WO2002020587A1 - Nouveau polypeptide, sous-unite humaine nadh deshydrogenase i-12.43, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, sous-unite humaine nadh deshydrogenase i-12.43, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002020587A1
WO2002020587A1 PCT/CN2001/001125 CN0101125W WO0220587A1 WO 2002020587 A1 WO2002020587 A1 WO 2002020587A1 CN 0101125 W CN0101125 W CN 0101125W WO 0220587 A1 WO0220587 A1 WO 0220587A1
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Prior art keywords
polypeptide
polynucleotide
human
sequence
nadh dehydrogenase
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PCT/CN2001/001125
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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 AU2002223371A priority Critical patent/AU2002223371A1/en
Publication of WO2002020587A1 publication Critical patent/WO2002020587A1/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)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide
  • MDH dehydrogenase subunits 1-12.43 MDH dehydrogenase subunits 1-12.43, and a polynucleotide sequence encoding this polypeptide.
  • the invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique
  • the respiratory chain MDH dehydrogenase (also known as complex I or NADH-coenzyme Q oxidoreductase) is a polymerase complex found in the inner mitochondrial inner membrane, chloroplast, and cyanobacteria. In cyanobacteria, it is also used as a NADH-plasmid quinone oxidoreductase.
  • the enzyme complex consists of 25-30 polypeptide chain subunits, of which 15 subunits are present on the membrane, and 7 of these 15 subunits are composed of mitochondrial and chloroplast genomes in many different species coding.
  • subunit I encoded by the ND1 gene in mitochondria and the NDH1 gene in chloroplasts
  • subunit II encoded by the ND1 gene in mitochondria and the NDH1 gene in chloroplasts
  • dehydrogenase 4 and subunit C of dehydrogenase 4 are highly similar.
  • Paracoccus denitrifying NQ0 8 protein and E. coli oH NADH coenzyme Q oxidoreductase subunits are also members of the MDH dehydrogenase subunit I family. Members of this protein family are involved in various respiratory chain reactions in the body. As intermediates for electron transfer, they are responsible for transferring electrons to oxygen, generating oxygen ions, and combining with 2H + to form H 2 0.
  • the respiratory chain is an important way of electron transfer and energy conversion in the body, and the role of various enzymes in the respiratory chain reaction process. These different enzymes coordinate their role in the respiratory chain process to facilitate the completion of respiratory metabolic processes. Mutation or abnormal expression of any of these substances will cause abnormal function of the respiratory chain. Therefore, the MDH dehydrogenase complex, as an important part of the respiratory chain, also plays an important regulatory role in this process. Abnormal expression of MDH dehydrogenase complex will lead to disturbances in the energy metabolism of the respiratory chain in the organism, thus causing various related metabolic disorders. .
  • the members of the enzyme subunit family also contain the two conserved sequences described above Fragment.
  • the two sequence fragments may be the active action centers of the enzyme complex subunit I. Mutations will cause abnormal expression of the subunit and cannot normally bind to coenzyme Q, thereby affecting its role in the respiratory chain.
  • the abnormal expression of members of this protein family is usually related to Chain-related developmental metabolic disorders, material metabolism disorders and other diseases are related.
  • the human NADH dehydrogenase subunit I-12.43 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. Identification of more human NADH dehydrogenase subunit I-12.43 proteins involved in these processes, and in particular the amino acid sequence of this protein. Isolation of the new human NADH dehydrogenase subunit I-12.43 protein encoding gene 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, so isolating its coding DNA is important. 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 MDH dehydrogenase subunit I-12.43.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human NADH dehydrogenase subunit I-12.43.
  • Another object of the present invention is to provide a method for producing human MDH dehydrogenase subunit I-12.43. Another object of the present invention is to provide antibodies against the polypeptide of the present invention-human MDH dehydrogenase subunit I-12.43.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the NADH dehydrogenase subunit I-12.43 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of human NADH dehydrogenase subunit I-12.43.
  • 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 The polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1735 to 2076 in SEQ ID NO: 1; and (b) a sequence having 1-2284 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human MDH dehydrogenase subunit 1-12.43 protein activity, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or disease susceptibility related to abnormal expression of human NADH dehydrogenase subunits 1-12.43 protein, comprising detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or 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 preparation of a polypeptide and / or polynucleotide of the present invention for the treatment of cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human MDH dehydrogenase subunit I-12.43. the use of.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic MA or RNA, which can be single-stranded or double-stranded, representing the sense strand or Antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants 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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human NADH dehydrogenase subunits 1-12.43, can cause 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 can bind to human NADH dehydrogenase subunit I-12.43.
  • Antagonist refers to a biological activity that can block or regulate human MDH dehydrogenase subunit I-12.43 when combined with human NADH dehydrogenase subunit 1-12.43. Or immunologically active molecules. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind human MDH dehydrogenase subunit I-12.43.
  • Regular refers to any change in the function of human NADH dehydrogenase subunit 1-12.43, including an increase or decrease in protein activity, a change in binding characteristics, and any of human MDH dehydrogenase subunit I-12.43. Changes in other biological, functional or immune properties.
  • 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 subunit I-12.43 using standard protein purification techniques.
  • Substantially pure human NADH dehydrogenase subunit I-12.43 produces a single main band on a non-reducing polyacrylamide gel.
  • Human NADH dehydrogenase subunit 1 -12. 43 The purity of the 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 A partially complementary sequence that at least partially inhibits the hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit 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 the same or similar in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, such as through the MEGALIGN program
  • the MEGA GN program can compare two or more sequences according to different methods such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Cluster method divides each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence 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 Clus ter method or using methods known in the art such as; Totun Hein to determine the percent identity between nucleic acid sequences (Hein J., (1990) Methods in emzumology 183: 625-645) 0 "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 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 DM 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 primary 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 human MDH dehydrogenase subunit I-12. 43 epitope.
  • 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 is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human NADH dehydrogenase subunit 1-12.43 means human NADH dehydrogenase subunit 1-12.43 is substantially free of other proteins, lipids, and sugars naturally associated with it. Or other substances. Those skilled in the art can purify human NADH dehydrogenase subunits 1-12.43 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human MDH dehydrogenase subunit I-12.43 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide-human MDH dehydrogenase subunit I-12. 43, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention 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). 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 initial methionine residues.
  • the invention also includes fragments, derivatives and analogs of human NADH dehydrogenase subunits 1-12.43.
  • fragments, derivatives and analogs of human NADH dehydrogenase subunits 1-12.43.
  • fragments refer to a polypeptide that substantially maintains the same biological function or activity of the human NADH dehydrogenase subunits 1-12.43 of the present invention.
  • a fragment, derivative or analog 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
  • the amino acid may or may not be encoded by the genetic code; or ( ⁇ ) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such a Species, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such an species in which the additional amino acid A polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) formed by fusion of a sequence into a mature polypeptide.
  • Such fragments, derivatives, and analogs are considered to be in the art according to the description herein.
  • 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 CDM library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 2284 bases and its open reading frame of 1735-2076 encodes 113 amino acids. According to the comparison of gene chip expression profiles, this peptide has a similar expression profile to human NADH dehydrogenase subunit I, and it can be inferred that the human NADH dehydrogenase subunit I-12. Similar functionality.
  • the polynucleotide of the present invention may be in the form of DNA or RM.
  • DNA forms include cDNA, genomic DNA, or synthetic DM.
  • DM can be single-stranded or double-stranded.
  • the DM can be a coding chain or a non-coding chain.
  • the coding region sequence encoding the 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 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 may 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, 6 (TC; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol l, 42 ° C, etc .; or
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human MDH dehydrogenase subunit I-12.43.
  • 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 MDH dehydrogenase subunits 1-12.43 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 CDM 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 MA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DM of the polypeptide.
  • genomic DM 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. There are many mature techniques for mRNA extraction. Kits are also commercially available (Qiagene).
  • the construction of a CDM library is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spruing 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.
  • the genes of the present invention can be screened from these CDM libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DM or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; ( 3 ) determination of human NADH dehydrogenase subunit I-12.43 transcription (4) Detecting protein products expressed by genes through immunological techniques or measuring 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 herein is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the gene or fragment of the present invention may of course Used as a probe.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human NADH dehydrogenase subunit I-12.43 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • the RACE method RACE-cDNA terminal rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / MA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to 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 'gene engineering using the vector of the present invention or directly using human NADH dehydrogenase subunit 1-12.43 coding sequence, and produced by recombinant technology A method of a polypeptide according to the invention.
  • a polynucleotide sequence encoding human NADH dehydrogenase subunit I-12.43 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as 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 recombinant expression vectors.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human MDH dehydrogenase subunit I-12.43 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DM technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua 1, cold Harbor Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide mRM synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • 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 adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human NADH dehydrogenase subunit I-12.43 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a gene containing the polynucleotide or the recombinant vector.
  • Engineered host cells 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 insect cells
  • insect cells such as flies S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM 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.
  • the alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human NADH dehydrogenase subunits 1-12.43 (Sc ience, 1984; 224: 1431). Generally speaking, there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional 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 gene chip expression profiles of the inventors' MDH dehydrogenase subunit I-12.43 and human NADH dehydrogenase subunit I.
  • the upper graph is a graph of the expression profile of human MDH dehydrogenase subunit 1-12.43, and the lower graph is the graph of the expression profile of human NADH dehydrogenase subunit I.
  • Figure 2 is a polyacrylamide gel electrophoresis image of isolated human NADH dehydrogenase subunit I-12. 43 (SDS-1)
  • lOkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Human fetal brain total MA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) raRNA 0 2ug poly (A) mRNA was isolated from total RM using Quik mRNA Isolat ion is t (product of Qiegene) to form cDNA by reverse transcription.
  • the Smart cDNA cloning kit purchased from Clontech l # cDNA fragment was inserted into the multicloning site of pBSK (+) vector (Clontech)) to transform DH5 ⁇ to form a cDNA library.
  • Dye terminate cycle react ion sequencing Kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequences were compared with the existing public DNA sequence database (Genebank). The comparison showed that the cDNA sequence of one of the clones 0540al2 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragment in both directions.
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imerl 5 '-CTGTAAAAATTTAAATCTGTATTT- 3' (SEQ ID NO: 3)
  • Pr imer2 5'- CTTTTGAAATGTCAGGTTTATAAT-3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 ⁇ l of KC1, 10 mmol / L Tri s-CI, (pH 8.5.5), 1.5 legs of ol / L MgCl 2 , 200 ⁇ ⁇ / in a reaction volume of 50 ⁇ 1 L dNTP, lOpmol primer, 1U Taq DNA polymerase (C 1 on tech company product). 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. During 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 using a TA cloning kit (Invitrogen).
  • the DNA sequence analysis results show that the DM sequence of the PCR product is exactly the same as the l-2284bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human MDH dehydrogenase subunit I-12.43 gene expression:
  • Total RM extraction in one step [Anal. Biochem 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), mixed After centrifugation. The aqueous phase was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain a MA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • RNA With 20 g of RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid ( ⁇ 7.0)-5 mM sodium acetate-ImM EDTA-2. 2M formaldehyde. It was then transferred to a nitrocellulose membrane. A- 32 P dATP was used to prepare 32 P-labeled DNA probes by random primers. The MA probe used was the PCR amplified human NADH dehydrogenase subunit I-12.43 coding region sequence (1735bp to 2076bp) shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7. 4)-5 x SSC-5 x Denhardt's solution and 200 g / ml salmon sperm DM. 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 subunits 1-12.43
  • Pr imer 3 5'-CATGCTAGCATGTGTGAGTTGGGGAATGATGTT-3 '(Seq ID No: 5)
  • Primer 4 5'-CATGGATCCTCAGGGATTATAAAAGTGATTTGA-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Mel and BamHI digestion respectively Site, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the restriction sites for Mel and BamHI correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • the pBS-0540al2 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0540al2 plasmid, primers Primer-3 and Primer-4 were 10 pmol, and Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into the colibacillus DH5 cx by the calcium chloride method. After being cultured on an LB plate containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1) overnight, positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0540al2) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) P lySs (product of Novagen) using the calcium chloride method. In containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1) in LB liquid medium, host strain BL21 (P ET-0540al2) at 37. C.
  • 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 It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under high-intensity washing conditions, the first type of probe and sample have the highest hybridization specificity and are retained.
  • the preferred range of probe size is 18-50 nucleotides
  • 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 unknown genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the sample film was placed in a plastic bag, was added 3- 10m g prehybridization solution (lOxDenhardt's;. 6xSSC, 0. lmg / ml CT DNA ( calf thymus the DM)), the sealed bag, 68 ° C shaking water Luo 2 hours.
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target DM for gene chip technology for high-throughput research of new gene functions; searching for and screening new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature, for example, see DeRis i, J. L., Lyer, V. & Brown, P. 0.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotides of the present invention. They were respectively amplified by PCR. After the purified amplified product was purified, the concentration was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ ⁇ . The spotted slides were hydrated, dried, and cross-linked in an ultraviolet cross-linker. After elution, the slides were fixed to fix the DM on the glass slides to prepare chips. The specific method steps have been reported in the literature in various ways. The post-spotting 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 the mRNA was purified with Ol igotex mRNA Midi Kit (purchased from QiaGen).
  • fluorescent reagent Cy3dUTP (5- Amino- propargyl-2'- deoxyur idine 5'- tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP was used.
  • the probes from the two tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Then use ScanArray 3000 scanner (purchased from General Scanning Company, USA) for scanning. The scanned images are analyzed and processed with Imagene software (Biodiscovery Company, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line, thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scar formation fc Growth factor stimulation, 1013HT, scar into fc without growth factor stimulation, 1G13HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, cardiac cancer. Draw a graph based on these 18 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profiles of human NADH dehydrogenase subunits 1-12.43 and human NADH dehydrogenase subunit I according to the present invention are very similar. Industrial applicability
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • the respiratory chain is an important way for electron transfer and energy conversion in the body, and the role of various enzymes in the respiratory chain reaction process. These different enzymes coordinate in the respiratory chain process to promote respiratory metabolism.
  • Suck NADH dehydrogenase is a polymerase complex that exists in the inner mitochondrial inner membrane.
  • MDH dehydrogenase subunit The protein I is a component thereof, and it has a conserved sequence fragment specific to members of the MDH dehydrogenase subunit I protein family to form its active mot if.
  • the abnormal expression of the specific MDH dehydrogenase subunit I mot if will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, resulting in abnormalities in the electron transfer, energy generation, and material conversion of the respiratory chain, and It further affects the metabolic processes of matter and energy, and produces related diseases such as disorders of substance and energy metabolism, disorders of embryonic development, disorders of growth and development, and tumors.
  • the abnormal expression of the human MDH dehydrogenase subunits 1-12.43 of the present invention will produce various diseases, especially material and energy metabolic disorders, embryonic development disorders, growth disorders, tumors, These diseases include, but are 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, Branched Amino Acid Metabolism Deficiency, Glycineemia, Hypersarcosinemia, Proline and Hydroxyproline Metabolism Defects, Glutamate Metabolism Defects, Urea Cycle Metabolism Defects, Histidine Metabolic Defective Disease, Lysine Metabolic Defective Disease, Mucopolysaccharidosis Type I ⁇ W, Rheumatoid Mucopolysaccharidosis, Mucolipid Storage Syndrome, Ray-ni Syndrome, Xanthineuria, Orotic Aciduria, Gland Purine nucleoside deaminase deficiency, hyperlipoproteinemia, familial hyperalpha-lipoproteinemia, congenital lactose intolerance,
  • Embryonic disorders congenital abortion, cleft palate, limb absentness, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney disease, double ureter, crypto, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias , Bisexual deformity, Atrial septal defect, Ventricular septal defect, Pulmonary stenosis, Arterial duct occlusion, Neural tube defect, Congenital hydrocephalus, Iris defect, Congenital cataract, Congenital glaucoma or cataract, Congenital deafness
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, familial cerebral nucleus dysplasia syndrome, skin, fat and muscular dysplasias such as congenital skin relaxation, premature aging, congenital horn Malnutrition, 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
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist J human NAM dehydrogenase subunit I-12. 43.
  • Agonists increase human NADH dehydrogenase subunit I-12 43 Stimulates biological functions such as cell proliferation, while antagonists prevent and treat those associated with excessive cell proliferation Disorders such as various cancers.
  • a mammalian cell or a membrane preparation expressing human NADH dehydrogenase subunit I-12.43 can be cultured with the labeled human NAM dehydrogenase subunit I-12.43 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human NADH dehydrogenase subunits 1-12.43 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human MDH dehydrogenase subunit 1-12.43 can bind to human NADH dehydrogenase subunit 1-12.43 and eliminate its function, or inhibit the production of the polypeptide, or with the activity of the polypeptide Site binding prevents the polypeptide from performing its biological function.
  • human MDH dehydrogenase subunit I-12.43 When screening compounds as antagonists, human MDH dehydrogenase subunit I-12.43 can be added to the bioanalytical assay, and by measuring the compounds against human NADH dehydrogenase subunit I-12.43 and its receptors The effect of this interaction is used to determine whether the compound is an antagonist. Receptor deletions and analogues that act as antagonists can be screened in the same way as the above-mentioned candidate compounds. Polypeptide molecules capable of binding to human NADH dehydrogenase subunit I-12.43 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human MDH dehydrogenase subunits 1-12.43 molecules should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human MDH dehydrogenase subunit I-12.43 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced using human NADH dehydrogenase subunits 1-12.
  • 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 subunit I-12.43 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975,
  • Antibodies against human NADH dehydrogenase subunit 1-12.43 can be used in immunohistochemical techniques to detect human NADH dehydrogenase subunit I-12.43 in biopsy specimens.
  • Monoclonal antibodies that bind to human NADH dehydrogenase subunits 1-12.43 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 Subunit I-12.43 High-affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human MDH dehydrogenase subunits 1- 12. 43 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human NADH dehydrogenase subunits 1-12.43.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human MDH dehydrogenase subunits 1-12.43.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human NADH dehydrogenase subunit I-12.43 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The levels of human NADH dehydrogenase subunit 1-12.43 detected in the test can be used to explain the importance of human NADH dehydrogenase subunit I-12.43 in various diseases and for the diagnosis of human NADH dehydrogenase. Diseases where catalase subunits 1 -12. 43 work.
  • 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.
  • Polynucleotides encoding human NADH dehydrogenase subunit I-12.43 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 MDH dehydrogenase subunits 1-12.43.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human NADH dehydrogenase subunit I-12.43 to inhibit endogenous human MDH dehydrogenase subunit 1-12.43 activity.
  • a variant human NADH dehydrogenase subunit I-12.43 may be a shortened human NADH dehydrogenase subunit I-12.43, although it may be associated with a downstream substrate Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human NADH dehydrogenase subunit I-12.43.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer polynucleotides encoding human NADH dehydrogenase subunits 1-12.43 into cells .
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human NADH dehydrogenase subunit I-12.43 can be found in existing literature (Sambrook, et al.).
  • a polynucleotide encoding human NADH dehydrogenase subunit 1-12.43 can be recombinantly 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 MA and DNA
  • ribozymes that inhibit human NADH dehydrogenase subunit 1-12.43 mRNA are also within the scope of the present invention.
  • Ribozyme is an enzyme-like MA that specifically breaks down specific MAs Molecules, whose mechanism of action is endonucleation after the ribozyme molecule specifically hybridizes to a complementary target RM.
  • Antisense RNA and DM and ribozymes can be obtained by any of the MA or DM synthesis techniques. For example, solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides has been widely used.
  • Antisense RM molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DM sequence has been integrated downstream of the MA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human MDH dehydrogenase subunit I-12.43 can be used for diagnosis of diseases related to human MDH dehydrogenase subunit I-12.43.
  • the polynucleotide encoding human MDH dehydrogenase subunit I-12.43 can be used to detect the expression of human NADH dehydrogenase subunit I-12.43 or human NADH dehydrogenase subunit I- in the disease state- 12.
  • Abnormal expression of 43 For example, a DNA sequence encoding human NADH dehydrogenase subunit I-12. 43 can be used to hybridize biopsy specimens to determine the expression of human NADH dehydrogenase subunit 1-12. 43.
  • 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. Some 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 MDH dehydrogenase subunit I-12.43 specific primers can also be used to detect the transcription products of human MDH dehydrogenase subunit 1-12.43 by MA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • Detection of mutations in the human NADH dehydrogenase subunit I-12.43 gene can also be used to diagnose human NADH dehydrogenase subunit 1-12.43-related diseases.
  • Human MDH dehydrogenase subunit 1-12.43 mutant forms include point mutations, translocations, deletions, recombinations, and any other mutations compared to normal wild-type human MDH dehydrogenase subunit 1-12.43 DM sequences Exception, etc. Mutations can be detected using existing techniques such as Southern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for labeling chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • the PCR primers (preferably 15-35b P ) are prepared according to the CDM, and the sequences can be mapped on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize MA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific CDM libraries.
  • Fluorescent in situ hybridization of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian 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 observed in any normal individual, the mutation may be the cause of the disease. Comparing diseased and diseased individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be 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 prompts permission for administration on the human body by government agencies that produce, use, or sell them.
  • 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 subunits 1-12.43 are administered in amounts effective to treat and / or prevent specific indications.
  • the amount and range of NADH dehydrogenase subunits 1 to 12.43 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 sous-unité humaine NADH déshydrogénase I-12.43, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment 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 la sous-unité humaine NADH déshydrogénase I-12.43.
PCT/CN2001/001125 2000-07-07 2001-07-02 Nouveau polypeptide, sous-unite humaine nadh deshydrogenase i-12.43, et polynucleotide codant ce polypeptide WO2002020587A1 (fr)

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AU2002223371A AU2002223371A1 (en) 2000-07-07 2001-07-02 A novel polypeptide-human nadh dehydrogenase subunit i-12.43 and the polynucleotide encoding said polypeptide

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CN00117080A CN1333347A (zh) 2000-07-07 2000-07-07 一种新的多肽——人还原型辅酶ⅰ脱氢酶亚单位ⅰ－12.43和编码这种多肽的多核苷酸
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1023896A (ja) * 1996-05-07 1998-01-27 Unitika Ltd 組換えプラスミド、それにより形質転換された大腸菌、その培養物及びそれを用いたアミノ酸又はその誘導体の製造方法
WO1998031815A2 (fr) * 1997-01-17 1998-07-23 Incyte Pharmaceuticals, Inc. Sous-unites de nadh deshydrogenase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1023896A (ja) * 1996-05-07 1998-01-27 Unitika Ltd 組換えプラスミド、それにより形質転換された大腸菌、その培養物及びそれを用いたアミノ酸又はその誘導体の製造方法
WO1998031815A2 (fr) * 1997-01-17 1998-07-23 Incyte Pharmaceuticals, Inc. Sous-unites de nadh deshydrogenase

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