+

WO2002020590A1 - Nouveau polypeptide, sucre phosphotransferase phosphoenolpyruvate-dependante 33, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, sucre phosphotransferase phosphoenolpyruvate-dependante 33, et polynucleotide codant ce polypeptide Download PDF

Info

Publication number
WO2002020590A1
WO2002020590A1 PCT/CN2001/001131 CN0101131W WO0220590A1 WO 2002020590 A1 WO2002020590 A1 WO 2002020590A1 CN 0101131 W CN0101131 W CN 0101131W WO 0220590 A1 WO0220590 A1 WO 0220590A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
sugar phosphotransferase
dependent sugar
sequence
Prior art date
Application number
PCT/CN2001/001131
Other languages
English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
Original Assignee
Biowindow Gene Development Inc. Shanghai
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biowindow Gene Development Inc. Shanghai filed Critical Biowindow Gene Development Inc. Shanghai
Priority to AU2002223374A priority Critical patent/AU2002223374A1/en
Publication of WO2002020590A1 publication Critical patent/WO2002020590A1/fr

Links

Classifications

    • 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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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, monoenolpyruvate phosphate-dependent sugar phosphotransferase 33, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique
  • TS enol pyruvate phosphate-dependent sugar phosphotransferase system
  • EI protein-enzyme I
  • HPr thermostable phosphoryl carrier protein
  • TS sugar-specific permease-enzyme I I complex.
  • EI protein-enzyme I
  • HPr thermostable phosphoryl carrier protein
  • PTS can catalyze the transport and transfer of sugar and the accompanying sugar phosphorylation process.
  • PTS permease is composed of different numbers of polypeptide chains.
  • some sugar-specific proteins will fuse to form domains with EI and / or HPr energy coupling functions. There is evidence that the entire EI I complex is required for sugar transport and phosphorylation.
  • permease has at least three easy-to-recognize functional domains: a hydrophobic transmembrane domain capable of binding and transporting sugar substrates; and a hydrophilic EI II-like domain Has a first phosphorylation site (always histamine); a second hydrophilic protein or protein domain has a second phosphorylation site.
  • This site is either a cysteamide residue corresponding to most homologous PTS permease, or a histamine residue of the three permease enzymes described below.
  • ⁇ is considered to be an important constituent protein of PTS, participating in the transmembrane, forming a membrane 'transfer channel and providing a sugar binding site.
  • BI I usually consists of two cytoplasmic domains ⁇ , ⁇ B and a transmembrane domain I IC.
  • ⁇ contains the first permease-specific phosphorylation site, which is a histidine that can be phosphorylated by phosphorylated HPr.
  • I IB contains a second phosphorylation site, which is phosphorylated by phosphorylated I IA, which is dependent on permease. Finally, the phosphoryl group is transferred from ⁇ B to the sugar as a substrate.
  • ⁇ and ⁇ can be connected through a polypeptide rich in alanine and proline to form a stable dimer structure ⁇ .
  • the secondary structure of I IA is usually an anti-parallel ⁇ -sheet consisting of five chains with a helix at both ends.
  • Histidine which serves as a phosphorylation site, is located in a shallow crack at the end of the third ⁇ -sheet chain with a hydrophobic surface composed of hydrophobic residues.
  • there is a histidine near the histidine that is the phosphorylation site (approximately 15 amino acid positions near the N-terminus), which can also interact with the phosphoryl group after ⁇ is phosphorylated.
  • the difference between the structural changes before and after phosphorylation is relatively small.
  • the enolpyruvate phosphate-dependent sugar phosphotransferase 33 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art. Identification of more enolpyruvate phosphate-dependent sugar phosphotransferase 33 proteins involved in these processes, especially the amino acid sequence of this protein. Isolation of the neoenol pyruvate phosphate-dependent sugar phosphotransferase 33 protein encoding gene also provides a basis for the study to determine the role of this protein in health and disease states. This protein may constitute a diagnostic and / or therapeutic The basis of the drug, so it is important to isolate its coding DNA. Disclosure of invention
  • 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 an enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding an enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Another object of the present invention is to provide a method for producing an enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Another object of the present invention is to provide an antibody against the polypeptide monoenolpyruvate phosphate-dependent sugar phosphotransferase 33 of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the mono-enol pyruvate phosphate-dependent sugar phosphotransferase 33 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conserved body, a biologically active fragment, or a 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 71 to 973 in SBQ ID NO: 1; and (b) a sequence having positions 1 to 1716 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of enolpyruvate phosphate-dependent sugar phosphotransferase 33 protein, which comprises utilizing 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 enolpyruvate phosphate-dependent sugar phosphotransferase 33 protein in vitro, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to polypeptides and / or polynucleotides of the present invention prepared for the treatment of ⁇ cancer, developmental or immune disease ⁇ or other diseases caused by abnormal expression of enolpyruvate phosphate-dependent sugar phosphotransferase 33 Use of drugs.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic DNA or RNA, which can be single-stranded or double-stranded, representing the sense strand or Antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similar The term “immunologically active” refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind to specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with an enolpyruvate phosphate-dependent sugar phosphotransferase 33, causes the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind an enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Antagonist refers to a biological activity that blocks or regulates enolpyruvate phosphate-dependent sugar phosphotransferase 33 when combined with enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • enolpyruvate phosphate-dependent sugar phosphotransferase 33 refers to a change in the function of enolpyruvate phosphate-dependent sugar phosphotransferase 33, including an increase or decrease in protein activity, a change in binding characteristics, and any of the enolpyruvate phosphate-dependent sugar phosphotransferase 33 Changes in other biological, functional or immune properties.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify enolpyruvate phosphate-dependent sugar phosphotransferase 33 using standard protein purification techniques.
  • the substantially pure enolpyruvate phosphate-dependent sugar phosphotransferase 33 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the enol pyruvate phosphate-dependent sugar phosphotransferase 33 peptide can be analyzed by amino acid sequence.
  • Complementary or “complementarity” refers to the natural binding of polynucleotides that are 'base-paired' under allowable salt concentration and temperature conditions.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madison Wis.). MEGAUGN The program can compare two or more sequences according to different methods, such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). The Cluster method arranges each group of sequences by checking the distance between all pairs. Clustering. 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 number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by Clus ter method or using methods known in the art such as Jotun Hein. L, (1990) Methods in emzuraology 183: 625-645) 0 "Similarity” refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions 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 RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ,) 2 and? , It can specifically bind to the epitope of enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated enolpyruvate phosphate-dependent sugar phosphotransferase 33 means that enolpyruvate phosphate-dependent sugar phosphotransferase 33 is substantially free of other proteins, lipids, and sugars naturally associated with it. Or other substances. Those skilled in the art can purify enol pyruvate phosphate-dependent sugar phosphotransferase 33 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the enol pyruvate phosphate-dependent sugar phosphotransferase 33 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide monoenolpyruvate phosphate-dependent sugar phosphotransferase 33, 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 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.
  • polypeptides of the invention may be glycosylated, or they may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives, and the like of enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the enolpyruvate phosphate-dependent sugar phosphotransferase 33 of the present invention.
  • a fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a type in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by the genetic code; 'or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III)
  • 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 SEQ ID NO: 1 Nucleotide sequence.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1,716 bases in length, and its open reading frames 71-973 encode 37 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile to enolpyruvate phosphate-dependent sugar phosphotransferase. It can be concluded that the enolpyruvate phosphate-dependent sugar phosphotransferase 33 has enolpyruvate phosphate. Similar functions for sugar-dependent phosphotransferases.
  • the polynucleotide of the present invention may be in the form of DNA or RM.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but having a sequence different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the present 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) 1 ° / ⁇ When hybridizing with a denaturant, such as 50% (v / v) formamide, 0.1 ° /. Calf serum / 0. l »/.
  • 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, preferably at least 100 nucleotides.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • 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 enolpyruvate phosphate-dependent sugar phosphotransferase 33 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 MA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DM 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. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua 1, Cold Spruing 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): (1) DM-DNA or DM-RM hybridization; (2) the presence or absence of marker gene functions; (3) determination of the enol pyruvate phosphate-dependent sugar phosphotransferase 33 transcription (4) Detecting protein products expressed by genes through immunological techniques or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably Is at least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product expressed by the enolpyruvate phosphate-dependent sugar phosphotransferase 33 gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay. Attached method (ELISA) and so on.
  • a method using DNA technology to amplify DNA / RM (Sa iki, et al. Sc ience 1985; 230: 1350-1354) 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.
  • the amplified DNA / RM 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 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, the 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 produced by genetic engineering using the vector of the present invention or directly using an enolpyruvate phosphate-dependent sugar phosphotransferase 33 coding sequence, and produced by recombinant technology A method of a polypeptide according to the invention.
  • a polynucleotide sequence encoding an enolpyruvate phosphate-dependent sugar phosphotransferase 33 may 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.
  • 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 an enol pyruvate phosphate-dependent sugar phosphotransferase 33 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua, 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 for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, 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 an enolpyruvate phosphate-dependent sugar phosphotransferase 33 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a gene containing the polynucleotide or the recombinant vector.
  • Engineered host cells 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 Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with the MA sequence according to the present invention or a recombinant vector containing the MA 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 absorbing DM may be harvested after exponential growth phase, treated with CaC l 2 method used in steps well known in the art. Alternatively, MgCl 2 is used.
  • transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant enolpyruvate phosphate-dependent sugar phosphotransferase 33 (Sc ience, 1984; 224: 1431). Generally there are the following steps:
  • step (3) Isolate and purify protein from culture medium or cells.
  • 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.
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion A combination of exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion A combination of exchange chromatography, high performance liquid
  • Fig. 1 is a comparison diagram of gene chip expression profiles of the enolpyruvate phosphate-dependent sugar phosphotransferase 33 and the enolpyruvate phosphate-dependent sugar phosphotransferase of the present invention.
  • the upper graph is a graph of the enol pyruvate phosphate-dependent sugar phosphotransferase 33
  • the lower graph is the graph of the enol pyruvate phosphate-dependent sugar phosphotransferase.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • 33kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Example 1 Cloning of enolpyruvate phosphate-dependent sugar phosphotransferase 33
  • 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 solat ion Kit (product of Qiegene). 2ug poly (A) mRNA forms CDM by reverse transcription.
  • the Smart cDM cloning kit purchased from Clontech was used to insert the 00 fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ , and the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Blmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with an existing public DM sequence database (Genebank), and it was found that the cDM sequence of one of the clones 0158c06 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • PCR amplification was performed with the following primers:
  • Pr imer 1 5, — — ATCACAGGAATCATCTTCTCATGA -3, (SEQ ID NO: 3)
  • Pr iraer2 5,-CTTGCTTTCATTGCCATTTTAATA -3, (SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10 mmol / L Tr is-Cl, (pH 8. 5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol in a reaction volume of 50 ⁇ 1 Primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55. C 30sec; 72. C 2min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit.
  • the DM sequence analysis results showed that the DM sequence of the PCR product was exactly the same as the 1-1717bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of the expression of the enolpyruvate phosphate-dependent sugar phosphotransferase 33 gene:
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate chloroform extraction.
  • the aqueous layer was aspirated, isopropanol (0.8 vol) was added, and the mixture was centrifuged to obtain a MA precipitate.
  • the resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.4)-5 x SSC-5 x Denhardt, s solution and 200 g / ml salmon sperm DNA. After hybridization, the filters were placed in 1 x SSC-0.1% SDS at 55. C wash 30rain. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant enolpyruvate phosphate-dependent sugar phosphotransferase 33 According to the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers was designed. The sequence is as follows:
  • Primer 3 5 '-CCCCATATGATGTCTGTTAAAAAAGGTGATGAC-3' (Seq ID No: 5)
  • Primer4 5 '-CATGGATCCTTAGGTTTCAGAGGCTGGTCTTCG-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Mel and BamHI restriction sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • the PCR reaction was performed using the pBS-0158c06 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS- 0158c06 plasmid, primers Primer-3 and Primer respectively; j is lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60. C 30s, 68 ° C 2 min, 25 cycles in total. Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed into colibacillus DH5ct by the calcium chloride method, cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), and positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0158C06) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BLn (DE3) plySs (product of Novagen) using the calcium chloride method.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • P harose total IgG isolated from the serum of rabbit antibodies with Protein A-Se.
  • 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 enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects.
  • 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. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment makes use of higher intensity membrane washing conditions (such as lower salt concentration and higher temperature) to enable hybridization
  • the background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types.
  • the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention.
  • Polynucleotide of the invention SEQ ID NO: 1 Identical or complementary oligonucleotide fragments.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other unknown genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SBQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41M) of the gene fragment or its complementary fragment of SBQ ID NO: 1: '5'-TGTCTGTTAAAAAAGGTGATCACCTACTGGAGACTAATAAT-3' (SEQ ID NO: 9 )
  • SBQ ID NO: 9 replacement mutation sequence
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • 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 analyzing biological information quickly, efficiently, and with high throughput.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature, for example, see the literature DeRis i, L L., Lyer, V. & Brown, P. 0.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as the target DM, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500 ng / ul after purification, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them 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 variously reported in the literature. The post-spotting processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen). The fluorescent reagent Cy3dUTP was separately reverse-transcribed.
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and a washing solution (1 ⁇ SSC, 0.2% SDS) was used at room temperature. After washing, scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed by Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line, thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar formation fc growth factor stimulation, 1013HT, scar into fc without growth factor stimulation, 1013HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma. Draw a chart based on these 18 Cy3 / Cy5 ratios. (figure 1 ) .
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • the enol pyruvate phosphate-dependent sugar phosphotransferase system is a sugar transport system. This system plays a vital role in regulating a variety of global metabolic pathways, and also involves the regulation of many metabolic and translation processes. It consists of two protein-enzyme I (EI) involved in energy metabolism, a thermostable phosphoryl carrier protein (HPr), and a sugar-specific permease-enzyme I I complex. The entire EI I complex is required for sugar transport and phosphorylation. It is considered to be an important constituent protein of PTS, involved in transmembrane, forming membrane transfer channels and providing sugar binding sites. EI I usually consists of two cytoplasmic domains IIA, IIB, and a transmembrane domain IIC.
  • the polypeptide of the present invention is a novel enzyme IIA protein in PTS, which is very important for sugar transportation and metabolism and phosphorylation of substances. Its specific conserved sequence is required to form its active mot if.
  • the abnormal function of the polypeptide containing the specific mot if of the present invention will lead to abnormal sugar transportation and metabolism, abnormal phosphorylation of metabolites, and produce related diseases such as disorders related to glucose metabolism disorders, tumors, and embryo development disorders. , Growth and development disorders.
  • the abnormal expression of the enolpyruvate phosphate-dependent sugar phosphotransferase 10 of the present invention will produce various diseases, especially diseases related to glucose metabolism disorders, tumors, embryonic development disorders, and growth and development disorders. These diseases include but are not Limited to:
  • Organic acidemia Propionic acidemia, methylmalonic aciduria, isovalerate, combined carboxylase deficiency, glutaric acid type I, congenital carbohydrate digestion and absorption Defects such as congenital lactose intolerance, hereditary fructose intolerance, monosaccharide metabolism defects such as galactosemia, fructose metabolism defects, glycogen metabolism diseases such as glycogen storage disease, mucopolysaccharidosis
  • Embryonic disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney, double ureter, cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, suburethral Fissure, hermaphroditism, atrial septal defect, ventricular septal defect, pulmonary stenosis, arterial duct occlusion, neural tube defect, congenital hydrocephalus, iris defect, congenital glaucoma or cataract, congenital deafness
  • Benign diseases such as congenital skin relaxation, Alzheimer's disease, congenital keratosis, various metabolic deficiencies, dementia, metaplasia Confucianism, sexual retardation
  • 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, gallbladder cancer, colon cancer, thymic tumor, tracheal tumor, fibroma, Fibrosarcoma, lipoma, liposarcoma, leiomyoma
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Agonists increase enolpyruvate phosphate-dependent sugar phosphotransferase 33 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing an enol pyruvate phosphate-dependent sugar phosphotransferase 33 can be cultured together with a labeled enol pyruvate phosphate-dependent sugar phosphotransferase 33 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of enol pyruvate phosphate-dependent sugar phosphotransferase 33 include selected antibodies, compounds, receptor deletions, and the like.
  • the antagonist of enolpyruvate phosphate-dependent sugar phosphotransferase 33 can bind to and eliminate the function of enolpyruvate phosphate-dependent sugar phosphotransferase 33, or it can inhibit the production of the polypeptide or the activity of the polypeptide Site binding prevents the polypeptide from performing its biological function.
  • enol propanoate phosphate-dependent sugar phosphotransferase 33 can be added to the bioanalytical assay, and by measuring the compound's enol pyruvate phosphate-dependent sugar phosphotransferase 33 and its receptor The effects of these interactions determine whether a compound is an antagonist.
  • Receptor deletions and analogues that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to enol pyruvate phosphate-dependent sugar phosphotransferase 33 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase.
  • 33 molecules of enolpropionate phosphate-dependent sugar phosphotransferase should 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 the enol pyruvate phosphate-dependent sugar phosphotransferase 33 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 enolpyruvate phosphate-dependent sugar phosphotransferase 33 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: It is not limited to Freund's adjuvant and the like.
  • Techniques for preparing monoclonal antibodies to enolpyruvate phosphate-dependent sugar phosphotransferase 33 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), three tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using conventional techniques (Morrison et al, PNAS, 1985, 81: 6851), while existing techniques for producing single-chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Antibodies against enolpyruvate phosphate-dependent sugar phosphotransferase 33 can be used in immunohistochemical techniques to detect enolpyruvate phosphate-dependent sugar phosphotransferase 33 in biopsy specimens.
  • Monoclonal antibodies that bind to enolpyruvate phosphate-dependent sugar phosphotransferase 33 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 against a specific bead site in the body.
  • enol pyruvate phosphate-dependent sugar phosphotransferase 33 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 enol pyruvate phosphate-dependent sugar phosphate Transferase 33 positive cells.
  • the antibodies of the present invention can be used to treat or prevent enolpyruvate phosphate-dependent sugar phosphotransferases.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • the present invention also relates to a diagnostic test method for quantitatively and locally detecting the level of enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • tests are well known in the art and include FISH and radioimmunoassays.
  • the level of enolpyruvate phosphate-dependent sugar phosphotransferase 33 detected in the test can be used to explain the importance of enolpyruvate phosphate-dependent sugar phosphotransferase 33 in various diseases and to diagnose enol acetone Diseases in which the acid phosphate-dependent sugar phosphotransferase 33 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding enol pyruvate phosphate-dependent sugar phosphotransferase 33 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 enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated enol pyruvate phosphate-dependent sugar phosphotransferase 33 to inhibit endogenous enol pyruvate phosphate-dependent sugar phosphotransferase 33 activity.
  • a variant enolpyruvate phosphate-dependent sugar phosphotransferase 33 may be a shortened enolpyruvate phosphate-dependent sugar phosphotransferase 33 that lacks a signal transduction domain, although it may interact with downstream substrates. Combined, but Lack of signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding an enol pyruvate phosphate-dependent sugar phosphotransferase 33 can be used to transfer a polynucleotide encoding an enol pyruvate phosphate-dependent sugar phosphotransferase 33 into a cell .
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding an enol pyruvate phosphate-dependent sugar phosphotransferase 33 can be found in the literature (Sambrook, et al.).
  • a polynucleotide encoding an enolpyruvate phosphate-dependent sugar phosphotransferase 33 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 enolpyruvate phosphate-dependent sugar phosphotransferase 33 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RM. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM and performs endonucleation.
  • Antisense RM, DNA and ribozymes can be obtained by any of the existing RNA or DM synthesis techniques, such as the technique of solid-phase phosphate amide synthesis of oligonucleotides, which is widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DM sequence encoding the RNA. This DM sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond rather than the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding the enolpyruvate phosphate-dependent sugar phosphotransferase 33 can be used for the diagnosis of diseases related to the enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Polynucleotides encoding enolpyruvate phosphate-dependent sugar phosphotransferase 33 can be used to detect the expression of enolpyruvate phosphate-dependent sugar phosphotransferase 33 or enolpyruvate phosphate-dependent sugar phosphate under disease conditions Abnormal expression of transferase 33.
  • the DM sequence encoding the enolpyruvate phosphate-dependent sugar phosphotransferase 33 can be used to hybridize biopsy specimens to determine the expression status of the enolpyruvate phosphate-dependent sugar phosphotransferase 33.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Enol pyruvate phosphate-dependent sugar phosphotransferase 33 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification. Enol pyruvate phosphate-dependent sugar phosphotransferase 33 transcription products can also be detected.
  • RT-PCR RNA-polymerase chain reaction
  • Detection of mutations in the enol pyruvate phosphate-dependent sugar phosphotransferase 33 gene can also be used to diagnose enol Pyruvate phosphate-dependent sugar phosphotransferase 33-related diseases.
  • Enol pyruvate phosphate-dependent sugar phosphotransferase 33 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type enol pyruvate phosphate-dependent sugar phosphotransferase 33 DNA sequence. Wait. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35b P ) are prepared according to cMA, and the sequences can be mapped on chromosomes. 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 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 pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data.
  • These data can be found in, for example, V. Mckusick, Mendel ian Inher i tance in Man (available through contact with Johns Hopkins Univers i ty Welch Medica l
  • 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 diseased and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technologies, The CDM of the disease-associated chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution and one gene per 20 kb).
  • 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.
  • Enolpyruvate phosphate-dependent sugar phosphotransferase 33 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of enolpyruvate phosphate-dependent sugar phosphotransferase 33 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un nouveau polypeptide, une sucre phosphotransférase phosphoénolpyruvate-dépendante 33, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment de diverses malformations survenant lors du développement, de maladies auto-immunes et de tumeurs. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la sucre phosphotransférase phosphoénolpyruvate-dépendante 33.
PCT/CN2001/001131 2000-07-07 2001-07-02 Nouveau polypeptide, sucre phosphotransferase phosphoenolpyruvate-dependante 33, et polynucleotide codant ce polypeptide WO2002020590A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002223374A AU2002223374A1 (en) 2000-07-07 2001-07-02 A novel polypeptide-phosphoenolpyruvate-dependent sugar phosphotransferase 33 and the polynucleotide encoding said polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN00117044A CN1333353A (zh) 2000-07-07 2000-07-07 一种新的多肽——烯醇丙酮酸磷酸依赖的糖磷酸转移酶33和编码这种多肽的多核苷酸
CN00117044.9 2000-07-07

Publications (1)

Publication Number Publication Date
WO2002020590A1 true WO2002020590A1 (fr) 2002-03-14

Family

ID=4586442

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2001/001131 WO2002020590A1 (fr) 2000-07-07 2001-07-02 Nouveau polypeptide, sucre phosphotransferase phosphoenolpyruvate-dependante 33, et polynucleotide codant ce polypeptide

Country Status (3)

Country Link
CN (1) CN1333353A (fr)
AU (1) AU2002223374A1 (fr)
WO (1) WO2002020590A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0549441A (ja) * 1991-08-27 1993-03-02 Kikkoman Corp ペントースの選択発酵方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0549441A (ja) * 1991-08-27 1993-03-02 Kikkoman Corp ペントースの選択発酵方法

Also Published As

Publication number Publication date
CN1333353A (zh) 2002-01-30
AU2002223374A1 (en) 2002-03-22

Similar Documents

Publication Publication Date Title
WO2002020590A1 (fr) Nouveau polypeptide, sucre phosphotransferase phosphoenolpyruvate-dependante 33, et polynucleotide codant ce polypeptide
WO2001066730A1 (fr) Nouveau polypeptide, proteine humaine rs3 12, et polynucleotide codant pour ce polypeptide
WO2001066578A1 (fr) Nouveau polypeptide, adn polymerase 10, et polynucleotide codant pour ce polypeptide
WO2001074876A1 (fr) Nouveau polypeptide, phosphatidylinositol-3 kinase humaine 14, et polynucleotide codant pour ce polypeptide
WO2001075101A1 (fr) Nouveau polypeptide, proteine humaine de regulation de la transcription 8, et polynucleotide codant pour ce polypeptide
WO2001070956A1 (fr) Nouveau polypeptide, proteine humaine de reparation 8 du mesappariement de l'adn, et polynucleotide codant pour ce polypeptide
WO2001070965A1 (fr) Nouveau polypeptide, facteur humain de regulation de la transcription 15, et polynucleotide codant pour ce polypeptide
WO2001081594A1 (fr) Nouveau polypeptide, proteine pax humaine 17, et polynucleotide codant pour ce polypeptide
WO2001075048A2 (fr) Nouveau polypeptide, proteine ribosomale humaine s11 23, et polynucleotide codant pour ce polypeptide
WO2001072801A1 (fr) Nouveau polypeptide, proteine ribosomale humaine s11 12, et polynucleotide codant pour ce polypeptide
WO2001079432A2 (fr) Nouveau polypeptide, facteur humain de transcription de la differentiation cellulaire 58, et polynucleotide codant pour ce polypeptide
WO2002012314A1 (fr) Nouveau polypeptide, proteine-1 humaine associee a la dihydropyrimidinase (drp-1) 9.68, et polynucleotide codant ce polypeptide
WO2001094371A1 (fr) Nouveau polypeptide, proteine ribosomale humaine s4-10, et polynucleotide codant ce polypeptide
WO2001073041A1 (fr) Nouveau polypeptide, adn ligase humaine 15, et polynucleotide codant pour ce polypeptide
WO2001087963A1 (fr) Proteine s18-12 ribosomale, polypeptide humain, et polynucleotide la codant
WO2001075102A1 (fr) Nouveau polypeptide, proteine humaine de regulation de la transcription 13, et polynucleotide codant pour ce polypeptide
WO2002000835A2 (fr) Nouveau polypeptide, proteine de transcription humaine 23, et polynucleotide codant ce polypeptide
WO2001070789A1 (fr) Nouveau polypeptide, proteine ribosomale humaine s5-17, et polynucleotide codant pour ce polypeptide
WO2001079508A1 (fr) Nouveau polypeptide, proteine porteuse mitochondriale humaine 18, et polynucleotide codant pour ce polypeptide
WO2002026971A1 (fr) Nouveau polypeptide, facteur de transcription humain lcr-f112.1, et polynucleotide codant ce polypeptide
WO2001090172A1 (fr) Nouveau polypeptide, proteine ribosomale l39 13, et polynucleotide codant ce polypeptide
WO2002020575A1 (fr) Nouveau polypeptide, proteine humaine 10 associee aux maladies auto-immunes, et polynucleotide codant ce polypeptide
WO2001075013A2 (fr) Nouveau polypeptide, kinase humaine cycline-dependante 14, et polynucleotide codant pour ce polypeptide
WO2001087916A1 (fr) Nouveau polypeptide, proteine ribosomale humaine s5-13, et polynucleotide codant pour ce polypeptide
WO2001090171A1 (fr) Nouveau polypeptide, proteine humaine ribosomale sii 12, et polynucleotide codant ce polypeptide

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CO CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载