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WO2001027285A1 - Nouveau polypeptide, methyltransferase humaine dpb 41 et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, methyltransferase humaine dpb 41 et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001027285A1
WO2001027285A1 PCT/CN2000/000316 CN0000316W WO0127285A1 WO 2001027285 A1 WO2001027285 A1 WO 2001027285A1 CN 0000316 W CN0000316 W CN 0000316W WO 0127285 A1 WO0127285 A1 WO 0127285A1
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Prior art keywords
polypeptide
methyltransferase
dpb
polynucleotide
human
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PCT/CN2000/000316
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bio Door Gene Technology Ltd.
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Priority to AU77686/00A priority Critical patent/AU7768600A/en
Publication of WO2001027285A1 publication Critical patent/WO2001027285A1/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.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a sequence of a gene encoding a novel human DPB methyltransferase 41, and a polypeptide encoded by the polynucleotide. The invention also includes the use and preparation of this polypeptide. Specifically, the polypeptide of the present invention is a novel dihydroxy polyisopentene benzoate methyltransferase, called DPB methyltransferase.
  • Methylation has important biological significance in the restricted modification system of organisms, especially higher eukaryotes.
  • some cytosine bases have a methyl group attached to the 5 'carbon, and the cytosine residue with methyl groups accounts for about 0.7-0.8 %, And it varies from tissue to tissue within the same animal.
  • the addition of methyl groups is due to the catalysis of methyltransferase.
  • DNA methylation has an effect on the regulation of some genes in higher eukaryotic cells.
  • Demethylation and methylation determine the activation and inactivation of transcription.
  • the polypeptide of the present invention is deduced to be human dihydroxypolyisopentene benzoate methyltransferase, which is called human DPB methyltransferase 41 (HDPBMT41), which is the result of amino acid homology comparison.
  • HDPBMT41 human DPB methyltransferase 41
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human DPB methyltransferase 41. It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding human DPB methyltransferase 41.
  • Another object of the present invention is to provide a method for producing human DPB methyltransferase 41. Another object of the present invention is to provide an antibody against the polypeptide of the present invention ⁇ human DPB methyltransferase 41. Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human DPB methyltransferase 41.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human DPB methyltransferase 41.
  • a novel isolated human DPB methyltransferase 41 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2; or a conservative variant polypeptide thereof; Or its active fragment, or its active derivative, analog.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 80 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human DPB methyltransferase 41; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 33-1 to 142 in SEQ ID NO: 1; and (b) a sequence having 1- in SEQ ID NO: 1 1267-bit sequence.
  • Fig. 1 is a comparison diagram of amino acid sequence homology between the DPB methyltransferase 41 of the present inventor and the mouse DPB methyltransferase.
  • the upper sequence is human DPB methyltransferase 41 and the lower sequence is murine DPB methyltransferase.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of an isolated human DPB methyltransferase. 41kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are the same as other substances existing in the natural state. It is separated and purified, and it is isolated and purified.
  • isolated means that human DPB methyltransferase 41 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 DPB methyltransferase 41 using standard protein purification techniques.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human DPB methyltransferase 41 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human DPB methyltransferase 41, 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 can be naturally purified products, or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the starting methionine residue.
  • the invention also includes fragments, derivatives and analogs of human DPB methyltransferase 41.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human DPB methyltransferase 41 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ )
  • Such a polypeptide sequence in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence)
  • such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes a 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 1267 bases in length and its open reading frame (33-1142) encodes 369 amino acids. Based on the amino acid sequence homology comparison, it was found that this peptide has 80% homology with murine DPB methyltransferase.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DM can be a coding chain or a non-coding chain.
  • 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 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 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) Add a denaturant during hybridization, such as 50 ° /.
  • polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores 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 human DPB methyltransferase 41.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding human DPB methyltransferase 41 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) Isolation of double-stranded DNA from genomic DNA Sequence; 2) chemically synthesize a DNA sequence to obtain 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 separation of cDM 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.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • 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.
  • 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-DM or DM-RNA hybridization; (2) the presence or absence of marker gene function; (3) determination of the level of transcripts of human DPB methyltransferase 41; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the human DPB methyltransferase 41 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-cDM terminal rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be 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.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using a human DPB methyltransferase 41 coding sequence, and a recombinant technology for producing a polypeptide of the present invention. method.
  • a polynucleotide sequence encoding human DPB methyltransferase 41 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.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human DPB methyltransferase 41 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis 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 operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers from 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human DPB methyltransferase 41 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or a recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS, or Bowes s melanoma cells, etc. .
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with (Method 12, using the procedure well known in the art.
  • Alternative is 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 liposomes Packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human DPB methyltransferase 41 by conventional recombinant DNA technology (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • Methylation and demethylation involve many areas of biology and medicine, especially for higher eukaryotes Is of great significance.
  • the DPB methyltransferase of the present invention is widely expressed in adult tumor cells, viscera, neural tissue, proliferative cells, secretory cells, and fetal viscera, placenta and other tissues
  • the DPB methyltransferase can be used with the above tissues or organs Diagnosis, prevention and treatment of related diseases.
  • Antagonists of human DPB methyltransferase 41 protein include screened antibodies, compounds, receptor deletions, and the like. Antagonists of human DPB methyltransferase 41 protein can bind to human DPB methyltransferase 41 protein and eliminate its function, or inhibit the production of human DPB methyltransferase 41 protein, or bind to the active site of a polypeptide Prevents the polypeptide from performing its biological function. Antagonists of human DPB methyltransferase protein can be used to treat neoplastic and immune diseases caused by methyltransferase overexpression.
  • neoplastic diseases include, but are not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, and adrenal, bone, bone marrow, brain, heart, lung, liver, muscle, skin and other tissues.
  • Immune diseases include, but are not limited to, AIDS, allergies, anemia, asthma, arteriosclerosis, bronchitis, cholecystitis, and the like.
  • the invention also provides a method for screening compounds to identify agents that increase (agonist) or suppress (antagonist human DPB methyltransferase 41).
  • Agonists enhance biological functions such as human DPB methyltransferase 41 to stimulate cell proliferation, and Antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human DPB methyltransferase 41 and labeled human DPB methyltransferases can be transferred in the presence of drugs Enzyme 41 is cultured together. The ability of the drug to increase or suppress this interaction is then determined.
  • Antagonists of human DPB methyltransferase 41 include screened antibodies, compounds, receptor deletions, and the like.
  • An antagonist of human DPB methyltransferase 41 can bind to human DPB methyltransferase 41 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 function. biological functions.
  • human DPB methyltransferase 41 When screening compounds as antagonists, human DPB methyltransferase 41 can be added to bioanalytical assays to determine whether a compound is affected by measuring the effect of the compound on the interaction between human DPB methyltransferase 41 and its receptor. Is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to human DPB methyltransferase 41 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human DPB methyltransferase 41 molecule should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides Antibodies against Human DPB Methyltransferase 41 Epitope
  • These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human DPB methyltransferase 41 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 DPB methyltransferase 41 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV -Hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies U.S. Pat No. 4946778, can also be used to produce single chain antibodies against human DPB methyltransferase 41.
  • Antibodies against human DPB methyltransferase 41 can be used in immunohistochemistry to detect human DPB methyltransferase 41 in biopsy specimens.
  • Monoclonal antibodies that bind to human DPB methyltransferase 41 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 DPB methyltransferase 41 high-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human DPB methyltransferase 41 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human DPB methyltransferase 41.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human DPB methyltransferase 41.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human DPB methyltransferase 41 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human DPB methyltransferase 41 detected in the test can be used to explain the importance of human DPB methyltransferase 41 in various diseases and to diagnose diseases in which human DPB methyltransferase 41 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.
  • the polynucleotide encoding human DPB methyltransferase 41 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat the disease caused by non-expression or abnormal / inactive expression of human DPB methyltransferase 41 Cell proliferation, development, or metabolism is abnormal. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human DPB methyltransferase 41 to inhibit endogenous human DPB methyltransferase 41 activity.
  • a mutated human DPB methyltransferase 41 may be a shortened human DPB methyltransferase 41 lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human DPB methyltransferase 41.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human DPB methyltransferase 41 into cells.
  • Methods for constructing a recombinant viral vector carrying a polynucleotide encoding human DPB methyltransferase 41 can be found in existing literature (Sambrook, etal.).
  • a recombinant polynucleotide encoding human DPB methyltransferase 41 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 DM
  • ribozymes that inhibit human DPB methyltransferase 41 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 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 RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human DPB methyltransferase 41 can be used for the diagnosis of diseases related to human DPB methyltransferase 41.
  • the polynucleotide encoding human DPB methyltransferase 41 can be used to detect the expression of human DPB methyltransferase 41 or the abnormal expression of human DPB methyltransferase 41 in a disease state.
  • the DNA sequence encoding human DPB methyltransferase 41 can be used to hybridize biopsy specimens to determine the expression status of human DPB methyltransferase 41.
  • Hybridization techniques include Sout hern blotting, Nor t hern blotting, in situ hybridization, and the like.
  • RNA-polymerase using human DPB methyltransferase 41-specific primers RT-PCR amplification can also detect the transcription product of human DPB methyltransferase 41.
  • Human DPB methyltransferase 41 mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human DPB methyltransferase 41 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern 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 specific loci of each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data can be used to mark chromosome locations.
  • an important first step is to locate these DM sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) 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 hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones to 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 in, for example, V. Mckusick, 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 differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first finding Structural changes in the chromosome, such as deletions or translocations that are visible from the chromosome 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 disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human DPB methyltransferase 41 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human DPB methyltransferase 41 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples
  • 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 I sola ti on Kit (product of Qiegene). 2ug po ly (A) mRNA was reverse transcribed to form cDNA.
  • the Smar t cDM cloning kit purchased from Clontech
  • the bacteria formed a cDNA library.
  • the sequences at the 5 'and 3' ends of all clones were determined using Dye termina te cyc le reac t ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer).
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and one of the clones was found.
  • the 0215E05 cDNA sequence is new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the BDP program (Basiclocal Alignment search tool) of the human DPB methyltransferase 41 sequence of the present invention and its encoded protein sequence were used [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] Perform homology search in Genbank, Swissport and other databases.
  • the gene most homologous to the human DPB methyltransferase 41 of the present invention is a known mouse DPB methyltransferase gene, and the protein encoded by it is accession number L20427 in Genbank.
  • the protein homology results are shown in Figure 1. The two are highly homologous, with 80% identity; 87% similarity.
  • Example 3 Cloning of a gene encoding human DPB methyltransferase 41 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5 '-GGGAAAAAAAAG AGGTGGGATCGTTTGT- 3' (SEQ ID NO: 3)
  • Primer2 5'- AGGTTTTAGCCCTTTTTATTGACCTTC- 3, (SEQ IDNO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10 mmol / L Tris-Cl, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers in a reaction volume of 50 ⁇ 1 1U of Taq DM 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.
  • RT-PCR set ⁇ -act in as a positive control and template blank as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector (Invitrogen product) using a TA cloning kit.
  • DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1267bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human DPB methyltransferase 41 gene expression
  • 0 acidic sulfur which comprises Guanidinium cyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH 4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA was prepared using 20 g of RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (PH7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DM probe used is the sequence of the coding region of human DPB methyltransferase 41 amplified by PCR shown in FIG. 1
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 ( P H7.4) -5 x SSC- 5 x Denhardt's solution and 200 ⁇ g / ml 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 5 In Vitro Expression, Isolation and Purification of Recombinant Human DPB Methyltransferase 41
  • Primer3 5'- CCCCCATGGATGTGGAGTGGCCGTAAGCTGGGC- 3, (Seq ID No: 5)
  • Primer 4 5,-CCCGGATCCTCATTATTTTTTCAGCTTTTCATGCACA- 3 '(Seq ID No: 6)
  • the pBS-0215E05 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0215E05 plasmid, , Advantage polymerase Mix
  • the following peptides specific for human DPB methyltransferase 41 were synthesized using a peptide synthesizer (product of PE): NH2-Met-Trp-Ser-G ly-Arg-Lys-Leu-Gly-Ser-Ser-G ly- Gly-Trp-Phe-Leu-0H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ral bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum.
  • Total I gG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human DPB methyltransferase 41.

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Abstract

L'invention concerne un nouveau polypeptide, une méthyltransférase humaine DPB 41 et un polynucléotide codant pour ce polypeptide ainsi que le procédé d'obtention de ce polypeptide par technique recombinante d'ADN. L'invention concerne aussi les applications de ce polypeptide dans les traitements de diverses maladies, notamment les tumeurs malignes, l'hémopathie, le VIH, les maladies immunitaires et différentes inflammations. L'invention concerne en outre les agonistes agissant contre le polypeptide et leur action thérapeutique. L'invention concerne finalement les applications de ce polynucléotide codant pour cette nouvelle méthyltransférase humaine DPB 41.
PCT/CN2000/000316 1999-10-13 2000-10-12 Nouveau polypeptide, methyltransferase humaine dpb 41 et polynucleotide codant pour ce polypeptide WO2001027285A1 (fr)

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AU77686/00A AU7768600A (en) 1999-10-13 2000-10-12 A novel polypeptide, a human dpb methyltransferase 41 and the polynucleotide encoding the polypeptide

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CN99116959.X 1999-10-13
CN99116959A CN1293253A (zh) 1999-10-13 1999-10-13 一种新的多肽－人dpb甲基转移酶41和编码这种多肽的多核苷酸

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 14 March 1994 (1994-03-14) *
GENE, vol. 138, no. 1-2, January 1994 (1994-01-01), pages 213 - 217 *

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