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WO2002012314A1 - Nouveau polypeptide, proteine-1 humaine associee a la dihydropyrimidinase (drp-1) 9.68, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine-1 humaine associee a la dihydropyrimidinase (drp-1) 9.68, et polynucleotide codant ce polypeptide Download PDF

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WO2002012314A1
WO2002012314A1 PCT/CN2001/001045 CN0101045W WO0212314A1 WO 2002012314 A1 WO2002012314 A1 WO 2002012314A1 CN 0101045 W CN0101045 W CN 0101045W WO 0212314 A1 WO0212314 A1 WO 0212314A1
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Prior art keywords
polypeptide
polynucleotide
drp
human
related protein
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Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU14903/02A priority Critical patent/AU1490302A/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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, human dihydropyrimidinase-related protein-1 (DRP-1) 9.68, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • Dihydropyrimase is the second enzyme involved in the catabolism of uracil and thymine.
  • DHPase catalyzes the hydrolysis of 5, 6-dihydrouracil to N-carbamyl-beta-alanine and catalyzes the hydrolysis of 5, 6-dihydrothymine to N-carbamyl-beta-aminoisobutyric acid.
  • DHPase also catalyzes the hydrolysis of various 5,6-dihydropyrimidines as well as hydantoin and succinamide.
  • DHPase is a tetrameric zinc-ion metalloenzyme that contains four closely linked zinc ions / molecules with enzymatic activity.
  • DHPase-associated proteins-1 DHPase-associated proteins-1
  • DHPase human dihydropyrimidinase related proteins
  • human DHPase is located in the liver and kidney; human DRP-1 is located in the brain; human DRP-2 is widely expressed in various tissues, but not in the liver.
  • Human DRP-3 is mainly located in the heart and skeletal muscle (Kikugawa, M., Kaneko, M., Fuj imoto-Sakata, S., Maeda, M., Kawasak i,., Takag i, T. and Tamaki , N. (1994) Purif icat ion, characteriza t ion and inhi bit ion of dihydropyr imidinase. Eur. J. Biochem. 219, 393-399).
  • the mRM of DRP-1 has a high expression level in the brain, and unlike other DRPs, DRP-1 has no corresponding protein in other species.
  • DRP-1 is a multifunctional protein with amidohydrolase and other types of enzymatic activities. It is involved in basic cellular processes and is not limited to functioning only in the nervous system.
  • the DRP-1 gene is located on chromosome 4 P 15-16. 1 near the Huntington's disease site.
  • DRP-1 Deletion or mutation of DRP-1 can cause various metabolic diseases related to uracil and thymine catabolism and diseases in various nervous systems, and is related to Huntington's disease (Naoki Hamaj ima, Koichi Matsuda, Shigeko Saka ta, Nanaya Tamaki, Makoto Sasak i, Masaru Nonaka, Gene 180 (1996) 157-163).
  • the human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so There is a continuing need in the art to identify more human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 proteins that are involved in these processes, and in particular the amino acid sequence of such proteins.
  • New human dihydropyrimidinase-associated protein-1 (DRP-1) 9. 68 The isolation of the protein-coding gene also provides a basis for research to determine the role of the protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs, so isolating its coding DNA is important. Object of the invention
  • DRP-1 human dihydropyrimidinase-related protein-1
  • 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 dihydropyrimidinase-related protein-1 (DP-1) 9.68.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • DRP-1 human dihydropyrimidinase-related protein-1
  • Another object of the present invention is to provide a method for producing human dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • Another object of the present invention is to provide an antibody against the polypeptide-human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 of the polypeptide of the present invention.
  • DRP-1 polypeptide-human dihydropyrimidinase-related protein-1
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the human dihydropyrimidin-related protein-1 (DRP-1) 9.68 of the polypeptide of the present invention.
  • DRP-1 human dihydropyrimidin-related protein-1
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68. Summary of invention
  • the invention relates to an isolated polypeptide, which is of human origin, and which comprises: SEQ ID No. 2 Amino acid sequence of a polypeptide, 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 254-520 in SEQ ID NO: 1; and (b) having a sequence of 1-2196 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68 protein activity, which comprises utilizing the polypeptide of the present invention.
  • DRP-1 human dihydropyrimidinase-associated protein-1
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or disease susceptibility related to abnormal expression of human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68 protein, which comprises detecting the polypeptide or its encoding in a biological sample. A mutation in a polynucleotide sequence, or the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • DRP-1 human dihydropyrimidinase-associated protein-1
  • 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 polypeptides and / or polynucleotides of the present invention that are prepared for use in the treatment of cancer, developmental or immune diseases, or other diseases due to human dihydropyrimidinase-related protein-1 (DRP-1) 9.68. Use of medicines that cause disease.
  • DRP-1 human dihydropyrimidinase-related protein-1
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 and human dihydropyrimidinase-related protein-1 (DRP-1). Pictured above is human dioxopyrimidase-related protein-1 (DRP- 1) A graph of the expression profile of 9. 68. The figure below is a graph of the expression profile of human dihydropyrimidinase-related protein-I (DRP-1).
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of human dihydropyrimidinase-related protein-I (DRP-1) 9.68.
  • OkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the substituted amino acid has similar crusting or chemical properties to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • Antagonist means when bound to human dihydropyrimidin-associated protein-1 (DRP-1) 9. 6S 'a A molecule that can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • Antagonist refers to a type that blocks or regulates human dihydropyrimidinase-related protein-1 (DRP) when combined with human dihydropyrimidinase-related protein-1 (DRP-1) 9.68. -1) 9. 68 biologically or immunologically active molecules.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • Regular refers to changes in the function of human dihydropyrimidinase-related protein-I (DRP-1) 9.68, including increased or decreased protein activity, changes in binding characteristics, and human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 any other biological, functional or immune change.
  • Substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68 using standard protein purification techniques.
  • Substantially pure human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 produces a single main band on a non-reducing polyacrylamide gel.
  • Human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 Polypeptide The purity of the peptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The 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. Two amino acid sequences such as The percent identity between sequence A and sequence B is calculated by:
  • the percentage identity Jotun Hein measured between nucleic acid sequences can also Clus ter or a method well known in the art (He in J., (1990) Methods in enzymol ogy 183: 625-645) 0
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or MA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa, F (ab ') 2 Fv, which can specifically bind to the epitope of human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 .
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • the polynucleotide may be part of a vector, or the 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 isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human dihydropyrimidinase-associated protein-1 (DRP-1) 9. 68) refers to human dihydropyrimidinase-associated protein-1 (DRP-1) 9. 68 is substantially free of nature and Related to other proteins, lipids, sugars or other substances. Those skilled in the art can purify humans using standard protein purification techniques. Dihydropyrimidinase-related protein-1 (DRP-1) 9. 68. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 The purity of the peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human dihydropyrimidinase-related protein-1 (DRP-1) 9.68, 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.
  • polypeptides of the invention may be glycosylated, or they 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 dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • fragment refers to substantially maintaining the human dihydropyrimidinase-related protein-1 (MP-1) 9.68 of the present invention or the same biological function or Active peptide.
  • 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 replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ )
  • Such a polypeptide sequence 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
  • a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as the leader or secretory sequence or the sequence used to purify the polypeptide or protease sequence).
  • such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 2196 bases, and its open reading frame 254-520 encodes 88 amino acids.
  • this polypeptide has a similar expression profile with human dihydropyrimidinase-related protein-1 (DRP-1), and it can be inferred that the human dihydropyrimidinase-related protein-1 (DRP-1) 9 68 has a similar function to human dihydropyrimidinase-related protein-1 (DRP-1).
  • the polynucleotide of the present invention may be in the form of DM or RM.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region shown in SEQ ID NO: 1
  • the sequences are identical or degenerate variants.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1 ° /.
  • the hybridizable polynucleotide has the same biological function as the mature polypeptide shown in SEQ ID NO: 2 And active.
  • nucleic acid fragments that hybridize to the sequences described above.
  • nucleic acid fragment having a length of at least 10 nucleotides, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, preferably at least 100 Nucleotides or more.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • 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 dihydropyrimidinase-related protein-1 (DRP-1) 9.68 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2 ) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of 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 cDM of interest is to isolate mRM 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 (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Col d Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the genes of the present invention can be screened from these CDM libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RM hybridization; ( 2 ) the appearance or loss of marker gene function; (3) determination of human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 transcript levels; (4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the human dihydropyrimidin-associated protein-1 (DRP-1) 9.
  • 68 gene expression protein product can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunoassay. Adsorption method (ELISA) and so on.
  • a method (Sa iki, et al. Science 1985; 230: 1350-1354) using PCR technology to amplify DNA / RM is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DM / 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 measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Fixed. Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length CDM sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell using the vector of the present invention or directly using human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 coding sequence, and Method for producing a polypeptide of the present invention by recombinant technology.
  • DRP-1 human dihydropyrimidinase-related protein-1
  • a polynucleotide sequence encoding human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pairs of the SV40 enhancer 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 for eukaryotic cell culture. And green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture.
  • GFP green fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide.
  • a 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with 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 eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 (Science, 1984; 224: 1431). Generally there are the following steps:
  • polynucleotide or variant encoding human human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 of the present invention, or transform or transform with a recombinant expression vector containing the polynucleotide Direct suitable host cells;
  • 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 isolated and purified by various separation methods using their physical, chemical, and other properties. These methods include but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic bacteria, Ultrasonication, 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.
  • 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.
  • Dihydropyrimase is the second enzyme involved in the catabolism of uracil and thymine.
  • DHPas e catalyzes the hydrolysis of 5, 6-dihydrouracil to N-carbamyl-beta-alanine and catalyzes the hydrolysis of 5, 6-dihydrothymine to N-carbamyl-be-ta-aminoisobutyric acid.
  • DHPase also catalyzes the hydrolysis of various 5,6-dihydropyrimidines as well as hydantoin and succinamide.
  • human dihydropyrimidinase-related protein DHPas e is a human dihydropyrimidinase-related protein-1 (DRP-1) of some DHPases, and they have high homology with DHPase.
  • DHPase and DRPs constitute a new gene family and have different tissue distributions.
  • human DHPase is located in the liver and kidney; human DRP-1 is located in the brain; human DRP-2 is widely expressed in various tissues, but not in the liver. Expression; human DRP-3 is mainly located in the heart and skeletal muscle.
  • DRP-1 Human DRP-1 has a high expression level of mMA in the brain, and unlike other DRPs, DRP-1 has no corresponding protein in other species.
  • DRP-1 is a multifunctional protein with amide hydrolase and other types of enzymatic activities. It is involved in basic cellular processes and is not limited to functioning only in the nervous system.
  • the DRP-1 gene is located on chromosome 4pl 5-16. 1 near the Huntington's disease site. Deletion or mutation of DRP-1 can cause various metabolic diseases related to uracil and thymine catabolism and various diseases in the nervous system, and is related to Huntington's disease.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human dihydropyrimidinase-related protein-1 (DRP-1), and both have similar biological functions.
  • the polypeptide of the present invention catalyzes the hydrolysis of 5, 6-dihydrouracil to N-carbamyl-beta-alanine and catalyzes the hydrolysis of 5, 6-dihydrothymine to N-carbamyl-be ta-amino in vivo.
  • Isobutyric acid the polypeptide of the present invention also catalyzes the hydrolysis of various 5, 6-dihydropyrimidines and hydantoin with succinamide. It is highly expressed in neural tissue, and its abnormal expression is usually closely related to the metabolic disorders of uracil and thymine, the pathological processes of various neuropsychiatric disorders, and related diseases.
  • DRP-1 human dihydropyrimase-related protein-1
  • Neurological diseases Alzheimer's disease, Parkinson's disease, Chorea, Depression, Amnesia, Huntington's disease, Epilepsy, Migraine, Dementia, Multiple sclerosis, Neuromuscular disease, Neurodermal syndrome, Nerve Phylogenetic disorders, gliomas, meningiomas, neurofibromas, Mental Illness: Schizophrenia, Depression, Paranoia, Anxiety, OCD, Phobia, Neurasthenia
  • Uracil and thymidine-related metabolic disorders Abnormalities of pyrimidine metabolism such as orotic aciduria, adenine nucleotide deaminase deficiency, metabolic deficiency of the urea cycle, abnormal purine metabolism, organic acidemia
  • DRP-1 human dihydropyrimidinase-associated protein-1
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially neuropsychiatric diseases, uracil and thymine-related metabolic disorders, and developmental disorders. Disease, certain hereditary, bloody diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • Agonists increase human dihydropyrimidinase-associated protein-1 (DRP-1) 9. 68 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 can be labeled with a labeled human dihydropyrimidinase-related protein-1 (DRP- 1) 9. 68 together. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human dihydropyrimidinase-associated protein-1 (DRP-1) 9. 68 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 can bind to human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 and eliminate its function, or inhibit the polypeptide Production, or binding to the active site of the polypeptide prevents the polypeptide from performing biological functions.
  • human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68 can be added to the bioanalytical assay to determine human dihydropyrimidinase-related protein-1 (DRP- 1) The effect of the interaction between 9.68 and its receptor to determine whether a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. Screening, the general response human dihydropyrimidine-related protein -1 (DRP-1) 9. 68 labeled molecule.
  • the present invention provides polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. To produce antibodies. These antibodies can be polyclonal or monoclonal antibodies.
  • the present invention also provides antibodies against human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 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 using human dihydropyrimidinase-associated protein-1 (DRP-1) 9. 68 by direct injection of 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 Immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), Three tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced by known techniques (Morrison et al, PMS, 1985, 81: 6851).
  • the existing technology for producing single-chain antibodies U.S. Pat No. 4946778, can also be used to produce single-chain antibodies against human dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • Anti-human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 antibody can be used in immunohistochemistry to detect human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 in biopsy specimens .
  • DRP-1 dihydropyrimidinase-related protein-1
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human dihydropyrimidinase-related protein-1 (DRP-1) 9.
  • 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 dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 positive cells.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68.
  • the present invention also relates to a diagnostic test method for quantitatively and locally detecting human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 level.
  • DRP-1 human dihydropyrimidinase-related protein-1
  • tests are well known in the art and include FISH and radioimmunoassays.
  • the level of human dihydropyrimidinase-associated protein-1 (DRP-1) 9. 68 detected in the test can be used to explain human dihydropyrimidinase-associated protein-1 (DRP-1) 9. 68 in various diseases
  • polypeptides of the present invention can also be used for peptide mapping, for example, the polypeptides can be physically, chemically or enzymatically Specific cleavage and one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, preferably mass spectrometry.
  • the polynucleotide encoding human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 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 dihydropyrimidinase-associated protein-1 (DRP-1) 9.68.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 to inhibit endogenous human dihydropyrimidinase-related protein-1 (DRP -1) 9.68 activity.
  • a mutated human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 may be a shortened human dihydropyrimidinase-related protein-1 (DRP-1) 9.68, although Can bind to downstream substrates, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human dihydropyrimidinase-related protein-1 (DRP-1) 9.68.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 can be used to transfer a polynucleotide encoding human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 Into the cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 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 dihydropyrimidinase-related protein-1 (DRP-1) 9.68 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RM, DNA, and ribozymes can be obtained using any existing RNA or DM synthesis techniques, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DM sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond rather than the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding human dihydropyrimidinase-associated protein-1 (MP-1) 9.68 can be used for the diagnosis of diseases related to human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68.
  • a polynucleotide encoding human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 can be used to detect the expression of human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 or human dihydrogen in a disease state Pyrimidinase-associated protein-1 (DRP-1) 9.68 Often expressed.
  • the DNA sequence encoding human dihydropyrimidinase-related protein-I (DRP-1) 9.68 can be used to hybridize biopsy specimens to determine the expression of human dihydropyrimidinase-related protein-1 (DRP-1) 9.68. situation.
  • 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.
  • Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissue.
  • DRP-1 Human dihydropyrimidinase-related protein-1
  • RT-PCR RNA-polymerase chain reaction
  • Detection of human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68 gene mutations can also be used to diagnose human dihydropyrimidinase-associated protein-1 (MP- 1) 9. 68-related diseases.
  • Human dihydropyrimidinase-associated protein-1 (DRP-D 9.68 mutant forms include point mutations compared to normal wild-type human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68 DNA sequences. Position, deletion, recombination and any other abnormalities, etc. Mutation can be detected using well-known techniques such as Southern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting, Wes The tern blot method can indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DM 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 of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the cD or genomic sequence differences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the CDM that is accurately mapped to a disease-related chromosomal region 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 dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 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
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) raRNA forms cDNA by reverse transcription.
  • the Smart cDNA cloning kit purchased from Clontech
  • the Smart cDNA cloning kit was used to insert the cDNA fragment into the multicloning site of pBSK (+) vector (Clontech) to transform DH5 ⁇ .
  • the bacteria formed a cDNA library.
  • Dye terminate cycle reaction 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 DM sequence database (Genebank), and it was found that the cDM sequence of one of the clones 0645c 01 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results show that the 0645c01 clone contains a full-length cDNA of 2196bp (as shown by Seq ID NO: l), and has a 266bp open reading frame (0RF) from 254bp to 520bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • 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:
  • Primerl 5 _ GGGAATTCTCACTGCAGTGCATGT -3, (SEQ ID NO: 3)
  • Primer2 5'- GAACATGTCTTTATTAAAGATGCA -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer 2 is the 3 'terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions 50 ⁇ l reaction volume contains 50 mmol / LK: Cl, 10 ramol / L Tri s-HCl pH 8.5, 1.5 mraol / L MgCl 2 , 200 mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -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.
  • DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-2196bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human dihydropyrimidinase-related protein-1 (DRP-1) 9. 68 gene expression
  • RNA extraction in one step [AnaL Biochem 1987, 162, 156-159].
  • the method includes acid sulfur Guanidinium cyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25raM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) and RNA-transferred nitrocellulose
  • the membrane was hybridized overnight at 42 ° C in a solution containing 50% formamide-25 mM KH 2 PO 4 (pH 7.4)-5 x SSC-5 x Denhardt's solution and 20 ( ⁇ 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, it was analyzed and quantified using a Phosphor Imager.
  • DRP-1 Recombinant human dihydropyrimidinase-related protein-1
  • Pr imer3 5'-CCCCATATGATGGCGTGGGAAGGGCCAGGGCTG-3 '(Seq ID No: 5)
  • Primer4 5'-CCCGAGCTCCTACTGGATGCGGCAGAACATGAC-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and Sacl restriction sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and Sacl restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • the PCR reaction was performed using the pBS-0645c01 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0645c01 plasmid, Primer-3 and Primer-4; Primer-3 and Primer-4; Primer polymerase Mix (Clontech) 1 ⁇ 1 in a total volume of 50 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles.
  • Ndel and Sacl 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 coliform bacteria DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 3 ( ⁇ g / ml)), positive clones were screened by colony PCR method and sequenced. A positive clone with the correct sequence (pET-0645c01) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Novagen) using calcium chloride method. LB containing kanamycin (final concentration 3 ( ⁇ g / ml)) In the liquid medium, the host bacteria BL21 (pET-0 645 c01) was cultured at 37 ° C.
  • NH2-Met-Ala-Trp-Glu-Gly-Pro-Gly-Leu-Pro-Val-Met-Pro-Arg-Ala-Val-C00H (SEQ ID NO: 7).
  • the peptide is coupled to hemocyanin and bovine serum albumin to form a complex.
  • 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.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers 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 proved that the purified antibody could specifically bind to human dihydropyrimidinase-associated protein-1 (DRP-1) 9.68.
  • DRP-1 human dihydropyrimidinase-associated protein-1
  • 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 using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • 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 labeled probes, and Incubation hybridizes 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, then the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 1 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt) :
  • PBS phosphate buffer solution
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the sample membrane was placed in a plastic bag, and 3 to 10 mg of prehybridization solution (10xDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DM)) was added. After sealing the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution 10xDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DM)
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature, for example, see the literature DeRis i, JL, Lyer, V. & Brown, P. 0. (1997) Science 278, 680-686. And the literature Hel le, RA, Schema , M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-21
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After the purified amplified product was purified, the concentration was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in an ultraviolet cross-linker. After elution, the DM was fixed on a glass slide to prepare chips. Its specific method steps There have been many reports in the literature. The sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • Reagent Cy 3dUTP (5-Am i no-pr opa r gy 1-2 ⁇ -deoxyur idi ne 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech Company) labeled mRM of human mixed tissue, using fluorescence Reagent Cy5dUTP (5-Amino-propargyl-2'- deoxyur idine 5 '-tripha te coupled to Cy5 f luorescent dye, purchased from Amersham Phamac ia Biotech) Marks specific tissues (or stimulated cell lines) mRNA, purified The probe was then prepared. For specific steps and methods, see:
  • the probes from the two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and then washed with a washing solution (lx SSC, 0.2 touch) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from the United States General Scanning Company), and the scanned images were analyzed and processed with Iraagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC :, Fibroblast, growth factor stimulation, 1024NT, scar formation fc growth factor stimulation, 1013HT, scar into fc without growth factor stimulation, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, Cardiac cancer. Based on these 18 Cy3 / Cy5 ratios, a bar graph is drawn ( Figure 1). It can be seen from the figure that the expression profile of human dihydropyrimidinase-related protein-1 (DRP-1) 9.68 and human dihydropyrimidinase-related protein-1 (DRP-1) according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine-1 humaine associée à la dihydropyrimidinase (DRP-1) 9.68, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment de maladies neuropsychologiques, de troubles du métabolisme associés à l'uracil et à la thymine. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine-1 humaine associée à la dihydropyrimidinase (DRP-1) 9.68.
PCT/CN2001/001045 2000-06-26 2001-06-25 Nouveau polypeptide, proteine-1 humaine associee a la dihydropyrimidinase (drp-1) 9.68, et polynucleotide codant ce polypeptide Ceased WO2002012314A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU14903/02A AU1490302A (en) 2000-06-26 2001-06-25 A novel polypeptide - human dihydropyrimidinase-related protein-1(drp-1) 9.68 and a polynucleotide encoding the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 00116757 CN1331329A (zh) 2000-06-26 2000-06-26 一种新的多肽——人二氢嘧啶酶相关蛋白－1(drp－1)9.68和编码这种多肽的多核苷酸
CN00116757.X 2000-06-26

Publications (1)

Publication Number Publication Date
WO2002012314A1 true WO2002012314A1 (fr) 2002-02-14

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PCT/CN2001/001045 Ceased WO2002012314A1 (fr) 2000-06-26 2001-06-25 Nouveau polypeptide, proteine-1 humaine associee a la dihydropyrimidinase (drp-1) 9.68, et polynucleotide codant ce polypeptide

Country Status (3)

Country Link
CN (1) CN1331329A (fr)
AU (1) AU1490302A (fr)
WO (1) WO2002012314A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7628989B2 (en) 2001-04-10 2009-12-08 Agensys, Inc. Methods of inducing an immune response

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DNA RES., vol. 6, no. 5, 1999, pages 291 - 297 *
GENE, vol. 180, no. 1-2, 1996, pages 157 - 163 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7628989B2 (en) 2001-04-10 2009-12-08 Agensys, Inc. Methods of inducing an immune response
US7641905B2 (en) 2001-04-10 2010-01-05 Agensys, Inc. Methods of inducing an immune response
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers

Also Published As

Publication number Publication date
AU1490302A (en) 2002-02-18
CN1331329A (zh) 2002-01-16

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