WO2001040482A1 - Nouveau polypeptide, eucaryote acetyltransferase 11, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, eucaryote acetyltransferase 11, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001040482A1
WO2001040482A1 PCT/CN2000/000499 CN0000499W WO0140482A1 WO 2001040482 A1 WO2001040482 A1 WO 2001040482A1 CN 0000499 W CN0000499 W CN 0000499W WO 0140482 A1 WO0140482 A1 WO 0140482A1
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polypeptide
polynucleotide
acetyltransferase
eukaryotic
sequence
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PCT/CN2000/000499
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Bioroad Gene Development Ltd. Shanghai
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Priority to AU16880/01A priority Critical patent/AU1688001A/en
Publication of WO2001040482A1 publication Critical patent/WO2001040482A1/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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, eukaryotic acetyltransferase 11, and a polynucleotide sequence encoding the polypeptide. The invention also relates to the preparation method and application of the polynucleotide and polypeptide. Background technique
  • the eukaryotic acetyltransferase superfamily has many members according to different substrates, among which choline 0-acetyltransferase (ChoAcTase), carnitine 0-acetyltransferase (COT), peroxisome carnitine caprylyl Transferases (PC0T), mitochondrial carnitine palmitoyltransferases, etc., due to sequence similarity, have become a family, and the most prominent two of the multiple similar sequence regions are: 1. Three [LIVM]-near the amino terminus There are a series of charged amino acid residues in the middle of the sequence of P dipeptide. Histidine is likely to be the catalytic center.
  • Choline 0-acetyltransferase Choline 0-acetyltransferase (ChAcTase) is involved in the biosynthesis of the neurotransmitter acetylcholine and is associated with certain choline neurological diseases such as Alzheimer's disease.
  • ChoAcTase cDNA of pigs and Drosophila is only 30% homologous, and the interval can reach 70%.
  • ChoAcTase from several different species of mammals has a common function, but the structures are very similar, and the lengths of their mRNAs are also very different.
  • Carnitine 0-acetyltransferase has three members: carnitine acetyltransferase (CAT), carnitine palmitoyl transferase (CPT) and carnitine octanoyl transferase (C0T).
  • CAT carnitine acetyltransferase
  • CPT carnitine palmitoyl transferase
  • C0T carnitine octanoyl transferase
  • the structure, immunogenicity, and catalytic substrate are all different, and the common motif: LPXLPXPXL does not exist in any other acetyltransferase members and is a carnitine binding site.
  • CAT exists in mitochondria and peroxisomes, and the substrate is short-chain acetyl groups (C2-C4). It maintains the relative balance of free CoA and acety-CoA.
  • CPT exists only in mitochondria, and mainly acts on medium and long chain acetyl groups (C2-C4), which promotes cytosolic long-chain fatty acetyl-CoA from mitochondrial inner membrane to ⁇
  • C2-C4 medium and long chain acetyl groups
  • COT exists only in the peroxisome
  • the preferred substrate is hexanoyl-CoAs, which catalyzes the detachment of medium-chain fatty acids from acetyl-CoA to carnitine, and in the medium-chain acetyl-CoA from Peroxisomes play a role in mitochondrial transport.
  • Carnitine acetyltransferase is involved in the metabolism of exogenous fatty acids.
  • the best proof of its metabolic significance is: CAT in patients with ataxia encephalopathy is significantly reduced.
  • the biosynthesis of choline 0-acetyltransferase (ChoAcTase) in the neurotransmitter acetylcholine is related to certain choline neurological diseases, such as Alzheimer's disease; Fatty acid metabolism and CAT in patients with ataxia encephalopathy are significantly reduced.
  • This gene can be applied to fat metabolic diseases and related cerebral neuropathy.
  • 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 eukaryotic acetyltransferase 11.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a eukaryotic acetyltransferase 11.
  • Another object of the present invention is to provide a method for producing eukaryotic acetyltransferase 11.
  • Another object of the present invention is to provide antibodies against the polypeptide __ eukaryotic acetyltransferase 11 of the present invention.
  • Another object of the present invention is to provide mimic compounds against the polypeptide of the present invention, eukaryotic acetyltransferase 11, Antagonists, agonists, inhibitors.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in eukaryotic acetyltransferase 11.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • the sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID NO: 1 A sequence of positions 730-1092; and (b) a sequence of positions 1-1203 in SEQ ID NO: 1.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of eukaryotic acetyltransferase 11 protein, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of a eukaryotic acetyltransferase 11 protein, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for the treatment of cancer, developmental or immune diseases or other diseases caused by abnormal expression of eukaryotic acetyltransferase 1 1.
  • the present invention Other aspects of the invention will be apparent to those skilled in the art due to the disclosure of the technology herein.
  • Nucleic acid sequence refers to an oligonucleotide
  • Nucleotides or polynucleotides and their fragments or parts can also refer to 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 oligopeptides, peptides, polypeptides or protein sequences and fragments or parts thereof.
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” means the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence
  • “Insertion” or “addition” refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule
  • “Replacement” refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with eukaryotic acetyltransferase 11, can cause changes in the protein and thereby regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to eukaryotic acetyltransferase 11.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of eukaryotic acetyltransferase 11 when combined with eukaryotic acetyltransferase 11.
  • Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other molecule that can bind to eukaryotic acetyltransferase 11.
  • Regular refers to a change in the function of eukaryotic acetyltransferase 11, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immunological changes in eukaryotic acetyltransferase 11.
  • 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 eukaryotic acetyltransferase 11 using standard protein purification techniques.
  • a substantially pure eukaryotic acetyltransferase 11 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the eukaryotic acetyltransferase 11 peptide can be analyzed by amino acid sequence.
  • “Complementary” or “complementary” refers to the natural binding of a nucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
  • the sequence "C-T-G-A” may be combined with the complementary sequence "G-A-C-T", and 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 blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow -specific binding, because the conditions of reduced stringency require that the two sequences bind to each other specifically or selectively.
  • Percent identity refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences.
  • the percentage identity can be determined electronically, such as by the MEGALIGN program (La sergene sof tware package, DNASTAR, Inc., Madi son 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 73: 237-244). 0
  • the Cluster method checks all The distances arrange 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 number of matching residues between sequence A and sequence B
  • 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 erazumo 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; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of eukaryotic acetyltransferase 11.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a vector, or such a nucleotide or polypeptide may be part of a composition. Since the carrier or composition is not a component 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 existing in the natural state. .
  • isolated eukaryotic acetyltransferase 11 means that eukaryotic acetyltransferase 11 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
  • Those skilled in the art can purify eukaryotic acetyltransferase 11 using standard protein purification techniques.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of the eukaryotic acetyltransferase 11 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, eukaryotic acetyltransferase 11, which is basically composed of the amino acid sequence shown in SEQ II) 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 may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells).
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude initial methionine residues.
  • the invention also includes fragments, derivatives and analogs of eukaryotic acetyltransferase 11. As used in the present invention, the terms "fragment”, “derivative” and “analog” refer to a polypeptide that substantially maintains the same biological function or activity of the eukaryotic acetyltransferase 11 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a type in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (II) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ ⁇ ) Such a type, 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 type, in which the additional amino acid sequence is fused into the mature polypeptide to form a polypeptide sequence (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) As set forth herein, such fragments, derivatives, and analogs are considered to be within the knowledge of those
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1038 bases in length and its open reading frame (730-1092) encodes 102 amino acids.
  • This polypeptide has a characteristic sequence of a eukaryotic acetyltransferase catalytic center, and it can be deduced that the eukaryotic acetyltransferase 11 has the structure and function represented by the eukaryotic acetyltransferase catalytic center.
  • the polynucleotide of the present invention may be in the form of MA or RNA.
  • 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 sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • degenerate variant in the present invention 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.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60'C; or (2) A denaturant was added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol l, 42.
  • 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, most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding eukaryotic acetyltransferase 11.
  • polypeptide and polynucleotide in the present invention are preferably provided in an isolated form, and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the eukaryotic acetyltransferase 11 of the present invention can be obtained by various methods, such as Polynucleotides are isolated using hybridization techniques well known in the art. These technologies include, but are not limited to:
  • DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring 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 selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of eukaryotic acetyltransferase 11 transcripts; (4) ) Detection of protein products expressed by genes through immunological techniques or determination of biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and 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 gene itself or the fragment of the present invention can of course be used as a probe.
  • the DNA probe can be labeled with a radioisotope, luciferin, or an enzyme (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the eukaryotic acetyltransferase 11 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method of applying a PCR technique to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize DNA / RNA fragments that can be isolated and purified by conventional methods such as gel electrophoresis
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence. ' 1 N
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a eukaryotic acetyltransferase 11 coding sequence, and a recombinant technology for producing a polypeptide of the present invention. method.
  • a polynucleotide sequence encoding a eukaryotic acetyltransferase 11 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, 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 expression vector (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vector expressed in mammalian cells (Lee and Nathans, J Bio Chem. 263: 3521, 1988) and in insect cells
  • a vector derived from a baculovirus In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding eukaryotic acetyltransferase 11 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, difficult-to-synthesize technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be effectively linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polytumor enhancers on the late side of the origin of replication, and adenoviral 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 used in E. coli, etc.
  • a polynucleotide encoding a eukaryotic acetyltransferase 11 or a recombinant vector containing the polynucleotide The body can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or a recombinant vector.
  • host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS, or Bowes s melanoma cells, etc. .
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl.
  • the steps used are well known in the art.
  • the alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the 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 eukaryotic acetyltransferase 11 (Scieence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • Figure 1 shows the eukaryotic acetyltransferase 11 of the present invention at 55-79 in total with 55 amino acids and eukaryotic acetyltransferase enzymes. Comparison of amino acid sequence homology of the chemokine center domain. The upper sequence is eukaryotic acetyltransferase 11, and the lower sequence is the eukaryotic acetyltransferase catalytic center domain. ⁇ "and”: “and” ⁇ "indicate that the probability of the occurrence of different amino acids at the same position between two sequences decreases in sequence.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the eukaryotic acetyltransferase 11 isolated.
  • llkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • RNA from human fetal brain was extracted by guanidine isothiocyanate / phenol / chloroform method. Using Quik mRNA Isolation Kit
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0415 g 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 full-length cDNA contained in the clone 0415g01 is 1203bp (as shown in Seq IDN0: 1), from 730b P to 1092bp there is a 363bp open reading frame (0RF), which encodes a new protein (such as Seq ID N0 : Shown in 2).
  • This clone pBS-0415g01 and the encoded protein was named eukaryotic acetyltransferase 11.
  • Example 2 Domain analysis of cDNA clones
  • the sequence of the eukaryotic acetyltransferase 11 of the present invention and the protein sequence encoded by the eukaryotic acetyltransferase 11 of the present invention were profiled using the GCG profile scan program (Basiclocal Alignment search tool) [Altschul, SF et al.
  • domain analysis was performed in a database such as Prote.
  • the eukaryotic acetyltransferase 11 of the present invention is homologous with the domain eukaryotic acetyltransferase catalytic center at 37-83, and the homology result is shown in FIG. 1.
  • the homology rate is 27%, and the score is 14.64; the threshold value is 12.62.
  • Example 3 Cloning of a gene encoding eukaryotic acetyltransferase 11 by RT-PCR
  • CDNA was synthesized using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction.
  • PCR amplification was performed with the following primers:
  • Primer 1 5,-CGAAGAAATAAAATAAATTGTGGAG- 3 '(SEQ ID NO: 3)
  • Primer2 5,-GAAAAAGGGAAAGGACAGGGAAGAG- 3, (SEQ ID NO: 4)
  • Prinierl is a forward sequence starting at lbp at the 5 end of SEQ ID NO: 1;
  • Primer 2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions A reaction volume of 50 ⁇ 1 contains 50 mmol / L KC1, 10 mmol / L Tris-Cl, (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 2rain thoroughlySet ⁇ _ac t in at the same time as RT-PC'R Positive control and template blank as negative control.
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. I.e. with 4M guanidinium isothiocyanate -25mM sodium citrate, 0.2M sodium acetate (P H4.0) of the tissue was homogenized phenol, 1 volume
  • RNA was synthesized by electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the PCR amplified eukaryotic acetyltransferase 11 coding region sequence (682bp to 945bp) shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpm / mi) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH 7.4)-5 x SSC- 5 x Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filters were washed in 1 x SSC-0.1% SDS at 55 ° C for 30 minutes. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant eukaryotic acetyltransferase 11
  • Primer 3 5'-CCCCATATGATGCTGCCATCGGCTCTCAGGAGCA-3 '(Seq ID No: 5)
  • Primer 4 5,-CATGGATCCCTAGACTCCCACCCGGGACATCCCT-3, (Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BamHI digestion sites, respectively Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Ndel and BaraHI restriction sites correspond to the selection on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Sex endonuclease site.
  • PCR was performed using pBS-0415g01 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: total volume 50 ⁇ 1 contains pBS- 0415g01 plasmid 10pg, primers Primer-3 and Primer- 4 points, and j is 10pmol, Advantage polymerase Mix
  • Cycle parameters 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, 25 cycles in total.
  • the ligation product was transformed into colibacillus DH50 by the calcium chloride method.
  • peptides specific for eukaryotic acetyltransferase 11 were synthesized using a peptide synthesizer (product of PE company): NH 2 -Met-Leu-Pro-Ser-Ala-Leu-Arg-Ser-Thr-Ser-Leu-Ser- Gly-Gly-Phe- C00H (SEQ ID NO: 7).
  • the peptide was coupled to hemocyanin and bovine serum albumin to form a complex. The method is known as: Avrameas, et al. Immunochemistry, 1969; 6: 43 StammUse 4mg of the above hemocyanin-white peptide complex with complete Freund's adjuvant.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same basic hybridization method after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first prehybridized 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. In this embodiment, higher intensity membrane washing conditions (such as lower salt concentration and higher temperature) are used to reduce the hybridization background and only Retain 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 same or complementary oligonucleotide fragment of the polynucleotide SEQ ID NO: 1 is used.
  • a dot blot method is used to fix the sample on the filter membrane. Under high-intensity washing conditions, the first type of probe is used. The hybridization with the sample has the strongest specificity and is retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used; 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.
  • Probe 1 (probel), 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):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • the sample membrane was placed in a plastic bag, and 3-10 mg of pre-hybridization solution (10xDenhardt's; 6xSSC, 0.1 mg / ml CT I) NA (calf thymus DNA) was added. ), After sealing the bag, 68. C water bath for 2 hours.
  • pre-hybridization solution 10xDenhardt's; 6xSSC, 0.1 mg / ml CT I
  • NA 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.
  • Example 10 DNA Microarray Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies.
  • 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 DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And the literature Helle, RA, Schema, M Chai, A., Shalom, I)., (1997) PNAS 94: 2150-2155.
  • 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 amplified by PCR respectively. After purification, the amplified product was adjusted to a concentration of about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip.
  • the specific method and step have been reported in the literature in various ways, and the specific method and step have been reported in the literature in various ways.
  • the sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from normal liver and liver cancer by a single method, and mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent C'y3dUTP (5-Amin () -propargy 1- 2'-deoxyur i dine 5'-tr iphate coupled to Cy3 fluorescent dye (purchased from Amersham Phamacia Biotech) was used to label the mRNA of normal liver tissue, and the fluorescent reagent Cy5dUTP (5-Araino-propargy l-2'-deoxyur idine 5 ' -tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech company) labeled liver cancer tissue mRNA, Probes were prepared after purification.
  • the probes from the two types of tissues and the chips were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000.
  • Scanner purchased from General Scanning Company, USA
  • the scanned image was analyzed and processed with Imagene software (Biodiscovery Company, USA), and the Cy3 / Cy5 ratio of each point was calculated. The points with the ratio less than 0.5 and greater than 2 were considered.
  • Genes with differential expression were used to analyze a UniHyb TM Hybridization Solution for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000.
  • Scanner purchased from General Scanning Company, USA
  • the scanned image was analyzed and processed with Imagene software (Biodiscovery Company, USA), and the Cy3
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immune diseases, etc.
  • Acetylcholine is an excitatory neurotransmitter at the neuromuscular junction of spinal thrusters. Nerve impulses are passed between nerve cells, or from nerve cells to muscle or glandular tissues, where they cause effects (eg, contraction or secretion). In this process, acetylcholine can increase the ion permeability of the post-synaptic membrane, thereby affecting the conduction of the entire nerve impulse.
  • the main function of the polypeptide of the present invention is to participate in the biosynthesis of the neurotransmitter acetylcholine. In addition, it can also play a role in the transport of medium-chain acetyl-CoA from peroxisomes to mitochondria, and also participate in the metabolism of exogenous fatty acids and other processes. . Therefore, the polypeptide of the present invention has an important role in the living body, and can be used to diagnose and treat many diseases, such as certain choline neurological diseases such as Alzheimer's disease and the like. Diseases treatable with the polypeptide of the present invention include other neurological diseases, malignant tumors, endocrine system diseases, development-related diseases, immune diseases, and human acquired immune deficiency syndrome (AIDS).
  • AIDS human acquired immune deficiency syndrome
  • polypeptides of the present invention can be used to treat common diseases of the nervous system of humans including:
  • Cerebrovascular disease transient ischemic attack, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage; intracranial space occupying lesions: glioma, meningiomas, neurofibromas, pituitary adenomas, intracranial Granuloma
  • Nervous system degenerative diseases Alzheimer's disease, Parkinson's disease, chorea, depression, amnesia, Huntington's disease, epilepsy, migraine, dementia, multiple sclerosis;
  • Neuromuscular diseases myasthenia gravis, spinal muscular atrophy, muscular pseudohypertrophy, Duchemie muscular dystrophy, tonic muscular dystrophy, myasthenia, retarded dyskinesia, dystonia;
  • Neurocutaneous Syndrome Neurofibromatosis, tuberous sclerosis, trigeminal neurohemangioma, ataxia telangiectasia;
  • Peripheral nerve disease trigeminal neuralgia, facial paralysis, bulbar palsy, sciatica, Glinger-Barre syndrome.
  • Developmental disorders that can be treated using the polypeptides of the present invention include: spina bifida, craniocerebral fissure, anencephaly, malocclusion, foramen forebral malformation, Down syndrome, congenital hydrocephalus, aqueduct malformation, cartilage dysgenesis Dwarfism, spinal epiphyseal dysplasia, pseudochondral dysplasia, Langer-Giedion syndrome, funnel chest, gonad hypoplasia, congenital adrenal hyperplasia, upper urethral tract, short stature syndrome (such as Conrad i syndrome And Danbolt-Closs syndrome), congenital glaucoma or cataract, congenital lens position abnormality, congenital blepharoplasia, retinal dysplasia, congenital optic nerve atrophy, congenital sensorineural hearing loss, cracked hands and cracked feet, Teratosis, Williams Syndrome, Alagille Syndrome, Baywet Syndrome, etc.
  • Various tumors that can be treated using the polypeptide of the present invention include: including epithelial tissue (such as basal epithelium, squamous epithelium, mucus cells, etc.), (such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue, blood vessels and lymphatic endothelial cells Tissue, etc.), hematopoietic tissue (such as B cells, T cells, histiocytes, etc.), tumors of central nervous tissue, peripheral nerve tissue, endocrine tissue, gonadal tissue, special tissue (such as dental tissue, etc.), for example, Stomach cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, esophageal cancer, etc.
  • epithelial tissue such as basal epithelium, squamous epithelium, mucus cells, etc.
  • fibrous tissue such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) eukaryotic acetyltransferase 11.
  • Agonists enhance eukaryotic acetyltransferase 11 to stimulate biological functions such as cell proliferation.
  • Antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing eukaryotic acetyltransferase 11 can be cultured with labeled eukaryotic acetyltransferase 11 in the presence of a drug, and then the ability of the drug to increase or block this interaction can be determined
  • Antagonists of eukaryotic acetyltransferase 11 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • Eukaryotic acetyltransferase 11 antagonists can bind to eukaryotic acetyltransferase 11 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to prevent the polypeptide from developing. Play biological functions.
  • eukaryotic acetyltransferase 11 When screening compounds as antagonists, eukaryotic acetyltransferase 11 can be added to the bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between eukaryotic acetyltransferase 11 and its receptor. . Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to eukaryotic acetyltransferase 11 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, the eukaryotic acetyltransferase 11 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against eukaryotic acetyltransferase 11 epitopes. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting eukaryotic acetyltransferase 11 directly into immunized animals (such as home immunity, mice, rats, etc.).
  • immunized animals such as home immunity, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for the preparation of monoclonal antibodies to eukaryotic acetyltransferase 11 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cells Hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morrson et al, PNAS, 1985, 81: 685 1). 0 Existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single chain antibodies against eukaryotic acetyltransferase 11.
  • Antibodies against eukaryotic acetyltransferase 11 can be used in immunohistochemical techniques to detect eukaryotic acetyltransferase 11 in biopsy specimens.
  • Monoclonal antibodies that bind to eukaryotic acetyltransferase 11 can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • eukaryotic acetyltransferase 11 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 eukaryotic acetyltransferase 11-positive cells .
  • the antibodies in the present invention can be used to treat or prevent diseases related to eukaryotic acetyltransferase 11.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of eukaryotic acetyltransferase 11.
  • W 1 The invention also relates to a diagnostic test method for quantitative and localized detection of eukaryotic acetyltransferase 11 levels. These tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of eukaryotic acetyltransferase 11 detected in the test can be used to explain the importance of eukaryotic acetyltransferase 11 in various diseases and to diagnose diseases in which eukaryotic acetyltransferase 11 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding eukaryotic acetyltransferase 11 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 eukaryotic acetyltransferase 11.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant eukaryotic acetyltransferase 11 to inhibit endogenous eukaryotic acetyltransferase 11 activity.
  • a variant eukaryotic acetyltransferase 11 may be a shortened eukaryotic acetyltransferase 11 lacking a signaling domain, and although it can bind to a downstream substrate, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of eukaryotic acetyltransferase 11.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses can be used to transfer polynucleotides encoding eukaryotic acetyltransferase 11 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a eukaryotic acetyltransferase 11 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding a recombinant eukaryotic acetyltransferase 1 1 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 eukaryotic acetyltransferase 11 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite 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 DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleosides are connected using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • Polynucleotide encoding eukaryotic acetyltransferase 11 can be used for diseases related to eukaryotic acetyltransferase 1 1 Diagnosis of the disease.
  • the polynucleotide encoding eukaryotic acetyltransferase 11 can be used to detect the expression of eukaryotic acetyltransferase 11 or the abnormal expression of eukaryotic acetyltransferase 11 in a disease state.
  • the DNA sequence encoding eukaryotic acetyltransferase 11 can be used to hybridize biopsy specimens to determine the expression status of eukaryotic acetyltransferase 1 1.
  • 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.
  • Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Eukaryotic acetyltransferase 11 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect eukaryotic acetyltransferase 11 transcription products.
  • Detection of mutations in the eukaryotic acetyltransferase 11 gene can also be used to diagnose eukaryotic acetyltransferase 1 1 related diseases.
  • Eukaryotic acetyltransferase 11 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type eukaryotic acetyltransferase 1 1 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific loci of genes on chromosomes need to be identified.
  • only few chromosome markers based on actual sequence data can be used to mark chromosomal locations.
  • the important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35 bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization (FI SH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in a single step.
  • FI SH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be To correlate with genetic map data. These data can be found, for example, in V. Mckusick, Mendel iaii Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Eukaryotic acetyltransferase 11 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of eukaryotic acetyltransferase 11 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une eucaryote acétyltransférase 11, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour l'eucaryote acétyltransférase 11.
PCT/CN2000/000499 1999-11-30 2000-11-27 Nouveau polypeptide, eucaryote acetyltransferase 11, et polynucleotide codant pour ce polypeptide WO2001040482A1 (fr)

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CN99124158.4 1999-11-30
CN 99124158 CN1298006A (zh) 1999-11-30 1999-11-30 一种新的多肽——真核乙酰转移酶11和编码这种多肽的多核苷酸

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DATABASE GENBANK [online] 4 August 1999 (1999-08-04), OHARA O., NAGASE T., XP002942603, accession no. NCBI Database accession no. BAA82988 *

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