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WO2002012303A1 - Nouveau polypeptide, proteine humaine de regulation 10.23 d'une ribonucleoside-diphosphate reductase, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine de regulation 10.23 d'une ribonucleoside-diphosphate reductase, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002012303A1
WO2002012303A1 PCT/CN2001/000983 CN0100983W WO0212303A1 WO 2002012303 A1 WO2002012303 A1 WO 2002012303A1 CN 0100983 W CN0100983 W CN 0100983W WO 0212303 A1 WO0212303 A1 WO 0212303A1
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Prior art keywords
polypeptide
polynucleotide
human
diphosphate reductase
ribonucleoside diphosphate
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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 AU93621/01A priority Critical patent/AU9362101A/en
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Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human ribonucleoside diphosphate reductase inhibitor protein 10.23, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • An important enzyme for ATP energy metabolism in the body which catalyzes the mutual conversion between ATP and AMP, plays an important catalytic role in the metabolism of many substances, provides the required energy for the progress of biological processes, and regulates Relevant metabolic processes in the body proceed normally.
  • the activity of this enzyme in vivo is also regulated by various proteins in the body, such as nucleic acid nucleoside diphosphate reductase inhibitory proteins.
  • the abnormal action of the inhibitory protein will cause abnormalities in the related energy metabolism pathways and material metabolism pathways in the body, and then cause various related metabolic disorders.
  • This protein is closely related to the occurrence of diseases such as young children's thyroid tumors in the body [Kot Chetkov R., Kr ivtchik AA et al., 1999, Fo ia Bi ol, 45 (5): 185-191].
  • the ribonucleoside diphosphate reductase inhibitory protein regulates the normal progress of the catalytic action of ribonucleoside diphosphate reductase in vivo in vivo.
  • the protein regulates the corresponding material metabolism and energy metabolism by regulating the activity of ribonucleotide reductase in vivo.
  • the mutation or abnormal expression of this protein will cause abnormal development and metabolism of related tissues in the body, and then cause various related diseases.
  • the protein is closely related to the development of related tissues and metabolic disorders in the body, especially diseases such as thyroid tumors in young children and adults. It can also be used to diagnose and treat the various related diseases mentioned above.
  • the human ribonucleoside diphosphate reductase inhibitor protein 10.23 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. There has been a need to identify more human ribonucleoside diphosphate reductase inhibitor protein 10.23 proteins involved in these processes, especially the amino acid sequence of this protein.
  • the new human ribonucleoside diphosphate reductase inhibitor protein 10.23 protein gene isolation 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. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human ribonucleoside diphosphate reductase inhibitory protein 10.23.
  • Another object of the present invention is to provide a protein containing a human ribonucleoside diphosphate reductase inhibitor.
  • a genetically engineered host cell of a 10.23 polynucleotide A genetically engineered host cell of a 10.23 polynucleotide.
  • Another object of the present invention is to provide a method for producing human ribonucleoside diphosphate reductase inhibitory protein 10.23.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human ribonucleoside diphosphate reductase inhibitory protein 10.0.2.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention-human ribonucleoside diphosphate reductase inhibitory protein 10.23.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of human ribonucleoside diphosphate reductase inhibitory protein 10.23.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 11 1 to 392 in SEQ ID NO: 1; and (b) a sequence having 1-392 in SEQ ID NO: 1 1556-bit sequence.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human ribonucleoside diphosphate reductase inhibitory protein 10.2 3 protein activity, which comprises using the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or disease susceptibility related to abnormal expression of human ribonucleoside diphosphate reductase inhibitory protein 10.23 protein, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to polypeptides and / or polynucleotides of the present invention prepared for use in the treatment of cancer, developmental or immune diseases, or other diseases caused by abnormal expression of human ribonucleoside diphosphate reductase inhibitor protein 10.23. Use of drugs.
  • 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
  • polypeptide or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants may have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as replacing tryptophan with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant, or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human ribonucleoside diphosphate reductase inhibitory protein 10.23, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate or any other molecule that binds to human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • Antagonist refers to a human ribonucleoside diphosphate reductase inhibitor protein 10.23, which can block or regulate human ribonucleoside diphosphate reductase inhibitor protein 10.23 when combined with human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • Regular refers to changes in the function of human ribonucleoside diphosphate reductase inhibitor protein 10.23, including an increase or decrease in protein activity, changes in binding characteristics, and human ribonucleoside diphosphate reductase inhibitor protein 10. 23 any other biological, functional or immune change.
  • Substantially pure means that it is essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human ribonucleoside diphosphate reductase inhibitory proteins using standard protein purification techniques.
  • 10. 23. Essentially pure human ribonucleoside diphosphate reductase inhibitory protein 10. 23. Can produce a single main band on a non-reducing polyacrylamide gel. Human ribonucleoside diphosphate reductase inhibitory protein 10. 23 The purity of the peptide can be analyzed by amino acid sequence.
  • Complementary refers to a polynucleotide that naturally binds by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence CGA
  • GACT complementary sequence
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • Homology refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid.
  • the inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (La sergene s of tware package, DNASTAR, Inc., Madi son Wis.). The MEGALI GN program can compare two or more sequences according to different methods such as Clus ter method (Higgins, D, G. and PM Sharp (1988) Gene 73: 237-244). 0 C luster method checks all The distance between the pairs arranges the groups of sequences into clusters. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by Cluster method or using methods known in the art such as Jotun He in. The percent identity between nucleic acid sequences (Hein J., (1990) Me thods in emzumol ogy 183: (625-645) 0
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be a substitution of a hydrogen atom with a fluorenyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules. -
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? (£ ⁇ ') 2 and? 7, its specificity The epitope that binds to human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • 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 matter 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 animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist in the natural system.
  • Such polynucleotides may be part of a vector, or such polynucleotides or polypeptides may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human ribonucleoside diphosphate reductase inhibitory protein 10.23 refers to human ribonucleoside diphosphate reductase inhibitory protein 10.23 is substantially free of other proteins and lipids naturally associated with it. , Sugar or other substances. Those skilled in the art can purify human ribonucleoside diphosphate reductase inhibitor protein 10.23 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. Human ribonucleoside diphosphate reductase inhibitory protein 10.23 The purity of the polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human ribonucleoside diphosphate reductase inhibitory protein 10.23, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention 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 ribonucleoside diphosphate reductase inhibitory protein 10.23.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human ribonucleoside diphosphate reductase inhibitor protein 10.23 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) one in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues, preferably conservative amino acid residues, And the substituted amino acid may or may not be encoded by a genetic codon; or (II) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or
  • polypeptide sequences (such as leader sequences or secretory sequences or sequences used to purify this polypeptide or protein sequences) As set forth herein, 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).
  • the transcript 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 a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1556 bases in length and its open reading frames 1 11 -392 encode 93 amino acids.
  • this polypeptide has a similar expression profile with human ribonucleoside diphosphate reductase inhibitory protein, and it can be inferred that the human ribonucleoside diphosphate reductase inhibitory protein 10.23 has human ribonucleoside two Phosphoreductase inhibits similar functions of the protein.
  • the polynucleotide of the present invention may be in the DM form or the MA 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.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention. '
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes 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.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least two sequences between 50%, preferably 70% identity).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the 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 co ll, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, 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 ribonucleoside diphosphate reductase inhibitory proteins 10.23.
  • 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 ribonucleoside diphosphate reductase inhibitory protein 10.23 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 DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the CDM of interest is to isolate tnRNA from donor cells that highly express the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction. Kits are also commercially available (Qi agene). Library is constructed cDM conventional method (Sambrook, et al., Mo l ecul ar Cl oni ng, A Labora tory Manua l, Co ld Spr ing Harbor Labora tory. New York, 1989) 0 may be a commercially available cDNA obtained Libraries, such as different cDNA files from Citech. 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 loss of marker gene function; (3) determination of human ribonucleoside diphosphate reductase inhibitory protein 10.23 ⁇ The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and has a length of at least 1Q nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of 10.23 gene expression of human ribonucleoside diphosphate reductase inhibitory protein 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 RACE method RACE- rapid cDNA end amplification method
  • 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 DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the human ribonucleoside diphosphate reductase inhibitor protein 10.23 coding sequence, and that the present invention is produced by recombinant technology Methods of the polypeptide.
  • a polynucleotide sequence encoding the human ribonucleoside diphosphate reductase inhibitor protein 10.23 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • DM sequence of protein 10.23 and expression vector for suitable transcription / translation regulatory elements are known to those skilled in the art. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, eta l.
  • the DM sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include SQ40 enhancer of 10Q to 270 base pairs on the late side of the origin of replication, polyoma enhancer and adenovirus enhancer 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 a human ribonucleoside diphosphate reductase inhibitor protein 10.23 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide containing the polynucleotide or the recombinant vector.
  • Genetically engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl.
  • the steps used are well known in the art.
  • the alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human ribonucleoside diphosphate reductase inhibitor protein 10.23 (Sc ience, 1984; 224: 1431).
  • ribonucleoside diphosphate reductase inhibitor protein 10.23 (Sc ience, 1984; 224: 1431).
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. 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.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salt praying 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 (salt praying method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (
  • Fig. 1 is a comparison diagram of gene chip expression profiles of human ribonucleoside diphosphate reductase inhibitory protein 10.23 and human ribonucleoside diphosphate reductase inhibitory protein of the present invention.
  • the upper graph is a graph of the expression profile of the human ribonucleoside diphosphate reductase inhibitory protein 10.23
  • the lower graph is the graph of the expression profile of the human ribonucleoside diphosphate reductase inhibitory protein.
  • 1-bladder mucosa 2- PMA + Ecv304 cell line, 3-LPS + Ecv304 cell line thymus, 4- normal fibroblasts 1 G24NC, 5- F ibrob l as t, growth factor stimulation, 1024NT, 6-scar Stimulated by fc growth factor, 1013HT, 7-scar scar without stimulation by fc, 1013HC, 8-bladder cancer cell EJ, 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12-liver cancer cell line , 13-fetus, 14-spleen, 15-prostate cancer, 16-jejunum adenocarcinoma, 17 cardia cancer.
  • FIG. 2 is a polyacrylamide gel electrophoresis diagram (SDS-PAGE) of the isolated human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • OkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Example 1 Cloning of human ribonucleoside diphosphate reductase inhibitor protein 10.23
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragments into the pCSK (+) vector (Clontech) at multiple cloning sites to transform DH5 ⁇ . 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 a public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1027G06 was new DNA.
  • a series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone.
  • the 1027G06 clone contained a full-length cDNA of 1556 bp (as shown in Seq ID NO: 1), and an open reading frame (0RF) of 281b P from lllbp to 392bp, encoding a new protein (such as Seq ID NO: 2).
  • This clone pBS-1027G06 and the encoded protein was named human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • Example 2 The gene encoding human ribonucleoside diphosphate reductase inhibitor protein 10.23 was cloned by RT-PCR method. The total RNA of fetal brain cells was used as a template, and ol igo-dT was used as a primer for reverse transcription reaction to synthesize cDNA.
  • PCR amplification was performed with the following primers:
  • Primerl 5'- GGAGGAGGAGGAGCAGCAGCAGCC -3 '(SEQ ID NO: 3)
  • Primer2 5'- TTCTTTGACTGAAAAAAAAGGTGA -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l reaction volume containing 50 ol / L KC1, 10 mmol / L Tris-Cl, ( P H8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer , 1U of 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.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • DNA sequence analysis results showed that The DNA sequence is exactly the same as 1-1556bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human ribonucleoside diphosphate reductase inhibitor protein 10.23 gene expression:
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate ( ⁇ 4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA was synthesized by electrophoresis on a 1.21 ⁇ 2 agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-IraM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the PCR amplified human ribonucleoside diphosphate reductase inhibitor protein 10.23 coding region sequence (lllbp to 392bp;) shown in FIG. 1.
  • the 32P- labeled probes (about 2 x 10 6 cpm / ml) and transferred to a nitrocellulose membrane RNA is hybridized overnight at 4 2 ° C in a solution, the solution containing 50% formamide - 2 5mM KH 2 P0 4 (pH7.4) -5 SSC- 5 x Denhardt, s solution and 20 (g / ml salmon sperm DNA. After hybridization, wash the filter in 1 x SSC- 0.1 ° / »SDS at 55 ° C for 30 min Analysis and quantification using Phosphor Imager.
  • Example 4 In vitro expression, isolation and purification of recombinant human ribonucleoside diphosphate reductase inhibitor protein 10.23 According to the sequence of the coding region shown in SEQ ID NO: 1 and FIG. 1, A pair of specific amplification primers was designed with the following sequence:
  • Primer 3 5'-CATGCTAGCATGCCTTCAGGAGGCAGAGAACTC-3 '(Seq ID No: 5)
  • Primer4 5,-CATGGATCCTCATAAATACTTCATCCCTCTTAT- 3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Nhel and BamHI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene Nhel and BamHI restriction sites correspond to selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • the 1027G06 plasmid was used as a template for the PCR reaction.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ l contains 10 pg of pBS-1027G06 plasmid, primers Primer-3 and Primer-4, and 1 J is 10 pmol, 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 BamHI were used to double-digest the amplified product and plasmid P ET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into E.
  • coli DH5a using the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 30 ⁇ g / ral), positive clones were screened by colony PCR and sequenced. Select positive clones with the correct sequence (pET-1027G06) to transform the recombinant plasmid into E. coli by calcium chloride method BL21 (DE3) plySs (product of Novagen). In LB liquid medium containing kanamycin (final concentration 30 ⁇ 8 / ⁇ 1), the host bacteria BL21 (pET-1027G06) was cultured at 37 C to the logarithmic growth phase, IPTG was added to a final concentration of Immol / L, and the culture was continued. 5 hours.
  • the cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidines (6His-Tag).
  • the purified human protein ribonucleoside diphosphate reductase inhibitor protein 10.23 was purified by SDS-PAGE electrophoresis, and a single band was obtained at 10 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method.
  • NH2-Met-Pro-Ser-Gly-Gly-Arg-Glu-Leu-Met-Tyr-Gly-Cys-Asp-Ile-Pro-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. I unochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex with complete Freund's adjuvant. After 15 days, the rabbit was immunized with hemocyanin-polypeptide complex and incomplete Freund's adjuvant once.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They are all performed after immobilizing a polynucleotide sample to be tested on a filter. Hybridization was performed using essentially the same procedure.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SBQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the 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 genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, 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 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so as to be used in the following experimental steps Wash the film with high strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared after combining the collection solutions of the first peak.
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target DM for gene chip technology for high-throughput research of new gene functions; searching for and screening new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, see DeRisi, J. L., Lyer, V. & Brown, P.0.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. Amplify them separately by PCR, and adjust the concentration of the amplified products to At about 500ng / ul, a Cartesian 7500 spotter (purchased from Cartesian, USA) was used to spot on the glass media, and the distance between the points was 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the raRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP 5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 fluorescent dye (purchased from Amersham Pharaacia Biotech) was used to label mRNA of human mixed tissues, and the fluorescent reagent Cy5dUTP (5- Amino- propargyl- 2'- deoxyuridine 5 --tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Pharaacia Biotech Company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Cy3dUTP 5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 fluorescent dye (purchased from Amersham Pharaacia Bio
  • bladder mucosa 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
  • scar into fc unused growth factor Stimulation 101 3HC
  • bladder cancer construct cells EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell lines, placenta, spleen, prostate cancer, jejunum adenocarcinoma, cardia cancer.
  • 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, HV infection and immune diseases.
  • Ribonucleoside diphosphate reductase is an important enzyme for ATP energy metabolism in the body. It catalyzes the mutual conversion between ATP and AMP, and plays an important catalytic role in the metabolism of many substances. It is the progress of biological processes. Provides the required energy and regulates the normal progress of relevant metabolic processes in the body. The activity of this enzyme in the body is also regulated by a variety of proteins in the body, such as nucleic acid nucleoside diphosphate reductase inhibitor protein.
  • the ribonucleoside diphosphate reductase inhibitor protein discovered in the study works synergistically with ribonucleoside diphosphate in the body to regulate the normal progress of related energy metabolism and material metabolism in the body. Studies have found that mutations or abnormal expression of this protein will cause abnormal metabolism pathways in the body, and then cause metabolic and developmental disorders of various related tissues. The protein is closely related to the occurrence of diseases such as thyroid tumors in young children in the body.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human ribonucleoside diphosphate reductase inhibitory protein, and both have similar biological functions.
  • the polypeptide of the present invention is related to ribonucleoside diphosphate reductase in vivo, and its abnormal expression is usually closely related to the occurrence of pathological processes such as metabolic disorder of some related substances, abnormal protein function, and tumors of related tissues, and cause related diseases.
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, sacral adenoma, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glial cells Tumor, Neurofibromatosis, Colon Cancer, Melanoma, Bladder Cancer, Uterine Cancer, Endometrial Cancer, Colon Cancer, Thymic Tumor, Nasopharyngeal Cancer, Laryngeal Cancer, Tracheal Tumor, Fibroma, Fibrosarcoma, Lipoma, Liposarcoma
  • Embryonic disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, brain development disorders, skin, fat, and muscular dysplasia, bone and joint dysplasia, various metabolic defects, stunting, dwarfism, Cushing's syndrome Sexual retardation
  • 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
  • the abnormal expression of the human ribonucleoside diphosphate reductase inhibitory protein 10.23 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 various tumors, embryonic development disorders, growth and development disorders, inflammation, and immunity. Sexual diseases, certain hereditary, blood diseases, etc. ⁇
  • the invention also provides a method for screening compounds to identify agents that increase (agonist) or repress (antagonist) human ribonucleoside diphosphate reductase inhibitory protein 1.02.
  • Agonists enhance human ribonucleoside diphosphate reductase inhibitory protein 10.23 to 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 ribonucleoside diphosphate reductase inhibitory protein 10.23 can be labeled with a human ribonucleoside diphosphate reductase inhibitory protein 10.23— ⁇ to cultivate. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human ribonucleoside diphosphate reductase inhibitory protein 10.23 include screened antibodies, compounds, receptor deletions, and the like.
  • An antagonist of human ribonucleoside diphosphate reductase inhibitor protein 10.23 can bind to human ribonucleoside diphosphate reductase inhibitor protein 10.23 and eliminate its function, or inhibit the production of the polypeptide, or with the polypeptide The active site binding prevents the polypeptide from performing biological functions.
  • human ribonucleoside diphosphate reductase inhibitory protein 10.23 can be added to the bioanalytical assay, and human ribonucleoside diphosphate reductase inhibitory protein 10.23 ⁇ ⁇ ⁇ The effects of interactions between humans to determine whether 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 ribonucleoside diphosphate reductase inhibitor protein 10.23 can be selected from various possible combinations by screening Amino acids are obtained by binding to a random peptide library consisting of a solid phase. When screening, the human ribonucleoside diphosphate reductase inhibitor protein 10.23 molecule should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human ribonucleoside diphosphate reductase inhibitor protein 10.23 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 produced by injecting human ribonucleoside diphosphate reductase inhibitor protein 10.23 directly 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 and the like.
  • Techniques for preparing monoclonal antibodies against human ribonucleoside diphosphate reductase inhibitor protein 10.23 include, but are not limited to, hybridoma technology (Kohler and Mil stein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cells Hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al, PMS, 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 ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • Antibodies against human ribonucleoside diphosphate reductase inhibitor protein 10.23 can be used in immunohistochemical techniques to detect human ribonucleoside diphosphate reductase inhibitor protein 10.23 in biopsy specimens.
  • Monoclonal antibodies that bind to human ribonucleoside diphosphate reductase inhibitor protein 10.23 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human ribonucleoside diphosphate reductase inhibitor protein 10.23 High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human ribonucleoside diphosphate reductase inhibition Protein 10.23 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human ribonucleoside diphosphate reductase inhibitor protein 10.23 detected in the test can be used to explain the importance of human ribonucleoside diphosphate reductase inhibitor protein 10.23 in various diseases and to diagnose humans Diseases where ribonucleoside diphosphate reductase inhibitor protein 10.23 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry.
  • Polynucleotides encoding human ribonucleoside diphosphate reductase inhibitory proteins 10.23 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 absence or abnormal / inactive expression of human ribonucleoside diphosphate reductase inhibitory protein 10.23. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutant human ribonucleoside diphosphate reductase inhibitor proteins
  • a variant human ribonucleoside diphosphate reductase inhibitor protein 10.23 may be a shortened human ribonucleoside diphosphate reductase inhibitor protein 10.23, although it can be related to downstream Substrate binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human ribonucleoside diphosphate reductase inhibitory protein 10.23.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding human ribonucleoside diphosphate reductase inhibitor protein 10.23 can be used to transfer a polynucleotide encoding human ribonucleoside diphosphate reductase inhibitor protein 10.23 to a cell Inside.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human ribonucleoside diphosphate reductase inhibitory protein 10.23 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding human ribonucleoside diphosphate reductase inhibitory protein 10.23 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 that inhibit human ribonucleoside diphosphate reductase inhibitory protein 10.23 mRNA and ribozymes are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes a specific MA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform endonucleation.
  • Antisense MA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis of oligonucleotides has been widely used.
  • Antisense MA molecules can be obtained by in vitro or in vivo transcription of DM sequences encoding the RNA. This DM sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human ribonucleoside diphosphate reductase inhibitor protein 10.23 can be used for diagnosis of diseases related to human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • a polynucleotide encoding human ribonucleoside diphosphate reductase inhibitor protein 10.23 can be used to detect human ribonucleoside diphosphate reductase inhibitor protein 10.23 expression or abnormal expression of human ribonucleoside diphosphate reductase inhibitory protein 10.23 in a disease state.
  • the DM sequence encoding human ribonucleoside diphosphate reductase inhibitor protein 10.23 can be used to hybridize biopsy specimens to determine the expression of human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also referred to as a "gene chip"), and used to analyze differential expression analysis and gene diagnosis of genes in tissues.
  • Human ribonucleoside diphosphate reductase inhibitor protein 10.23 specific primers can be used to perform MA-polymerase chain reaction (RT-PCR) in vitro amplification to detect the transcription product of human ribonucleoside diphosphate reductase inhibitor protein 10.23.
  • RT-PCR MA-polymerase chain reaction
  • Human ribonucleoside diphosphate reductase inhibitory protein mutations of the 0.23 gene can also be used to diagnose human ribonucleoside diphosphate reductase inhibitory protein-related diseases.
  • Human ribonucleoside diphosphate reductase inhibitor protein 10.23 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human ribonucleoside diphosphate reductase inhibitor protein 10.23 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.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization (FISH) of cDM clones and 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 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human ribonucleoside diphosphate reductase inhibitory protein 10.23 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount of human ribonucleoside diphosphate reductase inhibitory protein 10. 2 3 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine de régulation 10.23 d'une ribonucléoside-diphosphate réductase, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de troubles du développement, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine humaine de régulation 10.23 d'une ribonucléoside-diphosphate réductase.
PCT/CN2001/000983 2000-06-19 2001-06-18 Nouveau polypeptide, proteine humaine de regulation 10.23 d'une ribonucleoside-diphosphate reductase, et polynucleotide codant ce polypeptide Ceased WO2002012303A1 (fr)

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AU93621/01A AU9362101A (en) 2000-06-19 2001-06-18 A novel polypeptide-human ribonucleoside dephosphate reductase inhibitor 10.23 and the polynucleotide encoding said polypeptide

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CN00116569.0 2000-06-19
CN00116569A CN1329038A (zh) 2000-06-19 2000-06-19 一种新的多肽——人核糖核苷二磷酸还原酶抑制蛋白10.23和编码这种多肽的多核苷酸

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WO2002012303A1 true WO2002012303A1 (fr) 2002-02-14

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984000888A1 (fr) * 1982-09-01 1984-03-15 Univ Southern California N-benzenesulfonyloxyphthalimides substitues

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984000888A1 (fr) * 1982-09-01 1984-03-15 Univ Southern California N-benzenesulfonyloxyphthalimides substitues

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CN1329038A (zh) 2002-01-02

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