A novel polypeptide- marrow transposition gene related protein 12.32 and the polynucleotide encoding said polypeptide

Abstract

The invention discloses a new polypeptide- marrow transposition gene related protein 12.32, the polynucleotide encoding said polypeptide, and a process for producing the polypeptide by recombinant DNA technology. It also discloses methods of applying the polypeptide for the treatment of various diseases, such as hematopoietic system disease, tumor, development disorder, 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 marrow transposition gene related protein 12.32.

Description

[0001] A novel polypeptide—bone marrow translocation gene-related protein 12.32.

[0002] and the field of polynucleotide technology that encodes this polypeptide

[0003] This invention belongs to the field of biotechnology. Specifically, this invention describes a novel polypeptide—myeloid translocation gene-related protein 12.32—and the polynucleotide sequence encoding this polypeptide. This invention also relates to methods for preparing this polynucleotide and polypeptide, and their applications. Background Art

[0004] Many transcription factors encoded by chromosomal translocations of genes associated with human leukemia regulate normal hematopoiesis (Rabbitts, TH, 1994). The AML1-CBF transcription factor complex is crucial for the final hematopoiesis of all cell lineages and is the most frequent target of chromosomal translocations in human leukemia. In translocations associated with acute myeloid leukemia (AML), the AML1 (CBFA2 / PEBP2B) gene is parallel to the MTG8 (ET0 / CDR) gene, yielding the AML1-MTG8 gene product, which reacts strongly with an 85 kDa phosphoprotein. The AML1-MTG8 binding protein (myeloid translocation gene-associated protein 1, MTGR1) is highly similar to MTG8, and amino acid sequence comparisons reveal four evolutionarily conserved regions (NHR1-NHR4), including NHR2, which is crucial for the induction of G-CSF-dependent cell proliferation. Immunoprecipitation analysis showed that the R2 region is essential for the formation of a stable complex between AML1-MTG8 and MTGR1. Overexpression of MTGR1 promoted AML1-MTG8-induced G-CSF-dependent cell proliferation and disrupted AML1-dependent transcription. Therefore, the complex formed by MTGR1 and AML1-MTG8 plays a crucial role in promoting hematopoiesis.

[0005] MTGR1 is a member of the MTG8 family of proteins. Its cDNA contains an in-frame ATG at nucleotide 11, marking the beginning of the coding sequence, which terminates at position 1822. A poly-A additional signal appears at position 6387 of the cDNA, followed by continuous Alu sequences at positions 3207-3236 and 3690-3960. Transcripts of MTGR1 can be found in many human tissues and all cell lines, including L-G cells and Kasumi-1 cells.

[0006] The MTGR1 protein contains 604 amino acids and shares 61% identity with MTG8. It contains four conserved regions, NHR1-NHR4. NHR1 can react with homologous regions of TATA-binding protein-associated factors (TAFs). NHR2 is essential for the interaction between MTG8 and p85 proteins; its secondary structure demonstrates that NHR2 is a helical region, exhibiting characteristics of an amphipathic molecule. NHR4 is associated with two zinc finger domains (-C-X-X-C-7x-CX-X-C-, ―CXXXC-7XHXXXC-). Additionally, both MTG8 and MTGR1 contain three Pr-rich regions.

[0007] Rather than forming homologous complexes, AML1-MTG8 and MTGR1 tend to form hybrid complexes. Overexpression of MTGR1 can stimulate AML1-dependent cell proliferation and inhibit AML1-dependent transactivation, playing a very important regulatory role in hematopoiesis. Gene chip analysis revealed that the expression profile of the polypeptide of this invention is very similar to that of bone marrow translocation gene-associated protein 1 (MTGR1) in bladder mucosa, PMA+ ECV304 cell line, LPS+ ECV304 cell line, thymus, normal fibroblast 1024NC, Fibroblasts stimulated with growth factor 1024NT, scar fc stimulated with growth factor 1013HT, scar fc without growth factor stimulation 1013HC, bladder cancer cell line EJ, adjacent to bladder cancer, bladder cancer, liver cancer, liver cancer cell line, placenta, spleen, prostate cancer, jejunal adenocarcinoma, and gastric cardia cancer. Therefore, their functions may also be similar. This invention is named Bone Marrow Translocation Gene-Associated Protein 12.32.

[0008] As described above, the myeloid translocation gene-associated protein 12.32 plays a crucial role in regulating vital bodily functions such as cell division and embryonic development. Given that these regulatory processes are believed to involve numerous proteins, there has been a long-standing need in the art to identify more myeloid translocation gene-associated protein 12.32 proteins involved in these processes, particularly to identify their amino acid sequences. The isolation of the gene encoding a novel myeloid translocation gene-associated protein 12.32 provides a foundation for determining the protein's role in healthy and disease states. This protein may form the basis for developing disease diagnostics and / or therapeutics; therefore, isolating its encoding DNA is of paramount importance. Disclosure of the Invention

[0009] One object of the present invention is to provide an isolated novel polypeptide—myeloid translocation gene-associated protein 12.32. Another object of the present invention is to provide a polynucleotide encoding the polypeptide.

[0010] Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding protein 12.32, which is associated with myeloid translocation genes.

[0011] Another object of the present invention is to provide genetically engineered host cells containing a polynucleotide encoding a protein 12.32 associated with myeloid translocation genes.

[0012] Another object of the present invention is to provide a method for producing bone marrow translocation gene-related protein 12.32.

[0013] Another object of the present invention is to provide an antibody against the polypeptide of the present invention—myeloid translocation gene-associated protein 12.32.

[0014] Another object of the present invention is to provide mimicry compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention—myeloid translocation gene-associated protein 12.32.

[0015] Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of bone marrow translocation gene-associated protein 12.32.

[0016] 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. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

[0017] (a) A polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;

[0018] (b) A polynucleotide complementary to polynucleotide (a);

[0019] (c) A polynucleotide that has at least 70% identity with the polynucleotide sequence of (a) or (b).

[0020] More preferably, the sequence of the polynucleotide is selected from one of the following groups: (a) a sequence having positions 43-381 of SEQ ID NO: 1; and (b) a sequence having positions 1-882 of SEQ ID NO: 1.

[0021] 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.

[0022] The present invention also relates to an antibody that can specifically bind to the polypeptide of the present invention.

[0023] This invention also relates to a screening method for simulating, activating, antagonizing, or inhibiting myeloid translocation gene-related proteins.

[0024] 12.32 Methods for obtaining protein-active compounds, comprising utilizing polypeptides of the present invention. The present invention also relates to compounds obtained by this method.

[0025] The present invention also relates to a method for in vitro detection of diseases or disease susceptibility associated with abnormal expression of the bone marrow translocation gene-associated protein 12.32, 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.

[0026] 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.

[0027] 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 or immune diseases or other diseases caused by abnormal expression of bone marrow translocation gene-related protein 12.32.

[0028] Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.

[0029] Unless otherwise specified, the following terms used in this specification and claims have the following meanings:

[0030] "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.

[0031] A protein or polynucleotide "variant" is a protein or polynucleotide sequence that has one or more amino acid or nucleotide changes, or a polynucleotide sequence that encodes it. These changes may include deletions, insertions, or substitutions of amino acids or nucleotides in the amino acid or nucleotide sequence. Variants may have "conserved" changes. The substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

[0032] "Deficiency" refers to the absence of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

[0033] "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.

[0034] "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.

[0035] An "agonist" is a molecule that, when bound to myeloid translocation gene-associated protein 12.32, causes alterations in that protein, thereby regulating its activity. Agonists can include proteins, nucleic acids, carbohydrates, or any other molecules that can bind to myeloid translocation gene-associated protein 12.32.

[0036] An "antagonist" or "inhibitor" is a molecule that, when bound to myeloid translocation protein 12.32, blocks or modulates the biological or immunological activity of the protein. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules that can bind to myeloid translocation protein 12.32.

[0037] "Regulation" refers to changes in the function of myeloid translocation protein 12.32, including increases or decreases in protein activity, changes in binding properties, and any other changes in the biological, functional, or immunological properties of myeloid translocation protein 12.32.

[0038] "Substantially pure" means substantially free of other naturally occurring or associated proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify myeloid translocation-associated protein 12.32 using standard protein purification techniques. Substantially pure myeloid translocation-associated protein 12.32 produces a single master band on a non-reducing polyacrylamide gel. The purity of the myeloid translocation-associated protein 12.32 peptide can be determined by amino acid sequence analysis.

[0039] "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-CT". 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.

[0040] "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 hybridization between a completely complementary sequence and 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 two sequences... The interaction between the columns is a specific or selective interaction.

[0041] "Identity percentage" refers to the percentage of identical or similar sequences in a comparison of two or more amino acid or nucleic acid sequences. The identity percentage can be determined electronically, such as using the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences using different methods, such as the Cluster method (Higgins, DG, and PM Sharp (1988) Gene 73: 237-244). The Cluster method clusters sequences 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) / (Number of residues in sequence A - Number of spacer residues in sequence A - Number of spacer residues in sequence B). The percentage of similarity between nucleic acid sequences can also be determined using the Cluster method or methods known in the art, such as Jotun Hein's method (Hein J., (1990) Methods in emzuralogy 183: 625-645). "Similarity" refers to the degree of identical 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.

[0042] "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".

[0043] "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.

[0044] "Antibody" refers to the complete antibody molecule and its fragments, such as Fa, ? (^')2 and ? ^, which can specifically bind to the antigenic determinant of bone marrow translocation gene-related protein 12.32.

[0045] "Humanized antibody" refers to an antibody whose non-antigen binding region amino acid sequence has been replaced to make it more similar to human antibody, but still retains its original binding activity.

[0046] 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 existing in a living organism is not separated, but the same polynucleotide or polypeptide being 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... Its natural environment's components are still separate.

[0047] 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.

[0048] As used herein, "isolated myelogenous translocation-associated protein 12.32" means that myelogenous translocation-associated protein 12.32 is substantially free of other naturally occurring proteins, lipids, carbohydrates, or other substances associated with it. Those skilled in the art can purify myelogenous translocation-associated protein 12.32 using standard protein purification techniques. A substantially pure polypeptide produces a single master band on a non-reducing polyacrylamide gel. The purity of the myelogenous translocation-associated protein 12.32 polypeptide can be determined by amino acid sequence analysis.

[0049] This invention provides a novel polypeptide—myeloid translocation gene-related protein 12.32—which is essentially composed of the amino acid sequence shown in SEQ 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.

[0050] The present invention also includes fragments, derivatives, and analogs of myeloid translocation gene-associated protein 12.32. As used herein, the terms "fragment," "derivative," and "analyte" refer to polypeptides that substantially retain the same biological function or activity as myeloid translocation gene-associated protein 12.32 of the present invention. The fragments, derivatives, or analogs of the polypeptides 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 (II) in which a group on one or more amino acid residues is substituted by another group containing a substituent; or (III) in which the mature polypeptide is fused with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) in which an additional amino acid sequence is fused into the mature polypeptide to form a polypeptide sequence (e.g., a leader sequence, a secretory sequence, or a sequence used to purify the polypeptide 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.

[0051] This invention provides isolated nucleic acids (polynucleotides) that are essentially composed of polynucleotides encoding a polypeptide 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 cDM library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 882 bases, with open reading frames 43-381 encoding 112 amino acids. Comparison with gene chip expression profiles revealed that this polypeptide is associated with bone marrow translocation gene-associated protein 1 (MTGR1). Given their similar expression profiles, it can be inferred that this bone marrow translocation gene-related protein 12.32 has a similar function to bone marrow translocation gene-related protein 1 (MTGR1).

[0052] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. 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 or a degenerate variant of the coding region sequence shown in SEQ ID NO: 1. As used herein, "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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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, 60 °C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficol, 42 °C, etc.; or (3) hybridization only occurs when the similarity between the two sequences is at least 95%, preferably 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.

[0057] 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. The nucleic acid fragment can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate the polynucleotide encoding protein 12.32, a protein associated with the bone marrow translocation gene.

[0058] The polypeptides and polynucleotides in this invention are preferably provided in isolated form, and more preferably purified to homogenate. The specific polynucleotide sequence encoding myeloid translocation gene-related protein 12.32 of this invention can be used in multiple... Polynucleotides can be obtained by various methods. For example, they can be isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridizing 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.

[0059] The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating double-stranded DNA sequences from genomic DNA; 2) chemically synthesizing DNA sequences to obtain the double-stranded MA of the polypeptide.

[0060] Of the methods mentioned above, isolating genomic DNA is the least common. Direct chemical synthesis of DNA sequences is the frequently chosen method. An even more common method is the isolation of cDNA sequences. The standard method for isolating cDNA of interest is to isolate mRM from donor cells that highly express the gene and perform reverse transcription to form plasmid or phage cDNA libraries. Several mature techniques exist for mRM extraction, 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, such as those from Clontech, are also readily available. When combined with polymerase chain reaction (PCR) techniques, even very small amounts of expression products can be cloned.

[0061] Genes of the present invention can be screened from these cDM libraries using conventional methods. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) marker gene function presence or loss; (3) determination of the transcript level of bone marrow translocation gene-associated protein 12.32; and (4) detection of the protein products of gene expression by immunological techniques or by measuring biological activity. The above methods can be used alone or in combination.

[0062] 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 DNA 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.

[0063] In the fourth method, the protein product of the bone marrow translocation gene-related protein 12.32 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA).

[0064] The method of amplifying DNA / 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) 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 DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0065] The gene of the present invention obtained as described above, or the polynucleotide sequence of various DM fragments, etc., can be determined by conventional methods such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). These types of polynucleotide sequencing can also be performed using commercial sequencing kits. To obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes, it is necessary to sequence the cDNA of multiple clones before assembling them into a full-length cDNA sequence.

[0066] The present invention also relates to vectors containing the polynucleotides of the present invention, and host cells genetically engineered using the vectors of the present invention or directly using the coding sequence of the bone marrow translocation gene-related protein 12.32, as well as methods for generating the polypeptides of the present invention via recombinant technology.

[0067] In this invention, a polynucleotide sequence encoding the bone marrow translocation gene-related protein 12.32 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: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, JBio Chem. 263: 3521, 1988); and baculovirus-derived vectors expressed in insect cells. In short, any plasmid and vector can be used to construct a recombinant expression vector, provided it can replicate and stabilize within the host. An important characteristic of expression vectors is that they typically contain a replication initiation site, a promoter, a marker gene, and translational regulatory elements.

[0068] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a bone marrow translocation gene-related protein 12.32 and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques (Sambroook, et al. Molecular Cloning, a Laboratories Manual, Co. Spring Harbor Laboratories. New York, 1989). The DNA sequence can be efficiently ligated 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*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Inserting enhancer sequences into a vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors of DNA expression, typically ranging from 10 to 300 base pairs, that act on the promoter 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.

[0069] 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.

[0070] Those skilled in the art are well aware of how to select appropriate vectors / transcriptional regulatory elements (such as promoters). (e.g., enhancers, etc.) and selective marker genes.

[0071] In this invention, a polynucleotide encoding the bone marrow translocation gene-related protein 12.32, or a recombinant vector containing such a polynucleotide, can be transformed or introduced into host cells to constitute a genetically engineered host cell containing the polynucleotide or the 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.

[0072] Transformation of host cells using the DNA sequence described in this invention or a recombinant vector containing the DNA sequence 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 uptake DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps of which are well known in the art. MgCl2 is also an option. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc., can be used.

[0073] Using conventional recombinant DNA technology, the polynucleotide sequence of this invention can be used to express or produce recombinant myeloid translocation gene-related protein 12.32 (Science, 1984; 224: 1431). Generally, the following steps are involved:

[0074] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding human bone marrow translocation gene-related protein 12.32 of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0075] (2) Culture the host cells in a suitable culture medium;

[0076] (3) Isolate and purify proteins from culture media or cells.

[0077] 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.

[0078] 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, percolation, 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.

[0079] 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. Figure 1 is a comparison of the gene chip expression profiles of myeloid translocation gene-related protein 12.32 and myeloid translocation gene-related protein 1 (MTGR1) of this invention. The upper figure is a folded square diagram of the expression profile of myeloid translocation gene-related protein 12.32, and the lower figure is a folded square diagram of the expression profile of myeloid translocation gene-related protein 1 (MTGR1). Among them, 1 - fetal brain, 2 - bladder mucosa, 3 - PMA+ Ecv304 cell line, 4 - LPS+ Ecv304 cell line thymus, 5 - normal fibroblast 1024NC, 6 - Fibroblast, 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 - 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.

[0080] Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) image of the isolated bone marrow translocation gene-related protein 12.32. 10 kDa represents the molecular weight of the protein. The arrows indicate the isolated protein bands. The best mode for realizing this invention...

[0081] 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 Sarabrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0082] Example 1: Cloning of bone marrow translocation gene-related protein 12.32

[0083] Total amino acids (MA) were extracted from human fetal brain using a one-step method of guanidine isothiocyanate / phenol / chloroform. Poly(A) mRNA was isolated from the total RNA using the Quik mRNA Isolation Kit (Qiegene). 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) using the Smart cDNA Cloning Kit (Clontech), and the bacteria were transformed into DH5α bacteria to form a cDNA library. The 5' and 3' ends of all clones were sequenced using the Dye terminate cycle reaction sequencing kit (PerkinElmer) and an ABI 377 automated sequencer (PerkinElmer). The determined cDNA sequences were compared with existing public DNA sequence databases (Genebank), and the cDNA sequence of clone 0561B04 was found to be novel. A series of primers were synthesized to perform bidirectional sequencing of the inserted cDNA fragment contained in this clone. The results showed that the full-length cDNA of clone 0561B04 was 882 bp (as shown in Seq ID NO: 1), with a 338 bp open reading frame (ORF) from 43 bp to 381 bp, encoding a new protein (as shown in Seq ID NO: 2). We named this clone pBS_0561B04, and the encoded protein was named myeloid translocation gene-associated protein 12.32. Example 2: Cloning of the gene encoding myeloid translocation gene-associated protein 12.32 using RT-PCR.

[0084] Using total RNA from fetal brain cells as a template, cDNA was synthesized via reverse transcription using oligo-dT primers. After purification using a Qiagen kit, PCR amplification was performed using the following primers: Primer: 5'- GGCCGGGGACTCGTGGGTAACTTG-3' (SEQ ID NO: 3) Primer2: 5 GTCTGAAGTTTATAATATTTTATT- 3' (SEQ ID NO: 4)

[0085] Primerl is the forward sequence starting at the 5th lbp end of SEQ ID NO: 1;

[0086] Primer2 is the reversed 3' sequence of SEQ ID NO: 1.

[0087] Amplification reaction conditions: A 50 μL reaction volume containing 50 mmol / L KCl and 10 mmol / L Tris-

[0088] CI (pH 8.5), 1.5 mmol / L MgCl2, 200 μmol / L dNTP, 10 pmol primers, 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 1-882 bp as shown in SEQ ID NO: 1. Example 3: Northern blot analysis of the expression of bone marrow translocation gene-related protein 12.32:

[0089] Total RNA was extracted using a one-step method [Anal. Biochem 1987, 162, 156-159]. This method involves acidic guanidinium thiocyanate-phenol-chloroform extraction. Specifically, 4M guanidinium isothiocyanate-25mM sodium citrate and 0.2M sodium acetate were used. PH4.0) Homogenize the tissue, add 1 volume of phenol and 1 / 5 volume of chloroform-isopropyl alcohol (49:1), mix and centrifuge. Aspirate the aqueous phase, add isopropanol (0.8 volume) and centrifuge the mixture to obtain RNA precipitate. Wash the obtained RNA precipitate with 70% ethanol, dry and dissolve in water. jf 20μ § RNA was electrophoresed on a 1.2% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (H7.0), 5 mM sodium acetate, 1 mM EDTA, and 2.2 M formaldehyde. The RNA was then transferred to a nitrocellulose membrane. Using α- 32 P dATP was prepared using a random primer method. 32 P-labeled DNA probes. The DNA probes used were the PCR-amplified coding region sequence (43 bp to 381 bp) of the bone marrow translocation gene-related protein 12.32, as shown in Figure 1. 32P-labeled probes (approximately 2 x 10⁻⁶) were then used. 6 (cpm / ml) The RNA-transferred nitrocellulose membrane was hybridized overnight at 42°C in a solution containing 50% formamide-25mM KH2PO4 (pH 7.4)-5xSSC-5xDenhardt's solution and 200 μg / mL salmon sperm DNA. After hybridization, the membrane was washed in 1xSSC-0.1°C / mL SDS at 55°C for 30 min. Analysis and quantification were then performed using a Phosphor Imager. Example 4: In vitro expression, isolation, and purification of recombinant bone marrow translocation gene-associated protein 12.32.

[0090] Based on SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, with the following sequences:

[0091] Primer3: 5'- CCCCATATGATGGCGTCGGGCTGCAAGATTGGC- 3, (Seq ID No: 5)

[0092] Priraer4: 5'- CATGGATCCTTATTTATTTGAGACGGAGTCTTT-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 endonuclease sites on the expression vector plasmid pET-28b(+) (Novagen product, Cat. No. 69865.3). Using pBS- containing the full-length target gene... Using plasmid 0561B04 as a template, PCR was performed. The PCR reaction conditions were as follows: a total volume of 50 μL containing 10 pg of pBS-0561B04 plasmid, primers Primer-3 and Primer-4 at 10 pmol each, and Advantage polymerase Mix.

[0093] (Clontech product) 1 μL. Cycling parameters: 94°C 20s, 60°C 30s, 68°C 2 min, for a total of 25 cycles. The amplified product and plasmid pET-28(+) were double-digested with Ndel and BamHI, respectively. 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 kanamycin-containing medium.

[0094] (final concentration 30) g After overnight incubation on LB agar plates ( / nil), positive clones were screened using colony PCR and sequenced. The selected positive clone (pET-0561B04) was transformed into *E. coli* BL21(DE3)plySs (Novagen product) using the calcium chloride method with the recombinant plasmid. The plasmid was then added to a solution containing kanamycin (final concentration 30 μL). § In LB broth containing pET-0561B04, the host bacterium BL21 was cultured at 37°C to the logarithmic growth phase. IPTG was added to a final concentration of 1 μmol / L, and the culture was continued for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by sonication and centrifugation. The supernatant was then analyzed using an affinity chromatography column (His-Bind Quick Cartridge) that binds to six histidine residues (6His-Tag).

[0095] (Novagen product) Chromatography was performed to obtain purified target protein myeloid translocation gene-associated protein 12.32. SDS-PAGE electrophoresis yielded a single band at 10 kDa (Figure 2). This band was transferred to a PVDF membrane and analyzed for its N-terminal amino acid sequence 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: Production of Anti-Myeloid Translocation Gene-Associated Protein 12.32 Antibody

[0096] The following peptides specific to the bone marrow translocation gene-related protein 12.32 were synthesized using a peptide synthesizer (PE company product):

[0097] NH2-Met-Ala-Ser-Gly-Cys-Lys-Ile-Gly-Pro-Ser-Ile-Leu-Asn-Ser-Asp-C00H (SEQ ID NO: 7). This polypeptide was coupled with hemocyanin and bovine serum albumin to form complexes, as described in: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the above hemocyanin 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 bound to a cyanogen bromide-activated Sepharose 4B column, and anti-polypeptide antibodies were separated from total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody specifically binds to bone marrow translocation gene-associated protein 12.32. Example 6: Application of the polynucleotide fragment of the present invention as a hybridization probe.

[0098] Selecting suitable oligonucleotide fragments from the polynucleotides of this invention for use as hybridization probes has multiple applications. For example, these probes 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 this invention and to detect homologous polynucleotide sequences. Furthermore, these probes can be used to detect the polynucleotide sequence of this invention or its homologous polynucleotide sequences in normal or pathological tissues. Is the expression in tissue cells abnormal?

[0099] The purpose of this embodiment is to select suitable oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention 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 the filter membrane. Under high-intensity washing conditions, the first type of probe exhibits the strongest hybridization specificity with the sample and is thus retained.

[0100] I. Probe Selection

[0101] 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 followed:

[0102] 1. The preferred probe size range is 18-50 nucleotides;

[0103] 2. GC content should be 30% - 70%; exceeding this will increase non-specific hybridization.

[0104] 3. There should be no complementary regions inside the probe;

[0105] 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.

[0106] 5. Whether the initial probe is ultimately selected as a probe with practical application value should be further determined by experiments.

[0107] After completing the above analysis, the following two probes were selected and synthesized:

[0108] Probe 1 (probel), belonging to class I probes, is completely homologous to or complementary to the gene fragment of SEQ ID NO: 1 (41Nt).

[0109] 5'-TGGCGTCGGGCTGCAAGATTGGCCCGTCCATCCTCAACAGC-3' (SEQ ID NO: 8)

[0110] Probe 2 (probe2), belonging to the second class of probes, is a substitutional mutant sequence (41Nt) equivalent to the gene fragment of SEQ ID NO: 1 or its complementary fragment:

[0111] 5-TGGCGTCGGGCTGCAAGATTCGCCCGTCCATCCTCAACAGC-3' (SEQ ID NO: 9) For other commonly used reagents not listed and their preparation methods related to the specific experimental steps below, please refer to the document. References: DNA PROBES G. H, Keller; MM Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning laboratory manuals such as Molecular Cloning: A Laboratory Manual (2nd edition, 1998) [USA] Sambrook et al., Science Press.

[0112] Sample preparation:

[0113] 1. Extracting DNA from fresh or frozen tissues

[0114] 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 (approximately 10 ml / g) in cold homogenization buffer (0.25 mol / L sucrose; 25 rpm Tris-HCl, pH 7.5; 25 mol / L NaCl; 25 mol / L MgCl2). 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 (per 0.1 μL) g Initially, the tissue sample was treated with 1 ol (Il ol), followed by the phenol extraction method.

[0115] 2. Phenol extraction method for DM

[0116] 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 (1 x 10 ml). 8 (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 reaction 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) 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 DNA purification and ethanol precipitation.

[0117] 3. DNA purification and ethanol precipitation

[0118] Steps: 1) Add 1 / 10 volume of 2 mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DM 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 aspirate, while gradually increasing the TE, mixing until the DNA is fully dissolved, every 1-5 x 10⁻¹⁰ seconds. 6 The amount extracted from the cells is approximately 1 ul.

[0119] Steps 8-13 below are only used when it is necessary to remove contaminants; otherwise, proceed directly to step 14.

[0120] 8) Add RNase A to the DNA solution to a final concentration of 100 μg / ml, and incubate at 37°C for 30 minutes. 9) Add... Add SDS and proteinase K to final concentrations of 0.5% and 100 μg / ml, respectively. 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 aqueous phase, add 1 / 10 volume of 2 mol / L sodium acetate and 2.5 volumes of cold ethanol, mix well and incubate at -20°C for 1 hour. 13) Wash the precipitate with 70% ethanol and 100% ethanol, air dry, resuspend the nucleic acid, and repeat steps 3-6. 14) Determine A. 26 And A 28 To detect DNA purity and yield. 15) After aliquoting, store at -20°C. Sample membrane preparation:

[0121] 1) Take 4 x 2 appropriately sized nitrocellulose membranes (NC membranes), and lightly mark the spotting location and sample number on them with a pencil. Two NC membranes are required for each probe so that the membranes can be washed under high-intensity conditions and low-intensity conditions respectively in the subsequent experimental steps.

[0122] 2) Take 15 μL of each sample and control sample, spot them onto the sample film, and let them air dry at room temperature.

[0123] 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. 4) Place between clean filter paper, wrap with aluminum foil, and vacuum dry at 60-80°C for 2 hours.

[0124] Probe marking

[0125] 1) 3 μl Probe (0. IOD / Ιθμ 1), add 2 μl inase buffer, 8-10 uCi y- 32 P-dATP + 2U Kinase, to be added to a final volume of 20 μL.

[0126] 2) Keep warm at 37°C for 2 hours.

[0127] 3) Add 1 / 5 volume of bromophenol blue indicator (BPB).

[0128] 4) Pass through Sephadex G-50 column.

[0129] 5) To have 32 The first peak is collected before the P-Probe wash (it can be monitored using a monitor).

[0130] 6) Add 5 drops per tube, collect 10-15 tubes. 8) Combine the collected solutions of the first peak to obtain the desired solution. 32 P-Probe (the second peak is free γ-) 32 P-dATP).

[0131] prehybridization

[0132] 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 DNA (calf thymus DNA)), seal the bag, and incubate at 68°C for 2 hours.

[0133] Hybridization

[0134] Cut off a corner of the plastic bag, add the prepared probe, seal the bag, and incubate overnight in a 42°C water bath. Wash the membrane:

[0135] High-intensity film washing:

[0136] 1) Take out the hybridized sample membrane. 2) Wash 2xSSC, 0.1°C in SDS, at 40°C for 15 minutes (twice).

[0137] 3) Wash in 0.1xSSC and 0.1xSDS at 40°C for 15 minutes (twice).

[0138] 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:

[0139] 1) Take out the hybridized sample membrane.

[0140] 2) Wash with 2xSSC and 0.1%SDS at 37°C for 15 minutes (twice).

[0141] 3) Wash in 0.1xSSC, 0.1°C SDS for 15 minutes (twice).

[0142] 4) Wash in 0.1% SSC and 0.1% SDS at 40°C for 15 minutes (twice), then air dry at room temperature.

[0143] X-ray autoradiography:

[0144] -70°C, X-ray autoradiography (the compression time depends on the intensity of the radioactivity of the hybridization spot).

[0145] Experimental results:

[0146] Hybridization experiments conducted under low-intensity washing conditions showed no significant difference in radioactivity between the two probe hybridization spots; however, hybridization experiments conducted under high-intensity washing conditions showed that the radioactivity intensity of the hybridization spot of probe 1 was significantly stronger than that of the other probe hybridization spot. 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

[0147] Gene chips, or DNA microarrays, are new technologies that many national laboratories and large pharmaceutical companies are currently researching and developing. They involve arranging a large number of target gene fragments in an orderly, high-density manner on a carrier such as glass or silicon, and then using fluorescence detection and computer software to compare and analyze the data, achieving rapid, efficient, and high-throughput analysis of biological information. The polynucleotides of this invention 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. The specific methods and steps have been reported in various literatures, such as DeRi si , JL, Lyer, V. & Brown, P. O. (1997) Science 278, 680-686. and Hele , RA , Schema , M. , Chai , A. , Sha lom , D. , (1997) PNAS 94: 2150-2155.

[0148] (a) Spotting

[0149] A total of 4000 polynucleotide sequences of various full-length cDNAs 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 slide to prepare a chip. The specific steps are as follows: The following steps have been reported in various documents. The post-processing steps for spotting in this embodiment are as follows:

[0150] 1. Hydrate for 4 hours in a humid environment;

[0151] 2. Wash with 0.2% SDS for 1 minute;

[0152] 3. Wash twice with ddH2O, 1 minute each time;

[0153] 4. NaBH4 seal for 5 minutes;

[0154] 5. In 95°C water for 2 minutes;

[0155] 6. Wash with 0.2% SDS for 1 minute;

[0156] 7. Rinse twice with ddH2O;

[0157] 8. Let it air dry and store in a dark place at 25°C for later use.

[0158] (II) Probe Marking

[0159] Total mRNA was extracted from mixed human tissues and specific tissues (or stimulated cell lines) using a one-step method. The raRNA was purified using Oligotex raRNA MidiKit (purchased from QiaGen). The mRNA from the mixed human tissues was labeled with the fluorescent reagent Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech) via reverse transcription, while the mRNA from the specific tissues (or stimulated cell lines) was labeled with the fluorescent reagent Cy5dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech). The purified probes were then prepared. For detailed steps and methods, please refer to:

[0160] Schena,

[0161] M. , Shalon, D. , Hel ler, R. (1996) Proc. Nat l. Acad. Sci. USA. Vol. 93: 10614-

[0162] 10619. Schena, M., Shalon, Dar i., Davi s, RW (1995) Science. 270. (20): 467-480. (III) Hybridization

[0163] Probes from the two tissues were hybridized with the chip in UniHyb™ Hybridization Solution (purchased from T. eleChem) for 16 hours. After washing with washing solution (1 x 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.

[0164] 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, 1G13HC, 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 cross-sectional diagram was drawn based on these 18 Cy3 / Cy5 ratios (Figure 1). As can be seen from the figure, the expression profiles of bone marrow translocation gene-related protein 12.32 and bone marrow translocation gene-related protein 1 (MTGR1) described in this invention are very similar. Industrial applicability

[0165] 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 arsenoblastoma, adrenal insufficiency, skin diseases, various inflammations, HIV infection, and immune diseases.

[0166] The chromosomal translocations of genes associated with human leukemia encode numerous transcription factors that regulate normal hematopoiesis. The AML1-CBF transcription factor complex is crucial for the final hematopoiesis of all cell lineages and is the most frequent target of chromosomal translocations in human leukemia. In the translocations associated with acute myeloid leukemia (AML), the AML1 (CBFA2 / PEBP2B) gene is parallel to the MTG8 (ET0 / CDR) gene, and the AML1-MTG8 gene product has been obtained, which reacts strongly with an 85 kDa phosphoprotein. The AML1-MTG8 binding protein (myeloid translocation gene-associated protein 1, MTGR1) is highly similar to MTG8, and amino acid sequence comparisons reveal four evolutionarily conserved regions (NHR1–NHR4), including NHR2, which is crucial for the induction of G-CSF-dependent cell proliferation. Immunoprecipitation analysis showed that the NHR2 region is essential for the formation of a stable complex between AML1-MTG8 and MTGR1. Overexpression of MTGR1 promotes AML1-MTG8-induced G-CSF-dependent cell proliferation and disrupts AML1-dependent transcription. Therefore, the complex formed by MTGR1 and AML1-MTG8 plays a crucial role in promoting hematopoiesis.

[0167] MTGR1 is a member of the MTG8 family of proteins. Transcripts of MTGR1 can be found in many human tissues and all cell lines, including L-G cells and Kasmni-1 cells.

[0168] The MTGR1 protein contains four conserved domains, NHR1–NHR4. NHR1 can react with the homologous region of TATA-binding protein-associated factors (TAFs); NHR2 is essential for the interaction between MTG8 and p85 proteins; NHR4 is associated with two zinc finger domains. AML1-MTG8 and MTGR1 tend to form hybrid complexes. Overexpression of MTGR1 can stimulate G-CSF-dependent cell proliferation and inhibit AML1-dependent transactivation, playing a crucial regulatory role in hematopoiesis.

[0169] The expression profile of the polypeptide of this invention is consistent with that of human bone marrow translocation gene-associated protein 1 (MTGR1), and both have similar biological functions. The polypeptide of this invention can specifically bind to the AML1-MTG8 gene product in vivo, forming a complex. Overexpression of the polypeptide of this invention promotes AML1-MTG8-induced G-CSF-dependent cell proliferation and interrupts AML1-dependent transcription. The AML1-CBF transcription factor complex is crucial for the final hematopoiesis of all cell lineages and is the most frequent target of chromosomal translocations in human leukemia. Therefore, the polypeptide of this invention is closely related to the occurrence of pathological processes such as abnormal cell proliferation, especially hematopoietic cell division, proliferation, differentiation, and developmental disorders, and can lead to related diseases.

[0170] Therefore, it is evident that abnormal expression of the bone marrow translocation gene-related protein 12.32 in this invention will lead to various diseases, especially hematopoietic system diseases, tumors, and developmental disorders, including but not limited to:

[0171] Hematopoietic system diseases: 1. Anemia: Iron deficiency anemia, megaloblastic anemia, aplastic anemia, hemolytic anemia (hereditary spherocytosis, G-6-PD enzyme deficiency, thalassemia, hemoglobinopathies, autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria)

[0172] 2. Leukopenia and agranulocytosis

[0173] 3. Myelodysplastic syndrome

[0174] 4. Leukemia: Acute leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia

[0175] 5. Lymphoma: Lymphoreticular tissue lymphoma, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma

[0176] 6. Histiocytic disorders: Malignant histiocytosis, differentiated histiocytosis, plasma cell disease; tumors of various tissues: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, thyroid tumors, uterine fibroids, neurocytoma, astrocytoma, ependymoma, glioma, neurofibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, colon cancer, thymic tumors, nasopharyngeal carcinoma, laryngeal cancer, tracheal tumors, fibroma, fibrosarcoma, lipoma, liposarcoma.

[0177] Developmental disorders: congenital miscarriage, cleft palate, limb agenesis, limb differentiation disorders, atrial septal defect, neural tube defects, congenital hydrocephalus, intellectual disability, brain development disorders, skin, fat and muscle dysplasia, bone and joint dysplasia, various metabolic disorders, and sexual developmental delay.

[0178] Abnormal expression of the bone marrow translocation gene-related protein 12.32, as described in this invention, will also lead to certain hereditary and hematological diseases.

[0179] The polypeptides of the present invention, as well as their antagonists, agonists, and inhibitors, can be directly used for disease treatment, for example, to treat various diseases, especially various hematopoietic system diseases, tumors, developmental disorders, certain inflammatory diseases, and immune system diseases.

[0180] This invention also provides a method for screening compounds to identify agents that enhance (agonist) or inhibit (antagonist) myeloid translocation-associated protein 12.32. Agonists enhance biological functions of myeloid translocation-associated protein 12.32, such as stimulating cell proliferation, while antagonists inhibit and treat disorders associated with excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing myeloid translocation-associated protein 12.32 can be cultured together with labeled myeloid translocation-associated protein 12.32 in the presence of the drug. The ability of the drug to enhance or inhibit this interaction is then determined.

[0181] Antagonists of myeloid translocation gene-associated protein 12.32 include screened antibodies, compounds, receptor-deficient derivatives, and analogues. These antagonists can bind to myeloid translocation gene-associated protein 12.32 and eliminate its function, or inhibit the production of the peptide, or bind to the active site of the peptide, preventing it from exerting its biological function.

[0182] When screening compounds for potential antagonists, bone marrow translocation gene-associated protein 12.32 can be added. In bioanalytical assays, the antagonist status of a compound is determined by measuring its effect on the interaction between myeloid translocation gene-associated protein 12.32 and its receptor. The same method used to screen compounds can be used to screen for receptor deletions and analogs that act as antagonists. Peptides that bind to myeloid translocation gene-associated protein 12.32 can be obtained by screening a random peptide library consisting of various possible combinations of amino acids bound to a solid phase. During screening, myeloid translocation gene-associated protein 12.32 molecules are generally labeled.

[0183] 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 bone marrow translocation gene-associated protein 12.32 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.

[0184] Polyclonal antibodies can be produced by directly immunizing animals (such as rabbits, mice, and rats) with myeloid translocation protein 12.32. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Techniques for preparing monoclonal antibodies against myeloid translocation protein 12.32 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). Existing technology for producing single-chain antibodies (US Pat No. 4946778) can also be used to produce single-chain antibodies against myeloid translocation protein 12.32.

[0185] Antibodies against myeloid translocation gene-associated protein 12.32 can be used in immunohistochemistry to detect myeloid translocation gene-associated protein 12.32 in biopsy specimens.

[0186] Monoclonal antibodies that bind to the bone marrow translocation gene-associated protein 12.32 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.

[0187] Antibodies can also be used to design immunotoxins targeting specific sites in the body. For example, monoclonal antibodies with high affinity for bone marrow translocation gene-associated protein 12.32 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 cells positive for bone marrow translocation gene-associated protein 12.32.

[0188] The antibody of this invention can be used to treat or prevent diseases associated with myeloid translocation protein 12.32. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of myeloid translocation protein 12.32.

[0189] This invention also relates to diagnostic assays for the quantitative and localized detection of bone marrow translocation gene-associated protein 12.32. These assays are well known in the art and include FISH and radioimmunoassay assays. The levels of myeloid translocation protein 12.32 detected can be used to explain the importance of myeloid translocation protein 12.32 in various diseases and to diagnose diseases in which myeloid translocation protein 12.32 plays a role.

[0190] 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.

[0191] Polynucleotides encoding myeloid translocation protein 12.32 can also be used for various therapeutic purposes. Gene therapy can be used to treat abnormalities in cell proliferation, development, or metabolism caused by the absence or abnormal / inactive expression of myeloid translocation protein 12.32. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variants of myeloid translocation protein 12.32 to inhibit endogenous myeloid translocation protein 12.32 activity. For example, a variant of myeloid translocation protein 12.32 could be a shortened version lacking the signal transduction domain; while 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 myeloid translocation protein 12.32. Viral expression vectors, such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses, can be used to transfer polynucleotides encoding myeloid translocation gene-associated protein 12.32 into cells. Methods for constructing recombinant viral vectors carrying polynucleotides encoding myeloid translocation gene-associated protein 12.32 can be found in existing literature (Sambrook, et al.). Additionally, recombinant polynucleotides encoding myeloid translocation gene-associated protein 12.32 can be packaged into liposomes and transferred into cells.

[0192] 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.

[0193] Oligonucleotides (including antisense RNA and DM) and ribozymes that inhibit the mRNA of bone marrow translocation gene-related protein 12.32 are also within the scope of this invention. A ribozyme is an enzyme-like RNA molecule that specifically breaks down specific RNAs; its mechanism of action involves the ribozyme molecule specifically hybridizing with complementary target RNA and then performing endonuclease. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technique, such as the widely used technique of synthesizing oligonucleotides via solid-phase phosphoamide chemical synthesis. Antisense RNA molecules can be obtained by transcription in vitro or in vivo using the DM sequence encoding the RNA. This DNA sequence has been integrated downstream of the RM 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 ribonucleosides.

[0194] Polynucleotides encoding myeloid translocation gene-associated protein 12.32 can be used for the diagnosis of diseases related to myeloid translocation gene-associated protein 12.32. These polynucleotides can be used to detect the expression of myeloid translocation gene-associated protein 12.32 or to detect myeloid translocation in disease states. Aberrant expression of gene-associated protein 12.32. The DNA sequence encoding myeloid translocation gene-associated protein 12.32 can be used to hybridize biopsy specimens to determine the expression status of myeloid translocation gene-associated protein 12.32. 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 immobilized as probes on microarrays or DNA chips (also known as "gene chips") for analyzing differential gene expression in tissues and for gene diagnosis. In vitro amplification of the transcript of myeloid translocation gene-associated protein 12.32 using primers specific to myeloid translocation gene-associated protein 12.32 via RNA-polymerase chain reaction (RT-PCR) can also detect the transcript of myeloid translocation gene-associated protein 12.32.

[0195] Detection of mutations in the myeloid translocation protein 12.32 gene can also be used to diagnose diseases related to myeloid translocation protein 12.32. Mutations in myeloid translocation protein 12.32 can take the form of point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type myeloid translocation protein 12.32 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, Southern blotting and Western blotting can be used to indirectly determine the presence of gene mutations.

[0196] 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 (repetitive 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 DNA sequences on chromosomes.

[0197] In short, PCR primers (preferably 15-35 bp) are prepared based on cDNA, allowing the sequence to be located on the chromosome. These primers are then used for PCR screening of somatic hybrid cells containing each human chromosome. Only those hybrid cells containing the corresponding human genes from the primers will produce amplified fragments.

[0198] PCR localization of somatic hybrid cells is a rapid method for locating DNA 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 through 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.

[0199] 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 Manual of Basic Techniques, Pergamon Press, New York (1988).

[0200] Once a sequence is pinpointed to its precise chromosomal location, its 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 link to the Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the gene's association with the already located chromosomal region. The relationship between diseases.

[0201] 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 healthy individuals typically involves first looking for structural changes in the chromosomes, such as deletions or translocations visible at the chromosomal level or detectable by cDNA sequence-based PCR. 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).

[0202] 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.

[0203] 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.

[0204] The pharmaceutical composition can be administered in a convenient manner, such as via local, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes. Bone marrow translocation-associated protein 12.32 is administered in an amount effective for the treatment and / or prevention of a specific indication. The amount and dosage range of bone marrow translocation-associated protein 12.32 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.

[0205] 12. The compound of claim 11, characterized in that it is an antisense sequence of the polynucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof.

[0206] 1.3. An application of the compound of claim 11, characterized in that the compound is used in a method for regulating the activity of bone marrow translocation gene-related protein 12.32 in vivo and in vitro.

[0207] 14. A method for detecting a disease or susceptibility to a disease associated with the polypeptide claimed in any 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 nucleotide variations in a polynucleotide that cause abnormal expression or activity of the polypeptide.

[0208] 15. The application of the polypeptide as claimed in any one of claims 1-3, characterized in that it is used for screening mimics, agonists, antagonists or inhibitors of myeloid translocation gene-related protein 12.32; or for peptide fingerprint identification.

[0209] 16. The application of the nucleic acid molecule as described in any one of claims 4-6, characterized in that it is used as a primer for nucleic acid amplification reactions, or as a probe for hybridization reactions, or for the manufacture of gene chips or microarrays.

[0210] 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 is used in a safe and effective dose with a pharmaceutically acceptable carrier to form a pharmaceutical composition for the diagnosis or treatment of diseases associated with abnormalities of the bone marrow translocation gene-associated protein 12.32.

[0211] 18. The use of the polypeptide, polynucleotide, or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide, or compound is used to prepare a medicament for treating diseases such as malignant tumors, hematological diseases, HIV infection, immune diseases, and various types of inflammation.

Claims

Claims I. An isolated polypeptide-bone marrow translocation gene-related protein 12.32, 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 thereof, an analogue or a 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 polynucleotide encoding a polypeptide or fragment thereof, analogue, or derivative thereof having the amino acid sequence shown in SEQ ID NO: 2; (b) Polynucleotides complementary to polynucleotides; or (c) Polynucleotides that have at least 70% identity with (b) or (c) 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 claimed in claim 4, characterized in that... The polynucleotide sequence comprises the sequence of positions 43-381 in SEQ ID NO: 1 or the sequence of positions 1-882 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 of the patent 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 protein associated with bone marrow translocation genes. 12.32 A method for preparing active polypeptides, characterized in that... The method includes: (a) Expression of bone marrow translocation gene-related proteins 12. Under conditions of 32, culture the engineered host cells as described in claim 8; (b) Isolation of proteins associated with bone marrow translocation genes from cultures. 12.32 Active polypeptides.

10. An antibody capable of binding to a polypeptide, characterized in that... The antibody is a protein associated with bone marrow translocation genes. 12.32 Specific binding antibodies. II. A class of compounds that mimic or regulate the activity or expression of peptides, characterized in that... These are proteins that mimic, promote, antagonize, or inhibit bone marrow translocation gene-related proteins.

12. 32 active compounds.