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WO2002000830A2 - Nouveau polypeptide, sucre phosphotransferase phosphoenolpyruvate-dependante 12, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, sucre phosphotransferase phosphoenolpyruvate-dependante 12, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000830A2
WO2002000830A2 PCT/CN2001/000950 CN0100950W WO0200830A2 WO 2002000830 A2 WO2002000830 A2 WO 2002000830A2 CN 0100950 W CN0100950 W CN 0100950W WO 0200830 A2 WO0200830 A2 WO 0200830A2
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Prior art keywords
polypeptide
polynucleotide
dependent sugar
phosphate
sugar phosphotransferase
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PCT/CN2001/000950
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English (en)
Chinese (zh)
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WO2002000830A3 (fr
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU89536/01A priority Critical patent/AU8953601A/en
Publication of WO2002000830A2 publication Critical patent/WO2002000830A2/fr
Publication of WO2002000830A3 publication Critical patent/WO2002000830A3/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.)
    • C12N9/12—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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, monoenolpropionate phosphate-dependent sugar phosphotransferase 12, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique
  • the enol pyruvate phosphate-dependent sugar phosphotransferase system is a major sugar transport system in bacteria. 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 and a thermostable phosphoryl carrier protein (HPr), and a sugar-specific permease-enzyme I I complex. PTS can catalyze the process of sugar transport and the accompanying sugar phosphorylation. In different kinds of E. coli, PTS permease is composed of different numbers of polypeptide chains. In certain cases, some sugar-specific proteins will fuse to form domains with EI and / or HPr energy coupling functions. There is evidence that the entire ⁇ complex is required for sugar transport and phosphorylation.
  • EI protein-enzyme I
  • HPr thermostable phosphoryl carrier protein
  • permease has at least three easy-to-recognize functional domains: a hydrophobic transmembrane domain capable of binding and transporting sugar substrates; a hydrophilic EI II similar domain Has a first phosphorylation site (always histamine); a second hydrophilic protein or protein scab 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.
  • EI I is considered to be an important constituent protein of PTS, participating in transmembrane, forming membrane transfer channels and providing sugar binding sites.
  • EI I usually consists of two cytoplasmic domains I IA, ⁇ B, and a transmembrane domain I IC.
  • ⁇ contains the first permease-specific phosphorylation site, which is a histidine, which can be phosphorylated by phosphorylated HPr.
  • ⁇ B contains a second phosphorylation site, which is phosphorylated by phosphorylated I IA, which is dependent on permease.
  • the phosphoryl group is transferred from the IB to the sugar as a substrate.
  • I IA and ⁇ B can be linked by a polypeptide rich in alanine and proline to form a stable dimer structure ⁇ .
  • the secondary structure of ⁇ 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 of 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 structure changes before and after phosphorylation is relatively small.
  • the enolpyruvate phosphate-dependent sugar phosphotransferase 12 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art Identification of more enolpyruvate phosphate-dependent sugar phosphotransferase 12 proteins involved in these processes, especially the amino acid sequence of this protein. Isolation of the neoenol pyruvate phosphate-dependent sugar phosphotransferase 12 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 form the basis for the development of a diagnostic and / or therapeutic agent for disease 1 and it is therefore important to isolate its coding DM. 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 12.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding an enol pyruvate phosphate-dependent sugar phosphotransferase 12.
  • Another object of the present invention is to provide a method for producing an enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • Another object of the present invention is to provide an antibody against the polypeptide monoenolpyruvate phosphate-dependent sugar phosphotransferase 12 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the mono-enol pyruvate phosphate-dependent sugar phosphotransferase 12 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the 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 954-1286 in SEQ ID NO: 1; and (b) a sequence having 1-1424 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 screen for mimicking, activating, antagonizing or inhibiting enolpyruvate phosphate-dependent sugars
  • a method of a phosphotransferase 12 protein active compound comprising using a polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of enolpyruvate phosphate-dependent sugar phosphotransferase 12 protein in vitro, which comprises detecting the polypeptide or its encoding polynucleotide sequence in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the invention also relates to the preparation of a medicament of the polypeptide and / or polynucleotide of the invention for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of enolpyruvate phosphate-dependent sugar phosphotransferase 12 the use of.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DM 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 the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioly active refers to a protein that has the structure, regulatory, 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 to specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with enolpyruvate phosphate-dependent sugar phosphotransferase 12, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind an enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • Antagonist refers to a biological activity that blocks or regulates enol pyruvate phosphate-dependent sugar phosphotransferase 12 when combined with enol pyruvate phosphate-dependent sugar phosphotransferase 12.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind enolpyruvate phosphate-dependent sugar phosphate transfer ⁇ 12.
  • Regular refers to changes in the function of enolpyruvate phosphate-dependent sugar phosphotransferase 12, including any increase or decrease in protein activity, changes in binding characteristics, and any of the enolpyruvate phosphate-dependent sugar phosphotransferase ⁇ Changes in other biological, functional or immune properties.
  • Substantially pure '' means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify enolpyruvate phosphate-dependent sugar phosphotransferase 12 using standard protein purification techniques.
  • the substantially pure enolpyruvate phosphate-dependent sugar phosphotransferase 12 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the enol pyruvate phosphate-dependent sugar phosphotransferase 12 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of a polynucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
  • 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 achieved by hybridization under conditions of reduced stringency (Southern blotting or
  • 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 be combined with 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 percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madis on Wi s.).
  • the MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Clus ter method checks the distance between all pairs.
  • the groups of sequences are arranged into clusters. The clusters are then assigned in pairs or groups. Identity between two amino acid sequences such as sequence A and sequence B
  • the sex percentage is calculated by:
  • Number of residues matching between sequence A and sequence X 100 Number of residues in sequence A-number of spacer residues in sequence A-number of spacer residues in sequence B Methods such as Jo Um He in determine the percent identity between nucleic acid sequences (He in J., (1990) Methods in emzumo l ogy 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine. '
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RM sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be 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 characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa, F (ab ') ⁇ Fv, which can specifically bind to the epitope of enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a vector, or such a 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 enolpyruvate phosphate-dependent sugar phosphotransferase 12 means that enolpyruvate phosphate-dependent sugar phosphotransferase 12 is substantially free of other proteins, lipids, and sugars naturally associated with it. Class or other substances. Those skilled in the art can purify enolpyruvate phosphate-dependent sugar phosphotransferase 12 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 12 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, monoenolpyruvate phosphate-dependent sugar phosphotransferase 12, 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 present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the enolpyruvate phosphate-dependent sugar phosphotransferase 12 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-protective amino acid residues (preferably conservative amino acid residues), And the substituted amino acid may or may not be encoded by a genetic codon; or (II) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III ) A type in which the mature polypeptide is fused with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) a type in which an additional amino acid sequence is fused into a mature polypeptide Sequences (such as leader sequences or secretory sequences or sequences used to purify this polypeptide or protease sequences) 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 cDNA library of human fetal brain tissue. It contains a total nucleotide sequence of 1424 bases, and its open reading frame 954-1286 encodes 110 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 12 has enolpyruvate phosphate. Similar functions for sugar-dependent phosphotransferases.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DM forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding Code chain.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (there is 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% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Crosses occur at least 95% or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 nucleotides. More than 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 12.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the enolpyruvate phosphate-dependent sugar phosphotransferase 12 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) Isolating double-stranded DNA from genomic DNA Sequence; 2) chemically synthesize a DNA sequence to obtain double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDM of interest is to isolate mRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been developed for mRNA extraction, and kits are also commercially available (Qiagene :).
  • the construction of cDNA libraries is also a common method (Sanibrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, 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 DNA-A hybrids; (2) the presence or absence of marker gene functions; (3) determination of the enol pyruvate phosphate-dependent sugar phosphotransferase 12 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 At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2,000 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.
  • MA 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 enolpyruvate phosphate-dependent sugar phosphotransferase 12 gene. .
  • the RACE method RACE-rapid cDNA end rapid amplification method
  • the primers for PCR may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified MA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the 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 12 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 12 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMS D expression expressed in mammalian cells Carrier (Lee and Na thans, J Bio Chem.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • 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: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. 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 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.
  • the recombinant vector of the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as insect 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 DNA can be harvested after the exponential growth phase and treated with CaCl.
  • the steps used are well known in the art.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as Microinjection, electroporation, liposome packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant enolpyruvate phosphate-dependent sugar phosphotransferase 12 by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally speaking, there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. 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, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the enolpyruvate phosphate-dependent sugar phosphotransferase 12 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 12 and the lower graph is the graph of the enol pyruvate phosphate-dependent sugar phosphotransferase.
  • 1-bladder mucosa 2-PMA + Ecv304 cell line, 3-LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5-Fibroblas t, growth factor stimulation, 1024NT, 6-scars into fc growth factor Stimulation, 1013HT, 7-scar into fc without stimulation with growth factors, 1013HC, 8-bladder cancer cell EJ, 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12-liver cancer cell line, 13-fetus Skin, 14-spleen, 15-prostate cancer, 16-jejunum adenocarcinoma, 17 cardia cancer.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated enolpyruvate phosphate-dependent sugar phosphotransferase 12. 12kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • Total RM of human fetal brain was extracted by one step method with guanidine isothiocyanate / phenol / chloroform.
  • MRNA is formed by reverse transcription cDNA Quik mRNA Iso lat ion Ki t (Qiegene Co.) isolated from the total RNA poly (A) mRNA 0 2ug poly ( A) used.
  • a Smart cDNA cloning kit purchased from Clontecli was used to insert the cDNA fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5a.
  • the bacteria formed a cDNA library.
  • Example 2 Cloning of a gene encoding enolpyruvate phosphate-dependent sugar phosphotransferase 12 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and ol i go-dT as a primer for reverse transcription reaction. After purification of Qiagene's kit, PCR amplification was performed with the following primers:
  • Primerl 5,-AGCAAACAAACCCACAACCACCTCCA-3, (SEQ ID NO: 3)
  • Pr imer2 5'- GGTTTTGAAGGCAGTGAACCGATT -3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence starting at the lbp at the 5 'end of SEQ ID NO: 1;
  • Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l / L KC1, 10 ⁇ l / L in a 50 ⁇ l reaction volume
  • Tr is-Cl, (P H8 . 5), 1. 5mmol / LM g Cl 2, 200 ⁇ mol / L dNTP, l Opmo l primer, 1U Taq DNA polymerase (Clontech Co.).
  • 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.
  • ⁇ -acUn 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).
  • Example 3 Northern blot analysis of the expression of enol pyruvate phosphate-dependent sugar phosphotransferase 12 gene: Total RNA was extracted in one step [Ana l. Biochera 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. I.e. with 4M guanidinium isothiocyanate -25mM sodium citrate, 0. 2M sodium acetate (.
  • A-"P dATP was used to prepare labeled DNA probes by random primers.
  • the DM probe used was the PCR-encoded enol pyruvate phosphate-dependent sugar phosphotransferase 12 coding region sequence (954bp to 1286bp).
  • 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide- 25 mM KH 2 P0 4 ( ⁇ 4) -5 SSC-5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA.
  • Example 4 In vitro expression, isolation and purification of recombinant enolpyruvate phosphate-dependent sugar phosphotransferase 12
  • Pr imer3 5'-CCCCATATGATGCCAACTACTTGGCGGGGGAAT-3 '(Seq ID No: 5)
  • Pr imer4 5'- CATGGATCCTCATATATTTGTCCAAACCCAGGG- 3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5, and 3' ends of the target gene, respectively.
  • the Ndel and BaraHI digestion sites correspond to the expression vector plasmid pET-28b ( +) (Novagen, Cat. No. 69865. 3) selective endonuclease site.
  • the PCR reaction was performed using pBS-0977d02 plasmid containing the full-length target gene as a model.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0977d02 plasmid, primers Primer-3 and Primer-4 were 10 pmol, and Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. 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 ligation product was transformed into the colibacillus DH5 ct by the calcium chloride method.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. Chromatography was performed using an affinity chromatography column His s. Bind Quick Cartridge (product of Novagen) capable of binding to 6 histidines (6His-Tag). The purified protein enol pyruvate phosphate-dependent sugar phosphotransferase 12 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 12 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method.
  • polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 1 ⁇ 2 g of the hemocyanin-peptide complex described above with complete Freund's adjuvant.
  • the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once. ⁇ Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate as an ELISA to determine antibody titers in rabbit serum. Total Ig G was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • 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 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. 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 using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then use the same steps.
  • 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 synthetic 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 invention; the second type of probes are partially related to the 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;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The region is compared for homology. If the homology with the non-target molecule 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 to the gene fragment of SEQ ID NO: 1 Or complementary (41Nt):
  • ⁇ (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • 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 after combining the collection solutions of the first peak.
  • Pre-hybridization The sample film was placed in a plastic bag, was added 3 - 10mg prehybridization solution (l OxDenhardt 's;. 6xSSC , 0. Img / ml CT DNA ( the DNA calf thymus)), the sealed bag, 6 8 ° C Shui Luo shake for 2 hours.
  • 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 7 DNA Mi croar ray
  • Gene chip or gene micro matrix (DNA Mi croarray) is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high density arrangement of a large number of target gene fragments on glass. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps are in the literature ⁇ There are many reports, for example, see the literature DeRisi, JL, Lyer, V. & Brown, P.0.
  • 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 purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking apparatus. After elution, the slides were fixed to prepare the DNA on a glass slide to prepare a chip. The specific method steps have been reported in the literature in various ways. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) by one-step method, and mRNA was purified by Oligotex mRNA Midi Kit (purchased from QiaGen).
  • J Cy3dUTP (5-Araino-propargyl-2'-deoxyur idine 5'-triphate coupled to C 3 fluorescent dye, purchased from Amersham Phamacia Biotech) was used to label mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Araino-propargyl- 2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye (purchased from Amersham Phamacia Biotech) was used to label the mRNA of specific tissues (or stimulated cell lines) in the body, and probes were prepared after purification.
  • Cy3dUTP (5-Araino-propargyl-2'-deoxyur idine 5'-triphate coupled to C 3 fluorescent dye, purchased from Amersham Phamacia Biotech) was used
  • the above specific tissues are bladder mucosa, PMA + E CV 304 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, cardia cancer. Draw a chart based on these 17 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 (E I) and thermostable phosphoryl carrier protein (HPr) involved in energy metabolism, and a sugar-specific permease-enzyme I I complex. The entire ⁇ complex is required for sugar transport and phosphorylation. It is considered to be an important constituent protein of PTS, participates in transmembrane, forms membrane transfer channels and provides sugar-binding sites. EII usually consists of two cytoplasmic domains ⁇ , ⁇ B and a transmembrane domain I IC.
  • 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 necessary to form its active mo t i f.
  • 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 12 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:
  • Oxidative acidemia propionic acidemia, malonylmalonic aciduria, isovalerate, Combined carboxylase deficiency, glutaric acid type I, congenital sugar digestion and absorption defects such as congenital lactose intolerance, hereditary fructose intolerance, monosaccharide metabolism defects such as galactose, fructose metabolism defects Glycogen metabolism diseases such as glycogen storage disease, mucopolysaccharidosis
  • Fetal developmental disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney disease, double ureter, cryptorchidism, congenital abdominal sulcus hernia, double uterus, vaginal atresia, Hypospadias, amphoteric deformity, atrial septal defect, ventricular septal defect, pulmonary stenosis, open ductus arteriosus, neural tube defects, congenital hydrocephalus, iris defect, congenital glaucoma or cataract, congenital deafness, growth and development disorders : Mental retardation, cerebral palsy, mental retardation, mental retardation, familial cerebral nucleus hypoplasia syndrome, strabismus, skin, fat, and muscular dysplasia such as congenital skin sagging, premature senile, congenital horn Malnutrition, various metabolic defects, stunting, dwarfism, 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, 'fr 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, Fibroid, fibrosarcoma, lipoma, liposarcoma, leiomyoma
  • the abnormal expression of the enolpyruvate phosphate-dependent sugar phosphotransferase 12 of the present invention will also produce certain hereditary, hematological and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • Agonists increase enol pyruvate phosphate-dependent sugar phosphotransferase 12 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing enolpropionate phosphate-dependent sugar phosphotransferase 12 can be cultured in the presence of drugs with labeled enolpyruvate phosphate-dependent sugar phosphotransferase 12. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of enol pyruvate phosphate-dependent sugar phosphotransferase 12 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of enolpyruvate phosphate-dependent sugar phosphotransferase 12 can bind to enolpyruvate phosphate-dependent sugar phosphotransferase 12 and eliminate its function, or inhibit the production of the polypeptide, or with the activity of the polypeptide Site binding prevents the polypeptide from performing its biological function.
  • enolpyruvate phosphate-dependent sugar phosphotransferase 12 can be added to the bioanalytical assay. The effect of this interaction is used to determine whether the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Enolpyruvate Phosphate-dependent sugar phosphotransferase 12-bound polypeptide molecules can be obtained by screening a random peptide library consisting of various possible combinations of amino acids bound to a solid phase. In screening, 12 molecules of enolpyruvate phosphate-dependent sugar phosphotransferase should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the enol pyruvate phosphate-dependent sugar phosphotransferase 12 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 enol pyruvate phosphate-dependent sugar phosphotransferase 12 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 the preparation of monoclonal antibodies to enolpyruvate phosphate-dependent sugar phosphotransferase 12 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975,
  • Antibodies against enolpyruvate phosphate-dependent sugar phosphotransferase 12 can be used in immunohistochemical techniques to detect enolpyruvate phosphate-dependent sugar phosphotransferase 12 in biopsy specimens.
  • Monoclonal antibodies that bind to enolpyruvate phosphate-dependent sugar phosphotransferase 12 can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins against a specific bead site in the body.
  • enolpyruvate phosphate-dependent sugar phosphotransferase 12 High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill enol pyruvate phosphate-dependent sugar phosphate Transferase 12 positive cells.
  • the antibodies in the present invention can be used to treat or prevent diseases related to enolpyruvate phosphate-dependent sugar phosphotransferase I 2 .
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • Enol pyruvate phosphate-dependent sugar phosphotransferase 12 levels tested in the test can be used to explain enol The importance of pyruvate phosphate-dependent sugar phosphotransferase 12 in various diseases and for diagnosing diseases in which enolpyruvate phosphate-dependent sugar phosphotransferase 12 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry.
  • Polynucleotides encoding enolpyruvate phosphate-dependent sugar phosphotransferase 12 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of enolpyruvate phosphate-dependent sugar phosphotransferase 12. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated enol pyruvate phosphate-dependent sugar phosphotransferase 12 to inhibit endogenous enol pyruvate phosphate-dependent sugar phosphotransferase 12 activity.
  • a variant enolpyruvate phosphate-dependent sugar phosphotransferase 12 may be a shortened enolpyruvate phosphate-dependent sugar phosphotransferase 12 that lacks a signaling domain, although it can interact with downstream substrates. Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • polynucleotide of enzyme 12 can be used to transfer a polynucleotide encoding an enolpyruvate phosphate-dependent sugar phosphotransferase 12 into cells .
  • polynucleotide recombinant viral vectors 12 can be found in existing literature (Sarabrook, etal.) 0 Further recombinant encoding phosphoenolpyruvate-dependent sugar phosphotransferase
  • the polynucleotide of enzyme 12 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit enolpyruvate phosphate-dependent sugar phosphotransferase 12 m NA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like MA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RNA, DNA and ribozymes can be obtained by any existing or DNA synthesis technology, 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 the DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • Polynucleotides encoding enolpyruvate phosphate-dependent sugar phosphotransferase 12 can be used for the diagnosis of diseases related to enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • Enol pyruvate phosphate-dependent sugar phosphorus The polynucleotide of acid transferase 12 can be used to detect the expression of enol pyruvate phosphate-dependent sugar phosphotransferase 12 or the abnormal expression of enol pyruvate phosphate-dependent sugar phosphotransferase 12 in a disease state.
  • the DNA sequence encoding the enolpyruvate phosphate-dependent sugar phosphotransferase 12 can be used to hybridize biopsy specimens to determine the expression status of the enolpyruvate phosphate-dependent sugar phosphotransferase 12.
  • Hybridization techniques include Southern blotting, Nor thern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Enol pyruvate phosphate-dependent sugar phosphotransferase 12 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the enol pyruvate phosphate-dependent sugar phosphotransferase 12 transcription products.
  • Detection of mutations in the enol pyruvate phosphate-dependent sugar phosphotransferase 12 gene can also be used to diagnose enol propionate phosphate-dependent sugar phosphotransferase 12-related diseases.
  • Enol pyruvate phosphate-dependent sugar phosphotransferase 12 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type enol pyruvate phosphate-dependent sugar phosphotransferase 12 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 the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine the presence or absence of gene mutations.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position of a human chromosome and can hybridize with 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 (repeat polymorphisms) are available for labeling chromosomal 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.
  • PCR primers (preferably 15-35bp) are prepared based on the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization (FI SH) of cDNA clones and metaphase chromosomes allows precise chromosomal localization in one step.
  • Verara et al. Human Chromos omes: a Manua l of Basic Techniques, Pergaraon Pres s, New York (1988).
  • 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. cku si ck, Mende li an I nher i tance in Man (available online with Johns Hopk ins University Wetch Medica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected 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 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptide of the present 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 12 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 12 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une sucre phosphotransférase phosphoénolpyruvate-dépendante 12, 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 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 la sucre phosphotransférase phosphoénolpyruvate-dépendante 12.
PCT/CN2001/000950 2000-06-14 2001-06-11 Nouveau polypeptide, sucre phosphotransferase phosphoenolpyruvate-dependante 12, et polynucleotide codant ce polypeptide WO2002000830A2 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1998045451A1 (fr) * 1997-04-07 1998-10-15 University Of Florida Research Foundation, Inc. Micro-organismes de combinaison capables de produire la fermentation de la cellobiose

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Publication number Priority date Publication date Assignee Title
WO1998045451A1 (fr) * 1997-04-07 1998-10-15 University Of Florida Research Foundation, Inc. Micro-organismes de combinaison capables de produire la fermentation de la cellobiose

Non-Patent Citations (2)

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Title
DATABASE GENBANK [Online] 06 June 2000 GUIPPONI, M. ET AL. Retrieved from NCBI, accession no. GI:6681700 Database accession no. (AB017602.1) *
DATABASE GENBANK [Online] 12 January 2000 YU, Y. ET AL. Retrieved from NCBI, accession no. GI:6690248 Database accession no. (AAF24054.1) *

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