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WO2001038523A1 - Nouveau polypeptide, gene humain 14 lie a la surdite, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, gene humain 14 lie a la surdite, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001038523A1
WO2001038523A1 PCT/CN2000/000449 CN0000449W WO0138523A1 WO 2001038523 A1 WO2001038523 A1 WO 2001038523A1 CN 0000449 W CN0000449 W CN 0000449W WO 0138523 A1 WO0138523 A1 WO 0138523A1
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Prior art keywords
polypeptide
polynucleotide
related gene
deafness
sequence
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bioroad Gene Development Ltd
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Shanghai Bioroad Gene Development Ltd
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Priority to AU15095/01A priority Critical patent/AU1509501A/en
Publication of WO2001038523A1 publication Critical patent/WO2001038523A1/fr
Anticipated expiration legal-status Critical
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention belongs to the field of biotechnology, and specifically, the present invention describes a new polypeptide, a human ear 3 ⁇ 4 related gene 14, and a polynucleotide sequence encoding the polypeptide.
  • the invention also relates to a method and application for preparing the polynucleotide and polypeptide. Background technique
  • DFNB autosomal recessive inheritance method
  • the OT0F gene extends over 21 kb and contains 28 coding exons and a 5-terminal non-coding region exon. (Yasunaga, S. et al., 1999 Nat Genet 21 (4), 363-369)
  • the protein encoded by the OTOF gene contains 1230 amino acids and has a molecular weight of 140.5 kDa.
  • the 0TOF protein has a 33-amino acid hydrophobic C-terminus, which contains a sequence of leucine residues (amino acids 1198-1214) that may form a transmembrane domain.
  • the 0T0F protein has no leader peptide and target protein signals, and the remaining proteins (1-1197 amino acids) are localized in the cytoplasm.
  • the 0TOF protein has 4 N glycosylation sites and 13 potential protein kinase C phosphorylation sites.
  • the C-terminal cytoplasmic protein anchored on the cell membrane of the 0T0F protein has three C2 domains, namely C2A (196-329 amino acids), C2B (709-833 amino acids), and C2C (949-1104 amino acids).
  • the so-called C2 domain consists of two beta sheets, which are composed of four chains, and there is a high degree of homology in the structure between the sheets. So far, about 100 species have been found C2 domain.
  • the C2 domain of the 0T0F protein is similar to the Syt-1C2A type and binds to Ca ions.
  • the side chain of aspartyl is an ionic bidentate ligand (Sutton , RB et al., 1995 Cell 80, 929-938) (Rizo, J. et al., 1998 J. Biol. Chem. 273, 15879-15882).
  • C2 domain proteins react with phospholipids and proteins, and they fold inside It is used in the second messenger of lipids involved in transduction pathway or cell membrane transport.
  • the inventor's polypeptide has 48% identity and 74% homology with the TOF protein at the protein level, and has a similarity
  • the similar structure belongs to the 0TOF protein family, so it is named human deafness-related gene 14 and it is speculated that it has similar biological functions.
  • the human deafness-related gene 14 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. Therefore, there has been a need in the art to identify more involved in these processes.
  • Human deafness-related gene 14 protein especially the amino acid sequence of this protein. Isolation of the deafness-related gene 14 protein encoding genes in newcomers 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 developing diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is important.
  • 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 deafness-related gene 14.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human deafness-related gene 14.
  • Another object of the present invention is to provide a method for producing a deafness-related gene 14 in humans.
  • Another object of the present invention is to provide an antibody against the deafness-associated gene 14 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the deafness-related genes 14 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with a deafness-associated gene 14 in a human.
  • 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) having SEQ ID NO: 1 A sequence of positions 286 to 1272; and (b) a sequence of positions 1 to 1396 in SEQ ID NO: 1.
  • the present invention further relates to a vector containing the polynucleotide of the present invention, particularly an expression vector; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; The method for preparing a polypeptide of the present invention by describing a host cell and recovering an expressed product is described.
  • 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 deafness-related gene 14 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 susceptibility to a disease associated with abnormal expression of a human deafness-related gene 14 protein, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting a biological sample.
  • the amount or biological activity of a polypeptide of the invention comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting a biological sample.
  • 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 for the preparation of a medicament for treating cancer, developmental disease or immune disease, or other diseases caused by abnormal expression of human deafness-related genes 14.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes.
  • the amino acids that are replaced have structural or chemical properties similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • “Insertion” or “addition” refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • “Replacement” refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • 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 a deafness-associated gene 14, causes the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds to human deafness-related genes 14.
  • Antagonist refers to a molecule that, when combined with a human deafness-related gene 14, can block or regulate the biological or immunological activity of the human deafness-related gene 14.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human deafness-related genes 14.
  • Regular refers to a change in the function of human deafness-related gene 14, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of human deafness-related gene 14.
  • substantially pure ' means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human deafness-related genes 14 using standard protein purification techniques. Basically pure The human deafness-related gene 14 can generate a single main band on a non-reducing polyacrylamide gel. The purity of the human deafness-related gene 14 polypeptide can be analyzed 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 (Sout hern print or Nor t hern blot, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, such as through the MEGAL I GN 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). The Cluster 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 such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence B
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or by methods known in the art such as Jotun Hein. J., (1990) Methods in emzuraology 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.
  • 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 human deafness-related gene 14.
  • 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 a component 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 deafness-related gene 14 means that the human deafness-related gene 14 is substantially free of other proteins, lipids, sugars, or other substances naturally associated with it. Those skilled in the art can purify human deafness-related genes using standard protein purification techniques 14. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human deafness related gene 14 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a human deafness-related gene 14, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells).
  • the polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptide of the present invention may also include or exclude the initial methionine residue, and the present invention also includes fragments, derivatives and analogs of the human deafness-related gene 14.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human deafness-related gene 14 of the present invention.
  • Fragments, derivatives of the polypeptide of the present invention may be: U) a type 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 substituted 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 with another group to include a substituent; or (III) such a type in which the mature polypeptide and another A compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence (such as a leader sequence or secretory sequence or To purify the sequence of this polypeptide or protease sequence) as described herein, such fragments, derivatives Analogs are deemed to be within the knowledge of a skilled artisan.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1 396 bases in length and its open reading frame (286-1272) encodes 328 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 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 means 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 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 0.2 xSSC, 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 in two sequences Crosses occur only when the identity between them is at least 95%, and more preferably 97%.
  • 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, 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 human deafness-related genes.
  • 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 human deafness-related gene 14 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) expression The antibodies of the library are screened to detect cloned polynucleotide fragments having common structural characteristics.
  • 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 the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as 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 hybrids; (2) the presence or absence of marker gene functions; (3) determining the level of transcripts of human deafness-related genes 14; (4) Detection of gene-expressed protein products by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • 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 gene itself or the fragment of the present invention can of course be used as a probe.
  • the labeling of a DNA probe can be a radioisotope, luciferin, or an enzyme (such as alkaline phosphatase).
  • the protein products of the human deafness-related gene 14 gene 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 using PCR to amplify DNA / RNA 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 may 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 and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated.
  • 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 deafness-related gene 14 coding sequence, and a method for producing the polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding the human deafness-related gene 14 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 well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • DNA sequences encoding human deafness-related genes 14 and appropriate transcriptional / translational regulatory elements can be used to construct expression vectors containing DNA sequences encoding human deafness-related genes 14 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac 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, polytumor 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. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc.
  • 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 etc.
  • a polynucleotide encoding a human deafness-related gene 14 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 Sf9
  • 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 absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, 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 human deafness-related genes 14 (Science, 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). The cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide can be coated in the cell, or expressed on the cell membrane, or secreted outside the cell. If necessary, it can be separated and purified by various separation methods using its physical, chemical and other properties.
  • Recombinant protein 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
  • FIG. 1 is a comparison diagram of the amino acid sequence homology of the deafness-related gene 14 and the TOF protein of the present invention.
  • the upper sequence is the human deafness-related gene 14, and the lower sequence is the TOF protein.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human deafness-related gene 14.
  • 14kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band. The best way to implement the invention
  • RNA from human fetal brain was extracted by guanidine isothiocyanate / phenol / chloroform method.
  • Poly (A) mRNA was isolated from total RNA using Quik raRNA Isolation Kit (Qiegene). 2ug poly (A) raRNA is reverse transcribed to form cDNA.
  • the Smart cDNA 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, and the bacteria formed a cDNA library .
  • Dye terminate cycle react ion Sequencing kit Perkin-Eimer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the Blast program (Basiclocal Alignment search tool) of the human deafness-related gene 14 of the present invention and its encoded protein sequence [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], Homology search in databases such as Genbank and Swissport.
  • the gene with the highest homology to the human deafness-related gene 14 of the present invention is a known OTOF protein, and its accession number encoded in Genbank is AF107403.
  • the protein homology results are shown in Figure 1. The two are highly homologous, with an identity of 48% and a similarity of 743 ⁇ 4.
  • Example 3 Cloning of a gene encoding human deafness-related gene 14 by RT-PCR
  • RNA of fetal brain cells as a template and oligo-dT as a primer for reverse transcription to synthesize cDNA.
  • PCR amplification was performed with the following primers:
  • Primer2 5'- GGCTGTGATAGGTTTATTCAGAGG -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.
  • Conditions for the amplification reaction 50 ⁇ l of reaction volume containing 50 ⁇ l / L KC1, 10 mmol / L Tris-II, ( ⁇ 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ / L dNTP, lOpmoi primer , 1U of Taq DNA polymerase (Clomech).
  • 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.
  • RT-PCR set ⁇ -act in as a positive control and template blank as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit. DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1396bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human deafness-related gene 14 gene expression:
  • RNA extraction using a one-step method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. 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.
  • RNA was synthesized by electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the PCR-encoded human deafness-related gene 14 coding region sequence (286b P to 1272bp) shown in FIG. 1.
  • a 32P-labeled probe (about 2 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) -5 ⁇ SSC-5 ⁇ Denhardt's solution and 20 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 ⁇ SSC-0.1% SDS at 55 ° C for 30 minutes. Then, Phosphor Imager analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human deafness-related gene 14
  • Primer3 5'- CCCCATATGATGTGGATTGACATCTTTCCTCAAG _3, (Seq ID No: 5)
  • Primer4 5'- CCCGAATTCTCACTTGTGGAGGGGACGGAAGATG -3, (Seq ID No: 6)
  • Ndel and EcoRI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid P ET-28b (+) (Novagen, Cat. No. 69865.3).
  • the PCR reaction was performed using the pBS-0969a06 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 pg of P BS-0969aO6 plasmid, primers Primer-3 and Primer-4, and another 1 J is lOpmol, Advantage polymerase Mix
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total Ig G was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human deafness-related gene 14.
  • Example 7 Use of a 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 various aspects.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to ⁇ Determine whether it contains the 'polynucleotide sequence of the present invention and detect a homologous polynucleotide sequence, and further The probe is used to detect whether the expression of the polynucleotide sequence of the present invention or a homologous polynucleotide sequence thereof in cells of normal tissues or pathological tissues 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 imprinting, Nor thern blotting, and photocopying methods. They are all based on the use of substantially the same step of hybridization after the polynucleotide sample to be tested is fixed on the filter.
  • 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) 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 for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the probe size preferably ranges from 18 to 50 nucleotides
  • the total amount of GC 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 (probe l), 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 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: 5 '-CCTCAAATGCTGCCTGCTAGCTTCCCAGTAACTGTCAAGCC-3'
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotides of the present invention in different tissues. Practicality
  • 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.
  • 0TOF protein is mainly expressed in snail I HC and vestibular type I sensitive hair cells, and is indispensable to neurons. 0TOF protein may also be more commonly expressed in the vestibular cell membrane transport process and in the brain. Therefore, abnormal expression of 0T0F protein will cause a series of deafness and neurological diseases.
  • polypeptide o tofer l in 14 its abnormal expression will cause but is not limited to the following diseases: hearing loss, speech loss, neural tube insufficiency such as spina bifida, anencephaly, brain (meninges) ) Swelling, craniocerebral fissures, neural tube cysts, brain developmental abnormalities such as foramen malformations, forebrain, hydrocephalus, neuron migration disorders such as abnormal brain gyrus formation, aqueduct malformations, cerebellar hypoplasia, Down syndrome Syndrome, spinal deformity, congenital hydrocephalus, congenital cerebellar dysplasia syndrome, Alzheimer's disease, Parkinson's disease, multiple sclerosis, chorea, depression, amnesia, Huntington's disease, Epilepsy, migraine, dementia, myasthenia gravis, spinal muscular atrophy, muscular pseudohypertrophy, Duchenne muscular dystrophy, tonic muscular dystrophy, myasthenia
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human deafness related genes 14.
  • Agonists enhance human deafness-related genes 14 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing human deafness-related gene 14 can be cultured with the labeled human deafness-related gene 14 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human deafness-related genes 14 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human deafness-related gene 14 can bind to human deafness-related gene 14 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human deafness-related gene 14 When screening compounds as antagonists, human deafness-related gene 14 can be added to the bioanalytical assay. The effect of the compound on the interaction between human deafness-related gene 14 and its receptor is used to determine whether the 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 deafness-related genes 14 can be obtained by screening a random peptide library composed of a possible combination of amino acids bound to a solid phase. During screening, 14 molecules of human deafness-related genes 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 present invention also provides antibodies against human deafness-related genes 14 epitopes. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human deafness-related genes 14 directly into immunized animals (such as rabbits, mice, rats, etc.). Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. . Techniques for preparing monoclonal antibodies to human deafness-related gene 14 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybrid Cross tumor technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human-derived variable regions can be produced using existing techniques (Morri et al, 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 deafness-related gene 14.
  • Antibodies against human deafness-related gene 14 can be used in immunohistochemical techniques to detect human deafness-related gene 14 in biopsy specimens.
  • Monoclonal antibodies that bind to human deafness-related genes 14 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 deafness related genes 14 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 crosslinker such as SPDP, and toxins are bound to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human deafness-related gene 14 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases associated with human deafness-related genes 14. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human deafness-related genes 14.
  • the present invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human deafness-related gene 14.
  • diagnostic tests are well known in the art and include F I SH assays and radioimmunoassays.
  • the level of human deafness-related gene 14 detected in the test can be used to explain the importance of human deafness-related gene 14 in various diseases and to diagnose diseases in which human deafness-related gene 14 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, preferably mass spectrometry analysis.
  • the polynucleotide encoding human deafness-related gene 14 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell proliferation, development, or metabolism caused by the lack of expression or abnormal / inactive expression of human deafness-related gene 14.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human deafness-related genes 14 to inhibit endogenous human deafness-related genes 14 activity.
  • a mutated human deafness-related gene 14 may be a shortened human deafness-related gene 14 lacking a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of deafness-related gene 14.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human deafness-related gene 14 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human deafness-related gene 14 can be found in existing literature (Sambrook, et ai.).
  • a recombinant polynucleotide encoding human deafness-related gene 14 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 deafness-related genes 14 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 the solid-phase phosphoramidite chemical synthesis method for oligonucleotide synthesis.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human deafness-related gene 14 can be used for diagnosis of diseases related to human deafness-related gene 14.
  • the polynucleotide encoding human deafness-related gene 14 can be used to detect the expression of human deafness-related gene 14 or the abnormal expression of human deafness-related gene 14 in a disease state.
  • the DNA sequence encoding human deafness-related gene 14 can be used to hybridize biopsy specimens to determine the expression status of human deafness-related gene 14.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human deafness-related gene 14 specific primers can also be used to detect the transcription products of human deafness-related gene 14 by in vitro amplification of RNA-polymerase chain reaction (RT-PCR).
  • Detection of mutations in the human deafness-related gene 14 gene can also be used to diagnose human deafness-related gene 14 disease.
  • Human deafness-related gene 14 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human deafness-related gene 14 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 the expression of proteins, so Northern blotting and Western blotting can be used to indirectly determine the presence or absence of mutations in a gene.
  • 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 few chromosome markers based on actual sequence data (repeat polymorphisms) 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 hybrid pre-selection to construct chromosome-specific cDNA libraries,
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in a single 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, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). 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 diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing diseased and unaffected individuals usually involves first looking for structural changes in chromosomes, such as defects or bits 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 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 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 authorize them to be administered to humans by government agencies that manufacture, use, or sell them.
  • the polypeptides of the invention can be combined with other Of therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • the human deafness-related gene 14 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human deafness-related genes 14 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.
  • Trp lie Asp lie Phe Pro Gin Asp Val Pro Ala Pro Pro Pro

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Abstract

L'invention concerne un nouveau polypeptide, un gène humain 14 lié à la surdité, 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, 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 le gène humain 14 lié à la surdité.
PCT/CN2000/000449 1999-11-23 2000-11-20 Nouveau polypeptide, gene humain 14 lie a la surdite, et polynucleotide codant pour ce polypeptide Ceased WO2001038523A1 (fr)

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WO2017057308A1 (fr) * 2015-09-28 2017-04-06 国立大学法人岡山大学 Anticorps pour reconnaître et fixer spécifiquement la protéine reic/dkk-3 à structure active, et suivi de traitement du cancer dans lequel ledit anticorps anti-reic/dkk-3 est utilisé

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US8212017B2 (en) 2001-04-10 2012-07-03 Agensys, Inc. Nucleic acids and corresponding proteins entitled 158P3D2 useful in treatment and detection of cancer
EP1383922A4 (fr) * 2001-04-10 2005-03-30 Agensys Inc Acide nucleique et proteine correspondante intitule 158p3d2 utiles dans le traitement et la detection du cancer
US8524872B2 (en) 2001-04-10 2013-09-03 Agensys, Inc. Nucleic acids and corresponding proteins entitled 158P3D2 useful in treatment and detection of cancer
WO2002083928A2 (fr) 2001-04-10 2002-10-24 Agensys, Inc. Acide nucleique et proteine correspondante intitule 158p3d2 utiles dans le traitement et la detection du cancer
US7811575B2 (en) 2001-04-10 2010-10-12 Agensys, Inc. Nucleic acids and corresponding proteins entitled 158P3D2 useful in treatment and detection of cancer
US8039000B2 (en) 2001-04-10 2011-10-18 Agensys, Inc. Nucleic acid and corresponding protein entitled 158P3D2 useful in treatment and detection of cancer
EP2508606A1 (fr) 2006-10-24 2012-10-10 Huh, Nam-ho Fragment partiel du gene REIC/Dkk-3 et agent thérapeutique pour le cancer contenant celui-ci
WO2008050898A1 (fr) 2006-10-24 2008-05-02 Univ Okayama Nat Univ Corp FRAGMENT PARTIEL DU GÈNE REIC/Dkk-3 ET AGENT THÉRAPEUTIQUE POUR LE CANCER CONTENANT CELUI-CI
US8618273B2 (en) 2006-10-24 2013-12-31 Hiromi Kumon Partial fragment of REIC/Dkk-3 gene and cancer therapeutic agent comprising the same
US9475865B2 (en) 2006-10-24 2016-10-25 Hiromi Kumon Direct administration of REIC/Dkk-3 to mesothelioma
WO2009060982A1 (fr) 2007-11-05 2009-05-14 Momotaro-Gene Inc. Inducteur de mort de cellules cancéreuses ayant une activité de potentialisation d'agent anti-cancéreux dans un cancer présentant une résistance contre l'agent anti-cancéreux
US8946173B2 (en) 2007-11-05 2015-02-03 Momotaro-Gene Inc. Cancer cell death inducing agent having effects of potentiating anticancer drug against anticancer-drug-resistant cancer
US8658612B2 (en) 2008-07-30 2014-02-25 National University Corporation Okayama University Therapeutic agent for malignant mesothelioma and immunostimulant
WO2012002582A1 (fr) 2010-07-01 2012-01-05 国立大学法人岡山大学 Polypeptide de région partielle de la protéine reic/dkk-3
WO2012161352A1 (fr) 2011-05-25 2012-11-29 国立大学法人岡山大学 Vecteur adénoviral exprimant reic
KR20170012397A (ko) 2014-05-28 2017-02-02 고꾸리츠 다이가꾸 호우징 오까야마 다이가꾸 Reic 유전자를 발현하는 제한 증식형 아데노바이러스
US10071126B2 (en) 2014-05-28 2018-09-11 National University Corporation Okayama University Conditionally replicating adenovirus to express REIC gene
WO2017057308A1 (fr) * 2015-09-28 2017-04-06 国立大学法人岡山大学 Anticorps pour reconnaître et fixer spécifiquement la protéine reic/dkk-3 à structure active, et suivi de traitement du cancer dans lequel ledit anticorps anti-reic/dkk-3 est utilisé
JPWO2017057308A1 (ja) * 2015-09-28 2018-07-19 国立大学法人 岡山大学 活性構造のＲＥＩＣ／Ｄｋｋ−３タンパク質を特異的に認識して結合する抗体、及び該抗ＲＥＩＣ／Ｄｋｋ−３抗体を用いた癌治療のモニタリング

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