WO2001049857A1 - Nouveau polypeptide, serine transhydroxymethylase 15, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, serine transhydroxymethylase 15, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001049857A1
WO2001049857A1 PCT/CN2000/000646 CN0000646W WO0149857A1 WO 2001049857 A1 WO2001049857 A1 WO 2001049857A1 CN 0000646 W CN0000646 W CN 0000646W WO 0149857 A1 WO0149857 A1 WO 0149857A1
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polypeptide
polynucleotide
serine hydroxymethyltransferase
sequence
seq
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PCT/CN2000/000646
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Fudan University
Shanghai Bio Door Gene Technology Ltd.
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Priority to AU21420/01A priority Critical patent/AU2142001A/en
Publication of WO2001049857A1 publication Critical patent/WO2001049857A1/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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, serine hydroxymethyltransferase 15, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • Serine hydroxymethyltransferase catalyzes a reversible biosynthesis reaction from serine to glycine. This reaction relies on the aldol decomposition of pyridoxal phosphate to produce glycine and "active" formaldehyde, which is transferred to the tetrahydrofolate cofactor to form 5, 10-methylenetetrahydrofolate. 5, 10-methylenetetrahydrofolate plays a very important role in providing one carbon unit for other biosynthetic pathways.
  • SHMT exists in both the cytoplasm and mitochondria. Mammalian cells lacking mitochondrial SHMT activity are glycine auxotrophic. El sea et al. Found that the gene mapping of S1 T1 is closely related to Smith-Magenis syndrome.
  • Slim T contains pyridoxal phosphate.
  • the pyridoxal phosphate group is linked to a lysine residue.
  • the protein sequence near this residue is highly conserved:
  • K is the binding site of pyridoxal phosphate.
  • serine hydroxymethyltransferase 15 protein plays an important role in important body functions as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more serine hydroxymethyl transfers involved in these processes.
  • Enzyme 15 protein especially the amino acid sequence of this protein is identified. Isolation of the gene encoding the new serine hydroxymethyltransferase 15 protein also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for the disease, so it is important to isolate its coding for DM.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant protein comprising a polynucleotide encoding a serine hydroxymethyltransferase 15 Set of vectors. It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding a serine hydroxymethyltransferase 15.
  • Another object of the present invention is to provide a method for producing serine hydroxymethyltransferase 15.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, serine hydroxymethyltransferase 15.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, serine hydroxymethyltransferase 15.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with an abnormality of serine hydroxymethyltransferase 15.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1932 to 2342 in SEQ ID NO: 1; and (b) a sequence having 1-2425 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of serine hydroxymethyltransferase 15 protein, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of serine hydroxymethyltransferase 15 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.
  • the present invention also relates to a pharmaceutical composition, which contains the polypeptide of the present invention or a mimic, activator, antagonist Antibiotics or inhibitors and pharmaceutically acceptable carriers.
  • the present invention also relates to the use of the polypeptides and / or polynucleotides of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of serine hydroxymethyltransferase 15.
  • Figure 1 is a comparison diagram of the amino acid sequence homology of the serine hydroxymethyl transferase 15 of the present invention between a total of 56 amino acids from 3-69 and the serine hydroxymethyl transferase domain.
  • the upper sequence is the serine hydroxymethyltransferase 15 and the lower sequence is the serine hydroxymethyltransferase domain.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated serine hydroxymethyltransferase 15. 15kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RM, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” 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 a different amino acid Or nucleotides replace one or more amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with serine hydroxymethyltransferase 15, 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 serine hydroxymethyltransferase 15.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of serine methylol transferase 15 when combined with serine methylol transferase 15.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind serine hydroxymethyltransferase 15.
  • Regular refers to a change in the function of serine hydroxymethyltransferase 15, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of serine hydroxymethyltransferase 15. change.
  • Substantially pure ' means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify serine hydroxymethyltransferase 15 using standard protein purification techniques. Basic The pure serine hydroxymethyltransferase 15 can generate a single main band on a non-reducing polyacrylamide gel. The purity of the serine hydroxymethyltransferase 15 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 can be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern 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 MBGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.).
  • the MEGALIGN program can be Methods such as the Cluster method compare two or more sequences (Higgins, DG and PM Sharp (1988) Gene 73: 237-244).
  • the Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to HFP or a chemical modification of its nucleic acid. Such a chemical modification may be a substitution of a hydrogen atom with a fluorenyl group, an acyl group or an 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?, Which specifically bind to the epitope of serine hydroxymethyltransferase 15.
  • 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 matter 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 animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state.
  • isolated serine hydroxymethyltransferase 15 means that serine hydroxymethyltransferase 15 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify serine hydroxymethyltransferase 15 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the serine hydroxymethyltransferase 15 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, serine hydroxymethyltransferase 15, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the invention 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 serine hydroxymethyltransferase 15.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the serine hydroxymethyltransferase 15 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 (II) 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 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 the additional amino acid sequence is fused into the mature polypeptide and the polypeptide sequence is formed (Such as the leader or secretory sequence or the sequence used to purify this polypeptide or protein sequence). As set forth 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 a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 2425 bases, and its open reading frame (1932-2342) encodes 136 amino acids.
  • This polypeptide has a characteristic sequence of a serine hydroxymethyltransferase, and it can be deduced that the serine hydroxymethyltransferase 15 has the structure and function represented by the serine hydroxymethyltransferase.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DM forms include cDNA, genomic DM, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • 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.
  • "degenerate variant” means in the present invention that the code has A protein or polypeptide of SEQ ID NO: 2 but a nucleic acid sequence different from the coding region sequence shown in SEQ ID NO: 1.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70 identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60'C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol l, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 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 serine hydroxymethyltransferase 15. ,
  • 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 serine hydroxymethyltransferase 15 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 DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DM sequence from the DM of the genome; 2) chemically synthesizing the MA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic MA is the least commonly used. Direct chemical synthesis of DNA sequences Is the method of choice. The more commonly used method is the separation of the CDM sequences.
  • the standard method for isolating a CDM 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. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, eta l., Molecular Cloning, A Labora tory Manua, Cold Spruing 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 selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DM-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of the level of the serine hydroxymethyltransferase 15 transcript; ( 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 the protein product expressed by the serine hydroxymethyltransferase 15 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method (Sa iki, et al.-Sc ience 1985; 230: 1350-1354) using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize 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 DM fragments and the like obtained as described above can be determined by a conventional method such as a dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDM sequence of multiple clones in order to splice into a full-length cMA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a serine hydroxymethyltransferase 15 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding a serine hydroxymethyltransferase 15 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 (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells.
  • 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.
  • DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably contains one or more selectable marker genes that provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescence Protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes that provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescence Protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a serine hydroxymethyltransferase 15 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 Cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf9
  • 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 CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant serine hydroxymethyltransferase 15 by conventional recombinant DM technology (Science, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. 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
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immunological diseases.
  • Serine hydroxymethyltransferase catalyzes a reversible biosynthetic reaction from serine to glycine. This reaction relies on the aldol decomposition of pyridoxal phosphate to produce glycine and "active" formaldehyde, which is transferred to the tetrahydrofolate cofactor to form 5, 10-methylenetetrahydrofolate. 5, 10-Methylenetetrahydrofolate at Providing one carbon unit for other biosynthetic pathways plays a very important role.
  • SHMT exists in both the cytoplasm and mitochondria in the human body. Mammalian cells lacking mitochondrial SHMT activity are glycine auxotrophic. El sea et al. Found that the gene mapping of SHMT1 is closely related to Smith-Magenis syndrome. In addition, in rapidly proliferating cells, especially in tumor cells, SHMT has a very high activity to meet the needs of large amounts of DM synthesis. Serine hydroxymethyltransferase-specific conserved sequences are required to form its active mot if.
  • the abnormal expression of the serine hydroxymethyltransferase 15 of the present invention will produce various diseases, especially Smith-Magenis syndrome, glycine dystrophy, serine dystrophy, various tumors, and organic acidemia. And other amino acid metabolism disorders, these diseases include but are not limited to:
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma , Colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, cholangiocarcinoma, colon cancer, thymic tumor, nasal cavity and sinus tumor, Nasopharyngeal carcinoma, laryngeal carcinoma, tracheal tumors, fibromas, fibrosarcomas, lipomas, liposarcomas, leiomyomas
  • Organic acidemia propionic acidemia, methylmalonic aciduria, isovalerate, combined carboxylase deficiency, glutarate type I
  • Amino acid metabolism disorders Glycine nutritional deficiencies such as glycineemia, hypersarcosinemia, D-glyceridemia, trimethylamineuria, glycineuria and glucoglycineuria, primary oxalic acidosis, and oxalateuria , Serine nutritional deficiency disease, phenylketonuria, tyrosine metabolism deficiency disease, sulfur amino acid metabolism deficiency disease, tryptophan metabolism deficiency disease, branch amino acid metabolism deficiency disease, proline and hydroxyproline metabolism deficiency disease , Glutamate metabolism deficiency disease, urea cycle metabolism deficiency disease, histidine metabolism deficiency disease, lysine metabolism deficiency disease
  • Abnormal expression of the serine hydroxymethyltransferase 15 of the present invention will also cause certain hereditary, hematological and immune system diseases.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially Smith-Magenis syndrome, glycine dystrophy, serine dystrophy, each Tumors, organic acidemia, other amino acid metabolism disorders, some hereditary, hematological diseases and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) serine hydroxymethyltransferase 15. Agonists increase the biological functions of serine hydroxymethyltransferase 15 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing serine hydroxymethyltransferase 15 can be cultured with labeled serine hydroxymethyltransferase 15 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of serine hydroxymethyltransferase 15 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of serine hydroxymethyltransferase 15 can bind to serine hydroxymethyltransferase 15 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. ⁇
  • serine hydroxymethyltransferase 15 can be added to bioanalytical assays to determine whether a compound is a compound by measuring its effect on the interaction between serine hydroxymethyltransferase 15 and its receptor. 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 serine hydroxymethyltransferase 15 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 15 molecules of serine hydroxymethyltransferase should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against a serine hydroxymethyltransferase 15 epitope. 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 serine hydroxymethyltransferase 15 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 serine hydroxymethyltransferase 15 include, but are not limited to, hybridoma technology (Kohler and Mistein. 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) and existing techniques for producing single-chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against serine hydroxymethyltransferase 15.
  • Antibodies to serine methylol transferase 15 can be used in immunohistochemical techniques to detect serine methylol transferase 15 in biopsy specimens.
  • Monoclonal antibodies that bind to serine hydroxymethyltransferase 15 can also be labeled with radioisotopes. Injection into the body can track its 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.
  • Serine Hydroxymethyltransferase 15 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 serine hydroxymethyltransferase 15 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to serine hydroxymethyltransferase 15. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of serine hydroxymethyltransferase 15.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of serine hydroxymethyltransferase 15 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • Serine hydroxymethyltransferase 15 levels detected in the test can be used to explain the importance of serine hydroxymethyltransferase 15 in various diseases and to diagnose diseases in which serine hydroxymethyltransferase 15 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 serine hydroxymethyltransferase 15 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 serine hydroxymethyltransferase 15.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant serine methylol transferase 15 to inhibit endogenous serine methylol transferase 15 activity.
  • a variant serine hydroxymethyltransferase 15 may be a shortened serine hydroxymethyltransferase 15 that lacks a signal transduction domain. Although it can bind to downstream substrates, it lacks signal transduction activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of serine hydroxymethyltransferase 15.
  • 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 serine hydroxymethyltransferase 15 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a serine hydroxymethyltransferase 15 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding serine hydroxymethyltransferase 15 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) that inhibit serine hydroxymethyltransferase 15 mRNA, and Ribozymes are also within the scope of the invention.
  • a ribozyme is an enzyme-like MA molecule that can specifically decompose a specific RNA, and its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform endonucleation.
  • Antisense RNA, DM, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence is integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding serine hydroxymethyltransferase 15 can be used for the diagnosis of diseases related to serine hydroxymethyltransferase 15.
  • the polynucleotide encoding serine hydroxymethyltransferase 15 can be used to detect the expression of serine hydroxymethyltransferase 15 or the abnormal expression of serine hydroxymethyltransferase 15 in a disease state.
  • the DNA sequence encoding serine methylol transferase 15 can be used to hybridize biopsy specimens to determine the expression of serine methylol transferase 15.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Serine hydroxymethyltransferase 15 specific primers can also be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect serine hydroxymethyltransferase 15 transcripts.
  • Serine hydroxymethyltransferase 15 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type serine hydroxymethyltransferase 15 DNA sequence. Mutations can be detected using well-known techniques such as Sou thern blotting, DM 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. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • the PCR primers (preferably 15-35bp) are prepared based on 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 Heterozygous cells that contain human genes corresponding to the primers 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 difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Serine hydroxymethyltransferase 15 to effectively treat and / or predict Dosage for a specific indication.
  • the amount and dose range of serine hydroxymethyltransferase 15 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.
  • 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 t ion Kit (product of Qiegene). 2ug poly (A) mMA forms cDNA by reverse transcription. Smar t cDM cloning kit (purchased from C ontec) was used. The 0 fragment was inserted into the multiple cloning site of the pBSK (+) vector (Clontech), and transformed into DH5 oc. The bacteria formed a cDNA library.
  • the sequences at the 5 'and 3' ends of all clones were determined using a Dye terminate cycle reaction ion sequencing kit (Perkin-Blme): a company product and an ABI 377 automatic sequencer (Perkin-Elmer).
  • the determined cDNA sequence was compared with the existing "Co-DM sequence database (Genebank)", and it was found that the CDM sequence of one of the clones 0 663 c 0 3 was new DNA.
  • a series of primers were synthesized to insert the clone into the clone. The cDNA fragment was measured in both directions.
  • the sequence of the serine hydroxymethyltransferase 15 of the present invention and the protein sequence encoded by the serine hydroxymethyltransferase 15 were encoded using the profil i le scan program (Basic local al ignment search tool) in GCG [Altschul, SF et al. L Mol. Biol. 1990; 215: 403-10], domain analysis was performed in databases such as Prote.
  • the serine hydroxymethyltransferase 15 of the present invention is homologous to the domain of the serine hydroxymethyltransferase at 3-69. The results of the homology are shown in FIG. 1, the homology rate is 2 7%, and the score is 15. 16; The threshold is 15. 11.
  • CDM was synthesized using fetal brain cell total RNA as a template and ol igo-dT as 31 substances for reverse transcription reaction. After purification of Qiagene's kit, PCR amplification was performed with the following primers:
  • Primer 1 5 '-C AATGTGTCATTTTTTTTTCTTTT- 3' (SEQ ID NO: 3)
  • Primer2 5'-ATTAAAAAGGCCCAAAACTTTATT-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 ⁇ l of reaction volume containing 50 ol / L KC1, 10 mmol / L Tris-HCl, pH 8.5, 1.5 ⁇ l of ol / L MgCl 2 , 200 ⁇ ⁇ dNTP, lOpmol primers , 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector using a TA cloning kit (Invitrogen).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-2424bp shown in SEQ ID NO: 1.
  • Example 4 Analysis of Serine Hydroxymethyltransferase 15 Gene Expression by Northern Blotting Total RM was extracted by a one-step method [Anal. Biochem 1987, 162, 156-159] 0 This method involves acid guanidinium thiocyanate-chloroform extraction.
  • the tissue is homogenized with 4M guanidinium 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 ), Mix and centrifuge.
  • the aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RM precipitate.
  • the resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • 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 (pH7.4)-5xSSC-5xDenhardt, s solution and 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in lxSSC-0.1% SDS at 55 ° C for 30min. Then, analysis was performed with Phosphor Imager And quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant serine hydroxymethyltransferase 15
  • Primer3 5-CCCCATATGATGGCTGGGTGGGGACTGGTGGAT-3 '(Seq ID No: 5)
  • Primer4 5' -CATGGATCCTTACAGTCTAGAAGCATGCCAAGA- 3 '(Seq ID No: 6)
  • the 5 'ends of these two primers contain Mel and BamHI restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Ndel and BamHI restriction sites correspond to the expression vector plasmid pBT-28b ( +) (Novagen, Cat. No. 69865. 3).
  • the PCR reaction was performed using the pBS-0663c03 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions were: 1 in a total volume of 50 ⁇ plasmid pBS- 0663c03 containing 10pg, primer Pr imer- 3 and Pr imer-4 were lOpmol, Advantage polymerase Mix (Clontech Products) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles.
  • the amplified product and plasmid pET-28 (+) were double-digested with Mel and BamHI, respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed into colibacillus DH5 c (using calcium chloride method, cultured overnight on LB plates containing kanamycin (final concentration 3 (g / ml)), and positive clones were screened by colony PCR method and sequenced.
  • a positive clone (PET-0663c03) with the correct sequence was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Nova gen ) by calcium chloride method.
  • the following peptides specific to serine hydroxymethyltransferase 15 were synthesized using a peptide synthesizer (product of PE company): NH2-Met-Ala-Gly-Trp-Gly-Leu-Val-Asp-Va l-Ser-Gly-Ala -Pro-Glu-Pro-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled with 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 imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt)
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • the sample membrane was placed in a plastic bag, and 3-1 Omg pre-hybridization solution (1 OxDenhardt-s; 6xSSC, 0.1 lrag / ml CT DNA (calf thymus MA)) was added. After sealing the mouth of the bag, shake in a 68 C water bath for 2 hours.
  • 3-1 Omg pre-hybridization solution (1 OxDenhardt-s; 6xSSC, 0.1 lrag / ml CT DNA (calf thymus MA)
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Example 8 DNA icroarray
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research on the function of new genes; search for and screen new tissue-specific genes, especially diseases related genes such as tumors; diagnosis of diseases such as heredity disease.
  • the specific method steps have been reported in the literature. M., Chai, A., Sha lom, 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 respectively amplified by PCR. After the purified amplified product was purified, the concentration was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between the points is 280 ⁇ m. The spotted slide was hydrated, dried, and cross-linked in a UV cross-linker. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps have been reported in the literature. The sample post-processing steps of the embodiment are:
  • Total mRN A was extracted from normal liver and liver cancer by one-step method, and mRNA was purified with 01 i go t ex mRNA M idi K it (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP S-Amino- propargyH -deoxyur idine 5 '-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech company
  • Cy5dUTP 5-Amino-propargyl-2' -deoxyur idine 5 '-tr iphate coupled to Cy5 luorescent dye, purchased from Amersham Phamacia Biotech
  • Probes were prepared after purification. Specific steps and methods
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodi scovery, USA) to calculate the Cy3 / Cy5 ratio of each point, which was less than 0.5 Dots greater than 2 are considered genes with differential expression.

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Abstract

L'invention concerne un nouveau polypeptide, une sérine transhydroxyméthylase 15, 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 la sérine transhydroxyméthylase 15.
PCT/CN2000/000646 1999-12-29 2000-12-25 Nouveau polypeptide, serine transhydroxymethylase 15, et polynucleotide codant pour ce polypeptide WO2001049857A1 (fr)

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