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WO2001031022A1 - Nouveau polypeptide, arginyl arnt synthetase 44, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, arginyl arnt synthetase 44, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001031022A1
WO2001031022A1 PCT/CN2000/000373 CN0000373W WO0131022A1 WO 2001031022 A1 WO2001031022 A1 WO 2001031022A1 CN 0000373 W CN0000373 W CN 0000373W WO 0131022 A1 WO0131022 A1 WO 0131022A1
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WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
trna synthetase
arginyl
arginyl trna
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PCT/CN2000/000373
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bio Road Gene Development Ltd.
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Priority to AU12639/01A priority Critical patent/AU1263901A/en
Publication of WO2001031022A1 publication Critical patent/WO2001031022A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide ⁇ #aminoacyl tRNA synthetase 44 and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide.
  • Aminoacyl tRNA synthetase (aaRS) has three substrates namely amino acid, tRNA, and ATP.
  • aaRS catalyzes the esterification of amino acids to the 3 'end of tRNA. Each aaRS corresponds to one amino acid and several isotropic tRNAs.
  • ATP provides the energy required to activate amino acids.
  • the amino acids are first activated to aminoacyladenylic acid, and then tRNA is used to generate aminoacyl tRNA.
  • the interaction between aaRS and substrate involves issues of identification and binding.
  • aaRSs can be divided into two types with ten members each.
  • Arginyl tRNA synthetase belongs to aaRS type 1 (Eriani et al., 1990).
  • aaRS type 1 usually has the following domains (Cavarelli J et al., 1998):
  • Amino acid recognition region There are only two highly conserved amino acid residues on the aaRS type 1 amino acid binding gap, one is a tyrosine residue at 347 site, and the other is an aspartic acid residue at 191 site.
  • Tyr347 is involved in recognizing the n-nitrogen atom of the amino acid substrate.
  • tyrosine residues perform other functions.
  • P 191 is at the C-terminus of the 5th P-fold of the chain. It does not involve substrate binding but has a structural role. It stabilizes the interaction between the 5th p-fold of the chain and the 6th P-fold of the chain and the surrounding secondary structure. effect.
  • ATP binding site Two specific peptide domains, HIGH and KMSKS, are linked to the ATP binding site.
  • the invariant Gly on HIGH forms a platform at the N-terminus of the 6th alpha helix of the chain to bind ATP, the two Hiss and Lys are involved in the stability of the formation of aminoacylated transition state products.
  • tRNA anchoring platform four members of aaRS type 1 including ArgRS shared a strand 13 A domain consisting of 14 P folds and 14 folds of the chain, located after the Rossmann fold, this domain involves tRNA anchoring to the ArgRS platform.
  • RM-binding domain at the N-terminus of ArgRS The Add-1 domain of ArgRS recognizes tRNA and interacts most closely with D ⁇ ⁇ of tRNA. The naked P-fold of the Add-1 domain plays an important role on many nucleic acid binding proteins.
  • Anti-codon binding site The anti-codon binding site of ArgRS is located on the left-hand side of the C-terminus, on the amino acid residues of the 21st alpha helix and the 22nd alpha helix of the chain. The naked P-fold of the Add-1 domain also recognizes the anti-codon arm.
  • Arg, Asp, Gin, Glu, He, Leu, Lys. Met, and Pro are all encoded by a single gene (Ve ll ekamp etal., 1985).
  • the cDNA sequence of human ArgRS and Other mammals have 87% homology to the ArgRS gene.
  • the amino acid sequence has 87.7% homology with the Chinese rat ovary ArgRS and 37.7% homology with E. coli ArgRS.
  • ArgRS is highly conserved in mammalian cells (Girjes AA et al., 1995).
  • the human arginyl tR synthetase 44 gene of the present invention has 41% homology at the protein level with yeast's ArgRS gene (homologous protein number U5 128), and its domain is similar to the characteristic structure of the ArgRS gene family Domain-amino acid recognition region, ATP binding site, tRNA recognition and binding site, tRNA anchoring platform, ArgRS N-terminus RNA binding domain, anti-codon binding site.
  • the new gene of the present invention is considered to be a gene encoding the human ArgRS-based family, and is named human arginyl tRNA synthetase 44. It is inferred that its domain is similar to the domain of the ArglTs gene family and has similar biological functions.
  • ArgRS catalyzes arginine isoform tRM to carry arginine, and completes the initiation, extension and termination of the peptide chain with the participation of ribosomes and related factors.
  • the discovery of the polynucleotide encoding human arginyl tRNA synthetase 44 and its encoded human arginyl tRNA synthetase 44 provides a basis for studying the physiological and biochemical processes of cell differentiation and proliferation under normal and pathological conditions.
  • a method also provides a new way to diagnose, treat and treat diseases caused by cell differentiation and proliferation disorders, including cancer.
  • 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 an arginyl tRNA synthetase 44.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding an arginyl tRNA synthetase 44,
  • Another object of the present invention is to provide a method for producing arginyl tRNA synthetase 44.
  • Another object of the present invention is to provide an antibody against the polypeptide-arginyl tRNA synthetase 44 of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polypeptide-arginyl tR synthetase 44 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with an abnormality of arginyl tRNA synthetase 44.
  • a novel and isolated arginyl tRNA synthetase 44 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, Or its active fragment, or its active derivative, analog.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the above arginyl tR synthetase 44; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 714-1925 in SEQ ID NO: 1; and (b) a sequence having 1-1951 in SEQ ID NO: 1 Sequence of bits.
  • FIG. 1 is a comparison diagram of amino acid sequence homology between arginyl tRNA synthetase 44 and yeast ArgRS gene of the present invention.
  • the upper sequence is arginyl tRNA synthetase 44 and the lower sequence is yeast ArgRS gene.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated arginyl tRNA synthetase 44. 44kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • 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 arginyl tRNA synthetase 44 means that arginyl tRNA synthetase 44 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify arginyl tRNA synthetase 44 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of arginyl tRNA synthetase 44 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, arginyl tRNA synthetase 44, 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 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 arginyl tRNA synthetase 44.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the arginyl tRNA synthetase 44 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type 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 substituted
  • the amino acid may or may not be encoded by the genetic code; or ( ⁇ ) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such a Species, 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 (IV) such a polypeptide sequence (such as a leader sequence) formed by fusing an additional amino acid sequence into a mature polypeptide Or secreted sequences or sequences used to purify this polypeptide or protease sequences)
  • another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • 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 cD library of human fetal brain tissue. It contains a polynucleotide sequence that is 1,951 bases in length, and its open reading frame (714-1925) encodes 403 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 44% homology with the yeast ArgRS gene. It can be deduced that the arginyl tRNA synthetase 44 has similar structure and function to the yeast ArgRS gene.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DM forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is 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 polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60'C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fico ll, 42 'C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding arginyl tRM synthetase 44.
  • 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 arginyl tRNA synthetase 44 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 CDM 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 DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • mRNA extraction There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene). Construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua l, Cold Spr ing Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DM or DNA-RNA hybridization; (2) the appearance or loss of marker gene function; (3) measurement Determine the transcript level of arginyl tRNA synthetase 44; (4) Detect the protein product of gene expression by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually 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 product expressed by the arginyl tRNA synthetase 44 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method (Sa iki, et al. Science 1985; 230: 1350-1354) using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers 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 DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the arginyl tRNA synthetase 44 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding an arginyl tRNA synthetase 44 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, etal.
  • 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.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding arginyl tR synthetase 44 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, and the like (Sambroook, et al. Molecular Cloning, A Labora tory Manua, Collspring Harbor Labora tory. 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: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding arginyl tRNA synthetase 44 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 a 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: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transforming a host cell with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence may This is done using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with 0 & (Method 12, using the procedure well known in the art. Alternatively, it is a MgCl 2. 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 liposomes Packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant arginyl tRNA synthetase 44 (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. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention 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, H IV infection, and immune diseases.
  • ArgRS catalyzes the arginine isoform tRNA to carry arginine
  • the initiation, extension, and termination of the peptide chain are completed with the participation of ribosomes and related factors, and the protein biosynthesis is completed.
  • the abnormal expression of the human arginyl tR synthetase 44 of the present invention will cause a disorder of protein biosynthesis, and thus cause various diseases.
  • These diseases include, but are not limited to: Developmental disorders: spina bifida, craniocerebral fissure, anencephaly malformation, cerebral bulge, foramencephalic malformation, congenital hydrocephalus, aqueduct deformity, cartilage hypoplasia, dwarfism, spinal epiphyseal dysplasia,
  • Metabolic and nutritional diseases atrophy, hyperlipidemia and hyperlipoproteinemia, obesity, nutritional deficiencies
  • Inflammation allergic reaction, adult respiratory distress syndrome, pulmonary eosinophilia, rheumatoid arthritis, rheumatoid arthritis, cholecystitis, glomerulonephritis, dermatomyositis, polymyositis, Ah Dickson's disease
  • Basal epithelial tumor Basal epithelial tumor, squamous epithelial tumor, myxoid tumor, fibroma, lipoma, chondroma, hemangioma, lymphoma, hematopoietic tumor, neuroma, adenoma
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) arginyl tRNA synthetase 44.
  • Agonists increase arginyl tRNA synthetase 44 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 arginyl tRNA synthetase 44 can be cultured with labeled arginyl tRNA synthetase 44 in the presence of a drug. The ability of the drug to increase or suppress this interaction is then determined.
  • Antagonists of arginyl tRNA synthetase 44 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • An antagonist of arginyl tRNA synthetase 44 can bind to arginyl tRNA synthetase 44 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.
  • arginyl tRNA synthetase 44 can be added to a bioanalytical assay to determine whether the compound has an effect on the interaction between arginyl t RNA synthetase 44 and its receptor. Is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to arginyl tRNA synthetase 44 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, 44 molecules of arginyl tRM synthetase should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the arginyl tRNA synthetase 44 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 arginyl tRNA synthetase 44 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • 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 arginyl tRNA synthetase 44 include, but are not limited to, hybridoma technology (Koh ler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta- Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Embedding antibodies that bind human constant regions and 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 arginine tRNA synthetase 44.
  • Anti-arginyl tRNA synthetase 44 antibodies can be used in immunohistochemistry to detect arginyl tRNA synthetase 44 in biopsy specimens.
  • Monoclonal antibodies that bind to arginyl tR synthetase 44 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.
  • arginyl tRNA synthetase 44 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of the antibody with a thiol crosslinker such as SPDP and bind the toxin to the antibody through the disulfide exchange.
  • This hybrid antibody can be used to kill arginyl tRNA synthetase 44 cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to arginyl tR synthetase 44.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of arginyl tRNA synthetase 44.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of arginyl tRNA synthetase 44.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of arginyl tRNA synthetase 44 detected in the test can be used to explain the importance of arginyl tRNA synthetase 44 in various diseases and to diagnose diseases in which arginyl tRNA synthetase 44 functions.
  • the 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.
  • the polynucleotide encoding arginyl tRNA synthetase 44 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 arginyl tRNA synthetase 44.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated arginyl tRNA synthetase 44 to inhibit endogenous arginyl tRNA synthetase 44 activity.
  • a variant arginyl tRNA synthetase 44 may be a shortened arginyl tRNA synthetase 44 that lacks a signal transduction domain. Although it can bind to a downstream substrate, it lacks signal transduction activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of arginyl tRNA synthetase 44.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses can be used to transfer the polynucleotide encoding arginyl tRNA synthetase 44 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding an arginyl tR synthetase 44 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding arginyl tRNA synthetase 44 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 arginyl tRNA synthetase 44 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like R molecule that can specifically decompose a specific R. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA and D and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the solid-phase phosphoramidite synthesis method for oligonucleotide synthesis.
  • 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 RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding arginyl tR synthetase 44 can be used to diagnose diseases related to arginyl tRNA synthetase 44.
  • the polynucleotide encoding arginyl tRNA synthetase 44 can be used to detect arginyl tRNA synthetase 44 Expression or abnormal expression of arginyl tRNA synthetase 44 in disease states.
  • the DNA sequence encoding arginyl tRNA synthetase 44 can be used to hybridize biopsy specimens to determine the expression of arginyl tRNA synthetase 44.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • 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 of genes and genetic diagnosis in tissues.
  • the arginyl tRNA synthetase 44 specific primers can also be used to detect the transcription products of arginyl tRNA synthetase 44 by RM-polymerase chain reaction (RT-PCR) in vitro amplification.
  • RT-PCR RM-polymerase chain reaction
  • Detecting mutations in the arginyl tRNA synthetase 44 gene can also be used to diagnose arginyl tRNA synthetase 44-related diseases.
  • Arginyl tRNA synthetase 44 mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type arginyl tRNA synthetase 44 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.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome locations. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • 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 hybrid cells that contain 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 by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the cDNA or genomic sequence differences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all of the affected individuals and the mutation is not observed in any normal individual, the mutation may be Is the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, 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.
  • Arginyl tRNA synthetase 44 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of arginyl tRNA synthetase 44 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples
  • RNA Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RM using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRM was formed by reverse transcription. Smar t cDNA cloning kit (purchased from CI on tech) will be used. The 0 fragment was inserted into the multicloning site of the pBSK (+) vector (Clontech) and transformed into DH5 cc. The bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0539g02 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the sequence of the arginyl tR synthetase 44 of the present invention and the protein sequence encoded by the arginyl tR synthetase 44 were coded using the Blast program (Basic loca lal ignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], perform homology search in databases such as Genbank, Swissport, etc.
  • the gene most homologous to the arginyl tRNA synthetase 44 of the present invention is a known yeast ArgRS gene, and its accession number is 1151032 in Genbank.
  • the results of protein homology are shown in Figure 1. The two are highly homologous and their identity is 44%.
  • Example 3 Cloning of a gene encoding arginyl tRNA synthetase 44 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.
  • PCR amplification was performed with the following primers:
  • Primer2 5'- CATTTTAAAAGCCATTTTAATGG-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions A reaction volume of 50 ⁇ 1 contains 50mmol / L KC1, 10mmol / L Tris-Cl, (pH8.5), 1.5 legs ol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer, 1U of Taq DM polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the l-1951bp shown in SEQ ID NO: 1.
  • Example 4 Analysis of the expression of arginyl tRNA synthetase 44 gene by Northern blot
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate-chloroform extraction. I.e. with 4M guanidinium isothiocyanate -25fflM sodium citrate, 0.2M sodium acetate (P H4.0) of the tissue was homogenized, 1 volume of phenol and 1/5 volume of chloroform - isoamyl alcohol (49: 1) Centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA was prepared using 20 g of RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation cx- 32 P dATP with 32 P- DNA probe labeled by the random primer method. The DNA probe used is the PCR-amplified arginyl tRNA synthetase 44 coding region sequence (714bp to 1926bp) 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 ( pH7.4) -5 x SSC-5 x Denhardt, s solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant arginyl tRNA synthetase 44
  • Primer3 5'- CCCCATATGATGCAGTTTGGTCTTCTGGGAACTG-3 '(Seq ID No: 5)
  • Primer4 5'- CCCGGATCCTTACATCCTACATACAGGTGTTATTCC-3 '(Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BaraHI restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • 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 pBS-0539g02 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0539g02 plasmid in a total volume of 50 ⁇ l, Primer-3 and Primer-4 primers were 1 Opmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94 ° C 20s, 60. C 30s, 68. C 2 rain, a total of 25 cycles. Ndel 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 ligated product was transformed into E. coli bacteria DH50 using the calcium chloride method.
  • the polypeptide was coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avrameas, et al. Iramunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. ⁇ Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sephan) se4B column, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that purified antibodies can specifically interact with arginyl tRNA synthetase44 Combined.

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Abstract

L'invention concerne un nouveau polypeptide, une arginyl ARNt synthétase 44, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour l'arginyl ARNt synthétase 44.
PCT/CN2000/000373 1999-10-27 2000-10-27 Nouveau polypeptide, arginyl arnt synthetase 44, et polynucleotide codant pour ce polypeptide WO2001031022A1 (fr)

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AU12639/01A AU1263901A (en) 1999-10-27 2000-10-27 A novel polypeptide, an arginyl trna synthetase 44 and the polynucleotide encoding the polypeptide

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CN99119864.6 1999-10-27
CN 99119864 CN1303925A (zh) 1999-10-27 1999-10-27 一种新的多肽——精氨酰tRNA合成酶44和编码这种多肽的多核苷酸

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002044722A2 (fr) * 2000-11-28 2002-06-06 Subsidiary No. 3, Inc. Compositions et procedes d'inhibition des infections dues au vih par reduction du nombre des genes de cellules humaines

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0835936A2 (fr) * 1996-09-12 1998-04-15 Smithkline Beecham Plc Synthétase d'arginyl ARNt
US5965416A (en) * 1996-01-19 1999-10-12 Smithkline Beecham Plc Arginyl tRNA synthetase from staphyloccus aureus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5965416A (en) * 1996-01-19 1999-10-12 Smithkline Beecham Plc Arginyl tRNA synthetase from staphyloccus aureus
EP0835936A2 (fr) * 1996-09-12 1998-04-15 Smithkline Beecham Plc Synthétase d'arginyl ARNt

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002044722A2 (fr) * 2000-11-28 2002-06-06 Subsidiary No. 3, Inc. Compositions et procedes d'inhibition des infections dues au vih par reduction du nombre des genes de cellules humaines
WO2002044722A3 (fr) * 2000-11-28 2003-05-22 Subsidiary No 3 Inc Compositions et procedes d'inhibition des infections dues au vih par reduction du nombre des genes de cellules humaines

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