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WO2001073047A1 - Nouveau polypeptide, superoxyde dismutase humaine a cuivre et a zinc 17, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, superoxyde dismutase humaine a cuivre et a zinc 17, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001073047A1
WO2001073047A1 PCT/CN2001/000473 CN0100473W WO0173047A1 WO 2001073047 A1 WO2001073047 A1 WO 2001073047A1 CN 0100473 W CN0100473 W CN 0100473W WO 0173047 A1 WO0173047 A1 WO 0173047A1
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Prior art keywords
polypeptide
polynucleotide
superoxide dismutase
zinc superoxide
human copper
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU56088/01A priority Critical patent/AU5608801A/en
Publication of WO2001073047A1 publication Critical patent/WO2001073047A1/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.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, human copper / zinc superoxide dismutase 17, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides.
  • Cu / Zn SODC Copper / zinc superoxide dismutase
  • Cu / Zn SODC has a molecular weight of approximately 32kd. Its two subunits are relatively independent of each other and there is no covalent bond between each other. Each subunit contains 1-g atoms of Cu / Zn.
  • Cu / Zn SODC combines one zinc atom and one copper atom each.
  • Cu / Zn SODC has the following forms: Cytoplasmic form of eukaryotic cells, extra chloroplast form in plants, extracellular form of some eukaryotic cells, and periplasmic form of prokaryotic cells.
  • the metal binding sites are conserved. These binding sites are histidine 48, 50, 77, 135 that binds to copper atoms, and histidine 77, 86 that binds to zinc atoms. 95 and aspartic acid 98.
  • Histidine 77 is a bridge between copper and zinc atoms. The binding sites of copper and oxygen anions are four separate and highly intact.
  • the Cu / Zn SODC family has two characteristic sequences. [1] [GA]-[IFAT] -H- [LIVF] -H-X (2)-[GP]-[SDX]-X- [STAGD]. Among them, two H represent copper ligands. [2] G- [GN]-[SGA] -G- X-R-X- [SGA]-C-X (2)-[IV]. Among them, C is related to disulfide bonds. The previous sequence contains two histidine residues bound to a copper atom. The second sequence is located at the C-terminus of SODC and contains a cysteine residue associated with a disulfide bond.
  • the human Cu / Zn SODC gene is located on 21 human chromosomes, is 1 lkb long, and has 5 exons and four endogens.
  • the first foreign element had 5 '-GC instead of the highly conservative 5' -GT.
  • Cu / Zn S0DC abnormalities will cause but are not limited to the following diseases: oxidative hemolytic anemia, thalassemia, malaria, Fankang anemia, muscle dislocation, gallbladder fibrosis, various inflammations.
  • the invention is named human copper / zinc superoxide dismutase 17.
  • the human copper / zinc superoxide dismutase 17 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes. Therefore, it is always necessary to identify more Many human copper / zinc superoxide dismutase 17 proteins are involved in these processes, especially the amino acid sequence of this protein is identified. Isolation of the new gene encoding copper / zinc superoxide dismutase 17 protein also provides a basis for the study 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 isolating its coding DNA is very important. 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 recombinant vector containing a polynucleotide encoding human copper / zinc superoxide dismutase 17.
  • Another object of the present invention is to provide a method for producing human copper / zinc superoxide dismutase 17.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, human copper / zinc superoxide dismutase II.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human copper / zinc superoxide dismutase II.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human copper / zinc superoxide dismutase 17. Summary of invention
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 2, or a conservative variant, biologically active fragment, or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 430-891 in SEQ ID NO: 1; and (b) a sequence having 1-1244 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.
  • 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 copper / zinc superoxide dismutase 17 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 in vitro detection of a disease or susceptibility to disease associated with abnormal expression of a human copper / zinc superoxide dismutase ⁇ protein, which comprises detecting a mutation in the polypeptide or a coding polynucleotide sequence 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 preparation of a polypeptide and / or polynucleotide of the present invention for the treatment of cancer, developmental or immune diseases, or other drugs caused by abnormal expression of human copper / zinc superoxide dismutase 17 use.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of copper / zinc superoxide dismutase 17 and human copper / zinc superoxide dismutase 9 of the present invention.
  • the upper graph is a graph of human copper / zinc superoxide dismutase 17 expression profile
  • the lower graph is the human copper / zinc superoxide dismutase 9 expression profile.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates unstarved L02
  • 8 indicates L02 +, I hr, As 3+
  • 9 ECV304 PMA- 10
  • ECV304 PMA + 1 1 fetal liver, 12 normal liver, 13 3 thyroid
  • 14 skin 15 fetal lung, 16 lung, 17 lung cancer
  • 18 fetal spleen 19 indicates the spleen
  • 20 indicates the prostate
  • 21 indicates the fetal heart
  • 22 indicates the heart
  • 23 indicates muscle
  • 24 indicates the testis
  • 25 indicates the fetal thymus
  • 26 indicates the thymus.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human copper / zinc superoxide dismutase 17. 17 kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a succinic acid or a polynucleotide, and a fragment or part thereof. Refers to genomic or synthetic DNA or RNA, which 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 copper / zinc superoxide dismutase 17, causes a change in the protein to regulate the activity of the protein.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human copper / zinc superoxide dismutase 17.
  • Antagonist refers to a biological activity or immunity that can block or regulate human copper / zinc superoxide dismutase 17 when combined with human copper / zinc superoxide dismutase 17 Chemically active molecules. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind human copper / zinc superoxide dismutase 17.
  • Regular refers to a change in the function of human copper / zinc superoxide dismutase 17, including an increase or decrease in protein activity, a change in binding characteristics, and any other organism of human copper / zinc superoxide dismutase 17 Changes in nature, function, or immunity.
  • Substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human copper / zinc superoxide dismutase II using standard protein purification techniques.
  • Substantially pure human copper / zinc superoxide dismutase 17 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human copper / zinc superoxide dismutase 17 peptide can be analyzed by amino acid sequence.
  • Complementary refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-TG-A” can be combined with the complementary sequence "GA-C-T”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands The efficiency and strength of hybridization between nucleic acid strands has a significant effect.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern imprinting or Northern 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 identical or similar in the comparison of two or more amino acid or nucleic acid sequences.
  • the percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.).
  • the MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Cluster method divides each group of sequences by checking the distance between all pairs. Arranged in clusters. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun Hein (Hein L, (1990) Methods in enzymology 183: 625-645). 0
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Antibody refers to an intact antibody molecules and fragments thereof, such as Fa, F (a b ') 2 and F V, which is capable of specifically binding to human copper / zinc superoxide dismutase 17 antigenic determinants.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide exists in a living animal. It is not isolated, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.
  • isolated human copper / zinc superoxide dismutase 1 7 refers to human copper / zinc superoxide dismutase ⁇ which is substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. .
  • Those skilled in the art can purify human copper / zinc superoxide dismutase 17 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human copper / zinc superoxide dismutase 17 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human copper / zinc superoxide dismutase 17, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products, or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude initial methionine residues.
  • 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.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human copper / zinc superoxide dismutase 17.
  • 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 copper / zinc superoxide dismutase ⁇ of the present invention can be used in various ways Method to obtain.
  • 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.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • mRNA extraction There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the protein product of human copper / zinc superoxide dismutase 17 gene expression 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).
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using a human copper / zinc superoxide dismutase 17 coding sequence, and that the present invention is produced by recombinant technology Said method of polypeptide.
  • a polynucleotide sequence encoding human copper / zinc superoxide dismutase 17 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 expressed in bacteria (Rosenberg, et al.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human copper / zinc superoxide dismutase 17 and suitable transcription / translation regulatory elements. These methods include in vitro recombination DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pair SV40 enhancers 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.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human copper / zinc superoxide dismutase 17 (Scence, 1984; 224: 14 31). Generally 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 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 types of inflammation, HIV infection and immune diseases.
  • Cu / Zn SODC abnormalities will cause, but are not limited to, the following diseases: oxidative hemolytic anemia, thalassemia, malaria, Fankang anemia, muscle dislocation, gallbladder fibrosis, and various inflammations.
  • the abnormal expression of the specific copper / zinc superoxide dismutase mo tif will cause the abnormal function of the polypeptide containing this mo tif, which will cause the decomposition of H 2 0 2 and superoxide radicals which are harmful to the cells.
  • Glycogenogenesis abnormal lipid metabolism, produce related diseases such as anemia, abnormal inflammatory process, abnormal immune system function, organic acidemia, abnormal thyroid hormone production.
  • human copper / zinc superoxide dismutase 17 of the present invention will produce various diseases, especially anemia, abnormal inflammatory process, abnormal immune system function, organic acidemia, and thyroid diseases. These diseases include but not limited to:
  • inflammatory abnormalities caused by various infections and traumas such as viral hepatitis, Borrelia infection, tuberculosis, HIV, syphilis, allergic reactions, bronchial asthma, sarcoidosis, rheumatoid arthritis, rheumatoid arthritis, Dermatomyositis, urticaria, atopic dermatitis, hemochromatosis, Addison's disease, Graves' disease, chronic active hepatitis, intestinal emergency syndrome, atrophic gastritis, systemic lupus erythematosus, cerebral spinal cord sexual sclerosis, Guillain-Barre syndrome, intracranial granuloma, Wegener's granulomatosis, autoimmune thyroiditis, autoimmune interstitial nephritis, ulcerative colitis, pancreatitis, myocarditis, atherosclerosis, multiple Scleroderma
  • Immune system diseases antibody-based primary specific immunodeficiency, combined immunodeficiency, immunodeficiency with phagocytic deficiency, complement system deficiency, Down syndrome, biotin-dependent carboxylase deficiency, Dun Can syndrome, thymoma, chronic cutaneous mucosal candidiasis, aplastic anemia, Di George syndrome, Wisco t t A Al dr i ch syndrome, immunodeficiency with ataxia capillary dilatation Acquired immune deficiency syndrome
  • Organic acidemia propionic acidemia, methylmalonic aciduria, isovalerate, combined carboxylase deficiency, glutarate type I
  • 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 copper / zinc superoxide dismutase 17 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.
  • Inlay antibodies combining human constant regions and non-human variable regions can be produced using existing technologies (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 copper / zinc superoxide dismutase 17.
  • Monoclonal antibodies that bind to human copper / zinc superoxide dismutase 17 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.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human copper / zinc superoxide dismutase 17 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human copper / zinc superoxide dismutase 17 detected in the test can be used to explain the importance of human copper / zinc superoxide dismutase 17 in various diseases and to diagnose human copper / zinc superoxide Diseases in which dismutase ⁇ works.
  • Polynucleotides encoding human copper / zinc superoxide dismutase 17 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 copper / zinc superoxide dismutase ⁇ .
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human copper / zinc superoxide dismutase ⁇ to inhibit endogenous human copper / zinc superoxide dismutase 17 activity.
  • a mutated human copper / zinc superoxide dismutase ⁇ may be a shortened human copper / zinc superoxide dismutase 17 that lacks a signaling domain.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human copper / zinc superoxide dismutase 17.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human copper / zinc superoxide dismutase ⁇ into a cell.
  • recombinant viral vector carrying a polynucleotide encoding human copper / zinc superoxide dismutase ⁇ can be found in existing literature (Samb rook, et al.).
  • recombinant polynucleotides encoding human copper / zinc superoxide dismutase 17 can be packaged into liposomes and transferred into cells.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DM sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of 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 copper / zinc superoxide dismutase 17 can be used for the diagnosis of diseases related to human copper / zinc superoxide dismutase 17.
  • Polynucleotides encoding human copper / zinc superoxide dismutase ⁇ can be used to detect the expression of human copper / zinc superoxide dismutase 17 or abnormal expression of human copper / zinc superoxide dismutase 17 in disease states .
  • a DNA sequence encoding human copper / zinc superoxide dismutase 17 can be used to hybridize biopsy specimens to determine the expression of human copper / zinc superoxide dismutase 17.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • 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 tissue.
  • Human copper / zinc superoxide dismutase 17 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human copper / zinc superoxide dismutase ⁇ transcription products.
  • Detection of mutations in the human copper / zinc superoxide dismutase 17 gene can also be used to diagnose human copper / zinc superoxide dismutase 17-related diseases.
  • Human copper / zinc superoxide dismutase ⁇ mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human copper / zinc superoxide dismutase 17 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern 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 DNA 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 DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • 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 copper / zinc superoxide dismutase 17 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human copper / zinc superoxide dismutase 17 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. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik raRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Directional insertion of cDNA fragments into pBSK using the Smart cDNA Cloning Kit (purchased from Clontech) (+) The vector (Clontech) was transformed into DH5cc at multiple cloning sites, and the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0059C10 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • Primerl 5'— GCTCTTCCACCTGCGGAGCTCGCT-3 '(SEQ ID NO: 3)
  • Primer2 5,-TGCCACATAGATTTTAATTTGCCT- 3, (SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Amplification conditions 50 ⁇ l reaction volume containing 50 mmol / L C1, 10 ramol / L Tris-HCl, pH 8.5, 1.5 mmol / L MgCl 2 , 200 ⁇ 1 / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55. C 30sec; 72 ° C 2min solicitAt the same time, set ⁇ -act in as a positive control and template blank as a negative control at the time of RT-PCR.
  • Amplification products were purified using a QIAGEN kit, and connected to a pCR vector using a TA cloning kit. (Invitrogen product).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1244bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human copper / zinc superoxide dismutase 17 gene
  • One step method was used to extract total RM [AnaL Biochem 1987, 162, 156-159]. This method included acid guanidinium thiocyanate phenol-chloroform extraction.
  • 32P-labeled probe (about 2 x 10 6 cpm / ml)
  • the cellulose acetate membrane was hybridized overnight at 42 ° C in a solution containing 50% formamide-25mM H 2 PO 4 (pH 7.4)-5 SSC-5 Denhardt's solution and 200 ⁇ 8 / ⁇ 1 salmon sperm DNA. After hybridization, the filters were placed in 1 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 copper / zinc superoxide dismutase 17 Based on 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'- CATGCTAGCATGGAGGAAAAAAGACGGCGAGCC- 3, (Seq ID No: 5)
  • Primer4 5'- CATGGATCCTCAGCTTGTGTCGCTCCTTCCATG- 3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively.
  • the coding sequences for the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • the pBS-0059clO plasmid containing the full-length target gene was used as a template to perform a PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0059cl0 plasmid, primers Primer-3 and Primer-4 were 1 Opmol and Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Nde I and BamH I were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight in LB plates containing kanamycin (final concentration 3 (Vg / ml)), positive clones were screened by colony PCR and sequenced. The sequence was selected correctly The positive clone (pET-0059C10) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method.
  • Example 5 produced an anti-human antibody 17 Cu / Zn superoxide dismutase embodiment
  • a peptide synthesizer (product of PE) was used to synthesize the following human copper / zinc superoxide dismutase 17-specific peptides:
  • NH2-Met-Glu-Glu-Glu-Lys-Arg-Arg-Arg-Ala-Arg-Val-Gln-Gly-Ala-Trp-Ala-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin for methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4rag above-mentioned cyanin polypeptide complex plus complete Freund's adjuvant. Incomplete Freund's adjuvant boosts immunity once.
  • a titer plate coated with 15 g / ml bovine serum albumin peptide complex was used as EL I SA to determine the antibody titer in rabbit serum.
  • Protein A-Sepha rose was used to isolate total I gG from antibody-positive home-immunized serum.
  • the peptide was bound to a cyanogen bromide-activated Sepha rose4B column, and the anti-peptide antibody was separated from the total I gG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human copper / zinc superoxide dismutase 17.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Sou thern imprinting, Nor thern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity 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 invention; the second type of probes are partially related to the 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.
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements Region for homology comparison, if the homology with non-target molecular region is greater than 85% or more than 15 Two consecutive bases are completely the same, the primary probe should generally not be used;
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • the 32 P-Probe is prepared (the second peak is free ⁇ - 32 P- dATP).
  • the sample membrane was placed in a plastic bag and 3-10 mg of prehybridization solution (lOxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution lOxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance is 280 ⁇ ⁇ . 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. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • the probes from the two types of tissues and the chips were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000.
  • the scanner purchased from General Scanning Company, USA
  • the scanned image was analyzed and processed with Imagene software (Biodiscovery Company, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, Lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, arsenic stimulated L02 cell line for 1 hour, arsenic stimulated L02 cell line for prostate, heart, lung cancer , Fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, fetal lung, and fetal heart.

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Abstract

L'invention concerne un nouveau polypeptide, une superoxyde dismutase humaine à cuivre et à zinc 17, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la superoxyde dismutase humaine à cuivre et à zinc 17.
PCT/CN2001/000473 2000-03-28 2001-03-26 Nouveau polypeptide, superoxyde dismutase humaine a cuivre et a zinc 17, et polynucleotide codant pour ce polypeptide Ceased WO2001073047A1 (fr)

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AU56088/01A AU5608801A (en) 2000-03-28 2001-03-26 A novel polypeptide, a human copper/zinc superoxide dismutase 17 and the polynucleotide encoding the polypeptide

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CN00115218.1 2000-03-28
CN 00115218 CN1315522A (zh) 2000-03-28 2000-03-28 一种新的多肽——人铜/锌超氧化物歧化酶17和编码这种多肽的多核苷酸

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 23 November 1999 (1999-11-23), Database accession no. AC004382 *
DATABASE GENBANK [online] 24 November 1998 (1998-11-24), Database accession no. AF107342 *
DATABASE GENBANK [online] 26 September 1999 (1999-09-26), Database accession no. AF175966 *
DATABASE GENBANK [online] 27 January 1996 (1996-01-27), Database accession no. U38442 *

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