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WO2002004498A1 - Nouveau polypeptide, nouvelle sorte d'alpha 2,3-sialyltransferase humaine (st3 gal vi) 9.57, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, nouvelle sorte d'alpha 2,3-sialyltransferase humaine (st3 gal vi) 9.57, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002004498A1
WO2002004498A1 PCT/CN2001/001017 CN0101017W WO0204498A1 WO 2002004498 A1 WO2002004498 A1 WO 2002004498A1 CN 0101017 W CN0101017 W CN 0101017W WO 0204498 A1 WO0204498 A1 WO 0204498A1
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Prior art keywords
polypeptide
polynucleotide
sialyltransferase
gal
human alpha
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PCT/CN2001/001017
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Application filed by Biowindow Gene Development Inc. Shanghai filed Critical Biowindow Gene Development Inc. Shanghai
Priority to AU93643/01A priority Critical patent/AU9364301A/en
Publication of WO2002004498A1 publication Critical patent/WO2002004498A1/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)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • sialic acid-Lewis X determinant is a ligand for three known selection proteins (selective cell adhesion molecules) (E-, P-, and L-selectin).
  • glycosyltransferase involved in the synthesis of the sialic acid-Lewis X determinant structure is betal, 3-N-acetylglucosamine transferase; betal, 4-galactosyltransferase; alphal, 3-fucosyltransferase, And alpha2, 3-sialyltransferase.
  • ST3Gal I and II synthesis are common to many 0-linked oligosaccharides and the glycolipids like GMlb, GDla, and GTlb, such as NeuAc alpha2, 3Gal betal, 3GalNAc sequences.
  • ST3Gal III forms a relatively rare sequence
  • ST3Gal IV can form the ends of the glycosides of glycoproteins and glycolipids NeuAc alpha2, 3Gal betal, 3GalNAc and NeuAc alpha2, 3Gal betal, 4GlcMc sequence.
  • ST3Gal V (GM3 synthase), only active on LacCer.
  • human ST3Gal III and IV participate in the formation of sialic acid-Lewis X determinants (Sasaki, K., Watanabe, E., Kawashiraa, K., Sekine, S Dohi, T., Oshima, M., Hanai, N., Nishi, T., and Hasegawa, M. (1993) J. Biol. Chem. 268, 22782-22787).
  • ST3Gal VI is a new Gal betal, 4GlcNAc alpha2, 3-sialyltransferase.
  • Neolipid tetraceramide and neolactate hexaceramide are highly specific for lipid substrates.
  • 4GlcNAc alpha2 3-sialyltransferase.
  • Neolipid tetraceramide and neolactate hexaceramide are highly specific for lipid substrates.
  • ST3Gal VI prefers oligosaccharide enzymes containing Gal betal, 4GlcNAc structure rather than oligosaccharide enzymes containing Gal betal, 3GlcNAc structure, indicating that it is involved in the synthesis of sialic acid-paraglobulin, which is a glycoprotein and glycolipids.
  • ST3Gal VI is involved in the synthesis of the sialic acid-Lewis X determinant in melanoma and in the synthesis of SPG in the placenta.
  • ST3Gal VI gene is regulating the synthesis of sialic acid-Lewis X determinant in colon cancer and hematopoietic malignant cells
  • human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 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.
  • ST3 Gal VI human alpha 2,3-sialyltransferase
  • Newcomer alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 protein encoding gene isolation 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. 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 human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • Another object of the present invention is to provide an enzyme encoding human alpha 2, 3-sialyltransferase (ST3 Gal).
  • Another object of the present invention is to provide production of human alpha 2, 3-sialyltransferase (ST3 Gal VI) Method of 9.57.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention-human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormality of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a 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 200-463 in SEQ ID NO: 1; and (b) a sequence having 1-591 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 the activity of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 protein, which comprises utilizing the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to an in vitro detection with human alpha 2,3-sialyltransferase (ST3 Gal VI)
  • a method of disease or disease susceptibility related to abnormal expression of a protein comprising detecting a mutation in said polypeptide or a coding polynucleotide sequence thereof in a biological sample, or detecting the amount or biological activity of a polypeptide of the present 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 invention also relates to the polypeptides and / or polynucleotides of the invention in the preparation for the treatment of cancer, developmental or immune diseases or other due to human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 Use of medicines for diseases caused by abnormal expression.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RM, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human alpha 2, 3 -sialyltransferase (ST3Gal VI) 9.57, 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 binds human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • Antagonist refers to a type that can block or regulate human alpha 2, 3-saliva when combined with human alpha 2, 3-sialyltransferase (ST3Gal VI) 9.57.
  • Acid transferase (ST3Gal VI) 9. 57 biologically or immunologically active molecule.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human al pha 2, 3-sialyltransferase (ST3 Ga 1 VI) 9. 57.
  • Regulation refers to changes in the function of human a lpha 2, 3-sialyltransferase (ST3 Ga l VI) 9. 57, including increased or decreased protein activity, changes in binding characteristics, and human a lpha 2, 3 -Alteration of any other biological, functional or immune properties of sialyltransferase (ST3Gail VI) 9.57.
  • Substantially pure 11 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 alpha 2, 3-sialyltransferase using standard protein purification techniques.
  • ST3 Gal VI 9. 57. Basically pure human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9. 57 produces a single main band on a non-reducing polyacrylamide gel. Human a The purity of lpha 2, 3-sialyltransferase (ST3 Gal VI) 9. 57 peptide 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 Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are the same or similar in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, such as through the MEGALIGN program
  • 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 will check the distance between all pairs by Groups of 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: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein. Percentage identity (Hein J., (1990) Methods in emzumology 183: 625-645) 0 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. .
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, 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? 7, which can specifically bind to the epitope of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • 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 alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 refers to human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 which is substantially free of the natural associated with it Other proteins, lipids, sugars or other substances.
  • Those skilled in the art can purify human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 using standard protein purification techniques. Substantially pure peptides produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human alpha 2, 3-sialyltransferase (ST3 Gal VI). 9.57, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the 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 alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • fragment refers to substantially retaining the same biological function or activity of the human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 of the present invention Of peptides.
  • 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 the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by other groups to include a substituent; or (III) such One, in which the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused to the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • an additional amino acid sequence is fused to the mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protea
  • 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 593 bases in length and its open reading frames 200-463 encode 87 amino acids.
  • this peptide has a similar expression profile to human alpha 2, 3-sialyltransferase (ST3 Gal VI), and it can be deduced that the human alpha 2, 3-sialyltransferase (ST3 Gal VI) ) 9.57 has similar functions to human alpha 2, 3-sialyltransferase (ST3 Gal VI).
  • 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.
  • 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 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; The coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequences) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the present invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) 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, and most preferably at least 100 nucleotides. Nucleotides 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 alpha 2, 3-sialyltransferase (ST3 Ga l VI) 9. 57.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding human a lpha 2, 3-sialyltransferase (ST3 Ga l VI) 9.57 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 DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DM 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 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).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DM-RM hybridization; (2) the presence or absence of marker gene functions; (3) determination of human alpha 2, 3-sialyltransferase (ST3 Gal VI ) The level of transcript of 9.57; (4) Detection of the protein product of gene expression by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 gene expression protein product can be detected using 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.
  • the RACE method RACE-rapid cDNA end rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDN A sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a vector or a direct use of the vector of the present invention.
  • Human alpha 2, 3-sialyltransferase (ST3Gal VI) 9.57 coding sequence is used to produce genetically engineered host cells, and a method for producing the polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding human a lpha 2, 3-sialyltransferase (ST3 Ga l VI) 9. 57 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, 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 well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Labora tory Manua, cold Spring Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • coli the PL promoter of lambda phage
  • eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • Expression vectors also include ribosome binding sites and transcription terminators for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription.
  • Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers 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 multi-core encoding human a lpha 2, 3-sialyltransferase ST3 Gal VI 9.57
  • the nucleotide or the recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS, or Bowes 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 DNA uptake can be in the exponential growth phase were harvested, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human al pha 2, 3-sialyltransferase (ST3 Ga l VI) 9. 57 (Scence, 1984; 224 : 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • 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 chromat
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the present inventor alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57 and human alpha 2,3-sialyltransferase (ST3 Gal VI).
  • the upper graph is a graph of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57
  • the lower graph is a graph of human alpha 2, 3-sialyltransferase (ST3 Gal VI). .
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of isolated human alpha 2, 3 -sialyltransferase (ST3 Gal VI) 9.57.
  • LOkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein strip band.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • the Quik mRNA Isolation Kit (Qiegene) was used to separate poly (A) m from the total RM. 2ug poly (A) mRNA forms CDM by reverse transcription.
  • Use Smart cDNA Cloning Kit (purchased from Clontech). The 01 ⁇ fragment was inserted into the multicloning site of the pBSK (+) vector (Clontech), and transformed into DH5 ⁇ . The bacteria formed a cDNA library.
  • the Dye terminate cycle reaction sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and the cDNA sequence of one of the clones 0432d09 was found to be a new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain total RM as a template and ol igo-dT as a primer for reverse transcription reaction.
  • PCR amplification was performed with the following primers:
  • Primerl 5 — GAGACAGTGTTTACCATGTTGGCC -3 '(SEQ ID NO: 3)
  • Primer2 5,-AAAAATGAAACTTTATTACTACAA -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions A reaction volume of 50 ⁇ 1 contains 50 mmol / L KC1, 10 mmol / L Tris-CI, ( ⁇ 8.5 ⁇ ), 1.5 ⁇ l / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers, 1U of 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. Set RT-PCR (3-actin as a positive control and template blank as a negative control.
  • Amplification products were purified using a QIAGEN kit and TA cloning kits were connected to a PCR vector (Invitrogen product). DNA sequence The analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 593 bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57 gene expression :
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. 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.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which MA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 ( pH7.4)-5 x SSC- 5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was placed at lxSSC-0.1 ° /. 55 in SDS. C for 30 min. Then, Phosphor Imager was used for analysis and analysis. Quantitative.
  • Example 4 In vitro expression, isolation and purification of recombinant human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57
  • Primer3 5 '-CCCCATATGATGAGACAGAGTTTCGCCCTTGTC-3' (Seq ID No: 5)
  • Primer4 5'-CATGGATCCTCACGCCTGTAATCCTAGCACTTT-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BamHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • PCR was performed using the pBS-0432d09 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0432d09 plasmid, primers Primer-3 and Primer-4; ⁇ 1 ⁇ 10pmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. 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 ⁇ using the calcium chloride method. After being cultured overnight in LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive clones were screened by colony PCR method and sequenced. A positive clone (pET-0432d09) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) by the calcium gas method.
  • the polypeptide is coupled with 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 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml 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 serum using protein A-Sepharose.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method Acid sequence or a homologous polynucleotide sequence thereof.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • 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 size of the probe ranges from 18 to 50 nucleotides; 2, GC content is 30% -70%, 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 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, 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 1 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 ⁇ - 32P -dATP) is prepared.
  • 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 croarray
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, refer to the literature DeRi si, JL, Lyer, V.
  • 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 amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance from the point is 280 ⁇ ! ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:
  • Cy3 f luorescent dye purchased from Amersham Phamacia Biotech
  • Cy5dUTP 5 — Amino — propargyl — 2 '— deoxyur idine 5'-tr iphate coupled to Cy5 f luorescent dye, purchased Probes were prepared from Amersham Phamacia Biotech company) labeled with specific tissue (or stimulated cell line) mRNA.
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and then washed with a washing solution (lx SSC, 0.2 SDS) at room temperature before use.
  • ScanArray 3000 scanner purchased from General Scanning Company, USA was used for scanning. The scanned image was analyzed and processed with Imagene software (Biodicovery Company, 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. Draw a graph based on these 18 Cy3 / Cy5 ratios. (figure 1 ) .
  • 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 malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • sialic acid-Lewis X determinant is a ligand for three known selection proteins (selective cell adhesion molecules) (E-, P-, and L-selectin), and these selection proteins are involved in lymphoid tissues. Cell adhesion molecules that supplement leukocytes are also sites of inflammation. In addition, increased expression of sialic acid-Lewis X determinant results in metastasis of cancer cells. Human a lpha2, 3-sialyltransferase is involved in the synthesis of salivary Liquid acid-Lewis X is a glycosyltransferase that determines the structure of the factor. It is a family of more than 14 enzymes that are widely distributed in many cell types. In vivo, its abnormal expression can affect the synthesis of the sialic acid-Lewis X determinant structure, leading to the synthesis of the corresponding E-, P-, and L-selectin proteins, which in turn can cause related diseases.
  • selection proteins selective
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human alpha2, 3-sialyltransferase protein, both of which have similar biological functions.
  • the polypeptide of the present invention is involved in the synthesis of the sialic acid-Lewis X determinant structure, and the latter is a ligand of three known selection proteins (selective cell adhesion molecules) (E-, P- and L-selectin), These selectins have important regulatory roles in human immune regulation, inflammatory response, and cell canceration.
  • the abnormal expression of human al pha2, 3-sialyltransferase protein can affect the synthesis of the sialic acid-Lewis X determinant structure, leading to the synthesis of corresponding E-, P-, and L-selectin proteins, which leads to immune deficiency,
  • the occurrence of various inflammations and various tumor diseases including but not limited to:
  • Defects in cellular immune function can cause various intracellular parasitic infections and various viral infections. These diseases include but are not limited to:
  • Various viral infectious diseases such as measles virus (measles, measles bronchitis, pneumonia, otitis media, subacute sclerosing panencephalitis), herpes virus (shingles, chicken pox), etc .;
  • Humoral immune deficiency can cause various extracellular parasites and various viral infections. These diseases include but are not limited to:
  • polio virus poliomyelitis
  • hepatitis virus A, B, C, D, E, H, G
  • etc . Various viral infections: polio virus (poliomyelitis), hepatitis virus (A, B, C, D, E, H, G), etc .;
  • Serous inflammation tuberculous pleura, rheumatoid arthritis, acute rhinitis, etc .;
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenomas (carcinoma) [ovary], basal cell carcinoma [head and face Skin], (malignant) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .; 2.
  • Mesenchymal tissue :
  • Malignant lymphoma [Neck, mediastinum, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [push / thoracic / rib / skull and long bone], etc .;
  • Nerve fiber [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannoma [head, neck, limbs and other nerves], (malignant) glioblastoma [brain], medulloblastoma [ Cerebellum] (Malignant) Meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .;
  • malignant melanoma [skin, mucous membrane], (malignant) hydatidiform mole, chorionic epithelial carcinoma [uterine], (malignant) supporter cells, stromal cell tumor, (malignant) granulosa cell tumor [ovarian, testicular], fine Blastoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum and palate tail], etc .;
  • polypeptide of the present invention and the antagonist, agonist and inhibitor of the polypeptide can be directly used for the treatment of various diseases, such as immunodeficiency, various inflammations, and various tumor diseases.
  • the invention also provides screening compounds to identify increasing (agonist) or suppressing (antagonist) human alpha
  • Antagonists of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 include screened antibodies, compounds, receptor deletions, and the like. Antagonist of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 can bind to human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 and eliminate its function, or inhibit the production of the polypeptide Or, in combination with the active site of the polypeptide, the polypeptide cannot perform biological functions.
  • human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57 can be added to the bioanalytical assay, and the compound can be tested for human alpha 2, 3-sialyltransferase (ST3 Gal VI) The effect of the interaction between 9.57 and its receptor to determine if a 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.
  • Peptide molecules capable of binding to human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 can be obtained by screening a random peptide library consisting of various possible combinations of amino acids bound to a solid phase. During screening, 9.57 molecules of human alpha 2, 3-sialyltransferase (ST3 Gal VI) should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57 epitope.
  • ST3 Gal VI human alpha 2,3-sialyltransferase 9.57 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 using human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57 by direct injection in 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 immunity
  • the reaction includes, but is not limited to, Freund's adjuvant and the like.
  • Techniques for preparing monoclonal antibodies to human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57 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 to non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the unique technology for producing single chain antibodies U.S. Pat No. 4946778, can also be used to produce single chain antibodies against human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57.
  • Antibodies against human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 can be used in immunohistochemistry to detect human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 in biopsy specimens.
  • Monoclonal antibodies that bind to human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 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 alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 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 alpha 2, 3-sialic acid transfer Enzyme (ST3 Gal VI) 9.57 positive cells.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 level.
  • These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57 detected in the test can be used to explain the importance of human alpha 2,3-sialyltransferase (ST3 Gal VI) 9.57 in various diseases Sexual and diagnostic diseases for which human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 functions.
  • the polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzyme, and can be analyzed by one-dimensional or two-dimensional or three-dimensional gel electrophoresis, and more preferably by mass spectrometry coding.
  • the polynucleotide of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 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 alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • Recombinant gene therapy vectors can be designed to express mutated human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 to inhibit endogenous human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 activity.
  • mutated human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 may be a shortened human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 although it can bind to downstream substrates, it lacks signal transduction activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to encode a polynucleotide encoding human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 Transfer into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 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 human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM 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 using any existing RNA or DNA synthesis techniques, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA 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 RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified by various methods, such as increasing the sequence length on both sides, and the linkage between ribonucleosides should use phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 can be used for the diagnosis of diseases related to human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • Encoding Human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 polynucleotide can be used to detect the expression of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 or human alpha in disease states Aberrant expression of 2,3-sialyltransferase (ST3 Gal VI) 9.57.
  • a DNA sequence encoding human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 can be used to hybridize biopsy specimens to determine the expression of human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57.
  • Hybridization techniques include Southern blotting, Northern 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 microarray or a DM chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Detection of mutations in the human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 gene can also be used to diagnose human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57-related diseases.
  • Human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 mutant forms include point mutations and translocations compared to normal wild-type human alpha 2, 3-sialyltransferase (ST3 Gal VI) 9.57 DNA sequences , Deletions, reorganizations, and any other abnormalities. 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.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, 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 (FISH) of cDNA clones to metaphase chromosomes can be refined in one step Perform chromosomal mapping accurately.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel i an
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human a lpha 2,3-sialyltransferase (ST3Gal VI) 9. 57 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human a lpha 2, 3 -sialyltransferase (ST3 Gal VI) 9.57 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 nouvelle sorte d'alpha 2,3-sialyltransférase humaine (ST3 Gal VI) 9.57, 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 déficience immunitaire, de nombreuses inflammations et de toutes sortes de tumeurs. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la nouvelle sorte d'alpha 2,3-sialyltransférase humaine (ST3 Gal VI) 9.57.
PCT/CN2001/001017 2000-06-21 2001-06-19 Nouveau polypeptide, nouvelle sorte d'alpha 2,3-sialyltransferase humaine (st3 gal vi) 9.57, et polynucleotide codant ce polypeptide WO2002004498A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU93643/01A AU9364301A (en) 2000-06-21 2001-06-19 A novel polypeptide - the human alpha-2,3 sialyltransferase(st3 gal vi) 9.57 andthe polynucleotide encoding said polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN00116632A CN1330147A (zh) 2000-06-21 2000-06-21 一种新的多肽——人alpha2,3－唾液酸转移酶(ST3 GalVI)9.57和编码这种多肽的多核苷酸
CN00116632.8 2000-06-21

Publications (1)

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WO2002004498A1 true WO2002004498A1 (fr) 2002-01-17

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PCT/CN2001/001017 WO2002004498A1 (fr) 2000-06-21 2001-06-19 Nouveau polypeptide, nouvelle sorte d'alpha 2,3-sialyltransferase humaine (st3 gal vi) 9.57, et polynucleotide codant ce polypeptide

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CN (1) CN1330147A (fr)
AU (1) AU9364301A (fr)
WO (1) WO2002004498A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140248697A1 (en) * 2005-07-08 2014-09-04 Glykos Finland Oy Method for evaluating cell populations

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995004816A1 (fr) * 1993-08-04 1995-02-16 The Regents Of The University Of California Compositions et procedes de production de sialyltransferases
WO1998038315A1 (fr) * 1997-02-28 1998-09-03 Japan Tobacco Inc. GENE CODANT LA β-GALACTOSIDE α-2,6-SIALYLTRANSFERASE
WO1999024584A1 (fr) * 1997-11-12 1999-05-20 Neurotherapeutics Methodes de detection et de traitement de maladies utilisant une glycosyltransferase

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995004816A1 (fr) * 1993-08-04 1995-02-16 The Regents Of The University Of California Compositions et procedes de production de sialyltransferases
WO1998038315A1 (fr) * 1997-02-28 1998-09-03 Japan Tobacco Inc. GENE CODANT LA β-GALACTOSIDE α-2,6-SIALYLTRANSFERASE
WO1999024584A1 (fr) * 1997-11-12 1999-05-20 Neurotherapeutics Methodes de detection et de traitement de maladies utilisant une glycosyltransferase

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140248697A1 (en) * 2005-07-08 2014-09-04 Glykos Finland Oy Method for evaluating cell populations
US10000734B2 (en) 2005-07-08 2018-06-19 Glykos Finland Oy Method for evaluating cell populations

Also Published As

Publication number Publication date
CN1330147A (zh) 2002-01-09
AU9364301A (en) 2002-01-21

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