WO2001070809A1 - A novel polypeptide - protein 9 of growth hormone-family and a polynucleotide sequence encoding the same - Google Patents

Abstract

The present invention discloses a novel polypeptide - protein 9 of growth hormone-family and a polynucleotide encoding the same, as well as a method of producing the polypeptide by DNA recombinant technique. The present invention also discloses methods of using the polypeptide in treatment of various diseases, such as embryonic development disorder, growth disturbance, development disturbance, pregnancy abnormality, tumor and so on. The present invention also discloses an antagonist against the polypeptide and the therapeutic use of the same. Also disclosed is the use of such novel polynucleotide encoding protein 9 of growth hormone-family.

Description

 A new polypeptide, a growth hormone protein family 9 and a polynucleotide encoding the polypeptide. TECHNICAL FIELD

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide-growth hormone protein family 9 and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique

 Under the control of the nervous system, the pituitary gland regulates various endocrine glands throughout the body and is the promoter of various endocrine glands. The pituitary gland is divided into three parts: the anterior lobe, the middle lobe, and the posterior lobe. The anterior and middle lobe can synthesize hormones on its own, and the posterior lobe only stores and secretes hormones.

 The anterior pituitary gland plays a leading role in the endocrine system. It is directly stimulated by substances secreted by the hypothalamus and regulated by the nerves, thereby regulating the development and secretion of certain endocrine organs, and is therefore closely related to the growth, sex and metabolism of animals. There are at least 6 purified hormones secreted from the anterior pituitary: adrenocorticotropic hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone, and prolactin.

 Among them, growth hormone and prolactin have a high structural homology. They belong to a protein family, which we call the growth hormone protein family. This protein family includes the following: Prolactin in rodents; Cattle and sheep Prolactin; rat aspergillin; human growth hormones GH-II and GH-V2, etc. (Fa l l s M. J. Mol. Evol. 17: 10-18 (1981))

 Growth hormone (GH) plays an important role in the growth process of organisms. It has a wide range of functions, can stimulate the growth of bone and cartilage, promote the synthesis of mucopolysaccharides and collagen, and it also affects proteins, carbohydrates and lipids. This kind of metabolism ultimately affects weight gain. If auxin secretion is insufficient in young animals, dwarfism will occur; if auxin secretion is too much in young animals, giant disease will occur; if hyperpituitary function occurs in adult animals, acral hypertrophy will occur; if pituitary activity is too high, Causes hyperglycemia and glycosuria. This effect is caused by auxin promoting the adrenal cortex to secrete glucocorticoids. In addition, high pituitary activity has a large effect on many tissues, such as cartilage, connective tissue, muscle tissue and fat. The effect on tissues occurs at the nucleus level. Growth hormone can regulate the biosynthesis of various forms of MA, and may increase the formation of MA polymerase, thereby increasing RNA synthesis.

The physiological function of lactotropic hormone (LTH) is to stimulate the fully-developed breast glands to secrete milk, and to stimulate and maintain the progesterone secreted by the corpus luteum. LTH not only greatly promotes the synthesis of RNA and proteins in the breast, but also increases the activities of many enzymes in the metabolism of sugar and lipid in the breast. In summary, protein hormones containing the functional domains described here are important for the metabolism of proteins, carbohydrates, lipids, for the biosynthesis of various forms of RM, for some important physiological processes such as reproductive functions, etc. significance.

 Gene chip analysis revealed that in the thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, starved L 02 cell line, arsenic stimulated for 1 hour In the L 02 cell line and prostate tissue, the expression profile of the polypeptide of the present invention is very similar to the expression profile of the growth hormone protein family 10, so the functions of the two may also be similar. The invention is named the growth hormone protein family 9.

 As described above, the growth hormone protein family 9 protein plays an important role in regulating the body's important functions such as development, differentiation, and performing normal functions, and it is believed that a large number of proteins are involved in these regulatory processes, so more needs to be identified in the art The growth hormone protein family 9 proteins involved in these processes, and in particular the amino acid sequence of this protein is identified. Isolation of the new growth hormone protein family 9 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is very important. Disclosure of invention

 It is an object of the present invention to provide isolated novel polypeptides-the growth hormone protein family 9 and fragments, analogs and derivatives thereof.

 Another object of the invention is to provide a polynucleotide encoding the polypeptide.

 It is another object of the present invention to provide a recombinant vector containing a polynucleotide encoding a growth hormone protein family 9.

 Another object of the invention is to provide a genetically engineered host cell containing a polynucleotide encoding a growth hormone protein family 9.

 Another object of the present invention is to provide a method for producing growth hormone protein family 9.

 Another object of the present invention is to provide antibodies against the growth hormone protein family 9 of the polypeptide of the present invention.

 Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the growth hormone protein family 9 of the polypeptide of the present invention.

Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of the growth hormone protein family 9. The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

 The invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:

 (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;

 (b) a polynucleotide complementary to the polynucleotide (a);

 (c) A polynucleotide having at least 70% identity to a polynucleotide sequence of (a) or (b).

 More preferably, the sequence of the polynucleotide is one selected from: (a) a sequence having positions 422-691 in SEQ ID NO: 1; and (b) a sequence having 1-1885 in SEQ ID NO: 1 Sequence of bits.

 The present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.

 The invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.

 The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the growth hormone protein family 9 protein activity, which comprises utilizing the polypeptide of the invention. The invention also relates to compounds obtained by this method.

 The present invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of the growth hormone protein family 9 protein in vitro, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a biological sample The amount or biological activity of a polypeptide of the invention.

 The invention also relates to a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.

 The present invention also relates to the preparation of the polypeptide and / or polynucleotide of the present invention for the treatment of embryonic disorders, growth disorders, pregnancy abnormalities, tumors or other diseases caused by abnormal expression of the growth hormone protein family 9 use.

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.

The following terms used in this specification and claims have the following meanings unless specifically stated: "Nucleic acid sequence" refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or MA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule When listed, such a "polypeptide" or "protein" is not meant to limit the amino acid sequence to the complete natural amino acid associated with the protein molecule.

 A "variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

 "Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

 "Insertion" or "addition" refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

 "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.

 An "agonist" refers to a molecule that, when combined with the growth hormone protein family 9, can cause changes in the protein and thereby regulate the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds the growth hormone protein family 9.

 An "antagonist" or "inhibitor" refers to a molecule that, when combined with growth hormone protein family 9, can block or regulate the biological or immunological activity of growth hormone protein family 9. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind the growth hormone protein family 9.

 "Regulation" refers to a change in the function of growth hormone protein family 9, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of growth hormone protein family 9.

"Substantially pure ' ¹ means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify the growth hormone protein family 9 using standard protein purification techniques. Basically pure The growth hormone protein family 9 can generate a single main band on a non-reducing polyacrylamide gel. The purity of the growth hormone protein family 9 polypeptide can be analyzed by amino acid sequence.

"Complementary" or "complementary" refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-TG-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands The efficiency and strength of hybridization between nucleic acid strands has a significant effect.

 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.

"Percent identity" refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Laser gene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences based on different methods such as the Clus ter method (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244). _{0 The} Clus ter method compares each pair by checking the distance between all pairs. Group sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A Number of interval residues in a sequence B

The percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in emzumo logy 183: 625-645). ₀ "Similarity" means The degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitutions, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

 "Antisense" refers to a nucleotide sequence that is complementary to a particular DM or RNA sequence. "Antisense strand" refers to a nucleic acid strand that is complementary to a "sense strand."

 "Derivative" refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') ₂ and ^, which can be specific Antigenic determinants of sex-binding growth hormone protein family 9.

 A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.

 The term "isolated" refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.

 As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .

 As used herein, "isolated growth hormone protein family 9" means that the growth hormone protein family 9 is essentially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify the growth hormone protein family 9 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the growth hormone protein family 9 peptides can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, a growth hormone protein family 9, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

The invention also includes fragments, derivatives and analogs of the growth hormone protein family 9. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the growth hormone protein family 9 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (Π) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (Π Ι) Such a type, wherein the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); Or (IV) a polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) formed by fusing an additional amino acid sequence into a mature polypeptide, as described herein, such a fragment Derivatives and analogs are considered to be within the knowledge of those skilled in the art.

 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1885 bases, and its open reading frames 422-691 encode 89 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile to the growth hormone protein family 10, and it can be deduced that the growth hormone protein family 9 has similar functions to the growth hormone protein family 10.

 The polynucleotide of the present invention may be in the form of MA or RNA. DNA forms include cDNA, genomic MA, or synthetic DM. MA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.

 The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.

 The term "polynucleotide encoding a polypeptide" refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.

 The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences). The invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 »/. SDS, 6 (TC; or (2) hybridization When adding denaturants, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding growth hormone protein family 9.

 The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. The specific polynucleotide sequence encoding the growth hormone protein family 9 of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.

 The DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded MA sequence from the DM of the genome; 2) chemically synthesizing the DNA sequence to obtain the double-stranded MA of the polypeptide.

 Of the methods mentioned above, genomic DM is the least commonly used. Direct chemical synthesis of MA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating the cMA of interest is to isolate mMA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene). The construction of cDM libraries is also a common method (Sambrook, et al., Molecul ar Cloning, A Laboratory Manua, Colling Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

These genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; ( ³ ) determining the level of transcripts of the growth hormone protein family 9; (4) Detection of gene-expressed protein products by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.

In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used herein is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 Among the (4) methods, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein products expressed by the 9 genes of the growth hormone protein family.

 A method (Saiki, et al. Science 1985; 230: 1350-1354) for amplification of MA / RM using PCR technology is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length CDM from a library, the RACE method (RACE-rapid amplification of cDNA ends) may be preferably used. The primers for PCR may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods. The amplified DM / RM fragment can be isolated and purified by conventional methods such as by gel electrophoresis.

 The polynucleotide sequence of the gene or various fragments of the present invention obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDM sequences of multiple clones in order to splice into full-length cDM sequences.

 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a growth hormone protein family 9 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .

 In the present invention, a polynucleotide sequence encoding the growth hormone protein family 9 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Na thans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.

Methods known to those skilled in the art can be used to construct expression vectors containing DM sequences encoding the growth hormone protein family 9 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, MA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Mol ecular Cloning, a Laboratory Manua 1, cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRM synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, and early and late SV40 Promoters, LTRs of retroviruses, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pair enhancers on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication. .

 In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

 Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes.

 In the present invention, a polynucleotide encoding the growth hormone protein family 9 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS or Bowes melanoma cells.

Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing MA may be in exponential growth phase were harvested after the treatment with (¾ (1 ₂ method used in steps well known in the art. Alternatively MgCl ₂ is used If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Body packaging, etc.

 Using conventional recombinant DM technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant growth hormone protein family 9 (Sc i'ence, 1984; 224: 1431). Generally, the following steps are taken:

 (1) using the polynucleotide (or variant) encoding the human growth hormone protein family 9 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing host cells in a suitable medium;

 (3) Isolate and purify protein from culture medium or cells.

In step (2), depending on the host cell used, the medium used in the culture may be selected from various Conventional medium. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

 In step (3), the recombinant polypeptide may be coated in a cell, or expressed on a cell membrane, 'or secreted outside the cell. If desired, recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. ¾ of the drawings

 The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.

 FIG. 1 is a comparison diagram of gene chip expression profiles of growth hormone protein family 9 and growth hormone protein family 10 according to the present invention. The upper graph is a graph of the growth hormone protein family 9 expression profile, and the lower sequence is the graph of the growth hormone protein family 10 expression profile.

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated growth hormone protein family 9. Da is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention

 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Harbor Press, 1989), or Conditions recommended by the manufacturer.

 Example 1: Cloning of growth hormone protein family 9

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRM was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA forms CDM by reverse transcription. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragments into the 'pBSK (+) vector (Clontech) for multiple cloning sites to transform DH5 cc. The bacteria formed a cDNA library. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. CDM The sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0149B01 was a new DM. A series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone. The results show that the full-length CDM contained in the 0149B01 clone is 1885bp (as shown in Seq ID N0: l), and there is a 270bp open reading frame (0RF) from 422bp to 691bp, which encodes a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-0149B01, and named the encoded protein as growth hormone protein family 9. Example 2: Cloning of a gene encoding growth hormone protein family 9 by RT-PCR

 CDM was synthesized by reverse transcription reaction using total RNA from fetal brain cells as a template and ol igo-dT as a primer. After purification with Qiagene's kit, PCR was performed with the following primers:

 Pr imerl: 5,-AGACAAAATACTAGAAATTGCACC- 3 '(SEQ ID NO: 3)

 Pr imer 2: 5'- ACAAGAGTCTTGCTCTGTCACCCA-3 '(SEQ ID NO: 4)

 Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;

 Primer 2 is the 3, terminal reverse sequence of SEQ ID NO: 1.

 Amplification conditions: 50 fflfflol / L KC1, 10 fflfflol / L Tri s-

CI, (pH 8. 5), 1.5 mmol / L MgCl ₂ , 200 μmol / L dNTP, 1 Opmol primer, 1U Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Eimer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min. During RT-PCR, set β-act in as a positive control and template blank as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a PCR vector using a TA cloning kit (Invitrogen). The DM sequence analysis results showed that the DM sequence of the PCR product was exactly the same as that of 1 to 1885 bp shown in SEQ ID NO: 1. Example 3: Northern blot analysis of growth hormone protein family 9 gene expression:

Total A was extracted in one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain A precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water. j¾ 20 ug of RNA was electrophoresed on a 1.2% agarose gel containing 20 ηΜ 3-(N-morpholino) propanesulfonic acid (pH 7, 0)-5 mM sodium acetate-1 mM EDTA-2.2 M formaldehyde. It was then transferred to a nitrocellulose membrane. A- ³² P dATP was used to prepare ³² P-labeled DM probes by random primer method. The DNA probe used was the PCR amplified growth hormone protein family 9 coding region sequence (422bp to 691bp) shown in FIG. A 32P-labeled probe (approximately 2 x 10 ⁶ cpm / ml) and nitrocellulose with RM transferred 素 膜 在 42。 In a solution at 42. C hybridization overnight, the solution contains 50% formamide-25 mM KH ₂ PO ₄ (pH 7.4)-5 x SSC-5 x Denhardt's solution and 200 μg / ml salmon sperm DM. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 4: In vitro expression, isolation and purification of recombinant growth hormone protein family 9 '

 Based on SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:

Pr imer 3: 5'-CCCCATATGATGGGAAGGACAGGAGGGTGCCTA-3 '(Seq ID No: 5) Pr imer4: 5'- CATGGATCCCTAAACACCTCTGCACTTTTGTAG-3, (Seq ID No: 6) The 5' ends of these two primers contain Ndel and BamHI digestion respectively Site, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively. The Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site. The PCR reaction was performed using the pBS-0149B01 plasmid containing the full-length target gene as a template. The PCR reaction conditions are as follows: a total volume of 50 μ1, a plasmid containing 10 pg of pBS- Ol ^ BOl, primers? 1 ^ 1116]: -3 and? 1 ^ 1116]: -4 points and another!] Is 101) 11101, Advantage polymerase Mix (Clontech) 1 μ1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI were used to double-digest the amplified product and plasmid _P ET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into E. coli DH5 cc using the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 μ _§ / ηι1), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0149B01) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 μg / ml), the host strain BL21 (pET-0149B01) was at 37. C. Cultivate to the logarithmic growth phase, add IPTG to a final concentration of 1 cage ol / L, and continue to cultivate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation, and chromatography was performed using an His. Bind Quick Cartr idge (product of Novagen) which can bind 6 histidines (6His-Tag). A purified target growth hormone protein family 9 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 9 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 5 Production of Anti-HGH Protein Family 9 Antibodies

 Polypeptide synthesizer (product of PE company) was used to synthesize the following specific peptides of growth hormone protein family 9:

NH2- Me t-G 1 y ~ Arg-Thr-G 1 y-Gly-Cy s -Leu-G 1 y- A 1 a-Leu-Trp-G 1 y-A 1 a- A 1 a- OH

(SEQ ID NO: 7). The peptide was coupled to hemocyanin and bovine serum albumin to form a complex, respectively. See: Avrameas, et al. Immunochemistry, 1969; 6:43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used for BLISA to determine the titer of antibodies in rabbit serum. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibodies specifically bind to the growth hormone protein family 9. Example 6: Application of the polynucleotide fragment of the present invention as a hybridization probe

 Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.

 The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained. The probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention The polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment. In this example, the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.

First, the selection of the probe

 The selection of oligonucleotide fragments from the polynucleotide SBQ ID NO: 1 of the present invention as hybridization probes should follow the following principles and several aspects to be considered:

1. The preferred size of the probe ranges from 18 to 50 nucleotides; 2, GC content is 30% -70%, non-specific hybridization increases when it exceeds;

 3. There should be no complementary regions inside the probe;

 4. Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;

 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.

 After completing the above analysis, select and synthesize the following two probes:

 Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):

 5'-TGGGAAGGACAGGAGGGTGCCTAGGTGCACTGTGGGGAGCA-3 '(SEQ ID NO: 8)

 Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):

 5'-TGGGAAGGACAGGAGGGTGCCTAGGTGCACTGTGGGGAGCA-3 '(SEQ ID NO: 9) For other commonly used reagents and their preparation methods not related to the following specific experimental procedures, please refer to the literature: DNA PROBES GH Kel ler; MM anak; Stockton Press, 1989 ( USA) and more commonly used manuals of molecular cloning experiments such as "Molecular Cloning Experiment Guide" (Second Edition 1998) [US] Sambrook et al., Science Press.

 Sample Preparation:

1.Extract DM from fresh or frozen tissue

Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the pellet (approximately 10 ml / g) with a cold homogenate buffer (0.25 mol / L sucrose; 25 mmol / L Tris-HCl, pH 7.5; 25 mmol / LnaCl; 25 mmol / L MgCl ₂ ). 4) Homogenize the tissue suspension at 4 ° C at full speed with an electric homogenizer until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (add 1-5 ml per 0.1 g of the original tissue sample), and centrifuge at 1,000 g for 10 minutes. 7) Resuspend the pellet in lysis buffer (1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.

2, DM extraction of phenol

Steps: 1) Wash cells with 1-10 ml of cold PBS and centrifuge at 1000 g for 10 minutes. 2) Resuspend the pelleted cells (1 × ^{10 8} cells / ml) with cold cell lysate and apply a minimum of 100ul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is added directly to the cell pellet before resuspending the cells, the cells may Large agglomerates are formed which are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or at 37 ° C. C gently shake overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) Extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the aqueous phase containing MA to a new tube. Purification of DM and ethanol precipitation were then performed.

3, DNA purification and ethanol precipitation

Steps: 1) Add 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DNA, every 1- 5 xl0 ⁶ cells extracted about plus lul.

 The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.

8) Add RNase A to the DNA solution to a final concentration of 100 ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K, the final concentrations are 0.5% and 100ug / ml. Incubate at 37 ° C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 12) Carefully remove the water phase, force P 1/10 volume 2mol / L sodium acetate and 2.5 volume cold ethanol, and mix well-20 ° C for 1 hour. 13) Wash the precipitate with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-5. 14) Determine A _26Q and A ₂₈ . To detect the purity and yield of DNA. 15) Store at -20 ° C after dispensing. Preparation of sample membrane: '1) Take 4x2 sheets of nitrocellulose membrane (NC membrane) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are needed for each probe in order to In the following experimental steps, the film was washed with high-strength conditions and strength conditions, respectively.

 2) Pipette and control 15 microliters each, spot on the sample film, and dry at room temperature.

 3) Place on filter paper impregnated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place on filter paper impregnated with 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl Allow to dry for 5 minutes (twice).

4) Caught in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours. Labeling of probes

1) 3 μl Probe (0.1 IOD / Ιθμ 1), add 2 μ IKinase buffer, 8-10 uCi y- ³² P-dATP + 2U Kinase, to make up to a final volume of 20 μ 1.

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of bromophenol blue indicator (BPB).

 4) Pass through a Sephadex G-50 column.

5) Collect the first peak before ³² P-Probes are washed out (monitorable).

 6) 5 drops / tube, collect 10-15 tubes.

 7) Monitor the amount of isotope with a liquid scintillator

8) The ³² P- Probe (the second peak is free γ- ³² P-dATP) after the collection of the first peak is combined. ₀

 Pre-hybridization

 Put the sample film in a plastic bag, add 3- lOmg pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA).), Seal the bag, and shake at 68 ° C with water for 2 hours. hour.

 Cross

 Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake at 42 ° C in water overnight. Wash film:

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, 40. C Wash for 15 minutes (twice).

 3) 0. IxSSC, 0.1 ° / oSDS, wash at 0 ° C for 15 minutes (twice).

 4) 0.1xSSC, 0.1% SDS, 55. Wash for 30 minutes (twice) and dry at room temperature. Strength wash film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

 3) 0. IxSSC, 0.1¾SDS, 37. C wash for 15 minutes (twice).

 4) 0. IxSSC, 0.1¾SDS, wash at 40 ° C for 15 minutes (twice), and dry at room temperature.

X-ray autoradiography:

 ~ 70 ° C, X-ray autoradiography (pressing time depends on the radioactivity of the hybrid spot).

Experimental results: 釆 The hybridization experiments performed under low-intensity membrane washing conditions did not differ significantly in the radioactivity of the above two probe hybrid spots; while the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of hybridization spots of probe 1 was obvious. Stronger in radioactivity than the hybridization spot of another probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues. Example 7 DNA Microarray

 Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of fast, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as a target MA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases, such as hereditary diseases . The specific method steps have been reported in the literature. For example, see the literature DeRi si, JL, Lyer, V. feBrown, P. 0 (1997) Science 278, 680-686. And the literature Hel le, RA, Schema, M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.

 (A) spotting

 A total of 4,000 polynucleotide sequences of various full-length cDMs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500ng / ul after purification. The spots were spotted on a sloped glass using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 μm. The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps are widely reported in the literature. The post-spot processing steps of this embodiment are:

 1. Hydration in a humid environment for 4 hours;

 2. 0.2% SDS was washed for 1 minute;

3. (1 (¾ ₂ 0 wash twice, 1 minute each time;

4. NaBH _{4 is} blocked for 5 minutes;

 5. 95 ° C water for 2 minutes;

 6. 0. 2¾SDS wash for 1 minute;

7. Rinse twice with ddH ₂ 0;

 8. Dry and store at 25 ° C in the dark for future use.

 (Two) probe marking

One-step extraction from the human mixed tissue and the body's specific tissue (or stimulated cell lines) Total mRNA was extracted and fflRNA was purified with Oligotex mMA Midi Kit (purchased from QiaGen). Cy3dUTP (5-Amino-propargy 1-2 '-deoxyur idine 5'-tr iphate coupled was tested by reverse transcription. to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech Company) labeled mRNA of human mixed tissue, using a fluorescent reagent Cy5dUTP (5- Amino- propargyl- 2 '-deoxyur idine 5- -triphate coupled to Cy5 f luorescent dye, purchased from Amersham Phamacia Biotech Co., Ltd. labeled the body's specific tissues (or stimulated cell lines) with mRNA and purified the probes. For specific steps and methods, see:

Schena,

 M., Shalon, D., Heller, R. (1996) Proc. Nat l. Acad. Sci. USA. Vol. 93: 10614- 10619. Sc ena, M., Shalon, Dar i., Davis, R ¥. (1995) Science. 270. (20): 467-480.

(Three) cross

 The probes from the above two tissues and the chip were respectively hybridized in a UniHyb ™ Hybridization on Solution (purchased from TeieChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2 SDS) at room temperature. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Seaming, USA), and the scanned images were analyzed by Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.

 The above specific tissues (or stimulated cell lines) are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, L02 cell line to starve, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Draw a graph based on these 13 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profiles of the growth hormone protein family 9 and the growth hormone protein family 10 according to the present invention are very similar. Industrial applicability

 The polypeptides of the present invention, as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat embryonic developmental disorders, disorders of growth and development, abnormal pregnancy, tumors and the like.

 The growth hormone protein family is mainly secreted in the anterior pituitary gland and contains prolactin, growth hormones GH-V1 and GH-V2. Growth hormone protein family members share a specific functional domain in common.

Growth hormone has a wide range of functions. It can stimulate the growth of bone and cartilage and promote the synthesis of mucopolysaccharides and collagen. It also affects the metabolism of proteins, sugars and lipids, and ultimately affects the growth and development of the body. If auxin secretion is insufficient in young animals, dwarfism will occur; if auxin secretion is too much in young animals, giant disease will occur; if hyperpituitary function occurs in adult animals, acral hypertrophy will occur; if pituitary activity is too high, Promote the adrenal cortex to secrete glucocorticoids, causing hyperglycemia and diabetes. In addition, Physical activity has a large effect on many tissues, such as cartilage, connective tissue, muscle tissue, and fat. Its effect on tissue occurs at the level of the nucleus. Growth hormone can regulate the biosynthesis of various forms of RNA, which may increase The formation of MA polymerase increases the synthesis of RM.

 The physiological function of prolactin is to stimulate breast milk secretion from the fully developed breast, and to stimulate and maintain progesterone secretion from the corpus luteum.

 In addition, members of the growth hormone protein family play important roles in regulating body development, differentiation, and performing normal functions.

 It can be seen that the abnormal expression of specific growth hormone family proteins specific mot if will cause abnormal function of the polypeptide containing this mot if, resulting in embryo and body growth and development disorders, pregnancy abnormalities, and related diseases such as embryo development disorders Disease, growth disorder, pregnancy abnormalities, tumors, etc.

 It can be seen that the abnormal expression of the growth hormone protein family 10 of the present invention will produce various diseases, especially embryonic developmental disorders, growth disorders, pregnancy abnormalities, and tumors. These diseases include, but are not limited to:

 Embryonic disorders: congenital abortion, cleft palate, limb absentness, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney disease, double ureter, crypto, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias , Bisexual deformity, Atrial septal defect, Ventricular septal defect, Pulmonary stenosis, Arterial duct occlusion, Neural tube defect, Congenital hydrocephalus, Iris defect, Congenital cataract, Congenital glaucoma or cataract, Congenital deafness

 Growth and development disorders: dwarfism, giant disease, acromegaly, hyperglycemia, diabetic mental retardation, cerebral palsy, brain development disorder, mental retardation, familial cerebellar dysplasia syndrome, strabismus, Skin, fat, and muscular dysplasias such as congenital skin sagging, premature aging, congenital keratosis, various metabolic deficiencies such as various amino acid metabolic deficiencies, dementia, sexual retardation

 Tumors of various tissues: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, Colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, 'osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, biliary cancer, colon cancer, thymic tumor, nasal and sinus tumors, Nasopharyngeal carcinoma, laryngeal carcinoma, tracheal tumors, fibromas, fibrosarcomas, lipomas, liposarcomas, leiomyomas

 Pregnancy abnormalities: premature birth, miscarriage, pregnancy-induced hypertension

 The abnormal expression of the growth hormone protein family 9 of the present invention will also produce certain hereditary, hematological and immune system diseases.

The invention also provides screening compounds to identify increasing (agonist) or suppressing (antagonist) growth hormones. Method of medicament for protein family 9. Agonists increase biological functions such as growth hormone protein family 9 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing growth hormone protein family 9 can be cultured with labeled growth hormone protein family 9 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of the growth hormone protein family 9 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of the growth hormone protein family 9 can bind to the growth hormone protein family 9 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.

 When screening compounds that act as antagonists, the growth hormone protein family 9 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between the growth hormone protein family 9 and its receptor. Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to the growth hormone protein family 9 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 9 molecules of the growth hormone protein family should be labeled.

 The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies directed against the growth hormone protein family 9 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.

 Polyclonal antibodies can be produced by injecting growth hormone protein family 9 directly into immunized animals (such as rabbits, mice, rats, etc.). Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant . Techniques for preparing monoclonal antibodies to the growth hormone protein family 9 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma Technology, BBV-hybridoma technology, etc. Embedding antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against the growth hormone protein family 9.

 Antibodies against growth hormone protein family 9 can be used in immunohistochemistry to detect growth hormone protein family 9 in biopsy specimens.

Monoclonal antibodies that bind to the growth hormone protein family 9 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method The method is used to locate tumor cells and determine whether there is metastasis.

 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, high-affinity monoclonal antibodies from growth hormone protein family 9 can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol crosslinker such as SPDP, and toxins are bound to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill growth hormone protein family 9-positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases related to the growth hormone protein family 9. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of the growth hormone protein family 9.

 The invention also relates to a diagnostic test method for quantitative and localized detection of growth hormone protein family 9 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The levels of growth hormone protein family 9 detected in the test can be used to explain the importance of growth hormone protein family 9 in various diseases and to diagnose diseases in which growth hormone protein family 9 plays a role.

 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.

 Polynucleotides encoding the growth hormone protein family 9 can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat abnormal cell proliferation, development, or metabolism caused by non-expression or abnormal / inactive expression of growth hormone protein family 9. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated growth hormone protein family 9 to inhibit endogenous growth hormone protein family 9 activity. For example, a mutated growth hormone protein family 9 may be a shortened growth hormone protein family 9 lacking a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of the growth hormone protein family 9. Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding the growth hormone protein family 9 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a growth hormone protein family 9 can be found in the existing literature (Sambrook, et al.). In addition, a polynucleotide encoding the growth hormone protein family 9 can be packaged into liposomes and transferred into cells.

 Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit the growth hormone protein family 9 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that specifically breaks down specific RNAs. Its mechanism of action is that the ribozyme molecule specifically hybridizes to a complementary target RM to perform endonucleation. Antisense RNA, DNA, and ribozymes can be obtained by any of the existing RNA or DM synthesis techniques, such as the technique for the synthesis of oligonucleotides by solid-phase phosphate amide chemical synthesis, which is widely used. Antisense MA molecules can be obtained by in vitro or in vivo transcription of DM sequences encoding the RNA. This MA sequence is integrated downstream of the MA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length of the two hydrazones, and using phosphorothioate or peptide bonds instead of phosphodiester bonds for the linkage between ribonucleosides.

 Polynucleotides encoding growth hormone protein family 9 are useful in the diagnosis of diseases related to growth hormone protein family 9. A polynucleotide encoding growth hormone protein family 9 can be used to detect the expression of growth hormone protein family 9 or abnormal expression of growth hormone protein family 9 in a disease state. For example, a DNA sequence encoding growth hormone protein family 9 can be used to hybridize biopsy specimens to determine the expression status of growth hormone protein family 9. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. A part or all of the polynucleotide of the present invention can be fixed as a probe on a microarray or a DM chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes in a tissue and gene diagnosis. Transcription products of growth hormone protein family 9 can also be detected by RNA-polymerase chain reaction (RT-PCR) amplification using growth hormone protein family 9 specific primers.

 Detection of mutations in the growth hormone protein family 9 gene can also be used to diagnose growth hormone protein family 9-related diseases. Growth hormone protein family 9 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type growth hormone protein family 9 DNA sequences. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

 The sequences of the invention are also valuable for chromosome identification. The sequence specifically targets a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DM sequences on a chromosome.

 In short, PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.

PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes. Make Using the oligonucleotide primers of the present invention, sublocalization can be achieved by a similar method using a set of fragments from a specific chromosome or a large number of genomic clones. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.

 Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chrorao somes: a Manua l of Basic Techniques, Pergamon Press, New York (1988).

 Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopk ins University Wetch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

 Next, the differences in cDM or genomic sequences between the affected and the affected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the CDM that is accurately mapped to a disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).

 The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

 The invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds. '

 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. The growth hormone protein family 9 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of growth hormone protein family 9 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

Claims

 Claim 1, 1. An isolated polypeptide-growth hormone protein family 9, which is characterized in that it contains: a polypeptide having the amino acid sequence shown by SBQ ID NO.-2, or an active fragment, analog, or derivative of the polypeptide Thing.

 2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide, analog or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2.

 3. The polypeptide according to claim 2, further comprising a polypeptide having an amino acid sequence represented by SEQ ID NO: 2.

 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SBQ ID NO: 2 or a fragment, analog, or derivative thereof;

 (b) a polynucleotide complementary to polynucleotide (a); or

 (c) A polynucleotide that is at least 70% identical to (a) or (b).

 5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an amino acid sequence represented by SEQ ID NO: 2.

 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 422-691 in SBQ ID NO: 1 or the sequence of positions 1-1885 in SEQ ID NO: 1. .

7. A recombination vector containing an exogenous polynucleotide, characterized in that it is a recombination constructed by the polynucleotide according to any one of claims 4-6 and a plasmid, virus or a carrier expression vector Carrier.

 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:

 (a) a host cell transformed or transduced with the recombinant vector of claim 7; or

 (b) a host cell transformed or transduced with a polynucleotide according to any one of claims 4-6.

 9. A method for preparing a polypeptide having growth hormone protein family 9 activity, characterized in that the method includes:

 (a) culturing the engineered host cell according to claim 8 under conditions that express the growth hormone protein family 9;

(b) Isolating a polypeptide having growth hormone protein family 9 activity from the culture.

10. An antibody capable of binding to a polypeptide, characterized in that said antibody is an antibody capable of specifically binding to the growth hormone protein family 9.

 11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of the growth hormone protein family 9.

12. The compound according to claim 11, characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO: 1.

 13. Use of a compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of the growth hormone protein family 9 in vivo and in vitro.

 14. A method for detecting a disease or susceptibility to a disease associated with a polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide Or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

15. Use of a polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of growth hormone protein family 9; or for peptide fingerprinting Atlas identification.

16. The use of a nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or for manufacturing a gene chip Or microarray.

 17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or mimetic, agonist, antagonist is used Or the inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with a growth hormone protein family 9 abnormality.

 18. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Disease, HIV infection and immune diseases and drugs of various inflammations.