A novel polypeptide- human big protein 9.13 and the polynucleotide encoding saidpolypeptide

Abstract

The invention discloses a new polypeptide- human big protein 9.13, the polynucleotide encoding said polypeptide, and a process for producing the polynucleotide by recombinant DNA technology. It also discloses methods of applying the polypeptide for the treatment of various diseases, such as fetation abnormality, protein metabolism disorder, tumor disease, immune system disorder disease, etc. This invention also discloses antagonist against said polypeptide and thereof therapy uses. In addition, it refers to the use of said polynucleotide encoding this new human big protein 9.13.

Description

[0001] A novel polypeptide—human macroprotein 9.13 and the polynucleotide encoding this polypeptide. (Technical Field)

[0002] This invention belongs to the field of biotechnology. Specifically, this invention describes a novel polypeptide—human macroprotein 9.13—and the polynucleotide sequence encoding this polypeptide. This invention also relates to methods for preparing this polynucleotide and polypeptide, and their applications. Background Art

[0003] As part of a cDM protocol used to infer the coding sequences of undetermined human genes, we recently identified the sequences of 100 cDNA clones of varying sizes cloned from a human brain cDNA library and predicted the coding sequences of the corresponding genes. These clones showed an average intron size of 4.9 kb and a corresponding open reading frame size of 2.6 kb (882 amino acid residues). Computational data analysis revealed that 87 of the 100 clones contained sequences with high homology to known genes, and 53% of the genes (46 genes) encoded proteins related to cell signaling / communication, cell structure / motility, and nucleic acid control. These genes are expressed in 10 human tissues, 8 brain regions (amysalis, corpus callosum, cerebellum, caudate nucleus, hippocampus, basal thalamic nucleus, thalamus, substantia nigra), spinal cord, fetal brain, and fetal liver [Takahiro Nagase, Ken-ichi I shikawa et al., 1999, DNA Research., 6, 63-70].

[0004] In our research on these genes, we paid particular attention to those encoding large proteins, which likely play important roles in mammals [Ohara, O., Nagase, T., Ishikawa, KI et al., 1997, DNA Research., 4, 53-59] [Nagase, T., Ishikawa, KI, Suyama, M, et al., 1998, DNA Research., 5, 355-364]. Notably, these large proteins are expressed not only in 10 human tissues but also in various regions of the human central nervous system and the fetal brain, indicating their significant role in the human nervous system.

[0005] As mentioned above, the function of KIAA0072 protein is related to cell signaling / communication, cell structure / motility, and nucleic acid control. It is expressed in 10 human tissues and 8 brain regions, spinal cord, fetal brain, and fetal liver. Gene chip analysis revealed that the expression profiles of the polypeptides of this invention are very similar to those of KIAA0072 in bladder mucosa, PMA+ Ecv304 cell line, LPS+ Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblasts stimulated with growth factors 1024NT, scar fibroblasts stimulated with growth factors 1013HT, scar fibroblasts without growth factor stimulation 1013HC, bladder cancer cells (EJ), adjacent cells of bladder cancer, bladder cancer, liver cancer, liver cancer cell lines, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, and gastric cardia cancer. Therefore, their functions may also be similar. This invention is named Human Protein 9.13. As mentioned above, human macroprotein 9.13 plays a crucial role in regulating vital bodily functions such as cell division and embryonic development, and it is believed that numerous proteins are involved in these regulatory processes. Therefore, there has been a long-standing need in the art to identify more human macroprotein 9.13 proteins involved in these processes, particularly to identify their amino acid sequences. The isolation of the gene encoding this novel human macroprotein 9.13 protein provides a foundation for determining its role in healthy and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for diseases; therefore, isolating its encoding DNA is of paramount importance. Disclosure of the Invention

[0006] One object of the present invention is to provide a novel isolated polypeptide, human macroprotein 9.13, as well as its fragments, analogs and derivatives.

[0007] Another object of the present invention is to provide a polynucleotide encoding the polypeptide.

[0008] Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human macroprotein 9.13.

[0009] Another object of the present invention is to provide genetically engineered host cells containing a polynucleotide encoding human macroprotein 9.13.

[0010] Another object of the present invention is to provide a method for producing human macroprotein 9.13.

[0011] Another object of the present invention is to provide an antibody against the polypeptide of the present invention—human macroprotein 9.13. Another object of the present invention is to provide mimicry compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention—human macroprotein 9.13.

[0012] Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human macroprotein 9.13.

[0013] This invention relates to an isolated polypeptide, which is of human origin, comprising: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conserved variant thereof, a bioactive fragment, or a derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0014] The present invention also relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

[0015] (a) A polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;

[0016] (b) A polynucleotide complementary to polynucleotide (a);

[0017] (c) A polynucleotide that has at least 70% identity with the polynucleotide sequence of (a) or (b).

[0018] More preferably, the sequence of the polynucleotide is selected from one of the following groups: (a) having the sequence in SEQ ID NO: 1

[0019] (a) A sequence of 348-599 bits; and (b) A sequence having 1-1408 bits of SEQ ID NO: 1.

[0020] The present invention also relates to a vector containing the polynucleotides of the present invention, particularly an expression vector; a host cell genetically engineered using the vector, including a transformed, transduced, or transfected host cell; and a method for preparing the polypeptides of the present invention, including culturing the host cell and recovering the expression product. The present invention also relates to an antibody that can specifically bind to the polypeptide of the present invention.

[0021] The present invention also relates to a method for screening compounds that mimic, activate, antagonize, or inhibit the activity of human macroprotein 9.13 protein, comprising a polypeptide utilizing the present invention. The present invention also relates to compounds obtained by this method.

[0022] The present invention also relates to an in vitro method for detecting diseases or disease susceptibility associated with abnormal expression of human macroprotein 9.13 protein, comprising detecting mutations in the polypeptide or its encoding polynucleotide sequence in a biological sample, or detecting the amount or biological activity of the polypeptide of the present invention in a biological sample.

[0023] The present invention also relates to a pharmaceutical composition comprising the polypeptide of the present invention or an analogue thereof, an activator, an antagonist or inhibitor, and a pharmaceutically acceptable carrier.

[0024] The present invention also relates to the use of the polypeptides and / or polynucleotides of the present invention in the preparation of medicaments for treating cancer, developmental disorders or immune disorders or other diseases caused by abnormal expression of human macroprotein 9.13.

[0025] Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.

[0026] Unless otherwise specified, the following terms used in this specification and claims have the following meanings:

[0027] "Nucleic acid sequence" refers to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, and may also refer to genomic or synthetic DNA or RNA, which may be single-stranded or double-stranded, representing sense or antisense strands. Similarly, the term "amino acid sequence" refers to oligopeptides, peptides, polypeptides, or protein sequences and fragments or portions thereof. When "amino acid sequence" in this invention refers to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean limiting the amino acid sequence to the complete natural amino acids associated with said protein molecule.

[0028] A protein or polynucleotide "variant" is an amino acid sequence or polynucleotide sequence encoding it that has one or more amino acid or nucleotide changes. These changes may include the deletion, insertion, or substitution of amino acids or nucleotides in the amino acid or nucleotide sequence. Variants may have "conserved" changes, where the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine. Variants may also have non-conserved changes, such as replacing glycine with tryptophan.

[0029] "Deficiency" refers to the absence of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

[0030] "Insertion" or "addition" refers to a change in the amino acid or nucleotide sequence that results in the addition of one or more amino acids or nucleotides compared to the naturally occurring molecule. "Replacement" refers to the substitution of one or more amino acids or nucleotides by different amino acids or nucleotides.

[0031] "Bioactivity" refers to proteins that possess the structural, regulatory, or biochemical functions of natural molecules. Similarly, the term "immunologic activity" refers to the ability of natural, recombinant, or synthetic proteins and fragments thereof to induce specific immune responses and bind to specific antibodies in suitable animals or cells. An "agonist" is a molecule that, when bound to human macroprotein 9.13, causes alterations in the protein, thereby regulating its activity. Agonists can include proteins, nucleic acids, carbohydrates, or any other molecules that can bind to human macroprotein 9.13.

[0032] "Antagonist" or "inhibitor" refers to a molecule that, when bound to human macroprotein 9.13, blocks or modulates the biological or immunological activity of human macroprotein 9.13. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecules that can bind to human macroprotein 9.13.

[0033] "Regulation" refers to changes in the function of human macroprotein 9.13, including increases or decreases in protein activity, changes in binding properties, and any other changes in the biological, functional, or immune properties of human macroprotein 9.13.

[0034] "Substantially pure" means substantially free of other naturally occurring or associated proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify human macroprotein 9.13 using standard protein purification techniques. Substantially pure human macroprotein 9.13 produces a single master band on a non-reducing polyacrylamide gel. The purity of the human macroprotein 9.13 peptide can be determined by amino acid sequence analysis.

[0035] "Complementary" or "complementary" refers to the natural binding of polynucleotides through base pairing under permissible salt concentration and temperature conditions. For example, the sequence "C-T-G-A" can bind to the complementary sequence "G-A-C-T". Complementarity between two single-stranded molecules can be partial or complete. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0036] "Homology" refers to the degree of complementarity, which can be partial or complete homology. "Partial homology" refers to a partially complementary sequence that at least partially inhibits the hybridization of a completely complementary sequence with the target nucleic acid. This inhibition of hybridization can be detected by hybridization under reduced stringency conditions (such as Southern blotting or Nor thern blotting). Essentially, homologous sequences or hybridization probes can compete for and inhibit the binding of completely homologous sequences to the target sequence under reduced stringency conditions. This does not mean that reduced stringency conditions allow non-specific binding, because reduced stringency conditions require the binding of the two sequences to be a specific or selective interaction.

[0037] "Identity percentage" refers to the percentage of identical or similar sequences when comparing two or more amino acid or nucleic acid sequences. The identity percentage can be determined electronically, such as using the MEGALIGN program (Laser gene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences (Higgins, DG, and...) using different methods such as the Cluster method.

[0038] PM Sharp (1988) Gene 73: 237-244) The Cluster method arranges the sequences into clusters by examining the distances between all pairs. The clusters are then assigned in pairs or groups. The percentage of similarity between two amino acid sequences, such as sequence A and sequence B, is calculated using the following formula: Number of matching residues between sequence A and sequence B

[0039] The number of residues in sequence A minus the number of spacer residues in sequence A minus the number of spacer residues in sequence B can also be determined by the Cluster method or by methods known in the art, such as Jotun Hein (Hein L, (1990) Methods in emzumology 183: 625-645). "Similarity" refers to the degree of similarity or conserved substitution of amino acid residues at corresponding positions when comparing the arrangement of amino acid sequences. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; amino acids with similar hydrophilicity and uncharged head groups may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

[0040] "Antense" refers to a nucleotide sequence that is complementary to a specific DNA or RNA sequence. "Antense strand" refers to a nucleic acid strand that is complementary to the "sense strand".

[0041] "Derivatives" refer to chemical modifications of HFPs or the nucleic acids that encode them. These modifications can involve replacing hydrogen atoms with alkyl, acyl, or amino groups. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of the natural molecule.

[0042] "Antibody" refers to the complete antibody molecule and its fragments, such as Fa, ? (^')2 and ? ^, which can specifically bind to the antigenic determinant of human macroprotein 9.13.

[0043] "Humanized antibody" refers to an antibody whose non-antigen binding region amino acid sequence has been replaced to make it more similar to a human antibody, but still retains its original binding activity.

[0044] The term "separated" refers to the removal of a substance from its original environment (e.g., its natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living organism is not separated, but the same polynucleotide or polypeptide separated from some or all of the substances that coexist with it in the natural system is considered separated. Such a polynucleotide may be part of a carrier, or it may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still separated.

[0045] As used in this invention, "isolated" means that a substance has been separated from its original environment (or, if the substance is natural, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state within living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in their natural state.

[0046] As used herein, "isolated human macroprotein 9.13" means that human macroprotein 9.13 is substantially free of other naturally occurring proteins, lipids, carbohydrates, or other substances associated with it. Those skilled in the art can purify human macroprotein 9.13 using standard protein purification techniques. A substantially pure polypeptide produces a single master band on a non-reducing polyacrylamide gel. The purity of the human macroprotein 9.13 polypeptide can be determined by amino acid sequence analysis. This invention provides a novel polypeptide—human macroprotein 9.13—which is essentially composed of the amino acid sequence shown in SBQ ID NO: 2. The polypeptide of this invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptide of this invention can be a naturally purified product, a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptide of this invention can be glycosylated or non-glycosylated. The polypeptide of this invention may or may not include an initial methionine residue.

[0047] This invention also includes fragments, derivatives, and analogs of human macroprotein 9.13. As used herein, the terms "fragment," "derivative," and "analyte" refer to human macroprotein 9.13 that substantially retain the characteristics of this invention. 3 Peptides with the same biological function or activity. Fragments, derivatives, or analogs of the peptides of the present invention may be: (I) in which one or more amino acid residues are substituted by conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and the substituted amino acids may or may not be encoded by a genetic codon; or (π) in which a group on one or more amino acid residues is substituted by another group containing a substituent; or (II) in which the mature peptide is fused with another compound (e.g., a compound that extends the half-life of the peptide, such as polyethylene glycol); or (IV) in which an additional amino acid sequence is fused into the mature peptide to form a peptide sequence (e.g., a leader sequence, a secretory sequence, or a sequence used to purify the peptide or a proteogen sequence). As described herein, such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.

[0048] This invention provides isolated nucleic acids (polynucleotides) that are essentially composed of polynucleotides encoding polypeptides having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of this invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of this invention was discovered from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1408 bases, with open reading frames 348-599 encoding 38 amino acids. Based on gene chip expression profile comparison, this polypeptide has a similar expression profile to KIAA0072; it can be inferred that this human macroprotein 9.13 has a similar function to KIAA0072.

[0049] The polynucleotides of this invention can be in DM or RM form. DM form includes cDNA, genomic DNA, or artificially synthesized DM. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used in this invention, "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2, but differing from the coding region sequence shown in SEQ ID NO: 1.

[0050] 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) and non-coding sequences.

[0051] The term "polynucleotide encoding a polypeptide" refers to a polynucleotide that includes the polynucleotide encoding the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences. This invention also relates to variants of the polynucleotides described above, which encode polypeptides or polypeptide fragments, analogs, and derivatives having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0052] The present invention also relates to polynucleotides that hybridize with the sequences described above (having at least 50%, preferably 70%, similarity between the two sequences). The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2xSSC, 0.1% SDS, 6(TC); or (2) the use of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42 °C, etc.; or (3) hybridization only occurs when the similarity between the two sequences is at least 95%, preferably at least 97%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

[0053] This invention also relates to nucleic acid fragments that hybridize with the sequences described above. As used in this invention, the length of a "nucleic acid fragment" is at least 10 nucleotides, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate the polynucleotide encoding human macroprotein 9.13.

[0054] The polypeptides and polynucleotides of this invention are preferably provided in isolated form, and more preferably purified to homogenate. The specific polynucleotide sequence encoding human macroprotein 9.13 of this invention can be obtained by a variety of methods. For example, polynucleotides can be isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes with genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect polynucleotide fragments of clones with common structural features.

[0055] The DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DM sequence from the genomic DM; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.

[0056] Of the methods mentioned above, isolating genomic DM is the least common. Direct chemical synthesis of the DM sequence is a frequently chosen method. A more common method is the isolation of the cMA sequence. The standard method for isolating the cDM of interest is to isolate mRNA from donor cells that highly express the gene and perform reverse transcription to form a plasmid or phage cDM library. Several mature techniques exist for extracting mRM, and kits are commercially available (Qiagene). Constructing cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as various cDNA libraries from Clontech. When combined with polymerase chain reaction (PCR) technology, even very small amounts of expression products can be cloned.

[0057] Genes of the present invention can be screened from these cDM libraries using conventional methods. These methods include (but do not include) Limited to: (1) DNA-DM or DM-RNA hybridization; (2) the appearance or loss of marker gene function; 3 (3) Determine the level of the transcript of human macroprotein 9.13; (4) Detect the protein products of gene expression by means of immunological techniques or by measuring biological activity. The above methods can be used alone or in combination.

[0058] In 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, and most preferably at least 100 nucleotides. Furthermore, the length of the probe is generally within 2000 nucleotides, preferably within 1000 nucleotides. The probe used herein is typically a chemically synthesized MA sequence based on the gene sequence information of the present invention. The gene itself or a fragment of the present invention can, of course, be used as a probe. The DNA probe can be labeled with radioactive isotopes, fluorescein, or enzymes (such as alkaline phosphatase), etc.

[0059] In the fourth method, the protein products of human macroprotein 9.13 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA).

[0060] The method of amplifying MA / RNA using PCR technology (Saiki, et al. Science 1985; 230: 1350-1354) is preferred for obtaining the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA end amplification method) is preferred. The primers used for PCR can be appropriately selected based on the polynucleotide sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DM / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0061] The polynucleotide sequences of the gene or various DM fragments obtained as described above can be determined using conventional methods such as the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). These polynucleotide sequences can also be determined using commercial sequencing kits. To obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes, it is necessary to determine the cDNA sequences of multiple clones before they can be assembled into a full-length cDNA sequence.

[0062] The present invention also relates to vectors containing the polynucleotides of the present invention, and host cells generated by genetic engineering using the vectors of the present invention or directly using the human macroprotein 9.13 coding sequence, as well as methods for generating the polypeptides of the present invention via recombinant technology.

[0063] In this invention, a polynucleotide sequence encoding human macroprotein 9.13 can be inserted into a vector to construct a recombinant vector containing the polynucleotides described in this invention. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors applicable in this invention include, but are not limited to: expression vectors based on the T7 promoter expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem. 263: 3521, 1988); and vectors derived from baculoviruses expressed in insect cells. In short, any plasmid and vector can be used to construct a recombinant expression vector, provided it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain a replication origin, a promoter, a marker gene, and translational regulatory elements.

[0064] Methods well known to those skilled in the art can be used to construct DNA sequences encoding human macroprotein 9.13. Expression vectors containing suitable transcriptional / translational regulatory elements are used. These methods include in vitro recombinant DNA technology, DM synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory - New York, 1989). The DM sequence can be efficiently linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; promoters of λ phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. Expression vectors also include ribosome binding sites for translation initiation and transcription terminators. Inserting enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors of DNA expression, typically consisting of approximately 10 to 300 base pairs, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0065] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0066] Those skilled in the art are well aware of how to select appropriate vectors / transcriptional regulatory elements (such as promoters, enhancers, etc.) and selective marker genes.

[0067] In this invention, a polynucleotide encoding human macroprotein 9.13 or a recombinant vector containing such polynucleotide can be transformed or introduced into a host cell to constitute a genetically engineered host cell containing such polynucleotide or recombinant vector. The term "host cell" refers to prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: *Escherichia coli*, *Streptomyces*; bacterial cells such as *Salmonella typhimurium*; fungal cells such as yeast; plant cells; insect cells such as *Drosophila S2* or *Sf9*; and animal cells such as CHO, COS, or Bowes melanoma cells.

[0068] Transformation of host cells using the MA sequence or a recombinant vector containing the DM sequence described in this invention can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of DM uptake can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. MgCl2 can also be used. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, DM transfection methods such as calcium phosphate co-precipitation or conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used.

[0069] Using conventional recombinant DM technology, the polynucleotide sequence of this invention can be used to express or produce recombinant human macroprotein 9.13 (Science, 1984; 224: 1431). Generally, the following steps are involved:

[0070] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding human macroprotein 9.13 of the present invention, or using a recombinant expression vector containing the polynucleotide; (2) Culture the host cells in a suitable culture medium;

[0071] (3) Isolate and purify proteins from culture media or cells.

[0072] In step (2), the culture medium used in the culture can be selected from various conventional culture media, depending on the host cells used. The cells are cultured under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period.

[0073] In step (3), the recombinant polypeptide may be encapsulated intracellularly, expressed on the cell membrane, or secreted extracellularly. If desired, the recombinant protein may be separated and purified using various separation methods based on its 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 refolding treatment, protein precipitation (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. Brief description of the figures.

[0074] The following figures are used to illustrate specific embodiments of the present invention, and are not intended to limit the scope of the invention as defined by the claims.

[0075] Figure 1 is a comparison of the gene chip expression profiles of human macroprotein 9.13 and KIAA0072 of this invention. The upper figure is the expression profile diagram of human macroprotein 9.13, and the lower figure is the expression profile diagram of KIAA0072. Among them, 1-fetal brain, 2-bladder mucosa, 3-PMA+ Ecv304 cell line, 4-LPS+ Ecv304 cell line thymus, 5-normal fibroblast 1024NC, 6-Fibroblas t, growth factor stimulated, 1024NT, 7-scar fc growth factor stimulated, 1013HT, 8-scar fc unstimulated with growth factor, 1013HC, 9-bladder cancer cell line EJ, 10-bladder cancer adjacent to bladder cancer, 11-bladder cancer, 12-liver cancer, 13-liver cancer cell line, 14-fetal skin, 15-spleen, 16-prostate cancer, 17-jejunal adenocarcinoma, 18-cardia cancer.

[0076] Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) image of the isolated human macroprotein 9.13. 10 kDa represents the molecular weight of the protein. The arrows indicate the separated protein bands. The best mode for realizing this invention...

[0077] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Co ld Spr ing Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0078] Example 1: Cloning of human macroprotein 9.13

[0079] Total RNA was extracted from human fetal brain using a one-step method involving guanidine isothiocyanate / phenol / chloroform. The Quik mRNA isolation kit was used. (Qiegene product) Poly(A) mRNA was isolated from total RNA. 2 μg of poly(A) mRNA was reverse transcribed to form cDNA. The cDNA fragment was directionally inserted into the multiple cloning site of a pBSK(+) vector (Clontech product) using the Smart cDNA Cloning Kit (Clontech product), and the bacteria were transformed into DH5α to form a cDM library. The 5' and 3' ends of all clones were sequenced using a Dye terminate cycle reaction sequencing kit (PerkinElmer product) and an ABI 377 automated sequencer (PerkinElmer). The determined cDNA sequences were compared with existing public MA sequence databases (Genebank), and the cDNA sequence of clone 0540a04 was found to be novel. A series of primers were synthesized to perform bidirectional sequencing of the inserted cDM fragment contained in this clone. The results showed that the full-length cMA contained in clone 0540a04 was 1408 bp (as shown in Seq ID NO: l), starting from 348b. P At 599bp, there is a 251bp open reading frame (ORF) encoding a novel protein (as shown in Seq ID NO: 2). We named this clone pBS-0540a04, and the encoded protein was named human macroprotein 9.13. Example 2: Cloning the gene encoding human macroprotein 9.13 using the !^-?0 method.

[0080] Using total fetal brain cell MA as a template, cDNA was synthesized by reverse transcription with oligo-dT as a primer. After purification using a Qiagen kit, PCR amplification was performed using the following primers:

[0081] Primer 1: 5, - ATAGAAAGGMTACTATTATAGCA - 3, (SEQ ID NO: 3)

[0082] Primer2: 5, - AAAGCTATATATTTATTCAACTTA- 3' (SEQ ID NO: 4)

[0083] Pr imerl is the forward sequence starting at the ]th bp at the 5' end of SEQ ID NO: 1;

[0084] Pr imer2 is the reversed 3' end sequence of SEQ ID NO: 1.

[0085] ■ Amplification reaction conditions: A 50 μL reaction volume contained 50 mmol / L KCl, 10 mmol / L Tris-Cl (pH 8.5), 1.5 mmol / L MgCl2, 200 μmol / L dNTP, 1 Opmol primer, and 1 U Taq DNA polymerase (Clontech). The reaction was performed for 25 cycles on a PE9600 DNA thermal cycler (PerkinElmer) under the following conditions: 94°C for 30 sec; 55°C for 30 sec; 72°C for 2 min. β-actin was used as a positive control and template blank as a negative control during RT-PCR. The amplified products were purified using a QIAGEN kit and ligated into a PCR vector (Invitrogen) using a TA cloning kit. DNA sequence analysis showed that the DNA sequence of the PCR product was identical to 1-1408 bp as shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human macroprotein 9.13 gene expression:

[0086] Total amino acids (MA) were extracted using a one-step method [Anal. Biochem 1987, 162, 156-159]. This method involves acidic guanidinium thiocyanate-phenol-chloroform extraction. The tissue was homogenized with 4M guanidinium isothiocyanate, 25mM sodium citrate, and 0.2M sodium acetate (pH 4.0). One volume of phenol and 1 / 5 volume of chloroform-isoamyl alcohol (49:1) were added, and the mixture was centrifuged. The aqueous phase was aspirated, and isopropanol (0.8 volume) was added. The mixture was then centrifuged to obtain the MA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried, and dissolved in water. 20 g of RM was extracted onto a 1.2% agarose gel containing 20mM 3-(N-morpholino)propanesulfonic acid (pH 7.0), 5mM sodium acetate, 1mM EDTA, and 2.2M formaldehyde. Perform electrophoresis. Then transfer to a nitrocellulose membrane. Use α- 32 P dATP was prepared using a random primer method. 32 P-labeled DM probe. The DNA probe used was the human macroprotein 9.13 coding region sequence (348b) amplified by PCR, as shown in Figure 1. P Up to 599b P ). 32P-labeled probes (approximately 2 x 10) 6 The RNA-transferred nitrocellulose membrane (cpm / ml) was hybridized overnight at 42°C in a solution containing 50% formamide - 25mM KH2PO4 (pH 7.4) - 5× SSC - 5× Denhardt's solution and 20 g / ml salmon sperm DM. After hybridization, the membrane was washed in 1× SSC - 0.1% SDS at 55°C for 30 min. Then, it was analyzed and quantified using a Phosphor Imager. Example 4: In vitro expression, isolation, and purification of recombinant human macroprotein 9.13.

[0087] Based on the coding region sequence shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers were designed, with the following sequences:

[0088] Primer 3: 5'-CATGCTAGCATGCTTTCTTATAATGCATATTTC-3' (Seq ID No: 5) Primer : 5'-CATGGATCCCTAAAACGGTGAAACCCCGTCTCT-3' (Seq ID No: 6) These two primers contain Ndel and BamHI restriction sites at their 5' ends, respectively, followed by the coding sequences for the 5' and 3' ends of the target gene. The Ndel and BamHI restriction sites correspond to the selective restriction enzyme sites on the expression vector plasmid pBT-28b(+) (Novagen product, Cat. No. 69865. 3). PCR was performed using the pBS-0540a04 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μL containing 10 pg of pBS-0540a04 plasmid, primers Primer-3 and Primer-4 (10 pmol each), and 1 μL of Advantage polymerase Mix (Clontech). Cycling parameters: 94°C for 20 s, 60°C for 30 s, 68°C for 2 min, for a total of 25 cycles. The amplified product and plasmid pET-28(+) were double-digested with Ndel and BamHI, respectively, and the large fragments were recovered and ligated using T4 ligase. The ligation product was transformed into E. coli DH5α using the calcium chloride method in a solution containing kanamycin (final concentration 30 μL). § After culturing overnight on LB agar plates ( / ιη1), positive clones were screened using colony PCR and sequenced. Positive clones with correct sequences (pET-0540a04) were selected and transformed into *E. coli* BL21 (DE3) using the calcium chloride method. PlySs (Novagen product). The host bacterium BL21 (pET-0540a04) was cultured in LB broth containing kanamycin (final concentration 3 g / ml) at 37°C to the logarithmic growth phase. IPTG was added to a final concentration of 1 μL / L, and the culture was continued for 5 hours. The bacterial cells were collected by centrifugation, and the supernatant was collected by centrifugation. The purified target protein, human macroprotein 9.13, was obtained by His. Bind Quick Column (Novagen) affinity chromatography, which binds to six histidine residues (6His-Tag). SDS-PAGE electrophoresis showed a single band at 10 kDa (Figure 2). This band was transferred to a PVDF membrane, and the N-terminal amino acid sequence was analyzed using the Edams hydrolysis method. The results showed that the N-terminal 15 amino acids were identical to the N-terminal 15 amino acid residues shown in SEQ ID NO: 2. Example 5: Anti-human macroprotein 9. 13. Antibody production

[0089] The following human macroprotein 9.13-specific peptides were synthesized using a peptide synthesizer (PE company product):

[0090] NH2-Met-Leu-Ser-Tyr-Asn-Ala-Tyr-Phe-Phe-Ser-Val-Tyr-Phe-Leu-Phe-C00H (SEQ ID NO: 7). The polypeptide was coupled with hemocyanin and bovine serum albumin to form complexes, as described in: Avrameas, et al. Iramunochemi stry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex plus complete Freund's adjuvant, followed by a booster immunization with the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant 15 days later. The antibody titer in rabbit serum was determined by ELISA using a titration plate coated with 15 g / ml bovine serum albumin-polypeptide complex. Total IgG was separated from antibody-positive rabbit serum using protein A-Sepharose. The polypeptide was then bound to a cyanogen bromide-activated Se... P Antipeptide antibodies were isolated from total IgG using affinity chromatography on a Harose 4B column. Immunoprecipitation demonstrated that the purified antibody specifically bound to human macroprotein 9.13. Example 6: Application of the polynucleotide fragment of the present invention as a hybridization probe.

[0091] Selecting suitable oligonucleotide fragments from the polynucleotides of the present invention for use as hybridization probes has many applications. For example, the probe can be used to hybridize with genomic or cDNA libraries of normal or pathological tissues from different sources to identify whether they contain the polynucleotide sequence of the present invention and to detect homologous polynucleotide sequences. Furthermore, the probe can be used to detect whether the expression of the polynucleotide sequence of the present invention or its homologous polynucleotide sequences is abnormal in normal or pathological tissue cells.

[0092] The purpose of this embodiment is to select suitable oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes, and to identify whether some tissues contain the polynucleotide sequence of the present invention or its homologous polynucleotide sequence using a membrane hybridization method. Membrane hybridization methods include dot blot, Southern blotting, Northern blotting, and photocopying methods, all of which involve immobilizing the polynucleotide sample to be tested on a membrane and then hybridizing using essentially the same steps. These identical steps are: the membrane with the sample immobilized is first pre-hybridized with a hybridization buffer without probes to saturate the non-specific binding sites of the sample on the membrane with the carrier and the synthesized polymer. Then the pre-hybridization solution is replaced with a hybridization buffer containing labeled probes, and the membrane is incubated to allow the probes to hybridize with the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of washing steps. This embodiment utilizes high-intensity washing conditions (such as low salt concentration and high temperature) to reduce the hybridization background and retain only highly specific signals. The probes used in this embodiment include two types: the first type of probe is an oligonucleotide fragment that is completely identical to or complementary to the polynucleotide SEQ ID NO: 1 of this invention; the second type of probe is an oligonucleotide fragment that is partially identical to or complementary to the polynucleotide SEQ ID NO: 1 of this invention. In this embodiment, dot blot is used to immobilize the sample on a filter membrane. Under high-intensity washing conditions, the first type of probe exhibits the strongest hybridization specificity with the sample and is thus retained. When selecting oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of this invention for use as hybridization probes, the following principles and several aspects should be considered:

[0093] 1. The preferred probe size range is 18-50 nucleotides;

[0094] 2. GC content should be 30%-70%; exceeding this range will increase non-specific hybridization.

[0095] 3. There should be no complementary regions inside the probe; 4. Probes that meet the above conditions can be used as preliminary selection probes. Then, computer sequence analysis is performed, including comparing the homology of the preliminary selection probe with its source sequence region (i.e., SEQ ID NO: 1) and other known genomic sequences and their complementary regions. If the homology with the non-target molecule region is greater than 85% or there are more than 15 consecutive identical bases, then the preliminary selection probe should generally not be used.

[0096] 5. Whether the initial probe is ultimately selected as a probe with practical application value should be further determined by experiments.

[0097] After completing the above analysis, the following two probes were selected and synthesized:

[0098] Probe 1, belonging to class 1 probes, is completely homologous to or complementary to the gene fragment of SEQ ID NO: 1 (41Nt).

[0099] 5'- TGCTTTCTTATMTGCATATTTCTTCTCAGTTTATTTTTTA- 3' ( SEQ ID NO: 8 )

[0100] Probe 2, belonging to the second class of probes, is a substitutional mutant sequence (41Nt) corresponding to the gene fragment of SEQ ID NO: 1 or its complementary fragment:

[0101] 5'-TGCTTTCTTATAATGCATATCTCTTCTCAGTTTATTTTTTA-3' (SEQ ID NO: 9) For other commonly used reagents and their preparation methods related to the following specific experimental steps, please refer to: DNA PROBES GH Kel ler; MM Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning laboratory manuals such as Molecular Cloning: A Laboratory Manual (2nd edition, 1998) [US] Sambrook et al., Science Press.

[0102] Sample preparation:

[0103] 1. Extracting DM from fresh or frozen tissue

[0104] Steps: 1) Place fresh or freshly thawed normal liver tissue into a petri dish containing phosphate-buffered saline (PBS) and place it on ice. Cut the tissue into small pieces using scissors or a scalpel. Keep the tissue moist during the process. 2) Centrifuge at 1000g for 10 minutes to mince the tissue. 3) Resuspend the pellet in cold homogenization buffer (0.25 mol / L sucrose; 25 mmol / L Tris-HCl, pH 7.5; 25 mmol / L NaCl; 25 mmol / L MgCl2) (approximately 10 nil / g). 4) Homogenize the tissue suspension at full speed using an electric homogenizer at 4°C until the tissue is completely broken down. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (1-5 ml per 0.1g of the initial tissue sample) and centrifuge again at 1000g for 10 minutes. 7) Resuspend the precipitate with lysis buffer (1 ml for every 0.1 g of the initial tissue sample), and then proceed with the following phenol extraction method.

[0105] 2. Phenol extraction method for DNA

[0106] Steps: 1) Wash cells with 1-10 ml of cold PBS, centrifuge at 1000 g for 10 minutes. 2) Resuspend the precipitated cells in cold cell lysis buffer (IX 10). s (Cells / ml) Use at least 100ul of 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 form large clumps that are difficult to break down, reducing the overall yield. This is especially important when extracting >10... 7 The situation is particularly severe in cells. 4) Add proteinase K to a final concentration of 200 μg / ml. 5) Incubate at 50°C for 1 hour or gently shake overnight at 37°C. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1), and centrifuge for 10 minutes in a small centrifuge tube. The two phases should be clearly separated; otherwise, repeat the centrifugation. 7) Transfer the aqueous phase to a new tube. 8) Use Extract with an equal volume of chloroform:isoamyl alcohol (24:1) and centrifuge for 10 minutes. 9) Transfer the aqueous phase containing DNA to a new tube. Then perform DM purification and ethanol precipitation.

[0107] 3. Purification and ethanol precipitation of DM

[0108] Step 1: 1) Add 180 volumes of 2 mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix well. Incubate at -20°C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour off the ethanol. 4) Wash the precipitate with 500 μL of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour off the ethanol. Wash the precipitate with 500 μL of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour off the ethanol, then invert the container on absorbent paper to allow any remaining ethanol to drain. Air dry for 10-15 minutes to allow surface ethanol to evaporate. Do not allow the precipitate to dry completely, otherwise it will be difficult to redissolve. 7) Resuspend the DNA precipitate in a small volume of TE or water. Vortex at low speed or use a dropper to gradually increase the TE, mixing until the DNA is fully dissolved, adding 1-5 x 10⁻⁶ drops per 100 mL. 6 The amount extracted from the cells is approximately 1 ul.

[0109] Steps 8-13 below are only used when it is necessary to remove contaminants; otherwise, proceed directly to step 14.

[0110] 8) Add RNase A to the DM solution to a final concentration of 100 μg / ml and incubate at 37°C for 30 minutes. 9) Add SDS and proteinase K to final concentrations of 0.5 µg / ml and 100 μg / ml, respectively. o 10) Incubate at 37°C for 30 minutes. 11) Extract the reaction solution with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuge for 10 minutes. 12) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform:isoamyl alcohol (24:1) and centrifuge for 10 minutes. 13) Carefully remove the aqueous phase, add 1 / 10 volume of 2mol / L sodium acetate and 2.5 volume of cold ethanol, mix well, and incubate at -20°C for 1 hour. 14) Wash the precipitate with 70% ethanol and 100% ethanol, air-dry, and resuspend the nucleic acid, following steps 3-6. 15) Determine A. 26 And A 28 To detect DNA purity and yield. 15) After aliquoting, store at -20°C. Sample membrane preparation:

[0111] 1) Take 4x2 appropriately sized nitrocellulose membranes (NC membranes) and lightly mark the spotting locations and sample numbers on them with a pencil. Two NC membranes are needed for each probe so that the membranes can be washed under high-intensity and low-intensity conditions respectively in the subsequent experimental steps.

[0112] 2) Take 15 μL of each sample and control sample, spot them onto the sample film, and let them air dry at room temperature.

[0113] 3) Place on filter paper soaked in 0.1 mol / L NaOH and 1.5 mol / L NaCl for 5 minutes (twice), then air dry.

[0114] 4) Place it between clean filter paper, wrap it with aluminum foil, and vacuum dry at 60-80°C for 2 hours.

[0115] Probe marking

[0116] 1) Add 3 μl Probe (0.10D / 10 μl) to 2 μl Kinase buffer, then add 8-10 uCi γ- 32 P-dATP+2U Kinase, to be added to a final volume of 20 μL.

[0117] 2) Keep warm at 37 °C for 2 hours.

[0118] 3) Add 1 / 5 volume of bromophenol blue indicator (BPB).

[0119] 4) Pass through Sephadex G-50 column. 5) To have 32 The first peak is collected before the P-Probe wash (monitoring can be done with a Moni tor).

[0120] 6) 5 drops / tube, collect 10-15 tubes.

[0121] 7) Monitor isotope levels using a liquid scintillation analyzer

[0122] 8) The collected liquid from the first peak is the desired product. 32 P-Probe (the second peak is free γ-) 32 Ρ-dATP).

[0123] Prehybridization - Place the sample membrane in a plastic bag, add 3-10 mg of prehybridization solution (10x Denhardt's; 6x SSC, 0.1 mg / ml CT DM (calf thymus DNA)), seal the bag, and incubate at 68°C with shaking for 2 hours.

[0124] Hybridization

[0125] Cut off a corner of the plastic bag, add the prepared probe, seal the bag, and incubate overnight at 42°C with water and shaking. Wash the membrane:

[0126] High-intensity film washing:

[0127] 1) Take out the hybridized sample membrane.

[0128] 2) Wash at 40°C for 15 minutes (twice) in 2xSSC and 0.1%SDS.

[0129] 3) Wash at 40°C for 15 minutes (twice) in 0.1% SSC and 0.1% SDS.

[0130] 4) Wash in 0.1% SSC and 0.1% SDS at 55°C for 30 minutes (twice), then air dry at room temperature. Low-intensity washing:

[0131] 1) Take out the hybridized sample membrane.

[0132] 2) Wash with 2xSSC and 0.1%SDS at 37°C for 15 minutes (twice).

[0133] 3) In 0.1xSSC and 0.1¾SDS, wash at 37°C for 15 minutes (twice).

[0134] 4) 0.1xSSC, 0.1g medium, wash at 40°C for 15 minutes (twice), then air dry at room temperature.

[0135] X-ray autoradiography:

[0136] -70°C, X-ray autoradiography (the compression time depends on the intensity of the radioactivity of the hybridization spot).

[0137] Experimental results:

[0138] Hybridization experiments conducted under low-intensity washing conditions showed no significant difference in radioactivity intensity between the two probe hybridization spots. However, hybridization experiments conducted under high-intensity washing conditions showed that the radioactivity intensity of the hybridization spot from probe 1 was significantly stronger than that of the other probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotides of this invention in different tissues. Example 7 DNA Microarray

[0139] Gene chips, or DNA microarrays, are a new technology that many national laboratories and large pharmaceutical companies are currently researching and developing. They refer to the orderly, high-density arrangement of a large number of target gene fragments. The polynucleotides of this invention are arranged on supports such as glass and silicon, and then compared and analyzed using fluorescence detection and computer software to achieve rapid, efficient, and high-throughput analysis of biological information. These polynucleotides can be used as target DNA in gene chip technology for high-throughput research on the function of new genes; for finding and screening tissue-specific new genes, especially those related to diseases such as tumors; and for the diagnosis of diseases, such as hereditary diseases. Specific methods and steps have been reported in various literatures, such as DeRisi, JL, Lyer, V. & Brown, PO (1997) Science 278, 680-686, and Hele, RA, Schema, M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.

[0140] (a) Spotting

[0141] A total of 4000 polynucleotide sequences of various full-length cMAs were used as target DNA, including the polynucleotides of this invention. These sequences were amplified separately by PCR. After purification, the amplified products were adjusted to a concentration of approximately 500 ng / µl and spotted onto a glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA), with a spot distance of 280 μm. The spotted slides were then hydrated, dried, and cross-linked in a UV cross-linker. After elution and drying, the DNA was immobilized on the glass slides to prepare a chip. Specific methods and steps have been reported in various literatures; the post-spotting processing steps in this embodiment are as follows:

[0142] 1. Hydrate for 4 hours in a humid environment;

[0143] 2. Wash with 0.2% SDS for 1 minute;

[0144] 3. Wash twice with ddH2O, 1 minute each time;

[0145] 4. NaBH4 seal for 5 minutes;

[0146] 5.95°C water for 2 minutes;

[0147] 6. Wash with 0.2% SDS for 1 minute;

[0148] 7. Rinse twice with ddH2O;

[0149] 8. Let it air dry and store in a dark place at 25°C for later use.

[0150] (II) Probe Marking

[0151] Total niRNA was extracted from mixed human tissues and specific tissues (or stimulated cell lines) using a one-step method. The mRNA was purified using Oligotex mRNA MidiKit (purchased from QiaGen) and then separated by reverse transcription. 1 J labeled human mixed tissue mRNA with the fluorescent reagent Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech), and labeled specific tissue (or stimulated cell line) mRNA with the fluorescent reagent Cy5dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech). The probe was then purified to prepare the probe. For detailed steps and methods, please refer to:

[0152] Schena,

[0153] M., Shalon, D., Hel ler, R. (1996) Proc. Nat l. Acad. Sci. USA. Vol. 93: 10614- 10619. Schena, M., Shalon, Dar i., Davis, R. f. (1995) Science. 270. (20): 467-480. (III) Hybridization

[0154] Probes from the two tissues were hybridized with the chip in UniHyb™ Hybridization Solution (purchased from TeleChem) for 16 hours. After washing with washing solution (1 SSC, 0.2% SDS) at room temperature, the chips were scanned using a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned images were analyzed using Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio for each point.

[0155] The specific tissues (or stimulated cell lines) mentioned above are fetal brain, bladder mucosa, PMA+ Ecv304 cell line, LPS+ Ecv304 cell line, thymus, normal fibroblasts 1024NC, Fibroblasts t, growth factor stimulated, 1024NT, scar fc growth factor stimulated, 1013HT, scar fc without growth factor stimulation, 1Q13HC, bladder cancer cell line EJ, adjacent to bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, and gastric cardia cancer. A fold plot was drawn based on these 18 Cy3 / Cy5 ratios (Figure 1). As can be seen from the figure, the expression profiles of human macroprotein 9.13 and KIAA0072 described in this invention are very similar. Industrial Applicability

[0156] The polypeptides of the present invention, as well as their antagonists, agonists, and inhibitors, can be used directly for the treatment of diseases, such as malignant tumors, adrenal insufficiency, skin diseases, various inflammations, HIV infection, and immune diseases.

[0157] • Human KIAA protein is involved in cell signal transduction / communication, cell structure / motor function and nucleic acid control in the human body. Abnormal expression of KIAA protein can cause disorders in human cell signal transduction pathways, gene expression disorders and protein metabolism disorders, which can lead to related diseases.

[0158] The expression profile of the polypeptides of this invention is consistent with that of human KIAA protein, and both have similar biological functions. The polypeptides of this invention are involved in cell signaling / communication, cell structure / motor activity, and nucleic acid control in vivo. Abnormal expression of these polypeptides can cause disorders in human cell signaling pathways, gene expression disturbances, and protein metabolism disorders, leading to embryonic malformations, protein metabolism disorders, tumors, and immune system disorders, including but not limited to common embryonic malformations.

[0159] I. Common deformities of the face, neck, and limbs:

[0160] 1. Cleft lip (most common, may be accompanied by alveolar cleft and palate cleft), cleft palate, facial oblique cleft, cervical sac, cervical fistula, etc.;

[0161] 2. Common limb deformities:

[0162] 1) Limb absence: Transverse absence (congenital short limbs): no arm, no forearm, no hand, no finger, no leg, no toe, etc.; Longitudinal absence: radial / ulnar absence of the upper limb, tibial / fibular absence of the lower limb, etc.;

[0163] Phocomelia-like hand / foot deformities, etc.;

[0164] 2) Limb differentiation disorders: absence of a muscle or muscle group, joint dysplasia, bone deformities, bone fusion, polydactyly, syndactyly, tellurium varus foot, etc.

[0165] II. Common malformations of the digestive system:

[0166] Thyroglossal duct cyst, gastrointestinal atresia or stenosis, ileal diverticulum, umbilical fistula, congenital umbilical hernia, congenital nonganglionic megacolon, anal obstruction, intestinal rotation abnormality, bile duct atresia, annular pancreas, etc.

[0167] III. Common malformations of the respiratory system: laryngotracheal stenosis / atresia, tracheoesophageal fistula, hyaline membrane disease, unilateral pulmonary agenesis, ectopic lobes, congenital pulmonary cysts, pulmonary edema, etc.

[0168] IV. Common malformations of the urinary system: Polycystic kidney, ectopic kidney, horse tellurium kidney, double ureter, urachal fistula, bladder exstrophy, etc.

[0169] V. Common malformations of the reproductive system: cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, true / false hermaphroditism, testicular feminization syndrome, etc.

[0170] VI. Common malformations of the cardiovascular system: atrial septal defect, ventricular septal defect, abnormal septal septum (aortic and pulmonary artery transposition, aortic or pulmonary artery stenosis), patent ductus arteriosus, etc.; VII. Common malformations of the nervous system: neural tube defects (anencephaly, spina bifida, myelomeningocele, hydrocephalus), intracerebral / extracerebral hydrocephalus, etc.;

[0171] 8. Common eye and ear malformations: Iris defects, retained pupillary membrane, congenital cataracts, congenital glaucoma, unilateral / anophthalmia / microphthalmia, congenital deafness, auricular malformations, etc.; Common tumors of various tissues:

[0172] I. Epithelial tissue:

[0173] Papilloma, squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], muco / serous cystadenoma (carcinoma) [ovary], basal cell carcinoma [facial skin], (malignant) pleomorphic adenoma [medullodium], papilloma, transitional cell carcinoma [bladder, renal pelvis], etc.; II. Mesenchymal tissue:

[0174] Fibrosarcoma [limbs], (malignant) fibrous histiocytoma [limbs], lipoma [subcutaneous tissue, lower limbs, retroperitoneum], leiomyosarcoma [uterus and gastrointestinal tract], transverse striated myoma [head and neck, genitourinary tract, limbs], hemangiosarcoma, lymphangiosarcoma [skin, subcutaneous tissue, tongue, lips], bone sarcoma [skull, long bones], (malignant) giant cell tumor [upper femur / tibia / humerus], chondrosarcoma [short bones of hands and feet, pelvis / ribs / femur / humerus / scapula], synovial sarcoma [Near the knee / ankle / wrist / shoulder / elbow joints], (malignant) mesothelioma [pleura / peritoneum], etc.; III. Lymphohematopoietic tissue:

[0175] Malignant lymphoma [cervical, mediastinal, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphohematopoietic tissue], multiple myeloma [thoracic / rib / skull and long bones], etc.;

[0176] IV. Nervous tissue:

[0177] Neurofibroma [covering all cutaneous nerves / deep nerves and viscera], (malignant) schwannoma [covering nerves in the head, neck, limbs, etc.], (malignant) glioma [cerebrum], medulloblastoma [cerebellum], (malignant) meningioma [meninges], ganglion cell tumor / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc.

[0178] V. Other tumors:

[0179] Moles, malignant melanoma [skin, mucous membranes], (malignant) hydatidiform mole, choriocarcinoma [uterus], (malignant) Sertoli cell and stromal cell tumors, (malignant) granulosa cell tumors [ovary, testis], seminoma [testis], dysgerminoma [ovary], embryonal carcinoma [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum, and sacrococcygeal region], etc.; diseases related to protein metabolism disorders.

[0180] Disorders of protein metabolism can affect the following major physiological functions of proteins, thereby leading to the development of related diseases:

[0181] 1. Provides energy to the body, maintaining tissue growth, renewal, and repair:

[0182] Muscle atrophy, weakness in the limbs, emaciation; in severe cases, it can present as cachexia. II. Production of physiologically active substances, such as hormones, antibodies, and amines.

[0183] 1. Disorders of protein peptide hormone function can lead to the following diseases:

[0184] 1) Insulin and glucagon: Diabetes, hypoglycemia, etc.;

[0185] 2) Hypothalamic and pituitary hormones: gigantism, dwarfism, acromegaly, Cushing's syndrome, primary aldosteronism, secondary chronic adrenocortical insufficiency, hyperthyroidism, hypothyroidism (cretinism, juvenile hypothyroidism, adult hypothyroidism), male / female infertility, menstrual disorders (functional uterine bleeding, amenorrhea, polycystic ovary syndrome, premenstrual tension syndrome, menopausal syndrome), sexual development disorders, diabetes insipidus, inappropriate antidiuretic hormone secretion syndrome, abnormal lactation, etc.; 3) Parathyroid hormone: hyperparathyroidism, hypoparathyroidism, etc.;

[0186] 4) Gastrointestinal hormones: peptic ulcers, chronic indigestion, chronic gastritis, etc.;

[0187] 2. Disorders of amine metabolism can lead to the following diseases:

[0188] Cardiac arrhythmia, shock, mental confusion, epilepsy, chorea, hepatic encephalopathy (norepinephrine, γ-aminobutyric acid, serotonin, glutamine), kinesia, type I hypersensitivity diseases (urticaria, hay fever, allergic rhinitis, skin allergies), peptic ulcer (histamine), hypercholesterolemia (taurine), arsenopyramidal tumor (polyamine), etc. 3. Antibody deficiency makes individuals susceptible to various infections:

[0189] Sepsis, purulent meningitis, pneumonia, tracheitis, otitis media, pyoderma, etc.

[0190] III. Specific physiological functions of certain proteins:

[0191] Various hemoglobinopathies (anemia, jaundice, tissue hypoxia leading to organic acidemia), various coagulation factor deficiencies (hemorrhagic diseases), muscle spasms, muscle twitching, muscle paralysis (actinemia), hyperlipoproteinemia, etc.; diseases related to immune system dysfunction.

[0192] I. Various Infections

[0193] 1. Infections caused by cellular immunodeficiency:

[0194] 1) Intracellular parasitic infections: typhoid fever, paratyphoid fever (Salmonella typhi), tuberculosis (Mycobacterium tuberculosis), leprosy (Mycobacterium leprae), brucellosis, etc.;

[0195] 2) Various viral infectious diseases: such as measles virus (measles, measles-related bronchitis, pneumonia, otitis media, subacute sclerosing panencephalitis), herpesvirus (shingles, chickenpox), etc.;

[0196] 3) Others, such as certain fungal infections (Candida albicans), etc.

[0197] 2. Infections caused by humoral immune deficiency:

[0198] 1) Various extracellular parasitic infections: lobar pneumonia, otitis media, mastoiditis, paranasal sinusitis, meningitis, sepsis (pneumococcus), epidemic cerebrospinal meningitis (meningococcus), wound and burn infections (Pseudomonas aeruginosa), etc.

[0199] 2) Various viral infections: poliovirus (poliomyelitis), hepatitis viruses (hepatitis A, B, C, D, E, F, G, etc.);

[0200] 3) Other causes, such as parasitic infections (Pneumocystis carinii) etc.;

[0201] II. Malignant tumors: Leukemia, lymphoma, etc.;

[0202] III. Autoimmune Diseases: Systemic lupus erythematosus, rheumatoid arthritis, pernicious anemia, etc.; In summary, the polypeptides of the present invention, as well as their antagonists, agonists, and inhibitors, can be directly used to treat a variety of diseases, such as embryonic malformations, protein metabolism disorders, tumors, and immune system disorders. The present invention also provides a method for screening compounds to identify agents that enhance (agonist) or inhibit (antagonist) human macroprotein 9.13. Agonists enhance the biological functions of human macroprotein 9.13, such as stimulating cell proliferation, while antagonists inhibit and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing human macroprotein 9.13 can be cultured together with labeled human macroprotein 9.13 in the presence of the drug. The ability of the drug to enhance or inhibit this interaction can then be determined.

[0203] Antagonists of human macroprotein 9.13 include screened antibodies, compounds, receptor deletions, and analogues. Antagonists of human macroprotein 9.13 can bind to human macroprotein 9.13 and eliminate its function, or inhibit the production of the peptide, or bind to the active site of the peptide so that the peptide cannot perform its biological function.

[0204] When screening compounds for antagonistic effects, human macroprotein 9.13 can be added to a bioanalytical assay. The antagonist status is determined by measuring the compound's effect on the interaction between human macroprotein 9.13 and its receptor. Using the same method, receptor deletions and analogs that act as antagonists can be screened. Peptides that bind to human macroprotein 9.13 can be obtained by screening a random peptide library consisting of various possible combinations of amino acids bound to a solid phase. Generally, human macroprotein 9.13 molecules should be labeled during screening.

[0205] This invention provides a method for producing antibodies using polypeptides, fragments thereof, derivatives, analogs, or their cellular components as antigens. These antibodies may be polyclonal or monoclonal antibodies. This invention also provides antibodies against the human macroprotein 9.13 antigenic determinant. 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.

[0206] Polyclonal antibodies can be produced by directly injecting human macroprotein 9.13 into immunized animals (such as rabbits, mice, and rats). Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Techniques for preparing monoclonal antibodies against human macroprotein 9.13 include, but are not limited to, hybridoma technology (Kohl and Milstein. Nature, 1975, 256: 495-497), three-cell hybridoma technology, human β-cell hybridoma technology, and EBV-hybridoma technology. Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al., PNAS, 1985, 81: 6851), while existing techniques for producing single-chain antibodies (US Pat No. 4946778) can also be used to produce single-chain antibodies against human macroprotein 9.13.

[0207] Antibody against human macroprotein 9.13 can be used in immunohistochemistry to detect human macroprotein 9.13 in biopsy specimens.

[0208] Monoclonal antibodies that bind to human macroprotein 9.13 can also be labeled with radioactive isotopes and injected into the body to track their location and distribution. These radiolabeled antibodies can serve as a non-invasive diagnostic method for locating tumor cells and determining whether metastasis has occurred.

[0209] Antibodies can also be used to design immunotoxins targeting specific sites in the body. For example, monoclonal antibodies with high affinity for human macroprotein 9.13 can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, and erythrine). A common method is to use a thiol cross-linking agent, such as SPDP, to attack the amino group of the antibody, binding the toxin to the antibody through disulfide bond exchange. Such hybrid antibodies can be used to kill human macroprotein 9.13-positive cells.

[0210] The antibodies of this invention can be used to treat or prevent diseases associated with human macroprotein 9.13. Administration of appropriate doses of the antibodies can stimulate or block the production or activity of human macroprotein 9.13.

[0211] This invention also relates to diagnostic assays for the quantitative and localized detection of human macroprotein 9.13 levels. These assays are well known in the art and include FISH and radioimmunoassay assays. The human macroprotein 9.13 levels detected in these assays can be used to interpret the importance of human macroprotein 9.13 in various diseases and for diagnostic purposes. Diseases where human macroprotein 9.13 is ineffective.

[0212] The peptides of the present invention can also be used for peptide profiling analysis. For example, the peptides can be specifically cleaved by physical, chemical or enzymatic methods and analyzed by one-dimensional, two-dimensional or three-dimensional gel electrophoresis, or more preferably by mass spectrometry.

[0213] Polynucleotides encoding human macroprotein 9.13 can also be used for various therapeutic purposes. Gene therapy can be used to treat abnormal cell proliferation, development, or metabolism caused by the absence or abnormal / inactive expression of human macroprotein 9.13. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variants of human macroprotein 9.13 to inhibit endogenous human macroprotein 9.13 activity. For example, a variant of human macroprotein 9.13 may be a shortened form lacking the signal transduction domain; although it can bind to downstream substrates, it lacks signal transduction activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human macroprotein 9.13. Viral expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex viruses, and parvoviruses can be used to transfer polynucleotides encoding human macroprotein 9.13 into cells. Methods for constructing recombinant viral vectors carrying polynucleotides encoding human macroprotein 9.13 can be found in existing literature (Sambrook, et al.). Additionally, the recombinant polynucleotide encoding human macroprotein 9.13 can be packaged into liposomes and transferred into cells.

[0214] Methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in vivo; or introducing polynucleotides into cells in vitro using vectors (such as viruses, bacteriophages, or plasmids) and then transplanting the cells into the body.

[0215] Oligonucleotides (including antisense MA and MA) and ribozymes that inhibit human macroprotein 9.13 mRNA are also within the scope of this invention. A ribozyme is an enzyme-like RNA molecule that specifically breaks down a particular MA. Its mechanism of action involves the ribozyme molecule specifically hybridizing with a complementary target RM and then performing endonuclease. Antisense RNA, DNA, and ribozymes can be obtained using any existing RM or DNA synthesis technology, such as the widely used technique of synthesizing oligonucleotides via solid-phase phosphoramide chemical synthesis. Antisense RM molecules can be obtained by transcription in vitro or in vivo from the DNA sequence encoding the RNA. This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector. To increase the stability of nucleic acid molecules, they can be modified in various ways, such as increasing the length of the flanking sequences, and using phosphothioester or peptide bonds instead of phosphodiester bonds for the ribonucleoside linkage.

[0216] Polynucleotides encoding human macroprotein 9.13 can be used for the diagnosis of diseases related to human macroprotein 9.13. These polynucleotides can be used to detect the expression of human macroprotein 9.13 or its abnormal expression in disease states. For example, the DNA sequence encoding human macroprotein 9.13 can be used to hybridize biopsy specimens to determine its expression status. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques are publicly available and mature, and related kits are commercially available. Part or all of the polynucleotides of this invention can be used as probes immobilized on microarrays or DM chips (also known as "gene chips") for differential gene expression analysis and gene diagnosis in tissues. Human macroprotein 9.13-specific primers can be used for... RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the transcript of human macroprotein 9.13.

[0217] Detection of mutations in the human macroprotein 9.13 gene can also be used to diagnose diseases related to human macroprotein 9.13. Mutations in human macroprotein 9.13 can take the form of point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human macroprotein 9.13 DNA sequence. Existing techniques such as Southern blotting, DNA sequencing, PCR, and in situ hybridization can be used to detect mutations. Furthermore, mutations may affect protein expression; therefore, Northern blotting and Western blotting can be used to indirectly determine the presence of gene mutations.

[0218] The sequences of this invention are also valuable for chromosome identification. These sequences specifically target a particular location on a human chromosome and can hybridize with it. Currently, there is a need to identify the specific sites of genes on chromosomes. At present, only a few chromosome markers based on actual sequence data (duplication polymorphisms) are available for labeling chromosome locations. According to this invention, a crucial first step in associating these sequences with disease-related genes is to locate these DM sequences on chromosomes.

[0219] In short, PCR primers (preferably 15-35 bp) are prepared based on cMA, allowing sequences to be located on chromosomes. These primers are then used for PCR screening of somatic cell hybrids containing each human chromosome. Only hybrid cells containing the corresponding human genes from the primers will produce amplified fragments.

[0220] PCR localization of somatic hybrid cells is a rapid method for locating metasomes (MA) to specific chromosomes. Using the oligonucleotide primers of this invention, sub-localization can be achieved using a set of fragments from a specific chromosome or a large number of genomic clones via a similar method. Other similar strategies that can be used for chromosome localization include in situ hybridization, chromosome pre-screening using labeled flow cytometry, and hybridization pre-selection, thereby constructing chromosome-specific cDNA libraries.

[0221] Fluorescence in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows for precise chromosome localization in a single step. For a review of this technique, see Verma et al., Human Chromosomes: a

[0222] Manua l of Bas ic Techniques, Pergaraon Pres s, New York (1988).

[0223] Once a sequence is pinpointed to its precise chromosomal location, that physical location on the chromosome can be correlated with gene mapping data. This data can be found, for example, in V. McKusick, Mendelian Inheritance in Man (accessible via Johns Hopkins University Welch Medica).

[0224] (Library accessed online). Then, linkage analysis can be used to determine the relationship between the gene and the disease that has already been located on the chromosomal region.

[0225] Next, it is necessary to determine the differences in cDNA or genomic sequences between diseased and healthy individuals. If a mutation is observed in some or all diseased individuals, but not in any healthy individuals, then that mutation may be the cause of the disease. Comparing diseased and unaffected individuals typically involves first looking for structural changes in the chromosomes, such as deletions or translocations visible at the chromosomal level or detectable by PCR based on cDNA sequences. Depending on the resolution of current physical mapping and gene localization techniques, cDNA precisely located to the chromosomal region associated with the disease can be one of 50 to 500 potential pathogenic genes (assuming a mapping resolution of 1 megabase and one gene per 20 kb). The peptides, polynucleotides and their analogues, agonists, antagonists and inhibitors of the present invention can be used in combination with suitable drug carriers. These carriers can be water, glucose, ethanol, salts, buffer solutions, glycerol and combinations thereof. The compositions contain a safe and effective amount of the peptide or antagonist, as well as a carrier and excipients that do not affect the efficacy of the drug. These compositions can be used as drugs for the treatment of diseases.

[0226] The present invention also provides a kit or reagent kit containing one or more containers, wherein the containers contain one or more pharmaceutical composition ingredients of the present invention. Along with these containers, there may be indicative instructions from the government regulatory authority that manufactures, uses, or sells the pharmaceutical or biological product, reflecting the authority's permission for its administration to humans. Furthermore, the polypeptides of the present invention can be used in combination with other therapeutic compounds.

[0227] The pharmaceutical composition can be administered in a convenient manner, such as via local, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes. Human macroprotein 9.13 is administered in an amount that effectively treats and / or prevents a specific indication. The amount and dosage range of human macroprotein 9.13 administered to a patient will depend on many factors, such as the route of administration, the patient's health condition, and the diagnostic physician's judgment.

Claims

Claims I. An isolated polypeptide - human macroprotein 9.13, characterized in that It contains: a polypeptide of the amino acid sequence shown in SEQ ID NO: 2, or an active fragment of the polypeptide, analogue or derivative thereof.

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

2.

3. The polypeptide as described in claim 2, characterized in that... It contains a polypeptide having the amino acid sequence shown in SEQ ID NO:

2.

4. An isolated polynucleotide, characterized in that... The polynucleotide comprises one selected from the group consisting of: (a) a polypeptide or fragment thereof, analogue, or derivative thereof encoding the amino acid sequence shown in SEQ ID NO: 2; (b) A polynucleotide complementary to polynucleotide (a); or (c) A polynucleotide that is at least 70% identical to (a) or (b).

5. The polynucleotide as described in claim 4, characterized in that... The polynucleotide comprises a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:

2.

6. The polynucleotide as described in claim 4, characterized in that... The polynucleotide sequence comprises the sequence of positions 348-599 in SEQ ID NO: 1 or the sequence of positions 1-1408 in SEQ ID NO:

1.

7. A recombinant vector containing exogenous polynucleotides, characterized in that... It is a recombinant vector constructed from the polynucleotide described in any one of claims 4-6 and a plasmid, virus, or vector expression vector.

8. A genetically engineered host cell containing exogenous polynucleotides, characterized in that... It is selected from one of the following host cells: (a) Host cells transformed or transduced using the recombinant vector of claim 7; or (b) Host cells transformed or transduced with the polynucleotide as described in any one of claims 4-6.

9. A method for preparing a polypeptide with human macroprotein 9.13 activity, characterized in that the method comprises: (a) In the expression of human macroprotein 9.13 Under the conditions described in claim 8, the engineered host cells are cultured; (b) A polypeptide with human macroprotein 9.13 activity was isolated from the culture.

10. An antibody capable of binding to a polypeptide, characterized in that... The antibody is an antibody that specifically binds to human macroprotein 9.

13. II. A class of compounds that mimic or regulate peptide activity or expression, characterized in that they mimic, promote, antagonize, or inhibit human macroprotein. 9.13 active compounds.

12. The compound of claim 11, characterized in that... It is the antisense sequence of the polynucleotide sequence or a fragment thereof shown in SEQ ID NO:

1.

13. An application of the compound of claim 11, characterized in that... The compound is used in a method for regulating the in vivo and in vitro activity of human macroprotein 9.

13.

14. A method for detecting a disease or susceptibility to a disease associated with the polypeptide described in any one of claims 1-3, characterized in that... This includes detecting the expression level of the polypeptide, or detecting the activity of the polypeptide, or detecting nucleotide variations in the polynucleotide that cause abnormal expression or activity of the polypeptide.

15. The use of the polypeptide according to any one of claims 1-3, characterized in that... It is used to screen mimics, agonists, antagonists, or inhibitors of human macroprotein 9.13; or for peptide fingerprint identification.

16. The application of the nucleic acid molecule according to any one of claims 4-6, characterized in that... It can be used as a primer for nucleic acid amplification reactions, or as a probe for hybridization reactions, or for manufacturing gene chips or microarrays.

17. The use of the polypeptide, polynucleotide, or compound as claimed in any of claims 1-6 and 11, characterized in that... The polypeptide, polynucleotide or its analogue, agonist, antagonist or inhibitor, in a safe and effective dose with a pharmaceutically acceptable carrier, constitutes a pharmaceutical composition for the diagnosis or treatment of diseases associated with abnormalities of human macroprotein 9.

13.

18. The use of the polypeptide, polynucleotide, or compound according to any one of claims 1-6 and 11, characterized in that... Drugs for treating conditions such as malignant tumors, hematological diseases, HIV infection, immune disorders, and various types of inflammation can be prepared using the aforementioned polypeptides, polynucleotides, or compounds.