MXPA00008094A - Connective tissue growth factor homologs - Google Patents

Abstract

This present invention is directed to polypeptide and polynucleotide molecules that encode a connective tissue growth factor homolog polypeptide. The human polypeptides have been designated zCTGF4 and the mouse orthologs have been designated zCTGF2. The invention also includes antibodies, expression vectors, host cells expressing zCTGF4 and variants. Also included are methods for producing the polypeptides and using the polynucleotides and the polypeptides.

Description

HOMOLOGOUS OF FACTOR OF GROWTH OF CONNECTIVE TISSUE BACKGROUND OF THE INVENTION Connective tissue growth factor (CTGF) is a growth factor that is expressed by endothelial and fibroblastic cells. Two members of the CTGF family, CTGF, are known (U.S. Patent No. 5,585,270 and U.S. Patent No. 5,408.0.0) and CTGF-2 (WO 96/01896), incorporated herein by reference. CTGF belongs to the family of growth factors that include CTGF, CTGF-2, insulin binding proteins (IBP) 1 and 2. These growth factors have a cysteine-rich motif and several different structural protein domains in common, and they have been shown to have a role in cell proliferation, differentiation and chemotaxis. Studies have suggested that CTGF is immunologically similar to PDGF, with antibodies to PDGF from the A and B chains that bind to CTGF (Bradham et al., J. of Cell Biol. 11. (6): 1285-129 , 1991), and it has been reported that the biological activity of CTGF can be blocked using these antibodies. It has recently been shown that porcine CTGF isolated from the uterus is mitogenic for fibroblasts and smooth muscle cells, but not for endothelial cells. In addition, a REF: 122 93 l _-_ lril? l -_-- l truncated form in the N-terminal part of the protein is also active (Brigstock et al., J. Biol. Chem. 272: 2Q275-Q282, 1997). It has been suggested that this protein may play a role in the growth and remodeling of the endometrium and the placenta. The CTGF family is considered to play a role in the production of extracellular matrix components, such as collagen and fibronectin. Collagen and fibronectin are components of many connective tissues, for example ligaments, cartilages, tendons and vessel walls. Current therapy for ligament repair is limited to immobilizing damaged tissue, that is, stapling or replacing damaged tissue with synthetic or natural inserts. These tissues are notoriously difficult to heal, even in healthy individuals, and compositions that can improve recovery time would be very useful. The present invention is directed to novel polypeptides and polynucleotides that encode the polypeptide that predominantly shows high expression in testes, trachea and bone marrow, thereby providing a novel molecule to regulate growth, differentiation, chemotaxis and induction of cellular functions specialized in these tissues.

BRIEF DESCRIPTION OF THE INVENTION An objective of the present invention is to provide an isolated polynucleotide molecule comprising a polynucleotide sequence encoding a connective tissue growth factor homologous polypeptide that is at least 70% identical to the amino acid sequence as shown in SEQ. . FROM IDENT. NO: 2 from residue 2 to residue 354. In another embodiment, the present invention provides a polynucleotide molecule wherein the polynucleotide molecule comprises a region having the following motif, as shown in SEQ. FROM IDENT. NO: 23: Cx. { 8, 10) CxCCxxCx. { 7] Cx. { 5, 6 } Cx (5, 7) Cx. { 12, 13 } Cx [7, 8} Cx. { 20) CxCx. { 6.} Cx. { l2, l_} Cx. { 13.17} C where x. { } is the number of amino acid residues between cysteines (C). In another embodiment, the polynucleotide is 80% or 90% identical to the amino acid sequence as shown in SEQ. FROM IDENT. NO: 2 from residue 2 to residue 35. The aim of the present invention is also to provide an isolated polynucleotide acid molecule encoding a polypeptide homologue of connective tissue growth factor, wherein the polynucleotide molecule is selected from the group consisting of consists of (a) . ",., _t ____ 2M-- ~ - ~ - - a molecule that has the nucleotide sequence of the SEC. FROM IDENT. N0: 1, from nucleotide 17 or 86 to nucleotide 1078, (b) a molecule encoding the amino acid sequence of SEQ. FROM IDENT. NO: 3, from nucleotide 1 or 70 to nucleotide 1062, and (c) a molecule that hybridizes under stringent washing conditions to a polynucleotide molecule having nucleotide sequence 86 to 1078 of SEQ. FROM IDENT. NO: 1, or the complement of nucleotides 85 to 1078 of the SEC. FROM IDENT. NO: l. In another embodiment, the differences in the amino acid sequence encoded by the polynucleotide and SEQ. FROM IDENT. NO: 2 are conservative amino acid changes. In other aspects, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment comprising the isolated polynucleotide sequence encoding a homologous polypeptide of connective tissue growth factor that is at least 70% identical to the amino acid sequence as shown in SEQ. FROM IDENT. NO: 2 from residue 2 to residue 35; and a transcription terminator and a cultured host cell into which the expression vector has been introduced. In another embodiment, the present invention provides a method for producing a homologous connective tissue growth factor polypeptide comprising: (a) culturing the host cells expressing the homologous CGTF polypeptide; and (b) isolating the homologous polypeptide from the connective tissue growth factor of the cultured host cells. In another aspect, the present invention provides an isolated connective tissue growth factor polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence as shown in SEQ. FROM IDENT. NO: 2 from residue 24 to residue 35_. In other embodiments, the present invention provides homologous CTGF polypeptides wherein the amino acid sequence is at least 80% or 90% identical.

In another embodiment, the homologous polypeptide molecule of CTGF comprises a region having the following motif, as shown in SEQ. FROM IDENT. NO: 23: Cx. { 8, 10) CxCCxxCx. { 7] Cx. { 5, 6 } Cx (5, 7) Cx. { 12, 13 } Cx [7, 8} Cx. { twenty ) CxCx. { 6.} Cx. { 12.24} Cx. { 13.17} C where x. { } is the number of amino acid residues between cysteines (C). In another aspect, the present invention provides an antibody or antibody fragment that specifically binds to the homologous CTGF polypeptide. In another aspect, the present invention provides a method for detecting the presence of homologous polypeptide of connective tissue growth factor in a sample biological, comprising the steps of: (a) contacting the biological sample with an antibody or an antibody fragment of claim 14, wherein the contact is made under conditions that allow the binding of the antibody or the antibody fragment to the biological sample; and (b) detecting any bound antibody or bound antibody fragment. In another aspect, the present invention provides an anti-idiotypic antibody or an anti-idiotypic antibody fragment that specifically binds to the antibody or antibody fragment. In another aspect, the present invention provides a method for detecting an abnormality on chromosome 6q in a sample of an individual comprising: (a) obtaining zCTGF4 RNA from the sample; (b) generating zCTGF4 cDNA by polymerase chain reaction; and (c) comparing the nucleic acid sequence of the ZCTGF4 cDNA with the nucleic acid sequence as shown in SEQ. FROM IDENT. N0: 1 In another embodiment, the present invention provides indicators that the difference between the sequence of the ZCTGF4 cDNA or the zCGTF gene in the sample and the zCTGF sequence. as shown in the SEC. FROM IDENT. NO: 1 is indicative of an abnormality on chromosome 6q.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 a and 1 b illustrate a multiple alignment of human zCTGF4 (SEQ ID NO: 2) and other members of the family of connective tissue growth factor-instructing mouse zCTGF2 (FIG. SEQ ID NO: 5), human NOV (NOV HU; I DENT SEQ NO: 25), human CTGF 1 (CTGF H; ID SEQ ID NO: 26), insulin binding protein human 1 (IBP 1 H; S EC DE I DENT NO: 27) and human insulin protein 2 (IC P2 H; SEQ ID NO: 28).

DETAILED DESCRIPTION OF THE INVENTION Before establishing the invention in detail, it may be useful for the compression thereof to define the following terms: The term "affinity tag" is used herein to indicate a polypeptide segment that can be attached to a second polypeptide to provide purification or detection of the second polypeptide or to provide sites for binding the second polypeptide to a substrate. Primarily, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a polyhistidine tract, protein A (Nilsson et al., EMBO J. 4: 1075, 1985; Nilsson et al., Methods Enzvmol 198: 3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu affinity tag. -Glu (Grussenmeyer et al., Proc. Nati. Acad. Sci. USA 82: 7592-, 1985), substance P, Flag ™ peptide (Hopp et al., Biotechnoloay .6: 1204-10, 1988), streptavidin binding peptide or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ). The term "allelic variant" is used herein to indicate any of two or more alternative forms of a gene that occupy the same chromosomal locus. Allelic variation arises naturally by mutation, and can result in phenotypic polymorphism within populations. Mutations of genes can be silent (there is no change in the encoded polypeptide) or they can code for polypeptides having an altered amino acid sequence. The term allelic variant is also used herein to indicate a protein encoded by an allelic variant of a gene.

The terms "amino terminal" and "carboxyl terminal" are used herein to indicate positions within polypeptides. When the context permits, these terms are used with reference to a particular sequence or position of a polypeptide to indicate proximity or relative position. For example, a certain sequence placed carboxyl terminal to a reference sequence within a polypeptide is located close to the carboxyl terminal portion of the reference sequence, but not necessarily at the carboxyl terminus of the complete polypeptide. The term "complement / anticomplement pair" denotes non-identical portions that form a stable pair, not covalently associated under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypical members of a complement / anticomplement pair. Other exemplary complement / anti-complement pairs include receptor / ligand, antibody / antigen (or hapten or epitope) pairs, direct / antisense polynucleotide pairs and the like. When the subsequent dissociation of the complement / anticomplement pair is desirable, preferably the complement / anticomplement pair has a binding affinity of < 109 M "1. The term" complements of a polynucleotide molecule "indicates a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a sequence of references, For example, the 5 'sequence ATGCACGGG 3- is complementary to 5. 'CCCGTGCAT 3'.

The term "contiguous" indicates a polynucleotide having a contiguous chain of a sequence identical or complementary to another polynucleotide. The contiguous sequences are said to "overlap" in a given polynucleotide sequence chain either in its entirety or along a partial polynucleotide chain. The term "nucleotide degenerate sequence" denotes a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that codes for a polypeptide). Degenerate codons contain different triplets of nucleotides but code for the same amino acid residue (ie, each of the triplets GAU and GAC codes for Asp). The term "expression vector" is used to indicate a DNA molecule, linear or circular, comprising a segment consisting of 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 replication origins, one or more selectable markers, an extender, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The term "isolated", when applied to a polynucleotide, indicates that the polynucleotide has been removed from its natural genetic environment and which is therefore free of other foreign or unwanted coding sequences and which is in a form suitable for use within protein production systems undergoing genetic engineering. Such isolated molecules are those that are separated from their natural environment and that include cDNAs and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are usually 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 a person ordinarily skilled in the art (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 other than its active environment, for example separated from blood and 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 more than 95% pure, more preferably more 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", when referring to DNA segments, indicates that the segments are arranged in such a way that they function in concert for their intended purpose, for example, the transcription starts at the promoter and procethrough the coding segment to the terminator The term "ortholog" indicates a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The differences in sequence between orthologs are the result of species differentiation. The "paralogs" are different but structurally related proteins elaborated by an organ. It is considered that paralogs arise through the duplication of genes. For example, α-globin, β-globin and myoglobin are paralogs with each other. A "polynucleotide" is a single or double chain polymer of deoxyribonucleotide or ribonucleotide bases that are read from the 5 'to 3' end. The polynucleotides include RNA and DNA and can be isolated from natural sources, can be synthesized in vitro or can be prepared from a combination of natural and synthetic molecules. The sizes of the polynucleotides are expressed as base pairs ("pb"), nucleotides ("nt") or kilobases ("kb"). When the context permits, these last two terms can describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules, it is used to indicate the total length and it will be understood that it is 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 from the ends thereof may be dispersed as a result of enzymatic separation; therefore, all nucleotides within a double stranded polynucleotide molecule may not be paired. Such unpaired ends generally do not exceed 20 nt in length. A polypeptide is a polymer of amino acid residues linked by peptide bonds, whether produced naturally or synthetically. Peptides of less than about 10 amino acid residues are commonly referred to as "peptides." The term "promoter" is used herein for its recognized meaning in the art to indicate 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 comprising non-peptide components, such as carbohydrate groups. Carbohydrates and other nonpeptide substituents can be added to a protein by the cell in which the protein is produced and will vary with the cell type. Proteins are defined here in terms of their major amino acid structures; substituents such as carbohydrate groups are generally not specified, however they may be present. The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule (ie, a ligand) and a half of the effect of the ligand on the cell. Membrane-bound receptors are characterized by a structure of multiple peptides that comprise an extracellular ligand binding domain 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 molecules in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are related to receptor / ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, cellular calcium mobilization, membrane lipid mobilization, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane, cytosolic or nuclear bound; they can be monomeric (for example the thyroid stimulating hormone receptor, the beta adrenergic receptor) or multimeric (for example the PDGF receptor, the growth hormone receptor, the IL-3 receptor, the GM receptor) GSF, the G-CSF receptor, the erythropoietin receptor and the IL-6 receptor). The term "secretory signal sequence" refers to a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly removed to remove the secretory peptide during transit through the secretory pathway. The term "splice variant" is used herein to indicate alternative forms of RNA transcribed from a gene. The variation of splicing arises naturally by the use of alternative splicing sites within a transcribed RNA molecule, or less commonly between RNA molecules transcribed separately, and can result in several mRNAs transcribed from the same gene. The splice variants can encode polypeptides having altered amino acid sequences. The term splice variant it is also used herein to indicate a protein encoded by a splicing variant of an mRNA transcribed from a gene. It will be understood that the molecular weights and the lengths of the polymers determined by imprecise analytical methods (e.g., gel electrophoresis) are approximate values. When such value is expressed as "around" X or "approximately" X, it will be understood that the established value of X will be accurate with + 10%. All references mentioned herein are incorporated by reference in their entirety. The present invention is based in part on the discovery of a novel DNA sequence encoding a homologous polypeptide of connective tissue growth factor, ie, polypeptides having homologies by other members of a family of growth factors that are secreted and containing a cysteine motif of the formula: Cx. { 8, 10) CxCCxxCx. { 7] Cx. { 5, 6 } Cx (5, 7) Cx. { 12, 13 } Cx [7, 8} Cx. { 20) CxCx. { 6.} Cx. { 12.24} Cx. { 13.17} c where x. { } is the number of amino acid residues between cysteine (C), as shown in SEC. FROM IDENT. NO: 23 This motif represents a consecutive domain array known as an insulin-like growth factor binding domain followed by a von Willebrand c-factor domain (VWFc) (Bork, FEBS Letts, 327: 125-130, 1993). He Protein domain arrangement is unique to the protein structure family of CTGF / NOV. The binding domain of insulin-like growth factor is represented by the motif: Cx. { 8,10) CxCCxxCx. { 7] Cx (5,6.}. Cx (5,7) where x. { } is the number of amino acid residues between cysteines (C) as shown in SEC. FROM IDENT. NO: 24. This pattern is found in all known members of connective tissue growth factors and the insulin-binding protein family (eg, human CTGF, human CTGF-2, human NOV, human IBP 1 and 2) and it is unique for these proteins. The analysis of the tissue distribution of the mRNA corresponding to this novel DNA shows that the expression is higher in testes, followed by apparent but decreased levels of expression in the trachea, bone marrow and renal tissue. The polypeptide homologue of connective tissue growth factor has been called ZCTGF4. The mouse ortholog has also been identified and has been named zCTGF2. A DNA sequence and the corresponding sequence of putative amino acids are shown in SEQ. FROM IDENT. NO: 4 and 5, respectively.

The novel zCTGF4 polypeptide of the present invention initially it was identified by analyzing an EST database to determine homologous sequences with connective tissue growth factor and insulin-binding proteins. A single EST sequence was discovered and was predicted to be related to the connective tissue growth factor family. The sequence analysis of the clone from which EST is derived shows that the clone contains a defective cDNA with incorrect sequence. Isolation of an independent clone from a testicle library shows that the original cDNA clone contains an intronic sequence at the 5 'end of EST. The nucleotide sequence is described in SEQ. FROM IDENT. NO: 1 from nucleotide 17 to nucleotide 1078 and its deduced amino acid sequence is described in SEQ. FROM IDENT. NO: 2. Analysis of the DNA encoding the ZCTGF4 polypeptide (SEQ ID NO: 1) shows an open reading frame encoding 354 amino acids (SEQ ID NO: 2) comprising a signal peptide secretor of 23 amino acid residues (residue 1 (Met) to residue 23 (Gly) of SEQ ID NO: 2) and a mature 331 amino acid polypeptide (residue 24 (Thr) to residue 354 (Leu) of the SEC DE IDENT NO: 2). An alternately spliced variant has a 16 amino acid secretory signal peptide (residue 1 (Met) to residue 16 (GLy) of SEQ ID NO: 2, and a mature polypeptide of 338 amino acid residues (residue 17 (Phe) to residue 354 (Leu) of SEQ ID NO: 2). Members of the CTGF family are characterized by a multiple domain structure comprising an IBP domain (amino acid residue 59 (Pro) to 102 (Tyr) of the SEC. FROM IDENT. NO: 2) which has been suggested as an insulin growth factor binding domain (Kiefer et al., J. Biol. Chem. 266: 9043-9049, 1991), a von Willebrand factor C domain (residue amino acid 114 (Cys) to 179 (Cys) of SEQ ID NO: 2) which may be involved in multimerization, a variable domain (amino acid 180) (Ser) to 208 (Lys), which may be involved in specific interactions of tissue, matrix, growth factor or receptor; and a sulphated glycoconjugate binding motif domain (amino acid residue 209 (Cys) to 252 (Cys) of SEQ.

FROM IDENT. NO: 2) which is considered to be related in the binding of large macromolecules (Holt et al., J. Biol. Chem. 65: 2852-2855, 1990). It has recently been shown that CTGF binds to IGF with low affinity (Kim et al., Proc. Nat. Acad. Sci. 94: 12981-12986, 1997), and therefore expands the superfamily of the factor binding protein. insulin-like growth factor (IGFBP) to include CTGF. The IGFBP superfamily now comprises proteins that bind both high affinity IGF (eg IBFBP 1-5) as well as IGF binding proteins with low affinity (nov, cyr61 and CTGF), suggesting that proteins alter cell growth in both IGF-independent and IGF-independent ways. The IGF binding potential of members of the CTGF family, including zCTGF, can act as a competitive inhibitor of the biologically free component of IGF. The ZCTGF4 factor can alter other pathways where IGFBP can play a role, for example by competing with endogenous IGFBP for proteases that degrade the IGF / IGFBP complex, resulting in changes in circulating levels of IGF. As shown in Figure 1, a multiple alignment also shows that zCTGF4, like other known members of the CTGF family, have a heparin binding domain that has been suggested as a dimerization and receptor binding domain. The heparin binding domain is shown in SEC. FROM IDENT. NO: 2 from residue 262 (lie) to residue 295 (Phe). The structure of the domain has been reviewed by Brigstock et al., Ibid. , 1997 and Bork, FEBS Letts. 327: 125-130. 1993, both incorporated herein by reference. It is generally considered that under selective pressure on organisms to acquire new biological functions, members of the new CTGF family arise from the duplication of existing genes leading to the existence of families with multiple genes. Therefore, the members of the family they contain traces of the ancestral gene, and these characteristic traits can be exploited in the isolation and identification of additional family members. It has recently been shown that CTGF molecules truncated in the isolated N-terminal part of uterine secretory fluids have mitogenic activity and will bind heparin (Brigstock et al., Ibid., 1997) and may have activity. It would be expected that a ZCTGF4 molecule truncated in the N-terminal part would have similar activity, and would comprise molecules without the IBP domain and may or may not be truncated in the C-terminal part as well. The SEC. FROM IDENT. NO: 3 is a degenerate polynucleotide sequence encompassing all polynucleotides that can encode the ZCGTF4 polypeptide of SEQ. FROM IDENT. NO: 2 (amino acids 1 or 4 to 354). Therefore, the polynucleotides encoding the ZCTGF4 polypeptide vary from nucleotide 17 or 85 to nucleotide 1078 of SEQ. FROM IDENT. NO: 2, or from nucleotide 1 or 69 to 1062 of SEC. FROM IDENT. NO: 3 and are those contemplated by the present invention. Fragments and fusions as described herein with respect to SEC are also contemplated by the present invention. FROM IDENT. NO: 1, which are formed from analogous regions of the SEC. FROM IDENT. NO: 3, wherein nucleotides 191 to 322 of SEQ. FROM IDENT. NO: 1 correspond to nucleotides 175 to 306 of the SEC. FROM IDENT. NO: 3, for the IBP domain; wherein nucleotides 356 to 553 of SEQ. FROM IDENT. NO: 1 correspond to nucleotides 3 0 to 537 of the SEC. FROM IDENT. NO: 3, for the domain of von Willebrand factor C (VWFc); wherein nucleotides 554 to 640 of SEQ. FROM IDENT. NO: 1 correspond to nucleotides 538 to 624 of the SEC. FROM IDENT. NO: 3 for the variable region; wherein nucleotides 641 to 772 of SEQ. FROM IDENT. NO: 1 correspond to nucleotides 625 to 756 of the SEC. FROM IDENT. NO: 3, for the sulphated glycoconjugate binding domain; and wherein nucleotide 800 to nucleotide 904 of SEQ. FROM IDENT. NO: 1 correspond to nucleotide 784 to nucleotide 888 of SEC. FROM IDENT. NO: 3 for the heparin binding domain. Table 1 establishes the codes of a letter used within the SEC. FROM IDENT. NO: 3 to indicate degenerate nucleotide positions. The "resolutions" are the nucleotides indicated by the code of a letter. The term "complement" indicates the code for the complementary nucleotides. For example, the code Y indicates C or T, and its complement R indicates A or G, A is complementary to T and G is complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C c G G G C C T T A A C A G A C C G A C G A C G T A G C A G A C A G A C A G A C A T A A A C A A A A A A A A A A A A A A A A T A A A A A A A A A A A A A A T A A | G | T C | G T V A | C | G A | C G B C | G | T A | G T H A | c | T A | C G | T N A | C | G | T The degenerate codons used in SEC. FROM IDENT. NO: 2 and 5, encompass all possible codons for a given amino acid, as set forth in table 2.

TABLE 2 Amino Acid Codon Code Codon a degenerate letter Cys C TGC TGT TGY Be AGC AGT TCA TCC TCG TCT WSN Thr T ACÁ ACC ACG ACT 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 CGT MGN Lys K AAA AAG AAR Met M ATG ATG He I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TT 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 person ordinarily skilled in the art will appreciate that some ambiguity is introduced in determining a ^ degenerate codon, representative of all the possible codons that code for each amino acid. For example, the degenerate codon for serine (WSN) can in some circumstances code for arginine (AGR) and the degenerate codon for arginine (MGN) in some circumstances can code for serine (AGY). There is a similar relationship between codons that code for phenylalanine and leucine. Therefore, some polynucleotides encompassed by the degenerate sequence can encode variant amino acid sequences, but a person with ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ. FROM IDENT. NO: 2 and 5. Variant sequences can be tested with ease to determine their functionality as described herein. A person with ordinary skill in the art will also appreciate that different species can show a "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; Wain-Hobson, et al., Gene, 13: 355-64. 1981; Grosjean and Fiers, Gene, 18: 199-209, 1982; Holm, Nuc. Acids Res. 14: 3075-87, 1986; Ikemura, J. Mol. Biol. 158: 573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of the art that refers to protein translation codons that are used with more often in cells of certain species, and therefore favor one or some representatives of the possible codons that code for each amino acid (see table 2). For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG or ACT, but in mammalian cells the most commonly used codon is ACC; in other species, for example insect cells, yeast, virus or bacteria, different codons may be preferential for Thr. Preferred codons for a particular species can be introduced into the polynucleotides of the present invention by any of several methods known in the art. The introduction of preferential codon sequences within recombinant DNA can, for example, improve the production of the protein by making the translation of the protein more efficient within a particular cell type or species. Therefore, the degenerate codon sequence shown in SEC. FROM IDENT. NO: 3 serves as a template to optimize the expression of polynucleotides in various types and species of cells commonly used in the art and described herein. Sequences containing preferential codons can be tested and can be optimized for expression in various species, and can be tested for functionality as described herein. The present invention also provides variant polypeptides and nucleic acid molecules that represent counterparts of other species (orthologs). These species include, but are not limited to, mammalian cells, birds, amphibians, reptiles, fish, insects or other vertebrates or invertebrate species. Of particular interest are zCTGF4 polypeptides of other mammalian species, which include murine, porcine, ovine, bovine, canine, feline, equine and other primate polypeptides. Human ZCTGF4 orthologs can be cloned using information and compositions that are 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 zCTGF4 as described herein. Suitable sources of mRNA can be identified by Northern blotting with probes designed from the sequences described herein. A library is then prepared from the mRNAs of a positive cell line or tissue. The mouse sequence of zCTGF2 is an orthologous representative of human connective tissue growth factor zCTGF4, and is described herein as SEC. FROM IDENT. NO: 4 and 5. A cDNA encoding zCTGF4 can then be isolated by various methods, such as by probing with whole or partial human cDNA, or with one or more sets of degenerate probes based on the described sequences. A cDNA can also be cloned using the chain reaction of polymerase with primers designed from the representative human ZCTGF4 sequences described herein. Within a further method, a cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the zCTGF4 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. The present invention provides polynucleotide molecules that include DNA and RNA molecules that encode the zCTGF4 polypeptides described above. The zCTGF4 polynucleotide sequences described herein can also be used as probes or primers to clone 5 'non-coding regions of a ZCTGF4 gene. In view of the tissue-specific expression that is observed for zCTGF4 by Northern blotting, it is expected that this region of the gene will provide specific expression for testes, trachea, bone marrow and kidney. Promoter elements from the ZCTGF4 gene in this manner can be used to direct tissue-specific expression of heterologous genes, for example, in transgenic animals or patients treated with gene therapy. The cloning of the 5 'flanking sequences also facilitates the production of ZCTGF4 proteins by "gene activation", as described in U.S. Pat. No. 5,641,670. Briefly, the expression of an endogenous gene of ZCTGF4 in a cell is altered when it is introduced at the ZCTGF4 locus a DNA construct comprising at least one target sequence, a regulatory sequence, an exon and an unpaired splice donor site. The target sequence is a 5 'non-coding sequence of ZCTGF4 that allows homologous recombination of the construct with the endogenous ZCTGF4 locus, whereby the sequences within the construct are operably linked to the endogenous coding sequence of ZCTGF4. In this manner, an endogenous ZCTGF4 promoter can be substituted or supplemented with other regulatory sequences to provide improved expression, tissue specific or otherwise regulated. Those skilled in the art will recognize that the sequence described in SEQ. FROM IDENT. NO: 1 represents a single allele of human ZCTGF4 and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries of different individuals according to standard procedures. The allelic variants of the nucleotide sequence shown in SEC. FROM IDENT. NO: l, which include those that contain silent mutations and those in which mutations result in changes in the amino acid sequence, are within the scope of the present invention as they are proteins which are allelic variants of SEC. FROM IDENT. NO: 2. The cDNA molecules generated from allergenically spliced mRNA, the which retain the properties of the ZCTGF4 polypeptide are included within the scope of the present invention, since they are polypeptides encoded by such cDNA and mRNA. Allelic variants and splice variants of this sequence can be cloned by probing cDNA or genomic libraries of different individuals or tissues, according to standard procedures known in the art. The present invention provides methods for using ZCTGF4 polynucleotides and polypeptides to diagnose chromosomal disorders associated with abnormal expression of the ZCTGF4 protein. Detectable chromosomal mutations at the ZCTGF4 gene locus include, but are not limited to aneuploidy, changes in gene copy number, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be identified through the use of molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeated analysis (STR) using PCR techniques, and other genetic link analysis techniques. known in the art (Molecular Cloning: A Laboratorv Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biolos., John Wiley and Sons, Inc., NY, 1987; AJ Marian, Chest 108: 255-65, 1995). The analysis of DNA samples can detect deletions and insertions for changes in size in the amplified DNA products when comparing a DNA sample with a normal DNA standard for zCTGF4. Parallel errors of the duplex DNA can be detected by digestion with RNAse or differences in the melting temperature. Other methods to detect differences in sequences include, changes in electrophoretic motility, Southern analysis and direct DNA sequencing. Recently, techniques for accessing genetic information with high density arrays have been found available (Chee et al., Science 274: 610-614, 1996) and can analyze large fragments of genomic DNA with high resolution. The analysis of chromosomal DNA using the polynucleotide sequence zCTGF4 is useful for correlating diseases with abnormalities located on chromosome 6. The gene for zCTGF4 has been located on chromosome 6q22.1. Studies of the DNA, cDNA or genomic DNA sequences of some of the individuals that have disease that contains a mutation in the sequence of the gene for ZCTGF4 that is not present in normal individuals, can provide strong evidence that the mutation is causal of the illness. In one embodiment, the methods of the present invention provide a method for detecting an abnormality on chromosome 6q in a sample from an individual, comprising: (a) obtaining RNA for zCTGF4 from the sample; (b) generate cDNA for zCTGF4 by polymerase chain reaction; and (c) comparing the nucleic acid sequence of ZCTGF4 cDNA with the nucleic acid sequence as shown in SEQ. FROM IDENT. NO: 1. In additional embodiments, the difference between the cDNA sequence for zCTGF4 or the ZCTGF4 gene in the sample and the ZCTGF4 sequence as shown in SEQ. FROM IDENT. NO: 1, is indicative of an abnormality on chromosome 6q. Within the preferred embodiments of the invention, the isolated nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules having the nucleotide sequence of the SEC. FROM IDENT. NO: l, to nucleic acid molecules having the nucleotide sequence of nucleotides 86 to 1078 of SEQ. FROM IDENT. NO: 1, or to nucleic acid molecules having a nucleotide sequence complementary to the SEC. FROM IDENT. NO: 1. In general, astringent conditions are selected which are approximately 5 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA, can hybridize if the nucleotide sequences have a degree of complementarity. Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the individual is affected by the degree of mismatch. The Tm of the badly matched hybrid decreases by 1 ° C for every 1-1.5% of mismatch of base pairs. By varying the astringency of the hybridization conditions allows a control on the degree of mismatch that will be present in the hybrid. The degree of astringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases. The stringent hybridization conditions encompass temperatures of about 5-25 ° C below the Tm of the hybrid and a hybridization buffer having Na * up to 1 M. Higher degrees of astringency can be obtained at lower temperatures with the addition of formamide , which reduces the Tm of the hybrid by approximately 1 ° C for every 1% of formamide in the buffer solution. Generally, such astringent conditions include temperatures of 20-70 ° C and a hybridization buffer containing up to 6x SSC and 0-50% formamide. A higher degree of astringency can be obtained at temperatures of 40-70 ° C with a hybridization buffer having up to 4x SSC and 0-50% formamide. Highly stringent conditions typically encompass temperatures of 42-70 ° C with a hybridization buffer having up to lx SSC and 0-50% formamide. Different degrees of astringency can be used during hybridization and washing to obtain a specific binding maximum to the target sequence. Typically, the washings after hybridization are performed with increasing degrees of stringency to remove the unhybridized polynucleotide probes from the hybridized complexes. It is intended that the above conditions serve as a guide and are within the abilities of a person skilled in the art to adapt these conditions for use with a particular polypeptide hybrid. The Tm for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly matched probe sequence. Those conditions which influence Tn include the size and content of base pairs of the polynucleotide probe, the ionic strength of the hybridization solution and the presence of denaturing agents in the hybridization solution. Many equations for calculating Tm in the art are known, and are specific for DNA, RNA and DNA-RNA hybrids as well as variable length polynucleotide probes 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 Molecular Cloning Techniques, (Academic Press, Inc. 1987), and Wetmu, Crit. Rev. Biochem. Mol. Biol. 26: 227 (1990). of sequence such as OLIGO 6.0 (L? R; Long Lake, MN) and Prime Premier 4. 0 (Premier Biosoft International, Palo Alto, CA), as well as inert sites, are tools available to analyze a given sequence and calculate the Tm based on user-defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of larger polynucleotide sequences of > 50 base pairs are made at temperatures of approximately 20-25 ° C below the calculated Tm. For smaller probes, from < 50 base pairs, hybridization is typically carried out at Tm DE 5-10 ° C below. This allows a maximum hybridization rate for the DNA-DNA and DNA-RNA hybrids. The length of the polynucleotide sequence includes the speed and stability of hybrid formation. The smaller probe sequences, from < 50 base pairs, reach equilibrium with complementary sequences quickly, but can form less stable hybrids. Incubation times at any point from minutes to hours can be used to obtain lipid formation. Larger probe sequences reach equilibrium more slowly, but form more stable complexes even at lower temperatures. It is allowed that the incubation processes during the night or for a longer period.

Generally, incubations are carried out for a period equal to three times the calculated Cot time. The Cot time, the time that is required for the polynucleotide sequences to reassociate, can be calculated for a particular sequence by methods known in the art. The composition of base pairs of the polynucleotide sequence will alter the thermal stability of the hybrid complex, thereby influencing the choice of the hybridization temperature and the ionic strength of the hybridization buffer. The A-T pairs are less stable than the G-C pairs in aqueous solutions containing sodium chloride. Therefore, the higher the G-C content, the more stable the hybrid. A uniform distribution of G and C residues within the sequence also contributes positively to the stability of the hybrid. In addition, the composition of base pairs can be manipulated to alter the Tm of a given sequence. For example, 5-methyl deoxycytidine can be substituted for deoxycytidine and 5-bromo deoxyuridine can be substituted for thymidine to increase T ",, while 7-deaza-2 '-deoxyguanosine can be substituted for guanosine to reduce the dependence on Tm. The ionic concentration of the hybridization buffer also alters the stability of the hybrid. Hybridization buffers generally contain blocking agents such as Denhart's solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powder. { BLOTTO), heparin or SDS and a source of Na *, such as SSC (lx SSC: 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE: 1.8 m NaCl, 10 mM NaH2P04, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration of the buffer, the stability of the hybrid increases. Typically, hybridization buffers contain between 10 mM-1 mM Na *. The addition of destabilizing or denaturing agents such as formamide, tetraalkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution will alter the Tm of a hybrid. Formamide is typically used at a concentration of up to 50% to allow incubations to be carried out at more convenient and lower temperatures. Formamide also acts to reduce the non-specific background when using RNA probes. As an illustration, a nucleic acid molecule encoding a variant ZCTGF4 polypeptide can be hybridized to a nucleic acid molecule having the nucleotide sequence of SEQ. FROM IDENT. NO: 1 (or its complement) at 42 ° C overnight in a solution comprising 50% formamide, 5x SSC (Ix SSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhart's solution (lOOx of Denhardt's solution: Ficoll 400 2% (w / v), polyvinylpyrrolidone 2% (w / v) and serum albumin bovine 2% (v / v)) 10% dextran sulfate and 20 μg / ml of cut and denatured salmon sperm DNA. One skilled in the art can design variations of these hybridization conditions. For example, the hybridization mixture can be incubated at a higher temperature, for example at about 65 ° C in a solution that does not contain formamide. In addition, pre-mixed hybridization solutions are available (eg, EXPRESSHYB hybridization solution from CLONTECH Laboratories, Inc.), and hybridization can be carried out according to the manufacturer's instructions. After hybridization, the nucleic acid molecules can be washed to remove unhybridized nucleic acid molecules under stringent conditions or under highly stringent conditions. Typical conditions of astringent washing include washing in a 0.5x -2x SSC solution with 0.1% sodium dodecylsulfate (SDS) at 55-65 ° C, ie, nucleic acid molecules encoding a polypeptide variant of ZCTGF4 hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ. FROM IDENT. NO: 1 (or its complement) under conditions of astringent wash in which the wash astringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55-65 ° C, which includes 0.5x SSC with 0.1% SDS at 55 ° C or 2x SSC with 0.1% SDS at 65 ° C. A person skilled in the art can easily design conditions equivalents, for example, substituting SSPE for SSC in the wash solution. Typical highly stringent washing conditions include washing in a solution of 0. lx - 0.2x SSC with sodium dodecylsulfate (SDS 0.1% at 50-65 ° C). In other words, the nucleic acid molecules encoding a variant polypeptide of zCTGF4 hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ. FROM IDENT. NO: 1 (or its complement) under highly stringent washing conditions, in which the wash astringency is equivalent to 0. Ix - 0.2x SSC with 0.1% SDS at 50-65 ° C, and including 0. lx SSC with 0.1% SDS at 50 ° C or 0.2? SSC with SDS 0.1% at 65 ° C. The present invention also provides isolated ZCTGF4 polypeptides having a sequence identity substantially similar to that of the SEC polypeptides. FROM IDENT. NO: 2 or its orthologs. The term "substantially similar sequence identity" is used herein to mean polypeptides comprising at least 70%, at least 80%, at least 90%, at least 95% or more than 95% identity of sequence with the sequences shown in the SEC. FROM IDENT. NO: 2 or its orthologs. The present invention also includes polypeptides comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or more than 95% of sequence identity with the sequence of amino acid residues 1 to 24 to 354 of SEQ. FROM IDENT. NO: 2. The present invention further includes nucleic acid molecules that encode such polypeptides. Methods for determining percent identity are described below. The present invention also contemplates ZCTGF4 variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the polypeptide encoded with the amino acid sequence of SEQ. FROM IDENT. NO: 2, or a hybridization assay, as described above. Such variants of zCTGF4 include nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ. FROM IDENT. NO: 1 (or its complement) under conditions of astringent washing, in which the astringent wash is equivalent to 0.5x - 0.2x SSC with 0.1% SDS at 55-65 ° C, or (2) which code for a polypeptide that it has at least 70% at least 80%, at least 90%, at least 95% or more than 95% sequence identity with the amino acid sequence of the SEC. FROM IDENT. NO: 2. Alternatively, variants of zCTGF4 can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ. FROM IDENT. NO: 1 (or its complement) under conditions of highly astringent washing, wherein the wash stringency is equivalent to 0. lx -0.2x SSC with 0.1% SDS at 50-65 ° C, and (2) which code for a polypeptide having at least 70% at least 80%, at least 90%, at least 95% or more than 95% sequence identity with the amino acid sequence of SEC. FROM IDENT. NO: 2. Percent sequence identity is determined by conventional methods. See, for example Altscnul et al., Bull. Math. Bio 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 89: 10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment qualifications using an aperture separation penalty of 10, a separation extension penalty of 1, and the qualification matrix of "BLOSUM62" and Henikoff, and Henikoff (ibid.) As shown in Table 3. Amino acids are indicated by standard one-letter codes.

Total number of identical pairings _x 100 [Length of the longest sequence plus the number of separations entered in the longest sequence in order to align the two sequences] Table 3 A R N D C Q EGHILKMFPSTWYVA 4 R -1 5 N -2 0 6 D -2-2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3-3 -1 -3 -3 -4 -3 4 L -1 -2 - 3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 - 3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 - 1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 w -3-3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 - 3 -2 11 And -2-2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 Those skilled in the art will appreciate that there are many established algorithms available to align two amino acid sequences. The search algorithm "FASTA" similarity of Pearson and Lipman is an adequate method of protein alignment to examine the level of identity shared by the amino acid sequence shown herein and the amino acid sequence of a putative zCTGF4 variant. The FASTA algorithm is described by Person and Lipman Proc. Nati Acad. Sci. USA .85: 2444, 1988) and by Pearson (Meth. Enzymol., 183: 63, 1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the request sequence (eg, SEQ ID NO: 2) and a test sequence that has the highest density of identities (if the variable ktup is 1) or pairs of identities (if ktup = 2), without considering the substitutions, insertions or conservative deletions of amino acids. The ten regions with the highest identities densities are then re-classified by comparing the similarity of all paired amino acids using an amino acid distribution matrix, and the ends of the regions are "cut" to include only residues that contribute to the highest classification. If there are several regions with ratings higher than the "limit" value (calculated by the default formula, based on the length of the sequence and the ktup value), then the initial regions cut out are examined to determine if the regions can be enjoyed to form a approximate alignment with separations. Finally, the Higher scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 13: 444, 1970; Sellers, SIAM J. Appl. Math. 26: 787, 1974), which allows amino acid insertions and deletions. The illustrative parameters for analysis by FASTA are: ktup = 1, separation opening penalty = 10, separation extension penalty = 1, and substitution matrix = BLOSUM62. These parameters can be entered into a FASTA program by modifying the rating matrix file ("SMATRIX") as explained in Appendix 2 of Pearson, Meth. Enzymol. 183: 63 1990. FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as described above. For nucleotide sequence comparisons, the ktup value may vary between one to six, preferably four to six. ZCTGF4 variant polypeptides or polypeptides with substantially similar sequence identity are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is, conservative amino acid substitutions (see Table 4) and other substitutions which do not significantly affect the folding or activity of the polypeptide; the deletions | j ^ small, typically from one to about 30 amino acids; and terminal amino or carboxyl spreads, such as an amino terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. Therefore, the present invention includes polypeptides from about 28 to 354 amino acid residues comprising a sequence that is at least 70%, preferably at least 90%, and most preferably 95% more identical to the corresponding region of the SEC. FROM IDENT. NO: 2 In particular, the peptides and polypeptides corresponding to the domains in the zCTGF4 molecules as shown in SEQ. FROM IDENT. NO: 2 include the IBP domain (residues 59-102), the C domain of von Willebrand factor (residues 114-179), the variable domain (residues 180-08) and the glycoconjugate binding domain (residues 209-52) ) are within the scope of the present invention. Polypeptides comprising affinity tags may further comprise a proteolytic cleavage site between the zCTGF4 polypeptide and the affinity tag. Such preferred sites include thrombin separation sites and factor Xa separation sites.

TABLE 4 Conservative amino acid substitutions Basic: arginine lysine histidine Acid: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine The proteins of the present invention may also comprise amino acid residues that do not occur naturally. Amino acids that do not occur naturally include, without limitation, trans-3-methylproline, 2,4-ethanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-me ti lgl ic ina, ha lo-tr eonina, me ti 11 ronina, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and -methylproline, 3-dimethylproline, terleucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine. Various methods for incorporating amino acid residues that do not occur naturally in proteins are known in the art. For example, an in vi tro system where nonsense mutations are suppressed using chemically acylated amino suppressor tRNAs can be used. Methods for synthesizing amino acids and aminocilant tRNA are known in the art. The transcription and translation of plasmids containing nonsense mutations is typically carried out in a cell-free system comprising an extract of E. coli S30 and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113: 722, 1991; Ellman et al., Methods Enzymol. 202: 301, 1991; Chung et al., Science 259: 806 1993; and Chung et al., Proc. Nati Acad. Sci. USA 90: 10145 1993). In a second method, the translation is carried out in Xenopus oocytes by microinjection of the mutated mRNA and chemically suppressed tRNAs (Turcatti et al., J. Biol. Chem. 271: 19991 1996). In a t method, E. coli cells are grown in the absence of a natural amino acid that goes to be substituted (for example phenylalanine) and in the presence of the amino acid or amino acids that do not occur naturally, desired (for example 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine). The amino acid that does not occur naturally is incorporated into the protein instead of its natural counterpart. See Koide et al., Biochem. 33 .: 7470-7476, 1994. Naturally occurring amino acid residues can be converted to species that do not occur naturally by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitutions (Wynn and Richards, Protein Sci. 2: 395, 1993)). A limited amount of non-conservative amino acids, amino acids that are not encoded by the genetic code, amino acids that do not occur naturally and unnatural amino acids, can be substituted for the amino acid residues of ZCTGF4. Multiple substitutions of amino acids can be made and can be tested using known methods of mutagenesis and analysis, such as those described by Reidhaar-Olson and Sauer (Science 241: 53, 1988) or Bowie and Sauer (Proc. Nati. Acad. Sci. USA 86: 2152, 1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selected by polypeptide -ll ----------------- i ----_ functional, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions in such a position. Other methods that can be used include phage display (eg Lowman et al., Biochem 30: 1083, 1991, Ladner et al., US Patent number 5,223,409, Huse, International Publication, WO 92/06204) and mutagenesis. directed to the region (Derbys et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1983). The described variants of the zCTGF4 nucleotides and the polypeptide sequences can be generated through DNA release as described by Stemmer, Nature 370: 389 1994 and Stemmer, Proc. Nati Acad. Sci. USA 91: 10747 1994 and the international publication number WO 97/20078. Briefly, variant DNA molecules are generated by homologous recombination in vitro by random fragmentation of a parental DNA, followed by reassembly using PCR, resulting in point mutations introduced randomly. This technique can be modified using a family of parental DNA molecules, such as allelic variants or DNA molecules from different species, to introduce additional variability into the process. The selection or analysis to determine the desired activity, followed by additional iterations of mutagenesis and assay, provides a rapid "evolution" of sequences by selecting the desirable mutations while simultaneously being selected against harmful changes. Mutagenesis methods as described above can be combined with high throughput analysis methods to detect the activity of mutagenized and cloned polypeptides in host cells. Mutagenized DNA molecules that code for biologically active polypeptides, or polypeptides that bind with antibodies against ZCTGF4, can be recovered from the host cells and can be rapidly sequenced using modern equipment. These methods allow a rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. The essential amino acids in the polypeptides of the present invention can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244, 1081, 1989; Bass et al. , Proc. Nati, Acad. Sci. USA 88: 4498, 1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins: Analyeis and Design, Angeletti (ed.), Pages 259-311 ( Academic Press, Inc. 1998)). In the latter technique, unique alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested for determine their biological activity as described below to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699 (1996). The identities of the essential amino acids can also be inferred from the analysis of homologies with zCTGF4. The location of ZCTGF4 receptor binding domains can be identified by physical analysis of the structure, determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, together with the mutation of the amino acids of the site. Putative contact See, for example, de Vos et al. , Science 255: 306 (1992), Smith et al. , J. Mol. Biol. 224: 899 (1992) and Wlodaver et al. , FEBS Lett 309: 59 (1992). In addition, biotin-labeled zCTGF4 or FITC can be used for cloning of expression of zCTGF4 receptors.

The present invention also includes "functional fragments" of zCTGF4 polypeptides and nucleic acid molecules that encode such functional fragments. As previously described herein, zCTGF4 is characterized by a multiple domain structure comprising an IBP domain (amino acid residue 59 (Pro) to 102 (Tyr) of SEQ ID NO: 2) which has been suggested as a binding domain of insulin growth factor, a C domain of von Willebrand factor (amino acid residue 114 (Cys) to 179 (Cys) of SEC. FROM IDENT. NO: 2), a variable domain (amino acid 180 (Ser) to 208 (Lys), and a sulphated glycoconjugate binding motif domain (amino acid residue 209 (Cys) to 252 (Cys) of SEQ ID NO. 2) Therefore, the present invention further provides fusion proteins comprising: (a) polypeptide molecules comprising one or more of the domains described above, and (b) biologically active fragments comprising portions of one or more of The other polypeptide may be another domain of another CTGF, a non-native or unrelated secretory signal peptide to facilitate the secretion of the fusion protein, routine or systematic suppression of nucleic acid molecules may be carried out to obtain functional fragments of a nucleic acid molecule encoding a ZCTGF4 polypeptide As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO: 1 can be digested with n Bal 31 loop to obtain a series of suppressions housed. The fragments are then inserted into the expression vectors in the appropriate reading frame, and the polypeptides that are expressed are isolated and tested for ZCTGF4, or for the ability to bind antibodies to zCTGF4. An alternative to exonuclease digestion is the use of oligonucleotide-directed mutagenesis to introduce deletions or stop codons for specific production of a desired fragment.

Alternatively, particular fragments of a zCTGF4 gene can be synthesized using the polymerase chain reaction. Standard methods for identifying functional domains are well known to those skilled in the art. For example, studies regarding the cutting of either or both terminal portions of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66: 507 (1995). In addition, standard techniques for functional analysis of proteins have been described, for example, by Treuter et al., Molec. Gen Genet 240: 113 (1993). Content et al., "Expression and preliminary deletion analysis of the 32 kDa 2-5A synthetase mduced by human interferon," in Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.) Pages 65-72 ( Nijhoff 1987), Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation, Vol. 1, Boynton et al. , (eds.), pages 169-199 (Academic Press 1985), Coumailleau et al. , J. Biol. Chem. 270: 29270 (1995); Fukunaga et al. , J. Biol. Chem. 270: 25291 (1995); Yamagucho et al. , Biochem, Pharmacol. 50: 1295 (1995), and Meisel et al., Plant Molec Biol. 30: 1 (1996). The present invention also contemplates functional fragments of a gene for ZCTGF4 having amino acid changes, as compared to the amino acid sequence of SEQ. FROM IDENT. NO: 2. A variant gene of ZCTGF4 can be identify based on the structure when determining the identity level with the nucleotide and amino acid sequences of the SEC. FROM IDENT. NO: 1 and 2, as discussed earlier. An alternative solution for identifying a variant gene based on structure is to determine whether a nucleic acid molecule encoding a potential variant of the gene for ZCTGF4 can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ. FROM IDENT. NO: 1, as discussed before. The present invention also provides polypeptide fragments or peptides comprising a portion having an epitope of a ZCTGF4 polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope", which is a part of a protein that induces an antibody response when the whole of the protein is used as an immunogen. Peptides having immunogenic epitopes can be identified using standard methods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81: 3998 (1993)). In contrast, polypeptide fragments or peptides may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind. Some epitopes consist of a linear or contiguous chain of amino acids, and the antigenicity of such epitope is not interrupted by denaturing agents.

It is known in the art that relatively short synthetic peptides can mimic epitopes and a protein and can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al., Science 219: 660 (1983)) . Accordingly, the peptides presenting antigenic epitope and the polypeptides of the present invention are useful for increasing the antibodies that bind to the polypeptides described herein. Peptides having antigenic epitope and polypeptides preferably contain at least 4 to 10 amino acids, at least 10 to 15 amino acids, or about 15 to about 30 amino acids of SEQ. FROM IDENT. NO: 2. Such epitope-presenting peptides and polypeptides can be produced by fragmentation of the zCTGF4 polypeptide, or by chemical synthesis of peptides, as described herein. In addition, epitopes can be selected by phage display or random peptide libraries (see, for example, Lane and Stephen, Curr Opin. Immunol., 5: 268 (1993) and Cortese e al., Curr. Opin. Biotechnol. : 616 (1996)). Standard methods for identifying producing epitopes and antibodies from small peptides comprising an epitope are described, for example, by Mole, "Epitope Mapping," in Methods in Molecular Biology, Vol. 10, Manson (ed.), Pages 105-116 (The Humana Press, Inc. 1992), Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production Engineering, and Clinical Application, Rotter and Ladyman (eds.), Pages 60-84 (Cambridge University Press 1995), and Coligan et al. (eds.), Current Protocols in Immunology, pages 9.3.1 - 9.3.5. and pages 9.4.1 - 9.4.11 (John Wiley _ Sons 1997). Regardless of the particular nucleotide sequence of a variant gene of ZCTGF4, the gene encodes a polypeptide that is characterized by its proliferative or differentiating activity, or its ability to induce specialized cellular functions, or by the ability to specifically bind to an antibody to zCTGF4. . More specifically, variant genes for ZCTGF4 encode polypeptides which show at least 50% and preferably more than 70, 80 or 90% of the activity of the polypeptide encoded by the human ZCTGF4 gene described herein. For any ZCTGF4 polypeptide including variants and fusion proteins, one of those ordinarily skilled in the art readily generates a completely degenerate polynucleotide sequence coding for that variant using the information set forth in Tables 1 and 2 above. The present invention further provides a variety of other polypeptide fusions (and related multimeric proteins comprising one or more polypeptide fusions). For example, a ZCTGF4 polypeptide can be prepared as a fusion to a dimerizing protein as described in U.S. Pat. numbers 5,155,027 and 5,567,584. In this regard, preferred dimerizing proteins include immunoglobulin constant region domains. The immunoglobulin-zCTGF4 polypeptide fusions can be expressed in genetically engineered cells (to produce various multimeric analogues of ZCTGF4). Auxiliary domains can be fused to ZCTGF4 polypeptides to direct them to specific cells, tissues or macromolecules (e.g., collagen). For example, a ZCTGF4 polypeptide or protein can be targeted to a predetermined cell type by fusing a ZCTGF4 polypeptide with a ligand that specifically binds to a receptor on the surface of the target cell. In this manner, polypeptides and proteins can be engineered for therapeutic or diagnostic purposes. A ZCTGF4 polypeptide can be fused to two or more portions, such as an affinity tag for purification and a targeting domain. Polypeptide fusions may also comprise one or more separation sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34.:l-9, 1996. A Hoop / Woods hydrophilicity profile of the zCTGF4 protein sequence can be generated, as shown in SEQ. FROM IDENT. NO: 2 (Hoop et al., Proc. Nati, Acad. Sci. 78: 3824-3828, 1981; Hopp, J. Immun., Meth. 88.:1-18, 1986; Triquier et al., Protein Enqineering 11: 153-169, 1998). The profile is based on an interval of six slip residues. The buried G, S and T residues and the exposed H, Y and W residues are ignored. Hydrophilicity can be used to determine regions that have the most antigenic potential. For example, amino acid residues 239-244 of SEQ. Are included in the hydrophilic regions of ZCTGF4. FROM IDENT. NO: 2, amino acid residues 105-110 of SEC. FROM IDENT. NO: 2, amino acid residues 172-177 of SEC. FROM IDENT. NO: 2, amino acid residues 238-243 of SEC. FROM IDENT. NO: 2 and amino acid residues 171-176 of SEC. FROM IDENT. NO: 2 Using the methods discussed herein, a person of ordinary skill in the art can identify or prepare various polypeptides having substantially similar sequence identity to residues 1 or 4 to 354 of SEQ. FROM IDENT. NO: 2, or functional fragments and fusions thereof, and retain the properties of the wild type protein such as the ability to stimulate proliferation, differentiation and induce specialized cellular function. The polypeptides of the present invention, which include full length proteins, fragments thereof and fusion proteins, can be produced in genetically engineered host cells in accordance with conventional techniques. Suitable host cells are those types of cells which can be transformed or transfected with exogenous DNA and which can grow in culture, and which include bacterial, mycotic cells and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are described by Sambrook et al., Molecular Cloning: A Laboratorv 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 encoding the ZCTGF4 polypeptide is operably linked to other genetic elements necessary for its expression, which generally include a promoter. of transcription and a terminator, within an expression vector. The vector also commonly contains one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers can be provided in separate vectors, and the replication of the exogenous DNA can be provided by integration within the genome of the host cell. The selection of promoters, terminators, markers selectable, vectors and other elements is a matter of systematic design within the level of a person usually skilled in the art. Many such elements are described in the literature and are available through commercial providers. To direct a zCTGF4 polypeptide within the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zCTGF4, or it may be derived from another secreted protein (for example t-PA or it is synthesized de novo.) The secretory signal sequence is operably linked to the ZCTGF4 DNA sequence, i.e. two sequences are joined in the correct reading frame and placed to direct the newly synthesized polypeptide into the secretory pathway of the host cell.Sequences of secretory signal are commonly placed 5 'to the DNA sequence encoding the polypeptide of the host cell. interest, although certain secretory signal sequences may be placed elsewhere 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 sequence of the secretory signal contained in the polypeptides of the present invention is used to direct other polypeptides within the secretory pathway. The present invention provides such fusion polypeptides. A signal fusion polypeptide can be made where the secretory signal sequence derived from amino acid residue 1 to 23 of the? EC. FROM IDENT. NO: 2 is operably linked to a DNA sequence encoding 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 fused at its amino terminal portion with an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a protein that is not normally secreted. Such functions can be used in vivo or in vitro to direct peptides through the secretory pathway. Cultured mammalian cells are suitable hosts for use within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somati-c Cell Genetics 2: 603, 1981: Graham and Van der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841- i ^ fc-¿l-'i ^ a .. 845, 1982), transfection mediated by 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, Ciccarone et al., Focus 15:80, 1993), as well as viral vectors (Miller and Rosman, BioTechnisues 7: 980-90, 1989, Wang and Finer, Nature Med. 2: 714-6, 1996) . The production of recombinant polypeptides in cultured mammalian cells is described, for example, in Levinson et al., U.S. Pat. number 4,713,339; Hagen et al., U.S. Patent number 4,784,950; Palmiter et al-, U.S. Patent No. 4,579,821; and Ringold, U.S. Patent number 4,656,134. Suitable cultured mammalian cells include cell lines COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651) BHK (ATTC No. CRL 1632, BHK 570 (ATTC No. CRL 10314) , 293 (ATTC No. CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977) and Chinese hamster ovary (for example CHO-K1; ATTC No. CCL 61). of suitable cells are known in the art and are available from public repositories such as the American Type Culture Collection, Rockville, Md. In general, strong transcription promoters such as the SV-40 or cytomegalovirus promoters are preferred. , 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.

Drug selection is generally used to select cultured mammalian cells 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". An exemplary selectable marker is a gene that encodes resistance to the antibiotic neomycin. The selection is carried out in the presence of a neomycin-type medicament, 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 called "amplification." The amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select cells that produce high levels of the products of the introduced genes. An exemplary, amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other genes that provide drug resistance (eg, hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or proteins on the cell surface such as CD4, CD8, CPH Class I, placental alkaline phosphatase, can be used to classify transfected cells from untransfected cells by means of FACS classification or magnetic bed separation technology. Other higher eukaryotic cells, including insect cells, plant cells and bird cells, can also be used as hosts. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11: 47-58, 1987. The transformation of insect cells and the production of foreign polypeptides therein are described by Guarino et al., U.S. Pat. No. 5,162.22 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa califonica nuclear polyhedrosis virus (AcNPV). The DNA encoding the zCTGF4 polypeptide is inserted into the baculoviral genome instead of the sequence encoding the AcNPV polyhedrin gene by one of two methods. The first is the traditional method of recombination of homologous DNA between wild-type AcNPV and a transfer vector containing ZCTGF4 flanked by the AcNPV sequences. Suitable insect cells are infected, for example SF9 cells with wild type AcNPV and transfected with a transfer vector comprising a zCTGF4 polynucleotide operably linked to a promoter. of AcNPV polyhedrin gene, terminator and flanking sequences. See, King L.A: and Owns, The 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 Bioloqv, Totowa, NJ, Human Press 1995. Natural recombination within an insect cell will result in a recombinant baculovirus which contains zCTGF4 activated by the polyhedrin promoter. Recombinant viral concentrates are manufactured by methods commonly used in the art. The second method for making recombinant baculovirus uses a transposon-based system described by Luckow (Luckow, V.A, et al., J. Virol. 67: 4566-79, 1993). This system is sold in the Bac-to-Bac ™ equipment "(Life Technologies, Rockville, MD) This system uses a pFastBacl ™ transfer vector (Life Technologies) that contains a Tn7 transposon to move the DNA encoding the zCTGF4 polypeptide in a baculovirus genome maintained in E. coli as a large plasmid termed a "bacmid." The pFastBacl ™ transfer vector uses the AcNPV polyhedrin promoter to activate expression of the gene of interest, in this case ZCTGF4. pFastBacl ™ can be modified to a considerable degree, the polyhedrin promoter can be removed and replaced with the protein promoter baculovirus (also known as Peor, p6.9 or MP promoter) 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. 71: 971-976, 1990; Bonnmg, B.C. et al., J. Gen. Virol. 71: 971-976, 1990; and Chazenblak G.D., and Rapoport B., J. Biol. Chem. 270: 1543-1549, 1995. In such transfer vector constructs, a short or long version of the basic protein promoter can be used. In addition, transfer vectors can be constructed which replace the native secretory signal sequences of zCTGF4 with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdisteroid glucosyltransferase (EGT), honey bees (Invitrogen, Carlsbad, CA), or gp67 baculovirus (PharMingen, San Diego, CA) can be used in the constructs to replace the sequence of secretory signal native to ZCTGF4. In addition, transfer vectors can include frame fusion in DNA encoding an epitope tag in the C or N terminal part of the expressed ZCTGF4 polypeptide, eg, a Glu-Glu epitope tag (Grussenmeyer, T. et al. , Proc. Nati, Acad. Sci. 82: 7952-4, 1995). Using techniques known in the art, a transfer vector containing ZCTGF4 is transformed into E. coli. and it is analyzed to determine báemidos which contain a gene interrupted lacZ indicative of recombinant baculovirus. The bacmidic DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. The recombinant virus expressing ZCTGF4 subsequently produces. Recombinant vial concentrates are made by methods commonly used in the art. The recombinant virus is used to infect host cells, typically a cell line derived from an armed worm or autumn worm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnoloav: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C. 1994. Another suitable cell line is the High FiveO "cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435). Serum-free medium, commercially available, is used to grow and maintain the cells. Suitable media are Sf900 IIm (Life Technologies) or ESF 921 ™ (Expression Systems) by Sf9 cells; and Ex-cell0405 ™ (JRH Biosciences, Lenexa, KS) or Express FiveO ™ (Life Technologies) for T. ni cells. 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 concentrate is added to a multiplicity of infection (MOI) from 0.1 to 10, more typically, about 3. Recombinant cells infected with virus typically produce the ZCTGF4 recombinant polypeptide at 12-72 hours post infection and secrete it with varying efficiency within the medium. Culture is usually harvested at 48 hours after infection. Centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing the zCTGF polypeptide is filtered through micropore filters, usually with a pore size of 0.45 μm. The procedures used are generally described in available laboratory manuals (King L. A. and Posee, R. D., ibid, O'Reilly D.R. et al., Ibid, Richardson, C. D., ibid.). Subsequent purification of the ZCTGF4 polypeptide from the supernatant can be obtained using methods described herein. Fungal cells, including yeast cells, can also be used within the present invention. In this regard, the yeast species of particular interest include Saccharomyces cerevisiae, Pichia pastoris and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are described, for example, by Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent number 4,931,373; Brake, U.S. Patent number 4,870,008; Welch et al., U.S. Patent number 5,037,743; and Murray et al., U.S. Pat. number 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 Sacharomyces cerevisiae is the P0T1 vector system described by Kawasaki et al., (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in medium containing glucose. Promoters and terminators suitable for use in yeast include those of glycolytic enzyme genes (see, for example, Kawasaki, US Patent No. 4,599,311, Kingsman et al., US Patent No. 4,615,974, and Bitter, US Patent No. 4,977,092) and alcohol dehydrogenase. See also U.S. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts include transformation systems for other yeasts including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago mayis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltose are known in the art. See, for example, Gleeson et al., J. Gen. Micobiol. 132.: 3459-65, 1986 and Cregg, U.S. Pat. number 4,882,279. Aspergillus cells can be used according to with the methods of McKnight et al., U.S. Pat. number 4,935,349. Methods for transforming Acremonium chrysogenum are described by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are described by Lambowitz, U.S. Pat. No. 4,486,533. The use of Pichia methanolica as a 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 for use in the transformation of P. methanolica will commonly be prepared as double-stranded circular plasmids, which are preferably linearized before transformation. For the production of polypeptides in P.metanolica, it is preferred that the promoter and the terminator in the plasmid be those of the P.metanolica gene such as the alcohol utilization gene of P.metanolica (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 entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is the ADE2 gene of P.metanolica, which codes for phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells. -aj-ugly 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 methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P.metanolica cells. It is preferred to transform P.metanolica cells by electroporation using a pulsed electric field, which decays exponentially having a field strength of 2.5 to 4.5 kV / cm, preferably about 3.75 kV / cm and a time constant (i) from 1 to 40 milliseconds, more preferably from approximately 20 milliseconds. Prokaryotic host cells, which include strains of the bacteria Escherichia coli, Bacillus and other genera, are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign sequences of DNA cloned therein are well known in the art (see, for example, Sambrook et al., Ibid.). When a polypeptide of zCGTF4 is expressed in bacteria such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed to the perplastic space by a sequence of bacterial secretion. In the first case, the cells are used and the granules are recovered and denatured using, for example, guanidine isothiacyanate or urea. The denatured polypeptide can then be re-folded and dimerized by diluting the denaturant, for example by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by breaking the cells (for example, by sonication or osmotic shock) to release the content of the periplasmic space and recover the protein, so that it is eliminated the need for denaturing and refolding. The transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components necessary for the growth of the chosen host cells. Various suitable means are known in the art, including defined means and complex media, and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The media may also contain such components as growth factors or serum, as required. The growth medium will generally be selected from cells containing exogenously added DNA, for example, by selection by drugs or deficiency in an essential nutrient, which is complemented by the selectable marker that is presented in the expression vector or cotransfected in the host cell. The P.metanolica cells are grown in a medium comprising suitable sources of carbon, nitrogen and nutrients in larger amounts at a temperature of about 25 ° C to 35 ° C. Liquid cultures are provided with sufficient aeration by conventional means such as agitation or small flasks or blenders or fermentors. A preferred culture medium for P.metanolica is YEPD (D-glucose 2%, Bacto ™ Peptone 2% (Difco Laboratories, Detroit, MI), bacto ™ 1% yeast extract (Difco Laboratories), 0.004% adenine and L- leucine 0.006%). The expressed ZCTGF4 recombinant polypeptides (or zCTGF4 chimeric polypeptides can be used conventional fractionation and / or purification methods and media) Precipitation with ammonium sulfate and extraction with acid or chaotrope can be used for the fractionation of samples. Purification specimens may include hydroxyapatite, size exclusion, FPLC and reverse phase high resolution liquid chromatography. Suitable chromatographic medium includes derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silica and the like. PEI, DEAE, CAE and Q derivatives are preferred. The exemplary chromatographic medium includes those media derivatized with phenyl, butyl groups or octyl, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Motgomeryville, PA), octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass spheres, silica based resins, cellulosic resins, agarose spheres, crosslinked agarose spheres, polystyrene spheres, crosslinked polyacrylamide resins and the like which are insoluble under conditions in which they are to be used. These supports can be modified with reactive groups that allow the binding of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups or carbohydrate moieties. Examples of coupling chemistry include activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl, activation with hydrazide and carboxyl and amino derivatives for coupling chemicals with carbodiimide. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for joining receptor polypeptides to support medium are well known in the art. The selection of a particular method is a matter of systematic design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatoqraphv: Principies _ Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated by utilization of size, charge and hydrophobicity. For example, immobilized metal ion absorption chromatography (IMAC) can be used to purify histidine rich proteins (E. Sulkowski, Trends in Biochem. 3 ,: 1-7, 1985). 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 (eg, a maltose binding protein, an immunoglobulin domain) can be constructed to facilitate purification. The zCTGF4 molecule has a domain homologous to the heparin binding domain previously described for CTGF and the utilization of this property may be useful for purification of ZCTGF4. For a review, see Burgess et al., Ann. Rev. of Biochem. 58_: 575-606, 1989. Members of the FGF family, which also have a heparin-binding domain, can be purified to apparent homogeneity by heparin-Sheparose affinity chromatography (Gospodarowicz et al., Proc. Nati. Acad. Sci. £ 1: 6963-6967, 1984) and eluted using linear lid gradients of NaCl (Ron et al., J. Biol. Chem. 268 (4): 2984-2988. 1993; Cromatoqraphy: Principies & Methods, pp. 77-80, Pharmacia LKB Biotechnology, Upssala, Sweden, 1993; in "Immobilized Affinity Ligand Techniques", Hermanson et al., eds., pp. 165-167, Academic Press. San Diego, 1992; Kjellen et al., Ann. Rev. Biochem. Ann. Rev. Biochem. 60: 443-474, 1991; and Ke et al., Protein Expr. Purif. 3 (6): 497-507. 1992). The methods of protein refolding or renaturation (and optionally reoxidation) can be used advantageously. It is preferred to purify the protein at 80% purity, more preferably at > 90% purity, even more preferably > 95% and particularly preferably in a pharmaceutically pure state, that is, more than 99% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin.

ZCTGF4 polypeptides or fragments thereof can also be prepared by chemical synthesis (Merrifield, J. Am. Chem. Soc. 85: 2149, 1963). The ZCTGF4 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 activity of the molecules of the present invention can be measured using various tests that measurecell proliferation, differentiation, chemotaxis or the induction of specialized cellular functions. Of particular interest are changes in the proliferation or differentiation of endothelial cells, particularly endothelial cells isolated from the testes, trachea, bone marrow or renal tissue. Proliferation and differentiation can be measured using in vitro using cultured cells, or in vivo by administering molecules of the claimed invention to the appropriate animal model. Tests that measure cell proliferation and differentiation are well known in the art. For example, assays that measure proliferation include assays such as chemosensitivity to the neutral red dye (Cavanaugh et al., Investigational New Drugs 8: 347-354, 1990, incorporated herein by reference), incorporation of radiolabeled nucleotides (Cook et al., Analvtical Biochem. 179: 1-7, 1989, incorporated herein by reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of proliferating cells (Porstmann et al., J. Immunol Methods 8_2: 169-179, 1985, incorporated herein by reference) and the use of tetrazolium salts (Mosmann, J. Immunol. Methods £ 5: 55-63, 1983; Alley et al., Cancer Res. 48: 589-601 , 1988, Marshall et al., Growth Req. 5: 69-84, 1995, and Scudiero et al., Cancer Res. 48.:4827-4833, 1988, all incorporated herein by reference). Tests that measure differentiation include, for example, the measurement of markers of cell surface associated with cap-specific expression of a tissue activity, enzymatic, or functional activity or morphological changes (Watt, FASEB, 5: 281-284, 1991; Francis, Differentiation 57: 63-75, 1994; Raes, Adv. Ani. Cell Biol Technol. Bioprocesses, 161-171, 1989, all incorporated herein by reference). Examples of assays that measure the induction of specialized cellular functions include: extracellular matrix protein mRNA induction assay (Frazier et al., J. Invest, Dermatol 107: 404-411, 1996); 35S methionine pulse-box assays that measure the stimulation of matrix protein synthesis (Frazier et al., ibid., 1996); subcutaneous administration of growth factors to mice (Roberts et al., Proc. Nati, Acad. Sci. USA 83: 4167-4171, 1986) and in situ hybridization to measure changes in mRNA expression (Fava et al., Blood 76: 1946-1955, 1990). An exemplary in vivo assay is when expression of transfected (or cotransfected) mammalian host cells can be embedded in an alginate environment and injected (implanted) into recipient animals. The microencapsulation of alginate-poly-L-lysine, the encapsulation and diffusion of selective membrane chambers have been described as a means to trap transfected mammalian cells or primary mammalian cells. These types of non-immunogenic "encapsulations" or microenvironments allow the transfer of nutrients to the inside the microenvironment, and also allows the diffusion of proteins and other macromolecules secreted or released by the cells captured through the environmental barrier to the recipient animal. More importantly, the capsules of the microenvironments mask and protect the foreign embedded cells from the immune response of the recipient animal. Such microenvironments can prolong the life of cells injected from a few hours or pathways (bare cells) to several weeks (embedded cells). An in vivo approach to assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpes virus, retrovirus, vaccinia virus 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 Becker et al., Meth Cell Biol. 43: 161-89, 1994 and Douglas and Curiel, Science &Medicine 4: 44-53, 1997). The adenovirus system provides several advantages: (i) the adenovirus can harbor relatively large DNA inserts; (ii) can grow to a high degree; (iii) it infects a wide range of mammalian cell types; and (iv) it can be used with many different promoters including ubiquitous, tissue-specific and regulatable promoters. In addition, because the adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. The use of adenoviral vectors in which portions of the adenovirus genome have been deleted, the inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a cotransfected plasmid. In an exemplary system, the essential gene is deleted from the viral vector, and the virus is not replicated, unless the El gene is provided by the host cell (e.g., the human 293 cell line). When administered intravenously to intact animals, the adenovirus is directed primarily to the liver. If the adenoviral delivery system has a deletion of the El gene, the virus can not replicate in the host cells. However, host tissue (e.g. liver) will express and process (and, if a signal sequence is present, secretory, secrete) the heterologous protein. The secreted proteins will enter circulation in the highly vascularized liver, and the effects in the infected animal can be determined.

In addition, adenoviral vectors containing several deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector. Such adenoviruses are deleted for El, and also contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72: 2022- 2032, 1998; Raper, S.E. et al., Human Gene Therapy 9: 671-679, 1998). In addition, it has been reported that the suppression of E2b reduces immune responses (Amalfitano, A. et al., J. Virol 72; 926-933, 1998). In addition, by removing the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated. The generation of adenovirus denominated "without intestine" in which all the viral genes are suppressed, are particularly advantageous for insertion of large inserts of heterologous DNA. For a review see Yeh, P. and Perricauder, M., FASEB J. 11: 615-623, 10 1997). The adenovirus system can also be used for in vitro protein production. By culturing cells that are not 293 infected with adenovirus under conditions where the 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, and then exposed to the adenoviral vector encoding the secreted protein of interest. Then the cells are grown under free conditions serum, which allows infected cells to survive for several weeks without significant cell division. Alternatively, 293 infected cells can be grown with adenovirus vector, as adherent cells or in suspension culture at a relatively high cell density to produce important amounts of protein (see Garnier al., Cvtotechnol 5: 145-155, 1994). With either protocol, a secreted heterologous protein, expressed, can be repeatedly isolated from the cell culture supernatant, the lysate or the membrane fractions, depending on the arrangement of the protein expressed in the cell. Within the production protocol of infected 293 cells, the non-secreted proteins can also be obtained effectively. Assays can be used to measure other cellular responses that include chemotaxis, adhesion, changes in the ion channel input flux, regulation of second messenger levels, and neurotransmitter release. Such assays are well known in the art. See, for example, in "Basic &Clinical Endocrinology Ser., Vol. Vol. 3," Cvtochemical Bioassavs: Technjques & Applications, Chayen; Chayen, Bitensky, eds., Dekker, New York, 1983. In view of the tissue distribution observed for ZCTGF4, agonists (including the natural ligand) and antagonists have enormous potential in both in vitro and in vivo applications. Compounds identified as ZCTGF4 agonists are useful for stimulating the proliferation and / or differentiation of cells in culture. For example, agonist compounds are useful as components of defined cell culture media and can be used alone or in combination with other cytokines and hormones to substitute serum which is usually used in cell culture. Therefore, agonists are useful especially in promoting the growth or development of cells derived from testis, trachea, bone marrow or kidney tissues or from endothelial fibroblast cells and derived from cultured filament. Agonists (including ZCTGF4) will be useful for increasing the production of extracellular matrix components and can be used in the treatment of connective tissue. Particularly, the agonists will be useful as a treatment for ligaments, cartilages and tendons. By virtue of the tissue distribution for the expression of antler molecules, the agonists will be useful for improving the healing and stabilization of wounds and as a component for artificial skin. The presence of ZCTGF4 expression in bone marrow suggests that the molecules of the present invention play a role in hematopoiesis. That role is probably indirect, where the stromal cells within the architecture of the bone marrow secrete, ZCTGF4, modulate the production of cells of the hematopoietic line. Antagonists will be useful for inhibiting the expression of specialized cellular functions, such as the production of extracellular components and inhibition of cell proliferation. Genes encoding polypeptides having potential polypeptide binding domains of ZCTGF4 can be obtained by random library analysis of peptides shown in the phage (phage display) or in bacteria, such as E. coli. Nucleotide sequences encoding polypeptides can be obtained in numerous ways, for example by random mutagenesis and random synthesis of polynucleotides. These random peptide display libraries can be used to analyze peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and analyzing such random peptide display libraries are known in the art (Ladner et al., U.S. 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 random peptide display libraries and kits for analyzing 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). Random peptide display libraries can be analyzed using the ZCTGF4 sequences described herein to identify proteins which bind to ZCTGF4. These "binding proteins" which interact with ZCTGF4 polypeptides can be used to label cells; for isolation of homologous polypeptides by affinity purification; they can be conjugated directly or indirectly to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods for example for expression library analysis and neutralizing activity. The binding proteins can also be used for diagnostic assays to determine circulating levels of polypeptides; to detect or quantify soluble polypeptides as markers of the underlying pathology or disease. These binding proteins can also act as "antagonists" of ZCTGF4 to block the binding of ZCTGF4 and signal transduction in vitro and in vivo. These binding proteins against ZCTGF4 can be useful for inhibiting the expression of genes which result in the proliferation, differentiation or induction of specialized cell functions, such as the production of extracellular matrix. Such binding proteins against ZCTGF4 can be used for treatment in bone marrow fibrosis, modulate the production or differentiation of hematopoietic cells, prevention of scar tissue formation, cutaneous lupus erythematosus, scleroderma, dermatositis, and end-stage renal failure, alone or in combination with other therapies. ZCTGF4 can also be used to identify inhibitors (antagonists) of its activity. The test compounds are added to the assays described here to identify compounds that inhibit the activity of ZCTGF4. In addition to these assays described herein, samples can be tested to determine the inhibition of ZCTGF4 activity in various assays designed to measure receptor binding or stimulation / inhibition of ZCTGF4-dependent cellular responses. For example, cell lines that respond to zCTGF4 can be transfected with an indicator gene construct that responds to the cell pathway stimulated by ZCTGF4. Indicator gene constructs of this type are known in the art, and will generally comprise a ZCTGF4-DNA response element operably linked to a gene encoding a test able protein, such as luciferase. DNA response elements may include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE), insulin response elements (IRE) (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 response elements of cyclic AMP are reviewed in Roesteler 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 their ability to inhibit ZCTGF4 activity in the target cells, as evidenced by a decrease in ZCTGF4 stimulation of the expression of the indicator gene. Assays of this type will detect compounds that directly block the binding of zCTGF4 to cell surface receptors, as well as compounds that block processes in the cell pathway subsequent to receptor-ligand binding. Alternatively, compounds or other samples can be tested for direct blockade of zCTGF4 binding to the receptor using ZCTGF4 labeled with a detectable label (eg 125 I, biotin, horseradish peroxidase, FITC and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled ZCTGF4 to the receptor is indicative of the inhibitory activity, which can be confirmed by secondary assays. The receptors used within the binding assays can be cellular receptors or immobilized and isolated receptors. A ZCTGF4 polypeptide can be expressed as a fusion with a constant region of an immunoglobulin heavy chain, typically an Fc fragment, which contains two domains of constant region and lacks the variable region. Methods for preparing such fusions are described in U.S. Pat. numbers 5,155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules, wherein the Fc portions are disulfide linked together and two non-Ig polypeptides are arranged in close proximity to each other. Mergers of this type can be used to purify by affinity ligand, as an in vitro assay tool, or antagonist. For use in the assays, the chimeras are bound to a support via the Fc region and used in an ELISA format. A ligand-binding polypeptide of ZCTGF4 can also be used for ligand purification. The polypeptide is immobilized on a solid support, such as spheres of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide or similar materials that are stable under the conditions of use. Methods for linking 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 fluids containing the ligand are passed through the column one or more times to allow the ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine hydrochloride), or pH to interrupt ligand-receptor binding. A test system using a ligand-binding receptor (or an antibody, a complement / anti-complement 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 chip. The use of this instrument is described by Karlsson, J. Im unol. Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234: 554-63. 1993. A receptor, antibody, member or fragment is covalently linked using amine or sulfhydryl chemistry, to dextran fibers that bind to a gold film within the flow cell. 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 of the medium, which is detected as a change in the surface plasmon resonance of the gold film. This system allows the determination of activation and deactivation rates from which the binding affinity can be calculated, and determine the binding stoichiometry. Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include? Catchard analysis for binding affinity determination (see Scatchard, Ann. NYAcad. Sci. 51: 660-72, 1949) and assays. calorimetric (Cunningham et al., Science 53: 545-48, 1991; Cunningham et al., Sciences 245: 821-25, 1991). The zCTGF4 polypeptides can also be used to prepare antibodies that bind ZCTGF4 epitopes, peptides or polypeptides. The ZCTGF4 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and induce an immune response. A person skilled in the art will recognize that the antigenic epitope-presenting polypeptides 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 zCTGF polypeptide (e.g., SEQ ID NO: 2). Polypeptides comprising a larger portion of a ZCTGF4 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 attached labels, adjuvants and carriers, as described herein. Suitable antigens include the ZCTGF4 polypeptide encoded by SEC. FROM IDENT. NO: 2 from amino acid number 24 to amino acid number 354, or a contiguous fragment of amino acids 9 to 330 thereof. Other suitable antigens include the IBP domain (residues 59-102), von Willebrand factor C domain (residues 114-179), variable domain (residues 180-208), and a glycoconjugate binding motif domain sulphated (residues 209-252) as described herein. Preferred peptides for use as antigens are hydrophilic peptides such as those predicted by a person skilled in the art from a hydrophobicity plot (see figure). The hydrophilic peptides of ZCTGF4 include peptides comprising amino acid sequences that are selected from the group consisting of: residues 239-244, residues 105-110, residues 172-177, residues 238-243 and residues 171-176, all of the SEC . FROM IDENT. NO: 2. 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 polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunolo and, Cooligan, et al., (Eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloninq: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY, 1989; and Hurrell, JGR, Ed., Monoclonal Hvbridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton FL, 1982. As is evident to a person ordinarily skilled in the art, polyclonal antibodies can be generated from inoculating various homeothermic animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice and rats with a ZCGTF4 polypeptide or a fragment thereof. The immunogenicity of a zCGTF4 polypeptide can be increased by 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 ZCGTF4 fusions or a portion thereof with an immunoglobulin polypeptide or with a maltose binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be attached or advantageously be linked to a macromolecular carrier (such as keyhole limpet lock (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization . As used herein, the term "antibodies" includes polyclonal antibodies, polyclonal antibodies purified by affinity, monoclonal antibodies and antigen-binding fragments such as the proteolytic fragments F (ab ') 2 and Fab. Also included are engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic peptides and polypeptides that bind antigen. Non-human antibodies can be humanized by grafting non-human CDRs into human infrastructure and constant regions, or by incorporating all non-human variable domains (optionally "hiding" them with a human-like surface by substitution of exposed residues, where the result is a "coated" antibody). In some cases, humanized antibodies can retain non-human residues within human variable region infrastructure domains to improve the appropriate binding characteristics. Through humanized antibodies, the biological half-life can be increased, and the potential for adverse immune reactions when administered to humans is reduced. In addition, human antibodies can be produced in transgenic non-human animals that have been engineered to contain human immunoglobulin genes, described in WIPO publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, for example by homologous recombination. The antibodies are considered to bind specifically if (1) they show a threshold level of binding activity, and (2) they do not significantly cross-react with related polypeptide molecules. a threshold level of binding is determined if antibodies against ZCGTF4 to this bind to a polypeptide, peptide or epitope ZCGTF4 with an affinity at least 10 times greater than the binding affinity with a control polypeptide (other than ZCGTF4) . It preferred that the antibodies show a binding affinity (Ka) of 106 M "1 or greater, preferably 107 M'1 or greater, more preferably 10β M'1 or greater, and more preferably 109 M" 1 or greater. The affinity of a monoclonal antibody can be easily determined by a person ordinarily skilled in the art for example by Scatchard analysis (Scatchard, G.I. Ann.N.A.Acid.Sci.51: 660-672, 1949). The fact that antibodies against ZCGTF4 not give significant crossreactivity with related polypeptide molecules is shown, for example, by detection of antibody polypeptide of zCGTF4 but not known related polypeptides using a standard Western blot analysis (Western blot) (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, such as other human CTGFs (e.g., CTGF and CTGF-2). The analysis can also be performed using non-human zCGTF4 and mutant polypeptides of zCGTF4. In addition, the antibodies can be "analyzed against" known related polypeptides, to isolate a population that specifically binds to ZCGTF4 polypeptides. For example, the antibodies generated for ZCGTF4 are absorbed with related polypeptides adhered to an insoluble matrix; antibodies specific for ZCGTF4 will flow through the'- ^ s- • * -3e matrix under the appropriate buffer conditions. The analysis allows the isolation of polyclonal and monoclonal antibodies that are not cross-reactive with closely related related polypeptides (Antibodies: A Laboratorv Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunoloqy, Cooligan, et al, (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). The analysis 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., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984. Antibodies to ZCGTF4 that are specifically bound by numerous methods in the art can be detected and described below. Various assays known to those skilled in the art can be used to detect antibodies which bind ZCGTF4 proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboator Manual, Harlow and La e (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, immunosorbent assay bound to enzyme (ELISA), dot blot or assay Western blot, inhibition or competition test and interposition test (sandwich). In addition, the antibodies can be analyzed for binding to a wild type versus mutant zCGTF4 polypeptide. Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to the zCGTF4 protein or peptide, and selection of antibody display library in phage or in similar vectors (e.g., by the use of an immobilized or labeled protein or peptide of ZCGTF4). The genes coding for polypeptides having potential polypeptide binding domains for zCGTF4 can be obtained by random analysis of peptide libraries displayed on a phage (phage display) or on bacteria, such as E. coli. The nucleotide sequences encoding the polypeptides can be obtained in many ways, for example by random mutagenesis and random synthesis of polynucleotides. These random peptide display libraries can be used to analyze looking for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, or a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and analyzing such random peptide display libraries are known in the art (Ladner et al., U.S. 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. Pat. No. 5,571,698) and random peptide display libraries and kits for analyzing 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). Random peptide display libraries can be analyzed using the ZCTGF4 sequences described herein to identify proteins which bind to zCTGF. These "binding polypeptides" which interact with the ZCTGF4 polypeptide can be used to direct cells; for isolation of 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 analytical methods for example for analysis of expression libraries and neutralizing activity, for example, to block the interaction between the ligand and the receptor, or for viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays to determine circulating levels of ZCTGF4 polypeptides; to detect or quantify soluble zCTGF4 polypeptides as a marker of underlying pathology or disease. These binding polypeptides can also act as "antagonists" of zCTGF4 to block the binding of ZCTGF4 and signal transduction in vi tro and in vivo. These polypeptides that bind to zCTGF4 may be useful for inhibiting ZCTGF4 activity or protein binding. Antibodies to ZCTGF4 can be used to label cells that express zCTGF4; to isolate ZCTGF4 by affinity purification; for diagnostic assays to determine the circulating levels of ZCTGF4 polypeptides; to detect or quantify soluble ZCTGF4 as a marker of an underlying pathology or disease; in analytical methods that use FACS; for the analysis of expression libraries; to generate anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zCTGF4 in vi tro and in vivo. In particular, the antibodies will be useful for diagnosis, due to the association of proteins of the present invention with the extracellular matrix and the vessels, and the tagged proteins will be useful in the diagnosis of diseases such as bone marrow fibrosis, prevention of formation of scar tissue, cutaneous lupus erythematosus, scleroderma, dermatositis and renal failure of the final stage. The antibodies or polypeptides herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like and these conjugates can be used for in vivo diagnosis or for therapeutic applications. In addition, antibodies to zCTGF4 or fragments of them can be used in vi tro to detect denatured zCTGF4 or fragments thereof in assays, eg, Western blot or other assays known in the art. The antibodies or polypeptides herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates can be used for in vivo diagnosis or therapeutic applications. For example, the polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anticomplementary molecule (antigen receptor, respectively, for example). More specifically, ZCTGF4 polypeptides or antibodies against ZCTGF4, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anticomplementary molecule. For example, for the use of antibodies and polypeptides, labeled for detection by imaging technologies, they will be useful for diagnosing diseases associated with the extracellular matrix and vessels, such as bone marrow fibrosis, aberrant hematopoiesis, prevention of tissue formation. scarring, cutaneous lupus erythematosus, scleroderma, dermatositis and renal failure in the final stage.

Suitable detectable molecules can be linked directly or indirectly to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like. Suitable cytotoxic molecules can bind directly or indirectly to the polypeptide or antibody, and include bacterial or plant toxins (e.g., diphtheria toxins, Pseudomonas exotocin, ricin, abrin, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either linked directly to the polypeptide or antibody, or indirectly linked through a medium of a chelating moiety, for example). The polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect binding of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary / anticomplementary pair, wherein the other member is bound to the polypeptide or antibody portion. For these purposes, the pair of biotin / streptavidin is an exemplary complementary / anticomplementary pair. In another embodiment, polypeptide-toxin fusion proteins or antibody-toxin fusion proteins ee can be used to direct inhibition or suppression of cells or tissues (e.g., to treat diseases caused by the inappropriate growth of cells or tissues). Such fusion protein molecules therefore represent a genetic targeting vehicle for cell / tissue specific delivery of conjugates of detectable / cytotoxic anticomplementary generic molecules. In another embodiment, zCTGF4-cytokine fusion proteins or antibody-cytokine fusion proteins can be used to enhance the in vivo destruction of target tissues (e.g., cancers in the blood or bone marrow), if the ZCTGF4 polypeptide or the antibody against ZCTGF4 is directed to hyperproliferative blood or bone marrow cells (see generally, Hornick et al., Blood .89: 4437-47, 1997). Fusion proteins are described that allow targeting a cytokine to a desired site of action, whereby a high local concentration of cytokine is provided. Suitable ZCTGF4 polypeptides with antibodies against zCTGF4 directed against an undesirable cell or tissue (i.e., a tumor or a leukemia) and improved target cell lysis by means of cytokines fused by effector cells. Cytokines suitable for this purpose include interleukin 2 and granulocyte-macrophage colony stimulating factor (GM-CSF), for example. In another embodiment, if the ZCTGF4 polypeptide or the anti-ZCTGF4 antibody is directed to vascular cells or tissues, such a polypeptide or antibody can be conjugated to a radionuclide, and particularly with a radionuclide that mimics beta radiation, to reduce restenosis. Such a therapeutic solution generates less harm to the doctors who administer the radioactive therapy. For example, tapes impregnated with iridium-192 are placed in the enlarged vessels of patients until the required dose of radiation is delivered and a decrease in tissue growth in the vessel and larger luminal diameter is shown compared to the control group, which receives placebo tapes. In addition, revascularization and expanded thrombosis are significantly lower in the group undergoing treatment. Similar results are predicted by directing a bioactive conjugate containing a radionuclide, as described herein. The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or can be introduced locally at the proposed site of action. The molecules of the present invention can be used to identify and isolate receptors involved in the growth and differentiation of cells that respond to ZCTGF4. For example, the proteins and peptides of the present invention can be immobilized on a column and membrane preparations run on the column (Immobilized Affinitv Ligand Technisues, Hermanson et al., Eds., Academic Press, San Diego, CA, 1992, pp. 195-202). The proteins and * ____... -___ .. peptides can also be radiolabeled (Methods in Enzymol., vol.182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or can be labeled for photoaffinity (Brunner et al., Ann. Rev. Biochem. 62: 483-514, 1993 and Fedan et al., Biochem Pharmacol 33: 1167-1180, 1984) and can be identified in specific cells-surface proteins. The molecules of the present invention will be useful for regulating the growth and / or differentiation of cells that respond to ZCTGF4. The polypeptides, nucleic acids and / or antibodies of the present invention can be used in the treatment of disorders associated with the unregulated growth of tissues responsive to ZCTGF4. In particular, the molecules of the present invention can be used to produce antagonists to treat or prevent the development of pathological conditions in tissues such as testes, trachea, bone marrow and kidney. Some diseases such as bone marrow fibrosis, prevention of scar tissue formation, cutaneous lupus erythematosus, scleroderma, dermatositis and renal failure in the final stage, may be susceptible to such diagnosis, treatment or prevention. The polynucleotides encoding the ZCTGF4 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit the activity of ZCTGF4. If a mammal has mutated or lacks the gene for zCTGF4, the zCTGF4 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a ZCTGF4 polypeptide is introduced in vivo, in a viral reactor. 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, which are completely or almost completely free of viral genes, are preferred. A defective virus is not infective after its introduction into a cell. The use of defective viral vectors allows administration to cells in a specific localized area, without concern that the vector may infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 vector (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. 9J3: 626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61: 3096-101, 1987, Samulski et al., J. Virol. 63: 3822-28, 1989). In another embodiment, the ZCTGF4 gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell 31: 153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289, Markowitz et al., J. Virol. 62: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al .; and Kuo et al. Blood 82: 845-852, 1993. Alternatively, the vector can be introduced by ip vivo lipofection using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transaction of a gene encoding a marker (Felgner et al., Proc. Nati, Acad. Sci. USA 84: 7413-17, 1987; Mackey et al., Proc. Nati, Acad. Sci. USA 8_5: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. The molecular targeting of liposomes to specific cells presents an area of benefit. More particularly, transfection directed to particular cells represents an area of benefit. For example, directing transfection to particular cell types can be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney and brain. The lipids can be chemically coupled to other molecules for the purpose of directing them. Targeted peptides (for example hormones or neurotransmitters), 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 vectors as a plasmid of naked DNA, and then re-implant the transformed cells in the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, eg, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, the use of a gene cannon or the use of a transporter of a DNA vector. See, for example, Wu et al., J. Biol. Chem. 267: 963-67, 1992; Wu et al., J. Biol. Chem. 263: 14621-24. 1988. The antisense methodology can be used to inhibit transcription of the gene for ZCTGF4, so as to inhibit ip cell proliferation. Polynucleotides that are complementary to a segment of a polynucleotide encoding ZCTGF4 (e.g., a polynucleotide as set forth in SEQ ID NO: 1) are designated to bind to an mRNA encoding zCTGF4 and to inhibit translation. of such mRNA. Such antisense polynucleotides are used to inhibit the expression of genes encoding the ZCTGF4 polypeptide in cell cultures or in a subject. The present invention also provides reagents which will find use in diagnostic applications. For example, the gene for ZCTGF4, a probe comprising DNA or RNA for zCTGF4, or a subsequence thereof, can be used to determine if the ZCTGF4 gene is present on chromosome 6 or if a mutation has occurred. The chromosomal aberrations detectable at the locus of the zCTGF4 gene include, but are not limited to aneuploidy, change in the number of gene copies, insertions, deletions, restrictions, 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 tandem repeat analysis (STR) using PCR techniques, and other genetic binding analysis techniques known in the art (Sambrook et al., Ibid., Ausubel et al., Ibid., Marian, Chest 108: 255-65, 1995).

Mice engineered to express the ZCTGF4 gene, referred to as "transgenic mice," and mice showing a complete absence of the function of the zCTGF4 gene, referred to as "knockout mice" can also be generated (Snouwaert et al. ., Science 57: 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 overexpress ZCTGF4, either ubiquitously or under a tissue-specific or tissue-restricted promoter, can be used to analyze whether overexpression causes a phenotype. For example, overexpression of a polypeptide of wild-type zCTGF4, a polypeptide fragment or a mutant thereof, can alter normal cellular processes, resulting in a phenotype that identifies a tissue in which the expression of zCTGF4 is functionally relevant and may indicate a therapeutic target for ZCTGF4, its agonists or antagonists. In addition, such overexpression can result in a phenotype that shows similarity to human diseases. Similarly, the agonic mice for zCTGF4 can be used to determine if ZCTGF4 is absolutely required ip live. The phenotype of the athymic mice is a predictor of the live ip effects that could be had by this ZCTGF4 antagonist, such as those described herein. The cDNA for human zCTGF4 can be used to isolate mRNA, cDNA and murine genomic DNA for ZCTGF4, which is subsequently used to generate agénic mice. These mice can be used to study the ZCTGF4 gene and the protein encoded by it in an in vivo system, and can be used as in vivo models for corresponding human diseases. In addition, the transgenic expression in mouse of antisense polynucleotides of ZCTGF4 or ribosomes directed to ZCTGF4, described herein, can be used analogously to the transgenic mice described above. For pharmaceutical use, the proteins of the present invention are formulated for parenteral delivery, particularly intravenous or subcutaneous, in accordance with conventional methods. Intravenous administration will be by bolus injection or by infusion over a typical period of one to several hours. In general, the pharmaceutical formulations will include a ZCTGF4 protein in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water or the like. The formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss on bottle surfaces, etc. Formulation methods are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacv, Gennaro, ed. , Mack Publishing Co., Easton, PA, 19th ed., 1995. Therapeutic doses will generally be in the range of 0.1 to 100 μg / kg of patient weight per day, preferably 0.5-20 μg / kg per day, where The exact dose is determined by the doctor according to the affected standards, taking into consideration the nature and severity of the condition to be treated, the patient's features, etc. The determination of the dose is within the level of skill usual in the art. The proteins can be administered for acute treatment, for a week or less, often over a period of one to three days, or they can be used in chronic treatment, for several months or years. The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1 The scanning of a translated DNA database using the motifs of the CTGF family as a key, results in an identification of an EST sequence that is found to have some homology with the family of connective tissue growth factor. The plasmid DNA is isolated from a clone corresponding to EST, which has been called zCTGF4, and analyzed for the polynucleotide sequence. Upon alignment of the cDNA for ZCTGF4 and the CTGF family, the clone is shown to contain a truncated cDNA sequence with an mtron at the 5 'end of the sequence. Based on the tissue distribution by Northern blot analysis (see Example 2), a DNA library is constructed from human testes, and used to analyze a full-length clone. It is estimated that the library contains approximately 106 clones. A master plate containing 80 accumulations (each accumulated represents 2 accumulated of 1.25 x 104 clones) is analyzed using PCR to determine if the cDNA for ZCTGF4 is present. The reactions are established using: 1 μl of each accumulated, 20 pmol of each of the oligonucleotide primers ZC14,882 and ZC14,883 (SEQ ID NOS: 6 and 7 respectively), and 1 U of ExTaq DNA polymerase "(TaKaRa Shuzo Co., Ltd., Shiga, JP) in a volume of 25 μl PCR is carried out in a 96-well plate in a GenAmp 9700 PCR system (PE Applied Biosystems, Cheshire, UK) The reaction is carried out as follows: 94 βC for 1.5 minutes, then for 30 cycles of 94 ° C, 15 seconds, 55 ° C 20 seconds, 72 ° C 30 seconds, and finish with 7 minutes of incubation at 72 ° C. The accumulated substances named A3, A6, D3, D6 and F7 were positive for the presence of DNA for zCTGF4 and are selected for further analysis Positive accumulations are further analyzed with PCR to identify clones with larger 5 'end sequences The reaction mixture contains 1 μl of each accumulated, 20 pmoles of each of the primers oligonucleotides ZC15.909 (corresponding to a vector sequence) and ZC14.885 (corresponding to a gene-specific sequence (SEQ. FROM IDENT. NOS: 2 and 9, respectively), and 1 U of a mixture of ExTaq DNA polymerase (TaKaRa) and Pfum (Stratagene, La Jolla, CA) (2: 1), in a total volume of 25 μl. PCR is performed in a GenAmp 2400 PCR system (PE Biosystems) as follows: 94 ° C for 1.5 minutes, then for 25 cycles of 94 ° C, 15 seconds; 55 ° C 20 seconds; 72 ° C 30 seconds; incubation for 7 minutes at 72 ° C. A second anchored and hosted PCR is performed using 1 μl of the first round of PCR products diluted 1/50 as a template, 20 pmoles of each of the oligonucleotide primers ZC15,911 (corresponding to a vector sequence) and ZC14,884 (corresponding to a gene specific sequence (SEQ ID NO. NOS: 10 and 11, respectively), and 1 U of a mixture of ExTaq DNA polymerase (TaKaRa) and Pfum (Stratagene) (2: 1) in a total volume of 25 μl The reaction proceeds as follows: 94 ° C for 1.5 minutes, then for 25 cycles of 94 ° C, 15 seconds, 50 ° C 20 seconds, 72 ° C 30 seconds, incubation for 7 minutes at 72 ° C. The PCR products of A3, A6, D3 and D6 gel-purified with QIAquick gel extraction equipment (Qiagen Inc. Chatsworth, CA), are subcloned into a pCR2.1 vector of the TA Cloning ™ kit (Invitrogen, Carlsbad, CA), which provides direct linkage within expression vectors (Mead et al., Bio / Technology 9 (7): 657-663, 1991), and are designated CTGF4a3, CTGF4a6, CTGF4d3, CTGF4d6. The sequences of CTGF4a3, CTGF4a6, CTGF4d3 and CTGF4d6 show that the sequence of CTGF4a3 codes for an initial Met with a putative signal peptide, a large open reading frame (ORF) in which the first reading frame is interrupted by a codon of detention, and a short ORF subsequently in the second reading frame. The short ORF is identical to the coding region of the 5 'end of ZCTGF4, while the long ORF has a 45% similarity in sequence with human CTGF. The sequence of CTGF4a6 is almost identical to CTGF4a3 except that it is short in 16 amino acids from the N-terminal part in the peptide of the secretory signal. Both CTGF4d3 and CTGF4d6 have smaller inserts. The four clones code for a long ORF interrupted by a stop codon in the same place. A PCR approach is used, as previously described, to find a correctly coded sequence in other tissues. Human bone marrow marathon cDNA is used based on the results of Northern blot analysis (see Example 2). CDNA is prepared using the Clontech Marathon ™ cDNA kit (Clontech, Palo Alto, CA), according to the manufacturer's specifications. 5 μl of Marathon ™ bone marrow cDNA diluted 1/100, 20 pmoles of each of the oligonucleotide primers ZC15,910 and ZC14,885 (SEQ ID NOS: 12 and 9, respectively), and 1 U are used. of DNA polymerase ExTaq (TaKaRa) and Pfum (Stratagene) (2: 1) in 25 μl of reactions. The reactions are carried out as follows: 94 ° C for 1.5 minutes, and then for 25 cycles of 94 ° C, 15 seconds; 54 ° C 20 seconds; 72 ° C 30 seconds; and finish with a 7 minute incubation at 72 ° C. 1 μl of a first PCR product diluted 1/50 is used as template for PCR. 20 pmoles of each of the oligonucleotide primers ZC16,192 and ZC14,884 (SEQ ID NOS: 13 and 11, respectively) and 1U of ExTaq DNA polymerase (TaKaRa) are used. and Pfu ™ (Stratagene) (2: 1), in 25 μl of reactions. The reactions are carried out as follows: 94 ° C for 1.5 minutes, then for 25 cycles of 94 ° C, 15 seconds; 50"C 20 seconds, 72 ° C 30 seconds, and end with a 7 minute incubation at 72 ° C. The PCR product is gel purified, subcloned into the vector pCR2.1 (as previously described) and The sequence analysis shows that the 5 'and 3' end of the bone marrow clone overlaps with the testicular clones, with a discrepancy in the region that has a displacement in the reading frame in the clone of the testes The sequence of the bone marrow clone has a correct ORF, and is included in the full length sequence composed of ZCTGF 4. The initially identified EST, which originates from a penile library, as well as clones identified from two libraries of testicles, have introns or read frame shifts in the same region.A later analysis shows that this region is a less conserved area when compared with other members in the family, suggesting that the displacement of reading frame It can be a regulatory mechanism.

Example 2 Northern analysis is performed using samples of human multiple tissue spots (Human Mutliple Tissue Blots) I, II and III of Clontech (Palo Alto, CA). A probe is generated from a gel-purified PCR product, made from ZC14,883 (SEQ ID NO: 7) and ZC14,882 (SEQ ID NO: 6) as primers, and ZCTGF4 as a template, which has been radioactively labeled with a REDIPRIME ™ DNA labeling kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. The probe is purified using a NUCTRAP push column (Stratagene). An EXPRESSHYB ™ solution (Clontech) is used for prehybridization and as a hybridization solution for the Northern blot test. Hybridization is carried out overnight at 65 ° C and the spots are subsequently washed in 2X SSC and 0.05% SDS at RT (room temperature), followed by a wash at 0. IX SSC and 0.1% SDS at 50 ° C. . A larger transcript with a size of 1.4 kb is observed. The signs are present in the testicle, bone marrow, trachea, kidney, liver, stomach, small intestine, ovary, placenta, prostate and spinal cord. The expression of ZCTGF4 is also examined with a human RNA Master blot (Clontech) with a probe generated from a PCR product amplified with ZC16,192 (SEQ ID NO: 13) and ZC14,884 (SEQ ID NO: 11) as primers and ZCTGF4 as DNA template. The labeling conditions of the probe and hybridization were the same as those described above. He ZCTGF4 polynucleotide is positive in two other tissues, mammary gland and fetal kidney, in addition to those described above.

Example 3 ZCTGF4 is mapped to crosome 6 using the commercially available "GeneBridge 4 Radiation Hybrid Panel" (Research Genetics, Inc., Huntsville, AL). The hybrid GeneBridge 4 radiation equipment (Genebridge 4 Radiation Hybrid Panel) contains DNA from each of 93 hybrid radiation clones plus 2 control DNA (the donor HFL and the A23 receptor). A publicly available WWW server (http: // www-genome, wi .mit .edu / cgi-bin / contig / rhmapper.pl) allows mapping in relation to the hybrid radiation map of the human genome of Whitehead Institute / MIT Center for Genome Research (the hybrid radiation map of "WICGR") which is constructed with the hybrid GeneBridge 4 radiation equipment. For the mapping of zCTGF4 with the "GeneBridge 4 RH Panel", reactions are performed in 20 μl, in a 96-well microtiter plate (Stratagene, La Jolla, CA), and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the 95 PCR reactions consists of 2 μl of PCR reaction buffer (KlenTaq 10X (CLONTECH Laboratories, Inc., Palo Alto, CA), mixtures of 1.6 μl of dNTP (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl of direct primer, ZC15.089 (SEQ ID NO: 14), 1 μl of antisense primer, ZCL15.092 (SEQ ID NO: 15), 2 μl of "RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4 μl of polymerase mix (50X Advantage KlenTaq Polymerase Mix) (Clontech Laboratories, Inc.), 25 ng of DNA from a single hybrid clone or a control and ddH20 for a total volume of 20 μl. The reactions are covered with an equal amount of mineral oil and sealed. The conditions of the PCR cycler are as follows: an initial cycle of 5 minutes of denaturation at 95 ° C, 35 cycles of denaturation for 1 minute at 95 ° C, annealing of 1 minute at 56 ° C and extension from 1.5 minute to 72 ° C, followed by a final extension of a 7 minute cycle at 72 ° C. The reactions are separated by electrophoresis on a 2% agarose gel (GIBCO-BRL Life Technologies, Gaithersburg, MD). of zCTGF4 6.29 cR_3000 from main structure marker Wl-4792 on the hybrid radiation map of WICGR chromosome 6. The markers of proximal and distal infrastructure were CHLC.GATA31.100, respectively.The use of surrounding markers of ZCTGF4 positions in the region 6q22.1 on the map of integrated chromosome 6 LDB (The Genetic Location Data Base, University of Southhampton, WWW server: http- // cedar.genetics.soton.ac.uk/public_html/).

Example 4 Several expression constructs are elaborated for the eukaryotic expression of zCTGF4 cDNA. A mammalian expression vector is constructed with the dihydrofolate reductase gene under the control of the SV40 early promoter, an SV40 polyadenylation site, a cloning site for inserting the gene of interest under the control of the MT-1 promoter and a polyadenylation site of hGH. The expression vector is referred to as pZP9 and is deposited with the American Type Culture Collection, 12301 Parkiawn Drive, Rockville, MD. To facilitate purification, the pZP9 vector is modified by the addition of a tPA leader sequence (US Pat. No. 5,641,655, incorporated herein by reference) and a GluGlu tag (SEQ ID NO: 16) between the MT-promoter. 1 and the hGH terminator. The tPA leader replaces the sequence of the active secretory signal for the DNAs encoding polypeptides of interest and which are inserted into this vector, and the expression results in a protein labeled in the N-terminal part. The vector labeled in the N-terminal part is called pZP9NEE. Another vector with a C-terminal GluGlu tag (SEQ ID NO: 16) inserted just 5 'with respect to the hGH terminator and using the sequence of the native (or other fused) secretory signal for the secretion of the encoded polypeptide interest, and the expression results in a protein labeled in its C-terminal part. The vector labeled GluGlu in its C-terminal part is called pZP9CEE. A 5 'DNA fragment containing the coding region ZCTGF4 (nucleotide 86 to nucleotide 613 of SEQ ID NO: 1) is generated by PCR using oligonucleotide primers (ZC16, 422 (SEQ ID NO: 17) and ZC16,424 (SEQ ID NO: 18) and CTGF4A3 as the template, and a 3 'DNA fragment including nucleotide 590 to nucleotide 1078 of SEQ ID NO: 1 and ZC16 oligonucleotides, 421 (SEQ ID NO: 19) and ZC16,425 (SEQ ID NO: 20) as primers, and zCTGF4 as template PCR reactions are carried out as follows: 1 cycle at 94 ° C for 1.5 minutes, 3 cycles of 94 ° C for 15 seconds, 50 ° C for 30, 72 ° C for 30 seconds, 12 cycles of 94 ° C for 15 seconds, 50 ° C for 20 seconds, 72 ° C for 30 seconds and 1 cycle at 72 ° C for 2 minutes PCR products are gel purified and mixed together as the template for the following PCR reaction: 1 cycle at 94 ° C for 1.5 minutes, 3 cycles at 94 ° C for 15 seconds, 54 ° C for 20; 68 ° C for 45 seconds; 20 cycles of 94 ° C for 15 seconds; 68 ° C for 45 seconds; and 1 cycle at 72 ° C for 2 minutes. This PCR product, which contains the entire polynucleotide sequence encoding the mature polypeptide of ZCTGF4, is purified in gel and is digested by restriction with BamHI and Xho I for use with the vector labeled in its N-terminal part, pZP9NEE described above. The DNA sequence for ZCTGF4 is ligated into the pZP9NEE vector and the E. coli transformants are selected, and ee termed pZP9NEE / zCTGF4. Plasmid DNA is isolated and the region of the plasmid containing the polynucleotides encoding the tPA leader is cut followed by DNA encoding the GluGlu tag (SEQ ID NO: 16) and the mature CTGF4 polypeptide, using a site EcoRI 5 'and Xbal 3'. The insert is analyzed in terms of its sequence for verification. A similar cloning method is used to generate an expression construct labeled in its C-terminal part. This construct, designated pZP9CEE / cCTGF4, comprises the pZP9CEE vector with the DNAs encoding the native zCTGF4 secretory signal sequence, the mature polypeptide of ZCTGF4 (shown in SEQ ID NO: 1) and the GluGlu label in the C-terminal part (SEQ ID NO: 16).

The fragment of pZP9NEE / zCTGF4 which codes for the tPA secretory signal peptide, the GluGlu tag and mature ZCVTGF4 are ligated into the baculovirus vector designated pZBV4L. The vector pZBV4L is a baculovirus expression vector derived from the FASTBAC vector of the Bac-to-Bac ™ system (GIBCO-BRL, Gaithersburg, MD), described in Luckow et al., ÜZ Virol. 67: 4566-4579, 1993. The pFASTBAC vector is modified to Removing the polyhedrin promoter and substituting the baculovirus basic protein promoter (Hill-Perkins et al., J. Gen. Virol. 71: 971-976, 1990, Bonning et al., J. Gen. Virol. 1551-1556, 1994, and Chazenbalk et al., J. Biol. Chem. 270: 1543-1549, 1995). 1 μl of expression construct zCTGF4NEE / pZBV4L is used to transform 20 μl of DHlOBac (GIBCO-BRL, Gaithersburg, MD) into 980 μl of SOC (Bacto Tryptone 2%, Bacto 0.5% yeast extract, 10 ml of 1 mM NaCl , 1.5 mM KCl, 10 mM MgCl 2, 10 mM MgSO 4 and 20 mM glucose), according to the manufacturer's specifications. The cells are incubated for 48 hours at 37 ° C and identified (white) and 2 colonies are isolated in which the virus has incorporated the plasmid (referred to as a "bacmid"). The bacmidic DNA is isolated and used to transfect Spodoptera frugiperda (Sf9) cells using transfection liposomes from Cellfectin (GIBCO-BRL). The cells are cultured at 27 ° C in shake flasks using 50-100 ml of serum free medium Sf900II (GIBCO-BRL). After 3-4 days, the medium conditioned with the virus is harvested and used to infect Sf9 cells in medium-log growth at approximately 1 x 10 6 cells / ml. The cells are seeded on a larger scale by adding cell cultures at volumes of 15 liters when the cells have reached a density between 1-2 x 10d cells / ml, and then they become infected with an MOI of 1-3. After 2 days at 27 ° C, the medium containing the protein and virus is harvested.

Example 5 A. ZCTGF4 protein affinity tagged Purified as follows zCTGF4 expressed with a N-terminal or C-terminal GluGlu (EE) tag: A mixture of protease inhibitors is added to a 2000 ml sample of conditioned medium from baculovirus-infected Sf9 cells to final concentrations of ethylenediaminetetraacetic acid (EDTA). ) 2.5 mM (Sigma Chemical Co., St. Louis, MO), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, IN), and 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim). The sample is centrifuged at 10,000 rpm for 30 min at 4 ° C in a Beckman JLA-10.5 rotor (Beckman Instruments, Palo Alto, CA) in a Beckman Avanti J25I centrifuge (Beckman Instruments) to remove cell debris. To the fraction of the supernatant is added a 50.0 ml sample of anti-SE Sepharose, prepared as described below, and the mixture is gently shaken in a Wheaton rotary culture apparatus (Millville, NJ) for 18.0 h at 4 ° C.

The mixture is poured into a 5.0 x 20.0 cm Econo-Column column (Bio-Rad, Laboratories, Hercules, CA) and the gel is washed with 30 column volumes of phosphate buffered saline (PBS). The non-retained flow fraction is discarded. When the absorbance of the effluent at 280 nm is less than 0.05, the through flow of the column is reduced to 0 and the anti-SE Sepharose gel is washed with 2.0 column volumes of PBS containing 0.2 mg / ml of the EE peptide (AnaSpec , San José, CA). The peptide used has the sequence GluTyrMetGlu (SEQ ID NO: 16). After 1.0 h at 4 ° C, the flow is reinitiated and the eluted protein is collected. This fraction is the elution of the peptide. The anti-SE Sepharose gel is washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and the glycine wash is collected separately. The pH of the fraction eluted with glycine is adjusted to 7.0 by the addition of a small volume of 10X PBS, and stored at 4 ° C. The elution of the peptide is concentrated to 5.0 ml using a 15,000 molecular weight limit membrane concentrator according to the manufacturer's instructions. The elution of the concentrated peptide is separated from the free peptide by chromatography on a 1.5 x 50 cm Sephadex G-50 column (Pharmacia, Piscataway, NJ) in PBS at a flow rate of 1.0 ml / min using CLAP BioCad Sprint (PerSeptive BioSystems, Frammgham, MA). They are collected 2 ml fractions and the absorbance is monitored at 280 nm. The first peak of material that absorbs at 280 nm and that elutes near the empty volume of the column is collected. This fraction is zCTGF4 labeled in the N-terminal part, or zCTGF4 labeled in the C-terminal part pure. The pure material is concentrated as described above, analyzed by SDS-PAGE and by Western blotting with antibodies against EE, aliquots are formed and stored at -80 ° C. The anti-SE Sepharose preparation is carried out as follows: a 100 ml bed volume of G-Sepharose protein (Pharmacia, Piscataway, NJ) is washed 3 times with 100 ml of PBS containing 0.02% sodium azide using 500 ml of Nalgene and a 0.45 micron filter unit. The gel is washed with 6.0 volumes of 200 mM triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, MO), and an equal volume of EE antibody solution containing 900 mg of antibody is added. After an overnight incubation at 4 ° C, the unbound antibody is removed by washing the resin with 5 volumes of 200 mM TEA, as described above. The resin is resuspended in 2 volumes of TEA, transferred to a suitable vessel, and d-methylpimylimidate-2HCl (Pierce, Rockford, I) is added, dissolved in TEA, at a final concentration of 36 mg / ml of the gel. The gel is subjected to oscillation at room temperature for 45 min and the liquid is removed using a filter unit as described above. The non-specific sites in the gel after are blocked by incubation for 10 min at room temperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA. The gel is then washed with 5 volumes of PBS containing 0.02% sodium azide and stored in this solution at 4 ° C.

B. Non-labeled ZCTGF4 protein Protease inhibitors are added to the conditioned medium of Sf9 cells infected with baculovirus and the medium will be centrifuged as described above for protein labels with EE. The supernatant fraction is applied to a 50.0 ml column of POROS HE1 (PerSeptive BioSystems, Framingham, MS) pre-equilibrated in 20 mM Tris-HCl, 50 mM NaCl, pH 7.4 at a flow rate of 2.0 ml / min with a dilution in lines (triple final dilution) with water as diluent using CLAP BioBad Sprint (PerSeptive BioSystems, Framingham, MS). The heparin-bound proteins are eluted with a gradient of 0.1-1.0 M NaCl. Fractions containing proteins are identified by absorbance at 280 nM and by SDS-PAGE. Fractions containing zCTGF4 are identified by a band on the SDS-PAGE gels of the appropriate molecular weight for glycosylated ZCTGF4 of about 40 kDa. The accumulated zCTGF4 bound to heparin is concentrated, applied to a Sephadex-G50 or G-100 column and eluted as described above.

Purified ZCTGF4 is characterized by SDS-PAGE, amino acid analysis and N-terminal sequencing.

Example 6 A. Adenoviral expression of ZCTGF4 The protein coding region of ZCTGF4 is amplified by PCR using primers that add Fsel and AscI restriction sites at the 5 'and 3' end portions, respectively. The PCR primers ZC17948 (SEQ ID NO: 29) and ZC17949 (SEQ ID NO: 30) are used with a template containing the full-length zCTGF4 cDNA in a PCR reaction as follows: cycle at 95 ° C for 5 minutes; followed by 5 cycles at 95 ° C for 1 minute, 58 ° C for 1 minute, and 72 ° C for 1.5 min; followed by 72 ° C for 7 min; followed by rinsing at 4 ° C. The product of the PCR reaction is loaded on a 1.2% SeaPlaque GTG gel (low melt) (FMC, Rockland, ME) in TAE buffer. The zCTGF4 PCR product is cut from the gel and purified using the QIAquick ™ PCR Purification gel cleaning equipment following the instructions per kit (Qiagen). The PCR product is then digested with Fsel-Ascl, extracted with phenol / chloroform, precipitated with EtOH and rehydrated in 20 ml of TE (Tris / EDTA, pH 8). The fragment of 1065 bp of ZCTGF4 is then ligated into the Fsel-Ascl sites of the transgenic vector pTG12-8 (see description herein) and transformed into DH10B competent cells by electroporation. Clones containing ZCTGF4 are identified by minipreparation of plasmid DNA followed by digestion with Fsel-Ascl. A positive clone is confirmed by direct sequencing.

B. Preparation of the DNA construct for adenovirus generation The 1065 bp cDNA of zCTGF4 is released from the TGl-8 vector using Fsel and AscI enzymes. The cDNA is isolated on a SeaPlaque GTG ™ 1% low fusion gel (FMC, Rockland, ME) and then cut from the gel. The gel cut is melted at 70 ° C, extracted twice with an equal volume of phenol buffered with Tris, and precipitated with EtOH. The DNA is resuspended in 10 μl of H20. The ZCTGF4 cDNA is cloned into the Fsel-Ascl sites of modified pAdTrack CMV (He et al., PNAS 95: 2509-2514, 1998). This construct contains the GFP marker gene. The CMV promoters that activate GFP expression are replaced with the SV40 promoter and the SV40 polyadenylation signal is replaced with the polyadenylation signal of human growth hormone. In addition, the native polylinker with Fsel, EcoRV and Ascl sites. This modified form of CMV pAdTrach is called pZyTrack. The ligation is performed using a Fast-Link ™ DNA ligation and analysis kit (Epicenter Technologies, Madison, Wl). In order to linearize the plasmid, about 5 μg of the plasmid pZyTrack zctgf4 is digested with Pmel. Approximately 1 μg of the linearized plasmid is cotransformed with 200 ng of supercoiled pAdEasy (He et al., supra. ) in BJ5183 cells. Cotransformation is performed using a Bio-Rad gene switch at 2.5 kV, 200 ohms and 25 mFa. The complete cotransformation is seeded in plates on 4 LB plates containing 25 μg / ml kanamycin. The smallest colonies are taken and expanded in LB / kanamycin and the recombinant adenoviral DNA is identified by standard DNA mini-preparation procedures. The digestion of recombinant adenoviral DNA with Fsel-Ascl confirms the presence of ZCTGF4. The recombinant adenoviral minipreparation DNA is transformed into DH10B competent cells and the DNA prepared using the Qiagen maximpreparation equipment following the instructions per kit.

C. Transfection of 293A cells with recombinant DNA Approximately 5 μg of recombinant adenoviral DNA are digested with Pací enzyme (New England Biolabs) during 3 hours at 37 ° C in a reaction volume of 100 μl containing 20-30 U of Pací. The digested DNA is extracted twice with an equal volume of phenol / chloroform and precipitated with ethanol. The DNA pellet is suspended in 10 μl of distilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies, Inc. Mcntreal, Qc. Canada), inoculated the day before, is transfected and grown to 60-70% confluence, with DNA digested by Pac. The DNA digested with Paci is diluted to a total volume of 50 μl with sterile HBS (150 mM NaCl, 20 mM HEPES). In a separate tube, 20 μl of DOTAP (Boehringer Mannheim, 1 mg / ml) is diluted to a total volume of 100 μl with HBS. The DNA is added to DOTAP, mixed gently by pipetting up and down, and left at room tempearture for 15 minutes. The medium is removed from the 293A cells and washed with 5 ml of serum-free MEM-alpha (Gibco BRL) containing 1 mM sodium pyruvate (GibcoBRL), non-essential amino acids 0.1 mM MEM (GibcoBRL) and 25 mM HEPES buffer (GibcoBRL). 5 ml of serum-free MEM are added to the 293A cells and maintained at 37 ° C. The DNA / liquid mixture is added dropwise to the T25 flask of 293A cells, mixed gently and incubated at 37 ° C for 4 hours. After 4 h, the medium containing the DNA / lipid mixture is separated by aspiration and replaced with 5 ml of complete MEM containing 5% fetal bovine serum. The transfected cells are monitored for determine the expression of green fluorescent protein (GFP) and the formation of foci, ie, viral plaques. Seven days after the transfection of the 293A cells with the recombinant adenoviral DNA, the cells express the GFP protein and begin to form foci. These foci are viral "plaques" and the crude viral lysate is collected by using a cell scraper to collect all of the 293A cells. The lysate is transferred to a 50 ml conical tube. To release most of the virus particles from the cells, three freeze / reheat cycles are performed in a dry ice / ethanol bath and a 37 ° C water bath.

D. Amplification of recombinant adenovirus (rAdV) The crude lysate is amplified (primary amplification (1 °)) to obtain a "concentrate" of lysate work of ZCTGF4 rAdV. Ten 10 cm plates of almost confluent 293A cells (80-90%) are established for 20 hours previously, 200 ml of crude rAdV lysate are added to each 10 cm plate and monitored for 48 to 72 hours in search of low CPE. a white light microscope and the expression of GFP under a fluorescent microscope. When all 293A cells show CPE (cytopathic effect), this 1 ° concentrated lysate is collected and cycles of freezing / reheating as described under the crude rAdV lysate. The secondary amplification (2 °) of zCTGF4 rAdV is obtained as follows: twenty 15 cm tissue culture vessels of 293A cells are prepared, so that the cells are 80-90% confluent. The whole except 20 ml of 5% MEM medium is removed and each disk is inoculated with 300-500 ml of primary amplified rAdv lysate. After 48 hours, the 293A cells are lysed from virus production and this lysate is collected in 250 ml polypropylene centrifuge flasks and rAdV purified.

E. Purification of Adv / cDNA NP-40 detergent is added at a final concentration of 0.5% to the used raw jars in order to lyse all the cells. The bottles are placed on a rotating platform for 10 min, they are shaken as fast as possible without the bottles falling off. The residues are sedimented by centrifugation at 20,000 X G for 15 minutes. The supernatant is transferred to 250 ml polycarbonate centrifuge bottles and 0.5 volume 20% PEG800 / 2.5 M NaCl are added. The bottles are shaken overnight on ice. The bottles are centrifuged at 20,000 x G for 15 minutes and the supernatant is discarded in a bleaching solution. The precipitate white in the two vertical lines along the wall of the bottle on both sides of the spin mark is the virus / PEG precipitate. Using a sterile cell scraper, the precipitate of the two flasks is resuspended in 2.5 ml of PBS. The virus solution is placed in 2 ml microcentrifuge tubes and centrifuged at 14,000 X G in a microcentrifuge for 10 minutes to remove any additional cellular debris. The supernatant of the 2 ml microcentrifuge tubes is transferred to a 15 ml polypropylene snap-cap tube and adjusted to a density of 1.34 g / ml with cesium chloride (CsCl). The volume of the virus solution is estimated and 0.55 g / ml of CsCl are added. The CsCl is dissolved and 1 ml of this solution weighs 1.34 g. The solution is transferred to 3.2 ml polycarbonate thin-walled tubes (Beckman) and rotated at 80,000 rpm (348,000 XG) for 3-4 hours at 25 ° C in a Beckman Optimal TLX microcentrifuge with a TLA-100.4 rotor. . The virus forms a white band. Using the wide piercing pipette tips, the virus band is collected. The gradient virus has a lot of CsCl which must be removed before it can be used in cells. Pharmacia PD-10 columns pre-packed with Sephadex G-25M (Pharmacia) are used to remove the salt from the virus preparation. The column is equilibrated with 20 ml of PBS. The virus is loaded and allowed to run inside the column. 5 ml of PBS are added to the column and fractions of 8-10 drops are collected. The optical densities of 1:50 dilutions of each fraction at 260 nm are determined in a spectrophotometer. The peak of clear absorbance is present between fractions 7-12. These fractions are put together and the optical density (OD) of a 1:25 dilution is determined. A formula is used to convert OD to virus concentration: (OD 260 nm) (25) (1.1 x 1012 = virions / ml) The OD of a 1:25 dilution of zctgf4 rAdV is 0.221, which provides virus concentration of 6 X 1012 virions / ml.

To store the virus, glycerol is added to the purified virus to a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80 ° C.

F. Infectious dose of tissue culture at 50% CPE (TCID 50) Viral titration assay A protocol developed by Quantum Biotechnologies Inc. (Montreal, Qc. Canada) is followed to measure the infectivity of the virus. Briefly, two 96-well tissue culture plates are seeded with 1 X 10 4 293A cells per well in MEM containing 2% fetal bovine serum, for each recombinant virus to be tested. After 24 hours, decimal dilutions of each virus are made from 1 X 10'2 up to 1 X 10"14 in MEM containing 2% fetal bovine serum 100 μl of each dilution is placed in each of the 20 wells After 5 days at 37 ° C, the wells are read as positive or negative for cytopathic effect (CPE) and a value is calculated for the "plaque forming units / ml" (PFU) .The TCID50 of the formulation used is as indicated by Quantum Biotechnologies, Inc., before. from a plate where the diluted virus is used from 10'2 to 10'14 and read 5 days after infection.At each dilution the ratio (R) of positive wells for CPE is determined by the total number of wells. To calculate the title of the undiluted virus sample: the factor "F" = l + d (S-0.5) is used, where "S" is the sum of the relations (R), and "d" is LoglO of the series of dilutions, for example, "d" is equal to 1 for a 10-fold dilution series The title of the undiluted sample is T = 10 (1 * FI = TCID50 / ml.) To convert TCID50 / ml to pfu / ml, subtract 0.7 from the exponent in the calculation by the title (T). Adenovirus ZCTGF4 has a titer of 7.1 X 10 10 pfu / ml.

Example 7 Transgenic expression Transgenic animals are produced that express ZCTGF4 genes using adult mice (B6D2fl, 2-8 months, (Taconic Farms)), fertile prepubescent females (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females ( B6Dfl, 2-4 months, (Taconic Farms) as the parents.The donors are injected with approximately 8 IU / pregnant mare serum gonadotropin mouse (Sigma, St. Louis, MO) I.P. and 46-47 hours later, 8 IU / mouse human chorionic gonadotropin (hCG (Sigma)) is administered I.P. to induce superovulation. The fertilized eggs are collected and stored in an incubator with 37 ° C / C02 5% until the microinjection. An amount of 10-20 micrograms of plasmid DNA containing a cDNA of the ZCTGF4 gene is linearized, the gene is purified and resuspend in 10 mM Tris pH 7.4, 0.25 mM EDTA, pH 8.0 at a final concentration of 5-10 nanograms per microliter for microinjection. The plasmid DNA is microinjected into harvested eggs and penetrated with an injection needle, into one or both of the aploid pronuclei.

The next day, 2-cell embryos are transferred to pseudo-pregnant receptors. The recipients are returned to cages in pairs, and a gestation of 19-21 days is allowed. After birth, 19-21 days of postpartum period before weaning are allowed. An amount of 25 weaned animals are determined by sex and are placed in separate cages for sex, and a 0.5 cm biopsy (used to establish the genotype) is made in a section of the tail, with clean scissors. Genomic DNA is prepared from the tail cuts using the Quagen Dneasy equipment following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed for human growth hormone (hGH), the 3 'UTR portion of the transgenic vector. A region unique to the human sequence is identified from the alignment of human growth hormone and mouse DNA sequences 3 'UTR, which ensures that the PCR reaction does not amplify the mouse sequence. The primers ZC17251 (SEQ ID NO: 31) and acl7252 (SEQ ID NO: 32) amplify a fragment of 368 base pairs of hGH. In addition, the primers zcl7156 (SEQ ID NO: 33) and ZC17157 (SEQ ID NO: 34), which hybridizes with vector sequences and amplifies the DNA insert, is often used in conjunction with the primers of hGH. In these experiments, the DNA of the animals positive for the transgene generates two bands, a band of 368 base pairs corresponding to the 3 'UTR fragment of hGH, and a band of variable size corresponding to the cDNA insert. Once it is confirmed that 9 animals are transgenic (TG), they cross again with their wild type partners C57B1 / 6. As the young are born and weaned, they are separated by sex and the tails are cut to determine the genotype. To verify the expression of a transgene in a live animal, a small partial liver biopsy is collected. The collected liver biopsy is transferred to a 14 ml polypropylene round bottom tube and stored frozen and closed in liquid nitrogen and then stored on dry ice. Analysis of the mRNA expression level of each transgene is performed using an RNA solution hybridization assay. Furthermore, it is observed that the transgenic mice are smaller than the wild-type mice of the same bait. The weight of the thymus and the spleen is observed, which is lower when it is normalized with brain to organ weights. Histological examination shows that most transgenic mice have some pancreatic atrophy and some cardiomyopathy. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described in the present invention for purposes of illustration, various modifications can be made without deviating 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 to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (18)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: l. An isolated polynucleotide molecule, characterized in that it comprises a polynucleotide sequence which codes for a homologous polypeptide of connective tissue growth factor that is at least 70% identical to the amino acid sequence as shown in SEQ. C. DE I DENT. NO: 2 from residue 24 to residue 354.
2. 2. The polynucleotide molecule according to claim 1, characterized in that the polynucleotide molecule comprises a region having the following motif, as shown in SEQ. FROM IDENT. NO: 23: Cx. { 8, 10) CxCCxxCx. { 7] Cx. { 5,6} Cx (5,7) Cx. { 12, 13.}. Cx [7.8} Cx. { twenty) CxCx. { 6.} Cx. { 12, 14.}. Cx. { 13, 17.}. C where x. { } is the number of amino acid residues between the cysteines (C).
3. 3. The polynucleotide according to claim 1, characterized in that the polynucleotide is 80% identical to the amino acid sequence, as shown in SEQ. OF I DENT. NO: 2, from residue 24 to residue 354.
4. 4. The polynucleotide according to claim 1, characterized in that the polynucleotide is 90% identical to the amino acid sequence as shown in SEQ. FROM IDENT. NO: 2, from residue 24 to residue 354.
5. 5. An isolated polynucleotide acid molecule, characterized in that it encodes a polypeptide homologue of connective tissue growth factor, wherein the polynucleotide molecule is selected from the group consisting of: (a) molecules having the nucleotide sequence of SEQ. OF I DENT. NO: 1, nucleotide 17 or 86 to nucleotide 1078; (b) a molecule encoding the amino acid sequence in SEQ. OF I DENT. NO: 3, nucleotide 1 or 70 to nucleotide 1062; and (c) a molecule that hybridizes under stringent washing conditions to a polynucleotide molecule having the sequence of nucleotides from nucleotides 86 to 1078 of SEQ. FROM IDENT. NO: 1, or the complement of nucleotides 86 to 1078 of the SEC. OF I DENT. NO: 1.
6. 6. The polynucleotide molecule according to claim 5, characterized in that any difference in the amino acid sequence encoded by the polynucleotide and SEQ. ID ENT. NO: 2 are conservative amino acid changes.
7. 7. An expression vector, characterized in that it comprises the following operably linked elements: a transcription promoter; a DNA segment comprising the isolated polynucleotide molecule according to claim 1; and a transcription terminator.
8. 8. A cultured host cell, characterized in that the expression vector according to claim 7 has been iduced therein.
9. 9. A method for producing a polypeptide homologue of connective tissue growth factor, characterized in that it comprises: (a) culturing the host cells according to claim 8; and (b) isolating the homologous polypeptide from the connective tissue growth factor of the cultured host cells.
10. 10. A polypeptide of the isolated connective tissue growth factor, characterized in that it comprises an amino acid sequence that is at least 70% identical to the amino acid sequence as shown in SEQ. ID ENT. NO: 2, from residue 24 to residue 354. eleven . The polypeptide isolated, according to claim 10, characterized in that the amino acid sequence is at least 80% identical.

    12. The polypeptide isolated, according to claim 10, characterized in that the amino acid sequence is at least 90% identical.

    3. The isolated polypeptide according to claim 10, characterized in that the polypeptide molecule comprises a region having the following motif, as shown in SEQ. OF I DENT. NO: 23: Cx. { 8, 10) CxCCxxCx. { 7] Cx. { 5,6} Cx (5,7) Cx. { 12, 13.}. Cx [7.8} Cx. { twenty) CxCx. { 6.} Cx. { 12, 14.}. Cx. { 13, 1 7.}. C where x. { } is the number of amino acid residues between the cysteines (C).

    14. An antibody or antibody fragment, characterized in that it binds specifically with the polypeptide according to claim 10.

    15. A method for detecting the presence of a homologous polypeptide of connective tissue growth factor in a biological sample, characterized in that it comprises the steps of: (a) contacting the biological sample with an antibody, or an antibody fragment in accordance with claim 14, wherein the contact is carried out under conditions that allow the binding of the antibody or the antibody fragment to the biological sample, and (b) detecting either the bound antibody or the bound antibody fragment.

    16. An anti-idiotype antibody, or anti-idiotype antibody fragment, characterized in that it binds specifically to the antibody or antibody fragment according to claim 14.

    17. A method for detecting an abnormality on chromosome 6q in a sample from an individual, characterized in that it comprises: (a) obtaining zCTGF4 RNA from the sample; (b) generating zCTGF4 cDNA by polymerase chain reaction; and (c) comparing the nucleic acid sequence of the zCTGF4 cDNA with the nucleic acid sequence, as shown in SEQ. OF I DENT. NO: 1.

    18. The method according to claim 17, characterized in that the difference between the DNA sequence of ZCTGF4 or the zCTGF4 gene in the sample, and the sequence of ZCTGF4, is shown in SEC. FROM IDENT. NO: 1, is indicative of an abnormality on chromosome 6q.