including the ubiquitous, specific tissue-regulating promoters. Also, because the adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. Using the adenovirus vectors where the portions of the adenovirus genome have been removed, the inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential gene has been removed from the viral vector, and the virus is not replicated unless the El gene is provided via a host cell (the human 293 cell line is exemplary). When administered intravenously to intact animals, the adenovirus primarily targets the liver. If the adenoviral delivery system has a deletion in the El gene, the virus can not replicate in the host cells. However, the host tissue (e.g., the liver) will, for example, express and process (and, if a secretory signal sequence is secreted) the heterologous protein. The secreted proteins will go into circulation in the highly vascularized liver, and the effects can be determined in the infected animal. In addition, adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector. Such adenoviruses have the El deleted, and also contain deletions of E2A or E4 (Lusky, M. et al., J Virol 7_2_: 2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy _9: 671-679, 1998). In addition, the elimination of E2b is reported to reduce immune responses (Amalfítano, A. et al., J. Virol. 72: 926-933, 1988). In addition, by removing the entire adenovirus genome, very large inserts of the heterologous DNA can be accommodated. The generation of the so-called "stomach-free" adenoviruses in which all the viral genes are eliminated are particularly advantageous for the insertion of large inserts of heterologous DNA. For review, see Yeh, P. and Perricaudet, M., FASEB J. 11: 615-623, 1997. The adenovirus system can also be used for the production of proteins in vi t ro. By culturing cells that are not 293, infected with adenovirus, under conditions where cells do not divide rapidly, cells can produce proteins for extended 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. Alternatively, 293 cells infected with the adenovirus vector can be grown as adherent cells or in a suspension culture at relatively high cell densities to produce significant amounts of protein (See Garnier et al., Cytotechnol. -55, 1994). With any protocol, a secreted heterologous protein can be repeatedly isolated from the supernatant of the cell culture, used or membrane fractions, depending on the arrangement of the protein expressed in the cells. Within the production protocol of infected 293 cells, non-secreted proteins can also be obtained effectively. As a receptor, activation of the zsig28 polypeptide can be measured by a silicon-based biosensor microphysiometer, which measures the ratio of extracellular acidification or proton excretion associated with receptor binding and subsequent physiological cellular responses. An exemplary device 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, receptor and regulatory activation, and the like, can be measured by this method. See, for example, McConnell, H.M. et al. 'Science 257: 1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228: 84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212: 49-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346: 87-95, 1998. The microphysiometer can be used to test adherent or non-adherent prokaryotic or eukaryotic cells. By measuring the extracellular acidification changes in the cell medium during all the time, the microphysiometer measures the cellular responses to various stimuli, which include the agonists, ligands or antagonists of the zsig28 polypeptide. Preferably, the microphysiometer is used to measure the responses of a eukaryotic cell expressing zsig28, compared to a control eukaryotic cell that does not express the zsig28 polypeptide. Eukaryotic cells expressing zsig28 comprise cells within which zsig28 has been transfected, as described herein, creating a cell responsive to the stimulus that modulates zsig28; or cells that naturally express zsig28, such as cells expressing zsig28 derived from stomach tissue. The differences, measured by a change in extracellular acidification, for example, an increase or decrease in the response of cells expressing zsig28, relative to a control, are a direct measure of the modulated cellular responses of zsig28. In addition, such responses modulated by zsig28 can be tested under a variety of stimuli. As well, using the microphysiometer, a method is provided for identifying agonists and antagonists of the zsig28 polypeptide, which comprises providing cells expressing a zsig28 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, for example, an increase or decrease in a second portion of the cells compared to the first portion of the cells. The change in cellular response is shown as an extracellular acidification ratio of measurable change. Antagonists and agonists, which include the natural ligand for the zsig28 polypeptide, can be quickly identified using this method. A zsig28 polypeptide, or a polypeptide fragment thereof, can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an Fc fragment, which contains two constant region domains and lacks the variable region. Methods for preparing such fusions are described in U.S. Patent Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfides linked together and two non-Ig polypeptides are arranged in close proximity to each other. Mergers of this type can be used to affinity purify the ligand, as an in vi tro assay tool. For use in the assay, the chimeras are bound to a support via the Fc region and used in an immunosorbent assay format linked to the enzyme (ELISA). A zsig28 polypeptide can be used for the purification of the ligand that binds thereto. The zsig28 polypeptide or a fragment of the polypeptide linked to the ligand thereof can be used. The polypeptide is immobilized on an aolic support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or the like, materials that are stable under the conditions of use. Methods for linking the polypeptides to solid supports are known in the art, and include amine chemistry, activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl, and activation with hydrazide. The resulting medium will generally be configured in the form of a column, and the fluids containing the ligand are passed through the column one or more times to allow the ligand to bind to the polypeptide receptors. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl), or pH to break the ligand-receptor bond. An assay system using a ligand-binding receptor, such as zsig28 (or an antibody, a complement pair / anticomplement member) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway , NJ) can be used advantageously. Such a receptor, antibody, member of a complement / anticomplement pair or fragment is immobilized on the surface of a receptor particle. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cunningham and Wells, J.

Mol. Biol. 234: 554-63, 1993. A receptor, member antibody or fragment is covalently linked, using the chemistry of the amine or sulfhydryl, to the dextran fibers that are attached to a gold film within the cell flow. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement / anticomplement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index, which is detected as a change in the resonance of the superficial plasmon of the gold film. This system allows the determination of start and stop ratios, from which the affinity of the link can be calculated, and the valuation of the link is toquiometre. The ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for the determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 5JL_: 660-72, 1949) and calorimetric titrations (Cunningham et al., Science 253: 545-58, 199.1; Cunningham et al., Science 245: 821-25, 1991).

The zsig28 polypeptides can also be used to prepare antibodies that bind zsig28 epitopes, peptides or polypeptides. The zsig28 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and generate an immune response. One skilled in the art will recognize that the antigenic polypeptides, which carry the epitope, contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zsig28 polypeptide (e.g. , SEC ID NO: 2). Polypeptides comprising a larger portion of a zsig28 polypeptide, i.e. from 30 to 10 residues up to the full length of the amino acid sequence, are included. Immunogenic antigens or epitopes may also include the attached labels, adjuvants and carriers, as described herein. Suitable antigens include the zsig28 polypeptide encoded by SEC. ID NO: 2 from the amino acid number 24 (Ala) to 261 (Val) of the SEC. ID NO: 2 or a contiguous amino acid fragment 9 to 238 AA thereof. Preferred peptides for use as antigens are regions 1, 2, 3 and 4 described herein, and hydrophilic zsig28 peptides such as those predicted by someone skilled in the art from a hydrophobicity plot (See Figure 2). The hydrophilic peptides zsig28 include the peptides comprising the amino acid sequences selected from the group consisting of: (1) amino acid number 245 (Ala) to amino acid number 250 (Glu) of SEQ. ID NO: 2; (2) amino acid number 234 (Asn) to the amino acid number. 239 (Lys) of the SEC. ID NO: 2; (3) amino acid number 202 (Glu) to amino acid number 207 (Lys) of SEC. ID NO: 2; (4) amino acid number 254 (Lys) to amino acid number 259 (Asp) of SEC. ID NO: 2; and (5) amino acid number 110 (Glu) to amino acid number 115 (Ala) of SEC. ID NO: 2. In addition, the conserved portions, and the variable regions between the conserved portions of zsig28 are suitable antigens. Antibodies to an immune response generated by inoculating an animal with these antigens can be isolated and purified as described herein. Methods for preparing and isolating monoclonal and polyclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al., (Eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., ED., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL 1982. As will be evident to someone with ordinary skill in the art., polyclonal antibodies can be generated by inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zsig28 polypeptide or a fragment thereof. The immunogenicity of a zsig28 polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as zsig28 fusions or a portion thereof with an immunoglobulin polypeptide or with a protein that binds to maltose. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such a portion can be advantageously linked or bound to a macromolecular carrier (such as key limpet hemocyanin (KLH), bovine serum alumina (BSA) or tetanus toxoid) for the immunization. As used herein, the term "antibodies" includes polyclonal antibodies, polyclonal antibodies purified by affinity, monoclonal antibodies, and fragments linked to the antigen, such as F (ab ') 2 and the proteolytic fragments of Fab. Antibodies or intact fragments engineered, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-linked peptides and polypeptides, are also included. Non-human antibodies can be humanized by grafting non-human CRDs into the human structure and constant regions, or by incorporating the entire non-human variable domains (optionally by "coating" them with a human-like surface by replacing the exposed residues, where the result is a "coated" antibody). 0 in some cases, humanized antibodies can retain non-human residues within the domains of the human variable region structure to improve the binding characteristics, appropriate. Through humanizing antibodies, the biological half-life can be increased, and the potential for adverse immune reactions after administration to humans is reduced. In addition, human antibodies can be produced in non-human, transgenic animals that have been engineered to contain human immunoglobulin genes as described in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be activated or eliminated, such as by homologous recombination. The antibodies are considered to bind specifically if: 1) they exhibit a threshold level of binding activity, and 2) they do not cross-react significantly with the molecules of the related polypeptide. A binding threshold level is determined if the anti-zsig28 antibodies of the present bind to a zsig28 polypeptide, peptide or epitope with an affinity at least 10 times greater than the binding affinity to the control polypeptide (not zsig28). It is preferred that the antibodies show a binding activity (Ka) of 106 M "1 or greater, preferably 107 M-i greater, more preferably 10 M-i or greater, and more preferably 109 M" 1 or greater. The binding affinity of a. antibody can be easily determined by someone with ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 5_1_: 660-72, 1949). It is shown that the anti-zsig28 antibodies do not significantly cross-react with the molecules of the related polypeptide, for example, by detection of the antibody detecting the zsig28 polypeptide but not known, related polypeptides, using a standard Western blot analysis ( Ausubel et al., Ibid.). examples of known related polypeptides are those described in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family, polypeptide or fragments,. and similar. For example, an antibody specific for zsig28 would not bind to the human OSP-like protein, the murine CPE receptor claudin 1, claudin 2, or the like. The selection can also be made using the mutant zsig28, and zsig28 non-human polypeptides. In addition, the antibodies can be "screened against" known related polypeptides to isolate a population that specifically binds to the inventive polypeptides. For example, the antibodies generated for zsig28 are absorbed to the related polypeptides adhered to the insoluble matrix; the antibodies specific to zsig28 will flow through the matrix under the appropriate buffer conditions. The selection allows isolation of polyclonal and monoclonal antibodies, which do not cross-react with closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology , Cooligan, et al., (Eds.), National Institutes of Health, John Wiley and Sons, Inc. 1995). The selection and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principies and Practice, Goding J.W. (eds.), Academic Press Ltd. , nineteen ninety six; Benjamin et al., Ann. Rev. Immunol. 2_: 67-101, 1984. Anti-zsig28 antibodies that are specifically linked can be detected by a number of methods in the art, and are described below.

A variety of assays known to those skilled in the art can be used to detect antibodies that bind to zsig28 proteins or polypeptide. 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 assays include: concurrent immunoelect roforesis, radioimmunoassay, radioimmunoassay, assay enzyme-linked immunosorbent (ELISA), spot or Western spotting assay, inhibition or competition assay, and a sandwich assay. In addition, the antibodies can be selected to bind to a wild-type protein versus a mutant zsig28 protein or polypeptide. Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to the zsig28 protein or peptide, and selection of sample libraries of the antibody in the phage or similar vectors (e.g., through the use of the zsig28 protein or peptide immobilized or labeled). Genes encoding polypeptides having potential zsig28 polypeptide binding domains can be obtained by randomly selecting the peptide libraries displayed on the phage (phage sample) or on a bacterium, such as E. col i. The nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and synthesis of the random polynucleotide. These sample libraries of the random peptide can be used to select peptides that interact with a target that can be a protein or a polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for the creation and selection of such sample libraries of the random peptide are known in the art (Ladner et al., US Patent No. 5,223,409; Ladner et al., U.S. Patent No. 4,946,778; Ladner et al., U.S. Patent No. 5,403,484 and Ladner et al., U.S. Patent No. 5,571,698) and sample libraries, randomized peptide and kits for the selection of such libraries are commercially available, for example from Clontech (Palo. Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Sample libraries of the random peptide can be selected from the zsig28 sequences described herein to identify proteins that bind to zsig28. These "binding polypeptides" that interact with the zsig28 polypeptides can be used to label the cells; for isolating the homologous polypeptides by affinity purification; they can be conjugated directly or indirectly to drugs, toxins, radionuclides and the like. These binding polypeptides can also be used in methods such as selection of expression libraries and neutralizing activity, for example, the interaction of blocking between the ligand and the receptor, or viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays to determine the circulating levels of zsig28 polypeptides; to detect or quantify soluble zsig28 polypeptides as markers of underlying diseases or conditions. These binding polypeptides can also act as "antagonists" of zsig28 to block the zsig28 linkage and signal transduction in vi t ro and in vi vo. These anti-zsig28 binding polypeptides would be useful for inhibiting the activity of zsig28 or the binding of the protein. Antibodies to zsig28 can be used to label cells expressing zsig28; to isolate zsig28 by affinity purification; for assays diagnostic assays to determine the levels of zsig28 polypeptides in normal tissues; to detect or quantify zsig28 as a marker of the underlying pathology or condition; in the analytical methods that use the FACS; to select the expression libraries; to generate the anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block the activity of zsig28 in vi tro and in vivo. Direct labels or tags include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect brands or labels can characterize the use of biot ina-avidin or other complement / anti-complement pairs as intermediaries. The antibodies herein 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. In addition, antibodies to zsig28 or fragments thereof can be used to detect denatured zsig28 or fragments thereof in assays, for example, blots or Western blots or other assays known in the art. The antibodies or polypeptides herein can also be conjugated directly or indirectly to the drugs toxins, radionuclides and the like, and these conjugates used for therapeutic or in vivo diagnostic applications. For example, the polypeptides or antibodies or binding polypeptides that recognize zsig28 of the present invention can be used to identify or treat tissues or organs that express a corresponding ant i-complementary molecule (eg, a zsig28 receptor). More specifically the anti-zsig28 antibodies, or the bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs expressing the zsig28 molecule. Suitable detectable molecules can be linked directly or indirectly to polypeptides that are linked to zsig28 ("polypeptide linkage"), antibodies, or bioactive fragments or portions thereof. Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like. Suitable cytotoxic molecules can be linked directly or indirectly to the polypeptide or antibody, and include bacterial or plant toxins (eg, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131. , rhenium-188 or yttrium-90 (either directly bound to the polypeptide or antibody, or indirectly linked through means of a chelating moiety, for example). The polypeptides or binding antibodies can be conjugated to cytotoxic drugs, such as adriamycin. For direct binding of a cytotoxic or detectable molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary / ant i-complementary pair, wherein the other member is linked to the binding polypeptide or antibody portion. For these purposes, biotin / streptavidin is a complementary / anti- complementary pair. In another embodiment, the binding polypeptide-toxin fusion proteins or the antibody-toxin fusion proteins can be used for the inhibition or removal of the target tissue or cells (eg, to treat cancer cells or tissues). Alternatively, if the binding polypeptide has multiple functional domains (ie, an activation domain or a ligand binding domain, plus a target domain), a fusion protein that includes only the target domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a molecule complementary to a cell or tissue type of interest. In the examples where the fusion protein the domain only includes a complementary molecule, the anticomplementary molecule can be conjugated to a detectable or cytotoxic molecule. Such fusion proteins of the complementary domain molecule thus represent a generic target vehicle for the cell / tissue specific delivery of the conjugates of anti-complementary-detectable / cytotoxic, generic molecules. In another embodiment, the anti-cytokine fusion proteins or zsig28-binding linker can be used to improve target tissue death (eg, cancers of the blood, stomach, colon, and of bone marrow), whether the ido-cytokine or anti-zsig28 polypeptide binding antibody is directed toward the bone marrow or blood cells, hyperproliferatives (See, in general, Hornick et al., Blood 89: 4437-47, 1997 ). For example, Hornick et al. describes the fusion proteins that allow a cytokine to target a desired site of action, thus providing a high local concentration of cytokine. Suitable anti-zsig28 antibodies are directed towards an undesirable cell or tissue (i.e., a tumor or a leukemia), and improved objective cell lysis mediated by cytokines fused by effector cells. Suitable cytokines for this purpose include interleukin 2 and the stimulation factor of the granulocyte-macrophage colony (GM-CSF), for example. Alternatively, the polypeptide that binds to zsig28 or the antibody fusion proteins described herein can be used to improve in vivo death of target tissues by stimulation to an apoptotic pathway modulated with zsig28, resulting in cell death of the cells hypoproliferatives expressing zsig28. The conjugates of the bioactive, binding polypeptide or antibodies described herein can be delivered orally, intravenously, intially or intraductally, or can be introduced locally at the intended site of action. The molecules of the present invention can be used to identify and isolate the ligands involved in gastrointestinal function, peristalsis, mucous secretion and the like. For example, the proteins and peptides of the present invention can be immobilized on a column and the tissue preparations that run on the column (Immobilized Affinity Ligand Techniques, Hermanson et al., Eds., Academic Press, San Diego, CA, 1992 , pp. 195-202). Proteins and peptides can also be radiolabelled (Methods in Enzymol., Vol. 192, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or labeled by photoaffinity ( Brunner et al., Ann. Rev. Biochem. 62_: 483-514, 1993 and Fedan et al., Biochem Pharmacol 33: 1167-80, 1984) and proteins of the specific ligand can be identified. The polypeptides, nucleic acids and / or antibodies of the present invention can be used in the treatment of conditions associated with the contractibility of gastrointestinal cells of digestive enzymes and acids, gastrointestinal motility, the collection or recruitment of digestive enzymes.; inflammation, particularly as it affects the gastrointestinal system; reflux disease and regulation of nutrient absorption. The specific conditions that will benefit from the treatment with molecules of the present invention, but are not limited to, diabetic gastroparesis post-surgical gastroparesis, vagotomy, chronic idiopathic intestinal pseudo-obstruction and gastroesophageal reflux disease. Additional uses include, gastric emptying for radiological studies, contraction that stimulates the gallbladder and antrectomy. The motor and neurological effects of the molecules of the present invention make it useful for the treatment of obesity and other metabolic diseases where neurological feeding modulates nutritional absorption. The molecules of the present invention are useful for regulating satiety, glucose uptake, and metabolism, and gastrointestinal disorders associated with neuropathy. The molecules of the present invention are also useful as additives for glucose-containing anti-hypoglycemic preparations and as adsorption improvers for oral drugs that require rapid nutrient action. Additionally, the molecules of the present invention can be used to stimulate insulin release induced with glucose. The polypeptides, nucleic acids, antagonists, agonists and / or antibodies of the present invention can be used in the treatment, diagnosis or prevention of conditions associated with gastric ulcers, bacterial diseases, emptying and gastric function, repair and secretion of mucosa, cancer Stomach, nausea (for example, induced by cancer therapy and opioid pain control) stomach acid secretion, gastritis, trauma, diverticulitis, gastric mucositis or appetite. The molecules of the present invention can be used to isolate zsig28 modulars, including the natural ligand, used in gene therapy, or to stop or prevent the development of pathological conditions in various tissues such as the stomach and lung. In addition, the zsig28 polypeptides or agonists or antagonists thereof are expected to be useful in the modulation of mucosal production, composition or integrity or in a mucus-emptying paper. Such modulation may be useful in altering the composition or integrity of the mode for in vitro studies thereof, such as to reduce the integrity of the mucus to evaluate the involvement thereof in the bacterial-mucus interaction. In addition, such modulation may be useful in the treatment of disease states characterized by inappropriate production, composition or integrity of the mucus. For example, cystic fibrosis is associated with mucus dehydration, which results in thickening of the mucus (reduction in viscosity). Other conditions, such as chronic obstructive pulmonary disease, asthma, are associated with chronic mucosal hypersecretion. See, for example, Prescott et al., Ugeskr Laeger 158 (45): 6456-60, 1996; Gordon, Ear Nose Throat J. 75: (2): 97-101, 1996; and Jeffery, Am. J. Respir. Crit. Care Med. 150 (5 Pt 2): S6-13, 1994. Also, chronic obstructive pulmonary disease and inflammatory sinonasal disease are associated with changes in the ungovernable properties or mucus thickening. See, for example, Agliati, J. Int. Med. Res. 24 (3): 302-10, 1996 and Wippold et al., Allergy Proc. 16 (4): 165-9, 1995. In addition, the structural integrity of mucus is adversely impacted in inflammatory bowel disease, possibly via increased proteolysis. See, for example, Playford et al., Amer. J. Pathol. 146 (2): 310-6, 1995. Certain forms of chronic obstructive pulmonary disease are associated with increased acidic mucus. See, for example, Jeffery, supra. The emptying of the mucus can be useful in a number of these conditions as well. The zsig28 polypeptide is expressed in the stomach. As a receiver, the zsig28 play important roles in epithelial maintenance and normal gastric functioning. Thus, the zsig28 polypeptide pharmaceutical compositions, agonists and antagonists of the present invention may be useful in the prevention or treatment of gastric mucositis. Mucositis is manifested by damage and loss of the integrity of the gastric and oral epithelium. Such damage often provides a microbial entry gate that leads to sepsis. Mucositis is often induced by chemotherapy and radiation therapy, and is often a dose limiting side effect as well as a cause of mortality in cancer patients undergoing such treatment. The zsig28 polypeptides and the agonists and antagonists of the present invention can provide protection against gastric mucositis, analogous to some growth factors and cytokines, for example, interleukin-11 (Orazi, A. et al., Lab. Invest 7_5. : 33-42 '1996). The effect of zsig28, agonists and antagonists in the prevention or treatment of gastric mucositis can be measured in animal models, for example, the Syrian hamster model or in murine models using the methods described in the technique (Sonis, ST et al., Oral Surg. Oral Med. Oral Pathol. 69: 437-443, 1990; Farrell, C.L. et al., Cancer Res. 58: 933-939, 1998; Orazi, A. et al., Supra. ). In addition, the unconscious or transgenic mouse zsig28 can provide an additional in vivo model for gastric mucositis. To verify these capabilities in the zsig28 polypeptides of the present invention, agonists, or antagonists, zsig28 polypeptides, agonists or antagonists are evaluated for integrity maintenance activity according to methods known in the art. See, for example, Zahm et al., Eur. Respir. J. 8_: 381-6, 1995, which describes methods for measuring the viscoelastic properties and surface properties of mucus as well as for evaluating mucus transport through cough and through ciliary activity. Other tests to evaluate mucus properties are known to those with ordinary skill in the art. Such assays include those for determining mucin content, water content, carbohydrate content, buffering capacity, acidity, barrier properties, the ability to absorb water and the like. In addition, the detection of zsig28 polypeptides in the serum, mucosa or tissue biopsy of a patient who is under evaluation for conditions characterized by the deposition, composition or inappropriate properties of mucus, such as cystic fibrosis, asthma, bronchitis, Inflammatory bowel disease, Crohn's disease, obstructive pulmonary disease or the like, can be employed in a diagnostic application of the present invention. Such zsig28 polypeptides can be detected using immunoassay techniques and antibodies capable of recognizing an epitope of the zsig28 polypeptide. As an illustration, the present invention contemplates methods for detecting the zsig28 polypeptide comprising: displaying a sample possibly containing the zsig28 polypeptide to an antibody bound to a solid support, wherein the antibody binds to an epitope of the zs ig28 polypeptide: wash the immobilized anti- body-polypeptide to remove unbound contaminants; exposing the immobilized antigen-polypeptide to a second antibody directed to a second epitope of a zsig28 polypeptide, wherein the second antibody is associated with a detectable label; and detect the detectable label. An increase or decrease in zsig28 polypeptide concentrations (as compared to normal concentrations thereof) in the test sample appears to be indicative of dysfunction. One skilled in the art will appreciate that other assays known in the art can be used to detect zsig28 in the test sample, for example, a simple test solution with labeled anti-zsig28 antibody. In addition, pharmaceutical compositions containing such mucosal modulating agents can be used in the treatment of conditions associated with alterations in mucosal production, composition or integrity, such as those described above. Such patients will be given an effective amount of the zsig28 polypeptide or agonist or antagonist thereof having mucosal modulating activity to achieve a therapeutic benefit, generally manifested in a change in the production, composition or integrity of the mucosa in the direction of the normal physiological state of it. Also, the zsig28 polypeptides of the present invention are found in high abundance in the digestive tissues, such as in the stomach. Thus, the expression of zsig28 polypeptides can serve as a marker for digestive function or to promote the proliferation or differentiation of digestive organs. Also, the zsig28 polypeptides or agonists or antagonists thereof can be useful in the modulation of the lubrication or barrier properties of the mucosa of the digestive organ.

The zsig28 polypeptides of the present invention or agonists or antagonists thereof can be used as antimicrobial agents to protect against the pathological action of microorganisms. Such antibacterial agents are preferably active on microorganisms associated with the mucosa, such as C. a lbi ca n s, pneumon us, hemophi l us, Hel i coba cter pyl ori, and the like. An example of a condition associated with microbes, mucus involvement in humans, is the diminution of defensive properties of the mucosa by H. pylori ori, potentially resulting in the formation of ulcers. See, for example, Beligostskii et al., Klin. Khir. 8: 3-6, 1994. These antimicrobial protective agents may be acting directly or acting indirectly. Such agents that operate via membrane association or pore formation mechanisms of direct action bind to the invading microbe. The anti-microbial agents can also act via an enzymatic mechanism, breaking the protective substances of the microbes or the cell wall / membrane thereof. Anti-microbial agents, capable of inhibiting the proliferation or action of microorganisms or of breaking the integrity of microorganisms by either the mechanism established above, are useful in methods to prevent contamination in cell cultures by microbes susceptible to that antimicrobial activity. Such techniques involve culturing the cells in the presence of an effective amount of said polypeptide fragment from region 1, 2, 3, or 4 secreted from zsig28, or an agonist or zsig28 antagonist. Assays to evaluate the efficacy of zsig28 polypeptides, agonists or antagonists thereof as anti-microbial agents are well known in the art. In addition, the detection of zsig28 polypeptides in the serum, mucosa or tissue biopsy of a patient who is subjected to an evaluation due to microbial conditions, particularly those associated with the mucosa, can be employed in a diagnostic application of the present invention. . Such zsig28 polypeptides can be detected using immunoassay techniques and antibodies capable of recognizing an epitope of the zsig28 polypeptide, as described herein. Altered levels of the zsig28 polypeptides in a test sample, such as serum, saliva, saliva, biopsy, and the like, can be monitored as an indication of digestive function, gastric ulcer or cancer or condition, when compared against a normal control. In addition, pharmaceutical compositions containing such anti-microbial agents can be used in the treatment of microbial conditions, particularly those associated with the mucosa. Such patients will be given an effective amount of the soluble zsig28 polypeptide fragment or agonist or antagonist thereof having anti-microbial activity to achieve a beneficial therapeutic effect, generally manifested in a decrease in the proliferation or function of the pathogenic microbe. Other conditions to which it may be directed in accordance with the present invention are the conditions of the eyes, nasal, oral and rectal which involve the microbial agents of the mucosa and / or pathological, collateral effects of the chemotherapy that involve the mucosa, the AIDS complications related to the mucosa or similar. The anti- microbial activity of the soluble zsig28 polypeptide fragment, agonists or antagonists can be determined using known assays therefor. See, for example, Barsum et al., Eur. Breathe J. 8 (5): 709-14, 1995; Sandovs ky-Los ica et al., J. Med. Vet. Mycol (England) 28 (4): 279-87, 1990; Mehentee et al. , J. Gen. Microbiol (England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage, Journal of Medical and Veterinary Mycology 24: 477-479, 1986, and the like. Also, the zsig28 polypeptides of the present invention can be a component of a known tissue bond, which imparts additional anti-microbial and / or adhesive properties to it. In such applications, the purified zsig28 polypeptide could be used in combination with the collagen or in a form of gelatin, muscle adhesion protein, fibrinogen, thrombin, Factor XIII or the like. The different types of glue as well as the composition thereof are known in the art. The zsig28 can also be used to identify modulators (eg, antagonists) of its activity. The test compounds are added to the assays described herein to identify compounds that inhibit the activity of zsig28. In addition to those assays described herein, the samples can be tested for the inhibition of zsig28 activity within a variety of assays designed to measure binding or agglutination, oligomerization, or zsig28 stimulation / inhibition of cell-dependent cellular responses. zsig28 For example, cell lines expressing zsig28 can be transfected with a reporter gene construct that is responsive to a cell pathway stimulated with zsig28. Reporter gene constructs of this type are known in the art, and generally comprise a zsig28 DNA response element operably linked to a gene encoding the protein detectable in the assay, such as luciferase. DNA response elements may include, but are not limited to, AMP cyclic response elements (CRE), hormone response elements (HRE) insulin response elements (IRÉ) (Nasrin et al., Proc. Nati, Acad. Sci. USA 87: 5273-7, 1990) and serum response elements (SRE) (Shaw et al., Cell 56: 563-72, 1989). The AMP cyclic response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063-6; 1988 and Habener, Molec. Endocrinol 4 (8): 1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56: 335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit zsig28 activity in target cells as evidenced by a decrease in zsig28 stimulation of reporter gene expression. Tests of this type will detect compounds that directly block the binding of zsig28 to the cell's surface receptors, for example, through dimerization, as well as compounds that block processes in the cell pathway subsequent to such binding. As such, a method is provided for identifying the zsig28 polypeptide antagonists, which comprises providing the cells responsive to a zsig28 polypeptide, culturing a first portion of the cells in the presence of a zsig28 polypeptide, culturing a second portion of the cells in the presence of the zsig28 polypeptide and a test compound, and detect a decrease in a cellular response of the second portion of the cells when compared to the first portion of the cells. In addition, compounds or other samples compared to direct blocking of zsig28, or blocking of zsig28 that binds to other cell surface molecules, which use zsig28 labeled with a detectable label (eg, 125I, biotin, horseradish peroxidase, FITC , and similar). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zsig28 to another protein may be indicative of inhibitory activity, which can be confirmed through secondary assays. Antagonists are therefore useful for inhibiting or decreasing the function of the zsig28 polypeptide. Alternatively, there is provided a method for identifying zsig28 polypeptide agonists, which comprises providing cells expressing a zsig28 polypeptide as described above, culturing the cells in the presence of a test compound and comparing the response with the cultured cell in the presence of the zsig28 polypeptide, and selecting the test compounds for which the cellular response is of the same type. Agonists are therefore useful for mimicking a zsig28 ligand and / or increasing the function of the zsig28 polypeptides.

In view of the tissue distribution observed for zsig28, agonists (including natural ligand / substrate / cofactor / etc.) and antagonists have enormous potential in both applications in vi tro and in vi vo. Compounds identified as zsig28 agonists are used for the promotion of apoptosis in cells that over-express sig28, in vi tro and in vi vo, such as tumor cells. The compounds identified as agonists. zsíg28 are also employed for and that stimulate cell growth or differentiation, of various cell types. For example, the zsig28 agonist compounds are used as components of the defined cell culture medium, and many can be used alone or in combination with other cytokines and hormones to replace the serum that is commonly used in cell culture. Also, the zsig28 polypeptide can be hydrolyzed to provide a source of amino acids to the cultured cells. However, zsig28 polypeptides and zsig28 agonist polypeptides are employed as a research reagent, such as for the expansion of stomach derivatives, or intestinal cells. The zsig28 agonists can be added to the tissue culture medium for cell types that express the zsig28 polypeptide. Inhibitors of zsig28 activity (zsig28 antagonists), include anti-zsig28 antibodies and polypeptide linkage fragments, as well as other peptide and non-peptide agents (including ribozymes). The zsig28 can also be used to identify inhibitors (antagonists) of its activity. The test compounds are added to the assays described herein to identify compounds that inhibit the activity of zsig28. In addition to those assays described herein, samples can be tested for inhibition of zsig28 activity within a variety of assays designed to measure receptor binding or stimulation / inhibition of zsig28-dependent cellular responses. For example, the cell lines responsible for zsig28 can be transfected with a reporter gene construct that is responsible for a cell path stimulated by zsig28. Reporter gene constructs of this type are known in the art, and generally, they will comprise a DNA-zsig28 response element operably linked to a gene encoding a testable protein, such as luciferase. The DNA response elements may include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE), insulin response element (IRÉ) (Nasrin et al., Proc. Nati. Acad. Sci. USA B l: 5213-1, 1990) and serum response elements (SRE) (Shaw et al., Cell 56: 563-72, 1989). The cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063-6; 1988 and Habener, Molec. Endocrinol 4_ (8): 1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56: 355-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit zsig28 polypeptide activity in target cells as evidenced by a decrease in zsig28 stimulation of reporter gene expression. Tests of this type will detect compounds that directly block the zsig28 ligands from the binding to the zsig28 polypeptide receptors, or the mutation of the receptor, as well as the compounds that block the processes in the cell path subsequent to the binding of the ligand. -receiver. The molecules of the present invention can be used to identify and isolate zsig28 receptors involved or present in cancer metastasis. Thus, the zsig28 polypeptide can serve as a diagnostic for cancer metastasis. For example, the proteins and peptides of the present invention can be immobilized on a column and the membrane preparations run on the column (Immobilized Affinity Ligand Techniques, Hermanson et al., Eds., Academic Press, San Diego, CA, 1992, pp.195-202). (Methods in Enzymol., Vol.182, "Guide to Protein Purification," M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or labeled by photoaffinity (Brunner et al., Ann. Biochem 62: 483-514, 1993 and Fedan et al., Biochem Pharmacol 33: 1167-1180, 1984) and the zsig28 cell surface proteins, can be identified. However, using methods known in the art, antibodies to zsig28 can also be radiolabelled, fluorescently or chemically labeled and used in histological assays to detect elevated zsig28 present in biopsies. The polypeptides of the present invention are employed for the measurement of changes in the expression levels of zsig28 polypeptides. Because the zsig28 expression is restricted to specific tissues (ie, stomach and lung), changes in expression levels could be used to monitor the metabolism within these tissues. For example, the increase in expression and / or transcription of zsig28 polypeptides and polynucleotides may be predictive of increased cell proliferation of tumor cells. In addition, the expression of zsig28 in the tissue that does not normally express zsig28 may be indicative of the metastasis of the tumor cells. It has been shown that zsig28 is differentially expressed in certain epithelial tissues and carcinomas, particularly in the stomach, colon, esophagus, or intestine. The differential expression is the temporal expression or lack thereof, of specific genes, proteins or other phenotypic properties (known as differentiation markers) that occur during the progress of the mutation in a cell or tissue. A series of differentiation markers is defined as one or more phenotypic properties that can be identified and are specific to a particular cell type. Thus, pluripotent repression cells that can regenerate without being concomitant to a lineage express a series of differentiation markers that are lost when they are concomitant to a cell lineage. The precursor cells express a series of differentiation markers that may or may not continue to be expressed as the cells progress down the path of the cell line towards maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cellular products, enzymes to produce cellular products and receptors. The expression Zsig28 can be used as a marker of differentiation in tumor and normal tissues to determine the stage of the tumor or maturity of a cell. Zsig28 will be particularly valuable as a marker for epithelial and tumor cells of epithelial origin, and more particularly, epithelial cells and epithelial derived tumors from stomach tissues. A series of differentiation markers is defined as one or more phenotypic properties that can be identified and are specific to a particular cell type. Differentiation markers are temporarily displayed at various stages of the cell lineage. Pluripotent stem cells that can be regenerated without being concomitant to a lineage, express a series of differentiation markers that are lost when they are concomitant to a cell lineage. The precursor cells express a series of differentiation markers that may or may not continue to be expressed as the cell progresses down the path of the cell lineage towards maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cellular products, enzymes to produce cellular products and receptors. The activity of the molecules of the present invention can be measured using a variety of assays that measure the proliferation and / or differentiation of specific cell types, chemotaxis, adhesion, changes in the influx of the ion channel, regulation of the second messenger levels and release of the neurotransmitter. Such assays are well known in the art and are described herein. Additional methods using probes or primer derivatives, for example, of the nucleotide sequences described herein, can also be used to detect zsig28 expression in a patient sample, such as a tumor, stomach, lung, blood, biopsy. saliva, tissue sample or similar. For example, the probes can be hybridized to tumor tissues and the hybridized complex detected by in s i t u hybridization. The zsig28 sequences can also be detected by PCR amplification using cDNA generated by reverse translating the mRNA sample as a standard (PCR Primer A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Press, 1995). When compared to a normal control, both the increase and decrease of the zsig28 expression in a patient sample, relative to that of the control, can be monitored and used as an indicator or diagnosis for the condition. Polynucleotides encoding the zsig28 polypeptide are employed within gene therapy applications, where they are desired to increase or inhibit zsig28 activity. If a mammal has a mutated or absent zsig28 gene, the zsig28 gene can be introduced into the mammalian cells. However, using gene therapy applications, zsig28 can also be used directly as a chemotherapeutic agent. For example, using methods described herein, zsgi28 can be directly introduced into cancer cells to cause apoptosis and cell death. In one embodiment, a gene encoding a zsig28 polypeptide is introduced into a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, Herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses are preferred, which completely or almost completely lack viral genes. A defective virus is not infective 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 particular vectors include, but are not limited to, a vector of defective herpes simplex virus 1 (HSV1) (Kaplitt et al., Molec. Cell. Neurosci. 2_: 320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin.

Invest. 9_0_: 626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol., 6 ^: 3096-101, 1987, Samulski et al., J. Virol. 63: 3822-8, 1989). In another embodiment, a zsig28 gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 3_3: 153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995, by Dougherty et al .; and Kuo et al., Blood 8_2: 845, 1992. Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for the transfection of a gene encoding a marker (Felgner et al., Proc. Nati, Acad. Sci. USA 8 .7413-7, 1987; Mackey et al. ., Proc. Nati, Acad. Sci. USA 85: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs has no partial advantage. The molecular objectives of liposomes to specific cells represent an area of benefit. More particularly, the direction of transfection to particular cells represents an area of benefit. For example, the direction of transfection to particular cell types could be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids can be chemically coupled to other molecules for objective purposes. Target peptides (e.g. hormones or neutrot ransmitters), proteins such as antibodies, or non-peptide molecules, can be chemically coupled to the liposomes. It is possible to remove the target cells from the body; introduce the vector as a pure DNA plasmid; and then reimplant the transformed cells in the body. Pure DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of an injector gene or the use of a DNA vector transporter. See, for example, Wu et al., J. Biol. Chem. 267: 963-7, 1992; Wu et al.,. Biol. Chem. 263: 14621-4, 1988. The antisense methodology can be used to inhibit the transcription of the zsig28 gene, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a zsig28-encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ ID NO: 1), are designed to bind to an mRNA encoding zsig28 and inhibit translation of zsig28. MRNA. Such antisense polynucleotides are used to inhibit the expression of genes encoding the zsig28 polypeptide in cell cultures or in a subject. In addition, as a cell surface molecule, the zsig28 polypeptide can be used as a target to introduce a gene therapy into a cell. This application could be particularly appropriate for the introduction of therapeutic genes into cells in which zsig28 is normally expressed, such as stomach and lung tissue, or cancer cells which express the zsig28 polypeptide. For example, viral gene therapy, as described above, can be targeted to specific cell types in which a cellular receptor, such as the zsig28 polypeptide, is expressed in place of the viral receptor. Antibodies, or other molecules that recognize zsig28 molecules on the surface of target cells, can be used to target the virus to the therapeutic material of the gene administered and infected to that target cell. See, Woo, S. L. C, Nature Biotech. l4_: 1538, 1996; Wickman, T. J. et al., Nature Biotech. 14: 1570-1573, 1996; Douglas, J.T. et al., Nature Biotech. 1: 1574-1578, 1996; Rihova, B., Crit. Rev. Biotechnol. 1_7_: 149- 169, 1997; and Vile, R. G. et al., Mol. Med. Today 4_: 84-92, 1998. For example, a bispecific antibody containing a virus neutralizing Fab fragment, coupled to an antibody specific to zsig28, can be used to target the virus to cells expressing the zsig28 receptor and they allow the efficient entry of the virus that contains a genetic element in the cells. See, for example, Wickham, T. J., et al., J. Virol. 71: 7663-7669, 1997; and Wickham, T. J., et al., J. Virol. 70: 6831-6838, 1996. The present invention also provides reagents which will find use in diagnostic applications. For example, the zsig28 gene, a probe comprising the zsig28 DNA or RNA or a subsequence thereof, can be used to determine if the zsig28 gene is present on chromosome 3 or if a mutation has occurred. The zsig28 is located in the 3q22 region. l-3q22.2 of chromosome 3 (see Example 3). Chromosomal aberrations detectable at the site or locus of the zsig28 gene, include but are not limited to, aneuplidia, changes in gene copy number, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention by the use of molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short double repeat (STR) analysis, using PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid., Ausubel et al., ibid., Marian, Chest 108: 255-65, 1995). Accurate knowledge of a gene position can be used for a number of purposes, including: 1) determining whether a sequence is part of an existing contiguous contig and obtaining additional surrounding genetic sequences in various forms, such as YACs clones , BACs, or cDNA; 2) provide a possible candidate gene for a heritable condition, which shows a link to the same chromosomal region; and 3) cross-referenced model organisms, such as mice, which can help in determining what function a particular gene should have. The zsig28 gene is located in the 3q22 region. l-3q22.2 of chromosome 3. Several genes of known function map or correlate this region. For example, the angiotensin 1 receptor is mapped or correlated to 3q21-q25 and is related to hypertension in humans, since it controls blood pressure. In addition, the zsig28 polynucleotide probes can be used to detect abnormalities or genotypes associated with the tumor-associated L6 antigen, which maps or correlates to 3q21-q25, and is highly expressed in cancers of the lung, breast and colon (Marken et al, Proc. Nati, Acad. Sci. 3_9: 3503-3507, 1992). However, among other loci or genetic sites, those for alcaptoneuria (3q21-q23), Moebius syndrome 2 (3q21-q22), open-angle glaucoma (3q21-q24), calcium-sensitive receptor (3q21-q24), all they manifest themselves in states of human suffering, as well as map this region of the human genome. See the map of the Online Mendellian Inheritance of Man (OMIM) gene, and references here, for this region of chromosome 3 on a publicly available WWW server (http: // www3. Ncbi. Nlm. Nih. Gov / htbin-post / Omim /getmap?chromosome=3q22.1). All these serve as possible candidate genes for a heritable condition, which show the link to the same chromosomal region as the zsig28 gene. However, a gene related to zsig28, the human transmembrane protein suppressed in the velo-cardio-facial syndrome (TMVCF) (Sirotkin H. et al., Genomics 4_2_: 245- ^ 251, 1997) is a marker for a autosomal dominant human alteration of velo-cardio-facial syndrome (VCFS), characterized among other symptoms by facial deformation, mental retardation, and cardiac defects, and are suppressed in 80-85% of patients with this syndrome. However, mutations in other genes related to zsgi28, peripheral myelin protein-22 (PMP-22), are present in human Charcot-Marie-Tooth neuropathy (CTM), with a phenotypically similar neuropathy in the model. of transgenic murine mouse PMP-22 (Erdem, S. et al., J. Neuropathol., Exp. Neurol. 57: 635-642, 1998; Fabbretti, E. et al., Genes Dev. 15: 1846-1856, 1995; Magyar, J.P. et al., Neurosci. 16: 5351-5360, 1996; Adlkofer, K. et al., Nat. Genet. _1_1: 274-280, 1995). Similarly, defects at the zsig28 site or locus itself can result in a heritable human condition. The molecules of the present invention, such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention, could aid in the detection, diagnosis, prevention and treatment associated with a genetic defect of zsig28. Mice designed to express the zsig28 gene, referred to as a "transgenic mouse", and mice that exhibit a complete absence of zsig28 gene function, referred to as an "unconscious mouse", can also be generated (Snouwaert et al., Science 257: 1083, 1992; Lowell et al., Nature 366: 740-42, 1993; Capecchi, MR, Science 244: 1288-1292, 1989; Palmiter, RD et al., Annu Rev Genet., 20: 465-499, 1986). . For example, transgenic mice that over-express zsig28, either ubiquitously or under a specific tissue or restricted tissue promoter, can be used to ask whether over-expression causes a phenotype. For example, overexpression of a wild-type zsig28 polypeptide, polypeptide fragment or mutant thereof, can alter normal cellular processes, resulting in a phenotype that identifies a tissue, in which the expression zsig28 is functionally relevant and can indicate a therapeutic goal for zisg28, its agonist or antagonist. For example, a preferred transgenic mouse to be designed is one that over-expresses the mature polypeptide zsig28 (approximately amino acids 24 (Ala) to 261 (Val) of SEQ ID NO: 2). However, such over-expression may result in a phenotype that shows similarity to human conditions. Similarly, unconscious zsig28 mice can be used to determine if zsig28 is absolutely required in vi. The phenotype of the unconscious mouse is predictive of the effects vi n such that a zsig28 antagonist may have, such as those described herein. The human zsig28 cDNA can be used to isolate the murine zsig28 mRNA, cDNA and genomic DNA, which are subsequently used to generate unconscious mice. These unconscious mice can be used to study the zsig28 gene and the therapy encoded by the protein in an in vi ve system, and can be used in in vi vo models to correspond to human diseases. However, the expression of transgenic mice of the zsig28 antisense polynucleotides or ribosomes directed against zsig28, described herein, can be used analogously for the transgenic mice described above. The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1 Identification of Zsig28 A. Using an EST Sequence to Obtain Full-length Zsig28 Scanning of a DNA database of the translated lung library using a signal trap as an interrogation results in the identification of a sequence of label (EST) of the expressed sequence found to be homologous to a human secretory sequence signal. Confirmation of the EST sequence was made by sequence analysis of the cDNA from which EST originates. This cDNA was contained in a plasmid, and sequenced using the following primers: ZC447 (SEQ ID NO: 11), ZC12501 (SEQ ID NO: 12), ZC 12502 (SEQ ID NO: 13), and ZC 976 (SEQ. ID. NO: 14) The clone appears to be full length.

Example 2 Tissue Distribution Northern blot analyzes are performed using Northern ™ Multi-Human Tissue Stains (MTN I, MTN II, and MTN III) (Clontech). An insert of the full length clone described in Example 1 is removed using EcoRI and Notl (Boehringer) and gel purified using a commercially available set of elements (QiaexII ™; Qiagen) and then radiolabelled with 32P-dCTP using Rediprime ™ ( Amersham), a random priming labeling system, according to the manufacturer's specifications. The probe was then purified using a Nuc-Trap ™ column (Stratagene) according to the manufacturer's instructions. The ExpressHyb ™ solution (Clontech) was used for prehybridization and as a hybridization solution for Northern blots. Hybridization was carried out overnight at 42 ° C using 3 x 106 cpm / ml labeled probe. Then the spots were washed in 2X SSC / 1% SDS at room temperature, followed by a wash at 0. IX SSC / 0.1% SDS at 65 ° C. A full transcript of approximately 4 kb was detected strongly in the stomach and was detected weakly in the lung. There were no obvious signs in other tissues represented in the spots. Spot spots were also made using Human RNA Master Blots ™ (Clontech). The methods and conditions for Spot Spots are the same as for the Multi-Tissue Spots described above. The intensity of the strong signal was present in the stomach, with detectable but low expression in the adult or fetal lung.

Example 3 Preparation of PCR-based Chromosome Maps of the zsig28 Gene Zsig28 was mapped to chromosome 3 using the commercially available "GeneBridge 4 Hybrid Radiation Panel" (Research Genetics, Inc., Huntsville, AL). The Hybrid Radiation Panel GeneBridge 4 contains DNAs from each of the hybrid clones by radiation 93, plus two control DNAs (the HFL donor and the A23 receptor). A publicly available WWW server (ht tp: / / www-genome, wi.mult.edu/cgi-bin / contig / rhmapper.pl) allows the creation or creation of maps related to the hybrid hybrid map of Whitehead Institute / MIT Center for Genome Research of the human genome (the hybrid radiation map "WICGR") which was constructed with the Hybrid Radiation Panel 4 GeneBridge. For the mapping of Zsig 28 with the "RH 4 GeneBridge Panel", 20 μl reactions were placed in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycle former (Stratagene). Each of the 95 PCR reactions consists of a PCR reaction buffer of 2 μl 10X KlenTag (Clontech Laboratories, Inc., Palo Alto, CA), mixture of 1.6 μl dNTPs (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl of sense primer, ZC19,410 (SEQ ID NO: 15), 1 μl of antisense primer, ZC19,411 (SEQ ID NO: 16 =, 2 μl of "RediLoad" (Research Genetics) , 0.4 μl 50X Advantage KlenTaq ™ Polymerase Mix (Clontech), 25 ng DNA from a single hybrid clone or control and ddH20 for a total volume of 20 μl The reactions were coated with an equal amount of mineral oil and sealed. The conditions of the PCR cycle forming device were as follows: a denaturation of 5 minutes from 1 initial cycle at 95 ° C, 35 cycles of a 1 minute denaturation at 95 ° C, annealing for 1 minute at 60 ° C and 1.5 minutes extension at 72 ° C, followed by a final extension of 1 cycle of 7 minutes at 72 ° C. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD). The results show that the maps of Zsig28 4.40 cR_3000 from the marker of the structure D3S1576 on the hybrid radiation map of chromosome 3 WICGR. The near and far structure markers were D3S1576 and WI-3522, respectively. The use of the positions of markers around Zsig28 in the 3q22.1-3q22.2 region on the integrated LDB chromosome 3 map (The Genetic Location Database, University of Southhampton, server WWW: http: // cedar.genetics.soton ac. uk. / public_html /). 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 can be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims. 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 (20)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An isolated polynucleotide that encodes a polypeptide comprising an amino acid residue sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 24 (Ala), to amino acid number 261 (Val), and (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 261 (Val), characterized in that the percentage of amino acid identity is determined using a FASTA program with kTup = l, missing by interval opening = 10, lack by extension of interval = 1, and substitution matrix = BLOSUM62, with other parameters adjusted by default.

2. An isolated polynucleotide according to claim 1, characterized in that the polynucleotide is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 139 to nucleotide 853; and (b) a polynucleotide sequence as shown in SEQ ID NO: 1 from a nucleotide 70 to nucleotide 853.

3. A sequence of isolated polynucleotides according to claim 1, characterized in that the polynucleotide comprises from nucleotide 1 to nucleotide 783 of SEQ ID NO: 10- -

4. An isolated polynucleotide according to claim 1, characterized in that the polynucleotide encodes a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 24 (Ala), to amino acid number 261 (Val); and (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 261 (Val).

5. An expression vector characterized in that it comprises the following operably linked elements: a transcription promoter; a DNA segment encoding a zsig28 polypeptide as shown in SEQ ID NO: 2 from amino acid number 24 (Ala), to amino acid number 261 (Val); and a transcriptional terminator, wherein the promoter is operably linked to the DNA segment, and the DNA segment is operably linked to the transcriptional terminator.

6. An expression vector according to claim 5, characterized in that it also comprises a secretory signal sequence operably linked to the DNA segment.

7. A cultured cell comprising an expression vector according to claim 5, characterized in that the cell expresses a polypeptide encoded by the DNA segment.

8. A DNA construct or construct that encodes a fusion protein, the DNA construct characterized in that it consists of: (a) the amino acid sequence of SEQ ID NO: 2 of amino acid number 1 (Met), to amino acid number 23 (Ala); (b) the amino acid sequence of SEQ ID NO: 2 of amino acid number 24 (Ala) to amino acid number 82 (Leu); (c) the amino acid sequence of SEQ ID NO: 2 of amino acid number 101 (Leu) to amino acid number 122 (Gly); (d) the amino acid sequence of SEQ ID NO: 2 of amino acid number 141 (Asn) to amino acid number 174 (Ala); (e) the amino acid sequence of SEQ ID NO: 2 of amino acid number 193 (Cys), to amino acid number 261 (Val); and (f) the amino acid sequence of SEQ ID NO: 2 of amino acid number 24 (Ala), to amino acid number 261 (Val); and at least one other DNA segment encoding an additional polypeptide, wherein the first segment and other DNA segments are connected in the structure; and wherein the first and other DNA segments encode the fusion protein.

9. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA construct encoding a fusion protein according to claim 8; and a transcriptional terminator, characterized in that the promoter is operably linked to the DNA construct, and the DNA construct is operably linked to the transcriptional terminator.

10. A cultured cell comprising an expression vector according to claim 9, characterized in that the cell expresses a polypeptide encoded by the DNA construct.

11. A method of producing a fusion protein characterized in that it comprises: culturing a cell according to claim 10; and isolating the polypeptide produced by the cell.

12. An isolated polypeptide characterized in that it comprises a sequence of amino acid residues that is at least 90% identical to a sequence of amino acids selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 a starting from amino acid number 24 (Ala), to amino acid number 261 (Val); and (b) the amino acid sequence as shown in SEQ ID NO: 2 of amino acid number 1 (Met) to amino acid number 261 (Val), wherein the percentage of amino acid identity is determined using a FASTA program with ktup = l , missing by interval opening = 10, missing by interval extension = 1, and substi tution matrix = BLOSUM62, with other parameters adjusted by default.

13. An isolated polypeptide according to claim 12, characterized in that the polypeptide also contains portions of 1 to 4 spaced in a remote form from N-terminal to C-terminal in a configuration selected from the group consisting of: (a) Met- { 47-50} -Ml-. { 21-22} -M2-. { 73-92} -M3; Y (b) Met-. { 47-50} -Ml-. { 21-22} -M2-. { 73-92} - M3-. { 3} -M4, where M1 is "portion 1" an amino acid sequence as shown in amino acids 48 to 54 of SEQ ID NO: 2, M2 is "portion 2", an amino acid sequence as shown in amino acids 77 to 82 of SEQ ID NO: 2, M3 is "portion 3", an amino acid sequence as shown in amino acids 174 to 180 of SEQ ID NO: 2, M4 is "portion 4", an amino acid sequence as shown in amino acids 184 to 189 of SEQ ID NO: 2, and. { #} denotes the number of amino acids between the portions.

14. An isolated polypeptide according to claim 12, characterized in that the polypeptide comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 2 from the amino acid number 24 (Ala), to amino acid number 261 (Val); and (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met) to amino acid number 261 (Val).

15. A method for producing a zsig28 polypeptide characterized in that it comprises: culturing a cell according to claim 7; and isolating the zsig28 polypeptide produced by the cell.

16. A method for producing an antibody to zsig28 polypeptide characterized in that it comprises: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 39 to 238 amino acids, wherein the polypeptide is identical to a sequence contiguous amino acid in SEQ ID NO: 2 from amino acid number 24 (Ala), to amino acid number 261 (Val); (b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 24 (Ala) to amino acid number 82 (Leu); (c) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 101 (Leu) to amino acid number 122 (Gly); (d) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 141 (Asn) to amino acid number 174 (Ala); (e) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 193 (Cys) to amino acid number 261 (Val); (f) a polypeptide according to claim 12; (g) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 245 (Ala) to amino acid number 250 (Glu); (h) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 234 (Asn) to amino acid number 239 (Lys); (i) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 202 (Glu) to amino acid number 207 (Lys); (j) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 254 (Lys) to amino acid number 259 (Asp); and (k) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 110 (Glu) to amino acid number 115 (Ala); and wherein the polypeptide obtains an immune response in the animal to produce the antibody; and isolating the antibody from the animal.

17. An antibody produced by the method according to claim 16, characterized in that it binds to a zsig28 polypeptide.

18. The antibody according to claim 17, characterized in that the antibody is a monoclonal antibody.

19. An antibody characterized in that it binds specifically to a polypeptide according to claim 12.

20. A method for detecting, in a test sample, the presence of a zsig28 protein activity modulator, characterized in that it comprises: culturing a cell in which an expression vector has been introduced according to claim 5, wherein the cell expresses the zsig28 protein encoded by the DNA segment in the presence and absence of a test sample; and comparing zsig28 activity levels in the presence and absence of a test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the modulator of zsig28 activity in the test sample.