WO2002000866A1 - Nouveau polypeptide, aminoacyl-arnt-synthetase humaine 10 de type i, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, aminoacyl-arnt-synthetase humaine 10 de type i, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000866A1
WO2002000866A1 PCT/CN2001/000992 CN0100992W WO0200866A1 WO 2002000866 A1 WO2002000866 A1 WO 2002000866A1 CN 0100992 W CN0100992 W CN 0100992W WO 0200866 A1 WO0200866 A1 WO 0200866A1
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polypeptide
aminoacyl
polynucleotide
trna synthetase
human class
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PCT/CN2001/000992
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc.
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Priority to AU93627/01A priority Critical patent/AU9362701A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human class I aminoacyl-tRNA synthetase 10, 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
  • tRNAs function to map the codons of the mRNA to the corresponding amino acids. There is no structural correlation between the anti-codon on the tRNA molecule and its corresponding amino acid, and the tRNA itself cannot catalyze the load of the corresponding amino acid.
  • the reaction that combines tRNA with the corresponding amino acid is synthesized by aminoacyl-tRNA. Enzymatic.
  • aminoacyl tRM synthetase The reaction of aminoacyl tRM synthetase to synthesize aminoacyl-tRNA is divided into two steps: the amino acid is first activated into aminoacyl adenylate, and then aminoacyl adenylate and tRNA generate aminoacyl-tRNA.
  • Aminoacyl-tRNA synthetase recognizes different tRNAs through the "codon" on the tRM molecule, so that the correct amino acid and tRNA each amino acid corresponds to an aminoacyl-tRNA synthetase, so 20 amino acids
  • aminoacyl-tRNA synthetases There are 20 different aminoacyl-tRNA synthetases. But these 20 aminoacyl-tRNA synthetases are related to each other.
  • the 20 aminoacyl-tRNA synthetases can be divided into two classes, class I and class I.
  • the catalytic part of the class I enzyme contains a Rossmann fold backbone, which is a nucleic acid binding region; the backbone of the class I enzyme consists of a new antiparallel fold.
  • aminoacyl-tRNA synthetase Some N-terminal sequences of aminoacyl-tRNA synthetase are quite conserved, especially the peptide sequence Hi sI le-Gly-Hi s ('HIGH') o This sequence is in arginine, tryptophan, glutamic acid, isoleucine
  • aminoacyl-tRNA synthetases of acids, methionine, tyrosine, etc. are found. They are all class I synthetases.
  • Aminoacyl-tRNA synthetases provide a usable aminoacyl-tRNA for protein synthesis. If a certain aminoacyl-tRNA synthetase is deleted, the corresponding aminoacyl-tRNA will not be formed correctly, resulting in the premature termination of the protein synthesis process and the formation of a non-functional protein. If a certain aminoacyl-tRNA synthetase is mutated to recognize another amino acid, the resulting protein contains an incorrect amino acid sequence. If the mutation occurs at an amino acid site that is critical to the function of the protein, then The formed protein will not fold properly without function, or the function is weakened or enhanced, or the functioning substrate is changed to change its function Into. This functional change often leads to disease.
  • Ras-GTP cannot be converted into Ras-GDP, which results in Ras being in an active state for a long time, sending signals to the downstream without stopping, causing abnormal proliferation of cells, and thus causing cancer.
  • Mutations in aminoacyl-tRNA synthetase may also cause protein sorting and secretion disorders, and membrane receptor dysfunction, which can cause various endocrine diseases, immune system diseases, and neuromuscular system diseases.
  • the protein of the present invention contains a characteristic conserved region of class I aminoacyl-tRNA synthetase, and analysis of the expression profile reveals that the expression level of class II aminoacyl-tRNA synthetase in normal liver tissue is higher than that in liver cancer tissue. High volume, the same findings were found in normal laryngeal and laryngeal cancer tissues.
  • the polypeptide of the present invention is considered to be a new class I aminoacyl-tRNA synthetase, which has similar biological functions, and is named human class I aminoacyl-tRNA synthetase 10.
  • 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 class I aminoacyl-tRNA synthetase 10.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human class I aminoacyl-tRNA synthetase 10.
  • Another object of the present invention is to provide a method for producing human class I aminoacyl-tRM synthetase 10.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human class I aminoacyl-tRNA synthetase 10.
  • 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 214-501 in SEQ ID NO: 1; and (b) a sequence having positions 1-2004 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human Class I aminoacyl-tRM synthetase 10 protein activity, 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 class I aminoacyl-tRNA synthetase 10 protein in vitro, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human class I aminoacyl-tRNA synthetase 10 .
  • 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. Similar
  • 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 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 can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of 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” means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature. "Replacement” refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides. '
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human class I aminoacyl-tRNA synthetase 10, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human class I aminoacyl-tRNA synthetase 10.
  • Antagonist refers to a biological or immunological activity that can block or modulate human class I aminoacyl-tRNA synthetase 10 when combined with human class I aminoacyl-tRNA synthetase 10 Molecule.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds to a human class I aminoacyl-tRNA synthetase.
  • Regular refers to a change in the function of human class I aminoacyl-tRNA synthetase 10, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties of human class I aminoacyl-tRNA synthetase 10. , Functional or immune properties.
  • Substantially pure ' means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human class I aminoacyl-tRNA synthetases using standard protein purification techniques. 10. Basically pure human class I aminoacyl-tRNA synthetase 10 produces a single main band on a non-reducing polyacrylamide gel. The purity of human class I aminoacyl-tRNA synthetase 10 polypeptides can be analyzed by amino acid sequence .
  • Complementary or “complementary” refers to multicores that are base-paired by the allowed salt concentration and temperature conditions Nucleotides naturally bind.
  • the sequence "CT-GA” can be combined with the complementary sequence "GA-C-T”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands 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 or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other specifically or selectively.
  • 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 (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the 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 Clus ter method or using methods known in the art such as Jotun Hein.
  • the percent identity between nucleic acid sequences (Hein J., (1990) Methods in emzumology 183: 625-645) 0 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or MA 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. This chemical modification can be used An alkyl, acyl or amino group replaces a hydrogen atom. 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,? ( & 13 ') 2 &? ⁇ It can specifically bind to human epitope of class I aminoacyl-tRM synthase 10.
  • 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 occurs naturally).
  • 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 with it in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not separated and purified, but the same polynucleotides or polypeptides are separated and purified from other substances existing in the natural state. of.
  • isolated human class I aminoacyl-tRNA synthetase 10 means that human class I aminoacyl-tRNA synthetase 10 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify human class I aminoacyl-tRNA synthetase 10 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human class I aminoacyl-tRNA synthetase 10 polypeptides can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a human class I aminoacyl-tRNA synthetase 10, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of human class I aminoacyl-tRNA synthetase 10.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human class I aminoacyl-tMA synthetase 10 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 conserved or non- Conservative amino acid residues (preferably conservative amino acid residues) are substituted, and the substituted amino acid may or may not be encoded by the genetic codon; or ( ⁇ ) such a type in which one or more of the amino acid residues Each group is substituted with another group to include a substituent; or (III) a type in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or
  • a polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) formed by fusing additional amino acid sequences into a mature polypeptide.
  • a polypeptide sequence such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence
  • 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 with a full length of 2004 bases, and its open reading frame (214-501) encodes 87 amino acids.
  • This polypeptide has the characteristic sequence of human class I aminoacyl-tRNA synthetase, and it can be deduced that the human class I aminoacyl-tRNA synthetase 10 has the structure and function represented by human class I aminoacyl-tRM synthetase.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • DNA forms include cDM, 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 (having at least 50%, preferably 70% identity between the two sequences).
  • the invention particularly relates to polynucleosides according to the invention under stringent conditions Acid hybridizable polynucleotide.
  • “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% Ficol l, 42 ° C, etc .; or (3) only between the 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 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human class I aminoacyl-tRNA synthetase 10.
  • 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 class I aminoacyl-tRNA synthetase 10 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 DM sequence from the genome DM; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cMA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sarabrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spruing Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • 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 transcript of human class I aminoacyl-tRM synthetase 10 Level; (4) detecting protein products of gene expression by immunological techniques or measuring biological activity. The above methods can be used alone 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 10 nucleotides, preferably at least 3G nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides. It is 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).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein products of human class I aminoacyl-tMA synthase 10 gene expression.
  • ELISA enzyme-linked immunosorbent assay
  • a method using DNA technology to amplify DNA / RM (Sa iki, et al. Science 1985; 230: 1 350-1 354) is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-cDM terminal rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DM / RM 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 ai. 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 cDM 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 a human class I aminoacyl-tRNA synthetase 10 coding sequence, and recombinant technology to produce the present invention. Said method of polypeptide.
  • a polynucleotide sequence encoding human class I aminoacyl-tRNA synthetase 10 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 (Ros enberg, 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 replication origins, promoters, marker genes, and translational control methods. Methods that are well known to those skilled in the art can be used to construct DNA sequences containing human class I aminoacyl-tRNA synthetase 10 and Expression vectors for appropriate transcription / translation regulatory elements.
  • DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human class I aminoacyl-tRNA synthetase 10 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the MA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, 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.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human class I aminoacyl-tRNA synthetase 10 (Science, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • 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 amino acid sequence homology of 47 amino acids in a total of 3-49 of the class I aminoacyl-tRNA synthetase 10 of the present inventors and characteristic domains of the class I aminoacyl-tRNA synthetase of the present invention.
  • the upper sequence is human class I aminoacyl-tRNA synthetase 10
  • the lower sequence is the characteristic domain of human class I aminoacyl-tRNA synthetase.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".
  • Figure 1 is a polyacrylamide gel electrophoresis diagram of isolated human class I aminoacyl-tRM synthetase 10 (SDS-PAGE l OkDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Best way
  • RNA was isolated from total RM with Quik mRNA Isola t ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech
  • Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones.
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1325b06 was a new DM.
  • a series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone.
  • the results show that the 1325b06 clone contains a full-length CDM of 2004 bp (as shown in Seq ID NO: l), a 264 bp open reading frame (0RF) from 4 bp to 501 bp, and encodes a new protein (such as Seq ID NO : Shown in 2).
  • Example 2 Domain analysis of cDNA clones
  • the sequence of the human class I aminoacyl-tRNA synthetase 10 and the encoded protein sequence of the present invention were profiled by GCF using the prof i le scan program (Basiclocal Information Search tool) [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], domain analysis was performed in databases such as Prote. 07 , Score
  • the homology of human class I aminoacyl-tRNA synthetase 10 of the present invention is homologous to the domain human class I aminoacyl-tRNA synthetase at 3-49. The homology results are shown in FIG. Is 3. 43; the threshold is 3. 42.
  • Example 3 Cloning of a gene encoding human class I aminoacyl-tRNA synthetase 10 by RT-PCR
  • 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:
  • Primer 1 5'-CATCCTGAGAACTGAAATTGATCGC-3 '(SEQ ID NO: 3)
  • Primer2 5'-ATAAAATTTTTGAATTTATGTTCAA-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KCl, 10 mraol / L Tri s-Cl, (pH 8.5.5), 1.5 ramol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol in a reaction volume of 50 ⁇ 1 Primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2rain 0 ⁇ -act in was set as positive during RT-PCR Controls and template blanks are negative controls.
  • Amplification products were purified using QIAGEN kits and TA The cloning kit was ligated to a pCR vector (Invitrogen). DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-2004bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human class I aminoacyl-tRNA synthetase 10 gene expression:
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH 4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), 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.
  • 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with RM-transferred nitrocellulose membrane at 42 ° C overnight in a solution.
  • the solution Contains 50% formamide-25mM KHjPO, (pH7.4)-5xSSC-5xDenhardt's solution and 200 yg / ml salmon sperm DNA.
  • the filter was washed in 1xSSC-0.1% SDS at 55 ° C and washed at 30 ° C. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 Recombinant human class I aminoacyl-tRNA synthetase 10 in vitro expression, isolation and purification
  • Primer3 5-CCCCATATGATGCTCTGTCACCTTCAAAGGATGG-3 '(Seq ID No: 5)
  • Priraer4 5' -CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC- 3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and BaraHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are respectively followed by Nhel and BamHI restriction sites corresponding to the selective endonuclease sites on the expression vector plasmid pET28b (+) (Novagen, Cat. No, 69865.3). point.
  • the PCR reaction was performed using pBS-1325b06 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ l containing 10 pg of pBS-1325b06 wide grain, primers Primer-3 and Primer-4 l ⁇ l0praol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68. C 2 niin, a total of 25 cycles. Nhel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into the colibacillus DH5 ⁇ using the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive clones were selected by colony PCR method and sequenced. Selected positive clones with the correct sequence (P ET-1325b06) recombinant plasmids by the calcium chloride method to transform E. Bacillus BL21 (DE3) plySs (product of Novagen). In LB liquid medium containing kanamycin (final concentration 30 g / ral), the host strain BL21 (pET-1325b06) was at 37. C.
  • 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. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned jk cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • a titer plate coated with a 15 M g / ml bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method demonstrated that the purified antibody could specifically bind to human class I aminoacyl-tRNA synthetase 10.
  • Example 7 Use of a polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter 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 from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known 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 (41 Nt):
  • Probe 1 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 membrane nitrocellulose membrane
  • High-intensity washing film 1) Take out the hybridized sample membrane.
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • tRNA functions to map the codons of the mRNA to the corresponding amino acids.
  • the reaction that combines tRNA with the corresponding amino acid is the aminoacyl-tRNA synthetase Catalytic.
  • Each amino acid corresponds to an aminoacyl-tRNA synthetase, so there are 20 different aminoacyl-tMA synthetases for 20 amino acids.
  • This specific conserved sequence is necessary for the formation of aminoacyl-tRNA synthetase and its activity mot if .
  • the abnormal expression of this characteristic sequence will cause the loss of aminoacyl-tRNA synthetase function, cause protein translation failure, produce abnormal or / and non-functional proteins, and then cause various tumors, protein metabolism disorders and the like.
  • the novel polypeptide of the present invention has high homology and similarity with human aminoacyl-tRNA synthetase protein in structure and function, and the amino acid sequence contains the above-mentioned conserved characteristic sequence template.
  • the abnormal expression of the sequence will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, which will cause the translation failure of the protein, produce abnormal or / and non-functional proteins, and then cause various tumors, protein metabolism disorders, etc., These diseases include, but are not limited to: Common tumors in various tissues:
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix 1, adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenomas (carcinoma) [ovary], basal cell carcinoma [head and face Skin], (malignant) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .;
  • Fibrosarcoma [limb I, (malignant) fibrous histiocytoma [limb L liposarcoma [subcutaneous tissue, lower limb, retroperitoneum], smooth muscle (sarcoma) [uterine and gastrointestinal], striated muscle ( Sarcoma [Head and Neck, Genitourinary Tract, Limbs], Hemangio (sarcoma), Lymphatic (sarcoma) [Skin, Subcutaneous Tissue, Tongue, Lip], Bone (Meat) Tumor [Skull, Long Bone], (Evil) Giant cell tumor [femoral / tibia / upper humerus], cartilage (sarcoma) [hand and foot short bone, pelvis / rib / femoral / humerus / scapula], synovial (sarcoma) tumor [knee / ankle / wrist / shoulder / elbow] Near joints], (malignant) mesothelioma [thoracic /
  • Malignant lymphoma [Neck, mediastinum, mesentery and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [push / thoracic / rib / skull and long bone], etc .;
  • Nerve fiber [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannoma [nervous of head, neck, limbs, etc.], (malignant) glioblastoma [brain], myeloblastoma Cerebellum], (malignant) meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .; 5.
  • Other tumors :
  • malignant melanoma [skin, mucous membrane], (malignant) hydatidiform mole, chorionic epithelial cancer [uterine], (malignant) supporter cells, stromal cell tumor, (malignant) granulosa cell tumor [ovarian, testicular] Blastoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum and palate tail], etc .;
  • 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 Hypothyroidism (stingle disease, juvenile hypothyroidism, adult hypothyroidism), male / female infertility, menstrual disorders (functional uterine bleeding, amenorrhea, polycystic ovary syndrome, premenstrual tension syndrome, Menopause 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 .;
  • Arrhythmia shock, insanity, epilepsy, chorea, hepatic encephalopathy (norepinephrine, Y-aminobutyric acid, serotonin, glutamine), motion sickness, type I allergic disease (nettle Measles, hay fever, allergic rhinitis, skin allergies), peptic ulcer (histamine), hypercholesterolemia (taurine), tumors (polyamines), etc .;
  • hemoglobinopathy anemia, jaundice, tissue hypoxia-induced organic acidemia
  • various coagulation factor deficiency bleeding
  • muscle spasm muscle forcing
  • muscle paralysis actin
  • polypeptide of the present invention and the antagonist, agonist and inhibitor of the polypeptide can be directly used in the treatment of various diseases, such as various tumors, diseases of protein metabolism disorders, and the like.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human class I aminoacyl-tRNA synthetase 10.
  • Agonists enhance human Class I aminoacyl-tRNA synthetase 10 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human class I aminoacyl-tRNA synthetase 10 can be cultured with labeled human class I aminoacyl-tRNA synthetase 10 in the presence of a drug. The ability of the drug to increase or block this interaction is then measured.
  • Antagonists of human class I aminoacyl-tRNA synthetase 10 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human class I aminoacyl-tRNA synthetase 10 can bind to human class I aminoacyl-tRNA synthetase 10 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide such that The polypeptide cannot perform biological functions.
  • human class I aminoacyl-tRNA synthetase 10 When screening compounds as antagonists, human class I aminoacyl-tRNA synthetase 10 can be added In bioanalytical assays, whether a compound is an antagonist is determined by measuring the effect of the compound on the interaction between human class I aminoacyl-tRM synthase 10 and its receptor. 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 class I aminoacyl-tRNA synthetase 10 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 10 molecules of human class I aminoacyl-tRM synthetase should be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human class I aminoacyl-tRNA synthetase 10 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human Class I aminoacyl-tRNA synthetase 10 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 Freund's adjuvant, etc.
  • Techniques for preparing monoclonal antibodies to human class I aminoacyl-tRNA synthetase 10 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta cells Hybridoma technology, EBV-hybridoma technology, etc.
  • Inlay antibodies combining human constant regions and non-human variable regions can be produced using existing technologies (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies U.S. Pat No. 4946778, can also be used to produce single chain antibodies against human class I aminoacyl-tRNA synthetase 10.
  • Antibodies to human class I aminoacyl-tRNA synthetase 10 can be used in immunohistochemical techniques to detect human class I aminoacyl-tRM synthetase 10 in biopsy specimens.
  • Monoclonal antibodies that bind to human class I aminoacyl-tRNA synthetase 10 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 class I aminoacyl-tRNA synthetase 10 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 class I aminoacyl-tRM synthetase 10 positive cells.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human class I aminoacyl-tRNA synthetase 10.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human class I aminoacyl-tRM synthetase 10.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human class I aminoacyl-tRM synthase 10 levels. These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human class I aminoacyl-tRNA synthetase 10 detected in the test can be used to explain the importance of human class I aminoacyl-tRNA synthetase 10 in various diseases and to diagnose human class I aminoacyl- Diseases in which tRNA synthetase 10 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human class I aminoacyl-tRNA synthetase 10 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 class I aminoacyl-tRNA synthetase 10. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human class I aminoacyl-tRM synthetase 10 to inhibit endogenous human class I aminoacyl-tRNA synthetase 10 activity.
  • a variant human class I aminoacyl-tRNA synthetase 10 may be a shortened human class I aminoacyl-tRNA synthetase 10 that lacks a signaling domain. Although it can bind to a downstream substrate, it lacks Signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human class I aminoacyl-tRNA synthetase 10.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human class I aminoacyl-tRNA synthetase 10 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human class I aminoacyl-tRNA synthetase 10 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human class I aminoacyl-tRNA synthetase 10 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 class I aminoacyl-tRNA synthetase 10 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 MA and performs endonucleation.
  • Antisense RM, DNA and ribozymes can be obtained using any existing RNA or DNA synthesis technology. 'Synthetic oligonucleotide synthesis technology such as solid-phase phosphoramidite 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 has been integrated downstream of the vector's RNA polymerase promoter. 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 should use phosphorothioate or peptide bonds instead of Phosphodiester bond.
  • the polynucleotide encoding human class I aminoacyl-tRM synthetase 10 can be used for the diagnosis of diseases related to human class I aminoacyl-tRNA synthetase 10.
  • a polynucleotide encoding human class I aminoacyl-tRNA synthetase 10 can be used to detect the expression of human class I aminoacyl-tRNA synthetase 10 or abnormal expression of human class I aminoacyl-tRNA synthetase 10 in a disease state .
  • a DNA sequence encoding human class I aminoacyl-tRNA synthetase 10 can be used to hybridize biopsy specimens to determine the expression status of human class I aminoacyl-tRNA synthetase 10.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These technical methods are all mature technologies that are publicly available, 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 known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human Class I aminoacyl-tRNA synthetase 10 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also be used to detect human class I aminoacyl-tRM synthetase 10 transcription products.
  • Detection of mutations in the human class I aminoacyl-tRM synthetase 10 gene can also be used to diagnose human class I aminoacyl-tRM synthetase 10-related diseases.
  • Human form I aminoacyl-tRM synthetase 10 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild type human class I aminoacyl-tRNA synthetase 10 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 the expression of proteins. Therefore, Nor thern 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 DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, 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 cDNA clones to 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 Inherance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is 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 the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM 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 class I aminoacyl-tRNA synthetase 10 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dosage range of human Class I aminoacyl-tRNA synthetase 10 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.

Abstract

L'invention concerne un nouveau polypeptide, une aminoacyl-ARNt-synthétase humaine 10 de type I, 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 diverses tumeurs et des troubles du métabolisme protéique. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant l'aminoacyl-ARNt-synthétase humaine 10 de type I.
PCT/CN2001/000992 2000-06-19 2001-06-18 Nouveau polypeptide, aminoacyl-arnt-synthetase humaine 10 de type i, et polynucleotide codant ce polypeptide WO2002000866A1 (fr)

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KR101102485B1 (ko) 2008-10-10 2012-01-05 서울대학교산학협력단 Ｇｒｓ단백질 또는 이의 단편의 신규한 용도
WO2013055101A1 (fr) * 2011-10-10 2013-04-18 Medicinal Bioconvergence Research Center Procédé de criblage à la recherche d'un agent de prévention ou de traitement du cancer en utilisant une glycyl-arnt synthétase et une cadhérine
KR101398079B1 (ko) 2011-10-10 2014-05-27 재단법인 의약바이오컨버젼스연구단 글리실-티알엔에이 합성효소 및 캐드헤린을 이용한 암 예방 또는 치료제의 스크리닝 방법
US9274113B2 (en) 2011-10-10 2016-03-01 Medicinal Bioconvergence Research Center Method for screening an agent preventing or treating cancer using glycyl-tRNA synthetase and cadherin

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