WO2002012296A1 - Nouveau polypeptide, leucotriene b412-hydroxy deshydrogenase 36 nadp-dependante, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, leucotriene b412-hydroxy deshydrogenase 36 nadp-dependante, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002012296A1
WO2002012296A1 PCT/CN2001/001006 CN0101006W WO0212296A1 WO 2002012296 A1 WO2002012296 A1 WO 2002012296A1 CN 0101006 W CN0101006 W CN 0101006W WO 0212296 A1 WO0212296 A1 WO 0212296A1
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Prior art keywords
polypeptide
polynucleotide
hydroxydehydrogenase
nadp
dependent
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PCT/CN2001/001006
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English (en)
French (fr)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU93635/01A priority Critical patent/AU9363501A/en
Priority to US10/311,746 priority patent/US20040126858A1/en
Publication of WO2002012296A1 publication Critical patent/WO2002012296A1/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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, NADP-dependent leukot ene B412-hydroxydehydrogenase-36, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Leukotriene B 4 (LTB 4 ) is a very potent chemotactic pro-inflammatory factor present in many different tissues [Sarauelsson, B., Dehlen, SE, Lendgren, JA et al., 1987. Science237, 1171-1176].
  • Arachidonic acid is produced by various stimulation of cell membranes, and then converted to 5-peroxyhydroxy eicosadienoic acid and LTA 4 under the action of 5-lipoxygenase [Ruzer, CA, and Satsumoto, T., and Saamuelsson , B.1985. Proc. Natl. Acad. Sci. USA82, 60
  • 1 ⁇ 4 synthesizes LTB 4 under the catalysis of LTA 4 hydrolase [Orning, L., Jones, DA, and Fitzpatrick, FA1990. J. Biol. Chera. 265, 14911-14916]. * LTB 4 is expressed in other tissues in addition to white blood cells. In pig kidney, cytosolic LTB 4 NADP-dependent leukotriene B 4 12-hydroxydehydrogenase [Yokomizo,
  • LTB 4 is a lipid regulator that activates the transfer of white blood cells from blood vessels to produce superoxide anions and releases lysosomal enzymes.
  • the lipid modulator is produced in a variety of different tissues under pathophysiology conditions, such as the kidney or skin.
  • proline-rich domain There is a conserved proline-rich domain at the C-terminus of LTB 4- specific 12-hydroxydehydrogenase from three species.
  • proline-rich domain is involved in tyrosine kinase receptor signal transduction.
  • System and The SH3 domain plays a key role in binding [Pawson, T., and Gish, GD1992.71, 359- 362], the combination of the proline-rich domain and the SH3 domain is involved in the transfer of 5-lipoxygenase And activation, 5-lipoxygenase catalyzes the initial steps of lerkotrienes biosynthesis.
  • NADP-dependent leukotriene B 4 12-hydroxydehydrogenase is widely expressed in human kidney, liver, and intestine tissues.
  • the polypeptide of the present inventor is 99% identical and 99% similar to NADP-dependent leukotriene B 4 12-hydroxydehydrogenase at the protein level, and has similar structural characteristics, and both belong to MDP-dependent leukotriene B 4
  • the 12-hydroxydehydrogenase protein family is named NADP-dependent leukotriene B 4 12-hydroxydehydrogenase-36, and it is speculated that it has similar biological functions.
  • NADP-dependent leukotriene B412-hydroxydehydrogenase-36 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art Identification of more NADP-dependent leukotriene B412-hydroxydehydrogenase-36 proteins involved in these processes, especially the amino acid sequence of this protein.
  • the isolation of the new NADP-dependent leukotriene B412-hydroxydehydrogenase-36 protein encoding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is important. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Another object of the present invention is to provide a method for producing MDP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention-NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Another object of the present invention is to provide diagnostic treatment for NADP-dependent leukotriene B412-hydroxyl Methods for Dehydrogenase-36 Abnormal Associated Diseases.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • polynucleotide sequences of (c) and (a) or (b) have at least 99 '/. Identical polynucleotides.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID NO: 1
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit NADP-dependent leukotr iene B412-hydroxydehydrogenase-36 protein activity, 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 in vitro detection of a disease or disease susceptibility associated with abnormal expression of NADP-dependent eukot ri ene B412-hydroxydehydrogenase-36 protein. Mutations in the acid sequence, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to polypeptides and / or polynucleotides of the present invention prepared for the treatment of cancer, developmental or immune diseases, or other diseases caused by abnormal expression of NADP-dependent leukot ri ene B412-hydroxydehydrogenase-36. Use of drugs.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RM, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally-occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural sequence related to the protein molecule. Amino acid.
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioly active refers to a protein that has the structure, regulatory, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant, or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with MDP-dependent leuko trene ene B412-hydroxydehydrogenase-36, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to NADP-dependent eukotriene B412-hydroxydehydrogenase-36.
  • Antagonist refers to an eukot ri ene B412-hydroxydehydrogenase-blocking or regulating NADP-dependent leukotr iene B412-hydroxydehydrogenase- 36 biologically or immunologically active molecules.
  • Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to NADP-dependent leukot r iene B412-hydroxydehydrogenase-36.
  • Regular refers to changes in the function of MDP-dependent eukot ri ene B412-hydroxydehydrogenase-36, including increased or decreased protein activity, changes in binding characteristics, and NADP-dependent eukot r iene B412-hydroxydehydrogenase. Alteration of any other biological, functional or immune properties of catalase-36.
  • Substantially pure ' 1 means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify MDP-dependent leukotriene B412-hydroxydehydrogenation using standard protein purification techniques. Enzyme-36. Basically pure NADP-dependent leukotriene B412-hydroxydehydrogenase-36 produces a single main band on a non-reducing polyacrylamide gel. NADP-dependent leukotr iene B412-hydroxydehydrogenase-36 The purity of the polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to a polynucleotide that naturally binds by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences be combined with each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene s of tware package, DNASTAR, Inc. 'Madi son Wi s.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244). The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by the Cluster method or by a method known in the art such as Jotun He in (%). He In J., (1990) Methods in emzumo l ogy 183 : 625-645) 0
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? (813 ') 2 and? ⁇ It can specifically bind to the NADP-dependent eukotr iene B412-hydroxydehydrogenase-36 epitope.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of matter from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated NADP-dependent leukotr iene B412-hydroxydehydrogenase-36 means MDP-dependent leukotr iene B412-hydroxydehydrogenase-36 is substantially free of other proteins, lipids, Sugars or other substances.
  • Those skilled in the art can purify NADP-dependent leuko trene B412-hydroxydehydrogenase-36 using standard protein purification techniques.
  • Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of the NADP-dependent leukotr i ene B412-hydroxydehydrogenase-36 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, NADP-dependent leukotr iene B412-hydroxydehydrogenase-36, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of NADP-dependent leukot ri ene B412-hydroxydehydrogenase-36.
  • fragment refers to the basic A polypeptide that retains the same biological function or activity as the NADP-dependent leukotriene B412-hydroxydehydrogenase-36 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by other groups to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 1252 bases and its open reading frame 78-1067 encodes 329 amino acids.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding 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" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide Formula, it may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes.
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least two sequences between
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.13 ⁇ 4SDS, 6 (TC; or (2) during hybridization Add denaturants, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fico ll, 42 ° C, etc .; or (3) the identity between the two sequences only Hybridization occurs at least at least 95%, and more preferably at least 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding MDP-dependent leukotr iene B412-hydroxydehydrogenase-36.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the NADP-dependent eukotr iene B412-hydroxydehydrogenase-36 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDM of interest is to isolate niRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Moleclonal Cloning, A Labora tory Manua, Cold Spruing Harbor Labora tory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDM libraries from Clontech. When combined with polymerase reaction technology, even very small expression products can be cloned.
  • the genes of the present invention can be screened from these CDM libraries by conventional methods. These methods include (but are not limited to Yu): (1) DM-DM or DM-RM hybridization; (2) the presence or absence of marker gene function; (3) determination of NADP-dependent leukotr iene B412-hydroxydehydrogenase-36 transcript levels; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of MDP-dependent leukotr iene B412-hydroxydehydrogenase-36 gene expression can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA )Wait.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA )Wait.
  • a method using PCR technology to amplify bandit A / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a genetically engineered host cell using the vector of the present invention or directly using NADP-dependent leukot r iene B412-hydroxydehydrogenase-36 coding sequence, and recombinant Technology A method of producing a polypeptide of the invention.
  • a polynucleotide sequence encoding MDP-dependent eukotr iene B412-hydroxydehydrogenase-36 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cytopathic 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.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding NADP-dependent leukotr iene B412-hydroxydehydrogenase-36 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Labora tory Manua, Cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRM synthesis. Representative examples of these promoters are: the l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a NADP-dependent leukotr iene B412-hydroxydehydrogenase-36 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a gene containing the polynucleotide or the recombinant vector.
  • Genetically engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the steps used are in the art The domain is well known.
  • MgCl 2 is used.
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant NADP-dependent leukotriene B412-hydroxydehydrogenase-36 (Science, 1984; 224: 1431) by conventional recombinant DNA technology. Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromat
  • FIG. 1 is a comparison diagram of amino acid sequence homology of NADP-dependent leukotriene B412-hydroxydehydrogenase-36 and NADP-dependent leukotriene B412-hydroxydehydrogenase of the present invention.
  • the upper sequence is NADP-dependent leukotriene B412-hydroxydehydrogenase-36
  • the lower sequence is MDP-dependent leukotriene B412-hydroxydehydrogenase.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • 36kDa is the molecular weight of the protein.
  • the arrow points to the isolated protein ⁇ Bands.
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 2651f01 was new DNA.
  • the cloned insert cDNA fragment was bidirectionally determined by synthesizing a series of primers. The results show that the full-length cDNA contained in the 265U01 clone is 1252bp (as shown in Seq ID NO: 1), and there is a 990bp open reading frame (0RF) from 78bp to 1067bp, which encodes a new protein (such as Seq ID NO : Shown in 2).
  • Example 2 Homologous search of cDNA clones
  • NADP-dependent 1 euko triene B412-hydroxydehydrogenase-36 and the protein sequence encoded by the present invention were performed using the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990 215: 403-10], perform homology search in databases such as Genbank, Swissport.
  • the gene with the highest homology to the NADP-dependent leukotriene B412-hydroxydehydrogenase-36 of the present invention is a known NADP-dependent leukotriene B412-hydroxydehydrogenase, and the accession number of the encoded protein in Genbank is: D49387.
  • the results of protein homology are shown in Figure 1.
  • Example 3 Cloning of a gene encoding NADP-dependent leukotriene B412-hydroxydehydrogenase-36 by RT-PCR CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imerl 5'- TCCTTGGAGAGCTTGGAGCCGCGC-3, (SEQ ID NO: 3)
  • Primer2 5,-CATAGGCCGAGGCGGCCGACATGT -3, (SEQ ID NO: 4)
  • Pr imerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l KL of KC1, 10 mraol / L Tris-Cl, (pH 8.5.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L in a reaction volume of 50 ⁇ 1 dNTP, l Opmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a PCR vector using a TA cloning kit (Invitrogen).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-1252bp shown in SBQ ID NO: 1.
  • Example 4 Northern blot analysis of MDP-dependent leukotr iene B412-hydroxydehydrogenase-36 gene expression:
  • Total MA was extracted in one step [Anal. Biochem 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • Electrophoresis was performed on a 1.2% agarose gel containing 2 ⁇ g of RNA on 20 mM 3- (N-morphine) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2 M formaldehyde then transferred to nitrocellulose by a -.
  • 32 P dATP Preparation 32 P- DNA probe labeled by the random primer SYSTEM DNA probes used for PCR amplification shown in FIG NADP-dependent 1 euko. Triene B412-hydroxydehydrogenase-36 coding region sequence (78bp to 1067bp).
  • a pair of specific amplification primers is designed. as follows:
  • Primer 3 5'-CATGCTAGCATGGTTCGTACTAAGACATGGACC-3 '(Seq ID No: 5)
  • Priraer4 5'-CCCGAATTCTCATGCTTTCACTATTGTCTTCCC-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and EcoRI digestion sites, respectively, followed by the coding sequences of the 5 'and 3' ends of the target gene, respectively.
  • the Nhel and EcoRI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (product of Novagen, 0 & ⁇ . 69865.3).
  • the PCR reaction was performed using pBS-2651f01 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1, a plasmid containing 10 pg of pBS-2651f 01, primers ⁇ 1 ⁇ 1! 16: 1: -3 and]: 1 [116: 1: -4 points and another!] Is 1 ( ⁇ 11101, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, 25 cycles in total. Nhel and BcoRI were used for amplification products and plasmid pET-28, respectively. (+) Digestion with two enzymes to recover large fragments and ligase them with T4 ligase.
  • the ligation product was transformed into Escherichia coli DH5 CC by calcium chloride method, in LB plates containing kanamycin (final concentration 30 g / ml). After overnight culture, positive clones were screened by colony PCR and sequenced. Positive clones (pET-2651f01) with the correct sequence were selected. The recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the host bacteria BL21 (pET-2651f01) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mol / L, Continue to cultivate for 5 hours. Collect the cells by centrifugation, break the bacteria by ultrasound, collect the supernatant by centrifugation, and use 6 histidine (6His-Tag) binding affinity column His. Bind Quick Cartridge (Novagen) was chromatographed to obtain purified NADP-dependent leukotriene B412-hydroxydehydrogenase-36, a target protein of interest.
  • a peptide synthesizer (product of PE) was used to synthesize the following NADP-dependent leukotriene B412-hydroxydehydrogenase-36-specific peptides:
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avrameas, et al. Imm, chemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex with complete Freund's adjuvant, and 15 days later, the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Nor thern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps. In this embodiment, higher-intensity washing conditions (such as lower salt concentration and higher temperature) are used to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments which are completely identical or mutually identical to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention. Polynucleotide of the invention SEQ ID NO: 1 Identical or complementary oligonucleotide fragments.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • GC content is 30% -70 /. If it exceeds, non-specific hybridization increases;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements Region for homology comparison, if the homology with non-target molecular region is greater than 85% or more than 15 Two consecutive bases are completely the same, the primary probe should generally not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence (fiber) of the gene fragment of SEQ ID NO: 1 or its complementary fragment ...
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • 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 the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immunity. Disease, etc.
  • Leuko tri ene B 4 is a chemotactic pro-inflammatory factor present in many different tissues; at the same time, it is also a lipid that activates the transfer of white blood cells from blood vessels to produce superoxide anions and releases lysosomal enzyme lipids Regulator.
  • NADP-dependent eukotr iene B 4 12-hydroxydehydrogenase converts LTB 4 to 12-oxidized-LTB 4 , which is 100 times less active than LTB 4 .
  • Abnormal expression of NADP-dependent leukot r iene B 4 12 -hydroxydehydrogenase can cause metabolic disorders of LTB 4 and lead to the occurrence of related diseases.
  • the polypeptide of the present invention and NADP-dependent euko tri ene B 4 12-hydroxydehydrogenase are NADP-dependent leukot r iene B412-hydroxydehydrogenases, which contain characteristic sequences of the family, and both have similar biological functions.
  • this abnormal expression of the polypeptide in vivo metabolic disorders can cause LTB 4,
  • LTB 4 is a proinflammatory chemotactic factors associated with the body's metabolism and superoxide, which can lead to various inflammatory metabolic disorders, oxygen free radical injury Of sexually transmitted diseases, including but not limited to: various inflammations
  • Serous inflammation tuberculous pleura, rheumatoid arthritis, acute rhinitis, etc .;
  • Purulent inflammation superficial purulent and purulent (purulent urethritis, purulent bronchitis, purulent cholecystitis), cellulitis (skin, muscle, appendix), abscess ( ⁇ , ⁇ , sinus, sacral canal) ) Etc .; 2. Chronic inflammation:
  • Heart failure essential hypertension, atherosclerosis, coronary atherosclerotic heart disease, cardiomyopathy, viral myocarditis, etc .;
  • Iron deficiency anemia megaloblastic anemia, aplastic anemia, glucose-6-phosphate dehydrogenase deficiency, oceanic anemia, myelodysplastic syndrome, acute leukemia, lymphoma, multiple myeloma, diffuse Intravascular coagulation (DIC), etc .;
  • Cerebral edema head injury, hypertensive cerebral hemorrhage, subarachnoid hemorrhage, cerebral infarction, tremor paralysis, epilepsy, viral encephalitis, etc .;
  • Lung cancer esophageal cancer, gastric cancer, chest trauma, etc .
  • polypeptide of the present invention and the antagonist, agonist and inhibitor of the polypeptide can be directly used for the treatment of various diseases, such as various inflammations, oxygen free radical injury diseases, and the like.
  • the invention also provides screening compounds to identify increasing (agonist) or suppressing (antagonist) NADP dependence Method for the preparation of leukotriene B412-hydroxydehydrogenase-36.
  • Agonists increase NADP-dependent leukotriene B412-hydroxydehydrogenase_36 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing NADP-dependent leukotriene B412-hydroxydehydrogenase-36 and labeled NADP-dependent leukotriene B412-hydroxydehydrogenase-36 can be cultured in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of NADP-dependent leukotriene B412-hydroxydehydrogenase-36 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • the antagonist of NADP-dependent leukotriene B412-hydroxydehydrogenase-36 can bind to NADP-dependent leukotriene B412-hydroxydehydrogenase-36 and eliminate its function, or inhibit the production of the polypeptide, or the activity of the polypeptide Site binding prevents the polypeptide from performing its biological function.
  • MDP-dependent leukotriene B412-hydroxydehydrogenase-36 can be added to a bioanalytical assay, and by measuring the compounds' NADP-dependent leukotriene B412-hydroxydehydrogenase-36 and its receptor The effect of this interaction is used to determine whether the compound is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Peptide molecules capable of binding to NADP-dependent leukotriene B412-hydroxydehydrogenase-36 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the NADP-dependent leukotriene B412-hydroxydehydrogenase-36 molecule should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the NADP-dependent leukotriene B412-hydroxydehydrogenase-36 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by NADP-dependent leukotriene B412-hydroxydehydrogenase-36 direct injection in immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but It is not limited to Freund's adjuvant and the like.
  • Techniques for preparing MDP-dependent leukotriene B412-hydroxydehydrogenase-36 monoclonal antibodies include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta cells Hybridoma technology, EBV-hybridoma technology, etc.
  • the chimeric antibody variable region and a human constant region of non-human origin in combination produce the available prior art (Morrison et al, PNAS, 1985 , 81: 6851) 0 Ersi some technical production of single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Antibodies against NADP-dependent leukotriene B412-hydroxydehydrogenase-36 can be used in immunohistochemistry to detect NADP-dependent leukotriene B412-hydroxydehydrogenase-36 in biopsy specimens.
  • Monoclonal antibodies that bind to NADP-dependent leukotriene B412-hydroxydehydrogenase-36 can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • NADP-dependent leukotriene B412-hydroxydehydrogenase-36 high affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill NADP-dependent leukotriene B412-hydroxy dehydrogenation Enzyme-36 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of NADP-dependent leukotriene B412-hydroxydehydrogenase-36 levels.
  • tests are well known in the art and include FISH and radioimmunoassays.
  • the levels of NADP-dependent leukotriene B412-hydroxydehydrogenase-36 detected in the test can be used to explain the importance of NADP-dependent leukotriene B412-hydroxydehydrogenase-36 in various diseases and to diagnose NADP-dependent Diseases where leukotriene B412-hydroxydehydrogenase-36 works.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry.
  • a polynucleotide encoding a NADP-dependent leukotriene B412-hydroxydehydrogenase-36 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated NADP-dependent leukotriene B412-hydroxydehydrogenase-36 to inhibit endogenous NADP-dependent leukotriene B412-hydroxydehydrogenase-36 activity.
  • a variant of NADP-dependent leukotriene'B412-hydroxydehydrogenase-36 can be shortened and lack the signaling domain of NADP-dependent leukotriene B412-hydroxydehydrogenase-36. Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding a NADP-dependent leukotriene B412-hydroxydehydrogenase-36 can be used to transfer a polynucleotide encoding a NADP-dependent leukotriene B412-hydroxydehydrogenase-36 into a cell .
  • Method hydroxy dehydrogenase polynucleotide -36 recombinant viral vectors can be found in existing literature (Sambrook, e t al.) .
  • a recombinant polynucleotide encoding NADP-dependent leukotriene B412-hydroxydehydrogenase-36 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit NADP-dependent leukotriene B412-hydroxydehydrogenase-36 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology. For example, solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond is used instead of the phosphodiester bond to link the ribonucleosides.
  • a polynucleotide encoding MDP-dependent leukotriene B412-hydroxydehydrogenase-36 can be used for the diagnosis of diseases related to MDP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • a polynucleotide encoding MDP-dependent leukotriene B412-hydroxydehydrogenase-36 can be used to detect the expression of NADP-dependent leukotriene B412-hydroxydehydrogenase-36 or MDP-dependent leukotriene B412-hydroxydehydrogen in a disease state Abnormal expression of enzyme-36.
  • the DM sequence encoding NADP-dependent leukotriene B412-hydroxydehydrogenase-36 can be used to hybridize biopsy specimens to determine the expression status of NADP-dependent leukotriene B412-hydroxydehydrogenase-36.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and so on. These techniques and methods are publicly available and mature, and related kits are available commercially.
  • Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DM chip (also known as a "gene chip") for differential expression analysis and gene diagnosis of genes in tissues.
  • NADP-dependent leukotriene B412-hydroxydehydrogenase-36 specific primers can also be used to detect the transcription products of NADP-dependent leukotriene B412-hydroxydehydrogenase-36 in vitro amplification by RNA-polymerase chain reaction (RT-PCR).
  • RT-PCR RNA-polymerase chain reaction
  • MDP-dependent leukotriene B412-hydroxydehydrogenase-36 mutant forms include point mutations, translocations, deletions, recombination and recombination compared to the normal wild-type MDP-dependent leukotriene B412-hydroxydehydrogenase-36 DM sequence Any other anomalies, etc. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckus i ck, Mende l ian Inher i tance in Man (available online with Johns Hopk ins Un ivers i ty Wel ch Med ica l L brary). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDM or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be combined with Use after suitable drug carrier combination.
  • suitable drug carrier combination can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptide of the present invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • NADP-dependent leukot r ene B412-hydroxydehydrogenase-36 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of NADP-dependent leukot r iene B412-hydroxydehydrogenase-36 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

Description

一种新的多肽一一 NADP依赖性 leukotriene B412-羟基脱氢酶- 36
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 NADP 依赖性 leukot ene B412-羟基脱氢酶- 36, 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
Leukotriene B4 ( LTB4 ) 是一个存在于许多不同组织中非常有效的趋化性 促 炎 因 子 [Sarauelsson, B. , Dehlen, S. E. , Lendgren, J. A. et al. , 1987. Science237, 1171-1176]. 通过各种刺激细胞膜产生花生四烯酸， 然后 在 5-脂肪 氧化酶 的 作用 下 转 变 为 5-过氧羟基二 十碳二烯酸和 LTA4 [Ruzer, C. A. , and Satsumoto, T. , and Saamuelsson, B.1985. Proc. Natl. Acad. Sci. U. S. A.82, 60
40- 6044]。 1^ 4在 LTA4水解酶的催化下合成 LTB4 [Orning, L. , Jones, D. A. , and Fitzpatrick, F. A.1990. J. Biol. Chera.265, 14911-14916]。 *LTB4 除了在白血球中 表达外还表达于其他组织。 在猪的肾脏中， 已经提纯出胞液 LTB4 NADP 依赖性 leukotriene B412-羟基脱氢酶 [Yokomizo,
T. , Izumi, Τ. , Takahashi, Τ. , Kasama, Τ, et
al. , 1993. J. Biol. Chem.268, 18128-18135] 0 在 NADP 的参与下， 该酶将 LTB4转 换为 12-氧化- LTB4。 在人白血球钙积聚过程中， 12-氧化- LTB4 的活性比 LTB4 要低 100倍。 LTB4是一个激活白血球从血管转运产生超氧阴离子， 并释放溶酶体 酶的脂质调节物。 该脂质调节物在病理生理学的条件下产生于各种不同的组织， 例如肾脏或皮肤。
在猪肾脏中提取的 LTB4特异性 12 -羟基脱氢酶与猪的分叶核白细胞酶有本 质上的不同 [Wainwright, S. L. , and Powell, W. S.1991. J. Biol. Chem.266, 20899- 20906]。 该酶虽然也同样将 LTB4转换成 12-氧化- LTB4 , 但它是胞液酶并且 NADP 作为辅助因子。 猪的 LTB4特异性 12-羟基脱氢酶基因包括一个有 987bp的开放阅 读框， 编码 329个氨基酸残基， 含有所有的赖氨酸 -C- digested肽段， 分子量为 35, 761。 在三个物种的 LTB4特异性 12-羟基脱氢酶 C末端有一个保守的富含脯 氨酸的结构域， 实验表明该富含脯氨酸结构域在酪氨酸激酶受体信号转导系统与 SH3 结构域结合时起着非常关键的作用 [Pawson, T. , and Gish, G. D.1992.71, 359- 362], 富含脯氨酸结构域与 SH3 结构域的结合参与 5-脂肪氧化酶的转移和激活， 5 -脂肪氧化酶催化 lerkotrienes生物合成的起始步骤。
NADP依赖性 leukotriene B412-羟基脱氢酶广泛表达于人的肾脏、 肝脏和 肠等组织。
本发明人的多肽在蛋白质水平上与 NADP依赖性 leukotriene B412-羟基脱氢 酶有 99%的相同性和 99%的相似性， 并具有相似的结构特征， 同属于 MDP依赖性 leukotriene B412-羟基脱氢酶蛋白家族，故命名为 NADP依赖性 leukotriene B412- 羟基脱氢酶 -36， 同时推测其具有相似的生物学功能。
由于如上所述 NADP依赖性 leukotriene B412-羟基脱氢酶 -36 蛋白在调节细 胞分裂和胚胎发育等机体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 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36 蛋白， 特别是鉴定这种蛋白的氨基酸序列。 新 NADP依赖性 leukotriene B412-羟基脱氢酶- 36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 NADP依赖性 leukotriene B412- 羟基脱氢酶 -36 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 NADP依赖性 leukotriene B412-羟基脱 氢酶 -36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 NADP依赖性 leukotriene B412-羟基脱 氢酶- 36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 MDP依赖性 leukotriene B412-羟基脱氢酶 - 36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 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36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 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36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 NADP依赖性 leukotriene B412-羟基 脱氢酶 -36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 变体：
(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 99 '/。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
78-1067位的序列； 和（b)具有 SEQ ID NO: 1中 1-1252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 NADP依赖性 leukotr iene B412-羟基脱氢酶- 36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 NADP依赖性 l eukot r i ene B412-羟基脱氢酶- 36 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 NADP依赖性 leukot r i ene B412-羟基脱氢酶- 36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 RM, 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 ^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 MDP依赖性 leuko tr i ene B412-羟基脱氢酶- 36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 NADP依赖性 l euko t r i ene B412-羟基脱 氢酶- 36的分子。
"拮抗剂" 或 "抑制物" 是指当与 NADP依赖性 l eukotr iene B412-羟基脱氢酶 -36结合时， 一种可封闭或调节 NADP依赖性 l eukot r i ene B412-羟基脱氢酶- 36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 NADP依赖性 leukot r iene B412-羟基脱氢酶- 36的分子。
"调节" 是指 MDP依赖性 l eukot r i ene B412-羟基脱氢酶- 36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 NADP依赖性 l eukot r iene B412 - 羟基脱氢酶 -36的任何其它生物学性质、 功能或免疫性质的改变。
"基本上纯'1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 MDP依赖性 leukotriene B412- 羟基脱氢酶- 36。 基本上纯的 NADP依赖性 leukotriene B412-羟基脱氢酶 -36在非还 原性聚丙烯酰胺凝胶上能产生单一的主带。 NADP 依赖性 leukotr iene B412-羟基脱 氢酶 -36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 Nor thern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 s of tware package, DNASTAR, Inc. ' Madi son Wi s. ) 。 MEGALIGN程序可根据不同 的方法如 Clus ter法比较两种或多种序列（Higg ins , D. G. 和 P. M. Sharp (1988) Gene 73: 237-244)。 C lus 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 C lus ter法或用本领域周知的方法如 Jotun He in 测定核酸序列之 间的相同性百分率（He in J. , (1990) Methods i n emzumo l ogy 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、 ？(813') 2及？^ 其能特异性 结合 NADP依赖性 l eukotr iene B412-羟基脱氢酶- 36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 NADP依赖性 leukotr iene B412-羟基脱氢酶- 36" 是 指 MDP依赖性 leukotr iene B412 -羟基脱氢酶 - 36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 NADP 依赖性 leuko t r i ene B412-羟基脱氢酶- 36。 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。 NADP依赖性 leukotr i ene B412-羟基脱氢 酶- 36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 NADP依赖性 leukotr iene B412-羟基脱氢酶- 36 , 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 NADP依赖性 leukot r i ene B412-羟基脱氢酶- 36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 NADP依赖性 leukotriene B412-羟基脱氢酶 -36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 （ IV ) 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 cDM 文库中发现的。 它包含的 多核苷酸序列全长为 1252个碱基， 其开放读框 78-1067编码了 329 个氨基酸。 根据氨基酸序列同源比较发现， 此多肽与 NADP 依赖性 leukot riene B412-羟基 脱氢酶有 99%的同源性， 可推断出该 NADP依赖性 l eukotr iene B412 -羟基脱氢酶 -36具有 NADP依赖性 leukotriene B412-羟基脱氢酶相似的结构和功能。
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 DM或人工合成的 DNA。 DNA 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 NO: 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. 1¾SDS， 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%Fico l 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 MDP依赖性 leukotr iene B412-羟基脱氢酶- 36 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 NADP依赖性 l eukotr iene B412-羟基脱氢酶- 36 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 DM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 DNA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 cDM的 标准方法是从高表达该基因的供体细胞分离 niRM 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 iagene)。 而构建 cDNA文库也是通常的方法（Sambrook, et a l . , Mo l ecular C loning, A Labora tory Manua l , Cold Spr ing Harbor Labora tory. New York , 1989)。 还可得到商业供应的 cDNA文库， 如 C lontech公司的不同 cDM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 cDM文库中筛选本发明的基因。 这些方法包括（但不限 于）： (1) DM- DM或 DM- RM杂交; (2)标志基因功能的出现或丧失； (3)测定 NADP 依赖性 leukotr iene B412-羟基脱氢酶- 36 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 DM序列。 本发明的基因本身或者片段当然可以用作探针。 DNA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 MDP依赖性 leukotr iene B412-羟基脱氢酶- 36 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 匪 A/RNA 的 方 法 （Sa iki , et a l. Sc i ence 1985; 230: 1350- 1354)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 DM/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 NADP依赖性 leukot r iene B412-羟基脱氢酶 -36 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 MDP依赖性 l eukotr iene B412-羟基脱氢酶 -36 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 al. Gene, 1987, 56: 125)；在哺乳动物细胞中表达的 pMSXND表达载体 (Lee and Nathans , J Bi o 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 NADP 依赖性 leukotr iene B412-羟基脱氢酶- 36 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 DNA技术、 DM合成技术、 体内重组技术等（Sambroook, e t a l. Molecular Cloning, a Labora tory 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 mRM合成。 这些启动子的代表性例子有： 大肠杆菌的 l ac 或 trp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 核细胞或真核细胞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 10 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 100 到 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 NADP依赖性 leukotr iene B412-羟基脱氢酶- 36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 Sf 9 ; 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 DNA 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 MgCl2。 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 NADP依赖性 leukotrieneB412-羟基脱氢酶- 36 (Science, 1984; 224： 1431)。 一般来说有以下步骤：
(1).用本发 的编码人 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36 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， 而不用于限定由杈利要求书所 界定的本发明范围。
图 1是本发明 NADP依赖性 leukotriene B412-羟基脱氢酶 -36和 NADP依赖性 leukotriene B412-羟基脱氢酶的氨基酸序列同源性比较图。 上方序列是 NADP依 赖性 leukotriene B412-羟基脱氢酶- 36 , 下方序列是 MDP依赖性 leukotriene B412-羟基脱氢酶。 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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36的聚丙烯酰胺 凝胶电泳图 （SDS-PAGE)。 36kDa 为蛋白质的分子量。 箭头所指为分离出的蛋白 π 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 Spring 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: NADP依赖性 leukotriene B412-羟基脱氢酶 -36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 Isolation Kit
(Qiegene 公司产品） 从总 RNA中分离 poly (A) mRNA。 2ug poly (A) raRM经逆转录形 成 c賺。 用 Smart cDM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 cDNA文库。 用 Dye terminate cycle reaction sequencing Id 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 列与巳有的公共 DNA序列数据库（Genebank )进行比较，结果发现其中一个克隆 2651f01 的 cDNA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， 265U01克隆所含的全长 cDNA为 1252bp (如 Seq ID NO: 1所示） , 从第 78bp至 1067bp有一个 990bp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- 2651f 01, 编码的蛋白质命名为 NADP 依赖性 leukotriene B412-羟基脱氣酶 -36。 实施例 2: cDNA 克隆的同源检索
将本发明的 NADP依赖性 1 euko triene B412-羟基脱氢酶 -36的序列及其编码的蛋 白序列， 用 Blast程序（Basiclocal Alignment search tool) [Altschul, SF et al. J.Mol. Biol.1990; 215: 403-10], 在 Genbank、 Swissport等数据库进行同源检索。 与 本发明的 NADP依赖性 leukotriene B412-羟基脱氢酶 -36同源性最高的基因是一种已 知的 NADP依赖性 leukotriene B412-羟基脱氢酶， 其编码的蛋白在 Genbank的准入号 为 D49387。 蛋白质同源结果示于图 1， 两者高度同源， 其相同性为 99°/»; 相似性为 99%。 实施例 3: 用 RT-PCR方法克隆编码 NADP依赖性 leukotriene B412-羟基脱氢酶- 36的基 因 用胎脑细胞总 RNA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：
Pr imerl : 5'- TCCTTGGAGAGCTTGGAGCCGCGC—3， (SEQ ID NO: 3)
Primer2: 5，- CATAGGCCGAGGCGGCCGACATGT -3， (SEQ ID NO: 4)
Pr imerl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 50讓 ol/L KC1 , 10mraol/L Tr i s- Cl, (pH8. 5) , 1. 5mmol/L MgCl2, 200 μ mol/L dNTP, l Opmol引物， 1U的 Taq DNA聚合酶 (Clontech公司产品）。 在 PE9600型 DNA热循环仪（Perkin- Elmer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（Invi trogen公司产品） 。 DNA序列分析结果表明 PCR产物的 DNA序列与 SBQ ID NO: 1所示的 1- 1252bp完全相同。 实施例 4: Northern 印迹法分析 MDP依赖性 leukotr iene B412-羟基脱氢酶- 36基因 的表达：
用一步法提取总 MA [Anal. Biochem 1987, 162, 156-159]。 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 2 (^ g RNA, 在含 20mM 3- ( N-吗淋代） 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 32P-标记的 DNA探针。 所用 的 DNA探针为图 1所示的 PCR扩增的 NADP依赖性 1 euko triene B412-羟基脱氢酶- 36编码 区序列（78bp至 1067bp)。 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 χ SSC- 5 x Denhardt's溶液和 200 μ g/ml鲑精 DNA。 杂交之后， 将滤膜在 1 x SSC- 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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36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：
Primer 3: 5'-CATGCTAGCATGGTTCGTACTAAGACATGGACC-3' ( Seq ID No: 5)
Priraer4: 5'-CCCGAATTCTCATGCTTTCACTATTGTCTTCCC-3' (Seq ID No: 6 ) 此两段引物的 5'端分别含有 Nhel和 EcoRI酶切位点， 其后分别为目的基因 5'端和 3'端的编码序列， Nhel和 EcoRI酶切位点相应于表达载体质粒 pET- 28b(+) (Novagen公 司产品， 0&^^.69865.3)上的选择性内切酶位点。 以含有全长目的基因的 pBS- 2651f01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- 2651f 01 质粒 10pg、 引物卩1^1!16：1：-3和 ]：1[116：1：-4分另!]为1(^11101、 Advantage polymerase Mix (Clontech公司产品） 1μ1。 循环参数： 94。C 20s, 60°C 30s, 68°C 2 min,共 25个循 环。 用 Nhel和 BcoRI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 DH5 CC，在含卡那霉素 （终 浓度 30 g/ml) 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- 2651f01 ) 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 30μ§/ηι1) 的 LB液体培 养基中， 宿主菌 BL21 (pET- 2651f01 ) 在 37°C培养至对数生长期， 加入 IPTG至终浓度 1匪 ol/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 NADP依赖性 leukotriene B412 -羟基脱氢酶 - 36。 经 SDS- PAGE电泳， 在 36kDa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 MDP依赖性 leukotriene B412-羟基脱氢酶- 36抗体的产生
用多肽合成仪（PE公司产品） 合成下述 NADP依赖性 leukotriene B412 -羟基脱氢 酶 -36特异性的多肽：
NH2-Met-Val-Arg-Thr-Lys-Thr-Trp-Thr-Leu-Lys-Lys-His-Phe-Val-Gly-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. Imm画 chemistry, 1969; 6: 43。 用 4mg上述血蓝蛋白多肽复合物力口 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。 釆用经 15yg/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 IgG。 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 NADP依赖性 leukotriene B412-羟基脱氢酶 - 36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、 Nor thern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 相同或互朴的寡核苷酸片段； 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'-TGGTTCGTACTAAGACATGGACCCTGAAGAAGCACTTTGTT-3' ( SEQ ID NO: 8 )
探针 2 (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 (纖）··
5'-TGGTTCGTACTAAGACATGGCCCCTGAAGAAGCACTTTGTT-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 PROBBS G. H. Keller; M. M. 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》 U998 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。 搡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 ( 0.25mol/L蔗糖； 25mmol/L Tris-HCl, pH7.5; 25 ol/LnaCl; 25mmol/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 ) 1000g离心 10 分钟。 6 ) 用重悬细胞沉淀（每 O. lg最初组织样品加 1- 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。 1 )用冷细胞裂解液 重悬浮沉淀的细胞 （1 x lO8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 20(kg/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 DNA的水相转移至新管。 然后进行 DM 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 2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 TB, 混合至 DNA充分溶解， 每 1- 5χ 106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 RM酶 A加到 DNA溶液中, 终浓度为 100ug/ml, 37°C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 A2S。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. Imol/LNaOH, 1.5mol/LNaCl 的滤纸上 5 分钟 (两次）, 晾干置 于浸润有 0.5mol/L Tris-HCl ( pH7.0 ), 3mol/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 IProbe ( 0.10D/10 μ 1 ), 加入 2 μ IKinase缓冲液， 8-10 uCi γ- 32P- dATP+2U Kinase, 以补加至终体积 20 μ1。
2) 37 °C 保温 2小时。
3)加 1/5体积的溴酚蓝指示剂 (BPB X 4 ) 过 Sephadex G-50柱。
5 ) 至有 32P- Probe洗出前开始收集第一峰（可用 Moni tor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 DNA (小牛胸腺 DM )。 ), 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 )取出巳杂交好的样膜。
2 ) 2xSSC, 0. 1 SDS中， 40。C洗 15分钟 ( 2次）。
3 ) 0. lxSSC , 0. 1½SDS中， 40°C洗 15分钟 （ 2次）。
4 ) 0. lxSSC , 0. 1½SDS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：
釆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； 而桌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。
Leuko t r i ene B4 ( LTB4 ) 是一个存在于许多不同组织中的趋化性促炎因子； 同时， 它又是一个激活白血球从血管转运产生超氧阴离子， 并释放溶酶体酶的脂 质调节物。 在体内， NADP依赖性 l eukotr i ene B412-羟基脱氢酶将 LTB4转换为 12- 氧化- LTB4， 后者的活性比 LTB4要低 100倍。 NADP依赖性 leukot r iene B412 -羟 基脱氢酶的表达异常可引起 LTB4的代谢紊乱， 进而导致相关疾病的发生。
本发明的多肽与 NADP 依赖性 l euko t r i ene B412-羟基脱氢酶是 NADP 依赖性 leukot r iene B412-羟基脱氢酶， 含该家族的特征性序列， 两者具有相似的生物 学功能， 本多肽在体内的异常表达可引起 LTB4的代谢紊乱， LTB4是一种趋化性 促炎因子并与机体超氧化物的代谢有关， 其代谢紊乱可导致各种炎症、 氧自由基 损伤性疾病的发生， 这些疾病包括但不限于： 各种炎症
一. 急性炎症：
1. 浆液性炎： 结核性胸膜样， 风湿性关节炎， 急性鼻炎等；
2. 纤维素性炎： 粘膜纤维素性炎 （白喉， 细菌性痢疾） ， 浆膜纤维素性炎 （风 湿性心包炎， 大叶性肺炎） 等；
3. 化脓性炎症： 表面化脓和积脓 （化脓性尿道炎， 化脓性支气管炎， 化脓性胆 囊炎） ， 蜂窝织炎 （皮肤， 肌肉， 阑尾） ， 脓肿 （疖， 痈， 窦道， 痿管） 等； 二. 慢性炎症：
1. 一般类型： 慢性胆囊炎， 慢性肾盂肾炎， 结核病， 矽肺， 类风关等；
2. 特殊类型： 慢性肉芽肿性炎症 （结核性肉芽肿， 血吸虫性肉芽肿， 异物性肉 芽肿） 等； ' 三. 其它： 炎性息肉 （鼻粘膜， 宫颈） ， 炎性假瘤等；
氧自由基损伤相关疾病
一. 心血管疾病
心力衰竭， 原发性高血压， 动脉粥样硬化， 冠状动脉粥样硬化性心脏病， 心肌病， 病毒性心肌炎.等；
二. 消化系统疾病
胃炎， 消化性溃疡， 胃癌， 溃疡性结肠炎， Crohn 病， 肝硬化， 原发性肝 癌， 急性胰腺炎， 胆石病等； 三. 泌尿系统疾病
原发性肾小球疾病， 慢性肾小球肾炎， 肾病综合征， 阵发性睡眠性血红蛋 白尿， 溶血尿毒综合征， 肾盂肾炎， 急慢性肾功能不全， 肾移植， 慢性前列 腺炎等；
四. 呼吸系统疾病
上呼吸道感染， 支气管哮喘， 呼吸衰竭， 成人呼吸窘迫综合征 （ARDS ) , 阻塞性肺气肿， 慢性肺源性心脏病， 肺水肿， 肺炎等；
五. 代谢内分泌疾病
糖尿病， 肝豆状核变性， 慢性淋巴细胞性甲状腺炎， 类风湿关节炎， 系统 性红斑狼疮 （ SLE ) 等；
六. 血液系统疾病
缺铁性贫血， 巨幼细胞性贫血， 再生障碍性贫血， 葡萄糖 - 6 -磷酸脱氢 酶缺陷症， 海洋性贫血， 骨髓增生异常综合征， 急性白血病， 淋巴瘤， 多发 性骨髓瘤， 弥散性血管内凝血 （DIC ) 等；
七. 神经系统疾病
脑水肿， 颅脑损伤， 高血压性脑出血， 蛛网膜下腔出血， 脑梗死， 震颤麻 痹， 癫痫， 病毒性脑炎等；
八. 新生儿疾病
新生儿窒息， 新生儿吸入综合征， 新生儿缺血缺氧性脑病， 新生儿上呼吸 道感染， 新生儿肺炎， 新生儿透明膜病， 支气管肺发育不良， 新生儿败血症， 新生儿硬肿症， 地中海贫血等；
九. 外科疾病
肺癌， 食管癌， 胃癌， 胸部创伤等；
十. 妇产科疾病
流产， 胎膜早破， 子宫内膜异位症， 妊娠高血压综合症， 子宫颈癌， 外阴 恶性胂瘤， 子宫内膜癌等；
十一五官科疾病
复发性口腔溃疡， 牙髓炎， 口腔鳞癌， 喉癌， 突发性耳聋， 老年性白内障等； 十二其他， 如多脏器功能失调综合征、 休克等；
综合上述， 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 用于多种疾病的治疗， 例如各种炎症、 氧自由基损伤性疾病等。
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） NADP依赖性 leukotriene B412-羟基脱氢酶- 36 的药剂的方法。 激动剂提高 NADP 依赖性 leukotriene B412-羟基脱氢酶 _36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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36 的膜制剂与标记的 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36 —起培养。 然后测定药物提高或 阻遏此相互作用的能力。
NADP依赖性 leukotriene B412-羟基脱氢酶- 36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。 NADP依赖性 leukotriene B412-羟基脱氢酶- 36 的拮抗剂可以与 NADP依赖性 leukotriene B412-羟基脱氢酶 -36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 MDP依赖性 leukotriene B412-羟基 脱氢酶 -36 加入生物分析测定中， 通过测定化合物对 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36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 NADP依赖性 leukotriene B412-羟基脱氢酶 -36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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36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 MDP依赖性 leukotriene B412-羟基脱氢 酶- 36 的单克隆抗体的技术包括但不限于杂交瘤技术（Kohler and Milstein. 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 (Morrison et al , PNAS, 1985, 81: 6851) 0 而巳有的生产单链抗体的技术（U. S. Pat No.4946778)也可用于生产抗 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36 的 单链抗体。 抗 NADP依赖性 leukotriene B412-羟基脱氢酶- 36的抗体可用于免疫组织化 学技术中， 检测活检标本中的 NADP依赖性 leukotriene B412-羟基脱氢酶 -36。
与 NADP依赖性 leukotriene B412-羟基脱氢酶- 36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 NADP 依赖性 leukotriene B412-羟基脱氢酶 -36 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 NADP依赖性 leukotriene B412-羟基脱氢酶 -36阳性的细胞。
本发明中的抗体可用于治疗或预防与 NADP依赖性 leukotriene B412-羟基脱 氢酶 -36 相关的疾病。 给予适当剂量的抗体可以刺激或阻断 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36的产生或活性。
本发明还涉及定量和定位检测 NADP依赖性 leukotriene B412-羟基脱氢酶- 36 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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36水平， 可以用作解释 NADP依赖性 leukotriene B412-羟基脱氢酶- 36在各种疾病中的重 要性和用于诊断 NADP依赖性 leukotriene B412-羟基脱氢酶- 36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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36 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 NADP依赖性 leukotriene B412-羟基 脱氢酶 -36 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 NADP 依赖性 leukotriene B412-羟基脱氢酶 -36, 以抑制内源性的 NADP依赖性 leukotriene B412-羟基脱氢 酶 -36 活性。 例如， 一种变异的 NADP 依赖性 leukotriene'B412-羟基脱氢酶 -36 可以是缩短的、 缺失了信号传导功能域的 NADP依赖性 leukotriene B412-羟基脱 氢酶- 36， 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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36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 NADP依赖性 leukotriene B412-羟基脱 氢酶- 36的多核苷酸转移至细胞内。构建携带编码 NADP依赖性 leukotriene B412- 羟基脱氢酶 -36 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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36 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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36 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 DNA序列在体外或体内转录获得。 这种 DNA序列已整合到载 体的 RM 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 MDP 依赖性 leukotriene B412-羟基脱氢酶- 36 的多核苷酸可用于与 MDP依赖性 leukotriene B412-羟基脱氢酶- 36的相关疾病的诊断。 编码 MDP依 赖性 leukotriene B412-羟基脱氢酶- 36 的多核苷酸可用于检测 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36 的表达与否或在疾病状态下 MDP 依赖性 leukotriene B412-羟基脱氢酶- 36的异常表达。 如编码 NADP依赖性 leukotriene B412-羟基脱氢酶- 36 的 DM序列可用于对活检标本进行杂交以判断 NADP依赖性 leukotriene B412-羟基脱氢酶 -36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 DM 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 NADP依赖性 leukotriene B412-羟基脱氢酶- 36特 异的引物进行 RNA-聚合酶链反应（RT- PCR)体外扩增也可检测 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36的转录产物。
检测 NADP依赖性 leukotriene B412-羟基脱氢酶 -36基因的突变也可用于诊 断 NADP 依赖性 leukotriene B412-羟基脱氢酶- 36 相关的疾病。 MDP 依赖性 leukotriene B412-羟基脱氢酶- 36 突变的形式包括与正常野生型 MDP 依赖性 leukotriene B412-羟基脱氢酶- 36 DM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、 DNA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 Nor thern 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 DNA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, Mende l ian Inher i tance in Man (可通过与 Johns Hopk ins Un ivers i ty Wel ch Med ica 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 cDM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。 NADP 依赖性 leukot r i ene B412-羟基脱氢酶- 36 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 NADP 依赖性 leukot r iene B412-羟基脱氢酶- 36 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

杈 利 要 求 书
1、 一种分离的多肽- NADP依赖性 leukot ene B412-羟基脱氢酶- 36 , 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 99%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 SBQ ID NO: 2所示氨基酸序列的多核苷酸。
6、 如杈利要求 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 78-1 067位的序列或 SEQ ID NO: 1中 1-1252位的序列。
7、 一种含有外源多核苷酸的重组载体， 其特征在于它是由杈利要求 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) 用杈利要求 7所述的重组载体转化或转导的宿主细胞； 或
(b) 用杈利要求 4- 6中的任一权利要求所述多核苷酸转化或转导的宿主细胞。
9、 一种具有 NADP依赖性 l euko tr i ene B412-羟基脱氢酶 -36 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 NADP 依赖性 l eukot r i ene B412-羟基脱氢酶- 36 条件下， 培养杈 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 NADP 依赖性 leukotr iene B412-羟基脱氢酶- 36 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 MDP 依赖性 l eukotr i ene B412-羟基脱氢酶 -36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 NADP依赖性 leukot r iene B412-羟基脱氢酶- 36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。
13、 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 NADP 依赖性 l eukot r i ene B412-羟基脱氢酶 -36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、 如权利要求 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 NADP依赖性 leukot r i ene B412-羟基脱氢酶 -36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、 如杈利要求 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 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、 如杈利要求 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 NADP 依赖性 l euko t r i ene B412-羟基脱氢酶- 36异常相关的疾病的药物组合物。
18、 杈利要求 1-6及 1 1 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/CN2001/001006 2000-06-21 2001-06-19 Nouveau polypeptide, leucotriene b412-hydroxy deshydrogenase 36 nadp-dependante, et polynucleotide codant ce polypeptide WO2002012296A1 (fr)

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AU93635/01A AU9363501A (en) 2000-06-21 2001-06-19 A novel polypeptide-nadp dependent leukotriene b412-hydroxydehydrogenase-36 and the polynucleotide encoding said polypeptide
US10/311,746 US20040126858A1 (en) 2000-06-21 2001-06-19 Novel polypeptide-nadp dependent leukotriene b412-hydroxydehydrogenase-36 and the polynucleotide encoding said polypeptide

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CN00116628.X 2000-06-21
CN00116628A CN1329012A (zh) 2000-06-21 2000-06-21 一种新的多肽——烟酰胺腺嘌呤脱氧二核苷酸依赖性白三烯b412－羟基脱氢酶36和编码这种多肽的多核甘酸

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1611887A3 (en) * 2004-06-10 2006-02-08 Abgenomics Corporation Modulation of peroxisome proliferator-activated receptors

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CN108866142B (zh) * 2017-05-10 2022-08-16 中国科学院分子植物科学卓越创新中心 细胞色素p450、烟酰胺腺嘌呤二核苷酸-细胞色素p450还原酶及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010529A1 (en) * 1997-08-22 1999-03-04 Zeneca Limited Methods for analyzing ltc4 synthase polymorphisms and diagnostic use
WO1999052942A2 (en) * 1998-04-15 1999-10-21 Genset Genomic sequence of the 5-lipoxygenase-activating protein (flap), polymorphic markers thereof and methods for detection of asthma

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010529A1 (en) * 1997-08-22 1999-03-04 Zeneca Limited Methods for analyzing ltc4 synthase polymorphisms and diagnostic use
WO1999052942A2 (en) * 1998-04-15 1999-10-21 Genset Genomic sequence of the 5-lipoxygenase-activating protein (flap), polymorphic markers thereof and methods for detection of asthma

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1611887A3 (en) * 2004-06-10 2006-02-08 Abgenomics Corporation Modulation of peroxisome proliferator-activated receptors

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