WO2002026794A1 - Nouveau polypeptide, proteine ribosomale humaine l7a20.57, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine ribosomale humaine l7a20.57, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002026794A1
WO2002026794A1 PCT/CN2001/001137 CN0101137W WO0226794A1 WO 2002026794 A1 WO2002026794 A1 WO 2002026794A1 CN 0101137 W CN0101137 W CN 0101137W WO 0226794 A1 WO0226794 A1 WO 0226794A1
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polypeptide
polynucleotide
ribosomal protein
human ribosomal
sequence
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PCT/CN2001/001137
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU2002223376A priority Critical patent/AU2002223376A1/en
Publication of WO2002026794A1 publication Critical patent/WO2002026794A1/fr

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, human ribosomal protein L7a20.57, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique
  • the ribosomal protein L7a is an important component of the large ribosomal subunit.
  • domain 1, domain 2, and domain 3 There are three conserved characteristic domains in the amino acid sequence of the protein, domain 1, domain 2, and domain 3.
  • Each domain contains at least one nuclear localization signal fragment (NLS), which plays an important regulatory role in the nuclear localization of the protein.
  • Domain 2 regulates the aggregation and activity of proteins and their receptors in vivo. Mutations or abnormal expression of these domains will directly affect the protein's localization and action activity in the nucleus, and then affect the normal assembly and function of the cell's inner ribosomes.
  • the ribosomal protein L7a is an important component of the ribosome subunit in the body, and is involved in regulating the composition and action activity of the ribosome subunit.
  • the mutation or abnormal expression of this protein will lead to abnormal synthesis of the core ribosomes of the cell, and then affect the progress of the related protein synthesis process.
  • Modified protein is usually closely related to the developmental functions of some related tissues, such as developmental and metabolic disorders, related tumors, and cancer.
  • human ribosomal protein L7a20 The present invention is named human ribosomal protein L7a20. 57.
  • human ribosomal protein L7a20. 57 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art to identify more human ribosomal proteins L7a20 that are involved in these processes. 57 proteins, especially the amino acid sequence of this protein.
  • the isolation of the new human ribosomal protein L7a20. 57 protein encoding gene 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 agents for disease 1 and it is therefore important to isolate its coding DNA. Disclosure of invention
  • An object of the present invention is to provide an isolated novel polypeptide-human ribosomal protein L7a20. 57 and
  • 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 ribosomal protein L7a20.57.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human ribosomal protein L7a20.57.
  • Another object of the present invention is to provide a method for producing human ribosomal protein L7a20.57.
  • Another object of the present invention is to provide antibodies against the polypeptide of the present invention, human ribosomal protein L7a20.57.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human ribosomal protein L7a20.57.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human ribosomal protein L7a20.57.
  • 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 724-1287 in SEQ ID NO: 1; and (b) having a sequence of 1-1597 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing a polynucleotide of the invention;
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell; a method for preparing a polypeptide of the present invention comprising culturing the host cell and recovering an 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 ribosomal protein L7a20.57 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of human ribosomal protein L7a 20.57 protein in vitro, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or Detection of 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 other polypeptides and / or polynucleotides of the present invention prepared for the treatment of ⁇ cancer, developmental or immune disease ⁇ or other diseases of the present invention caused by abnormal expression of human ribosomal protein L7a20.57. Aspects will be apparent to those skilled in the art from the disclosure of the techniques herein.
  • 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 RM, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • “Insertion” or “addition” 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.
  • Biological activity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • the term “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 ribosomal protein L7a20.57, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind the human ribosomal protein L7a20.57.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human ribosomal protein L7a20.57 when combined with human ribosomal protein L7a20.57.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human ribosomal protein L7a20.57.
  • Regular refers to a change in the function of human ribosomal protein L7a20.57, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of human ribosomal protein L7a20.57. change.
  • Those skilled in the art can purify human ribosomal protein L7a20. 57 using standard protein purification techniques. Basic The pure human ribosomal protein L7a20. 57 can generate a single main band on a non-reducing polyacrylamide gel. The purity of human ribosomal protein L7a20. 57 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • 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 (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: Number of residues matching between sequence A and sequence B
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or using methods known in the art such as Jo tun He in. (He in J., (1990) Me thods in emzurao l 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 may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? 0 ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of human ribosomal protein L7 a20. 57.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human ribosomal protein L7a20. 57 means that the human ribosomal protein Ja L is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. 57. Those skilled in the art can purify human ribosomal protein L7 a20. 57 using standard protein purification techniques. Essentially pure peptide A single main band can be generated on a non-reducing polyacrylamide gel. The purity of human ribosomal protein L7a20. 57 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human ribosomal protein L7a20.57, 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 invention can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the human ribosomal protein L7a20.57.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human ribosomal protein L7a20.57 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) such a type in which one or more amino acid residues are substituted with other groups to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1597 bases in length and its open reading frame 724-1287 encodes 187 amino acids.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DM forms include cDNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the present invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% ( ⁇ / ⁇ ) formamide, 0.1% calf serum / 0.1% F ico 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 nucleotides. Nucleotides 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 ribosomal protein L7a20.57.
  • 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 ribosomal protein Ua20.57 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. Isolate cDNA of interest The standard method is to isolate raRNA from donor cells that highly express the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many proven techniques for extracting mRM, and kits are also commercially available (Qiagene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Labora tory Manua, Co. Harbor Labora tory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of the human ribosomal protein L7a20.57 transcript; ( 4) Detecting gene-expressed protein products 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 its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2,000 nucleotides, and preferably within 1,000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of the human ribosomal protein L7a20.57 gene can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method (Sa ik i, et al. Science 1985; 230: 1350-1354) using DNA technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DM / RNA fragment 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 produced by genetic engineering using the vector of the present invention or directly using human ribosomal protein L7a20.57 coding sequence, and a recombinant technology to produce a polypeptide of the present invention. method.
  • a polynucleotide sequence encoding human ribosomal protein L7a20.57 can be inserted into a vector, In order 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.
  • 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.
  • An expression vector for DNA sequences and appropriate transcriptional / translational regulatory elements include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Labora tory Manua l, cod Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human ribosomal protein L7a20.57 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Representative examples are: Large intestine Bacillus, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as flies S2 or Sf 9; animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl, the steps used are well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, 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 ribosomal protein L7 a20. 57 (Scence, 1984; 224: 1431). 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.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human ribosomal protein L7a20.57 and human ribosomal protein L7a according to the present invention.
  • the upper graph is a graph of the expression profile of human ribosomal protein L7a20. 57, and the lower graph is the graph of the expression profile of human ribosomal protein L7a.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human ribosomal protein L7a20.57.
  • 21 kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Example 1 Cloning of human ribosomal protein L7a20.57
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Quik mRNA Isolat ion Kit product of Qiegene was used to isolate poly (A) raRNA 0 2ug poly (A) mRNA from reverse transcription to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragments into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5a. 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 an existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0301d03 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imer l 5 '-GGCACAGTGACAGCTTCCTTTCTC -3' (SEQ ID NO: 3)
  • Pr imer2 5, — TTTCTGTCCACCTACCATTAGGTG- 3,
  • Pr imerl is the 5th, lbp terminal of SEQ ID NO: 1
  • Start forward sequence Pr iraer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 ⁇ l of ol / L C1, 10 mmol / L of Tris-CI, (pH 8.5.5), 1.5 ⁇ l / L MgCl 2 , 200 ⁇ raol in a reaction volume of 50 ⁇ 1 / L dNTP, Opmo primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -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 using a TA cloning kit (Invitrogen).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-1597bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human ribosomal protein L7a20. 57 gene expression: Total RNA was extracted in one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. With 20 U g RNA, containing 2 0mM 3- (N - morpholino) propanesulfonic acid (pH7, 0) - 5 mM sodium acetate -.
  • ImM EDTA-2 2M on a 1.2% formaldehyde agarose gel Perform electrophoresis. It was then transferred to a nitrocellulose membrane.
  • A- 32 P dATP was used to prepare 32 P-labeled DM probes by random primers.
  • the DNA probe used was the PCR amplified human ribosomal protein L7a20.57 coding region sequence (724bp to 1287bp) shown in FIG.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.
  • Example 4 In vitro expression, isolation and purification of recombinant human ribosomal protein L7a20. 57 According to the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:
  • Pr imer 3 5 '-CCCCATATGATGGATTATGCAGCTCAGAAGTAT -3, (Seq ID No: 5)
  • Pr imer 4 5,-CCCGAGCTCTTATGACAGAGGGCTTAACATTTC -3, (Seq ID No: 6)
  • the 5' ends of these two primers contain Mel and BamHI respectively.
  • Site, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • the PCR reaction was performed using the pBS-0301d03 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0301d03 plasmid, primers? 1 "1016: 1: -3 and? 1: 111161--4 minutes!] Is 10 0101, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, 25 cycles in total.
  • Example 5 Production of anti-human ribosomal protein L7a20.57 antibody
  • the following polypeptide specific to human ribosomal protein L7a20.57 was synthesized using a peptide synthesizer (product of PE Company): NH2- Met-Asp-Tyr-Ala-Ala- Gln- Ly s-Ty r-Va 1-G 1 y-Thr-H i s-As p-Phe-Ar -COOH (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • 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 membrane hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods, etc., all of which fix the polynucleotide sample to be tested on the filter The membranes were hybridized 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 SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments 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, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate 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 that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • High-intensity washing film 1) Take out the hybridized sample membrane.
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, see DeRi si, JL, Lyer, V. & Brown, P. 0 (1997) Science 278, 680-686. And He l le, RA, Schema. , M., Cha i, A., Sha lom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDM are used as target DM, 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 Company, USA) was used to spot on the glass medium, and the distance between the spots was 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DM on the glass slides to prepare chips. The specific method steps are widely 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 tnRNA was purified by Ol igotex mRNA Midi Ki t (purchased from QiaGeri).
  • the fluorescent reagent Cy3dUTP (5-Amino-propargy 1-2 ⁇ -deoxyur i dine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amershara Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP ( 5- Amino-propargyl-2'- deoxyuridine 5'-tr ip ate coupled to Cy5 f luorescent dye, purchased from Amershara Phamacia Biotech company, labeled the body's specific tissue (or stimulated cell line) mRNA, purified and prepared to detect Please refer to the specific steps and methods:
  • Solut ion (purchased from TeleChem) was used for hybridization for 16 hours, and washed with a washing solution (lx SSC, 0.2 Jan.) at room temperature, and then scanned with a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned image For example, the data were analyzed and processed by Iraagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line, thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar formation fc growth factor stimulation, 1013HT, scar into unused fc Long factor stimulation, 101 3HC, bladder cancer construct cells EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell lines, placenta, spleen, prostate cancer, jejunal adenocarcinoma, and cardia cancer. Draw a chart based on these 18 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profile of human ribosomal protein L7a20. 57 and human ribosomal protein L7a according to the present invention are very similar. Industrial applicability
  • 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 malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • Ribosomal protein L7a is a new ribosomal protein, which is an important component of the ribosome subunit in the body; it is involved in regulating the composition and action of the ribosome subunit.
  • the mutation or abnormal expression of this protein will lead to abnormal synthesis of the core ribosomes of the cell, and then affect the progress of the protein synthesis process related to it.
  • This protein is usually related to the developmental disorders of some related tissues and the development of disorders of protein metabolism.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human ribosomal protein L7a protein, and both have similar biological functions.
  • the polypeptide of the present invention is an important component of the ribosome subunit in vivo, and is involved in regulating the composition and action activity of the ribosome subunit.
  • the mutation or abnormal expression of this protein will lead to abnormal synthesis of nuclear ribosomes, and then affect the progress of related protein synthesis processes. In turn, it leads to developmental disorders of related tissues and disorders of protein metabolism. These diseases include, but are not limited to:
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus dysplasia syndrome, strabismus, skin, fat and muscular dysplasia such as congenital skin laxity, premature aging Dysfunction, congenital keratosis, various metabolic defects such as stunting, dwarfism, sexual retardation, etc .;
  • Disturbances in protein metabolism can affect the following major physiological functions of proteins, which can lead to the occurrence of related diseases: 1. Provide body energy; maintain tissue growth, renewal and repair:
  • 1 protein peptide hormone dysfunction can cause the following diseases:
  • Insulin and glucagon diabetes, hypoglycemia, etc .;
  • Hypothalamus and pituitary hormones giant disease, dwarfism, acromegaly, cortisol syndrome (Cushing's syndrome), primary aldosteronism, secondary chronic adrenal insufficiency, hyperthyroidism Onset, Hypothyroidism (Small disease, Juvenile hypothyroidism, Adult hypothyroidism), Male / female infertility, Menstrual disorders (functional uterine bleeding, amenorrhea, polycystic ovary syndrome, premenstrual stress syndrome) Disease, menopausal syndrome), sexual development disorder, diabetes insipidus, inappropriate antidiuretic hormone secretion syndrome, abnormal lactation, etc .;
  • parathyroid hormone hyperparathyroidism, hypoparathyroidism, etc .
  • Gastrointestinal hormones peptic ulcer, chronic indigestion, chronic gastritis, etc .;
  • hemoglobinopathy anemia, jaundice, tissue hypoxia-induced organic acidemia
  • various coagulation factor deficiency bleeding
  • muscle spasm muscle forcing
  • muscle paralysis actin
  • Ribosomal protein L7a protein is a new type of ribosomal protein. Mutation or abnormal expression of this protein will lead to abnormal synthesis of the core ribosomes of the cell, and then affect the progress of the related protein synthesis process. It is known that tumor tissues can synthesize some tumor-associated proteins; such as a fetal protein (liver cancer), fetal saccharoprotein (gastric cancer), etc., ribosomal protein L7a protein. Mutation or abnormal expression of this protein will lead to abnormal synthesis of cell core glycosomes. This in turn affects the synthesis of proteins, which can produce some heterogeneous proteins, which may be related to the occurrence of some tumors.
  • the expression profile of the polypeptide of the present invention is consistent with the expression of the human ribosomal protein L7a protein, and both have similar biological functions. Mutation or abnormal expression of the polypeptide of the present invention will lead to abnormal synthesis of the core ribosomes of the cell, and then affect protein synthesis. Some heterogeneous proteins may be produced, which may be related to the occurrence of some tumors. These diseases include, but are not limited to:
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine myometrium, neuroblastoma, astrocytoma, ependymoma, glioblastoma, Colon cancer, malignant histiocytosis, melanoma, teratoma, sarcoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, Colon cancer, thymic tumor, nasal cavity and sinus tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, pleural mesothelioma, fibroid, fibrosarcoma, lipoma, liposarcoma, leiomyoma, etc .;
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human ribosomal protein L7a20.57.
  • Agonists enhance human ribosomal protein L7a20.
  • 57 stimulates biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • 57 can be cultured together with labeled human ribosomal protein L7a20. 57 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human ribosomal protein L7a20.57 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human ribosomal protein L7a20. 57 can bind to human ribosomal protein L7a20. 57 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot exert its biology Features.
  • human ribosomal protein L7a20. 57 can be added to bioanalytical assays to determine whether a compound is a compound by measuring the effect of the compound on the interaction between human ribosomal protein L7a20.57 and its receptor. Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above.
  • Polypeptide molecules capable of binding to human ribosomal protein L7a20.57 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human ribosomal protein L7a20. 57 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human ribosomal protein L7a20.57 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 ribosomal protein L7a20. 57 directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's Agent. Techniques for preparing monoclonal antibodies to human ribosomal protein L7a20.57 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridization Tumor 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, 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 ribosomal protein L7a20.57.
  • Antibodies against human ribosomal protein L7a20. 57 can be used in immunohistochemical techniques to detect human ribosomal protein L7a20. 57 in biopsy specimens.
  • Monoclonal antibodies bound to human ribosomal protein L7a20.57 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.
  • L7a20 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 ammonia of the antibody with a thiol crosslinker such as SPDP.
  • SPDP thiol crosslinker
  • 57 through the exchange of disulfide bonds to bind toxins to antibodies, this hybrid antibody can be used to kill human ribosomal protein L7a20.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human ribosomal protein L7a20.57.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human ribosomal protein L7a20.57.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human ribosomal protein L7a20.57 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human ribosomal protein L7a20.57 detected in the test can be used to explain the importance of human ribosomal protein L7a20.57 in various diseases and to diagnose diseases in which human ribosomal protein L7a20.57 plays a role.
  • the polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis. '
  • the polynucleotide encoding human ribosomal protein L7a20.57 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 ribosomal protein L7a20.57. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human ribosomal protein L7a20.57 to inhibit endogenous human ribosomal protein L7a20.57 activity.
  • a mutated human ribosomal protein L7a20.57 may be a shortened human ribosomal protein L7a20.57, which lacks a signaling functional domain.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human ribosomal protein L7a20.57.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the polynucleotide encoding human ribosomal protein L7a20.57 into cells.
  • Methods for constructing a recombinant viral vector carrying a polynucleotide encoding human ribosomal protein L7a20.57 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human ribosomal protein L7a20.57 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human ribosomal protein L7a20.57 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RM. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence is integrated downstream of the vector's RNA polymerase promoter. To increase the stability of nucleic acid molecules, they can be modified in a variety of ways. For example, if the sequence length on both sides is increased, the linkage between ribonucleosides should use phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human ribosomal protein L7a20. 57 can be used for the diagnosis of diseases related to human ribosomal protein L7a20. 57.
  • the polynucleotide encoding human ribosomal protein L7a20. 57 can be used to detect the expression of human ribosomal protein L7a20. 57 or the abnormal expression of human ribosomal protein L7a20. 57 in a disease state.
  • a DNA sequence encoding human ribosomal protein L7a20. 57 can be used to hybridize biopsy specimens to determine the expression status of human ribosomal protein L7a20. 57.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • 57 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human ribosomal protein L7a20.
  • 57 transcripts Detection of mutations in the human ribosomal protein L7a20.
  • 57 gene can also be used to diagnose human ribosomal protein L7a20. 57-related diseases.
  • Human ribosomal protein L7a20.57 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human ribosomal protein L7a20.57 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, the mutation may affect the expression of the protein, so the Nor thern blotting and Wes tern blotting can be used to indirectly determine whether there is a mutation in the gene.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • 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 (FISH) of cDNA clones with metaphase chromosomes can be refined in one step Perform chromosomal mapping accurately.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.
  • the difference in cDM or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human ribosomal protein L7a20. 57 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human ribosomal protein L7a20.57 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 ribosomale humaine L7a20.57, 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 de maladies liées aux troubles du métabolisme des protéines et aux troubles du développement et d'études menées en vue du traitement de diverses tumeurs de même que les méthodes thérapeutiques apparentées. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine ribosomale humaine L7a20.57.
PCT/CN2001/001137 2000-07-07 2001-07-02 Nouveau polypeptide, proteine ribosomale humaine l7a20.57, et polynucleotide codant ce polypeptide WO2002026794A1 (fr)

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CN 00117098 CN1333278A (zh) 2000-07-07 2000-07-07 一种新的多肽——人核糖体蛋白L7a20.57和编码这种多肽的多核苷酸
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4865970A (en) * 1986-02-28 1989-09-12 Hoffmann-La Roche Inc. Method of detecting ribosomal protein antibodies in systemic lupus erythematosus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4865970A (en) * 1986-02-28 1989-09-12 Hoffmann-La Roche Inc. Method of detecting ribosomal protein antibodies in systemic lupus erythematosus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COLOMBO P. ET AL., NUCLEIC ACIDS RES., vol. 20, no. 13, 11 July 1992 (1992-07-11), pages 3367 - 3373 *
GIALLONGO A. ET AL., MOL. CELL. BIOL., vol. 9, no. 1, January 1989 (1989-01-01), pages 224 - 231 *
WANG Y. ET AL., INT. J. ONCOL., vol. 16, no. 4, April 2000 (2000-04-01), pages 757 - 762 *

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