WO2001094537A2 - Nouveau polypeptide, nadh-ubiquinone oxydoreductase humaine 21.89, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, nadh-ubiquinone oxydoreductase humaine 21.89, et polynucleotide codant ce polypeptide Download PDF

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WO2001094537A2
WO2001094537A2 PCT/CN2001/000854 CN0100854W WO0194537A2 WO 2001094537 A2 WO2001094537 A2 WO 2001094537A2 CN 0100854 W CN0100854 W CN 0100854W WO 0194537 A2 WO0194537 A2 WO 0194537A2
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polypeptide
polynucleotide
ubiquinone oxidoreductase
human
sequence
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PCT/CN2001/000854
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Chinese (zh)
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WO2001094537A3 (fr
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU89496/01A priority Critical patent/AU8949601A/en
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Publication of WO2001094537A3 publication Critical patent/WO2001094537A3/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)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, human MDH ubiquinone oxidoreductase 21. 89, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • Enzyme is an internal mitochondrial membrane-bound multi-subunit enzyme complex.
  • Complex I consists of at least 41 subunits, seven of which are encoded by the mitochondrial genome.
  • the function of NADH ubiquinone oxidoreductase is to catalyze the sensitive oxidation of NADH rotenone and reduce ubiquinone.
  • the complex is composed of two hydrophilic portions and one hydrophobic portion.
  • the hydrophilic portion includes a flavin protein portion and a ferritin portion.
  • the flavin protein consists of 51-, 24-, and 10-KD subunits, all of which are encoded by nuclear genes.
  • This part plays a catalytic role in oxidizing MDH because it is related to the binding of flavin protein and NAD.
  • the four-nucleus Fe-S center in the 51-KD subunit and the two-nucleus Fe-S center in the 24-KD subunit participate in electron transfer (de Coo RF, Budd i ger P, Smeet s HJ, van Oos t BA, Genomi cs 1997 Oct 15; 45 (2): 434-7).
  • the human gene encoding the 10-KD flavin protein subunit of complex I is named NDUFV3 gene, which is located on chromosome 21q22.3. This gene defect will cause Down's syndrome and is related to the deterioration of dementia. Down syndrome, also known as 21-trisomy syndrome or Mongolian dementia, is one of the most common autosomal diseases caused by autosomal aberrations. Three 21 chromosomes contain the same genes and disrupt the balance of genetic material. Causes fetal developmental abnormalities. It is characterized by a small head shape and flat front and back, low nose bridges, short finger (toe) bones, large gaps between the first and second fingers (toes) of hands and feet, mental retardation, and chromosomal abnormalities.
  • the new NADH ubiquinone oxidoreductase 21. 89 protein isolation 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 the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM. Object of the invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding human NADH ubiquinone oxidoreductase 21.89.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of NADH ubiquinone oxidoreductase 21.89 against 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 ubiquinone oxidoreductase 21.89.
  • Invention
  • 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:
  • 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 ubiquinone oxidoreductase 21.89 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 MDH ubiquinone oxidoreductase 21.89 protein in vitro, comprising detecting mutations in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample Or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the 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 ubiquinone oxidoreductase 21. 89 .
  • Fig. 1 is a comparison diagram of gene chip expression profiles of NADH ubiquinone oxidoreductase 21.89 of the present inventor and human NADH ubiquinone oxidoreductase.
  • the upper graph is a graph of the expression profile of human NADH ubiquinone oxidoreductase 21. 89, and the lower graph is the graph of the expression profile of human NADH ubiquinone oxidoreductase.
  • 1-bladder mucosa 2- PMA + Ecv304 cell line, 3- LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5-Fibroblas t, growth factor stimulation, 1024NT, 6-scars into fc growth factor Stimulation, 1013HT, 7_ scar into fc without stimulation with growth factor, 1013HC, 8-bladder cancer cell EJ, 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12- liver cancer cell line, 13-fetus Skin, 14-spleen, 15-prostate cancer, 16-jejunum adenocarcinoma, 17 cardia cancer.
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of human NADH ubiquinone oxidoreductase 21.89. 22kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted 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 ubiquinone oxidoreductase 21. 89, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human NADH ubiquinone oxidoreductase 21.89.
  • Antagonist refers to a biological or immunological activity that can block or modulate human MDH ubiquinone oxidoreductase 21. 89 when combined with human MDH ubiquinone oxidoreductase 21. 89 Molecule. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind human NADH ubiquinone oxidoreductase 21.89.
  • Regular refers to a change in the function of human NADH ubiquinone oxidoreductase 21. 89, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties of human NADH ubiquinone oxidoreductase 21. 89 , Functional or immune properties.
  • substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Quality. Those skilled in the art can use standard protein purification techniques to purify human NADH ubiquinone oxidoreductase 21. 89 0 substantially pure human NADH ubiquinone oxidoreductase 21. 89 can be produced on non-reducing polyacrylamide gels Single main band. The purity of human NADH ubiquinone oxidoreductase 21. 89 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that 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. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higg ins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein L, (1990) Methods in enzymology 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 substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand means A nucleic acid strand complementary to a “sense strand”.
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ,) 2 and? It can specifically bind to the epitope of human NADH ubiquinone oxidoreductase 21.89.
  • 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 certain vector, or such a polynucleotide or polypeptide may be part of a certain 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 in the natural state .
  • isolated human NADH ubiquinone oxidoreductase 21. 89 refers to human NADH ubiquinone oxidoreductase 21. 89 that is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated with it. Those skilled in the art can purify human NADH ubiquinone oxidoreductase 21.89 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 ubiquinone oxidoreductase 21. 89 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human NADH ubiquinone oxidoreductase 21. 89, 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.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of human NADH ubiquinone oxidoreductase 21.89.
  • fragment As used in the present invention, the terms “fragment”, “derivative” and “analog” mean substantially maintaining the present invention Human MDH ubiquinone oxidoreductase 21. 89 Peptides with the same biological function or activity.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such One, in which the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as leader sequences or secretory sequences or sequences used to purify this polypeptide).
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human brain tissue. It contains a polynucleotide sequence of 2097 bases in length and its open reading frame of 599-1160 encodes 199 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile to human NADH ubiquinone oxidoreductase. It can be inferred that the human NADH ubiquinone oxidoreductase 21. 89 has similar functions as human NADH ubiquinone oxidoreductase.
  • the polynucleotide of the present invention may be in a DNA 'form or a RM form.
  • 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.
  • the "degenerate variant” refers to a fusic 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 a polynucleotide A replacement form, which 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, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fi col 1, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, 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 NADH ubiquinone oxidoreductase 21.89.
  • 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 ubiquinone oxidoreductase 21.89 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 DM is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua 1, Cold Spoon 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 can be screened 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) measurement Set the transcript level of human NADH ubiquinone oxidoreductase 21. 89; (4) Detect the protein product of gene expression by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • the D probe can be labeled with a radioisotope, luciferin, or an enzyme (such as alkaline phosphatase).
  • the protein products of the human NADH ubiquinone oxidoreductase 21. 21. gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method (Saiki, et al. Science 1985; 230: 1350-1354) using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be measured 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 cDM sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDM sequences of multiple clones in order to splice into full-length cDM sequences.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using human NADH ubiquinone oxidoreductase 21. 89, and to produce the present invention by recombinant technology Said method of polypeptide.
  • a polynucleotide sequence encoding human NADH ubiquinone oxidoreductase 21.89 can be inserted into a vector to form 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 DM sequence encoding human MDH ubiquinone oxidoreductase 21.89 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 Manual, Cold Spiring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an 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 for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors expressed by DM, 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 and adenovirus enhancers on the late side of the origin of replication.
  • 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 MDH ubiquinone oxidoreductase 21.89 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence 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. It is well known in the art. Alternatively, M g Cl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DM 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 MDH ubiquinone oxidoreductase 21 ⁇ 89 (Science, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), 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 isolated 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
  • 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 inflammations, HIV infections, and immune diseases.
  • NADH ubiquinone oxidoreductase (complex I) is an internal mitochondrial membrane-bound multi-subunit enzyme complex and one of the mitochondrial respiratory chain complexes. Its function is to catalyze the sensitive oxidation of MDH rotenone and the reduction of ubiquinone.
  • the body's gene encoding the 10-KD flavin protein subunit of complex I is named NDUFV3 gene, and this gene defect will cause Down's syndrome (trisomy 21) and is related to the worsening of dementia.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human NADH ubiquinone oxidoreductase, and both have similar biological functions.
  • the polypeptide of the present invention regulates ubiquinone metabolism on the inner mitochondrial membrane in the body, and is an important component of the mitochondrial respiratory chain. Its abnormal expression is usually associated with some related disorders of substance metabolism disorders, protein metabolism disorders and tumors of related tissue And cancer and other diseases Related and produce related diseases, especially Down syndrome, dementia, etc.
  • developmental disorders congenital abortion, cleft palate, lack of limbs, limb differentiation disorders, atrial septal defect, neural tube defects, congenital hydrocephalus, mental retardation, brain development disorders, skin, fat and muscular dysplasia, Bone and joint dysplasia, various metabolic defects, sexually retarded tumors of various tissues: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, nerve cells Tumor, astrocytoma, ependymal tumor, glioblastoma, neurofibromas, colon cancer, bladder cancer, endometrial cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • Abnormal expression of the human NADH ubiquinone oxidoreductase 21.89 of the present invention will also produce certain hereditary, hematological diseases and the like.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially Down syndrome, dementia, tumors, developmental disorders, inflammation, and immune diseases. , Some hereditary, hematological diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human NADH ubiquinone oxidoreductase 21.89.
  • Agonists enhance human NADH ubiquinone oxidoreductase 21. 89 Stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human NADH ubiquinone oxidoreductase 21.89 can be cultured with labeled human NADH ubiquinone oxidoreductase 21.89 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human MDH ubiquinone oxidoreductase 21. 89 include screened antibodies, compounds, receptor deletions and the like. Antagonist of human NADH ubiquinone oxidoreductase 21. 89 can bind to human NADH ubiquinone oxidoreductase 21. 89 and eliminate its function, or inhibit the production of the polypeptide, or combine with the active site of the polypeptide to make The polypeptide cannot perform biological functions. When screening compounds as antagonists, human MDH ubiquinone oxidoreductase 21. 89 can be added to the bioanalytical assay, and the interaction between human NADH ubiquinone oxidoreductase 21.
  • 89 and its receptor can be determined by determining the compound Influence to determine if a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human MDH ubiquinone oxidoreductase 21. 89 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 NADH ubiquinone oxidoreductase 21. 89 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human NADH ubiquinone oxidoreductase 21.89 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • polyclonal antibodies can be obtained by direct injection of human NADH ubiquinone oxidoreductase 21. 89 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc.
  • Techniques for preparing monoclonal antibodies to human NADH ubiquinone oxidoreductase 21. 89 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human B -Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al., PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human NADH ubiquinone oxidoreductase 21.8.
  • Antibodies against human NADH ubiquinone oxidoreductase 21. 89 can be used in immunohistochemical techniques to detect human NADH ubiquinone oxidoreductase 21. 89 in biopsy specimens.
  • Monoclonal antibodies that bind to human NADH ubiquinone oxidoreductase 21. 89 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 against a specific bead site in the body. Such as human MDH ubiquinone oxidoreductase 21.
  • 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 toxin is bound to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human NADH ubiquinone oxidoreductase 21. 89 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human MDH ubiquinone oxidoreductase 21.89. Administration of appropriate doses of antibodies can stimulate or block human M ubiquinone oxidoreductase 21. 89 Production or activity.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human NADH ubiquinone oxidoreductase 21.89.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human NADH ubiquinone oxidoreductase 21. 89 detected in the test can be used to explain the importance of human NADH ubiquinone oxidoreductase 21. 89 in various diseases and to diagnose human NAM ubiquinone oxidoreductase 21. 89 diseases at 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 MDH ubiquinone oxidoreductase 21. 89 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 ubiquinone oxidoreductase 21.89.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutant human NADH ubiquinone oxidoreductase 21.89 to inhibit endogenous human MDH ubiquinone oxidoreductase 21. 89 activity. For example, a mutated human NADH ubiquinone oxidoreductase 21.
  • 89 may be a shortened human NADH ubiquinone oxidoreductase 21. 89 lacking a signaling domain, although it can bind to downstream substrates, 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 ubiquinone oxidoreductase 21.89.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human MDH ubiquinone oxidoreductase 21.89 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human NADH ubiquinone oxidoreductase 21.89 can be found in the literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human MDH ubiquinone oxidoreductase 21.89 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DM
  • ribozymes that inhibit human NADH ubiquinone oxidoreductase 21.
  • 89 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA and ribozymes can be obtained by any existing RM or DM synthesis technology, such as the technology of solid phase phosphate amide synthesis of oligonucleotides, which is 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. To increase the stability of nucleic acid molecules, they can be modified in a variety of ways. For example, if the sequence length on both sides is increased, the linkage between ribonucleosides should use phosphorothioate or peptide bonds instead of phosphodiester bonds. .
  • the polynucleotide encoding human NADH ubiquinone oxidoreductase 21.89 can be used for the diagnosis of diseases related to human NADH ubiquinone oxidoreductase 21.89.
  • a polynucleotide encoding human NADH ubiquinone oxidoreductase 21. 89 can be used to detect the expression of human NADH ubiquinone oxidoreductase 21. 89 or abnormal expression of human NADH ubiquinone oxidoreductase 21. 89 in a disease state. .
  • 89 can be used to hybridize biopsy specimens to determine the expression of human NADH ubiquinone oxidoreductase 21.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. 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 (Microarray) or a DM chip (also called a "gene chip”) for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • 89 specific primers can be used to perform RM-polymerase chain reaction (RT-PCR) in vitro amplification to detect the transcription products of human NADH ubiquinone oxidoreductase 21. 89.
  • 89 gene can also be used to diagnose human MDH ubiquinone oxidoreductase 21. 89-related diseases.
  • 89 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human MDH ubiquinone oxidoreductase 21.
  • 89 DM sequences Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect protein expression. Therefore, 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 marking 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 DM sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. 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 DM 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 a chromosome-specific CDM library.
  • Fluorescent in situ hybridization (FISH) of cDM clones and metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH fluorescent in situ hybridization
  • the difference in cDM 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).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human NADH ubiquinone oxidoreductase 21. 89 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dosage range of human NADH ubiquinone oxidoreductase 21.89 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.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Quik mRNA Isolat ion Kit product of Qiegene was used to isolate poly (A) raRNA 0 2ug poly (A) mRNA from reverse transcription to form CDM.
  • the Smart cD cloning kit purchased from Clontech ⁇ cDM fragment was inserted into the multiple cloning site of pBSK (+) vector (Clontech)) to transform DH5 ⁇ to form a CDM library.
  • Dye terminate cycle reaction sequencing kit Perkin-Blmer company
  • ABI 377 automatic sequencer Perkin-Elmer company
  • the determined CDM sequence was compared with the public DM sequence database (Genebank), the results
  • the CDM sequence of one of the clones 0449B05 was found to be new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragment of the clone in both directions.
  • 89 total fetal brain cell RN A was used as a template, and oligo-dT was used as a primer for reverse transcription reaction to synthesize cDNA. After purification with Qiagene's kit, PCR amplification was performed with the following primers:
  • Primerl 5 — GAAAAAAAAAGATTTAATTGGTCT -3, (SEQ ID NO: 3)
  • Primer2 5'- TTGAGAGTGCAGAGTCTACAACTG -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Pr imer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions containing 50mmol / L KCl in a reaction volume of 50 ⁇ 1, the legs 10 ol / L Tr i s- HC1 pH8 5, 1. 5mmol / L MgCl 2, 20 ( ⁇ mol / L dNTP. , lOpmol primer, 1 ⁇ Taq DM polymerase (product of Clontech).
  • the reaction was performed on a PE9600 MA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55. C 30sec; 72 ° C 2min. Set ⁇ -act in as positive control and template blank as negative control at the same time during RT-PCR.
  • Amplification products were purified with QIAGEN kit, and connected to pCR vector with TA cloning kit (Invitrogen product)
  • the DNA sequence analysis results show that the DNA sequence of the PCR product is exactly the same as that of 1-2097bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human NADH ubiquinone oxidoreductase 21. Expression of 89 genes. Total RM was extracted in one step [AnaL Biochem 1987, 162, 156-159] 0 This method involves acid guanidine thiocyanate phenol-chloroform extraction mention.
  • the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate ( PH 4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49 : 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA probes were the PCR amplified human NADH ubiquinone oxidoreductase 21. 89 coding region sequence (599bp to 1160bp) shown in FIG.
  • a 32P-labeled probe (approximately 2 x 10 fi cpm / ml) was hybridized with a nitrocellulose membrane to which RM was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (. P H7 4) - 5 x SSC- 5 x Denhardt, s 200 ⁇ ⁇ 1 solution and salmon sperm DM. After hybridization, the filter was placed at 1 x SSC-0.1 ° /. 55 in SDS. C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human MDH ubiquinone oxidoreductase 21. 89 According to the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:
  • Primer3 5 '-CATGCTAGCATGCACTGCAGCAGGCAGCAC AGA- 3' (Seq ID No: 5)
  • Primer 4 5'-CCCAAGCTTCTACTCAGGAAGGCGAGGTAGGGG-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and Hindlll digestion sites, respectively Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Nhel and Hindll l digestion sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • PCR reaction PCR reaction conditions were: 1 in a total volume of 50 ⁇ containing pBS - 0449 ⁇ 05 10 pg plasmid, primer Primer-3 and Primer - 4 were l Opmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60. C 30s, 68. C 2 min, a total of 25 cycles. Nhel and Hindlll were used for amplification products and plasmid pET-28, respectively. (+) Digestion with two enzymes, recovering large fragments separately, and ligating with T4 ligase.
  • the ligation product was transformed into Escherichia coli DH5 cx by the calcium chloride method on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1). after overnight incubation, colonies were screened by PCR and positive clones and sequenced. the correct sequence was selected positive clones (PET-0449B05) the recombinant plasmid by the calcium chloride method to transform E. coli BL21 (DE3) P lySs (Novagen Co.).
  • LB liquid medium containing kanamycin final concentration 30 ⁇ / ⁇ 1
  • the host bacteria BL21 pET-0449B05
  • IPTG was added to a final concentration of 1 leg ol / L, continue to incubate for 5 hours.
  • Set cells, broken by ultrasonic The supernatant was collected by centrifugation and chromatographed using an affinity column His.
  • Bind Quick Cartridge product of Novagen capable of binding to 6 histidines (6His-Tag) to obtain purified human NADH protein. Quinone oxidoreductase 21. 89.
  • NH2-Met-His-Cys-Ser-Arg-Gln-His-Arg-Pro-Thr-Val-Cys-Tyr-His-Ser-C00H SEQ ID NO: 7
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Imraunochemis try, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sephar 0Se 4B column, and the anti-peptide antibody was separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human NADH ubiquinone oxidoreductase 21. 89.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using 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 labeled probes, and Incubation hybridizes the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genome sequences and their complement 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, then 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 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffer solution
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the sample membrane was placed in a plastic bag, and 3-1 Omg pre-hybridization solution (lOxDenhardt-s; 6xSSC, 0.1 lrng / ml CT DNA (calf thymus DNA)) was added. After sealing the bag, shake at 68 ° C for 2 hours.
  • 3-1 Omg pre-hybridization solution lOxDenhardt-s; 6xSSC, 0.1 lrng / ml CT DNA (calf thymus DNA)
  • Gene chip 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 slopes. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as 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.
  • 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 respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500 ng / ul after purification, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slide to prepare a chip. Its specific method steps There have been many reports in the literature. The sample post-processing steps in this embodiment are:
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridiza ionization solution (purchased from TeleChem) for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scar-like fc growth factor stimulation 1013HT, scar into fc not stimulated with growth factors, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, cardia cancer. Based on these 17 Cy3 / Cy5 ratios, a histogram is drawn (Figure 1). It can be seen from the figure that the expression profiles of human MDH ubiquinone oxidoreductase 21.89 and human NADH ubiquinone oxidoreductase according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, une NADH-ubiquinone oxydoréductase humaine 21.89, 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 du syndrome de Down, de démence, de tumeurs, des troubles du développement, d'inflammations et de maladies immunitaires etc. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la NADH-ubiquinone oxydoréductase humaine 21.89.
PCT/CN2001/000854 2000-05-24 2001-05-21 Nouveau polypeptide, nadh-ubiquinone oxydoreductase humaine 21.89, et polynucleotide codant ce polypeptide WO2001094537A2 (fr)

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WO1999047666A1 (fr) * 1998-03-18 1999-09-23 Shanghai Second Medical University Gene humain homologue de ci-mnll: cbdfmd04
CN1261104A (zh) * 1999-01-20 2000-07-26 复旦大学 一种人氧化还原酶亚基及其编码序列,以及制法和用途

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Publication number Priority date Publication date Assignee Title
WO1999047666A1 (fr) * 1998-03-18 1999-09-23 Shanghai Second Medical University Gene humain homologue de ci-mnll: cbdfmd04
CN1261104A (zh) * 1999-01-20 2000-07-26 复旦大学 一种人氧化还原酶亚基及其编码序列,以及制法和用途

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