WO2001079280A1 - Nouveau polypeptide, proteine humaine 16 associee au represseur catabolique du carbone, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 16 associee au represseur catabolique du carbone, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001079280A1
WO2001079280A1 PCT/CN2001/000531 CN0100531W WO0179280A1 WO 2001079280 A1 WO2001079280 A1 WO 2001079280A1 CN 0100531 W CN0100531 W CN 0100531W WO 0179280 A1 WO0179280 A1 WO 0179280A1
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polypeptide
polynucleotide
protein
carbon metabolism
repressor
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PCT/CN2001/000531
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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 AU60035/01A priority Critical patent/AU6003501A/en
Publication of WO2001079280A1 publication Critical patent/WO2001079280A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 novel polypeptide, a human carbon metabolism by-product repressor protein 16 and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique
  • Carbon metabolism by-product repressor protein is a transcription regulation protein commonly found in organisms. By forming a multi-subunit complex in the body, it can affect the expression of many genes.
  • CCR4 does not bind to DNA, it can stimulate the transcription process when combined with abnormal DNA-binding domains.
  • CCR4 is glucose-regulated.
  • the two transcriptional regulatory domains one is glucose-related and contains a glutamate-proline-rich structural region, which is very similar to the structural regions contained in many transcription factors found in eukaryotes. similar.
  • Studies have found that if CCR4 lacks a leucine repeat structure and a C-terminus, its two transcriptional regulatory domains cannot form a functional CCR4. Therefore, the leucine repeat structure and C-terminus play a key role in linking two transcriptional regulatory domains to form a complete CCR4.
  • the yeast CCR4 protein is an indispensable protein in the expression of some non-fermentative growth regulating genes (such as glucose-suppressing ethanol dehydration, ADH2, etc.), and it is also a correction gene for the SPT6 and SPT10 mutant genes.
  • SPT6 and SPT10 are It is useful to maintain the stability of chromatin. (Mol Cell Biol 1994 Jul; 14 (7): 4522-31)
  • CCR4-related protein CAF1 (P0P2) is a component protein of the CCR4 complex. Its homologous protein has been found in many organisms, such as humans, nematodes, xenopus, mice, yeast, and so on.
  • the main function of CCR4-related protein (CAF1) in organisms is to regulate the transcription process.
  • CAF1 CCR4-related protein
  • CCR4 can affect the expression of genes in many yeasts by forming multi-subunit complexes.
  • DBF2 Cell cycle-regulated kinase
  • DBF2 plays an important role in the process of transition from the end of mitosis to G1.
  • DBF2 forms a complex with CCR4 and CAF-1 / P0P2.
  • Gene expression in the cell cycle and subsequent mitotic gene transcription Has an important role in it.
  • Human B-cell translocation protein (BTG1) is a tumor suppressor because its overexpression inhibits the proliferation of NIH 3T3 cells.
  • the carbon metabolism by-product repressor protein (CCR4) binding factor (hCAF-1) is homologous to murine CAF-1 and Saccharomyces cerevisiae CAF-1 / P0P2.
  • CCR4 binding factor hCAF-1
  • BTG1 The carbon metabolism by-product repressor protein binding factor
  • hCAF-1 / BTG1 complex plays an important and obvious role in regulating cell division and cell proliferation.
  • the human carbon metabolism by-product repressor-related protein 16 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, so the identification of Many human carbon metabolism byproducts, repressor protein 16 protein, are involved in these processes, and especially the amino acid sequence of this protein is identified. Isolation of newcomer carbon metabolism by-product repression protein-related protein 16 protein-encoding genes also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic agents for diseases, so it is important to isolate its coding DM. It is an object of the present invention to provide isolated novel polypeptides-human carbon metabolism by-product repressor protein 16 and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human carbon metabolism byproduct repressor related protein 16.
  • Another object of the present invention is to provide a protein containing a human carbon metabolism byproduct repressor related protein 16 Polynucleotide genetically engineered host cells.
  • Another object of the present invention is to provide a method for producing a human carbon metabolism by-product repressor related protein 16.
  • Another object of the present invention is to provide antibodies against the polypeptide of the present invention-human carbon metabolism by-product repressor protein 16-.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human carbon metabolism by-product repressor protein 16.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human carbon metabolism by-product repressor protein 16.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 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 84-371 in SEQ ID NO: 1; and (b) a sequence having 1-502 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said 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 present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human carbon metabolism by-product repressor protein 16 protein activity, which comprises utilizing the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or disease susceptibility related to abnormal expression of human carbon metabolism by-product repressor-related protein 16 protein, comprising detecting mutations in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample Or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the invention also relates to the polypeptides and / or polynucleotides of the invention for use in the treatment of cancer, developmental disorders Wo
  • 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 in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of 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 and to bind to specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human carbon metabolism by-product repressor-related protein 16, 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 binds human carbon metabolism byproduct repressor-related protein 16.
  • Antagonist or “inhibitor” refers to a biological or immunological activity that can block or regulate human carbon metabolism byproduct repressor protein 16 when combined with human carbon metabolism byproduct repressor protein 16 Molecule.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to human carbon metabolism byproduct repressor-related protein 16.
  • Regulation refers to a change in the function of human carbon metabolism by-product repressor protein 16, including an increase or decrease in protein activity, a change in binding properties, and any other biological properties of human carbon metabolism by-product repressor protein 16 , Functional or immune properties.
  • Substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human carbon metabolism by-product repressor protein 16 using standard protein purification techniques.
  • Substantially pure human carbon metabolism byproduct repressor-related protein 16 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human carbon metabolism byproduct repressor-related protein 16 peptide can be analyzed by amino acid sequence.
  • Complementary or “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 can be partial or complete.
  • the degree of complementarity 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. Inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences be combined with each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Cluster method divides each group of sequences by checking the distance between all pairs. Arranged in clusters. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by the Cluster method or by methods known in the art such as Jotun Hein. Percentage of identity (He in J., (1990) Methods in emzumo l ogy 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? (& 1) ') 2 and? ⁇ It can specifically bind to the epitope of human carbon metabolism byproduct repressor-related protein 16.
  • 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 vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part 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 existing in the natural state. .
  • isolated human carbon metabolism byproduct repressor protein 16 refers to human carbon metabolism byproduct repressor protein 16 which is substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human carbon metabolism by-product repressor protein 16 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human carbon metabolism by-product repressor-related protein 16 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human carbon metabolism by-product repressor protein 16
  • the above is 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 present invention also includes fragments, derivatives and analogs of human carbon metabolism by-product repressor related protein 16.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human carbon metabolism by-product repressor-related protein 16 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) such a type in which one or more amino acid residues are substituted with 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 a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 502 bases, and its open reading frame of 84-371 encodes 95 amino acids.
  • this polypeptide has a similar expression profile with human CCR4 related protein 31, and it can be inferred that the human carbon metabolism byproduct repressor protein 16 has similar functions to human CCR4 related protein 31.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genome
  • DNA or synthetic 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 it may be a degenerate variant.
  • the "degenerate variant” refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) Coded sequences) and non-coded sequences.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes 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.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • 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 invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least two sequences between
  • 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) only between the two sequences Crosses occur at least 95% or more, and more preferably 97% or more.
  • the 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 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human carbon metabolism byproduct repressor-related protein 16.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • polynucleotide sequence encoding the human carbon metabolism by-product repressor related protein 16 of the present invention can be obtained by various methods.
  • 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 DNA 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 cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage CDM library.
  • Q i agene There are many mature techniques for mRNA extraction, and kits are also commercially available (Q i agene).
  • the construction of cDNA library is also a common method (Sambrook, etal., Molecula ar Cloning, A Labora tory Manua 1, Cold Spr ing Harbor Labora tory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the transcript of human carbon metabolism byproduct repressor protein 16 Level; (4) detecting protein products of gene expression by immunological techniques or 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 2,000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM 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 human carbon metabolism by-product repressor related protein 16 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELI SA). .
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELI SA).
  • a method for amplifying DM / RNA by PCR (Sa i k i, e t a l. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers 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 / RNA 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 DNA 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. 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 a human carbon metabolism by-product repressor-related protein 16 coding sequence, and recombinant technology to produce the present invention. Said method of polypeptide.
  • a polynucleotide sequence encoding a human carbon metabolism by-product repressor related protein 16 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenopathies, which are well known in the art. Virus, retrovirus or other vector.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • pMSXND expression vectors expressed in mammalian cells Lee and Na thans, J Bio Chem. 263: 3521, 1988
  • baculovirus-derived vectors expressed in insect cells in short, as long as it can be replicated and stabilized in a host, any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing DM sequences encoding human carbon metabolism byproduct repressor related protein 16 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecule Cloning, Laboratory Manua, Cod Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an 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, a transcription terminator, and the like. 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, polyoma 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 human carbon metabolism by-product repressor protein 16 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • DNA sequence of the present invention or a recombinant vector containing the DNA sequence to transform a host cell is useful
  • the conventional techniques are 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 harvested after exponential growth phase, with (: Treatment 1 2, steps well known in the art used alternative is to use MgCl 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 liposomes Packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human carbon metabolism by-product repressor protein 16 (Science, 1984; 224: 1431). Generally speaking, there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. 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. If necessary, recombinant proteins can be isolated 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, ionization 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, ionization Exchange chromatography, high performance
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human carbon metabolism by-product repressor protein 16 and human CCR4 related protein 31 of the present invention.
  • the upper graph is a graph of the expression profile of human carbon metabolism by-product repressor protein 16 and the lower graph is the graph of expression profile of human CCR4 related protein 31.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates non-starved L02
  • 8 indicates L02 +, lhr, As 3+
  • 9 indicates ECV304 PMA-, 10 for ECV304 PMA +, 11 for fetal liver, 12 for normal liver, 13 for thyroid, 14 for skin, 15 for fetal lung, 16 for lung, 17 for lung cancer, 18 for fetal spleen, 19 is the spleen
  • 20 is the prostate
  • 21 is the fetal heart
  • 22 is the heart
  • 23 is the muscle
  • 24 is the testis
  • 25 is the fetal thymus
  • 26 is the thymus.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human carbon metabolism byproduct repressor-related protein 16. 16kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • Example 1 Cloning of human carbon metabolism byproduct repressor related protein 16
  • RNA Human fetal brain total RNA 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 was formed into cDNA by reverse transcription. A Smart cDM cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multicloning site of the pBSK (+) vector (Clontech) to transform DH5 cc. The bacteria formed a cDNA library.
  • Dye terminate cycle reaction ion 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 it was found that the cDNA sequence of one of the clones 0176cl2 was new DNA.
  • the inserted cDNA fragment contained in this clone was 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, the following primers were used for PCR amplification:
  • Primerl 5'- GTTTGTTTACCTTCTGAGCTGCAG-3 '(SEQ ID NO: 3)
  • Primer2 5'- CCAAGACAACCTCTTTATTTAATG-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ 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-Cl, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers in a 50 ⁇ 1 reaction volume, 1U of Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-502bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human carbon metabolism byproduct repressor-related protein 16 gene expression: Total RNA was extracted by a one-shot method. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70 % ethanol, dried and dissolved in water.
  • Primer 3 5,-CCCCATATGATGGGGCAAATACTGCTGTGTGGC- 3 '(Seq ID No: 5)
  • Primer4 5'-CATGGATCCTCAGAAGAGAAGCCGTTTATTGGT-3 '(Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Nde I and BamH I restriction sites correspond to the expression vector plasmid pET- 28 b (+) (Nova gen, Cat. No. 69865.3).
  • PCR was performed using the pBS-0176cl2 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 p g of pBS-0176cl2 plasmid, primer Primer-3 and Pr iraer-4, and another 1 J lOpmol, Advantage polymerase Mix (Clontech) Product) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into E. coli DH5CC using the calcium chloride method.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following 16 specific peptides related to human carbon metabolism by-product suppressor protein:
  • NH2-Met-Gly-Gln-Ile-Leu-Leu-Cys-Gly- et-Val-Val-Pro-Thr-Pro-Met- C00H SEQ ID NO: 7
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin for methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex plus complete Freund's adjuvant. After 15 days, the rabbit was immunized with hemocyanin polypeptide complex plus incomplete Freund's adjuvant 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 Sepharos B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method demonstrated that the purified antibody specifically binds to human carbon metabolism by-product repressor protein 16.
  • 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.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • 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
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • 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 14 Resuspend the DNA pellet in a small volume of TE or water. Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DM every 1-5 X 10 6 cells extracted about plus lul.
  • steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that it can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and 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, for example, see the literature DeRi s i, J. L., Lyer, V. & Brown, P. 0.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the present invention.
  • Polynucleotides They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance is 280 ⁇ m. 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.
  • the specific method steps have been variously reported in the literature.
  • the post-spot processing steps of this embodiment are:
  • the probes from the two types of tissues and the chips were hybridized in a UniHyb TM Hybridization Solution (purchased from TeieChem) hybridization solution for 16 hours, washed with a washing solution (1> SSC, 0.2 SDS) at room temperature and scanned with ScanArray 3000.
  • the instrument purchased from General Scanning Company, USA
  • the scanned image was analyzed and processed with Imagene software (Biodiscovery Company, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, and non-starved L02 Cell line, arsenic stimulated L02 cell line for 1 hour, arsenic stimulated L02 cell line for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, Fetal brain, fetal lung, and fetal heart.
  • 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 malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • the polypeptide (human carbon metabolism by-product repressor protein 16) of the present invention is an interaction protein of carbon metabolism by-product repressor protein (CCR4). Its main function is to regulate the transcription process of genes. According to existing research, the polypeptide of the present invention can play an important role in cell cycle gene expression and subsequent mitotic gene transcription process; the polypeptide of the present invention can interact with human The B-cell translocation protein BTG1 forms the hCAF-1 / BTG1 complex, which has an important and obvious regulatory effect on cell division and cell proliferation; the polypeptide of the present invention can also regulate some non-fermentative growth through its interaction with CCR4 Regulating the expression process of genes (such as glucose-inhibiting alcohol dehydrogenase ADH2, etc.); The polypeptide of the present invention can also be used indirectly as a correction gene for SPT6 and SPT10 mutant genes, and therefore has the effect of maintaining the stability of chromatin.
  • CCR4 carbon metabolism by-product repressor protein
  • the polypeptide of the present invention can be used for the diagnosis and treatment of many diseases, such as malignant tumors, endocrine system diseases, neurological diseases, immune diseases, human acquired immune deficiency syndrome (AIDS), and the like.
  • diseases such as malignant tumors, endocrine system diseases, neurological diseases, immune diseases, human acquired immune deficiency syndrome (AIDS), and the like.
  • AIDS human acquired immune deficiency syndrome
  • Developmental disorders that can be treated using the polypeptides of the present invention include: spina bifida, craniocerebral fissure, anencephaly, malocclusion, foramen forebral malformation, Down syndrome, congenital hydrocephalus, aqueduct malformation, cartilage dysgenesis Dwarfism, spinal epiphyseal dysplasia, pseudochondral dysplasia, Langer-Gyed ion syndrome, funnel chest, gonad hypoplasia, congenital adrenal hyperplasia, upper urethral tract, crypt, accompanied by short stature deformity syndrome (Such as Conrad i syndrome and Danbo l tC los s syndrome), congenital glaucoma or cataract, congenital lens position abnormality, congenital blepharoplasia, retinal dysplasia, congenital optic atrophy, congenital sensory neurological hearing loss , Cracked hand and foot, teratosis, Williams syndrome, A lag il
  • Various tumors that can be treated using the polypeptide of the present invention include: including epithelial tissue (such as basal epithelium, squamous epithelium, mucus cells, etc.), (such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue, blood vessels and lymphatic endothelial cells Tissue, etc.), hematopoietic tissue (such as B cells, T cells, histiocytes, etc.), tumors of central nervous tissue, peripheral nerve tissue, endocrine tissue, gonadal tissue, special tissue (such as dental tissue, etc.), for example, Gastric cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, food Road cancer and so on.
  • epithelial tissue such as basal epithelium, squamous epithelium, mucus cells, etc.
  • fibrous tissue such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue, blood vessels
  • the polypeptide of the present invention is also an immunomodulatory agent and has an immune promoting or immunosuppressing effect.
  • the polypeptide of the present invention can be used for the treatment of diseases including non-response of immune response, or abnormal immune response, or ineffective host defense.
  • the polypeptide of the present invention and its antibody also have effects on damage, defects or disorders of immune tissue, especially for diseases of the hematopoietic system (such as malignant anemia), skin diseases (such as psoriasis), and autoimmune diseases (such as rheumatoid arthritis). ), Radiation diseases and the production and regulation of immune lymphocytes are extremely closely related.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human carbon metabolism by-product repressor protein 16.
  • Agonists enhance human carbon metabolism by-product suppressor-related protein 16 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human carbon metabolism byproduct repressor protein 16 can be cultured with labeled human carbon metabolism byproduct repressor protein 16 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human carbon metabolism by-product repressor-related protein 16 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human carbon metabolism by-product repressor protein 16 can bind to human carbon metabolism by-product repressor protein 16 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide. The polypeptide cannot perform biological functions.
  • human carbon metabolism by-product repressor protein 16 can be added to bioanalytical assays. By measuring the compounds' interactions with human carbon metabolism by-product repressor protein 16 and its receptors, Influence to determine if a compound is an antagonist. 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 human carbon metabolism by-product repressor-related protein 16 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, 16 molecules of human carbon metabolism by-product repressor related protein should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human carbon metabolism by-product repressor-related protein 16 epitope. 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 injecting human carbon metabolism by-product repressor protein 16 directly into immunized animals (such as home immunity, mice, rats, etc.).
  • immunized animals such as home immunity, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to 'S adjuvant and so on.
  • Preparation of monoclonal antibodies against human carbon metabolism by-product repressor protein 16 Technologies include, but are not limited to, hybridoma technology (Kohler and Milestein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma technology, and EBV-hybridoma technology.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morr is on etal, PNAS, 1985, 81: 6851).
  • the existing technology for producing single-chain antibodies (US Pat No. 4946778) can also be used to produce single-chain antibodies against human carbon metabolism by-product repressor-related protein 16.
  • Antibodies against human carbon metabolism byproduct repressor protein 16 can be used in immunohistochemical techniques to detect human carbon metabolism byproduct repressor protein 16 in biopsy specimens.
  • Monoclonal antibodies that bind to human carbon metabolism byproduct repressor-related protein 16 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.
  • human carbon metabolism by-product repressor protein 16 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 human carbon metabolism by-product repressor protein. 16 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human carbon metabolism by-product repressor protein 16. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human carbon metabolism by-product repressor protein 16.
  • the present invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human carbon metabolism by-product suppressor protein 16.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of human carbon metabolism byproduct repressor protein 16 detected in the test can be used to explain the importance of human carbon metabolism byproduct repressor protein 16 in various diseases and to diagnose human carbon metabolism byproduct repressor protein. Diseases where related protein 16 plays a role.
  • 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.
  • Polynucleotides encoding human carbon metabolism byproduct repressor related protein 16 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 non-expression or abnormal / inactive expression of human carbon metabolism by-product repressor-related protein 16.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human carbon metabolism byproduct repressor protein 16 in order to inhibit endogenous human carbon metabolism byproduct repressor protein 16 activity.
  • a variant human carbon metabolism byproduct repressor protein 16 may be a shortened human carbon metabolism byproduct repressor protein 16 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks Signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human carbon metabolism by-product suppressor protein 16.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a human carbon metabolism byproduct repressor protein 16 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human carbon metabolism byproduct repressor-related protein 16 can be found in the existing literature (Sambrook, eta l.).
  • a recombinant polynucleotide encoding human carbon metabolism by-product repressor protein 16 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 DNA
  • ribozymes that inhibit human carbon metabolism by-product repressor-related protein 16 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and then performs endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DM sequence has been integrated downstream of the vector's RNA polymerase promoter. 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 human carbon metabolism by-product repressor protein 16 is useful for the diagnosis of diseases related to human carbon metabolism by-product repressor protein 16.
  • Polynucleotides encoding human carbon metabolism by-product repressor related protein 16 can be used to detect the expression of human carbon metabolism by-product repressor related protein 16 or the abnormal expression of human carbon metabolism by-product repressor related protein 16 in disease states .
  • the DNA sequence encoding human carbon metabolism byproduct repressor protein 16 can be used to hybridize biopsy specimens to determine the expression of human carbon metabolism byproduct repressor protein 16.
  • Hybridization techniques include Sout hern blotting, Nor thern blotting, and in situ hybridization.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray (Microarray) or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in a tissue.
  • Human carbon metabolism by-product repressor-related protein 16 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human carbon metabolism by-product repressor-related protein 16 transcription products.
  • Human carbon metabolism by-product repressor-related protein 16 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human carbon metabolism by-product repressor-related protein 16 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position of a human chromosome and can hybridize with it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeat polymorphisms) are available for labeling chromosomal positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on the cDNA, and the sequence can be mapped on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers 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 by a similar method, 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 (FI SH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FI SH 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. Mckus i ck, Mende l i an Inher i tance i n Man (available online with Johns Hopk ins Univer s Wet ch Med ica l L brary). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • 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 of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, 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 chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be combined with Use after suitable drug carrier combination.
  • suitable drug carrier combination 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptide of the present 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.
  • Human carbon metabolism byproduct repressor-related protein 16 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human carbon metabolism by-product repressor-related protein 16 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.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 16 associée au répresseur catabolique du carbone, et un polynucléotide codant pour 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 des tumeurs malignes, de l'hémopathie, des troubles du développement, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine 16 associée au répresseur catabolique du carbone.
PCT/CN2001/000531 2000-03-29 2001-03-26 Nouveau polypeptide, proteine humaine 16 associee au represseur catabolique du carbone, et polynucleotide codant pour ce polypeptide WO2001079280A1 (fr)

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CN00115260A CN1315438A (zh) 2000-03-29 2000-03-29 一种新的多肽——人碳代谢副产物阻遏蛋白相关蛋白16和编码这种多肽的多核苷酸

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CN113388640A (zh) * 2020-06-11 2021-09-14 百奥赛图(北京)医药科技股份有限公司 Ccr4基因人源化的非人动物及其构建方法和应用

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WO2000032783A1 (fr) * 1998-12-02 2000-06-08 E.I. Du Pont De Nemours And Company Facteurs de tanscription de vegetaux

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000032783A1 (fr) * 1998-12-02 2000-06-08 E.I. Du Pont De Nemours And Company Facteurs de tanscription de vegetaux

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113388640A (zh) * 2020-06-11 2021-09-14 百奥赛图(北京)医药科技股份有限公司 Ccr4基因人源化的非人动物及其构建方法和应用

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