WO2002020781A1 - Nouveau polypeptide, proteine associee au gene de translocation de la moelle osseuse 12.32, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine associee au gene de translocation de la moelle osseuse 12.32, et polynucleotide codant ce polypeptide Download PDF

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WO2002020781A1
WO2002020781A1 PCT/CN2001/001140 CN0101140W WO0220781A1 WO 2002020781 A1 WO2002020781 A1 WO 2002020781A1 CN 0101140 W CN0101140 W CN 0101140W WO 0220781 A1 WO0220781 A1 WO 0220781A1
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polypeptide
polynucleotide
bone marrow
related protein
gene
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PCT/CN2001/001140
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU2002210344A priority Critical patent/AU2002210344A1/en
Publication of WO2002020781A1 publication Critical patent/WO2002020781A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a bone marrow translocation gene-related protein 12.32, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique ''
  • AML1-CBF transcription factor complex is important for eventual hematopoiesis in all cell lineages and is the most frequent target for chromosomal translocation in human leukemia.
  • AML acute myeloid leukemia
  • CBFA2 / PEBP2B the AML1 gene and the MTG8 (ET0 / CDR) gene are juxtaposed.
  • the AML1-MTG8 gene product is obtained, which can be combined with an 85KDa phosphorus The protein reacts strongly.
  • AML1-MTG8 binding protein (Bone Marrow Translocation Gene Related Protein 1, MTGR1) is highly similar to MTG8, and the amino acid sequence comparison shows four regions conserved during evolution (Rl-NHR4), which contains NHR2 for G-CSF The induction of dependent cell proliferation is very important.
  • Immunoprecipitation analysis shows that the R2 region of the dish is necessary for AML1-MTG8 and MTGR1 to form a stable complex.
  • MTGR1 overexpression promotes AML1-MTG8 to induce G-CSF-dependent cell proliferation and disrupts AML1-dependent transcription. It can be seen that the complex formed by MTGR1 and AML1-MTG8 plays a very important role in promoting the production of blood cells.
  • MTGR1 is a protein member of the MTG8 family. There is an in-frame ATG at the eleventh nucleotide of its cDNA, from which the coding sequence starts and ends at 1822. A poly A additional signal began to appear at position 6387 of the cDNA, and contained Alu contiguous sequences at 3207-3236 and 3690-3960. MTGR1 transcripts can be found in many human tissues and all cell lines, including L-G cells and Kasumi-1 cells.
  • MTGR1 protein contains 604 amino acids, which is 61% identical to MTG8. It contains four conserved regions, NHR1- NHR4, and NHR1 can react with homologous regions of TATA binding protein-related factors (TAFs); NHR2 is the interaction between MTG8 and p85 protein Necessary, the secondary structure proves that NHR2 is a helical region and has the characteristics of an amphoteric molecule; NHR4 is related to two zinc finger domains (-C- X- X- C-7x- CX- X- C-, ⁇ CXXXC -7 XHXXXC-). In addition, MTG8 and MTGR1 both contain three Pr. Rich area.
  • TAFs TATA binding protein-related factors
  • AML1-MTG8 and MTGR1 tend to form complexes.
  • MTGR1 overexpression can stimulate dependent cell proliferation and suppress AML1-dependent transactivation, and plays a very important regulatory role in hematopoietic cells.
  • the bone marrow translocation gene-related protein 12.32 protein plays an important role in regulating important functions of the body, such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, more needs to be identified in the art.
  • New bone marrow translocation gene-related protein 12. Isolation of the protein-coding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a bone marrow translocation gene-related protein 12.32.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a bone marrow translocation gene-related protein 12.32.
  • Another object of the present invention is to provide a method for producing a bone marrow translocation gene-related protein 12.32.
  • Another object of the present invention is to provide antibodies against the polypeptide-to-bone marrow translocation gene-related protein 12.32 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide-to-bone marrow translocation gene-related protein 12.32 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities in the bone marrow translocation gene-related protein 12.32.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 43-381 in SEQ ID NO: 1; and (b) a sequence having 1-882 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a screened protein that mimics, activates, antagonizes or inhibits bone marrow translocation genes.
  • a method of a 32-protein active compound comprising utilizing a polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of a bone marrow translocation gene-related protein 12.32 protein in vitro, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of bone marrow translocation gene-related protein 12.32.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic DNA or RNA, which can be single-stranded or double-stranded, representing the sense strand or Antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, which The amino acid substituted in the amino acid has a structural or chemical property similar to that of the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with the bone marrow translocation gene-related protein 12.32, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to a bone marrow translocation gene-related protein 12.32.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of bone marrow translocation gene-related protein 12.32 when combined with bone marrow translocation gene-related protein 12.32.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to the bone marrow translocation gene-related protein 12.32.
  • “Regulation” refers to a change in the function of the bone marrow translocation gene-related protein 12.32, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of the bone marrow translocation-gene protein 12.32. Or changes in immune properties.
  • substantially pure ' means essentially free of other proteins, lipids, sugars or other substances naturally associated with it.
  • Those skilled in the art can use standard protein purification techniques to purify bone marrow translocation gene-related proteins 12.32
  • the substantially pure bone marrow translocation gene-related protein 12.32 can generate a single main band on a non-reducing polyacrylamide gel.
  • the bone marrow translocation gene-related protein 12. 32 can be analyzed for its purity by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require two sequences Columns are bound to each other as specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are the same or similar in a comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg ins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences is calculated by The interval in B can also be determined by Clus ter method or by the method known in the art such as Jotun Hein.
  • the percent identity between nucleic acid sequences He in J., (1990) Methods in emzurao logy 183: 625-645) "similar "Sexuality” refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment between amino acid sequences.
  • negatively charged amino acids may include aspartic acid and glutamic acid; Positively charged amino acids can include lysine and arginine; amino acids with similarly charged head groups that have similar hydrophilicity can include leucine, isoleucine, and valine; glycine and alanine Acids; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of bone marrow translocation gene-related protein 12.32.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not Components of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated bone marrow translocation gene-related protein 12.32 refers to bone marrow translocation gene-related protein 12.32 which is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can use standard protein purification techniques to purify bone marrow translocation gene-related proteins 12.32. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Bone marrow translocation gene-related protein 12. 32 The purity of the polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a bone marrow translocation gene-related protein 12.32, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products, or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives, and analogs of bone marrow translocation gene-related protein 12.32.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the bone marrow translocation gene-related protein 12.32 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by other groups to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • Polynucleotides of the invention are found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 882 bases in length and its open reading frame 43-381 encodes 112 amino acids.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but having a sequence different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the present invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3).
  • polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides. Nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding bone marrow translocation gene-related proteins 12.32.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the bone marrow translocation gene-related protein 12.32 of the present invention can be used in many ways Method to obtain.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded MA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many proven techniques for extracting mRM. Kits are also commercially available (Qiagene).
  • CDNA library is constructed in a conventional method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989) 0 may be obtained commercially available cDNA library such as cDNA library from Clontech is different. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be screened from these cDM libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the transcript level of bone marrow translocation gene-related protein 12.32; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of 12.32 gene expression of the bone marrow translocation gene-related protein can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method for amplifying DNA / .RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RM fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the bone marrow translocation gene-related protein 12.32 coding sequence, and the recombinant technology to produce the Said method of polypeptide.
  • a polynucleotide sequence encoding a bone marrow translocation gene-related protein 12.32 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Ros enberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • 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 appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Mo l ecul ar Cloning, a Labora tory Manua l, co l d Harbor Harbora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: the l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, tumorigenic enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a bone marrow translocation gene-related protein 12.32 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant bone marrow translocation gene-related protein 12.32 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of gene chip expression profiles of bone marrow translocation gene-related protein 12.32 and bone marrow translocation gene-related protein 1 (MTGR1) according to the present invention.
  • the upper graph is a graph of the expression profile of bone marrow translocation gene-related protein 12.32, and the lower graph is the graph of the expression profile of bone marrow translocation gene-related protein 1 (MTGR1).
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of the isolated bone marrow translocation gene-related protein 12.32.
  • lOkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Example 1 Cloning of bone marrow translocation gene-related protein 12.32
  • Human fetal brain total MA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using the Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragments into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ .
  • the bacteria formed a cDNA library.
  • the Dye terminate cycle reaction sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and the cDNA sequence of one of the clones 0561B04 was found to be new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, PCR was performed using the following primers: Primerl: 5'- GGCCGGGGACTCGTGGGTAACTTG-3 '(SEQ ID NO: 3) Primer2: 5 GTCTGAAGTTTATAATATTTTATT- 3' (SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10 mmol / L Tris- in a reaction volume of 50 ⁇ 1
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.4) - 5xSSC- 5xDenhardt, s solution, and 200 ⁇ 8 / ⁇ 1 salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1 ° / »SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant bone marrow translocation gene-related protein 12.32
  • Primer3 5'- CCCCATATGATGGCGTCGGGCTGCAAGATTGGC- 3, (Seq ID No: 5)
  • Priraer4 5'- CATGGATCCTTATTTATTTGAGACGGAGTCTTT- 3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Ndel and BamHI restriction sites correspond to selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • pBS containing the full-length target gene-
  • the 0561B04 plasmid was used as a template for the PCR reaction.
  • PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS- 0561B04 plasmid, primers Primer-3 and Primer- 4 points, and 'J is lOpmol, Advantage polymerase Mix
  • Cycle parameters 94. C 20s, 60 ° C 30s, 68 ° C 2 min, 25 cycles. Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into Ca. bacillus DH5a by the calcium chloride method.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following specific peptides related to bone marrow translocation gene 12.32:
  • NH2-Met-Ala-Ser-Gly-Cys-Lys-Ile-Gly-Pro-Ser-Ile-Leu-Asn-Ser-Asp-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the above-mentioned * cyanin polymorph complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit sera.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to the bone marrow translocation gene-related protein 12.32.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method Acid sequence or a homologous polynucleotide sequence thereof.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • GC content is 30 ° /. -70%, non-specific hybridization increases
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • Gene chip or gene micro matrix (DNA Mi croarray) is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high density arrangement of a large number of target gene fragments on glass. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotides of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500ng / ul after purification.
  • the spots were spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ ⁇ .
  • the spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. Its specific method steps There have been various reports in the literature, and the post-sampling processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and raigo was purified using Oligotex raRNA Midi Kit (purchased from QiaGen).
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5- Amino-propargyl- 2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Araersham Phamacia Biotech, was used to label the mRNA of specific tissues (or stimulated cell lines) of the body, and probes were prepared after purification.
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy3 f l
  • the probes from the above two tissues were hybridized with the chip in a UniHyb TM Hybridizat ion Solut ion (purchased from T.eleChem) hybridization solution for 16 hours, and the washing solution (1 x SSC, 0.2%) was used at room temperature. SDS) After washing, scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line, thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar formation fc growth factor stimulation, 1013HT, scar into fc without growth factor stimulation, 1 G13HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma Cardiac cancer. Draw a chart based on these 18 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the bone marrow translocation gene related protein 12.32 and the bone marrow translocation gene related protein 1 (MTGR1) expression profiles described in the present invention are very similar. Industrial applicability
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • AML1-CBF transcription factor complex is important for eventual hematopoiesis in all cell lineages and is the most frequent target for chromosomal translocations in human leukemia.
  • the AML1 (CBFA2 / PEBP2B) gene is parallel to the MTG8 (ET0 / CDR) gene in the translocation associated with acute myeloid leukemia (AML), and the AMLl-MTG8 gene product has been obtained, which can be strongly associated with an 85Kda phosphoprotein reaction.
  • AML1-MTG8 binding protein (Bone Marrow Translocation Gene Related Protein 1, MTGR1) is highly similar to MTG8, and amino acid sequence comparisons show four regions conserved during evolution (NHR1-NHR4), which contains NHR2 that is G-CSF-dependent The induction of cell proliferation is very important. Immunoprecipitation analysis showed that the NHR2 region is required for AML1-MTG8 and MTGR1 to form a stable complex. MTGR1 overexpression promotes AML1-MTG8 to induce G-CSF-dependent cell proliferation and disrupts AML1-dependent transcription. It can be seen that the complex formed by MTGR1 and AML1-MTG8 plays a very important role in promoting blood cell production.
  • MTGR1 is a protein member of the MTG8 family. MTGR1 transcripts are found in many human tissues and all cell lines, including L-G cells and Kasmni-1 cells.
  • MTGR1 protein contains four conserved regions NHR1- NHR4, NHR1 can react with TATA-binding protein related factors (TAFs) homologous regions; NHR2 is required for MTG8 to interact with p85 protein; NHR4 is related to two zinc finger domains AMLl- MTG8 and MTGR1 tend to form complexes.
  • TAFs TATA-binding protein related factors
  • MTGR1 superexpression can stimulate G-CSF-dependent cell proliferation and curb AML1-dependent transactivation, which plays a very important regulatory role in blood cell production.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human bone marrow translocation gene-related protein 1 (MTGR1), and both have similar biological functions.
  • the polypeptide of the present invention can specifically bind to the AML1-MTG8 gene product in vivo to form a complex.
  • the overexpression of the polypeptide of the present invention promotes AML1-MTG8 to induce G-CSF-dependent cell proliferation and interrupts AML1-dependent transcription.
  • the AMLl-CBF transcription factor complex is very important for the final blood cell generation of all cell lineages, and is the most frequent target for chromosomal translocation in human leukemia. It can be seen that the polypeptide of the present invention is useful for cell proliferation, especially blood cell division, The occurrence of pathological processes such as abnormal proliferation, differentiation, and developmental disorders are closely related and produce related diseases.
  • the abnormal expression of the bone marrow translocation gene-related protein 12.32 of the present invention will produce various diseases, especially hematopoietic system diseases, tumors, and development disorders. These diseases include, but are not limited to:
  • Hematopoietic diseases 1. Anemia: iron deficiency anemia, megaloblastic anemia, aplastic anemia, hemolytic anemia (hereditary spherocytosis, G-6-PD enzyme deficiency, thalassemia, hemoglobinopathy, autoimmune hemolysis Anemia, paroxysmal nocturnal hemoglobinuria)
  • Leukemia acute leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, chronic mononuclear leukemia
  • Lymphoma Lymphatic reticulum, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma
  • Histiocytosis Malignant histiocytosis, differentiated histiocytosis, plasma cell disease Tumors of various tissues: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, thyroid tumor, uterine fibroids, neurocytoma , Astrocytoma, ependymal tumor, glioblastoma, neurofibromas, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, colon cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, Tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma
  • Developmental disorders congenital abortion, cleft palate, lack of limbs, limb differentiation disorders, atrial septal defect, neural tube defects, congenital hydrocephalus, mental retardation, brain development disorders, skin, fat and muscular dysplasia, Bone and joint dysplasia, various metabolic defects, sexual retardation.
  • the abnormal expression of the bone marrow translocation gene-related protein 12.32 of the present invention will also produce certain hereditary, hematological diseases and the like.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially various hematopoietic system diseases, tumors, development disorders, certain inflammations, and the immune system. Illness, etc.
  • the invention also provides a method for screening compounds to identify agents that increase (agonist) or suppress (antagonist bone marrow translocation gene-related protein 12.32.
  • Agonists increase bone marrow translocation gene-related protein 12.32 to stimulate cell proliferation and the like Biological function, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing the bone marrow translocation gene-related protein 12.32 can be prepared and labeled The bone marrow translocation gene-related protein 12.32 is cultured together. The ability of the drug to increase or suppress this interaction is then determined.
  • Antagonists of bone marrow translocation gene-related proteins 12.32 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of the bone marrow translocation gene-related protein 12.32 can bind to the bone marrow translocation gene-related protein 12.32 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the polypeptide Cannot perform biological functions.
  • bone marrow translocation gene-related protein 12.32 can be added in bioanalytical assays, whether a compound is an antagonist is determined by determining the effect of the compound on the interaction between the bone marrow translocation gene-related protein 12.32 and its receptor. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to bone marrow translocation gene-related protein 12.32 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the 12.32 molecule of the bone marrow translocation gene-related protein should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the 12.32 epitope of the bone marrow translocation gene-related protein. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • polyclonal antibodies can be obtained by direct injection of bone marrow translocation gene-related protein 12.32 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to 'S adjuvant and so on.
  • Techniques for preparing a monoclonal antibody against bone marrow translocation gene-related protein 12.32 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta -Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Inlay antibodies combining human constant regions and non-human-derived variable regions can be produced using existing techniques (Morri et al, PNAS, 1985, 81: 6851).
  • the unique technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against bone marrow translocation gene-related protein 12.32.
  • Antibodies against bone marrow translocation gene-related protein 12.32 can be used in immunohistochemical techniques to detect bone marrow translocation-gene related protein 12.32 in biopsy specimens.
  • Monoclonal antibodies that bind to bone marrow translocation gene-associated protein 12.32 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body. Such as bone marrow translocation gene-related proteins 12.
  • 32 High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP, and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill the bone marrow translocation gene-related protein 12. 32 Positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the bone marrow translocation gene-related protein 12.32.
  • Administration of the appropriate dose of antibody can stimulate or block the production or activity of bone marrow translocation gene-related protein 12.32.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the bone marrow translocation gene-related protein 12.32 level.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • Test The level of bone marrow translocation gene-associated protein 12.32 detected in the study can be used to explain the importance of bone marrow translocation gene-associated protein 12. 32 in various diseases and for the diagnosis of bone marrow translocation-gene-related protein 12.32 up Effect of disease.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding the bone marrow translocation gene-related protein 12.32 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the absence or abnormal / inactive expression of bone marrow translocation gene-related protein 12.32.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated bone marrow translocation gene-related proteins 12.32 to inhibit endogenous bone marrow translocation gene-related proteins 12.32 activity.
  • a mutant bone marrow translocation gene-related protein 12.32 may be a shortened bone marrow translocation gene-related protein 12.32 that lacks a signaling domain, although it can bind to downstream substrates, but lacks signal transduction. active. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of the bone marrow translocation gene-related protein 12.32.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a bone marrow translocation gene-related protein 12.32 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a bone marrow translocation gene-related protein 12.32 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding the bone marrow translocation gene-related protein 12.32 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DM
  • ribozymes that inhibit bone marrow translocation gene-related protein 12.32 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of DM sequences encoding the RNA. This DNA sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding the bone marrow translocation gene-related protein 12.32 can be used for the diagnosis of diseases related to the bone marrow translocation gene-related protein 12.32.
  • the polynucleotide encoding the bone marrow translocation gene-related protein 12.32 can be used to detect the expression of the bone marrow translocation gene-related protein 12.32 or the bone marrow translocation in a disease state Abnormal expression of gene-associated protein 12.32.
  • the DNA sequence encoding the bone marrow translocation gene-related protein 12.32 can be used to hybridize biopsy specimens to determine the expression status of the bone marrow translocation gene-related protein 12.32. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization.
  • Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Bone marrow translocation gene-related protein 12.32 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the bone marrow translocation gene-related protein 12.32 transcription product.
  • Bone marrow translocation gene-related protein 12.32 mutations can also be used to diagnose bone marrow translocation gene-related protein 12.32-related diseases.
  • Bone marrow translocation gene-related protein 12.32 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type bone marrow translocation gene-related protein 12.32 DNA sequence. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inherance in Man (available online with Johns Hopkins University Wetch Medical Library). Linkage analysis can then be used to determine where genes and genes are located in chromosomal regions Relationship between diseases.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping Resolving power and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Bone marrow translocation gene-related protein 12.32 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of bone marrow translocation gene-related protein 12.32 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.
  • the compound according to claim 11 characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO: 1.
  • a method for detecting a disease or disease susceptibility related to the polypeptide comprises detecting the expression amount of the polypeptide, or detecting the polypeptide Activity, or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.
  • polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimics, agonists, antagonists or inhibitors of bone marrow translocation gene-related protein 12.32; Or used for peptide fingerprint identification.
  • nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or for manufacturing a gene chip Or microarray.
  • the agent or inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with a bone marrow translocation gene-related protein 12.32 abnormality.
  • polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, 'characterized in that said polypeptide, polynucleotide or compound is used for preparing a treatment such as a malignant tumor, Hematological diseases, HIV infection and immune diseases and drugs of various inflammations.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine associée au gène de translocation de la moelle osseuse 12.32, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment de maladies affectant le système hématopoïétique, de tumeurs et de troubles du développement. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine associée au gène de translocation de la moelle osseuse 12.32.
PCT/CN2001/001140 2000-07-07 2001-07-02 Nouveau polypeptide, proteine associee au gene de translocation de la moelle osseuse 12.32, et polynucleotide codant ce polypeptide WO2002020781A1 (fr)

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CN 00119402 CN1333230A (zh) 2000-07-07 2000-07-07 一种新的多肽——骨髓易位基因相关蛋白12.32和编码这种多肽的多核苷酸
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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN [online] 3 April 2000 (2000-04-03), AFONSO C.L. ET AL., retrieved from GI:9631447 accession no. NCBI Database accession no. (NP_048270.1) *
DATABASE PROTEIN [online] 3 April 2000 (2000-04-03), AFONSO C.L. ET AL., retrieved from GI:9631448 accession no. NCBI Database accession no. (NP_048269.1) *

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