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WO2001066701A1 - Nouveau polypeptide, nadh-deshydrogenase humaine 13, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, nadh-deshydrogenase humaine 13, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001066701A1
WO2001066701A1 PCT/CN2001/000154 CN0100154W WO0166701A1 WO 2001066701 A1 WO2001066701 A1 WO 2001066701A1 CN 0100154 W CN0100154 W CN 0100154W WO 0166701 A1 WO0166701 A1 WO 0166701A1
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Prior art keywords
polypeptide
seq
sequence
polynucleotide
nadh dehydrogenase
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PCT/CN2001/000154
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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 AU42219/01A priority Critical patent/AU4221901A/en
Publication of WO2001066701A1 publication Critical patent/WO2001066701A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • a new polypeptide one human NADH dehydrogenase 13 and a polynucleotide encoding the polypeptide TECHNICAL FIELD
  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide—human NADH dehydrogenase 13, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide. Background technique
  • the respiratory chain NADH dehydrogenase (also known as coenzyme Q oxidoreductase) is a complex of oligomeric enzymes located on the inner surface of the mitochondrial membrane. It is also found in chloroplasts and cyanobacteria. The entire NADH dehydrogenase complex includes 25-30 peptide subunits, and electrons from NADH pass through this complex to coenzyme Q.
  • This 75Kd subunit has three highly conserved regions, which are related to the binding of ferritin: (1) P— X (2) — C— [YWS] —X (7) _G— X— C— R— X— C; (2) C—P— X— C— [DE] — X— [GS] (2) —X— C—X— L—Q; (3) R— C— [LI VM ] _X_C— X— R— C— [L IVM]-X— [FY]. Each of them has three Cys residues, and the sulfhydryl group on it can bind to the protein containing iron and sulfur atoms.
  • the electrons are initially accepted by the FMN prosthetic group on NADH dehydrogenase, and then passed to the coenzyme Q through the iron-sulfur complex. It exists in an oxidized state (Fe3 +) or a reduced state (Fe2 +) and is actually an acceptor or donor of electrons.
  • NADH dehydrogenase complex In organisms, respiratory metabolism is an important metabolic process, which ultimately generates ATP through electron transfer and oxidative phosphorylation to meet the cell's energy needs.
  • the first step in electron transfer is through the NADH dehydrogenase complex.
  • certain toxins can cause defects in NADH dehydrogenase, leading to the development of some brain diseases. More common diseases such as Parkinson syndrome. Some people develop this symptom after the age of 40. It is due to the weakened function of the NADH dehydrogenase complex in the mitochondria; in addition, low NADH dehydrogenase activity can cause Huntington's disease.
  • the human MDH dehydrogenase 1 3 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 there has been a need in the art to identify more involved in these The process of human NADH dehydrogenase 1 3 protein, especially the amino acid sequence of this protein is identified. Isolation of the new NADH dehydrogenase 1 3 protein encoding gene also provides a basis for research to determine the role of the protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the 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 human NADH dehydrogenase 1 3.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human NADH dehydrogenase 1 3.
  • Another object of the present invention is to provide a method for producing human NADH dehydrogenase 1 3.
  • Another object of the present invention is to provide an antibody against the human NADH dehydrogenase 1 3 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of NADH dehydrogenase 1 3 to the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human NADH dehydrogenase 1 3.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from: (a) a sequence having positions 439-804 in SEQ ID NO: 1; and (b) a sequence having 1-241 in SEQ ID NO: 1 3-bit sequence.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human NADH dehydrogenase 13 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of human NADH dehydrogenase 13 protein in vitro, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in 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 in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human NADH dehydrogenase 13.
  • 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- or double-stranded and represent 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 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 may have "conservative" changes in which the substituted amino acid has a structural or chemical property 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 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 human NADH dehydrogenase 1 3, causes a change in the protein and thereby regulates the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human NADH dehydrogenase 1 3.
  • Antagonist refers to a molecular antagonist and inhibitor that can block or regulate the biological or immunological activity of human NADH dehydrogenase 13 when combined with human NADH dehydrogenase] 3
  • the substance may include a protein, a nucleic acid, a carbohydrate or any other molecule that can bind human NADH dehydrogenase 13.
  • Regular refers to a change in the function of human NADH dehydrogenase 13, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human NADH dehydrogenase 13 change.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated.
  • Those skilled in the art can purify human NADH dehydrogenase 1 3 using standard protein purification techniques. Essentially pure human NADH dehydrogenase 1 3 produces a single main band on a non-reducing polyacrylamide gel. The purity of human NADH dehydrogenase 1 3 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” can be combined with the 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. Inhibition of such hybridization can be detected by performing hybridization (Sou thern blot or Nort he rn blot, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to 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 percentage 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).
  • the 0 C 1 us ter method will check the distance between all pairs by Groups of sequences are arranged in clusters. The clusters are then assigned in pairs or groups.
  • sequence A and sequence B 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 Number of interval residues in a sequence B
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art, such as Jotun He in (Hein J., (1990) Methods in emzumology 183: 625-645) c. "Similarity” refers to amino acids The degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment between sequences.
  • Amino acids used for conservative substitutions 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, F (ab ') 2 and Fv, which can specifically bind to the epitope of human NADH dehydrogenase 13.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not Components of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state.
  • isolated human NADH dehydrogenase 1 3 means that human NADH dehydrogenase 1 3 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 NADH anhydrogenase 1 3 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human NADH dehydrogenase 1 3 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human NADH dehydrogenase 13, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human NADH dehydrogenase 13.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human NADH dehydrogenase 1 3 of the present invention.
  • the present invention provides an isolated nucleic acid (polynucleoside hinge), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • Polynucleotides of the invention are found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 2413 bases in total length and its open reading frame 4 39-804 encodes 121 amino acids. Based acid.
  • this peptide has a similar expression profile to human NADH dehydrogenase 12, and it can be deduced that the human NADH dehydrogenase 13 has similar functions to human NADH dehydrogenase 12.
  • 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 in the present invention, but which differs 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); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6trC; or (2) added during hybridization Use a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95% Above, and preferably above 97 »/ «, crosses only occur.
  • 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 cores. 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 NADH dehydrogenase 13.
  • 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 NADH dehydrogenase 13 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 separating cDNAs 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. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene).
  • 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 hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of human NADH dehydrogenase 13 transcripts; (4) ) Detection of protein products expressed by genes through 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 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).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the human NADH dehydrogenase 13 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method using DNA technology to amplify DNAZRNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE_cDNA terminal rapid amplification method
  • the primers used for PCR may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • Polynucleotide sequences of the gene of the present invention obtained as described above, or various DNA fragments can be used It is determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence. ,
  • the present invention also relates to a vector comprising a 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 NADH dehydrogenase 13 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .
  • a polynucleotide sequence encoding human NADH dehydrogenase 13 ' may 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.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. 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 for eukaryotic cell culture. And green fluorescent protein (GFP)., Or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture. And green fluorescent protein (GFP)., Or tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human NADH dehydrogenase. 13 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or a recombinant carrier.
  • 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 insect 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 CaCl ⁇ .
  • the steps used are well known in the art.
  • MgCl 2 is used.
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human NADH dehydrogenase 13 by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally there are the following steps:
  • step (3) Isolate and purify protein from culture medium or cells.
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • 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, 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 chromat
  • FIG. 1 is a comparison diagram of gene expression profiles of human NADH dehydrogenase 13 and human NADH dehydrogenase 12 in the present invention.
  • the above figure is a graph of the expression profile of human NADH dehydrogenase 13. Expression spectrum chart.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human NADH dehydrogenase 13 isolated.
  • lOkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Use Smart cDNA Cloning Kit (purchased from Clontech). The 0 fragment was inserted into the multicloning site of pBSK (+) vector (Cloiuech) and transformed into DH5 ⁇ to form a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 An automatic sequencer (Perki nElmer) determined the 5 'and 3' end sequences of all clones. The determined c DN A sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 029 Oe 07 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, PCR was performed using the following primers: Primerl: 5-GCAGGAAGCGTGTCCGTGCTTAGG -3 "(SEQ ID NO: 3)
  • Primer2 5-TTAAACAAAACACATTTATTTCTC —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 ⁇ l / L KC1, 10mmol / L Tris in a reaction volume of 50 ⁇ 1-
  • This method involves acid guanidinium thiocyanate phenol-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 probes were the PCR-encoded human NADH dehydrogenase 13 coding region sequence ( 4 39 bp to 804 bp) shown in FIG.
  • the 32P- labeled probe (approximately 2 X 10 6 cpm / ml) and transferred to a nitrocellulose membrane RNA is hybridized overnight at 42 ° C in a solution, the solution contains 50% amide Yue -25mM ⁇ 2 ⁇ 0, ( ⁇ 7.4) -5 x SSC-5 x Denhardt's solution and 20Q pg / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human NADH dehydrogenase 13
  • Primer3 5'- CATGCTAGCATGACATCCACGTGTTCCTGTCGG-3 '(Seq ID No: 5)
  • Primer4 5'- CATGGATCCTCACTGCTCCAAGTGCTCCACAGG- 3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the 5 'end of the target gene And 3 'coding sequences, the Nde I and BamH I restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28 b (+) (Novagen product, Cat. No. 69865.3).
  • the PCR reaction was performed using pBS-0290e07 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 pBS-0290e07 plasmid, primer Primer-3 and Primer- 4 points, and 1 J being lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60. C 30s, 68 ° C 2 min, a total of 25 cycles. Nde I and BamH I were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E. coli DH5a by the calcium chloride method.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation.
  • the purified protein NADH dehydrogenase ⁇ was purified. After SDS-PAGE electrophoresis, a single band was obtained at 10 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2.
  • 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. Immunochemi stry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polymorphic 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. 15A titer plate coated with 15 g / ml bovine serum albumin polypeptide complex was used as an ELISA to determine the antibody titer in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Seph arOS e4B 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 NADH dehydrogenase 13.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • the 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 the genome or CDM library of normal tissue or pathological tissue from different sources to Identify whether it contains a polynucleotide sequence of the present invention and detect a homologous polynucleotide sequence; further use the probe to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or no.
  • 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. Or a homologous polynucleotide sequence.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods, etc. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • 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 membrane washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and only Retain 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 SEQ ID NO:] identical or complementary oligonucleotide fragments.
  • 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 preferred range of probe size is 18-50 nucleotides
  • GC content is 30% -70 ° /. If it exceeds, non-specific hybridization increases;
  • Those that meet the above conditions can be used as primary selection probes. Then further computer sequence analysis is performed, including the primary selection probes and their source sequence regions (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 identical to: 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 of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41 Nt): 5'-TGACATCCACGTGTTCCTGTCGGACGCAGCGGCTTCGGGAA-3 '(SEQ ID NO: 9)
  • SEQ ID NO: 9 For other commonly used reagents and their preparation methods related to the following specific experimental procedures, please refer to the literature: DNA PROBES GH Keller; MM Manak; Stockton Press, 1989 ( USA) and more commonly used manuals of molecular cloning experiments such as "Molecular Cloning Experiment Guide” (Second Edition 1998) [US] Sambrook et al., Science Press.
  • 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
  • the sample membrane was placed in a plastic bag, and 3- 10 mg of prehybridization solution (10xDenhardfs; 6xSSC, 0.1 mg / ml) was added. CT ON Calf thymus DJ). ), After sealing the bag, shake at 68 ° C for 2 hours.
  • Gene microarray or gene microarray is a new technology 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 D for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and a Cartesian 7500 spotter (purchased from Cartesian, USA) was used to spot the glass medium. The distance is 280 ⁇ ⁇ 1 . 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:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP 5-Amino-propargyl-2'-deoxyuridine 5'_triphate coupled to Cy3 fluorescent dye (purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissues.
  • the fluorescent reagent Cy5dUTP (5_Amino-propargyl_2'-deoxyuridine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech, was used to label the mRNA of specific tissues (or stimulated cell lines) in the body, and probes were prepared after purification. For specific steps and methods, see:
  • Probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000.
  • Scanner purchased from General Scanning Company, USA
  • Imagene software Biodiscovery, USA
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Based on these 13 Cy 3 / Cy5 ratios, a bar graph is drawn. (figure 1 ) . It can be seen from the figure that the expression profiles of human NADH dehydrogenase 13 and human NADH dehydrogenase 12 according to the present invention are very similar. Industrial applicability
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases-for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections, and immune diseases.
  • the respiratory chain NADH dehydrogenase is an oligomeric enzyme complex.
  • the 75Kd peptide of the enzyme complex is the largest subunit.
  • respiratory metabolism is an important metabolic process, which ultimately generates ATP through electron transfer and oxidative phosphorylation to meet the cell's energy needs.
  • the first step in electron transfer is through the NADH dehydrogenase complex. To be done.
  • NAM dehydrogenase can cause defects in NAM dehydrogenase, leading to the development of some brain diseases. More common diseases such as Parkinson syndrome, and some people have this after the age of 40. Symptoms are due to the weakened function of the NADH dehydrogenase complex in mitochondria; in addition, low NADH dehydrogenase activity can cause Huntington's disease.
  • the abnormal expression of the specific NADH dehydrogenase 75Kd subunit motif will cause the function of the polypeptide containing the motif of the present invention to be abnormal, which will cause the abnormal function of the respiratory chain, affect the metabolism of substances and energy, and generate correlations.
  • Diseases such as Parkinson's syndrome, Huntington's disease, physical and energy metabolism disorders, growth and development disorders, tumors, etc.
  • the abnormal expression of the human NADH dehydrogenase 13 of the present invention will produce various diseases, especially Parkinson's syndrome, Henry's disease, physical and energy metabolic disorders, growth and development disorders, tumors, and these diseases. including but not limited to:
  • disorders related to energy and substance metabolism disorders isovaleric acidemia, propionic acidemia, methylmalonic aciduria, combined carboxylase deficiency, glutaric acid type I, phenylketonuria, albinism, color Aminoemia, Glycineemia, Hypersarcosineemia, Metabolism of glutamate, Metabolism of urea cycle, Metabolism of histidine, Metabolism of lysine, Mucopolysaccharidosis Types I to VII , Mucolipidosis, Ray-niney syndrome, xanthineuria, orotic aciduria, adenine deaminase deficiency, hyperlipoproteinemia, familial hyperalpha-lipoproteinemia, Congenital lactose intolerance, hereditary fructose intolerance, galactosemia Disease, deficiency of fructose metabolism, glycogen storage disease
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus dysplasia syndrome, strabismus, skin, fat and muscular dysplasia such as congenital skin laxity, premature aging Disease, congenital keratosis, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism, sexual retardation
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, Colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymic tumor, nasal cavity and sinus tumor, nose Pharyngeal cancer, Laryngeal cancer, Tracheal tumor, Fibroma, Fibrosarcoma, Lipoma, Liposarcoma, Leiomyoma
  • Abnormal expression of the human NADH dehydrogenase 1 of the present invention will also produce certain hereditary, hematological and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human NADH dehydrogenase 13.
  • Agonists enhance human NADH dehydrogenase 13 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 membrane preparations expressing human NADH dehydrogenase 1 3 can be cultured with labeled human NADH dehydrogenase 1 3 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human NADH dehydrogenase 1 3 include selected antibodies, compounds, receptor deletions, and the like. Antagonists of human NADH dehydrogenase 1 3 can bind to human NADH dehydrogenase 13 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 exert its biology Features.
  • human NADH dehydrogenase 1 3 When screening compounds as antagonists, human NADH dehydrogenase 1 3 can be added to a bioanalytical assay 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 NADH dehydrogenase 1 3 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human NADH dehydrogenase 13 molecules 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 against human NADH dehydrogenase 1 3 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries. It can be obtained by NADH enzyme 13 (, mouse, rat, etc.).
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human NADH dehydrogenase 13 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma technology, EBV -Hybridoma technology, etc. Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). 0 Existing techniques for producing single-chain antibodies (US Pat No. .4946778) can also be used to produce single chain antibodies against human NADH dehydrogenase 13.
  • Antibodies against human NADH dehydrogenase 13 can be used in immunohistochemistry to detect human NADH dehydrogenase 13 in biopsy specimens.
  • Monoclonal antibodies that bind to human NADH dehydrogenase 13 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 NADH dehydrogenase 13 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 the antibody with a thiol crosslinker such as SPDP, and toxins are bound to the antibody by disulfide exchange.
  • This hybrid antibody can be used to kill human NADH dehydrogenase 13 positive cells .
  • the antibodies of the present invention can be used to treat or prevent diseases related to human NADH dehydrogenase 13. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human NADH dehydrogenase 13.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human NADH dehydrogenase 13 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the levels of human NADH dehydrogenase 13 detected in the test can be used to explain the importance of human NADH dehydrogenase 13 in various diseases and to diagnose diseases in which human NADH dehydrogenase 13 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 analysis.
  • the polynucleotide encoding human NADH dehydrogenase 13 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 NADH dehydrogenase 13.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human NADH dehydrogenase 13 to inhibit endogenous human NADH dehydrogenase 13 activity.
  • a mutated human NADH dehydrogenase 13 may be a shortened human NADH dehydrogenase 13, although it can bind to downstream substrates, it lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human NADH dehydrogenase 13.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human NADH dehydrogenase 13 into a cell.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human NADH dehydrogenase 13 can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human NADH dehydrogenase 13 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 NADH dehydrogenase 13 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 for endonucleation.
  • Antisense A, DNA and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis technology for oligonucleotide synthesis has been widely used.
  • 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. 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 phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human NADH dehydrogenase 13 can be used for the diagnosis of diseases related to human NADH dehydrogenase 13.
  • a polynucleotide encoding human NADH dehydrogenase 13 can be used to detect the expression of human NADH dehydrogenase 13 or the abnormal expression of human NADH dehydrogenase 13 in a disease state.
  • the DNA sequence encoding human NADH dehydrogenase 13 can be used to hybridize biopsy specimens to determine the expression of human NADH dehydrogenase 13.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available mature technologies, 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 (Microa ay) or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human NADH dehydrogenase 13 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect human NADH dehydrogenase 13 transcription products.
  • Detection of mutations in the human NADH dehydrogenase 13 gene can also be used to diagnose human NADH dehydrogenase 13-related diseases.
  • Human NADH dehydrogenase 13 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human NADH dehydrogenase 13 DNA sequence.
  • Available technologies such as Southern blot, DNA sequence analysis, PCR and in situ hybridization were used to detect mutations.
  • mutations may affect the expression of proteins. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, 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 affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the 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).
  • polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be combined with Use 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human NADH dehydrogenase 1 3 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of NADH dehydrogenase 1 3 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 NADH-déshydrogénase humaine 13, 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 la nouvelle NADH-déshydrogénase humaine 13.
PCT/CN2001/000154 2000-03-07 2001-02-26 Nouveau polypeptide, nadh-deshydrogenase humaine 13, et polynucleotide codant pour ce polypeptide WO2001066701A1 (fr)

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CN00111932A CN1312376A (zh) 2000-03-07 2000-03-07 一种新的多肽——人nadh脱氢酶13和编码这种多肽的多核苷酸

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998031815A2 (fr) * 1997-01-17 1998-07-23 Incyte Pharmaceuticals, Inc. Sous-unites de nadh deshydrogenase
US5925543A (en) * 1997-09-12 1999-07-20 Incyte Pharmaceuticals, Inc. Isolated polynucleotide sequence encoding NADH dehydrogenase B17 subunit
US5976804A (en) * 1997-12-12 1999-11-02 Incyte Pharmaceuticals, Inc. NADH dehydrogenase PDSW subunit

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998031815A2 (fr) * 1997-01-17 1998-07-23 Incyte Pharmaceuticals, Inc. Sous-unites de nadh deshydrogenase
US5925543A (en) * 1997-09-12 1999-07-20 Incyte Pharmaceuticals, Inc. Isolated polynucleotide sequence encoding NADH dehydrogenase B17 subunit
US5976804A (en) * 1997-12-12 1999-11-02 Incyte Pharmaceuticals, Inc. NADH dehydrogenase PDSW subunit

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 13 May 1999 (1999-05-13), ROWEN L. ET AL.: "Homo sapiens chromosome 14 clone BAC 316E14 map 14q24.3", Database accession no. AC006530 *
SULSTON J.E. AND WATERSTON R.: "Toward a complete human genome sequence", GENOME RES., vol. 8, no. 11, 1998, pages 1097 - 1108 *

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