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WO2001075022A2 - Nouveau polypeptide, eucaryon acetyl transferase humaine 12, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, eucaryon acetyl transferase humaine 12, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001075022A2
WO2001075022A2 PCT/CN2001/000360 CN0100360W WO0175022A2 WO 2001075022 A2 WO2001075022 A2 WO 2001075022A2 CN 0100360 W CN0100360 W CN 0100360W WO 0175022 A2 WO0175022 A2 WO 0175022A2
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Prior art keywords
polypeptide
polynucleotide
acetyltransferase
human eukaryotic
sequence
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WO2001075022A3 (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 AU50257/01A priority Critical patent/AU5025701A/en
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Publication of WO2001075022A3 publication Critical patent/WO2001075022A3/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, a human eukaryotic acetyltransferase 12, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a 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 palmitoyl transferase, etc. have become a family member due to sequence similarity.
  • the most prominent two of its multiple similar sequence regions are: First, there are three [LIVM] -P dipeptides near the amino terminus. Second, there is a series of charged amino acid residues in the middle of the sequence, and 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 from several different mammals has a common function, but the structures are very similar, and the lengths of their raRNAs also vary greatly.
  • 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, mainly acting on medium and long-chain acetyl groups (C2-C4), which promotes cytosolic long-chain fatty acetyl-CoA from mitochondrial inner membrane to ⁇ Transport of oxidation sites
  • 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 evidence for its metabolic significance is: CAT in patients with ataxia encephalopathy is significantly reduced.
  • Choline 0-acetyltransferase (ChAcTase) biosynthesis in the neurotransmitter acetylcholine it is related to certain choline neurological diseases, such as Alzheimer's disease;
  • Carnitine acetyltransferase is involved in the metabolism of exogenous fatty acids, and the CAT in patients with ataxia encephalopathy is significantly reduced.
  • the research and development of this gene can be applied to fat metabolic diseases and related cerebral neuropathy.
  • human eukaryotic acetyltransferase 12 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so the identification of more involved in these Processes for the identification of human eukaryotic acetyltransferase 12 proteins, especially the amino acid sequence of this protein. Isolation of the new human eukaryotic acetyltransferase 12 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the diagnosis and / or treatment of a disease, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human eukaryotic acetyltransferase 12.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human eukaryotic acetyltransferase 1 2.
  • Another object of the present invention is to provide a method for producing human eukaryotic acetyltransferase 12.
  • Another object of the present invention is to provide an antibody against the human eukaryotic acetyltransferase 1 2 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the human eukaryotic acetyltransferase 12 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with an abnormality of human eukaryotic acetyltransferase 12.
  • 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 multiple The peptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 670-987 in SEQ ID NO: 1; and (b) a sequence having 1-1517 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 the activity of human eukaryotic acetyltransferase 12 protein, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of human eukaryotic acetyltransferase 12 protein in vitro, comprising detecting mutations 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 for the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human eukaryotic acetyltransferase 12.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genome or a synthetic DM or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule
  • polypeptide or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants may have "conservative" changes in which the substituted amino acid 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 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 human eukaryotic acetyltransferase 12, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human eukaryotic acetyltransferase 12.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human eukaryotic acetyltransferase 12 when combined with human eukaryotic acetyltransferase 12.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind human eukaryotic acetyltransferase 12.
  • Regular refers to a change in the function of human eukaryotic acetyltransferase 12, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human eukaryotic acetyltransferase 12 Change.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human eukaryotic acetyltransferase 12 using standard protein purification techniques. Essentially pure human eukaryotic acetyltransferase 12 produces a single main band on a non-reducing polyacrylamide gel. The purity of human eukaryotic acetyltransferase 12 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology A partially complementary sequence that at least partially inhibits the hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods, such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in 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 X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by Clus ter method or by methods known in the art such as; Totun Hein to determine the percent identity between nucleic acid sequences (Hein J., (1990) Methods in emzumology 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 a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. 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,? ( ⁇ ) 2 and? It can specifically bind to the epitope of human eukaryotic acetyltransferase 12.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • 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 eukaryotic acetyltransferase 12 means that human eukaryotic acetyltransferase 12 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human eukaryotic acetyltransferase 12 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human eukaryotic acetyltransferase 12 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human eukaryotic acetyltransferase 12, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the 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). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human eukaryotic acetyltransferase 12.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human eukaryotic acetyltransferase 12 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 replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) such a type in which one or more amino acid residues are substituted with other groups to include a substituent; or (III) such A type in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a type in which an additional amino acid sequence is fused into the mature polypeptide Polypeptide sequences (such as leader sequences or secreted sequences or sequences used to purify this polypeptide) or protease sequences As described herein, 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 polynucleotide sequence with a total length of 1,517 bases, and its open reading frame 670-987 encodes 105 amino acids.
  • this peptide has a similar expression profile to human eukaryotic acetyltransferase 11, and it can be deduced that the human eukaryotic acetyltransferase 12 has similar functions to human eukaryotic acetyltransferase 11.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DM can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding the 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 having a sequence different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% ( ⁇ / ⁇ ) formamide, 0.1% calf serum / 0.1% Fico ll, 42 ⁇ , etc .; or (3) Hybridization occurs only when the identity between the two sequences 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, 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 eukaryotic acetyltransferase 12.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human eukaryotic acetyltransferase 12 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) separating the double-stranded DNA sequence from the DM of the genome; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the 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.
  • Kits are also commercially available (Q i agene).
  • the construction of cDNA libraries is also a common method (Sarabrook, et al., Molecular Cloning, A Laboratory Manua, Cold Harbor Labora tory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be screened from these cDM 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 human eukaryotic acetyltransferase 12 transcripts; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by human eukaryotic acetyltransferase 12 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method for amplifying DNA / RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RM fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human eukaryotic acetyltransferase 12 coding sequence, and a recombinant technology for producing a polypeptide of the present invention. method.
  • a polynucleotide sequence encoding human eukaryotic acetyltransferase 12 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • pMSXND expression vectors expressed in mammalian cells Lee and Na thans, J Bio Chem. 263: 3521, 1988
  • baculovirus-derived vectors expressed in insect cells 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.
  • An expression vector for DNA sequences and appropriate transcriptional / translational regulatory elements include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua, Cold 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: the l ac or trp promoter of E.
  • 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 eukaryotic acetyltransferase 12 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • 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 DM can be harvested after the exponential growth phase and treated with the ⁇ 01 2 method.
  • the steps used are well known in the art.
  • MgC 12 If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipids. Body packaging, etc.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human eukaryotic acetyltransferase 12 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • polynucleotide or variant
  • encoding human human eukaryotic acetyltransferase 12 of the present invention or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. When host cells grow to proper After inducing the cell density, the appropriate promoter (such as temperature conversion or chemical induction) is used to induce the selected promoter, and the cells are cultured for a period of time.
  • the appropriate promoter such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human eukaryotic acetyltransferase 12 and human eukaryotic acetyltransferase 11 of the present invention.
  • the upper graph is a graph of the expression profile of human eukaryotic acetyltransferase 12, and the lower graph is the graph of expression profile of human eukaryotic acetyltransferase 11.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates non-starved L02
  • 8 indicates L02 +, l hr, As 3+
  • 9 ECV304 PMA-, 10 ECV304 P A +, 11 fetal liver, 12 normal liver, 1 3 thyroid, 14 skin, 15 fetal lung, 16 lung, 17 lung cancer, 18 fetal spleen, 19
  • the spleen, 20 indicates the prostate
  • 21 indicates the fetal heart
  • 22 indicates the heart
  • 23 indicates muscle
  • 24 indicates testis
  • 25 indicates fetal thymus
  • 26 indicates thymus.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human eukaryotic acetyltransferase 12 isolated. 12kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isola ti on Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed CDNA is formed.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragment into the multicloning site of pBSK (+) vector (Clontech) to transform DH5 ⁇ .
  • the bacteria formed a CDM library.
  • the sequences at the 5 'and 3' ends of all clones were determined using Dye terminate cyc le react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer).
  • the determined cDNA sequence was compared with an existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0516el2 was a new DNA.
  • a series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone.
  • the 0516el 2 clone contains a full-length cDNA of 1517bp (as shown in Seq ID NO: 1), a 317bp open reading frame (0RF) from 670bp to 987bp, and encodes a new protein (such as Seq ID NO: 2).
  • This clone pBS-0516el2 and the encoded protein was named human eukaryotic acetyltransferase 12.
  • Example 2 Cloning of a gene encoding human eukaryotic acetyltransferase 12 by RT-PCR
  • CDM was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction.
  • PCR amplification was performed with the following primers:
  • Pr imerl 5
  • one GTATTTGCTACTTTTACTTTCATC-3 (SEQ ID NO: 3)
  • Pr iraer2 5'- CAAGCAATTTTTTCCCTTTATTTT-3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Amplification conditions 50 ⁇ l / L KC1, l Ommo l / L Tris s-CI, (pH 8.5.5), 1. 5 ramo l / L MgCl 2 , 200 ⁇ in a reaction volume of 50 ⁇ 1 mol / L dNTP, l Opraol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-1515bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human eukaryotic acetyltransferase 12 gene expression:
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • RNA in 20 mM 3- (N-morpholino) Propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA-2.2% formaldehyde was run on a 1.2% agarose gel. It was then transferred to a nitrocellulose membrane.
  • A- 32 P dATP was used to prepare 32 P-labeled DNA probes by random primers.
  • the DNA probe used was the PCR amplified human eukaryotic acetyltransferase 12 coding region sequence (670bp to 987bp) shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RM was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7. 4)-5 x SSC-5 x Denhardt's solution and 200 ⁇ ⁇ / ⁇ 1 salmon sperm DNA. After hybridization, place the filter at 1 x SSC-0. 1 ° /. Wash in SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human eukaryotic acetyltransferase 12
  • Pr imer 3 5 '-CATGCTAGCATGCAAGCCATTTGCACTCTGGAA- 3' (Seq ID No: 5)
  • Pr imer4 5 '-CCCGAATTCTCATAATAAGGACATTGTTGTTTG- 3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and EcoRI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Ndel and EcoRI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3).
  • PCR was performed using the pBS-0516el2 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0516el2 plasmid, Primer-3 and Primer-4 was divided into 1 Opmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60 U C 30s, 68 U C 2 min, a total of 25 cycles. Ndel and EcoRI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into E. coli DH5 ct using the calcium chloride method.
  • peptides specific for human eukaryotic acetyltransferase 12 were synthesized using a peptide synthesizer (product of PE): NH2-Met-Gln-Ala-I le-Cys-Thr-Leu-Glu-Leu-H i s-Ala -Val-Lys-Thr-Pro-COOH (SEQ ID NO: 7).
  • the peptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • 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 blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained. First, the selection of the probe
  • 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;
  • 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 (probe2), 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):
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of large numbers of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature, for example, refer to the literature DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And the literature Helle, RA, Schema, M. , Chai, A., Shalom, D., (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 concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in an ultraviolet cross-linker. After elution, the slides were fixed to fix the DM on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spotting processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified using OHgotex mRNA Midi Kit (purchased from QiaGen).
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label mRNA of human mixed tissues, and the fluorescent reagent Cy5dUTP (5-Amino- Propargy 2'-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amershara Pharaacia Biotech Company, was used to label the mRNA of specific tissues (or stimulated cell lines) of the body, and probes were prepared after purification.
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased
  • Solut ion (purchased from TeleChem) was used for hybridization for 16 hours, and then washed with a washing solution (lx SSC, 0.2% SDS) at room temperature, and then scanned with a ScanArray 3000 scanner (purchased from General Scanning, USA). Images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, Fetal lung and fetal heart.
  • 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 inflammations, HIV infections and immune diseases.
  • Acetylcholine is an excitatory neurotransmitter at the neuromuscular junction of spinal thrusters. Nerve impulses are transmitted between nerve cells, or from nerve cells to muscle or glandular tissues, so that they have an effect (eg (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 . 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, neurofibroma, pituitary adenoma, intracranial granulation Swollen
  • 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, Duchenne muscular dystrophy, tonic muscular dystrophy, myasthenia, retarded dyskinesia, dystonia;
  • Neurocutaneous Syndrome Neurofibromatosis, Tuberous Sclerosis, Cerebral Trigeminal Hemangiomatosis, Ataxia Capillary Dilatation;
  • Peripheral nerve diseases trigeminal neuralgia, facial paralysis, bulbar palsy, sciatica, Guillain-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, recessive, with short stature syndrome (such as Conradi Syndrome and Danbol t_Closs syndrome), congenital glaucoma or cataract, congenital lens position abnormality, congenital blepharoplasia, retinal dysplasia, congenital optic atrophy, congenital sensorineural hearing loss, cracked hands and feet, Teratosis, Williams Syndrome, Alagille Syndrome, Baywet Syndrome, etc.
  • stature syndrome
  • 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) human eukaryotic acetyltransferase 12.
  • Agonists enhance human eukaryotic acetyltransferase 12 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human eukaryotic acetyltransferase 12 can be cultured with labeled human eukaryotic acetyltransferase 12 in the presence of drugs. The ability of the drug to increase or block this interaction is then measured.
  • Antagonists of human eukaryotic acetyltransferase 12 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human eukaryotic acetyltransferase 12 can bind to human eukaryotic acetyltransferase 12 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot exert its biology Features.
  • human eukaryotic acetyltransferase 12 When screening compounds as antagonists, human eukaryotic acetyltransferase 12 can be added to the bioanalytical assay to determine whether the compound is a compound by measuring the effect of the compound on the interaction between human eukaryotic acetyltransferase 12 and its receptor. Antagonist. Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to human eukaryotic acetyltransferase 12 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, 12 molecules of human eukaryotic acetyltransferase 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 invention also provides antibodies against human eukaryotic acetyltransferase 12 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human eukaryotic acetyltransferase 12 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's Agent.
  • Techniques for preparing monoclonal antibodies to human eukaryotic acetyltransferase 12 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.
  • Chimera combining human constant region and non-human variable region Antibodies can be produced using existing techniques (Morr i son et al, PNAS, 1985, 81: 6851). 0 Existing techniques for producing single-chain antibodies (US Pat No. 4946778) can also be used to produce anti-human eukaryotic acetyltransfers. Single-chain antibody to enzyme 12.
  • Antibodies against human eukaryotic acetyltransferase 12 can be used in immunohistochemical techniques to detect human eukaryotic acetyltransferase 12 in biopsy specimens.
  • Monoclonal antibodies that bind to human eukaryotic acetyltransferase 12 can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human eukaryotic acetyltransferase 12 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 disulfide exchange.
  • This hybrid antibody can be used to kill human eukaryotic acetyltransferase 12 positive cell.
  • the antibodies of the present invention can be used to treat or prevent human eukaryotic acetyltransferase 12-related diseases. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human eukaryotic acetyltransferase 12.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human eukaryotic acetyltransferase 12 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human eukaryotic acetyltransferase 12 detected in the test can be used to explain the importance of human eukaryotic acetyltransferase 12 in various diseases and to diagnose diseases in which human eukaryotic acetyltransferase 12 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.
  • the polynucleotide encoding human eukaryotic acetyltransferase 12 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 eukaryotic acetyltransferase 12 '.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutant human eukaryotic acetyltransferase 12 to inhibit endogenous human eukaryotic acetyltransferase 12 activity.
  • a variant human eukaryotic acetyltransferase 12 may be a shortened human eukaryotic acetyltransferase 12 lacking a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human eukaryotic acetyltransferase-12.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human eukaryotic acetyltransferase 12 into cells.
  • a recombinant viral vector carrying a polynucleotide encoding human eukaryotic acetyltransferase 12 can be seen In existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human eukaryotic acetyltransferase 12 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human eukaryotic acetyltransferase 12 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RM, DM and ribozymes can be obtained by any existing RNA or DM synthesis technology, such as solid-phase phosphoramidite chemical synthesis technology for oligonucleotide synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DM sequence encoding the RM. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human eukaryotic acetyltransferase 12 can be used for the diagnosis of diseases related to human eukaryotic acetyltransferase 12.
  • the polynucleotide encoding human eukaryotic acetyltransferase 12 can be used to detect the expression of human eukaryotic acetyltransferase 12 or the abnormal expression of human eukaryotic acetyltransferase 12 in a disease state.
  • the DNA sequence encoding human eukaryotic acetyltransferase 12 can be used to hybridize biopsy specimens to determine the expression status of human eukaryotic acetyltransferase 12.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human eukaryotic acetyltransferase 12 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect human eukaryotic acetyltransferase 12 transcription products.
  • RT-PCR RNA-polymerase chain reaction
  • Detection of mutations in the human eukaryotic acetyltransferase 12 gene can also be used to diagnose human eukaryotic acetyltransferase 12-related diseases.
  • Human eukaryotic acetyltransferase 12 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human eukaryotic acetyltransferase 12 DNA sequences. 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 protein expression. Therefore, the Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification.
  • This sequence will specifically * target a specific position on a human chromosome and can hybridize to it.
  • the specificity of each gene on the chromosome needs to be identified Site.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DM 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 construct 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 physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found, for example, in V. Mckus ck, Mendelian 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 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.
  • these 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 via a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human eukaryotic acetyltransferase 12 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dosage range of human eukaryotic acetyltransferase 12 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 eucaryon acétyl transférase humaine 12, 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'eucaryon acétyl transférase humaine 12.
PCT/CN2001/000360 2000-03-22 2001-03-19 Nouveau polypeptide, eucaryon acetyl transferase humaine 12, et polynucleotide codant pour ce polypeptide Ceased WO2001075022A2 (fr)

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CN 00115060 CN1314483A (zh) 2000-03-22 2000-03-22 一种新的多肽——人真核乙酰转移酶12和编码这种多肽的多核苷酸
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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LEITE J.P. ET AL. GENE vol. 41, no. 2-3, 1986, pages 207 - 215 *
STRADA O. ET AL. PROC. NATL. ACAD. SCI. USA vol. 89, no. 20, 15 October 1992, pages 9549 - 9553 *
THOMPSON J.F. ET AL. GENE vol. 103, no. 2, 22 July 1991, pages 171 - 177 *
WU Y.J. ET AL. PROG. CLIN. BIOL. RES. vol. 316A, 1989, pages 313 - 321 *

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