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WO2001094535A2 - Nouveau polypeptide, uracil desoxyribonucleotide glycosylase humaine 9.9, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, uracil desoxyribonucleotide glycosylase humaine 9.9, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2001094535A2
WO2001094535A2 PCT/CN2001/000850 CN0100850W WO0194535A2 WO 2001094535 A2 WO2001094535 A2 WO 2001094535A2 CN 0100850 W CN0100850 W CN 0100850W WO 0194535 A2 WO0194535 A2 WO 0194535A2
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Prior art keywords
polypeptide
polynucleotide
deoxyribonuclease
uracil
human
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WO2001094535A3 (fr
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 AU89494/01A priority Critical patent/AU8949401A/en
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Publication of WO2001094535A3 publication Critical patent/WO2001094535A3/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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2497—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing N- glycosyl compounds (3.2.2)

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide ⁇ ⁇ uracil deoxyribonuclease 9.9, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • Uracil- DM glycosylase catalyzes the first step in this repair pathway, removing the mutant uracil. Firstly, the N-glycosyl bond between uracil and deoxyribose is hydrolyzed, and then the rest of the repair work is performed sequentially by endo-AP, enzyme, phosphodiesterase, DM polymerase and DM ligase [Prog. Nucleic Acids Res. Mol Biol. 22, 135-192].
  • UDG also plays a role in DNA replication. Found in vaccinia virus and bacterial megalovirus, UDG is necessary in DNA synthesis and virus replication [Virol. 67, 2503-2512] [Virology 198, 504-513] [J. Virol. 70, 3018-3025] . In addition, highly conserved human N-terminal UDG can interact with replicating protein A2, suggesting its role in DM replication-repair [J. Biol. Chem. 272, 6561-6566].
  • UDG is ubiquitous from viruses to humans. Two types of UDG have been found in humans, nuclear UDG and mitochondrial UDG, which repair nuclear DNA and mitochondrial DM, respectively. Except that they are different at the N terminal, the other parts are exactly the same. In fact, they are transcribed from the same gene [Nucleic Acids Res 1997 Feb 15; 25 (4): 750-51.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human uracil deoxyribonuclease 9.9.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human uracil deoxyribonuclease 9.9.
  • Another object of the present invention is to provide a method for producing human uracil deoxyribonuclease 9.9.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, human uracil deoxyribonucleic acid glycosylase 9.9.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, human uracil deoxyribonucleic acid glycosylase 9.9.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human uracil deoxyribonuclease 9.9. Summary of invention
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SBQ 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 560 to 832 in SEQ ID NO: 1; and (b) a sequence having positions 1 to 1760 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 invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human uracil deoxyribonuclease glycosylase 9.9 protein activity, which comprises utilizing the polypeptide of the invention.
  • 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 associated with abnormal expression of human uracil deoxyribonuclease 9.9 protein, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the 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 prepared for use in the treatment of cancer, developmental or immune diseases, or other diseases caused by abnormal expression of human uracil deoxyribonuclease 9.9. Use of medicine.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human uracil deoxyribonuclease 9.9 and human UDG according to the present invention.
  • the upper graph is a graph of the expression profile of human uracil deoxyribonuclease 9.9
  • the lower graph is the graph of the expression profile of human UDG.
  • 1-bladder mucosa 2-PMA + Ecv304 cell line
  • 3-LPS + Ecv304 cell line thymus 3-normal fibroblasts 1024NC
  • 5-Fibroblas t growth factor stimulation
  • Figure 2 is a polyacrylamide gel electrophoresis diagram of isolated human uracil deoxyribonuclease 9.9 (SDS-PAGE). OkDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also be 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 a fragment or portion thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion 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.
  • An "agonist” refers to a molecule that, when combined with human uracil deoxyribonuclease 9.9, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human uracil deoxyribonuclease 9.9.
  • Antagonist refers to an organism that can block or regulate human uracil deoxyribonucleyl glycosylase 9.9 when combined with human uracil deoxyribonuclease 9.9 Molecularly active or immunologically active molecule. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human uracil deoxyribonuclease 9.9.
  • Regular refers to changes in the function of human uracil deoxyribonuclease 9.9, including an increase or decrease in protein activity, changes in binding characteristics, and human uracil deoxyribonuclease Any other changes in biological, functional or immune properties.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify human uracil deoxyribonuclease by standard protein purification techniques 9.9.
  • Substantially pure human uracil deoxyribonuclease 9.9 produces a single main band on a non-reducing polyacrylamide gel.
  • Human uracil deoxyribonuclease 9.9 The purity of the polypeptide can be analyzed by amino acid sequence analysis.
  • Complementary refers to the natural binding of polynucleotides that are base-paired under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to target sequences under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (La sergene s of tware package, DNASTAR, Inc., Madis on Wi s.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 7 3: 237-244). 0 Cluster method by checking all pairs The distance between them arranges the groups of sequences into clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun He in (He in J., (1990) Methods in enzymo logy 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine Acids 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 RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “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,? ( & 13 ') 2 &? 7, which can specifically bind to the human uracil deoxyribonuclease 9.9 epitope.
  • 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 exists in a living thing, but the same polynucleotide or polypeptide is in the natural system with some or all of it. Separation with the material that coexists with it is separation.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human uracil deoxyribonuclease 9.9 refers to human uracil deoxyribonuclease 9.9 which is substantially free of other proteins, lipids, Sugars or other substances. Those skilled in the art can purify human uracil deoxyribonuclease by standard protein purification techniques 9.9. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. Human uracil deoxyribose glycosylase 9. 9 The purity of the polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human uracil deoxyribonucleyl glycosylase 9.9, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the starting Methionine residue.
  • the invention also includes fragments, derivatives and analogs of human uracil deoxyribonuclease 9.9.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human uracil deoxyribonucleyl glycosylase 9.9 of the present invention.
  • a fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) such a type in which one or more amino acid residues are substituted with other groups to include a substituent; or (III) such One, in which the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences).
  • such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1760 bases, and its open reading frame of 560-832 encodes 90 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile to human UDG, and it can be inferred that the human uracil deoxyribonuclease 9.9 has similar functions to human UDG.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DM forms include cDNA, genomic DM or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DM can be coded or non-coded.
  • 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 can be days Naturally occurring allelic 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 a denaturant during hybridization, such as 503 ⁇ 4 (v / v) formamide, 0.1% calf serum / 0.1% Fi col 1, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides. Nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human uracil deoxyribonuclease 9.9.
  • 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 human uracil deoxyribonuclease 9.9 according to 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 MA sequence to obtain the double-stranded DM of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the separation of the CDM sequences.
  • the standard method for isolating cDNA 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. There are many mature techniques for fflRNA extraction. Kits are also commercially available (Qiagene). CDM libraries are also commonly used (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries such as different cDNAs from Clontech library. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • 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-DM or DM-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human uracil deoxyribonucleyl glycosylase 9.9's The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and 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 protein product of the human uracil deoxyribonuclease 9.9 gene can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA )Wait.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA )Wait.
  • a method for amplifying DNA / RNA by PCR is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be 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 produced by genetic engineering using the vector of the present invention or directly with human uracil deoxyribonuclease 9.9, and produced by recombinant technology.
  • a method of a polypeptide according to the invention is also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly with human uracil deoxyribonuclease 9.9, and produced by recombinant technology.
  • a polynucleotide sequence encoding human uracil deoxyribonuclease 9.9 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Suitable carriers in the present invention include, but are not limited to: T7 promoter-based expression vector expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human uracil deoxyribonuclease 9.9 and appropriate transcriptional / translational regulatory elements. These methods include in vitro f-group DNA technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Mo lecu lar Cloning, Laboratory Manua l, Coll d Harbor Harbora 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: E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pair enhancers on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human uracil deoxyribonucleyl glycosylase 9.9 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide or recombinant vector containing the Genetically engineered host cells.
  • 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.
  • Transformation of a host cell with a DM 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 DM 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.
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human uracil deoxyribonucleyl glycosylase 9. 9 (Science, 1984; 224: 1431). Generally speaking, there are the following steps:
  • polynucleotide or variant encoding human uracil deoxyribonuclease 9.9 according to the present invention, or a suitable host transformed or transduced with a recombinant expression vector containing the polynucleotide Cell
  • 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, egg 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 egg 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
  • 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.
  • Uracil DM glycosylase catalyzes the first step in this repair pathway, removing the mutant uracil.
  • the N-glycosyl bond between uracil and deoxyribose is firstly hydrolyzed, and then the rest of the repair work is performed in order by AP endonuclease, phosphodiesterase, DM polymerase and DM ligase.
  • UDG also plays a role in DM replication. Human nuclear UDG and mitochondrial UDG have been discovered. They repair nuclear DM and mitochondrial DNA, respectively, and they are transcribed from the same gene.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human uracil deoxyribonuclease, both of which have similar biological functions.
  • the mutation of the polypeptide of the present invention in the process of deamination of cytosine to uracil can be recognized and repaired before replication to maintain the stability of the cell genome. Abnormal expression will cause some related metabolic disorders such as pyrimidine metabolism disorder; abnormal expression will lead to mutations in the process of deamination of cytosine to uracil that cannot be identified and repaired before replication, resulting in genetic information errors It will produce wrong protein signals; its abnormal expression will also cause tumors, immune system disorders and other pathological processes, and produce related diseases.
  • the abnormal expression of the human uracil deoxyribonuclease 9.9 of the present invention will produce various diseases, especially pyrimidine metabolism deficiency diseases, tumors, development disorders, inflammation, and immune diseases. These diseases include But not limited to:
  • Pyrimidine and Purine Metabolism Defects Abnormal pyrimidine metabolism such as orotic aciduria, adenosine deaminase deficiency, abnormal purine metabolism such as Ray-niney syndrome, xanthineuria
  • Tumors gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, astrocytoma, ependymal tumor, glioblastoma, neurofibromas, colon cancer, melanoma , Endometrial cancer, fibroma, fibrosarcoma
  • Developmental disorders congenital abortion, cleft palate, lack of limbs, limb differentiation disorders, atrial septal defect, neural tube defects, congenital hydrocephalus, mental retardation, brain development disorders, skin, fat and muscular dysplasia, Bone and joint dysplasia, various metabolic defects, stunting inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, multiple sclerosis of the cerebral spinal cord, glomerulonephritis , Myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, various infectious inflammations
  • Immune diseases systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • Abnormal expression of the human uracil deoxyribonuclease 9.9 of the present invention will also produce certain genetic and blood diseases.
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can treat various diseases, especially pyrimidine metabolism deficiency diseases, tumors, development disorders, inflammation, immune diseases, Some hereditary, bloody diseases, etc.
  • the present invention also provides screening compounds to identify improving (agonist) or suppressing (antagonist) human uracil.
  • Agonists increase human uracil deoxyribonuclease 9.9 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human uracil deoxyribonuclease 9.9 can be cultured with a labeled human uracil deoxyribonuclease 9.9 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human uracil deoxyribonuclease 9.9 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of human uracil deoxyribonuclease 9.9 can bind to human uracil deoxyribonuclease 9.9 and eliminate its function, or inhibit the production of the polypeptide, or with the polypeptide The active site binding prevents the polypeptide from performing biological functions.
  • human uracil deoxyribonuclease 9.9 can be added to a bioanalytical assay, and human uracil deoxyribonuclease 9.9 and its receptors can be determined by measuring compounds The effects of interactions between humans to determine whether a compound is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human uracil deoxyribonuclease 9.9 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 9.9 molecules of human uracil deoxyribonuclease are generally labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the present invention also provides antibodies against human uracil deoxyribonuclease 9.9 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 obtained by direct injection of human uracil deoxyribonuclease 9.9 into animals (such as rabbits, mice, rats, etc.).
  • animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including But it is not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human uracil deoxyribonuclease 9.9 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple 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 using known techniques (Morrison et al., PNAS, 1985, 81: 6851).
  • the existing technology for the production of single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human uracil deoxyribonuclease 9.9.
  • Antibodies against human uracil deoxyribonuclease 9.9 can be used in immunohistochemical techniques to detect human uracil deoxyribonuclease 9.9 in biopsy specimens.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • 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 uracil deoxyribonucleic acid glycosylation Enzyme 9.9 positive cells.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human uracil deoxyribonuclease 9.9.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of human uracil deoxyribonuclease 9.9.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human uracil deoxyribonucleyl glycosylase 9.9.
  • tests are well known in the art and include FISH and radioimmunoassays.
  • the level of human uracil deoxyribonuclease 9.9 detected in the test can be used to explain the importance of human uracil deoxyribonuclease 9.9 in various diseases and to diagnose humans Diseases where uracil deoxyribonuclease 9.9 works.
  • 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 human uracil deoxyribonuclease 9.9 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 uracil deoxyribonuclease 9.9.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human uracil deoxyribonuclease 9.9 to inhibit endogenous human uracil deoxyribonuclease 9.9 active.
  • a mutated human uracil deoxyribonuclease 9.9 may be shortened and a human uracil deoxyribonuclease 9.9 lacking a signaling domain, although it can be related to downstream Substrate binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human uracil deoxyribonuclease 9.9.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a recombinant viral vector carrying a polynucleotide encoding human uracil deoxyribonuclease 9.9 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human uracil deoxyribonuclease 9.9 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly 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 uracil deoxyribonuclease 9.9 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific A. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target MA to perform endonucleation.
  • Antisense RNA, MA, and ribozymes can be obtained by any existing MA or DM synthesis technology, such as the technology of solid phase phosphate amide synthesis of oligonucleotides has been widely used.
  • Antisense molecules can be obtained by in vitro or in vivo transcription of DM sequences encoding the RNA. This DM sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding human uracil deoxyribonuclease 9.9 can be used to diagnose diseases related to human uracil deoxyribonuclease 9.9.
  • a polynucleotide encoding human uracil deoxyribonuclease 9.9 can be used to detect the expression of human uracil deoxyribonuclease 9.9 or human uracil deoxyribonucleic acid sugar in a disease state Aberrant expression of basylase 9.9.
  • the DM sequence encoding human uracil deoxyribonuclease 9.9 can be used to hybridize biopsy specimens to determine the expression of human uracil deoxyribonuclease 9.9.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These technical methods are all mature technologies that are publicly available, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microairay) or a DM chip (also known as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human uracil deoxyribose glycosylase 9.9 specific primers for RM-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human uracil deoxyribonuclease glycosylation 9.9 transcripts .
  • Human uracil deoxyribonuclease 9.9 gene mutations can also be used to diagnose human uracil deoxyribonuclease 9.9-related diseases.
  • Human uracil deoxyribonuclease 9.9 mutant forms include point mutations, translocations, deletions, recombinations, and others compared to normal wild-type human uracil deoxyribonuclease 9.9 DM sequences Any exceptions etc. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins, so the Nort hern blotting and Wes tern blotting can be used to indirectly determine whether the 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. At present, the specificity of each gene on the chromosome needs to be identified Site. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for labeling 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 DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to build chromosome-specific cDM libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDM or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the 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 present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • containers there can be medicines manufactured, used or sold by Instructions given by the government regulatory agency for the product or biological product, which reflects the permission of the government regulatory agency for production, use, or sale to be administered to the human body.
  • 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 uracil deoxyribonuclease 9.9 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human uracil deoxyribonucleotide glycosylase 9.9 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
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0763H03 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer. After purification of Qiagene's kit, PCR amplification was performed with the following primers:
  • Pr imer 1 5'- TAGTCAAGAGCTGTGCCCCAAGGT-3, (SEQ ID NO: 3)
  • Pr imer2 5'- GCACGGCTGCGAGAAGACGAAGCT -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 the implicit ol / L KCl at 5 0, 10ramol / L Tri s -HCl pH8 5, 1. 5mmol / L MgCl 2, 2 (mol / L dNTP, l. Opmol primer, 1U Taq DM polymerase (product of 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. Set ⁇ -act in as a positive control and template blank as a negative control at the same time during RT-PCR.
  • the amplified product was purified using a QIAGEN kit, and connected to a pCR vector using a TA cloning kit (Invi trogen). DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1760bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human uracil deoxyribonucleyl glycosylase 9.9 gene expression
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] rempliThis method involves acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidinium isothiocyanate-25 mM sodium citrate, 0.21 ⁇ 1 Sodium acetate (114.0) homogenize the tissue, add 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), mix and centrifuge. Aspirate the aqueous layer and add isopropyl Alcohol (0.8 vol) and the mixture was centrifuged to obtain MA precipitate. The resulting RNA precipitate was washed with 70% ethanol, dried and dissolved in water.
  • RNA was used in 20 mM 3- (N-morpholino) propane Sulfuric acid (H7. 0)-5 mM sodium acetate-ImM EDTA-2. 2M formaldehyde on a 1. 23 ⁇ 4 agarose gel for electrophoresis. Then transfer to a nitrocellulose membrane. Randomized primer method using a- 32 P dATP A 32 P-labeled DNA probe was prepared. The DNA probe used was the PCR amplified human uracil deoxyribonucleyl glycosylase 9.9 coding region sequence (56 ( ⁇ to 8321 ⁇ ) shown in Figure 1.
  • a 32P-labeled probe (approximately 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 P 0 4 (pH7. 4)-5 x SSC- 5 x Denhardt 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, analysis and quantification were performed using Phosphor Imager.
  • Example 4 Recombinant human uracil deoxyribonucleyl glycosylase 9.9 in vitro expression, isolation and purification
  • Pr imer3 5 '-CCCCATATGATGCTTAAAGAAATGCTCACTGGA-3' (Seq ID No: 5)
  • Pr imer4 5 '-CCCGAGCTCTCAAGGACTCTGGACTACCCTAGT-3' (Seq ID No: 6)
  • the two ends of the two primers contain Ndel and Sacl restriction sites Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Ndel and Sacl restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • PCR reaction was performed using the pBS-0763H03 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions were: total volume of combined P BS-0763H03 plasmid 10pg 50 ⁇ 1, primer? 3: 11116]: -3 and ⁇ ]: 1 [116: 1: -4 points and another!] Is 1 ( ⁇ 11101, Advantage polymerase Mix
  • Cycle parameters 94 ° C 20s, 60 ° C 30s, 68 ° C 2 ⁇ , 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 the colibacillus DH5 ⁇ by the calcium chloride method.
  • the LB plate with a final concentration of 3 was cultured overnight, and the positive clones were selected by colony PCR method and sequenced.
  • the positive clones with the correct sequence were selected to transform the recombinant plasmid into the large intestine by the calcium chloride method.
  • Bacterium BL21 (DE3) p lySs product of Novagen.
  • the host bacteria BL21 (pET-0763H03) was cultured at 37 ° C. to logarithmic growth.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following specific human uracil deoxyribonuclease 9.9 peptides:
  • ELISA Use a 15 g / ml bovine serum albumin-polypeptide complex-coated titer plate for ELISA to determine rabbit serum Antibody titer.
  • Total IgG was isolated from antibody-positive rabbit sera with protein A-Sepharose.
  • Peptide was bound to a cyanogen bromide-activated Sepharose 4B column and affinity chromatography was performed from total IgG. Isolation of anti-peptide antibodies. 9 ⁇
  • the immunoprecipitation method demonstrated that the purified antibody could specifically bind to human uracil deoxyribonucleyl glycosylase 9.9.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • the probes can be used to hybridize to the genome or cMA library of normal tissues or pathological tissues from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal 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 imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements Region for homology comparison, if the homology with non-target molecular region is greater than 85% or more than 15 Two consecutive bases are completely the same, the primary probe should generally not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • cold homogenization buffer (0.25 raol / L sucrose; 25 mmol / L Tris-HCl, pH 7.5; 25 mmol / L NaCl; 25 mraol / L MgCl 2 ).
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (OxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution OxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Gene chip or gene microarray is a new technology that many national laboratories and large pharmaceutical companies are currently researching and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on slopes. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, 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 select new tissue-specific new genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as heredity disease. The specific method steps have been reported in the literature.
  • 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 purification, the concentration of the amplified product was adjusted to about 500 ng / ul.
  • a Cartesian 7500 spotter (purchased from Cartesian Company, USA) was spotted on the glass medium. The distance between them is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to prepare DNA on a glass slide to prepare a chip.
  • the specific method steps have been reported 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) by one-step method, and the mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen), and the fluorescent reagents were separately reverse-transcribed.
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) labeled mMA of human mixed tissue, using the fluorescent reagent Cy5dUTP (5- Amino-propargyl- 2'- deoxyuridine 5'-tr iphate coupled to Cy5 f luorescent dye, purchased from Amersham Phamacia Biotech The company) labeled raMA, a specific tissue (or stimulated cell line) of the body, and prepared probes after purification.
  • fluorescent reagent Cy5dUTP (5- Amino-propargyl- 2'- deoxyuridine 5'-tr iphate coupled to Cy5 f luorescent dye, purchased from Amersham Phamacia Biotech The company labeled raMA, a specific tissue (or stimulated cell line) of the body, and
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridizat ion Solution (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar-like fc growth factor Stimulation, 1013HT, scar into fc without stimulation with growth factor, 1 G13HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunum adenocarcinoma, cardia cancer . Based on these 17 Cy3 / Cy5 ratios, a histogram is drawn ( Figure 1). It can be seen from the figure that the expression profile of human uracil deoxyribonuclease 9.9 according to the present invention is very similar to that of human UDG.

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Abstract

L'invention concerne un nouveau polypeptide, une uracil désoxyribonucléotide glycosylase humaine 9.9, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des troubles du métabolisme de la pyrimidine, des tumeurs, des troubles du développement, des inflammations et des maladies immunitaires. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant l'uracil désoxyribonucléotide glycosylase humaine 9.9.
PCT/CN2001/000850 2000-05-24 2001-05-21 Nouveau polypeptide, uracil desoxyribonucleotide glycosylase humaine 9.9, et polynucleotide codant ce polypeptide Ceased WO2001094535A2 (fr)

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AU89494/01A AU8949401A (en) 2000-05-24 2001-05-21 A novel polypeptide, human glycosylase deoxyribonucleotide uracil 9.9 and the polynucleotide encoding the polypeptide

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CN 00115838 CN1324950A (zh) 2000-05-24 2000-05-24 一种新的多肽——人尿嘧啶脱氧核糖核酸糖基化酶9.9和编码这种多肽的多核苷酸
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WO2001096560A1 (fr) Nouveau polypeptide, proteine humaine stat2, et polynucleotide codant ce polypeptide
WO2001092319A1 (fr) NOUVEAU POLYPEPTIDE, RECEPTEUR HUMAIN 19.68 DE L'INTERFERON α, ET POLYNUCLEOTIDE CODANT CE POLYPEPTIDE
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WO2001092515A1 (fr) Nouveau polypeptide, facteur humain de transcription 29.26, et polynucleotide codant ce polypeptide
WO2001092518A1 (fr) Nouveau polypeptide, proteine humaine 9.5 associee a la ccr4, et polynucleotide codant ce polypeptide
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WO2001090177A1 (fr) Nouveau polypeptide, activateur humain de la mort naturelle des cellules b13.64, et polynucleotide codant ce polypeptide
WO2001094401A1 (fr) Nouveau polypeptide, proteine npat humaine 15, et polynucleotide codant pour ce polypeptide
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WO2001094534A2 (fr) Nouveau polypeptide, facteur humain de transcription 9.57, et polynucleotide codant ce polypeptide
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WO2001090379A1 (fr) Nouveau polypeptide, nucleoproteine basophile humaine 22.55, et polynucleotide codant ce polypeptide
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WO2001090131A1 (fr) Nouveau polypeptide, proteine humaine 10.56 du gene cancerigene tre, et polynucleotide codant ce polypeptide
WO2001092324A1 (fr) Nouveau polypeptide, nucleoproteine humaine 10.78 basophile, et polynucleotide codant ce polypeptide

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