WO2001048167A1 - Nouveau polypeptide, lipoxygenase 10, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, lipoxygenase 10, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001048167A1
WO2001048167A1 PCT/CN2000/000701 CN0000701W WO0148167A1 WO 2001048167 A1 WO2001048167 A1 WO 2001048167A1 CN 0000701 W CN0000701 W CN 0000701W WO 0148167 A1 WO0148167 A1 WO 0148167A1
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polypeptide
polynucleotide
lipoxygenase
sequence
seq
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PCT/CN2000/000701
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU21457/01A priority Critical patent/AU2145701A/en
Publication of WO2001048167A1 publication Critical patent/WO2001048167A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, lipoxygenase 10, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • Lipids contain many different types of compounds, which have the following biological functions: U) In all cells, the main structural component of the membrane is lipids; (2) some lipids can effectively store energy; (3) Many vitamins and hormones found in animals are lipids or lipid derivatives; (4) Bile acids help dissolve other lipids during digestion.
  • Arachidonic acid is the main C 2Q polyunsaturated fatty acid in most mammals. It can be catalyzed by enzymes into many compounds, such as prostaglandins, thromboxane, and leukotrienes. In the above-mentioned catalytic process, lipoxygenase plays an important role.
  • Lipoxygenase is a type of iron-bound dioxygenase, which can catalyze the hydroperoxidation of lipids. Its catalytic substrate includes cis, cis-1, 4-pentane in addition to arachidonic acid. Ene and so on. Lipoxygenase has an important catalytic function for the oxidation of lipids, so it is of great significance for the metabolism of lipids and the physiological functions of lipids and their derivatives.
  • soybean lipoxygenase The nucleotide sequence of the gene encoding soybean lipoxygenase has been obtained. Soybean lipoxygenase has been found in three types, L0X1, L0X2, L0X3. Although they differ in nucleotide sequence, they all have an approximate It is a conserved sequence of 40 amino acid residues. This conserved sequence contains 6 histidines and 2 tyrosines. This conserved sequence is highly conserved among the three lipoxygenases in soybean, and is speculated to be possible Iron atom binding is involved (J Biol Chem 1988 May 15; 263 (14): 6816-21).
  • lipoxygenase activity plays an important role (Boyington JC, Gaffney BJ, Amzel LM Science 260: 1482-1486 (1993)); (Steczko J., Donoho GP, Clements JC, Dixon JE, Axe l rod B. Biochemi st ry 31: 4053-4057 (1992)) e
  • lipoxygenase In plants, the role of lipoxygenase is related to many physiological processes in plants, such as growth, development, resistance to diseases and insect pests, and damage repair (Vick BA, Zimmerman DC (In) Biochemistry of plants: A comprehensive treatise, Stumpf PK, Ed. , Vol. 9, pp.53-90, Academic Press, New-York, (1987)) 9
  • Leukotriene is a very strong muscle contractor, which can shrink the lung trachea, which is of great significance for the treatment of asthma.
  • leukotrienes are found in leukocytes and mast cell tumor cells.
  • the mechanism of action of prostaglandins and thromboxane is not clear, but they have important effects on blood coagulation and activation of adenylate cyclase.
  • lipoxygenase is involved in the metabolism of substances such as prostaglandins, thromboxane and leukotriene. Therefore, lipoxygenase can regulate some of the important physiological processes mentioned above (Needleman P., Turk J., Jakschik B. A., Morrison AR, Lefkowith LB. Annu. Rev. Biochem. 55: 69-102 (1986)) 0
  • lipoxygenase 10 protein plays an important role in important functions of the body, 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 to identify more lipoxygenase 10 proteins involved in these processes, especially The amino acid sequence of this protein was identified.
  • the isolation of the new lipoxygenase 10 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 the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of lipoxygenase 10.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 163-432 in SEQ ID NO: 1; and (b) a sequence having 1-1640 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of lipoxygenase 10 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of lipoxygenase 10 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a biological sample.
  • the amount or biological activity of a polypeptide of the invention comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of lipoxygenase 10.
  • Fig. 1 is a comparison diagram of amino acid sequence homology of a total of 55 amino acids and domain lipoxygenase characteristic proteins of lipoxygenase 10 at 12-66 of the present invention.
  • the upper sequence is lipoxygenase 10
  • the lower sequence is the characteristic protein domain of lipoxygenase.
  • ⁇ "and": "" and ".” Indicate that the probability of the same amino acid appearing between two sequences decreases in sequence.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated lipoxygenase 10.
  • OkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a 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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with lipoxygenase 10, causes a change in the protein to regulate the activity of the protein.
  • Agonists can include proteins, nucleic acids, carbohydrates or any other A molecule that can bind lipoxygenase 10.
  • Antagonist refers to a molecule that, when combined with lipoxygenase 10, can block or regulate the biological or immunological activity of lipoxygenase 10.
  • Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind lipoxygenase 10.
  • “Regulation” refers to a change in the function of lipoxygenase 10, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of lipoxygenase 10.
  • Substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify lipoxygenase 10 using standard protein purification techniques. Substantially pure lipoxygenase 10 produces a single main band on a non-reducing polyacrylamide gel. The purity of the lipoxygenase 10 peptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T
  • the complementarity between two single-stranded molecules can be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern 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 bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences.
  • the percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Mad Son Wis.).
  • the MEGALIGN program can compare two or more sequences (Higgins, D. G., and P. M. Sharp (1988) according to different methods, such as the Cluster method.
  • 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:
  • amino acid substitutions such as negatively charged amino acids can include aspartic acid and glutamic acid; positively charged amino acids can include lysine and arginine; have uncharged head groups
  • Amino acids with similar hydrophilicity may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine acid.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? It can specifically bind to the epitope of lipoxygenase 10.
  • 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 lipoxygenase 10 means that lipoxygenase 10 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify lipoxygenase 10 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the lipoxygenase 10 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, lipoxygenase 10, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be obtained from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammals) using recombinant technology. Cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of lipoxygenase 10.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the lipoxygenase 10 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted An amino acid may or may not be encoded by a genetic code; or ( ⁇ ) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such a Species, wherein the mature polypeptide is fused with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as Leader or secreted sequence or the sequence used to purify the polypeptide or protease sequence).
  • conservative amino acid residues preferably conservative amino acid residues
  • An amino acid may or may not be encoded by a genetic code
  • ⁇ such a type in which
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a 1640 base polynucleotide sequence, and its open reading frame 163-432 encodes 89 amino acids.
  • This polypeptide has a characteristic sequence of a lipoxygenase characteristic protein, and it can be deduced that the lipoxygenase 10 has the structure and function represented by the lipoxygenase characteristic protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 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 can be naturally occurring Allelic or non-naturally occurring variants.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the 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, 0.1% SDS, 6 (TC; or (2) added during hybridization) Use a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95% Above, it is more preferable that the hybridization occurs at 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. 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 lipoxygenase 10.
  • 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 lipoxygenase 10 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • mRNA extraction There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDM libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DM or DNA-RNA hybridization; (2) the appearance or loss of marker gene function; (3) measurement Determine the level of lipoxygenase 10 transcripts; (4) Detect 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 DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the lipoxygenase 10 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid cDNA end rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to 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 host cell genetically engineered using the vector of the present invention or directly using a lipoxygenase 10 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • the polynucleotide sequence encoding the lipoxygenase 10 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements. Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding lipoxygenase 10 and appropriate transcriptional / translational regulatory elements.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site 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 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral 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 lipoxygenase 10 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as insect cells such as Fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used for expression or production Recombinant lipoxygenase 10 (Science, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immunological diseases.
  • Lipoxygenase is a type of iron-bonded dioxygenase, which can catalyze the hydroperoxidation of lipids.
  • its catalytic substrate includes cis, cis-1, 4-pentane Ene and so on. It is of great significance to the metabolism of lipids and the physiological functions of lipids and their derivatives.
  • Arachidonic acid can be converted into many compounds by the catalysis of lipoxygenase, such as prostaglandins, thromboxane, and leukotrienes. It plays an important role in regulating human inflammation and allergic diseases (Proc Na t l Acad Sci USA 1988 Jan; 85 (2): 416-20).
  • Prostaglandin is the most widely distributed and most effective biologically active substance in the human body. It has effects on the reproductive, cardiovascular, respiratory, digestive, and nervous systems. It does not act as a hormone in the body, but through certain hormones. The adjustment works. Abnormal metabolism of prostaglandins in the human body can cause uterine dysfunction, dystocia, persistent corpus luteum, vasodilation-related cardiovascular disease, bronchospasm, gastric ulcer caused by excessive gastric acid, and impaired blood supply to the organ. Leukotriene is involved in various physiological and pathological processes such as inflammatory response, coagulation, and asthma. Thromboxane is also involved in the coagulation process.
  • abnormal expression of the specific lipoxygenase mot if will cause abnormal function of the polypeptide containing the mot if, resulting in metabolic disorders of lipids and their derivatives, affecting the metabolism of arachidonic acid, and It produces abnormal metabolism of prostaglandins, thromboxane and leukotriene, and produces related diseases such as lipid metabolism disorders, inflammation, coagulopathy, respiratory diseases, and diseases related to abnormal prostaglandin metabolism.
  • lipoxygenase 10 of the present invention will produce various diseases, especially lipid metabolic disorders, inflammation, coagulopathy, respiratory diseases, and diseases related to abnormal prostaglandin metabolism. These diseases include but are not Limited to:
  • Fatty deposition disease fatty liver, steatosis cardiomyopathy, steatosis nephropathy
  • Cardiovascular diseases coronary atherosclerotic heart disease such as occult heart disease, angina pectoris, myocardial infarction, dying coronary heart disease, hypertension
  • Sterol derivatives such as bile acids, sex hormones (testosterone, estradiol, estriol, progesterone)
  • Metabolic disorders (1) Bile acid metabolic disorders such as biliary cirrhosis, cholelithiasis ( 2) Sexual developmental disorders in the growth and development stages: precocious puberty, delayed sexual development, sexual differentiation disorders, other defects in external genital development (3) endocrine and metabolic syndromes: hyperadrenocortical diseases such as Cushing syndrome, hyperaldosteronism, Adrenal insufficiency diseases such as acute adrenal insufficiency and chronic adrenal insufficiency
  • Various inflammations allergic reactions, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immune complex glomerulonephritis, acute anterior uveitis, bone Porphyria, dermatomyositis, urticaria, atopic dermatitis, hemochromatosis, polymyositis, Addison's disease, Graves' disease, chronic active hepatitis, bowel emergency syndrome, atrophic gastritis, system Lupus erythematosus, myasthenia gravis, cerebrospinal multiple sclerosis, Guillain-Barre syndrome, intracranial granulomatosis, Wegener granulomatosis, autoimmune thyroiditis, autoimmune interstitial nephritis, ulcerative colitis, anemia , Pancreatitis, segmental ileitis, myocarditis, atherosclerosis
  • Respiratory diseases bronchial asthma, allergic pneumonia, adult respiratory distress syndrome, pulmonary eosinophilia, sarcoidosis
  • dystocia dystocia
  • persistent corpus luteum vasoconstriction
  • vasodilation-related cardiovascular disease vasodilation-related cardiovascular disease
  • bronchospasm gastric ulcer caused by hyperacidity
  • Abnormal expression of the lipoxygenase 10 of the present invention will also cause certain hereditary and immune diseases.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially lipid metabolic disorders, inflammation, coagulopathy, respiratory Systemic diseases, diseases related to abnormal prostaglandin metabolism, certain hereditary and immune diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or inhibit (antagonist) lipoxygenase 10.
  • Agonists increase biological functions such as lipoxygenase 10, which stimulates cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing lipoxygenase 10 can be cultured with labeled lipoxygenase 10 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of lipoxygenase 10 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • An antagonist of lipoxygenase 10 can bind to lipoxygenase 10 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • lipoxygenase 10 When screening compounds as antagonists, lipoxygenase 10 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between lipoxygenase 10 and its receptor. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to lipoxygenase 10 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 10 molecules of lipoxygenase should be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against a lipoxygenase 10 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting lipoxygenase 10 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to lipoxygenase 10 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV-hybridoma Technology, etc.
  • Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). 0
  • Existing techniques for producing single-chain antibodies US Pat No. .4946778) can also be used to produce single chain antibodies against lipoxygenase 10.
  • Anti-lipoxygenase 10 antibodies can be used in immunohistochemistry to detect lipoxygenase 10 in biopsy specimens.
  • Monoclonal antibodies that bind to lipoxygenase 10 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 There is metastasis due to the localization and judgment of tumor cells.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • lipoxygenase 10 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill lipoxygenase 10-positive cells.
  • the antibodies in the present invention can be used to treat or prevent diseases related to lipoxygenase 10. Administration of an appropriate amount of antibody can stimulate or block the production or activity of lipoxygenase 10.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of lipoxygenase 10 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of lipoxygenase 10 detected in the test can be used to explain the importance of lipoxygenase 10 in various diseases and to diagnose diseases in which lipoxygenase 10 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding lipoxygenase 10 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 lipoxygenase 10.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant lipoxygenase 10 to inhibit endogenous lipoxygenase 10 activity.
  • a mutated lipoxygenase 10 may be a shortened lipoxygenase 10 lacking a signal transduction domain. Although it can bind to downstream substrates, it lacks signal transduction activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of lipoxygenase 10.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding lipoxygenase 10 into a cell.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding lipoxygenase 10 can be found in the literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding lipoxygenase 10 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 lipoxygenase 10 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform endonucleation.
  • Antisense RNA DNA and ribozymes can be obtained by any existing RNA or DNA synthesis technology. For example, the technique of solid phase phosphoramidite chemical synthesis to synthesize oligonucleotides has been widely used.
  • Antisense RM molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • Polynucleotides encoding lipoxygenase 10 can be used to diagnose diseases related to lipoxygenase 10.
  • Polynucleotides encoding lipoxygenase 10 can be used to detect the expression of lipoxygenase 10 or the abnormal expression of lipoxygenase 10 in a disease state.
  • a DNA sequence encoding lipoxygenase 10 can be used to hybridize biopsy specimens to determine the expression of lipoxygenase 10.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These technical methods are all mature technologies that are publicly available, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in a tissue.
  • RNA-polymerase chain reaction (RT-PCR) in vitro amplification with lipoxygenase 10 specific primers can also detect lipoxygenase 10 transcript products.
  • Detection of mutations in the lipoxygenase 10 gene can also be used to diagnose lipoxygenase 10-related diseases.
  • Forms of lipoxygenase 10 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type lipoxygenase 10 DNA sequences. Mutations can be detected using existing techniques such as Southern imprinting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosome localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybrids to construct chromosome-specific cDNA library.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in 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 used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Lipoxidase 10 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of lipoxygenase 10 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.
  • RNA Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (product of Qiegene). 2ug poly (A) mRNA was reverse transcribed to form cDM. Smart cDNA cloning kit (purchased from Clontech ⁇ cDNA fragment was inserted into the multiple cloning site of pBSK (+) vector (Clontech)) to transform DH5 ⁇ to form a cDNA library.
  • Dye terminate cycle reaction sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) to determine the 5 'and 3' ends of all clones. Compare the determined cDNA sequence with the existing public DNA sequence database (Genebank), It was found that the cDNA sequence of one of the clones 1050e06 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragment of the clone in both directions.
  • the sequence of the lipoxygenase 10 of the present invention and the protein sequence encoded by the lipoxygenase 10 of the present invention were profiled using the GCG profile scan program (Basic local alignment search tool) [Altschul, SF et a 1. J. Mol. Biol. 1990; 215: 403 -10], perform domain analysis in databases such as prosite.
  • the lipoxygenase 10 of the present invention is homologous with the domain lipoxygenase characteristic protein at 12-66, and the homology result is shown in FIG. 1.
  • the homology rate is 0.26, and the score is 13.99; the threshold value is 13.92.
  • Example 3 Cloning of a gene encoding lipoxygenase 10 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'-GATCATAGAACTATTGCATAAAAC-3 '(SEQ ID NO: 3)
  • Primer2 5-AGTTTTGTTTGTTTAATGAATAAT-3 '(SEQ ID NO: 4)
  • Primerl 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 mmol / L C1, 10 mmol / L in a 50 ⁇ l reaction volume Tris-HCl, pH 8.5, 1.5 mmol / L gCl 2 , 200 raol / L dNTP, lOpmol primer, 1U Taq DMA polymerase (Clontech).
  • PE9600 DM thermal cycler Perkin-Elmer reacts for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -actin 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 (Invitrogen product) using a TA cloning kit. DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1640bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of lipoxygenase 10 gene expression
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA was precipitated at 70 ° / °. Wash with ethanol, dry and dissolve in water.
  • a 32P-labeled probe (approximately 2 ⁇ 10 6 cpm / ml) and a nitrocellulose membrane to which RNA was transferred were placed in a solution at 42 ° C. C hybridization overnight, the solution contains 50% formamide-25mM KH 2 P0 4 (pH7.4) -5 ⁇ SSC-5 ⁇ Denhardt's solution and 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, the filter membrane is at 1 ⁇ SSC-0.1 ° /. Wash in SDS for 30 min at 55 ° C. Then, analyze and quantify with Phosphor Imager.
  • Example 5 In vitro expression, isolation and purification of recombinant lipoxygenase 10
  • Primer 3 5'- CCCCATATGATGATTTTAAACAGCATTTCCATT- 3 '(Seq ID No: 5)
  • Primer4 5-CATGGATCCCTAAATGTATGGCTGACACTGTAC-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively.
  • the coding sequences for the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • PCR was performed using the pBS-1050e06 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions were: 1 in a total volume of 50 ⁇ plasmid pBS- 1050e06 containing 10pg, primer Primer- 3 and Pr imer- 4 are lOpmol, Advantage polymerase Mix (Clontech Products) 1 ⁇ 1.
  • Cycle parameters 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed into Escherichia coli DH5 C by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 3 (g / ml)), positive clones were selected by colony PCR method and sequenced. The correct positive clone (pET-1050e06) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following lipoxygenase 10 specific peptides:
  • NH 2 -Met-I le-Leu-Asn-Ser-I le-Ser-I le-Phe-Leu-Pro-Met-Met-Cys-Leu-COOH SEQ ID NO: 7
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin for methods, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned jk cyanoprotein complex and complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex and incomplete Freund's adjuvant were used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method demonstrated that the purified antibody could specifically bind to lipoxygenase 10.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select suitable oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes, and to identify whether some tissues contain the multinucleus of the present invention by using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • 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
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt)
  • Probe 1 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • PBS phosphate buffered saline
  • step 14 Resuspend the DM pellet in a small volume of TE or water.
  • the following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared after the collection solutions of the first peak are combined.

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Abstract

L'invention concerne un nouveau polypeptide, une lipoxygénase 10, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la lipoxygénase 10.
PCT/CN2000/000701 1999-12-27 2000-12-25 Nouveau polypeptide, lipoxygenase 10, et polynucleotide codant pour ce polypeptide WO2001048167A1 (fr)

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CN99125795A CN1301847A (zh) 1999-12-27 1999-12-27 一种新的多肽——脂肪氧化酶10和编码这种多肽的多核苷酸

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003265026B2 (en) * 2002-09-20 2009-01-22 Pure Depth Limited Multi-view display

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0038750A1 (fr) * 1980-04-17 1981-10-28 Merck & Co. Inc. Dérivés dipeptidiques de 4-0-, 6-0-acyl-2-amino-2-désoxy-D-glucose à activité immunologique et méthodes pour leur préparation
WO1994005777A1 (fr) * 1992-08-28 1994-03-17 City Of Hope Inhibition de la formation ou de l'activite du processus de 12-lipoxygenase dans les leucocytes humains
WO1996034943A1 (fr) * 1995-05-04 1996-11-07 City Of Hope 12-lipoxygenase leucocytaire humaine et son role dans la pathogenese d'etats pathologiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0038750A1 (fr) * 1980-04-17 1981-10-28 Merck & Co. Inc. Dérivés dipeptidiques de 4-0-, 6-0-acyl-2-amino-2-désoxy-D-glucose à activité immunologique et méthodes pour leur préparation
WO1994005777A1 (fr) * 1992-08-28 1994-03-17 City Of Hope Inhibition de la formation ou de l'activite du processus de 12-lipoxygenase dans les leucocytes humains
WO1996034943A1 (fr) * 1995-05-04 1996-11-07 City Of Hope 12-lipoxygenase leucocytaire humaine et son role dans la pathogenese d'etats pathologiques

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003265026B2 (en) * 2002-09-20 2009-01-22 Pure Depth Limited Multi-view display

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