WO2001098505A1 - Nouveau polypeptide, proteine 11 de liaison d'une udp-glycosyltransferase (ugt) et d'une cobalamine, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine 11 de liaison d'une udp-glycosyltransferase (ugt) et d'une cobalamine, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2001098505A1
WO2001098505A1 PCT/CN2001/000784 CN0100784W WO0198505A1 WO 2001098505 A1 WO2001098505 A1 WO 2001098505A1 CN 0100784 W CN0100784 W CN 0100784W WO 0198505 A1 WO0198505 A1 WO 0198505A1
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Prior art keywords
polypeptide
polynucleotide
ugt
binding protein
udp glycosyltransferase
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PCT/CN2001/000784
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English (en)
French (fr)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc.
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Priority to AU87511/01A priority Critical patent/AU8751101A/en
Publication of WO2001098505A1 publication Critical patent/WO2001098505A1/zh

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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)

Definitions

  • the present invention is in the field of biotechnology, and in particular, the present invention describes a novel polypeptide- UDP glycosyltransferase (UGT) and cobalamin binding protein 11, and a polynucleotide sequence encoding the polypeptide.
  • UDP glycosyltransferase UDP glycosyltransferase
  • the invention also relates to methods and uses for the preparation of such polynucleotides and polypeptides. Background technique
  • UDP glycosyltransferase is a superfamily of enzymes that catalyze the addition of a sugar-based group of UTP-sugars to small hydrophobic molecules.
  • the family includes the following members: Mammalian UDP-glucuronide Transferase (UDPGT), an enzyme that is an autosomal recessive trait that causes Crigler-Najjar syndrome (Dutton GJ, (In) Glucoronidation of drugs and other compounds, Dutton GJ, Ed. Pp 1-78, CRC Press, Boca Raton, (1980) ), a large family of membrane-bound microsomal enzymes that catalyze the conversion of glucuronic acid to various exogenous and endogenous lipophilic substrates.
  • UDP glycosyltransferase UDP glycosyltransferase
  • This enzyme plays an important role in detoxification and subsequent removal of heterologous substances like drugs and carcinogenic sources (Burchell B., Nebert DW, Nelson DR, Bock KW, lyanagi T., Jans en PL, Lancet D., Mulder GJ, Chowdhury JR, Siest G., Tephly TR, Mackenzie PI, DNA Cell Biol.
  • Mammalian 2-hydroxysphingosine-1-beta-galactosyltransferase also Known as UDP-galactosylceramide galactosyltransferase
  • UDP-galactosylceramide galactosyltransferase UDP-galactosylceramide galactosyltransferase
  • vitamin B12 cobalamin
  • TC1 transcobalamin I
  • TC2 transfer of cobalamin II
  • TC2 transports cobalamin from blood to cells
  • binds porphyrin also known as R protein or troponin
  • binds porphyrin to cobalamin and In combination with a cobalamin derivative such as a cobalt olinamide, it lifts in the prevention of absorption of cobalamin analogs To be effective.
  • glycoproteins are polypeptides of approximately 400 amino acids with many similar regions and a highly conserved signature sequence template in the central portion: [SN] -VDT- [GA] -A- [LIVM] -AxLA- [ LIVMF] -TC (Plat ica 0., Janeczko R., Quadros EV, Regec A., Roma in R., Rothenberg SP, J. Biol. Chem. 266: 7860-7863 (1991); Li N. , Seetharam S . , Lindemans J. , Alpers DH , Arwer t F. , Seetharam B. , Biochim. Biophys. Acta 1172: 21-30 (1993) ) 0
  • the polypeptide of the present invention contains the characteristic sequence templates of the above two families and has similar biological functions, and thus is considered to be a new member of the UDP glycosyltransferase (UGT) and the cobalamin binding protein family, and the polypeptide and its activation Agents, inhibitors, and antagonists can be used for the diagnosis and prevention of diseases associated with catalyzing the addition of sugar-based groups of UTP-sugars to small hydrophobic molecules and to the binding and transport of vitamin B12 (cobalamin), such as gram-sodium (Cr Igler — Naj jar ) Syndrome, etc.
  • UTP UDP glycosyltransferase
  • cobalamin cobalamin
  • UDP glycosyltransferase (UGT) and cobalamin binding protein 11 proteins play important roles in the important functions in the body as described above, and it is believed that these regulatory processes involve a large amount of proteins, it has been necessary in the art to identify more UDP glycosyltransferase (UGT) and cobalamin binding protein 11 proteins involved in these processes, in particular, identify the amino acid sequence of this protein.
  • the isolation of the novel UDP glycosyltransferase (UGT) and cobalamin binding protein 11 protein encoding genes also provides a basis for studying the role of this protein in health and disease states. This protein may form the basis for the development of disease diagnosis and/or therapeutic drugs, so it is important to isolate the DNA encoding it. Disclosure of invention
  • UGT glycosyltransferase
  • 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 comprising a polynucleotide encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11. .
  • UDP glycosyltransferase UDP glycosyltransferase
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a UDP glycosyltransferase (UGT) and a cobalamin binding protein 11.
  • UDP glycosyltransferase UDP glycosyltransferase
  • Another object of the present invention is to provide a method for producing UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, UDP glycosyltransferase (UGT) and cobalamin binding protein 11. ,
  • Another object of the present invention is to provide a polypeptide of the present invention, a UDP glycosyltransferase (UGT) and A mimetic compound, antagonist, agonist, inhibitor of cobalamin binding protein 11.
  • UDP glycosyltransferase UDP glycosyltransferase
  • Another object of the present invention is to provide a method for the diagnosis and treatment of diseases which are abnormally associated with UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • UDP glycosyltransferase UDP glycosyltransferase
  • the present invention relates to an isolated polypeptide comprising human polypeptide comprising: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant thereof, a biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the present invention also relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of or
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 91-1201 of SEQ ID NO: 1; and (b) having 1 - 2194 of SEQ ID NO: 1.
  • sequence of bits is one selected from the group consisting of: (a) a sequence having positions 91-1201 of SEQ ID NO: 1; and (b) having 1 - 2194 of SEQ ID NO: 1.
  • the invention further relates to a vector comprising a polynucleotide of the invention, in particular an expression vector; a host cell genetically engineered with the vector, comprising a transformed, transduced or transfected host cell; Host cells and methods of preparing the polypeptides of the invention for the recovery of expression products.
  • the invention also relates to an antibody that specifically binds to a polypeptide of the invention.
  • the invention also relates to a method of screening for a compound that mimics, activates, antagonizes or inhibits the activity of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 protein, which comprises utilizing a polypeptide of the invention.
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 11 protein which comprises utilizing a polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility associated with abnormal expression of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 in vitro, comprising detecting the polypeptide or its encoding polynucleoside in a biological sample Mutations in the acid sequence, or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.
  • UDP glycosyltransferase UDP glycosyltransferase
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic, activator, antagonist or inhibitor thereof, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the production of the polypeptides and/or polynucleotides of the present invention in the treatment of cancer, developmental diseases or immune diseases or other abnormalities due to abnormal expression of UDP glycosyltransferase (UGT) and cobalamin binding protein 11
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 11 The use of drugs for the disease.
  • Nucleic acid sequence refers to an oligonucleotide, nucleotide or polynucleotide and fragments or portions thereof, and may also refer to Due to group or synthetic DNA or RNA, they may be single-stranded or double-stranded, representing the sense strand or the 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 limiting the amino acid sequence to the complete natural amino acid associated with the protein molecule. .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acid or nucleotide changes or a polynucleotide sequence encoding the same. Such alterations may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants may have a "conservative" change in which the substituted amino acid has structural or chemical properties similar to those of the original amino acid, such as replacement of isoleucine with leucine. Variants may also have non-conservative changes, such as the replacement of glycine with tryptophan.
  • “Deletion” refers to the absence of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that an alteration in an amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides as compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides by a different amino acid or nucleotide.
  • Bio activity refers to a protein that has the structural, regulatory or biochemical functions of a natural molecule.
  • immunological activity refers to the ability of a natural, recombinant or synthetic protein and its fragments to induce a particular immune response and bind to a specific antibody in a suitable animal or cell.
  • Agonist refers to a molecule that, when combined with UDP glycosyltransferase (UGT) and cobalamin binding protein 11, causes changes in the protein to modulate the activity of the protein.
  • Agonists can include proteins, nucleic acids, carbohydrates, or any other molecule that binds to UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • Antagonist refers to a cloning or regulation of UDP glycosyltransferase (UGT) and cobalamin binding when combined with UDP glycosyltransferase (UGT) and cobalamin binding protein 11
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that binds to UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • Modulation refers to changes in the function of UDP glycosyltransferase (UGT) and cobalamin binding protein 11, including increased or decreased protein activity, altered binding properties, and UDP glycosyltransferase (UGT) and cobalamin Any other biological, functional or immunological property of the binding protein 11 .
  • Substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances naturally associated therewith. Those skilled in the art will be able to purify UDP glycosyltransferase (UGT) using standard protein purification techniques and Cobalamin binding protein 11. Substantially pure UDP glycosyltransferase (UGT) and cobalamin binding protein 11 A single primary band can be produced on a propolyacrylamide gel. The purity of the UDP glycosyltransferase (UGT) and the cobalamin binding protein 11 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of a polynucleotide by base pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single chain molecules can be partial or total.
  • 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 may be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits the hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of such hybridization can be detected by hybridization (Southern blot or Northern blot, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit binding of a completely homologous sequence to a reduced degree of stringency of the target sequence. This does not mean that the condition of reduced stringency allows for non-specific binding, since the reduced degree of stringency requires 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, 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 (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges the sets of sequences into clusters by examining the distance between all pairs. The clusters are then distributed in pairs or in 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 matched between sequence A and sequence B X 100
  • nucleic acid sequences can also be determined by the Clus ter method or by methods well known in the art such as Jotun Hein ( Hein J., (1990) Methods in emzumology 183: 625-645) 0
  • Similarity refers to the degree of identical or conservative substitution of amino acid residues at corresponding positions when aligned between amino acid sequences.
  • Amino acids 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 uncharged head groups are 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 DM or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the "sense strand”.
  • Derivative means a chemical modification of HFP or a nucleic acid encoding the same. This chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the major biological characteristics of the natural molecule.
  • Antibody refers to intact antibody molecules and fragments thereof, such as Fa, ⁇ and ? It specifically binds to the determinants of UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • UDP glycosyltransferase UDP glycosyltransferase
  • Humanized antibody means an antibody in which the amino acid sequence of the non-antigen-binding region is replaced to become more similar to the human antibody, but retaining the original binding activity.
  • the term "separated” refers to the removal of a substance from its original environment (for example, if it is naturally occurring, its natural environment).
  • a naturally occurring polynucleotide or polypeptide that is present in a living organism is not isolated, but the same polynucleotide or polypeptide is separated from some or all of the material with which it coexists in the natural system.
  • Such polynucleotides may be part of a vector, and it is also possible that such polynucleotides or polypeptides are part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • isolated UDP glycosyltransferase (UGT) and cobalamin binding protein 11 means that UDP glycosyltransferase (UGT) and cobalamin binding protein 11 are substantially free of other proteins associated with nature. , lipids, sugars or other substances.
  • UDP glycosyltransferase (UGT) and cobalamin binding protein 11 are substantially free of other proteins associated with nature. , lipids, sugars or other substances.
  • One skilled in the art can purify UDP glycosyltransferase (UGT) and cobalamin binding protein 11 using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel. The purity of the UDP glycosyltransferase (UGT) and the cobalamin-binding protein 11 can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-UDP glycosyltransferase (UGT) and cobalamin binding protein 11, which consist essentially of the amino acid sequence set forth in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptides of the invention may be naturally purified products, either chemically synthesized or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells).
  • the polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of UDP glycosyltransferase (UGT) and cobalamin binding protein 11. As used herein, the terms "fragment,””derivative,” and “analog” mean substantially maintaining the same biological function or activity of the UDP glycosyltransferase (UGT) of the invention and cobalamin binding protein 11. Peptide.
  • a fragment, derivative or analog of a polypeptide of the invention may be: (I) wherein one or more amino acid residues are substituted by a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue) and substituted The amino acid may or may not be encoded by the genetic code; or (II) wherein one of the one or more amino acid residues is substituted by another group to contain a substituent; or (III) a polypeptide sequence in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or UV), wherein the additional amino acid sequence is fused to the mature polypeptide (eg, A leader sequence or a secretory sequence or a sequence or proprotein sequence used to purify the polypeptide)
  • a leader sequence or a secretory sequence or a sequence or proprotein sequence used to purify the polypeptide 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) consisting essentially of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found in a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 2194 bases in length and an open reading frame (911-1201) encoding 87 amino acids.
  • the polypeptide has a characteristic sequence of UDP glycosyltransferase (UGT) and cobalamin binding protein, and it can be inferred that the UDP glycosyltransferase (UGT) and cobalamin binding protein 11 have UDP glycosyltransferase (UGT) and The structure and function represented by cobalamin binding proteins.
  • the polynucleotide of the present invention may be in the form of DNA or in the form of RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DM can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a protein or polypeptide having SEQ ID NO: 2 but differs from the coding region sequence set forth in SEQ ID NO: 1.
  • Polynucleotides encoding the mature polypeptide of SEQ ID NO: 2 include: 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 optionally the additional coding sequence) and Coding sequence.
  • polynucleotide encoding a polypeptide is meant to include a polynucleotide encoding 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 fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deficiency Loss variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the invention also relates to polynucleotides that hybridize to the sequences described above (having at least between the two sequences
  • the invention particularly relates to polynucleotides which hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0. 2xSSC, 0. 1°/»SDS, 6(TC; or ( 2) Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum/0. l%Ficol l, 42 °C, etc.; or (3) only in two sequences Hybridization occurs when the identity is at least 95% or more, more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function as the mature polypeptide represented by SEQ ID NO: 2. Active.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” is 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. More than the glycosidic acid.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques (e.g., PCR) to identify and/or isolate polynucleotides encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • UDP glycosyltransferase UDP glycosyltransferase
  • polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11 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 a probe to a genomic or cDNA library to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides having common structural features. Acid fragment.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating a double-stranded DNA sequence from genomic DNA; 2) chemically synthesizing a DM sequence to obtain a double-stranded DNA of the polypeptide.
  • the isolation of genomic DNA is the least common.
  • Direct chemical synthesis of DNA sequences is a frequently chosen method.
  • a more frequently chosen method is the separation of the cDNA sequences.
  • a standard method for isolating a cDNA of interest is to isolate mRNA from a donor cell that highly expresses the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • cDNA libraries are also a common method (Sambrook, et al., Mo l ecu lar C l oning, A Labora tory Manua l, Co ld Spr ing Harbor Labora tory. New York, 1989).
  • Commercially available cDNA libraries such as different cDNA libraries from Cl ontech, are also available. When a polymerase reaction technique is used in combination, even very few expression products can be cloned.
  • the genes of the present invention can be screened from these cDNA libraries by a conventional method. These methods include (but are not limited to): (1) 0 -0 or 0 -1 ⁇ human cross; (2) appearance or loss of marker gene function; (3) determination The level of transcripts of UDP glycosyltransferase (UGT) and cobalamin binding protein 11; (4) detection of protein products of gene expression by immunological techniques or determination of biological activity.
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 11 cobalamin binding protein 11
  • the above method can be used alone or in combination of various methods.
  • the probe for hybridization is homologous to any part of the polynucleotide of the present invention, and has a length of 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 DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the gene itself or fragment of the present invention can of course be used as a probe.
  • the label of the DM probe may be a radioisotope, a fluorescein or an enzyme such as an alkaline phosphatase or the like.
  • the protein product of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and the like.
  • a method of expanding DNA/RNA using a PCR technique (Sa ik i , et al . Sc ience 1985; 230: 1350 - 1 354) is preferably used to obtain the gene of the present invention.
  • RACE method RACE-cDNA end rapid amplification method
  • primers for PCR can be appropriately selected according to the polynucleotide sequence information of the present invention disclosed herein. Ground selection, and can be synthesized by conventional methods.
  • the amplified DNA/RNA fragment can be isolated and purified by a conventional method such as by gel electrophoresis.
  • polynucleotide sequence of the present invention obtained as described above, or a polynucleotide sequence of various DNA fragments or the like can be determined by a conventional method such as the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequence determinations can also be carried out using commercial sequencing kits and the like. In order to obtain a full-length cDNA sequence, the sequencing needs to be repeated. It is sometimes necessary to determine the cDNA sequences of multiple clones in order to splicing into full-length cDNA sequences.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using UDP glycosyltransferase (UGT) and cobalamin binding protein 11 coding sequences, and Recombinant techniques produce methods of the polypeptides of the invention.
  • UDP glycosyltransferase UDP glycosyltransferase
  • a polynucleotide sequence encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • the term "vector” refers to bacterial plasmids, phage, 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 expressed in bacteria (Rosenberg, et al.
  • pMSXND expression vectors expressed in mammalian cells Lee and Na thans, J Bio Chem. 263: 3521, 1988
  • baculovirus-derived vectors expressed in insect cells Any plasmid and vector can be used to construct a recombinant expression vector as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational regulatory element.
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 11 suitable transcriptional/translational regulatory elements.
  • These methods include in vitro recombinant DM techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or tr rp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of an enhancer sequence into a vector will enhance its transcription in higher eukaryotic cells.
  • An enhancer is a cis-acting factor for DNA expression, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Illustrative examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation site, and an adenovirus enhancer.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • vector/transcriptional regulatory elements e.g., promoters, enhancers, etc.
  • selectable marker genes e.g., promoters, enhancers, etc.
  • a polynucleotide encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide or recombinant Vector engineered host cells of the 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells such as flies S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence of the invention or a recombinant vector containing the DNA sequence can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM can be harvested after the exponential growth phase, and treated with the CaCl 2 method. It is well known in the art.
  • An alternative is to use MgC l 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant UDP glycosyltransferase (UGT) and cobalamin binding protein 11 by conventional recombinant DNA techniques (Sc ence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide may be coated in a cell, or expressed on a cell membrane, or secreted outside the cell.
  • 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 method), centrifugation, osmotic sterilizing, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ions 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 method), centrifugation, osmotic sterilizing, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ions Exchange chromatography, high performance liquid chromat
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 11 amino acid sequence of the UDP glycosyltransferase (UGT) and cobalamin binding protein 11 of the present invention in a total of 43 amino acids of 10-52 and UDP glycosyltransferase (UGT) and cobalamin binding protein.
  • Source comparison chart The upper sequence is UDP glycosyltransferase (UGT) and cobalamin binding protein 11
  • the lower sequence is the UDP glycosyltransferase (UGT) and cobalamin binding protein domain.
  • the same amino acid is represented by a single-character amino acid between the two sequences, and the similar amino acid is represented by "+".
  • Figure 2 is a polyacrylamide gel electrophoresis pattern (SDS-PAGE) of isolated UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • UDP glycosyltransferase UDP glycosyltransferase
  • UDT UDP glycosyltransferase
  • cobalamin binding protein 11.
  • l lkDa is the molecular weight of the protein.
  • the arrow indicates the separated protein band.
  • RNA Human fetal brain total RNA was extracted by one-step method using guanidinium isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA I solation Ki t (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. The cDNA fragment was inserted into the multiple cloning site of the pBSK (+) vector (Clontech) using the Smart cDNA Cloning Kit (purchased from Clontech) to transform DH5 ⁇ , and the bacteria formed a cDM library.
  • sequences of the 5' and 3' ends of all clones were determined using Dye terminate cycle reaction sequencing ki t (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDM sequence of one of the clones 1235b07 was a new DNA. The inserted cDNA fragment contained in the clone was subjected to two-way measurement by synthesizing a series of primers.
  • the sequence of the UDP glycosyltransferase (UGT) and the cobalamin binding protein 11 of the present invention and the encoded protein sequence thereof are used in the rof i lee search program of the GCG [Al tschul, SF Et al. J. Mol. Biol. 1990; 215: 403-10], domain analysis in databases such as pros i te.
  • the UDP glycosyltransferase (UGT) and cobalamin binding protein 11 of the present invention are homologous to the domain UDP glycosyltransferase (UGT) and cobalamin binding protein in 10 - 52 , and the homologous results are shown in Fig. 1 0 ⁇ The homology rate was 0. 12, the score was 5. 32; the threshold was 5. 30.
  • Example 3 Cloning of a gene encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11 by RT-PCR
  • PCR amplification was performed using the following primers:
  • Primerl 5-CATCCTGAGAACTGAAATTGATCGC-3' (SEQ ID NO: 3)
  • Primer2 5-ATAAAATTTTTGAATTTATGTTCAA-3' (SEQ ID NO: 4)
  • Primerl is the forward sequence starting at the lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3, end inverted sequence of SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 mmol/L C1, 10 mmol/L Tris-Cl, (pH 8.5), 1.5 ⁇ ol/L MgCl 2 , 200 ⁇ mol/L dNTP, lOpmol primer in a reaction volume of 50 ⁇ l , 1 U of Taq DNA polymerase (product of C 1 on t ech).
  • the PE9600 DM thermal cycler (Per ki nE lmer) was reacted for 25 cycles under the following conditions: 94. C 30 sec; 55 ° C 30 sec; 72. C 2min. At RT-PCR, both ⁇ -act in was used as a positive control and the template blank was used as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated into a pCR vector (product of Invitrogen) using a TA cloning kit.
  • the DM sequence analysis revealed that the DNA sequence of the PCR product was identical to the l-2194 bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of HDP glycosyltransferase ((JGT) and cobalamin binding protein 11 gene expression:
  • RNA precipitate was washed with 70% ethanol, dried and dissolved in water.
  • Electrophoresis was carried out with 20 ⁇ g of RNA on a 1.2% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0) - 5 mM sodium acetate - 1 mM EDTA - 2.2 M formaldehyde. It is then transferred to a nitrocellulose membrane.
  • a 32 ⁇ -labeled DNA probe was prepared by random primer method using a- 32 P dATP. The DNA probe used was the PCR amplified UDP glycosyltransferase (UGT) and the cobalamin binding protein 11 coding region sequence (911 bp to 1201 bp) shown in Fig. 1.
  • UDP glycosyltransferase UDP glycosyltransferase
  • the 32P-labeled probe (about 2 x 10 6 cpm/ml) was hybridized with the transferred nitrocellulose membrane in a solution at 42 ° C overnight, the solution containing 50% formamide - 25 mM KH 2 P0 4 (pH 7.4 ) - 5 x SSC-5 x Denhardt, s solution and 200 g/ml salmon sperm DNA. After hybridization, the filters were washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, use the Phosphor Imager for analysis and determination.
  • Example 5 In vitro expression, isolation and purification of recombinant UDP glycosyltransferase (UGT) and cobalamin binding protein 11
  • the PCR reaction was carried out using the pBS-l 2 35b07 plasmid containing the full-length gene of interest as a template.
  • the PCR reaction conditions were: total volume 50 ⁇ 1 containing pBS-1235b07 plasmid 10pg, primer 1:11116:1:-3 and ? :!:10161-4 points!] is 10 11101, Advantage polymerase Mix (Clontech products) 1 ⁇ 1. Cycle parameters: 94 ° C 20 s, 60 ° C 30 s, 68 ° C 2 min, a total of 25 cycles.
  • the amplified product and plasmid pET-28(+) were digested with Ndel and BaraHI, respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed with the calcium chloride method, Escherichia coli DH5a, and cultured overnight on an LB plate containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), and positive clones were screened by colony PCR and sequenced.
  • the positive clone ( P ET-1235b07) with the correct sequence was selected and transformed into Escherichia coli BL21(DE3)plySs (product of Novagen) by the calcium chloride method.
  • host strain BL21 (pET-1235b07) was at 37.
  • C was cultured to logarithmic growth phase, IPTG was added to a final concentration of 1 IMO1/L, and incubation was continued for 5 hours.
  • the cells were collected by centrifugation, disrupted by ultrasonication, and the supernatant was collected by centrifugation, and chromatographed with an affinity chromatography column His.
  • Bind Quick Cartridge product of Novagen which can bind to 6 histidine (6His-Tag).
  • the purified target protein UDP glycosyltransferase (UGT) and cobalamin binding protein 11 were purified.
  • a single band was obtained at llkDa by SDS-PAGE electrophoresis (Fig. 2).
  • the band was transferred to a PVDF membrane and subjected to N-terminal amino acid sequence analysis by Edams hydrolysis.
  • the N-terminal 15 amino acids were identical to the N-terminal 15 amino acid residues shown by SEQ ID NO: 2.
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 11 The polypeptides specific for UDP glycosyltransferase (UGT) and cobalamin binding protein 11 were synthesized using a peptide synthesizer (product of PB):
  • the polypeptide is complexed with hemocyanin and bovine serum albumin, respectively. See, for example, Avrameas, et al. Iraraunochemistry, 1969; 6:43. Rabbits were immunized with 1 ⁇ 2 g of the above hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex was intensified with incomplete Freund's adjuvant.
  • the titer of antibodies in rabbit serum was determined by ELISA using a titer plate coated with a 15 g/ml bovine serum albumin polypeptide complex.
  • Total IgGo was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the polypeptide was bound to a cyanogen bromide-activated Se P harose 4B column and the anti-polypeptide antibody was isolated from total IgG by affinity chromatography.
  • Immunoprecipitation demonstrated that the purified antibody specifically binds to UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • UDP glycosyltransferase UDP glycosyltransferase
  • a suitable oligonucleotide fragment from a polynucleotide of the present invention for use as a hybridization probe has various uses, such as hybridization of a genomic or cDNA library of normal tissues or pathological tissues of different origins with the probe. Whether it contains the polynucleotide sequence of the present invention and the homologous polynucleotide sequence, and further, the probe can be used to detect the polynucleotide sequence of the present invention or a homologous polynucleotide sequence thereof in normal tissues or pathologies. Whether the expression in the tissue cells is abnormal.
  • the purpose of this example is to select a suitable oligonucleotide fragment from the polynucleotide of the present invention SEQ ID NO: 1 for use as a hybridization probe, and to identify whether some tissues contain the multinuclear of the present invention by a membrane hybridization method.
  • the membrane hybridization method includes dot blotting, Southern blotting, Northern blotting, and copying methods, etc., which all hybridize using substantially the same procedure after immobilizing the sample of the polynucleotide to be tested on the filter.
  • the filter to which the sample is immobilized 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 synthesized polymer.
  • the prehybridization 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 probe is removed by a series of membrane washing steps. This example utilizes higher strength wash conditions (e.g., lower salt concentration and higher temperature) to reduce the hybridization background and retain only a highly specific signal.
  • the probes selected in this embodiment include two types: the first type of probe is an oligonucleotide fragment which is identical or complementary to the polynucleotide of the present invention SEQ ID NO: 1; the second type of probe is a part of the present invention.
  • the oligonucleotide of SEQ ID NO: 1 is the same or a complementary oligonucleotide fragment.
  • the sample was fixed on the filter by dot blotting. Under the condition of high-strength washing, the first type of probe and the sample had the strongest hybridization specificity and were retained.
  • the probe size preferably ranges from 18 to 50 nucleotides
  • GC content is 30% - 70%, more than non-specific hybridization increases
  • the above conditions can be used as a primary probe, and then further computer sequence analysis, including the primary probe and its source sequence region (ie SEQ ID NO: 1) and other known genomic sequences and their complementary The region performs homology comparison. If the homology with the non-target molecule region is greater than 85% or more than 15 consecutive bases are identical, the primary probe should generally not be used;
  • Probe 1 belonging to the first class of probes, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • probe 1 belonging to the second type of probe, corresponding to the substitution mutation sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • cold homogenization buffer (0.25fflol/L sucrose; 25 Saol/L Tris-HCl, pH 7.5; 25 sec ol/LnaCl; 25 leg ol/L MgCl 2 .
  • Steps 8-13 below are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two probes are required for each probe, so as to be in the subsequent experimental steps.
  • the membrane was washed with high strength conditions and strength conditions, respectively.
  • CT DM calf thymus DNA
  • 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.
  • polypeptide of the present invention and antagonists, agonists and inhibitors of the polypeptide can be directly used for the treatment of diseases, for example, malignant tumors, adrenal deficiencies, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • diseases for example, malignant tumors, adrenal deficiencies, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • UDP glycosyltransferase family catalyze the addition of a sugar-based group of UTP-sugars to small hydrophobic molecules
  • UDP-glucuronyltransferase UDP glycosyltransferase
  • UDP glycosyltransferase is a type of membrane-bound microsomes.
  • mammalian 2-hydroxysphingosine-1 - beta-galactosyltransferase is a key step in the biosynthesis of galactocerebroside, which is the central nervous system and the peripheral nervous system.
  • galactocerebroside which is the central nervous system and the peripheral nervous system.
  • UDP glycosyltransferases have a highly conserved signature sequence template.
  • a conserved sequence specific for the UDP glycosyltransferase family is required for the formation of its active domain.
  • the polypeptide of the present invention also has a conserved sequence specific for UDP glycosyltransferase.
  • abnormal expression of a specific UDP glycosyltransferase domain will result in abnormal function of the polypeptide containing the domain of the present invention, thereby causing the conversion of glucuronic acid into various exogenous and endogenous pro
  • the process of the lipid substrate is dysfunctional, and the process of biosynthesis of galactocerebroside is abnormal.
  • the polypeptide of the present invention also has a conserved sequence specific for cobalamin binding protein. Cobalamin binding proteins are extremely important for the binding, transport, and metabolism of cobalamin. Therefore, abnormal expression of the polypeptide of the present invention may also cause abnormal function of cobalamin binding, affect cobalamin metabolism, and cause related diseases.
  • UDP glycosyltransferase (UGT) and cobalamin binding protein 11 of the present invention will cause various diseases, especially the Cr-igler-Naj jar syndrome, a nervous system developmental disease. , cobalamin deficiency, cobalamin poisoning syndrome, certain tumors, inflammation, immune diseases, including but not limited to:
  • neural tube insufficiency such as spina bifida, no brain malformation, brain (meningeal) bulging, cranial brain fissure, neural tube cyst
  • brain developmental malformations such as cerebral malformation, full forebrain, hydrocephalus Neuronal migration disorders such as abnormal cerebral gyrus
  • other malformations such as aqueduct malformation, cerebellar hypoplasia, Down syndrome, spinal deformity, congenital hydrocephalus, congenital cranial dysplasia syndrome
  • Some tumors astrocytoma, ependymoma, glioma, neurofibroma, gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid neoplasms, uterine fibroids, colon cancer, Melanoma, bladder cancer, endometrial cancer, colon cancer, thymic tumor, tracheal neoplasm, fibroid, fibrosarcoma
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammation
  • Immune diseases systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barré syndrome, general susceptibility to immunity Defective disease, primary B lymphocyte immunodeficiency disease, acquired immunodeficiency syndrome
  • UDP glycosyltransferase (UGT) and cobalamin binding protein 11 of the present invention may also produce certain hereditary, hematological diseases and the like.
  • the polypeptide of the present invention and antagonists, agonists and inhibitors thereof can be directly used for the treatment of diseases, for example, can treat various diseases, especially gram-naj jar syndrome, nervous system development. Disease, cobalamin deficiency, cobalamin poisoning syndrome, certain tumors, inflammation, immune diseases, certain hereditary, blood diseases, etc.
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or repress (antagonist) UDP glycosyltransferase (UGT) and cobalamin binding protein 1 1 .
  • Agonists increase UDP glycosyltransferase (UGT) and cobalamin binding protein 11 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders associated with excessive cell proliferation such as various cancers.
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 1 labeled UDP glycosyltransferase (UGT) and cobalamin in the presence of a drug. Protein 1 1 was cultured together. The ability of the drug to increase or repress this interaction is then determined.
  • Antagonists of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 include selected antibodies, compounds, receptor deletions, and the like. Antagonists of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can bind to and eliminate the function of UDP glycosyltransferase (UGT) and cobalamin binding protein 1 or inhibit the production of the polypeptide. Or binding to the active site of the polypeptide renders the polypeptide incapable of functioning biologically.
  • UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can be added to the bioassay to determine the compound versus UDP glycosyltransferase (UGT) and cobalamin binding protein. The effect of the interaction between 1 1 and its receptor to determine if the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • a polypeptide molecule capable of binding to UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, UDP glycosyltransferase (UGT) and cobalamin binding protein 11 molecules should generally be labeled.
  • the present invention provides a method of producing an antibody using the polypeptide, and fragments, derivatives, analogs thereof or cells thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against UDP glycosyltransferase (UGT) and cobalamin binding protein 1 1 antigenic determinants.
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 1 1 antigenic determinants 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 obtained by direct injection of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 into immunized animals (such as rabbits, mice, rats, etc.), and various adjuvants can be used to enhance the immune response. , including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to UDP glycosyltransferase (UGT) and cobalamin binding protein 11 include, but are not limited to, hybridoma technology (Kohler and Mi s te in. Nature, 1975, 256: 495-497), trioma Technology, human sputum-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morr i son et al, PNAS, 1985, 81: 6851) 0 and existing techniques for producing single-chain antibodies (US Pa) t No. 4946778) can also be used to produce single-chain antibodies against UDP glycosyltransferase ( UGT ) and cobalamin binding protein 11
  • Antibodies against UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can be used in immunohistochemistry to detect UDP glycosyltransferase (UGT) and cobalamin binding protein 11 in biopsy specimens.
  • Monoclonal antibodies that bind to UDP glycosyltransferase (UGT) and cobalamin binding protein 11 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 for the localization of tumor cells and for the determination of metastasis.
  • Antibodies can also be used to design immunotoxins directed to a particular site in the body.
  • UDP glycosyltransferase UDP glycosyltransferase
  • UGT UDP glycosyltransferase
  • Cobalamin binding Protein 11 High affinity monoclonal antibodies can be covalently bound to bacterial or phytotoxins such as diphtheria toxin, ricin, and erythromycin.
  • a common method is to use a sulfhydryl-based cross-linking agent such as SPDP to attack the amino group of the antibody, and to bind the toxin to the antibody through exchange of disulfide bonds.
  • the hybrid antibody can be used to kill UDP glycosyltransferase (UGT) and Cobalamin-binding protein 11 positive cells.
  • the antibody of the present invention can be used for the treatment or prevention of diseases associated with UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • Administration of an appropriate dose of the antibody stimulates or blocks the production or activity of UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • the invention also relates to a diagnostic assay for the quantitative and localization of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 levels.
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 11 levels UDP glycosyltransferase and cobalamin binding protein 11 levels.
  • assays are well known in the art and include FISH assays and radioimmunoassays.
  • the levels of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 detected in the assay can be used to explain the importance of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 in various diseases.
  • polypeptide of the present invention can also be used as a peptide profile analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably to mass spectrometry.
  • UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat UDP glycosyltransferase (UGT) and cobalamin knots Cell proliferation, developmental or metabolic abnormalities due to no expression or abnormal/inactive expression of the protein 11.
  • Recombinant gene therapy vectors eg, viral vectors
  • UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can be designed to express variant UDP glycosyltransferase (UGT) and cobalamin binding protein 11 to inhibit endogenous UDP glycosyltransferase (UGT) and cobalamin Protein binding protein 11 activity.
  • a variant of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 may be a shortened, UDP-transferase (UGT) and cobalamin-binding protein 11 lacking a signaling domain, although It binds to downstream substrates but lacks signaling activity.
  • a recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • Viral-derived expression vectors such as retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, parvoviruses, etc.
  • UDP glycosyltransferase UDP glycosyltransferase
  • cobalamin binding protein 11 A method for constructing a recombinant viral vector carrying a polynucleotide encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can be found in the literature (Sambrook, et al.).
  • a polynucleotide encoding a UDP glycosyltransferase (UGT) and a cobalamin binding protein 11 can be packaged into a liposome and transferred into a cell.
  • the method of introducing a polynucleotide into a tissue or a cell comprises: injecting a polynucleotide directly into a tissue in vivo; or introducing a polynucleotide into a cell by a vector (such as a virus, a phage or a plasmid, etc.) in vitro, and then transplanting the cell. Go to the body and so on.
  • a vector such as a virus, a phage or a plasmid, etc.
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit UDP glycosyltransferase (UGT) and cobalamin binding protein 11 mRNA are also within the scope of the invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes a specific RNA. The mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for nucleic acid endo-cutting.
  • Antisense RNA and DNA and ribozymes can be obtained by any existing RNA or DNA synthesis technique, such as solid phase phosphoric acid amide chemical synthesis, which has been widely used.
  • Antisense RNA molecules can be obtained in vitro or in vivo by DM sequences encoding the RNA. This DNA sequence has been integrated downstream of the vector's MA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified by various methods, such as increasing the length of the sequences on both sides, and the linkage between the ribonucleosides is a phosphate thioester bond or a peptide bond instead of a phosphodiester bond.
  • a polynucleotide encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can be used for the diagnosis of diseases associated with UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • Polynucleotides encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can be used to detect the expression of UDP glycosyltransferase (UGT) and cobalamin binding protein 11 or UDP glycosylation in disease states Abnormal expression of transferase (UGT) and cobalamin binding protein 11.
  • a DNA sequence encoding UDP glycosyltransferase (UGT) and cobalamin binding protein 11 can be used to hybridize biopsy specimens to determine the expression of UDP glycosyltransferase (UGT) and cobalamin binding protein 11.
  • Hybridization techniques include Southern blotting, Northern blotting Trace method, in situ hybridization, etc. These technical methods are all open and mature technologies, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray
  • UDP glycosyltransferase (UGT) and cobalamin binding can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using UDP glycosyltransferase (UGT) and cobalamin binding protein 11 specific primers. Transcription product of protein 11.
  • UDP glycosyltransferase (UGT) and cobalamin binding protein 11 genes can also be used to diagnose UDP glycosyltransferase (UGT) and cobalamin binding protein 11 related diseases.
  • Mutant forms of UDP glycosyltransferase ( UGT ) and cobalamin binding protein 11 include point mutations, translocations, deletions compared to normal wild-type UDP glycosyltransferase ( UGT ) and cobalamin binding protein 11 DNA sequences , reorganization and any other anomalies, etc. Mutations can be detected using existing techniques such as Southern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of the protein, so Northern blotting, Western blotting can be used to indirectly determine whether the gene has mutations.
  • sequences of the invention are also valuable for chromosome identification.
  • the sequence will specifically target and hybridize to a particular human chromosome.
  • specific sites for each gene on the chromosome need to be identified.
  • chromosomal markers based on actual sequence data (repetitive polymorphisms) that can be used to label chromosomal locations.
  • an important first step is to localize these DM sequences to the chromosome.
  • sequences can be mapped to chromosomes based on cDNA preparation of PCR primers (preferably 15-35 bp). These primers were then used for PCR screening of somatic cell hybrid cells containing individual human chromosomes. Only those hybrid cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic cell hybrid cells is a quick way to locate DNA to specific chromosomes.
  • sub-localization can be achieved by a similar method using a set of fragments from a particular chromosome or a large number of genomic clones.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct a chromosome-specific cDNA library.
  • Fluorescence in situ hybridization of cDNA clones with metaphase chromosomes allows for precise chromosomal localization in one step.
  • FISH Fluorescence 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, for example, in V. Mckus i ck, Mendelian Inher i tance in Man (available with Johns Hopkins Univers i ty Wel ch Medica l L i brary is obtained online). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • cDNA or genomic sequence it is necessary to determine the difference in cDNA or genomic sequence between the diseased and unaffected individuals. If a mutation is observed in some or all of the affected individuals, and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing both afflicted and unaffected individuals usually involves first looking for structural changes in the chromosome, such as missing or translocations that are detectable at the chromosomal level or detectable using cDNA sequence-based PCR. Based on the current resolution of physical mapping and gene mapping techniques, cDNA that is pinpointed to the chromosomal region associated with the disease can be one of 50 to 500 potentially disease-causing genes (assuming 1 megabase mapping) Ability and corresponds to one gene per 20 kb).
  • polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors thereof of the invention may be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the compositions comprise a safe and effective amount of the polypeptide or antagonist and carriers and excipients which do not interfere with the effect of the drug. These compositions can be used as a medicament for the treatment of diseases.
  • the invention also provides a kit or kit comprising one or more containers containing one or more of the pharmaceutical composition ingredients of the invention.
  • a kit or kit comprising one or more containers containing one or more of the pharmaceutical composition ingredients of the invention.
  • these containers there may be an indicative cue given by a government regulatory agency that manufactures, uses, or sells the drug or biological product that reflects the administration of the human body by the government agency that manufactures, uses, or sells it.
  • the polypeptides of the invention may be used in combination with other therapeutic compounds.
  • the pharmaceutical compositions may be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes of administration.
  • UDP glycosyltransferase (UGT) and cobalamin binding protein 11 are administered in an amount effective to treat and/or prevent a particular indication.
  • the amount and dosage range of UDP glycosyltransferase (UGT) and cobalamin binding protein 1 1 administered to a patient will depend on a number of factors, such as the mode of administration, the health conditions of the subject to be treated, and the judgment of the diagnosing physician.

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Description

一种新的多肽一一 UDP糖基转移酶（UGT)及钴胺素结合蛋白 11
和编码这种多肽的多核苷酸 技术领域
本发明属于生物技术领域， 具体地说， 本发明描述了一种新的多肽一一 UDP 糖 基转移酶 （UGT)及钴胺素结合蛋白 11, 以及编码此多肽的多核苷酸序列。 本发明 还涉及此多核苷酸和多肽的制备方法和应用。 背景技术
UDP糖基转移酶 （UGT)是一种酶类的超家族， 这种酶催化 UTP-糖的糖基集团加 成至小的疏水分子， 该家族包括以下成员： 哺乳动物 UDP-葡糖苷酸基转移酶 (UDPGT), 此酶缺乏为一种常染色体隐性性状， 可致克-钠 （Crigler- Najjar ) 综 合征 ( Dutton G. J. , (In) Glucoronidation of drugs and other compounds, Dutton G. J.， Ed. , pp 1-78, CRC Press, Boca Raton, (1980) ), 这是一类膜结合微粒体 酶类大家族， 它催化葡糖醛酸转化成各类外源及内源性亲脂底物， 这种酶类在解 毒以及而后的去处类似药物及致癌源的异源物质中起重要作用（ Burchell B. , Nebert D. W. , Nelson D. R. , Bock K. W. , lyanagi T. , Jans en P. L. , Lancet D. , Mulder G. J. , Chowdhury J. R. , Siest G. , Tephly T. R. , Mackenzie P. I. , DNA Cell Biol. 10: 487-494 (1991)) ; 哺乳动物 2-羟酰鞘氨醇 -1- beta-半乳糖基转移酶（也被称为 UDP-半乳糖神经酰胺半乳糖基转移酶）， 该酶催化转移半乳糖至神经酰胺,这是生物 合成半乳糖脑苷脂中一步关键的酶促步骤， 半乳糖脑苷脂是中枢神经系统以及外 周神经系统的髓磷脂膜的丰富的鞘脂（Scl lte S. , Stoffel W. , Proc. Natl. Acad. Sci. U.S.A. 90: 10265-10269 (1993)) 0 该家族成员都有一个位于 C-端部分， 约为 50 个氨基酸残基的保守域： [FW] -X (2) -Q-x (2) - [LIVMYA]一 [LIMV] - x (4, 6) - [LVGAC]― [LVFYA] - [LIVMF] - [STAGCM] - [HNQ] - [STAGC] - G-x (2) - [STAG] -x (3) - [STAGL] - [LIVMFA] -X (4) - [PQR]一 [LIVMT]—x (3) - [PA] -x (3) - [DES] - [QEHN]。
在真核生物中，有一些蛋白参与结合并转运维生素 B12(钴胺素）（ Seetharam B. , Alpers D. H. , Annu. Rev. Nutr. 2: 343-369 (1982) )： 内因子 （IF), 其功能为通过 特异性受体-介导的内吞作用促进回肠中钴胺素的吸收； 转钴胺素 I (TC1 ) (也被称 为 R -粘合剂蛋白）， TC1转运钴胺素自血液到细胞中； 转钴胺素 II (TC2 ), TC2转运 钴胺素自血液到细胞中； 结合咕啉（又称 R蛋白或嗜钴素）， 结合咕啉与钴胺素结 合， 并与钴胺素衍生物例如钴啉醇酰胺结合， 它在防止吸收钴胺素类似物中起重 要作用。 这些糖蛋白是约含 400个氨基酸的多肽， 它们具有许多相似的区域， 在中 心部分有一个高度保守的特征序列模板： [SN] -V-D-T- [GA] -A- [LIVM] -A-x-L-A- [LIVMF] -T-C (Plat ica 0. , Janeczko R. , Quadros E. V. , Regec A. , Roma in R. , Rothenberg S. P. , J. Biol. Chem. 266: 7860-7863 (1991) ; Li N. , Seetharam S. , Lindemans J. , Alpers D. H. , Arwer t F. , Seetharam B. , Biochim. Biophys. Acta 1172: 21-30 (1993) ) 0
本发明的多肽中含有上述两个家族的特征序列模板， 且具有相似的生物功能， 因而被认为是 UDP糖基转移酶 （UGT )及钴胺素结合蛋白家族的新成员， 该多肽及其 激动剂， 抑制剂， 拮抗剂可用于诊断兼防治与催化 UTP-糖的糖基集团加成至小的疏 水分子以及参与结合并转运维生素 B12 (钴胺素） 有关的疾病， 例如克-钠 ( Cr igler— Naj jar ) 综合征等。
由于如上所述 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11蛋白在机体内重要 功能中起重要作用， 而且相信这些调节过程中涉及大量的蛋白， 因而本领域中一 直需要鉴定更多参与这些过程的 UDP糖基转移酶（UGT )及钴胺素结合蛋白 11蛋白， 特别是鉴定这种蛋白的氨基酸序列。 新 UDP 糖基转移酶 （UGT )及钴胺素结合蛋白 11 蛋白编码基因的分离也为研究确定该蛋白在健康和疾病状态下的作用提供了基 础。 这种蛋白可能构成开发疾病诊断和 /或治疗药的基础， 因此分离其编码 DNA 是 非常重要的。 发明的公开
本发明的一个目的是提供分离的新的多肽一一 UDP 糖基转移酶 （UGT )及钴胺 素结合蛋白 11以及其片段、 类似物和衍生物。
本发明的另一个目的是提供编码该多肽的多核苷酸。
本发明的另一个目的是提供含有编码 UDP糖基转移酶 （UGT )及钴胺素结合蛋 白 11的多核苷酸的重组载体。 .
本发明的另一个目的是提供含有编码 UDP糖基转移酶 （UGT )及钴胺素结合蛋 白 11的多核苷酸的基因工程化宿主细胞。
本发明的另一个目的是提供生产 UDP糖基转移酶（UGT )及钴胺素结合蛋白 11 的方法。
本发明的另一个目的是提供针对本发明的多肽一一 UDP 糖基转移酶 （UGT ) 及 钴胺素结合蛋白 11的抗体。 ，，
本发明的另一个目的是提供了针对本发明多肽一一 UDP 糖基转移酶 （UGT ) 及 钴胺素结合蛋白 11的模拟化合物、 拮抗剂、 激动剂、 抑制剂。
本发明的另一个目的是提供诊断治疗与 UDP 糖基转移酶 （UGT )及钴胺素结合 蛋白 11异常相关的疾病的方法。
本发明涉及一种分离的多肽， 该多肽是人源的， 它包含： 具有 SEQ ID No. 2 氨基酸序列的多肽、 或其保守性变体、 生物活性片段或衍生物。 较佳地， 该多肽 是具有 SEQ ID NO: 2氨基酸序列的多肽。
本发明还涉及一种分离的多核苷酸， 它包含选自下组的一种核苷酸序列或其
(a)编码具有 SEQ ID No. 2氨基酸序列的多肽的多核苷酸；
(b)与多核苷酸（a)互补的多核苷酸；
(c)与（a)或（b)的多核苷酸序列具有至少 70%相同性的多核苷酸。
更佳地， 该多核苷酸的序列是选自下组的一种： （a)具有 SEQ ID NO: 1 中 911- 1201位的序列； 和（b)具有 SEQ ID NO: 1中 1 - 2194位的序列。
本发明另外涉及一种含有本发明多核苷酸的载体， 特别是表达载体； 一种用 该载体遗传工程化的宿主细胞， 包括转化、 转导或转染的宿主细胞； 一种包括培 养所述宿主细胞和回收表达产物的制备本发明多肽的方法。
本发明还涉及一种能与本发明多肽特异性结合的抗体。
本发明还涉及一种筛选的模拟、 激活、 拮抗或抑制 UDP糖基转移酶 （UGT )及 钴胺素结合蛋白 11蛋白活性的化合物的方法， 其包括利用本发明的多肽。 本发明 还涉及用该方法获得的化合物。
本发明还涉及一种体外检测与 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 蛋白异常表达相关的疾病或疾病易感性的方法， 包括检测生物样品中所述多肽或 其编码多核苷酸序列中的突变， 或者检测生物样品中本发明多肽的量或生物活性。
本发明也涉及一种药物组合物， 它含有本发明多肽或其模拟物、 激活剂、 拮 抗剂或抑制剂以及药学上可接受的载体。
本发明还涉及本发明的多肽和 /或多核苷酸在制备用于治疗癌症、 发育性疾病 或免疫性疾病或其它由于 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11表达异常 所引起疾病的药物的用途。
本发明的其它方面由于本文的技术的公开， 对本领域的技术人员而言是显而 易见的。
本说明书和权利要求书中使用的下列术语除非特别说明具有如下的含义： "核酸序列" 是指寡核苷酸、 核苷酸或多核苷酸及其片段或部分， 也可以指基 因组或合成的 DNA或 RNA, 它们可以是单链或双链的， 代表有义链或反义链。 类似地， 术语 "氨基酸序列" 是指寡肽、 肽、 多肽或蛋白质序列及其片段或部分。 当本发 明中的 "氨基酸序列" 涉及一种天然存在的蛋白质分子的氨基酸序列时， 这种 "多 肽" 或 "蛋白质" 不意味着将氨基酸序列限制为与所述蛋白质分子相关的完整的 天然氨基酸。
蛋白质或多核苷酸 "变体" 是指一种具有一个或多个氨基酸或核苷酸改变的 氨基酸序列或编码它的多核苷酸序列。 所述改变可包括氨基酸序列或核苷酸序列 中氨基酸或核苷酸的缺失、 插入或替换。 变体可具有 "保守性" 改变， 其中替换 的氨基酸具有与原氨基酸相类似的结构或化学性质， 如用亮氨酸替换异亮氨酸。 变体也可具有非保守性改变， 如用色氨酸替换甘氨酸。
"缺失" 是指在氨基酸序列或核苷酸序列中一个或多个氨基酸或核苷酸的缺 失。
"插入" 或 "添加" 是指在氨基酸序列或核苷酸序列中的改变导致与天然存 在的分子相比， 一个或多个氨基酸或核苷酸的增加。 "替换" 是指由不同的氨基酸 或核苷酸替换一个或多个氨基酸或核苷酸。
"生物活性" 是指具有天然分子的结构、 调控或生物化学功能的蛋白质。 类似 地， 术语 "免疫学活性" 是指天然的、 重组的或合成蛋白质及其片段在合适的动 物或细胞中诱导特定免疫反应以及与特异性抗体结合的能力。
"激动剂" 是指当与 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11结合时， 一 种可引起该蛋白质改变从而调节该蛋白质活性的分子。 激动剂可以包括蛋白质、 核酸、 碳水化合物或任何其它可结合 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 的分子。
"拮抗剂" 或 "抑制物" 是指当与 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 结合时， 一种可封闭或调节 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的生物学活 性或免疫学活性的分子。 拮抗剂和抑制物可以包括蛋白质、 核酸、 碳水化合物或 任何其它可结合 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的分子。
"调节" 是指 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的功能发生改变， 包 括蛋白质活性的升高或降低、 结合特性的改变及 UDP糖基转移酶 （UGT )及钴胺素结 合蛋白 11的任何其它生物学性质、 功能或免疫性质的改变。
"基本上纯' '是指基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化 UDP 糖基转移酶 （UGT )及钴胺 素结合蛋白 11。 基本上纯的 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 在非还 原性聚丙烯酰胺凝胶上能产生单一的主带。 UDP 糖基转移酶 （UGT )及钴胺素结合 蛋白 11多肽的纯度可用氨基酸序列分析。
"互补的" 或 "互补" 是指在允许的盐浓度和温度条件下通过碱基配对的多核 苷酸天然结合。 例如， 序列 "C- T- G-A" 可与互补的序列 "G- A- C-T" 结合。 两个单 链分子之间的互补可以是部分的或全部的。 核酸链之间的互补程度对于核酸链之 间杂交的效率及强度有明显影响。
"同源性" 是指互补的程度， 可以是部分同源或完全同源。 "部分同源" 是指 一种部分互补的序列， 其至少可部分抑制完全互补的序列与靶核酸的杂交。 这种 杂交的抑制可通过在严格性程度降低的条件下进行杂交 （Southern印迹或 Northern 印迹等） 来检测。 基本上同源的序列或杂交探针可竟争和抑制完全同源的序列与 靶序列在的严格性程度降低的条件下的结合。 这并不意味严格性程度降低的条件 允许非特异性结合， 因为严格性程度降低的条件要求两条序列相互的结合为特异 性或选择性相互作用。
"相同性百分率" 是指在两种或多种氨基酸或核酸序列比较中序列相同或相似 的百分率。 可用电子方法测定相同性百分率， 如通过 MEGALIGN程序 （Lasergene sof tware package, DNASTAR, Inc. , Madi son Wis. )。 MEGALIGN程序可根据不同的 方法如 Clus ter法比较两种或多种序列（Higgins, D. G. 和 P. M. Sharp (1988) Gene 73: 237-244)。 Clus ter法通过检查所有配对之间的距离将各组序列排列成簇。 然后 将各簇以成对或成组分配。 两个氨基酸序列如序列 A和序列 B之间的相同性百分率 通过下式计算： 序列 A与序列 B之间匹配的残基个数 X 100
(序列 A的残基数一序列 A中间隔残基数一序列 B中间隔残基数） 也可以通过 Clus ter法或用本领域周知的方法如 Jotun Hein 测定核酸序列之 间的相同性百分率（Hein J.， (1990) Methods in emzumology 183: 625-645) 0
"相似性" 是指氨基酸序列之间排列对比时相应位置氨基酸残基的相同或保 守性取代的程度。 用于保守性取代的氨基酸例如， 带负电荷的氨基酸可包括天冬 氨酸和谷氨酸； 带正电荷的氨基酸可包括赖氨酸和精氨酸； 具有不带电荷的头部 基团有相似亲水性的氨基酸可包括亮氨酸、 异亮氨酸和缬氨酸； 甘氨酸和丙氨酸; 天冬酰胺和谷氨酰胺； 丝氨酸和苏氨酸； 苯丙氨酸和酪氨酸。 "反义" 是指与特定的 DM或 RNA序列互补的核苷酸序列。 "反义链" 是指与 "有 义链" 互补的核酸链。
"衍生物" 是指 HFP或编码其的核酸的化学修饰物。 这种化学修饰物可以是用 烷基、 酰基或氨基替换氢原子。 核酸衍生物可编码保留天然分子的主要生物学特 性的多肽。
"抗体" 是指完整的抗体分子及其片段， 如 Fa、 ^ 及？ 其能特异性结 合 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的抗原决定簇。
"人源化抗体" 是指非抗原结合区域的氨基酸序列被替换变得与人抗体更为相 似， 但仍保留原始结合活性的抗体。
"分离的" 一词指将物质从它原来的环境 （例如， 若是自然产生的就指其天然 环境） 之中移出。 比如说， 一个自然产生的多核苷酸或多肽存在于活动物中就是 没有被分离出来， 但同样的多核苷酸或多肽同一些或全部在自然系统中与之共存 的物质分开就是分离的。 这样的多核苷酸可能是某一载体的一部分， 也可能这样 的多核苷酸或多肽是某一组合物的一部分。 既然载体或组合物不是它天然环境的 成分， 它们仍然是分离的。
如本发明所用， "分离的" 是指物质从其原始环境中分离出来 （如果是天然的 物质， 原始环境即是天然环境）。 如活体细胞内的天然状态下的多聚核苷酸和多肽 是没有分离纯化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物 质中分开， 则为分离纯化的。
如本文所用， "分离的 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 " 是指 UDP 糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 基本上不含天然与其相关的其它 蛋白、 脂类、 糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技术 纯化 UDP 糖基转移酶 （UGT ) 及钴胺素结合蛋白 11。 基本上纯的多肽在非还原 聚丙烯酰胺凝胶上能产生单一的主带。 UDP 糖基转移酶 （UGT )及钴胺素结合蛋 白 11多肽的纯度能用氨基酸序列分析。
本发明提供了一种新的多肽一一 UDP糖基转移酶（UGT )及钴胺素结合蛋白 11， 其基本上是由 SEQ ID NO: 2所示的氨基酸序列组成的。 本发明的多肽可以是重组多 肽、 天然多肽、 合成多肽， 优选重组多肽。 本发明的多肽可以是天然纯化的产物， 或是化学合成的产物， 或使用重组技术从原核或真核宿主 (例如， 细菌、 酵母、 高 等植物、 昆虫和哺乳动物细胞）中产生。 根据重组生产方案所用的宿主， 本发明的 多肽可以是糖基化的， 或可以是非糖基化的。 本发明的多肽还可包括或不包括起 始的甲硫氨酸残基。 · 本发明还包括 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的片段、 衍生物和 类似物。 如本发明所用， 术语 "片段"、 "衍生物" 和 "类似物" 是指基本上保持 本发明的 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11相同的生物学功能或活性的 多肽。 本发明多肽的片段、 衍生物或类似物可以是： （I ) 这样一种， 其中一个或 多个氨基酸残基被保守或非保守氨基酸残基 （优选的是保守氨基酸残基） 取代， 并且取代的氨基酸可以是也可以不是由遗传密码子编码的； 或者 （I I ) 这样一种， 其中一个或多个氨基酸残基上的某个基团被其它基团取代包含取代基； 或者 （I I I ) 这样一种， 其中成熟多肽与另一种化合物 （比如延长多肽半衰期的化合物， 例如 聚乙二醇） 融合； 或者 UV ) 这样一种， 其中附加的氨基酸序列融合进成熟多肽 而形成的多肽序列 （如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序 列） 通过本文的阐述， 这样的片段、 衍生物和类似物被认为在本领域技术人员的 知识范围之内。
本发明提供了分离的核酸（多核苷酸）， 基本由编码具有 SEQ ID NO: 2 氨基酸 序列的多肽的多核苷酸组成。 本发明的多核苷酸序列包括 SEQ ID N0: 1的核苷酸序 列。 本发明的多核苷酸是从人胎脑组织的 cDNA 文库中发现的。 它包含的多核苷酸 序列全长为 2194个碱基， 其开放读框 （911-1201 )编码了 87个氨基酸。 此多肽具 有 UDP糖基转移酶 （UGT )及钴胺素结合蛋白的特征序列， 可推断出该 UDP糖基转 移酶 （UGT )及钴胺素结合蛋白 11 具有 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 所代表的结构和功能。
本发明的多核苷酸可以是 DNA形式或是 RNA形式。 DNA形式包括 cDNA、 基因组 DNA或人工合成的 DNA。 DM可以是单链的或是双链的。 DNA可以是编码链或非编码 链。 编码成熟多肽的编码区序列可以与 SEQ ID NO: 1所示的编码区序列相同或者是 简并的变异体。 如本发明所用， "简并的变异体" 在本发明中是指编码具有 SEQ ID N0: 2的蛋白质或多肽， 但与 SEQ ID NO: 1所示的编码区序列有差别的核酸序列。
编码 SEQ ID NO: 2 的成熟多肽的多核苷酸包括： 只有成熟多肽的编码序列； 成熟多肽的编码序列和各种附加编码序列； 成熟多肽的编码序列 （和任选的附加 编码序列） 以及非编码序列。
术语 "编码多肽的多核苷酸" 是指包括编码此多肽的多核苷酸和包括附加编 码和 /或非编码序列的多核苷酸。
本发明还涉及上述描述多核苷酸的变异体， 其编码与本发明有相同的氨基酸 序列的多肽或多肽的片断、 类似物和衍生物。 此多核苷酸的变异体可以是天然发 生的等位变异体或非天然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺 失变异体和插入变异体。 如本领域所知的， 等位变异体是一个多核苷酸的替换形 式， 它可能是一个或多个核苷酸的取代、 缺失或插入， 但不会从实质上改变其编 码的多肽的功能。
本发明还涉及与以上所描述的序列杂交的多核苷酸（两个序列之间具有至少
50%, 优选具有 70%的相同性）。 本发明特别涉及在严格条件下与本发明所述多核苷 酸可杂交的多核苷酸。 在本发明中， "严格条件" 是指： （1)在较低离子强度和较高 温度下的杂交和洗脱， 如 0. 2xSSC, 0. 1°/»SDS，6(TC ;或（2)杂交时加用变性剂， 如 50% (v/v)甲酰胺， 0. 1%小牛血清 /0. l%Ficol l , 42 °C等; 或（3)仅在两条序列之间的 相同性至少在 95%以上，更好是 97%以上时才发生杂交。 并且， 可杂交的多核苷酸编 码的多肽与 SEQ ID NO: 2所示的成熟多肽有相同的生物学功能和活性。
本发明还涉及与以上所描述的序列杂交的核酸片段。 如本发明所用， "核酸片 段"的长度至少含 10个核苷酸， 较好是至少 20- 30个核苷酸， 更好是至少 50- 60个 核苷酸，最好是至少 100个核苷酸以上。核酸片段也可用于核酸的扩增技术（如 PCR) 以确定和 /或分离编码 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的多核苷酸。
本发明中的多肽和多核苷酸优选以分离的形式提供， 更佳地被纯化至均质。 本发明的编码 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 的特异的多核 苷酸序列能用多种方法获得。 例如， 用本领域熟知的杂交技术分离多核苷酸。 这些技术包括但不局限于： 1)用探针与基因组或 cDNA文库杂交以检出同源的多 核苷酸序列， 和 2)表达文库的抗体筛选以检出具有共同结构特征的克隆的多核 苷酸片段。
本发明的 DNA片段序列也能用下列方法获得： 1)从基因组 DNA分离双链 DNA 序列； 2)化学合成 DM序列以获得所述多肽的双链 DNA。
上述提到的方法中， 分离基因组 DNA 最不常用。 DNA 序列的直接化学合成 是经常选用的方法。 更经常选用的方法是 cDNA序列的分离。 分离感兴趣的 cDNA 的标准方法是从高表达该基因的供体细胞分离 mRNA并进行逆转录， 形成质粒或 噬菌体 cDNA 文库。 提取 mRNA 的方法已有多种成熟的技术， 试剂盒也可从商业 途径获得（Q i agene)。 而构建 cDNA 文库也是通常的方法（Sambrook, et a l.， Mo l ecu lar C l oning, A Labora tory Manua l , Co ld Spr ing Harbor Labora tory. New York , 1989)。 还可得到商业供应的 cDNA 文库， 如 Cl ontech公司的不同 cDNA 文库。 当结合使用聚合酶反应技术时， 即使极少的表达产物也能克隆。
可用常规方法从这些 cDNA 文库中筛选本发明的基因。 这些方法包括（但不 限于）： （1) 0 -0 或 0 -1^人杂交； （2)标志基因功能的出现或丧失； （3)测定 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 的转录本的水平； （4)通过免疫学 技术或测定生物学活性， 来检测基因表达的蛋白产物。 上述方法可单用， 也可 多种方法联合应用。
在第（1)种方法中， 杂交所用的探针是与本发明的多核苷酸的任何一部分同 源， 其长度至少 10个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷 酸， 最好是至少 100个核苷酸。 此外， 探针的长度通常在 2000个核苷酸之内， 较佳的为 1000个核苷酸之内。 此处所用的探针通常是在本发明的基因序列信息 的基础上化学合成的 DNA序列。 本发明的基因本身或者片段当然可以用作探针。 DM探针的标记可用放射性同位素， 荧光素或酶（如碱性磷酸酶）等。
在第（4)种方法中， 检测 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11基因 表达的蛋白产物可用免疫学技术如 Wes tern 印迹法， 放射免疫沉淀法， 酶联免 疫吸附法（ELISA)等。
应 用 PCR 技 术 扩 增 DNA/RNA 的 方 法 （Sa ik i , et a l . Sc ience 1985; 230: 1350- 1 354)被优选用于获得本发明的基因。 特别是很难从文库中得到 全长的 cDNA 时， 可优选使用 RACE法（RACE - cDNA末端快速扩增法）， 用于 PCR 的引物可根据本文所公开的本发明的多核苷酸序列信息适当地选择， 并可用常 规方法合成。 可用常规方法如通过凝胶电泳分离和纯化扩增的 DNA/RNA片段。
如上所述得到的本发明的基因， 或者各种 DNA 片段等的多核苷酸序列可用 常规方法如双脱氧链终止法（Sanger et a l. PNAS , 1977 , 74 : 5463- 5467)测定。 这类多核苷酸序列测定也可用商业测序试剂盒等。为了获得全长的 cDNA序列， 测 序需反复进行。 有时需要测定多个克隆的 cDNA 序列， 才能拼接成全长的 cDNA 序列。
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或直接 用 UDP 糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 编码序列经基因工程产生的宿 主细胞， 以及经重组技术产生本发明所述多肽的方法。
本发明中， 编码 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 的多核苷酸 序列可插入到载体中， 以构成含有本发明所述多核苷酸的重组载体。 术语 "载 体" 指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物 细胞病毒如腺病毒、 逆转录病毒或其它载体。 在本发明中适用的载体包括但不 限于：在细菌中表达的基于 T7启动子的表达载体（Rosenberg, et a l . Gene, 1987, 56: 125)； 在哺乳动物细胞中表达的 pMSXND表达载体（Lee and Na thans, J Bio Chem. 263: 3521 , 1988)和在昆虫细胞中表达的来源于杆状病毒的载体。 总之， 只要能在宿主体内复制和稳定， 任何质粒和载体都可以用于构建重组表达载体。 表达载体的一个重要特征是通常含有复制起始点、 启动子、 标记基因和翻译调 控元件。
本领域的技术人员熟知的方法能用于构建含编码 UDP糖基转移酶 （UGT )及 钴胺素结合蛋白 11 的 DNA序列和合适的转录 /翻译调控元件的表达载体。 这些 方法包括体外重组 DM技术、 DNA合成技术、体内重组技术等（Sambroook, et a l . Molecular C loning, a Laboratory Manua l , cold Spr ing Harbor Laboratory. New York, 1989)。 所述的 DNA序列可有效连接到表达载体中的适当启动子上， 以指 导 mRNA合成。 这些启动子的代表性例子有： 大肠杆菌的 lac或 t rp启动子； λ 噬菌体的 PL启动子； 真核启动子包括 CMV立即早期启动子、 HSV胸苷激酶启动 子、 早期和晚期 SV40启动子、 反转录病毒的 LTRs 和其它一些已知的可控制基 因在原核细胞或真核细袍或其病毒中表达的启动子。 表达载体还包括翻译起始 用的核糖体结合位点和转录终止子等。 在载体中插入增强子序列将会使其在高 等真核细胞中的转录得到增强。 增强子是 DNA 表达的顺式作用因子， 通常大约 有 1 0到 300个碱基对， 作用于启动子以增强基因的转录。 可举的例子包括在复 制起始点晚期一侧的 1 00到 270个碱基对的 SV40增强子、 在复制起始点晚期一 侧的多瘤增强子以及腺病毒增强子等。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择 转化的宿主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗 性以及绿色荧光蛋白（GFP) , 或用于大肠杆菌的四环素或氨苄青霉素抗性等。
本领域一般技术人员都清楚如何选择适当的载体 /转录调控元件 （如启动 子、 增强子等） 和选择性标记基因。
本发明中， 编码 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 的多核苷酸 或含有该多核苷酸的重组载体可转化或转导入宿主细胞， 以构成含有该多核苷 酸或重组载体的基因工程化宿主细胞。 术语 "宿主细胞" 指原核细胞， 如细菌 细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 细菌细胞如鼠伤寒沙门氏菌； 真菌细胞 如酵母； 植物细胞； 昆虫细胞如果蝇 S2或 Sf9; 动物细胞如 CH0、 COS或 Bowes 黑素瘤细胞等。
用本发明所述的 DNA序列或含有所述 DNA序列的重组载体转化宿主细胞可 用本领域技术人员熟知的常规技术进行。 当宿主为原核生物如大肠杆菌时， 能 吸收 DM 的感受态细胞可在指数生长期后收获， 用 CaCl2法处理， 所用的步骤 在本领域众所周知。 可供选择的是用 MgC l2。 如果需要， 转化也可用电穿孔的方 法进行。 当宿主是真核生物， 可选用如下的 DNA 转染方法： 磷酸钙共沉淀法， 或者常规机械方法如显微注射、 电穿孔、 脂质体包装等。
通过常规的重组 DNA 技术， 利用本发明的多核苷酸序列可用来表达或生产 重组的 UDP糖基转移酶（UGT )及钴胺素结合蛋白 11 (Sc i ence , 1984; 224： 1431)。 一般来说有以下步骤：
(1) .用本发明的编码人 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 的多 核苷酸（或变异体） , 或用含有该多核苷酸的重组表达载体转化或转导合适的宿 主细胞；
(2) .在合适的培养基中培养宿主细胞；
(3) .从培养基或细胞中分离、 纯化蛋白质。
在步骤 （ 2 ) 中， 根据所用的宿主细胞， 培养中所用的培养基可选自各种 常规培养基。 在适于宿主细胞生长的条件下进行培养。 当宿主细胞生长到适当 的细胞密度后， 用合适的方法（如温度转换或化学诱导）诱导选择的启动子， 将 细胞再培养一段时间。
在步骤 （ 3 ) 中， 重组多肽可包被于细胞内、 或在细胞膜上表达、 或分泌到 细胞外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分 离和纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法包括但 并不限于： 常规的复性处理、 蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超声波处理、 超离心、 分子筛层析（凝胶过滤）、 吸附层析、 离子交换层析、 高 效液相层析（HPLC)和其它各种液相层析技术及这些方法的结合。 附图的简要说明
下列附图用于说明本发明的具体实施方案， 而不用于限定由权利要求书所 界定的本发明范围。
图 1是本发明 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11在 10- 52共 43个氨 基酸和 UDP糖基转移酶 （UGT )及钴胺素结合蛋白特征结构域氨基酸序列的同源性 比较图。 上方序列是 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 , 下方序列是 UDP糖基转移酶 （UGT )及钴胺素结合蛋白特征结构域。 相同氨基酸在两个序列间 用单字符氨基酸表示， 相似氨基酸用 "+" 表示。
图 2为分离的 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的聚丙烯酰胺凝 胶电泳图 （SDS-PAGE )。 l lkDa为蛋白质的分子量。 箭头所指为分离出的蛋白条带。 实现本发明的最佳方式
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件如 Sambrook等人， 分子克隆：实验室手册（New York: Cold Spr ing Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1 : UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的克隆
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA。 用 Quik mRNA I solat ion Ki t ( Qiegene 公司产品） 从总 RNA中分离 poly (A) mRNA。 2ug poly (A) mRNA经逆转录 形成 cDNA。用 Smart cDNA克隆试剂盒（购自 Clontech )将 cDNA片段定向插入到 pBSK (+) 载体 (Clontech公司产品）的多克隆位点上， 转化 DH5 α， 细菌形成 cDM文库。 用 Dye terminate cycle react ion sequencing ki t (Perkin - Elmer公司产品) 和 ABI 377 自动测序仪 (Perkin- Elmer公司）测定所有克隆的 5'和 3'末端的序列。 将测定的 cDNA 序列与已有的公共 DNA序列数据库 （Genebank )进行比较， 结果发现其中一个克隆 1235b07的 cDM序列为新的 DNA。 通过合成一系列引物对该克隆所含的插入 cDNA片段 进行双向测定。 结果表明， 1235b07克隆所含的全长 cDNA为 2194bp (如 Seq ID NO: 1 所示） ， 从第 911bp至 1201bp有一个 264bp的开放阅读框架 ( 0RF ) ， 编码一个新的 蛋白质 （如 Seq ID NO: 2所示） 。 我们将此克隆命名为 pBS - 1235b07， 编码的蛋白质 命名为 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11。 实施例 2: cDNA 克隆的结构域分析
将本发明的 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的序列及其编码的蛋 白 序 列 , 用 GCG 中 的 rof i le scan程序 (Bas iclocal Al ignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] , 在 pros i te等数 据库进行结构域分析。 本发明的 UDP糖基转移酶（UGT )及钴胺素结合蛋白 11在 10 - 52 与结构域 UDP糖基转移酶 （UGT )及钴胺素结合蛋白有同源， 同源结果示于图 1， 同 源率为 0. 12 , 得分为 5. 32; 阈值为 5. 30。 实施例 3: 用 RT-PCR方法克隆编码 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的基 因
用胎脑细胞总 RM为模板，以 ol igo-dT为引物进行逆转录反应合成 cDNA，用 Qiagene的试剂盒纯化后，用下列引物进行 PCR扩增：
Primerl: 5-CATCCTGAGAACTGAAATTGATCGC-3' (SEQ ID NO: 3)
Primer2: 5-ATAAAATTTTTGAATTTATGTTCAA-3' (SEQ ID NO: 4)
Primerl为位于 SEQ ID NO: 1的 5，端的第 lbp开始的正向序列;
Primer2为 SEQ ID NO: 1的中的 3，端反向序列。
扩增反应的条件： 在 50 μ 1的反应体积中含有 50mmol/L C1, 10mmol/L Tris- Cl, (pH8.5), 1.5隱 ol/L MgCl2, 200 μ mol/L dNTP, lOpmol引物， 1U的 Taq DNA聚合酶 (C 1 on t ech公司产品）。 在 PE9600型 DM热循环仪（Per k i n-E lmer公司）上按下列条件 反应 25个周期： 94。C 30sec; 55°C 30sec; 72。C 2min。 在 RT- PCR时同时设 β -act in 为阳性对照和模板空白为阴性对照。 扩增产物用 QIAGEN公司的试剂盒纯化， 用 TA克 隆试剂盒连接到 pCR载体上 （Invitrogen公司产品） 。 DM序列分析结果表明 PCR产 物的 DNA序列与 SEQ ID NO: 1所示的 l-2194bp完全相同。
实施例 4: Northern 印迹法分析 HDP糖基转移酶 （ (JGT )及钴胺素结合蛋白 11基因的 表达：
用一步法提取总 RNA [Anal. Biochem 1987, 162, 156-159] 0 该法包括酸性硫氰 酸胍苯酚-氯仿抽提。 即用 4M异硫氰酸胍- 25mM柠檬酸钠， 0.2M乙酸钠 （ pH4.0 ) 对 组织进行匀浆， 加入 1倍体积的苯酚和 1/5体积的氯仿-异戊醇 （49: 1) , 混合后离 心。 吸出水相层， 加入异丙醇 （0.8体积） 并将混合物离心得到 RNA沉淀。 将得到的 RNA沉淀用 70%乙醇洗涤， 干燥并溶于水中。 用 20yg RNA， 在含 20mM 3- ( N-吗啉代） 丙磺酸 （pH7.0) - 5mM乙酸钠 - ImM EDTA- 2.2M甲醛的 1.2%琼脂糖凝胶上进行电泳。 然后转移至硝酸纤维素膜上。 用 a-32P dATP通过随机引物法制备 32Ρ-标记的 DNA探 针。 所用的 DNA探针为图 1所示的 PCR扩增的 UDP糖基转移酶 （ UGT )及钴胺素结合蛋 白 11编码区序列（911bp至 1201bp)。 将 32P-标记的探针 （约 2 x 106cpm/ml ) 与转移 了 RNA的硝酸纤维素膜在一溶液中于 42°C杂交过夜， 该溶液包含 50%甲酰胺 - 25mM KH2P04 ( pH7.4 ) - 5 x SSC-5 x Denhardt，s溶液和 200 g/ml鲑精 DNA。 杂交之后， 将 滤膜在 1 xSSC- 0.1%SDS中于 55°C洗 30min。 然后， 用 Phosphor Imager进行分析和定 '量。 实施例 5: 重组 UDP糖基转移酶 （UGT)及钴胺素结合蛋白 11的体外表达、 分离和纯 化
根据 SEQ ID NO: 1和图 1所示的编码区序列， 设计出一对特异性扩增引物， 序 列如下： Primer3: 5'- CCCCATATGATGCTCTGTCACCTTCAAAGGATGG— 3' ( Seq ID No: 5 ) Primer4: 5-CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC-3' (Seq ID No: 6 ) 此两段引物的 5'端分别含有 Mel和 BamHI酶切位点， 其后分别为目的基因 5'端 和 3'端的编码序列， Ndel和 BamHI酶切位点相应于表达载体质粒 pET 28b (+) (Novagen 公司产品， Cat. No.69865.3)上的选择性内切酶位点。 以含有全长目的基因的 pBS- l235b07质粒为模板， 进行 PCR反应。 PCR反应条件为： 总体积 50 μ 1中含 pBS- 1235b07 质粒 10pg、 引物 1：11116：1：-3和？：!：10161-4分另!]为10 11101、 Advantage polymerase Mix (Clontech公司产品） 1 μ 1。 循环参数： 94°C 20s, 60°C 30s, 68°C 2 min,共 25个 循环。 用 Ndel和 BaraHI分别对扩增产物和质粒 pET- 28(+)进行双酶切，分别回收大片 段，并用 T4连接酶连接。 连接产物转化用氯化钙法大肠杆细菌 DH5a,在含卡那霉素 (终浓度 30μ§/πι1 ) 的 LB平板培养过夜后， 用菌落 PCR方法筛选阳性克隆， 并进行 测序。 挑选序列正确的阳性克隆 (PET-1235b07)用氯化钙法将重组质粒转化大肠 杆菌 BL21(DE3)plySs (Novagen公司产品）。 在含卡那霉素 （终浓度 30 g/ml ) 的 LB 液体培养基中， 宿主菌 BL21 (pET- 1235b07) 在 37。C培养至对数生长期， 加入 IPTG 至终浓度 1IMO1/L， 继续培养 5小时。 离心收集菌体， 经超声波破菌，离心收集上清， 用能与 6个组氨酸（ 6His-Tag )结合的亲和层析柱 His. Bind Quick Cartridge( Novagen 公司产品）进行层析， 得到了纯化的目的蛋白 UDP糖基转移酶 （UGT)及钴胺素结合 蛋白 11。 经 SDS- PAGE电泳， 在 llkDa处得到一单一的条带 （图 2 ) 。 将该条带转移至 PVDF膜上用 Edams水解法进行 N-端氨基酸序列分析， 结果 N-端 15个氨基酸与 SEQ ID NO: 2所示的 N-端 15个氨基酸残基完全相同。
实施例 6 抗 UDP糖基转移酶 （UGT)及钴胺素结合蛋白 11抗体的产生
用多肽合成仪 （PB公司产品）合成下述 UDP糖基转移酶 （UGT)及钴胺素结合 蛋白 11特异性的多肽：
NH2-Met-Leu-Cys-His-Leu-Gln-Arg-Met-Val-Ser-Glu-Gln-Cys-His-Leu-C00H (SEQ ID NO: 7)。 将该多肽分别与血蓝蛋白和牛血清白蛋白耦合形成复合， 方法参 见： Avrameas, et al. Iraraunochemistry, 1969; 6: 43。 用 ½g上述血蓝蛋白多肽复合 物加上完全弗氏佐剂免疫家兔， 15天后再用血蓝蛋白多肽复合物加不完全弗氏佐 剂加强免疫一次。 采用经 15 g/ml牛血清白蛋白多肽复合物包被的滴定板做 ELISA 测定兔血清中抗体的滴度。 用蛋白 A-Sepharose从抗体阳性的家兔血清中分离总 IgGo 将多肽结合于溴化氰活化的 SePharose4B柱上， 用亲和层析法从总 IgG中分离 抗多肽抗体。 免疫沉淀法证明纯化的抗体可特异性地与 UDP糖基转移酶（UGT)及钴 胺素结合蛋白 11结合。 实施例 7: 本发明的多核苷酸片段用作杂交探针的应用
从本发明的多核苷酸中挑选出合适的寡核苷酸片段用作杂交探针有多方面的 用途， 如用该探针可与不同来源的正常组织或病理组织的基因组或 cDNA文库杂交 以鉴定其是否含有本发明的多核苷酸序列和检出同源的多核苷酸序列，进一步还可 用该探针检测本发明的多核苷酸序列或其同源的多核苷酸序列在正常组织或病理 组织细胞中的表达是否异常。 . 本实施例的目的是从本发明的多核苷酸 SEQ ID NO: 1 中挑选出合适的寡核苷 酸片段用作杂交探针， 并用滤膜杂交方法鉴定一些组织中是否含有本发明的多核 苷酸序列或其同源的多核苷酸序列。 滤膜杂交方法包括斑点印迹法、 Southern 印 迹法、 Northern 印迹法和复印方法等， 它们都是将待测的多核苷酸样品固定在滤 膜上后使用基本相同的步骤杂交。 这些相同的步骤是： 固定了样品的滤膜首先用 不含探针的杂交缓冲液进行预杂交， 以使滤膜上样品的非特异性的结合部位被载 体和合成的多聚物所饱和。 然后预杂交液被含有标记探针的杂交缓冲液替换， 并 保温使探针与靶核酸杂交。 杂交步骤之后， 未杂交上的探针被一系列洗膜步骤除 掉。 本实施例利用较高强度的洗膜条件（如较低盐浓度和较高的温度）， 以使杂交 背景降低且只保留特异性强的信号。 本实施例选用的探针包括两类： 第一类探针 是完全与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段； 第二类探 针是部分与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段。 本实施 例选用斑点印迹法将样品固定在滤膜上， 在较高强度的的洗膜条件下， 第一类探 针与样品的杂交特异性最强而得以保留。
一、 探针的选用
从本发明的多核苷酸 SEQ ID NO: 1 中选择寡核苷酸片段用作杂交探针， 应遵 循以下原则和需要考虑的几个方面：
1 , 探针大小优选范围为 18-50个核苷酸；
2 , GC含量为 30%- 70%， 超过则非特异性杂交增加；
3 , 探针内部应无互补区域；
4 , 符合以上条件的可作为初选探针， 然后进一步作计算机序列分析， 包括将该 初选探针分别与其来源序列区域 （即 SEQ ID NO: 1 )和其它已知的基因组序列 及其互补区进行同源性比较， 若与非靶分子区域的同源性大于 85%或者有超过 15个连续碱基完全相同， 则该初选探针一般就不应该使用；
5 , 初选探针是否最终选定为有实际应用价值的探针还应进一步由实验确定。
完成以上各方面的分析后挑选并合成以下二个探针： 探针 1 (probel), 属于第一类探针， 与 SEQ ID NO: 1的基因片段完全同源或 互补（41Nt)：
5 '-TGAGTAAGGAAATGCTTCTCACCTTCTCTGCTCCAAAGAGA- 3' ( SEQ ID NO: 8 ) 探针 1 ( probe2 ), 属于第二类探针， 相当于 SEQ ID NO: 1 的基因片段或其 互补片段的替换突变序列 （41Nt):
5-TGAGTAAGGAAATGCTTCTCCCCTTCTCTGCTCCAAAGAGA-3' ( SEQ ID NO: 9 ) 与以下具体实验步骤有关的其它未列出的常用试剂及其配制方法请参考文 献： DNA PROBES G. Η· Keller; M. Μ· Manak; Stockton Press, 1989 (USA)以及更常 用的分子克隆实验手册书籍如 《分子克隆实验指南》（1998 年第二版） 〖美]萨姆 布鲁克等著， 科学出版社。 '
样品制备：
1 , 从新鲜或冰冻组织中提取 DNA
步骤： 1) 将新鲜或新鲜解冻的正常肝组织放入浸在冰上并盛有磷酸盐缓冲液 (PBS) 的平皿中。 用剪刀或手术刀将组织切成小块。 操作中应保持组织湿润。 2) 以 lOOOg离心切碎组织 10分钟。 3)用冷匀浆缓冲液 (0.25fflol/L蔗糖； 25薩 ol/L Tris-HCl,pH7.5; 25隱 ol/LnaCl; 25腿 ol/L MgCl2 ) 悬浮沉淀 （大约 10ml/g)。 4) 在 4°C用电动匀浆器以全速匀浆组织悬液， 直至组织被完全破碎。 5) lOOOg 离心 10分钟。 6)用重悬细胞沉淀（每 0. lg最初组织样品加 l-5ml)， 再以 lOOOg离心 10分钟。 7)用裂解缓冲液重悬沉淀（每 O.lg最初组织样品加 lml), 然后接以下 的苯酚抽提法。
2, DNA的苯酚抽提法
步骤： 1)用 1- 10ml冷 PBS洗细胞， lOOOg离心 10分钟。 2)用冷细胞裂解液 重悬浮沉淀的细胞 （l xlO8细胞 /ml) 最少应用 lOOul 裂解缓冲液。 3)加 SDS 至 终浓度为 1%, 如果在重悬细胞之前将 SDS直接加入到细胞沉淀中， 细胞可能会形 成大的团块而难以破碎， 并降低的总产率。 这一点在抽提 >107细胞时特别严重。 4) 加蛋白酶 K至终浓度 200ug/ml。 5) 50°C保温反应 1小时或在 37°C轻轻振摇过夜。 6)用等体积苯酚： 氯仿： 异戊醇 （ 25: 24: 1 )抽提， 在小离心机管中离心 10分 钟。 两相应清楚分离， 否则重新进行离心。 7) 将水相转移至新管。 8)用等体积 氯仿： 异戊醇 （24: 1)抽提， 离心 10分钟。 9)将含 DM的水相转移至新管。 然 后进行 DNA的纯化和乙醇沉淀。
3, DNA的纯化和乙醇沉淀
步骤： 1 )将 1/10体积 2mol/L醋酸钠和 1倍体积冷 100%乙醇加到 DNA溶液中， 混匀。 在- 20°C放置 1小时或至过夜。 2) 离心 10分钟。 3)小心吸出或倒出乙醇。 4)用 70%冷乙醇 500ul洗涤沉淀， 离心 5分钟。 5)小心吸出或倒出乙醇。 用 500ul 冷乙醇洗涤沉淀， 离心 5分钟。 6)小心吸出或倒出乙醇， 然后在吸水纸上倒置使 残余乙醇流尽。 空气干燥 10-15 分钟， 以使表面乙醇挥发。 注意不要使沉淀完全 干燥， 否则较难重新溶解。 7) 以小体积 TE或水重悬 DNA沉淀。 低速涡旋振荡或 用滴管吹吸， 同时逐渐增加 TE, 混合至 DNA充分溶解， 每 1-5 xlO6细胞所提取的 大约加 lul。
以下第 8-13步骤仅用于必须除去污染时， 否则可直接进行第 14步骤。
8 )将 RNA酶 A加到 DNA溶液中， 终浓度为 lOOug/ml, 37DC保温 30分钟。 9 )加 入 SDS和蛋白酶 K, 终浓度分别为 0.5%和 100ug/ml。 37°C保温 30分钟。 10)用 等体积的苯酚： 氯仿： 异戊醇 （ 25: 24: 1 )抽提反应液， 离心 10 分钟。 11)小 心移出水相， 用等体积的氯仿： 异戊醇 （24: 1) 重新抽提， 离心 10 分钟。 12) 小心移出水相， 加 1/10体积 2mol/L醋酸钠和 2.5体积冷乙醇， 混匀置 -20°C 1小 时。 13)用 70%乙醇及 100%乙醇洗涤沉淀， 空气干燥， 重悬核酸， 过程同第 3-6 步骤。 14 ) 测定 A26。和 A28。以检测 DNA的纯度及产率。 15 )分装后存放于 - 20°C。 样膜的制备：
1)取 4x2 张适当大小的硝酸纤维素膜（NC膜）， 用铅笔在其上轻轻标出点样 位置及样号， 每一探针需两张 NC膜， 以便在后面的实验步骤中分别用高强度条件 和强度条件洗膜 。
2 ) 吸取及对照各 15微升， 点于样膜上， 在室温中晾干。
3) 置于浸润有 0. lmol/LNaOH, 1.5raol/LNaCl 的滤纸上 5分钟 (两次）, 晾干置 于浸润有 0.5mol/L Tris- HC1 (pH7.0), 3raol/LNaCl 的滤纸上 5分钟 （两次）， 晾 干。
4) 夹于干净滤纸中， 以铝箔包好， 60- 80°C真空干燥 2小时。
探针的标记
1 ) 3μ lProbe ( 0.10D/10 μ 1 ), 加入 2 μ IKinase缓冲液， 8-10 uCi y-32P-dATP+2U Kinase, 以补加至终体积 20 μ 1。
2) 37°C 保温 2小时。
3)加 1/5体积的溴酚蓝指示剂 （BPB)。
4 ) 过 Sephadex G-50柱。
5) 至有 32P- Probe洗出前开始收集第一峰（可用 Monitor监测）。
6) 5滴 /管， 收集 10-15管。 7 )用液体闪烁仪监测同位素量
8 ) 合并第一峰的收集液后即为所需制备的 32P- Probe (第二峰为游离 γ- 32P- dATP )。
预杂交
将样膜置于塑料袋中，加入 3- 10mg预杂交液（l OxDenhardt' s ; 6xSSC 0. lmg/ml
CT DM (小牛胸腺 DNA )。）, 封好袋口后， 68 C水浴摇 2小时。
杂交
将塑料袋剪去一角， 加入制备好的探针， 封好袋口后， 42°C水浴摇过夜。
洗膜：
高强度洗膜：
1 )取出已杂交好的样膜。
2 ) 2xSSC, 0. 1%SDS中， 40°C洗 15分钟 ( 2次）。
3 ) 0. lxSSC, 0. 1%SDS中， 40°C洗 15分钟 （ 2次）。
4 ) 0. lxSSC, 0. 1%SDS中， 55 C洗 30分钟 （2次）， 室温晾干。
低强度洗膜：
1 )取出已杂交好的样膜。
2 ) 2xSSC, 0. 1%SDS中， 37 C洗 15分钟 （ 2次）。
3 ) 0. lxSSC, 0. 1%SDS中， 37°C洗 15分钟 （ 2次）。
4 ) 0. lxSSC, 0. 1%SDS中， 40°C洗 15分钟 （ 2次）， 室温晾干。
X-光自显影：
- 70°C X-光自显影 （压片时间根据杂交斑放射性强弱而定）。
实验结果：
采用低强度洗膜条件所进行的杂交实验， 以上两个探针杂交斑放射性强弱没 有明显区别； 而采用高强度洗膜条件所进行的杂交实验， 探针 1 的杂交斑放射性 强度明显强于另一个探针杂交斑的放射性强度。 因而可用探针 1 定性和定量地分 析本发明的多核苷酸在不同组织中的存在和差异表达。 工业实用性
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗恶性肿瘤、 肾上腺缺乏症、 皮肤病、 各类炎症、 HIV感染和免疫性疾 病等。
UDP糖基转移酶 （UGT )家族成员催化 UTP-糖的糖基集团加成至小的疏水分子， 如哺乳动物 UDP-葡糖苷酸基转移酶 （UDPGT ) 缺乏可形成一种常染色体隐性性状即 克-钠（Cr igler- Naj jar )综合征； UDP糖基转移酶是一类膜结合微粒体酶类大家族， 它催化葡糖醛酸转化成各类外源及内源性亲脂底物， 这种酶类在解毒以及而后的 去处类似药物及致癌源的异源物质中起重要作用， 如哺乳动物 2-羟酰鞘氨醇- 1 - beta-半乳糖基转移酶是生物合成半乳糖脑苷脂中一步关键的酶促步骤， 半乳糖脑 苷脂是中枢神经系统以及外周神经系统的髓磷脂膜的丰富的鞘脂。 UDP糖基转移酶 具有一个高度保守的特征序列模板。
UDP糖基转移酶家族特异的保守序列是形成其活性功能域所必需。 本发明的多 肽也具有 UDP糖基转移酶特异的保守序列。
由此可见， 特异的 UDP 糖基转移酶功能域的表达异常， 将致使本发明的含此 功能域的多肽的功能异常， 从而导致催化葡糖醛酸转化成各类外源及内源性亲脂 底物的过程发生障碍， 生物合成半乳糖脑苷脂的过程异常。 本发明的多肽还具有 钴胺素结合蛋白特异的保守序列。 钴胺素结合蛋白对于钴胺素的结合、 转运、 代 谢是极为重要的。 因此， 本发明的多肽的表达异常， 也会使钴胺素结合功能异常， 影响钴胺素代谢， 并产生相关的疾病。
由此可见， 本发明的 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的表达异常 将产生各种疾病尤其是克-钠 （Cr igler- Naj jar ) 综合征、 神经系统发育性疾病、 钴胺素缺乏症、 钴胺素中毒综合症、 某些肿瘤、 炎症、 免疫性疾病， 这些疾病包 括但不限于：
神经系统的发育紊乱性疾病： 神经管闭合不全如脊柱裂， 无脑畸形， 脑（脑膜） 膨出， 颅脑裂， 神经管囊肿； 大脑发育畸形如孔脑畸形， 全前脑， 水脑畸形； 神经 元迁徙障碍如脑回形成异常； 其它畸形如导水管畸形， 小脑发育不全， Down 综合 症， 脊髓畸形， 先天性脑积水， 先天性脑神经核发育不全综合症
某些肿瘤： 星形细胞瘤， 室管膜瘤， 胶质细胞瘤， 神经纤维瘤， 胃癌， 肝癌， 肺癌， 食管癌， 乳腺癌， 白血病， 淋巴瘤， 甲状腺肿瘤， 子宫肌瘤， 结肠癌， 黑 色素瘤， 膀胱癌， 子宫内膜癌， 结肠癌， 胸腺肿瘤， 气管肿瘤， 纤维瘤， 纤维肉 瘤
炎症： 慢性活动性肝炎， 结节病， 多肌炎， 慢性鼻炎， 慢性胃炎， 脑脊髓多发 性硬化， 肾小球性肾炎， 心肌炎， 心肌病， 动脉粥样硬化， 胃溃疡， 子宫颈炎， 各种感染性炎症
免疫性疾病： 系统性红斑狼疮， 类风湿性关节炎， 支气管哮喘， 荨麻疹， 特异 性皮炎， 感染后心肌炎， 硬皮病， 重症肌无力， 格林-巴利综合症， 普通易变免疫 缺陷病， 原发性 B淋巴细胞免疫缺陷病， 获得性免疫缺陷综合症
本发明的 UDP糖基转移酶（UGT )及钴胺素结合蛋白 11的表达异常还将产生某些 遗传性， 血液性疾病等。
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗各种疾病尤其是克-纳 （Cr i g ler- Naj jar ) 综合征、 神经系统发育性 疾病、 钴胺素缺乏症、 钴胺素中毒综合症、 某些肿瘤、 炎症、 免疫性疾病， 某些 遗传性， 血液性疾病等。 本发明也提供了筛选化合物以鉴定提高（激动剂）或阻遏（拮抗剂） UDP 糖基 转移酶 （UGT )及钴胺素结合蛋白 1 1 的药剂的方法。 激动剂提高 UDP 糖基转移 酶 （UGT ) 及钴胺素结合蛋白 11 刺激细胞增殖等生物功能， 而拮抗剂阻止和治 疗与细胞过度增殖有关的紊乱如各种癌症。 例如， 能在药物的存在下， 将哺乳 动物细胞或表达 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 1 1 的膜制剂与标记 的 UDP 糖基转移酶 （UGT )及钴胺素结合蛋白 1 1 一起培养。 然后测定药物提高 或阻遏此相互作用的能力。
UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 的拮抗剂包括筛选出的抗体、 化合物、 受体缺失物和类似物等。 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 的拮抗剂可以与 UDP 糖基转移酶 （UGT ) 及钴胺素结合蛋白 1 1 结合并消除其功 能， 或是抑制该多肽的产生， 或是与该多肽的活性位点结合使该多肽不能发挥 生物学功能。
在筛选作为拮抗剂的化合物时， 可以将 UDP糖基转移酶 （UGT ) 及钴胺素结 合蛋白 11 加入生物分析测定中， 通过测定化合物对 UDP糖基转移酶 （UGT )及 钴胺素结合蛋白 1 1 和其受体之间相互作用的影响来确定化合物是否是拮抗剂。 用上述筛选化合物的同样方法， 可以筛选出起拮抗剂作用的受体缺失物和类似 物。 能与 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 结合的多肽分子可通过 筛选由各种可能组合的氨基酸结合于固相物组成的随机多肽库而获得。 筛选时， 一般应对 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11分子进行标记。
本发明提供了用多肽， 及其片段、 衍生物、 类似物或它们的细胞作为抗原 以生产抗体的方法。 这些抗体可以是多克隆抗体或单克隆抗体。 本发明还提供 了针对 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 1 1 抗原决定簇的抗体。 这些 抗体包括（但不限于）： 多克隆抗体、 单克隆抗体、 嵌合抗体、 单链抗体、 Fab 片段和 Fab表达文库产生的片段。 多克隆抗体的生产可用 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 直接 注射免疫动物 （如家兔， 小鼠， 大鼠等） 的方法得到， 多种佐剂可用于增强免 疫反应， 包括但不限于弗氏佐剂等。 制备 UDP糖基转移酶 （UGT ) 及钴胺素结合 蛋白 11 的单克隆抗体的技术包括但不限于杂交瘤技术（Kohler and Mi l s te in. Nature, 1975, 256: 495-497) , 三瘤技术， 人 Β-细胞杂交瘤技术， EBV-杂交瘤技 术等。 将人恒定区和非人源的可变区结合的嵌合抗体可用已有的技术生产 (Morr i son e t a l , PNAS, 1985, 81: 6851) 0 而已有的生产单链抗体的技术（U. S. Pa t No. 4946778)也可用于生产抗 UDP糖基转移酶 （ UGT ) 及钴胺素结合蛋白 11 的单 链抗体》
抗 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 的抗体可用于免疫组织化 学技术中， 检测活检标本中的 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11。
与 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 结合的单克隆抗体也可用 放射性同位素标记， 注入体内可跟踪其位置和分布。 这种放射性标记的抗体可 作为一种非创伤性诊断方法用于肿瘤细胞的定位和判断是否有转移。
抗体还可用于设计针对体内某一特殊部位的免疫毒素。 如 UDP 糖基转移酶
( UGT ) 及钴胺素结合蛋白 11 高亲和性的单克隆抗体可与细菌或植物毒素（如白 喉毒素， 蓖麻蛋白， 红豆碱等）共价结合。 一种通常的方法是用巯基交联剂如 SPDP , 攻击抗体的氨基， 通过二硫键的交换， 将毒素结合于抗体上， 这种杂交 抗体可用于杀灭 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11阳性的细胞。
本发明中的抗体可用于治疗或预防与 UDP糖基转移酶 （UGT ) 及钴胺素结合 蛋白 11相关的疾病。给予适当剂量的抗体可以刺激或阻断 UDP糖基转移酶（UGT ) 及钴胺素结合蛋白 11的产生或活性。
本发明还涉及定量和定位检测 UDP糖基转移酶（UGT )及钴胺素结合蛋白 11 水平的诊断试验方法。 这些试验是本领域所熟知的， 且包括 FISH测定和放射免 疫测定。 试验中所检测的 UDP 糖基转移酶 （UGT )及钴胺素结合蛋白 11 水平， 可以用作解释 UDP 糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 在各种疾病中的重 要性和用于诊断 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11起作用的疾病。
本发明的多肽还可用作肽谱分析， 例如， 多肽可用物理的、 化学或酶进行 特异性切割， 并进行一维或二维或三维的凝胶电泳分析，更好的是进行质谱分 析。
编码 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 的多核苷酸也可用于多 种治疗目的。 基因治疗技术可用于治疗由于 UDP糖基转移酶 （UGT )及钴胺素结 合蛋白 11 的无表达或异常 /无活性表达所致的细胞增殖、 发育或代谢异常。 重 组的基因治疗载体（如病毒载体）可设计用于表达变异的 UDP糖基转移酶 （UGT) 及钴胺素结合蛋白 11， 以抑制内源性的 UDP 糖基转移酶 （UGT) 及钴胺素结合 蛋白 11 活性。 例如， 一种变异的 UDP糖基转移酶 （UGT)及钴胺素结合蛋白 11 可以是缩短的、 缺失了信号传导功能域的 UDP糖基转移酶 （UGT) 及钴胺素结合 蛋白 11, 虽可与下游的底物结合， 但缺乏信号传导活性。 因此重组的基因治疗 载体可用于治疗 UDP 糖基转移酶 （UGT) 及钴胺素结合蛋白 11 表达或活性异常 所致的疾病。 来源于病毒的表达载体如逆转录病毒、 腺病毒、 腺病毒相关病毒、 单纯疱疹病毒、 细小病毒等可用于将编码 UDP糖基转移酶 （UGT)及钴胺素结合 蛋白 11 的多核苷酸转移至细胞内。 构建携带编码 UDP 糖基转移酶 （UGT) 及钴 胺素结合蛋白 11 的多核苷酸的重组病毒载体的方法可见于已有文献 (Sambrook,et al.)。 另外重组编码 UDP糖基转移酶（ UGT )及钴胺素结合蛋白 11 的多核苷酸可包装到脂质体中转移至细胞内。
多核苷酸导入组织或细胞内的方法包括： 将多核苷酸直接注入到体内组织 中； 或在体外通过载体（如病毒、 噬菌体或质粒等）先将多核苷酸导入细胞中， 再将细胞移植到体内等。
抑制 UDP糖基转移酶 （UGT)及钴胺素结合蛋白 11 mRNA 的寡核苷酸 (包括 反义 RNA和 DNA)以及核酶也在本发明的范围之内。 核酶是一种能特异性分解特 定 RNA 的酶样 RNA分子， 其作用机制是核酶分子与互补的靶 RNA特异性杂交后 进行核酸内切作用。 反义的 RNA和 DNA及核酶可用已有的任何 RNA或 DNA合成 技术获得， 如固相磷酸酰胺化学合成法合成寡核苷酸的技术已广泛应用。 反义 RNA分子可通过编码该 RNA 的 DM序列在体外或体内转录获得。 这种 DNA序列 已整合到载体的 MA 聚合酶启动子的下游。 为了增加核酸分子的稳定性， 可用 多种方法对其进行修饰， 如增加两侧的序列长度， 核糖核苷之间的连接应用磷 酸硫酯键或肽键而非磷酸二酯键。
编码 UDP糖基转移酶（UGT)及钴胺素结合蛋白 11的多核苷酸可用于与 UDP 糖基转移酶 （UGT) 及钴胺素结合蛋白 11 的相关疾病的诊断。 编码 UDP 糖基转 移酶（UGT)及钴胺素结合蛋白 11的多核苷酸可用于检测 UDP糖基转移酶（UGT) 及钴胺素结合蛋白 11 的表达与否或在疾病状态下 UDP糖基转移酶 （UGT) 及钴 胺素结合蛋白 11 的异常表达。 如编码 UDP 糖基转移酶 （UGT)及钴胺素结合蛋 白 11 的 DNA序列可用于对活检标本进行杂交以判断 UDP糖基转移酶 （UGT)及 钴胺素结合蛋白 11 的表达状况。 杂交技术包括 Southern 印迹法， Northern 印 迹法、 原位杂交等。 这些技术方法都是公开的成熟技术， 相关的试剂盒都可从 商业途径得到。 本发明的多核苷酸的一部分或全部可作为探针固定在微阵列
(Microarray)或 DNA 芯片（又称为 "基因芯片" ）上， 用于分析组织中基因的差 异表达分析和基因诊断。 用 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 特异 的引物进行 RNA-聚合酶链反应（RT- PCR)体外扩增也可检测 UDP 糖基转移酶 ( UGT ) 及钴胺素结合蛋白 11的转录产物。
检测 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 基因的突变也可用于诊 断 UDP 糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 相关的疾病。 UDP 糖基转移酶 ( UGT ) 及钴胺素结合蛋白 11 突变的形式包括与正常野生型 UDP 糖基转移酶 ( UGT ) 及钴胺素结合蛋白 11 DNA 序列相比的点突变、 易位、 缺失、 重组和其 它任何异常等。 可用已有的技术如 Southern 印迹法、 DM序列分析、 PCR 和原 位杂交检测突变。 另外， 突变有可能影响蛋白的表达， 因此用 Northern印迹法、 Wes tern印迹法可间接判断基因有无突变。
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人 染色体具体位置且并可以与其杂交。 目前， 需要鉴定染色体上的各基因的具体 位点。 现在， 只有很少的基于实际序列数据（重复多态性）的染色体标记物可用 于标记染色体位置。 根据本发明， 为了将这些序列与疾病相关基因相关联， 其 重要的第一步就是将这些 DM序列定位于染色体上。
简而言之， 根据 cDNA制备 PCR引物（优选 15- 35bp) , 可以将序列定位于染色 体上。 然后， 将这些引物用于 PCR筛选含各条人染色体的体细胞杂合细胞。 只 有那些含有相应于引物的人基因的杂合细胞会产生扩增的片段。
体细胞杂合细胞的 PCR定位法， 是将 DNA定位到具体染色体的快捷方法。 使 用本发明的寡核苷酸引物， 通过类似方法， 可利用一组来自特定染色体的片段 或大量基因组克隆而实现亚定位。 可用于染色体定位的其它类似策略包括原位 杂交、 用标记的流式分选的染色体预筛选和杂交预选， 从而构建染色体特异的 cDNA库。
将 cDNA克隆与中期染色体进行荧光原位杂交（FISH) , 可以在一个步骤中精 确地进行染色体定位。 此技术的综述， 参见 Verma等， Human Chromosomes: a Manua l of Bas ic Techniques, Pergamon Pres s, New York (1988)。
一旦序列被定位到准确的染色体位置， 此序列在染色体上的物理位置就可 以与基因图数据相关联。 这些数据可见于例如， V. Mckus i ck，Mendel ian Inher i tance in Man (可通过与 Johns Hopkins Univers i ty Wel ch Medica l L i brary联机获得）。 然后可通过连锁分析， 确定基因与业已定位到染色体区域 上的疾病之间的关系。
接着， 需要测定患病和未患病个体间的 cDNA或基因组序列差异。 如果在一 些或所有的患病个体中观察到某突变， 而该突变在任何正常个体中未观察到， 则该突变可能是疾病的病因。 比较患病和未患病个体， 通常涉及首先寻找染色 体中结构的变化， 如从染色体水平可见的或用基于 cDNA序列的 PCR可检测的缺 失或易位。 根据目前的物理作图和基因定位技术的分辨能力， 被精确定位至与 疾病有关的染色体区域的 cDNA , 可以是 50至 500个潜在致病基因间之一种（假定 1兆碱基作图分辨能力和每 20kb对应于一个基因）。
可以将本发明的多肽、 多核苷酸及其模拟物、 激动剂、 拮抗剂和抑制剂与 合适的药物载体组合后使用。 这些载体可以是水、 葡萄糖、 乙醇、 盐类、 缓冲 液、 甘油以及它们的组合。 组合物包含安全有效量的多肽或拮抗剂以及不影响 药物效果的载体和赋形剂。 这些组合物可以作为药物用于疾病治疗。
本发明还提供含有一种或多种容器的药盒或试剂盒， 容器中装有一种或多 种本发明的药用组合物成分。 与这些容器一起， 可以有由制造、 使用或销售药 品或生物制品的政府管理机构所给出的指示性提示， 该提示反映出生产、 使用 或销售的政府管理机构许可其在人体上施用。 此外， 本发明的多肽可以与其它 的治疗化合物结合使用。
药物组合物可以以方便的方式给药， 如通过局部、 静脉内、 腹膜内、 肌内、 皮下、 鼻内或皮内的给药途径。 UDP 糖基转移酶 （UGT ) 及钴胺素结合蛋白 11 以有效地治疗和 /或预防具体的适应症的量来给药。 施用于患者的 UDP糖基转移 酶 （UGT ) 及钴胺素结合蛋白 1 1 的量和剂量范围将取决于许多因素， 如给药方 式、 待治疗者的健康条件和诊断医生的判断。

Claims

权 利 要 求 书
1、 一种分离的多肽- UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11 , 其特征在于它 包含有： SEQ ID NO: 2 所示的氨基酸序列的多肽、 或其多肽的活性片段、 类似物或 衍生物。
2、 如权利要求 1 所述的多肽， 其特征在于所述多肽、 类似物或衍生物的氨基酸序 列具有与 SEQ ID NO: 2所示的氨基酸序列至少 95%的相同性。
3、 如杈利要求 2所述的多肽， 其特征在于它包含具有 SEQ ID NO: 2所示的氨基酸 序列的多肽。
4、 一种分离的多核苷酸， 其特征在于所述多核苷酸包含选自下组中的一种：
(a) 编码具有 SEQ ID NO: 2 所示氨基酸序列的多肽或其片段、 类似物、 衍生物 的多核苷酸；
(b) 与多核苷酸（a ) 互补的多核苷酸； 或
(c) 与 （a )或 （b )有至少 70%相同性的多核苷酸。
5、 如权利要求 4所述的多核苷酸，其特征在于所述多核苷酸包含编码具有 SEQ ID NO: 2所示氨基酸序列的多核苷酸。
6、 如权利要求 4所述的多核苷酸， 其特征在于所述多核苷酸的序列包含有 SEQ ID NO: 1中 911 - 1201位的序列或 SEQ ID NO: 1中 1-2194位的序列。
7、 一种含有外源多核苷酸的重组载体， 其特征在于它是由权利要求 4-6 中的任一 权利要求所述多核苷酸与质粒、 病毒或运载体表达载体构建而成的重组载体。
8、 一种含有外源多核苷酸的遗传工程化宿主细胞， 其特征在于它是选自于下列一 种宿主细胞：
(a) 用权利要求 7所述的重组载体转化或转导的宿主细胞； 或
(b) 用权利要求 4- 6中的任一杈利要求所述多核苷酸转化或转导的宿主细胞。
9、 一种具有 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11活性的多肽的制备方法， 其特征在于所述方法包括：
(a) 在表达 UDP 糖基转移酶 （UGT )及钴胺素结合蛋白 11 条件下， 培养权利要 求 8所述的工程化宿主细胞；
(b) 从培养物中分离出具有 UDP糖基转移酶（UGT )及钴胺素结合蛋白 11 活性 的多肽。
10、 一种能与多肽结合的抗体,其特征在于所述抗体是能与 UDP糖基转移酶（UGT ) 及钴胺素结合蛋白 11特异性结合的抗体。
11、 一类模拟或调节多肽活性或表达的化合物， 其特征在于它们是模拟、 促进、 拮抗或抑制 UDP糖基转移酶 （UGT )及钴胺素结合蛋白 11的活性的化合物。
12、 如权利要求 11 所述的化合物， 其特征在于它是 SEQ ID N0: 1所示的多核苷酸 序列或其片段的反义序列。
1 3、 一种杈利要求 11所述化合物的应用， 其特征在于所述化合物用于调节 UDP糖 基转移酶 （UGT )及钴胺素结合蛋白 11在体内、 体外活性的方法。
14、 一种检测与权利要求 1-3中的任一权利要求所述多肽相关的疾病或疾病易感性 的方法， 其特征在于其包括检测所述多肽的表达量， 或者检测所述多肽的活性， 或者检测多核苷酸中引起所述多肽表达量或活性异常的核苷酸变异。
15、 如杈利要求 1-3中的任一权利要求所述多肽的应用， 其特征在于它应用于筛选 UDP糖基转移酶（UGT )及钴胺素结合蛋白 11的模拟物、 激动剂， 拮抗剂或抑制剂； 或者用于肽指紋图谱鉴定。
16、 如权利要求 4-6中的任一权利要求所述的核酸分子的应用， 其特征在于它作为 引物用于核酸扩增反应， 或者作为探针用于杂交反应， 或者用于制造基因芯片或 微阵列。
17、 如权利要求 1-6及 11 中的任一权利要求所述的多肽、 多核苷酸或化合物的应 用， 其特征在于用所述多肽、 多核苷酸或其模拟物、 激动剂、 拮抗剂或抑制剂以 安全有效剂量与药学上可接受的载体组成作为诊断或治疗与 UDP糖基转移酶 （UGT ) 及钴胺素结合蛋白 11异常相关的疾病的药物组合物。
18、 权利要求 1-6及 11中的任一权利要求所述的多肽、 多核苷酸或化合物的应用， 其特征在于用所述多肽、 多核苷酸或化合物制备用于治疗如恶性肿瘤， 血液病， HIV 感染和免疫性疾病和各类炎症的药物。
PCT/CN2001/000784 2000-05-19 2001-05-14 Nouveau polypeptide, proteine 11 de liaison d'une udp-glycosyltransferase (ugt) et d'une cobalamine, et polynucleotide codant ce polypeptide WO2001098505A1 (fr)

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CN107012156A (zh) * 2016-01-28 2017-08-04 刘春生 甘草酸生物合成相关的糖基转移酶基因及其编码产物与应用
CN110462033A (zh) * 2017-05-16 2019-11-15 中国科学院上海生命科学研究院 糖基转移酶、突变体及其应用

Non-Patent Citations (2)

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Title
DATABASE GENBANK [online] 21 December 1999 (1999-12-21), SULSTON J.E. AND WATERSTON R., retrieved from GI:4753279 accession no. NCBI Database accession no. AC006006.2 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107012156A (zh) * 2016-01-28 2017-08-04 刘春生 甘草酸生物合成相关的糖基转移酶基因及其编码产物与应用
CN110462033A (zh) * 2017-05-16 2019-11-15 中国科学院上海生命科学研究院 糖基转移酶、突变体及其应用

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