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WO2002040519A1 - NOUVEAU POLYPEPTIDE, β-1,4 GALACTOSIDE TRANSFERASE HUMAINE 13.2, ET POLYNUCLEOTIDE CODANT CE POLYPEPTIDE - Google Patents

NOUVEAU POLYPEPTIDE, β-1,4 GALACTOSIDE TRANSFERASE HUMAINE 13.2, ET POLYNUCLEOTIDE CODANT CE POLYPEPTIDE Download PDF

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Publication number
WO2002040519A1
WO2002040519A1 PCT/CN2001/001042 CN0101042W WO0240519A1 WO 2002040519 A1 WO2002040519 A1 WO 2002040519A1 CN 0101042 W CN0101042 W CN 0101042W WO 0240519 A1 WO0240519 A1 WO 0240519A1
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Prior art keywords
polypeptide
polynucleotide
human
galactosyltransferase
seq
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PCT/CN2001/001042
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU2002212041A priority Critical patent/AU2002212041A1/en
Publication of WO2002040519A1 publication Critical patent/WO2002040519A1/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.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01038—Beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase (2.4.1.38)
    • 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, human P-1,4-galactosyltransferase 13.2, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • Enzyme is a type I membrane-bound glycoprotein and is widely distributed in spinal animals. These enzymes constitute a large family of proteins in the organism, namely the 4-galactosyl transporter protein family. Members of this enzyme family transport galactose to N-acetylglucosamine residues in the body to form ⁇ 4-N-acetylglucosamine or poly-N-acetylglucosamine.
  • P 4-N-acetylglucosamine or poly-N-acetylglucosamine is the basic structural unit that constitutes glycolipids, glycoproteins, and proteoglycans, and its mutation or abnormal expression will affect the composition of these proteins and their in vivo Effect activity. In mammals, P-1,4-galactosyltransferase has various tissue-specific biosynthetic activities, and it plays an important role in regulating breast secretion and lactose synthesis.
  • ⁇ -1,4 galactosyl transporter plays an important role in the regulation of lactose synthesis in living organisms. It catalyzes the synthesis of lactose and various glycoproteins in related tissues in vivo.
  • the mutation or abnormal expression of this protein will cause abnormalities in related tissues, cell signaling pathways and metabolic pathways in the body, and then cause various related metabolic disorders.
  • the protein is usually closely related to the occurrence of many related disorders of glycoprotein metabolism in the body. It can also be used to diagnose and treat various diseases mentioned above.
  • the human ⁇ -1,4 galactosyltransferase 13.2 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 human ⁇ -1,4 galactosyltransferase 13.2 proteins involved in these processes, especially the amino acid sequence of this protein. Newcomer 4 Galactosyltransferase 13.2 The isolation of protein-coding genes also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Object of the invention
  • An object of the present invention is to provide an isolated novel polypeptide, human P-1,4-galactosyl transporter 13.2, and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human 4-galactosyltransferase 13.2.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human 4-galactosyltransferase 13.2.
  • Another object of the present invention is to provide a method for producing human P-1,4-galactoside transporter 13.2. Another object of the present invention is to provide an antibody against the polypeptide-human galactosyltransferase 13.2 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the human polypeptide P-1,4 galactosyltransferase 13.2 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 in human P-1, galactosyl transporter 13.2. Summary of invention
  • the invention relates to an isolated polypeptide, which is of human origin, and which comprises: SEQ ID No. 2 Amino acid sequence of a polypeptide, 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 410-772 in SEQ ID NO: 1; and (b) a sequence having positions 1-1343 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 present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human ⁇ -1,4-galactosyltransferase 13.2 protein activity, which comprises utilizing the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of human 4 galactosyltransferase 13.2 protein, comprising detecting mutations in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample. Or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human 4-galactosyltransferase 13.2 .
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the human beta-1, 4-galactosyltransferase 13.2 and human p-1,4-galactosyltransferase of the present invention.
  • the upper graph is a graph of the expression profile of human P-1,4-galactosyltransferase 13.2, the lower graph The figure is a graph of the expression profile of human P-1,4-galactosyltransferase.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human P-1,4 galactosyltransferase 13.2. 13kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • 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 DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, 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 “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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human P-1,4-galactosyltransferase 13.2, can cause the protein to change, thereby regulating the activity of the protein.
  • Agonists can include proteins, nucleic acids, Carbohydrate or any other molecule that can bind to human P-1,4-galactoside transporter 13.2.
  • Antagonist refers to a biological activity that can block or regulate human 4-galactosyl transporter 13.2 when combined with human beta-1,4-galactosyl transporter 13.2 Or immunologically active molecules.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human beta-1, 4-galactosyl transporter 13.2.
  • Regular refers to a change in the function of human 4-galactosyltransferase 13.2, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties of human 4-galactosyltransferase 13.2 , 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 human ⁇ -1,4 galactosyltransferase 13.2 using standard protein purification techniques.
  • Substantially pure human ⁇ -1,4 galactosyltransferase 13.2 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human P-1,4-galactosyltransferase 13.2 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology 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 Northern 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 the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi ggins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method compares each pair by checking the distance between all pairs. Group sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: Number of residues that match between sequences
  • the assay may be Jotun Hein percent identity between nucleic acid sequences Clus ter or a method well known in the art (Hein J., (1990) Methods in enzyraology 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 substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to HFP or a chemical modification of its nucleic acid. 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 human 4-galactosyltransferase 13.2.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human beta-1, 4-galactosyl transporter 13.2 refers to human beta-1, 4-galactosyl transporter 13.2, which is substantially free of other proteins and lipids naturally associated with it. Class, sugar or other substance.
  • Those skilled in the art can purify human ⁇ -1, galactosyltransferase 13.2 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human ⁇ -1,4-galactosyltransferase 13.2 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human 4-galactosyltransferase 13.2, 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. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human 4-galactosyltransferase 13.2.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human ⁇ -1,4 galactosyl transporter 13.2 of the present invention .
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) such a type in which one or more amino acid residues are substituted with other groups to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide ( Such as leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences).
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1343 bases in length and its open reading frame 410-772 encodes 120 amino acids.
  • this polypeptide has a similar expression profile with human 4-galactosyl transporter, and it can be inferred that the human ⁇ -1, 4-galactosyl transporter 13.2 has human 4-galactosyl transporter Similar functionality.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • 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 NO: 1 or a degenerate variant.
  • "degenerate variant" in the present invention refers to a coding region that encodes a protein or polypeptide having SEQ ID NO: 2 but is identical to the coding region shown in SEQ ID NO: 1 Sequences with different nucleic acid sequences.
  • 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 (there is at least 50% identity between the two sequences, preferably 70% identity).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2. '
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human ⁇ -1,4 galactosyl transporter 13.2.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human P-1,4-galactosyltransferase 13.2 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 the double-stranded MA sequence from the DM of the genome; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA 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 mRNA extraction. Kits are also commercially available (Qiagene).
  • cDNA libraries are also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua, 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): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human beta-1, 4-galactosyltransferase 13.2 The level of transcripts; (4) Detecting gene-expressed protein products by immunological techniques or by 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 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein is usually a D 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 of human ⁇ -1,4 galactosyltransferase 13.2 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) and so on.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) and so on.
  • a method (Sa iki, et al. Science 1985; 230: 1350-1354) using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified 'DM / 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 that is genetically engineered using the vector of the present invention or directly using human ⁇ -1, 4-galactosyltransferase 13.2 coding sequence, and recombinant technology A method for producing a polypeptide according to the invention.
  • a polynucleotide sequence encoding human ⁇ -1,4 galactosyltransferase 13.2 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 human ⁇ -1,4 galactosyl transporter 13.2 and appropriate transcription / translation regulatory elements. These methods include in vitro recombination DM technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spiring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in the 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 for translation initiation and a transcription terminator. 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 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.
  • a polynucleotide encoding human ⁇ -1, 4-galactosyltransferase 13.2 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide or the recombinant vector. Genetically engineered host cells.
  • 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • 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; 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 D may be in exponential growth phase were harvested after the treatment with (Method 12, using the procedure well known in the art. Alternatively, it is a MgCl 2. If If necessary, transformation can also be performed by electroporation.
  • 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, and liposome packaging Wait.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human ⁇ -1,4 galactosyl transporter 13. 2 (Sc ience, 1984; 224: 1431). Generally speaking, there are the following steps:
  • polynucleotide or variant encoding human human beta-1, 4-galactosyltransferase 13.2 of the present invention, or a recombinant expression vector containing the polynucleotide for transformation or transduction Host cell
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention and the 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, etc.
  • 4-galactosyl transporter is a type I membrane-bound glycoprotein. These enzymes constitute the 4-galactosyl transporter protein family in vivo. Members of the enzyme family transport galactose to
  • N-acetylglucosamine residues to form ⁇ 4-N-acetylglucosamine or poly-N-acetylglucosamine.
  • P 4-N-acetylglucosamine or poly-N-acetylglucosamine is the basic structural unit that constitutes glycolipids, glycoproteins, and proteoglycans, and its mutation or abnormal expression will affect the composition of these proteins and their in vivo Effect activity.
  • P-1,4-galactosyltransferase has various tissue-specific biosynthetic activities, and it plays an important role in regulating breast secretion and lactose synthesis.
  • a new ⁇ -1, 4-galactosyl transporter can be used as an upstream regulatory factor in the body, regulating the biosynthesis of lactose in the body.
  • the enzyme is expressed in a variety of tissues including the heart, muscle, pancreas, and brain of an organism, but the expression level in the brain is significantly higher than in other tissues.
  • the mutation or abnormal expression of this protein will cause abnormal lactose synthesis in related tissues in the body, and then affect a series of related abnormalities in intracellular signaling pathways and glycoprotein metabolism pathways, which will cause various related diseases.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human ⁇ -1, galactosyl transporter, and both have similar biological functions.
  • the polypeptide of the present invention can transport galactose to N-acetylglucosamine residues in the body to form ⁇ 4-N-acetylglucosamine or poly-N-acetylglucosamine, known as P 4-N-acetylglucosamine Amines or poly-N-acetylglucosamine are the basic structural units that make up glycolipids, glycoproteins, and proteoglycans.
  • the polypeptide of the present invention as an upstream regulatory factor, regulates the biosynthesis of lactose in the body. It is abundantly expressed in the brain. Its abnormal expression is usually closely related to the occurrence of pathological processes such as some related metabolic disorders, protein dysfunction, and tumors of related tissues, and related diseases.
  • human ⁇ -1,4 galactosyltransferase 13.2 of the present invention will produce various diseases, especially glucose metabolism disorders, tumors, embryonic development disorders, growth disorders, and inflammation.
  • Immune diseases including but not limited to:
  • Glucose metabolism disorders Congenital sugar 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 Backlog
  • Tumors of various tissues stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma
  • Embryonic disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, brain development disorders, skin, fat and muscular dysplasia, bone and joint dysplasia, various metabolic defects, stunting, dwarfism, Cushing's syndrome, sexual retardation
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • Abnormal expression of the human P-1,4 galactosyltransferase 13.2 of the present invention will also cause certain hereditary, hematological diseases and the like.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially glucose metabolism disorders, various tumors, embryonic development disorders, and developmental disorders. Diseases, inflammation, immune diseases, certain hereditary, blood diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or inhibit (antagonist) human P-1,4-galactosyl transporter 13.2.
  • Agonists enhance human 4-galactosyltransferase 13.2 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 human 13-1, 4-galactosyltransferase 13.2 and a labeled human beta-1, 4-galactosyltransferase 13.2 can be present in the presence of a drug. From cultivation. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human 4-galactosyltransferase 13.2 include screened antibodies, compounds, receptor deletions, and the like. Antagonist of human
  • 3 -1, 4-galactosyl transporter 13. 2 can bind to human P-1, 4-galactosyl transporter 13. 2 and eliminate its function, or inhibit the production of the polypeptide, or Binding to the active site of the polypeptide prevents the polypeptide from performing its biological function.
  • human beta-1, 4-galactosyl transporter 13.2 When screening compounds as antagonists, human beta-1, 4-galactosyl transporter 13.2 can be added to the bioassay, and by measuring the compounds against human beta-1, 4-galactosyl transporter 13.2 and The effect of their receptor interactions to determine whether a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above.
  • Polypeptide molecules capable of binding to human ⁇ -1,4 galactoside transporter 13.2 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, the human ⁇ -1,4 galactosyltransferase 13.2 molecule should generally be labeled.
  • the present invention provides polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. To produce antibodies. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies directed against the human ⁇ -1,4 galactosyltransferase 13.2 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human galactosyltransferase 13.2 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's Agent.
  • Techniques for preparing monoclonal antibodies to human Pl, 4-galactosyltransferase 13.2 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple 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 existing techniques (Morrison et al, PNAS, 1985, 81: 6851) and existing techniques for producing single-chain antibodies ( ⁇ .S Pat No. 4946778) can also be used to produce single chain antibodies against human Pl, 4-galactosyltransferase 13.2.
  • Antibodies against human beta-1,4-galactosyltransferase 13.2 can be used in immunohistochemical techniques to detect human p-1,4-galactosyltransferase 13..2. In biopsy specimens.
  • Monoclonal antibodies that bind to human P-1,4-galactosyltransferase 13.2 can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human 4 galactoside transportase 13.2 High affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human ⁇ -1,4 galactosides. Transportase 13.2 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human ⁇ -1,4-galactosyltransferase 13.2.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of human 4-galactosyltransferase 13.2.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human P-1,4-galactosyltransferase 13.2.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human 1,4-galactosyltransferase 13.2 detected in the test can be used to explain the importance of human ⁇ -1,4-galactosyltransferase 13.2 in various diseases and to diagnose human ⁇ -1, 4 Diseases where galactosyl transportase 13.2 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. Analysis.
  • the polynucleotide encoding human ⁇ -1,4 galactosyl transporter 13.2 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human ⁇ -1,4-galactosyltransferase 13.2.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human ⁇ -1,4 galactosyl transporter 13.2 to inhibit endogenous human ⁇ -1,4 galactosyl transporter 13.2 activity.
  • a mutated human ⁇ -1,4 galactosyl transporter 13.2 may be a shortened human ⁇ -1,4 galactosyl transporter 13.2 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 to treat diseases caused by abnormal expression or activity of human ⁇ -1, galactosyltransferase 13.2.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding human ⁇ -1,4 galactosyltransferase 13.2 can be used to transfer a polynucleotide encoding human ⁇ -1,4 galactosyltransferase 13.2 into cells .
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human ⁇ -1,4 galactosyltransferase 13.2 can be found in the literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human ⁇ -1,4 galactosyltransferase 13.2 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell via a vector (such as a virus, phage, or plasmid) in vitro,
  • Oligonucleotides including antisense R and DNA
  • ribozymes that inhibit human ⁇ -1,4 galactosyltransferase 13.2 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically breaks down specific MAs. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense MA molecules can be obtained by in vitro or in vivo transcription of a DM sequence encoding the RM. This DNA sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human ⁇ -1, galactosyltransferase 13.2 can be used for the diagnosis of diseases related to human ⁇ -1,4-galactosyltransferase 13.2.
  • a polynucleotide encoding human ⁇ -1,4 galactosyl transporter 13.2 can be used to detect the expression of human ⁇ -1, galactosyl transporter 13.2 or human ⁇ -1,4 galactoside transport under disease conditions Abnormal expression of enzyme 13.2.
  • a DNA sequence encoding human ⁇ -1,4 galactosyl transporter 13.2 can be used to hybridize biopsy specimens to determine the expression of human ⁇ -1,4 galactosyl transporter 13.2.
  • Hybridization techniques include Southern blotting, Northern blotting, Bit hybridization and so on. 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 (Microarray) or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human ⁇ -1,4-galactosyltransferase 13.2 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human ⁇ -1,4-galactosyltransferase 13.2 Transcription products.
  • Detection of mutations in the human beta-1, 4-galactosyltransferase 13.2 gene can also be used to diagnose human beta-1, 4-galactosyltransferase 13.2-related diseases.
  • Human beta-1, 4-galactosyltransferase 13.2 mutant forms include point mutations, translocations, deletions, and recombinations compared to normal wild-type human beta-1, 4-galactosyltransferase 13.2 DNA sequences And any other exceptions. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA 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 cDM 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 V. Mckusick, Mendel ian Inherance in Man (available online with Johns Hopkins University Welch Medica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions. Next, the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease.
  • Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR.
  • the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human ⁇ -1,4-galactosyltransferase 13.2 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human beta-1,4 galactosyltransferase 13.2 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples
  • Human fetal brain total MA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Directional insertion of cDNA fragments into pBSK using Smart cDNA Cloning Kit (purchased from Clontech) (+) At the multiple cloning site of the vector (Clontech), DH5 ⁇ was transformed, and the bacteria formed a cDNA library.
  • Dye terminate cyc le react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3 ends of all clones. Comparing the determined cDNA sequence with an existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one of the clones, 0502b09, was new DNA. A series of primers were synthesized to perform bidirectional determination of the inserted cMA fragments contained in the clone.
  • Pr imerl 5'- TTTTGTTAGAAACACCTGCCTTGC -3 '(SEQ ID NO: 3)
  • Pr imer2 5'- TATTGAGACGGAGCCTGGGCGACA -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'-end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 mmol / L KCl, 10 mmol / L Tris-HCl pH 8.5, 1. 5 ramol / L MgCl 2 , 200 ⁇ 1/1 dNTP, 1 Opmol primer, 1 U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55. C 30sec; 72 ° C 2min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector using a TA cloning kit (Invitrogen). DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1343bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human ⁇ -1,4 galactosyltransferase 13.2 gene expression Total RNA extraction in one step [Ana l. Biochem 1987, 162, 156-159] 0 This method includes acid thiocyanate Guanidine phenol-chloroform extraction.
  • the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate ( ⁇ 4.0 ⁇ ), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70 % ethanol, dried and dissolved in water. 20 ⁇ 8 RNA was applied on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA-2.
  • Example 4 In vitro expression, isolation and purification of recombinant human ⁇ -1, 4-galactosyltransferase 13.2 According to the coding shown in SEQ ID NO: 1 and Figure 1 Region sequence, a pair of specific amplification primers were designed, the sequence is as follows:
  • Pr iraer3 5'-CATGCTAGCATGTTTATATTTTTAGTAGAGATA-3 '(Seq ID No: 5)
  • Pr imer4 5' -CCCGAATTCCTAATCAGGGAGCAGCTGGAAAGG-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and EcoRI digestion sites, respectively Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Nhel and EcoRI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • the PCR reaction was performed using the pBS-0502b09 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: the total volume of 50 ⁇ 1 contains 10 pg of pBS-5052b09 plasmid, primers? 1 ⁇ 1116]: -3 and? 1 ⁇ 1116]: -4 are 10 11101, Advantage polymerase Mix (Clontech) 1 ⁇ 1.
  • the ligation product was transformed into coliform bacteria DH5C using the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0502b09) with the correct sequence was selected, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • NH2-Met-Phe-I le-Phe-Leu-Val-Glu-I le-Glu-Phe-His-Hi s-Val-Gly-Gln-C00H SEQ ID NO: 7
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avrameas, et al. I Jean unochemi stry, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned jk cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • a titer plate coated with a 15 ⁇ ⁇ / ⁇ 1 bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit sera 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.
  • Immunoprecipitation demonstrated that the purified antibody could specifically bind to human 4-galactosyltransferase 13.2.
  • 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.
  • 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 of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained. First, the selection of the probe
  • oligonucleotide fragments 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
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation 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 membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the sample membrane was placed in a plastic bag, and 3-1 Omg pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ral CT DM (calf thymus DNA)) was added. After sealing the bag, shake at 68 ° C for 2 hours.
  • 3-1 Omg pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ral CT DM (calf thymus DNA)
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target MA 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 methods and steps have been reported in the literature. For example, see DeRis i, L L., Lyer, V. & Brown, P. O.
  • 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 slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) using a one-step method, and the mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5 '-triphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5- Amino-propargyl- 2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Pharaacia Biotech Company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Cy3dUTP 5-Amino-propargyl-2'-deoxyur idine 5 '-triphate coupled to Cy3
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (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 cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, Cardiac cancer. Based on these 18 Cy3 / Cy5 ratios, a bar graph is drawn ( Figure 1). It can be seen from the figure that the expression profile of human P-1,4 galactosyl transporter 13.2 and human P-1,4 galactoside transporter according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, une β-1,4 galactoside transférase humaine 13.2, 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 troubles du métabolisme des protéines. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la β-1,4 galactoside transférase humaine 13.2.
PCT/CN2001/001042 2000-06-26 2001-06-25 NOUVEAU POLYPEPTIDE, β-1,4 GALACTOSIDE TRANSFERASE HUMAINE 13.2, ET POLYNUCLEOTIDE CODANT CE POLYPEPTIDE WO2002040519A1 (fr)

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AU2002212041A AU2002212041A1 (en) 2000-06-26 2001-06-25 A novel polypeptide, a human beta -1,4 galactosyl transferase 13.2 and the polynucleotide encoding the polypeptide

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CN00116775.8 2000-06-26
CN 00116775 CN1331292A (zh) 2000-06-26 2000-06-26 一种新的多肽——人β－1,4半乳糖苷转运酶13.2和编码这种多肽的多核苷酸

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990007000A2 (fr) * 1988-12-13 1990-06-28 La Jolla Cancer Research Foundation Nucleotides codant la b1,4-galactosyltransferase humaine et leurs utilisations
US5308769A (en) * 1989-01-11 1994-05-03 Konica Corporation Cancer-related human galactosyltransferase GT-II
US5821117A (en) * 1993-03-16 1998-10-13 The Austin Research Institute Xenotransplantation therapies

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990007000A2 (fr) * 1988-12-13 1990-06-28 La Jolla Cancer Research Foundation Nucleotides codant la b1,4-galactosyltransferase humaine et leurs utilisations
US5308769A (en) * 1989-01-11 1994-05-03 Konica Corporation Cancer-related human galactosyltransferase GT-II
US5821117A (en) * 1993-03-16 1998-10-13 The Austin Research Institute Xenotransplantation therapies

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AU2002212041A1 (en) 2002-05-27

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