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WO2001055380A1 - Nouveau polypeptide, 3-beta-hydroxysteroide deshydrogenase/5-ene-4-ene isomerase 57, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, 3-beta-hydroxysteroide deshydrogenase/5-ene-4-ene isomerase 57, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001055380A1
WO2001055380A1 PCT/CN2001/000077 CN0100077W WO0155380A1 WO 2001055380 A1 WO2001055380 A1 WO 2001055380A1 CN 0100077 W CN0100077 W CN 0100077W WO 0155380 A1 WO0155380 A1 WO 0155380A1
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Prior art keywords
ene
polypeptide
polynucleotide
human
dehydrogenase
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Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai BioDoor Gene Technology Ltd
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Shanghai BioDoor Gene Technology Ltd
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Priority to AU2001231491A priority Critical patent/AU2001231491A1/en
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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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12N9/90—Isomerases (5.)
    • 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, a human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57, and a multinucleus encoding the polypeptide. Nucleotide sequence. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • PSS precursor sex steroids
  • DHEA dehydroepiandrosterone
  • Adrenal PSS requires 3-betahydroxydentanol dehydrogenase / 5-ene-4-ene isomerase, 17-betahydroxydentanol dehydrogenase / 5-ene-4-ene isomerase, 5 cc reductase and And / or the activity of enzymes such as aromatase to synthesize the male hormone dihydrotestosterone (DHT) or the estrogen 17 beta estradiol and androstenyl glycol.
  • DHT dihydrotestosterone
  • 3-beta 3 -hydroxys teroid dehydrogenase / 5-ene-4-ene isomerase (3 beta-HSD) catalyzes 5-refining-3- beta
  • the sterol precursors of menstrene and 5-ene-hydroxyandrostene are the corresponding 4-ene-sterones, the latter of which is necessary for the synthesis of various steroid hormones.
  • the activity of 3 beta-HSD can be measured not only in the gonads and adrenals, but also in many other peripheral tissues, such as adipose tissue, breast, kidney, Liver, prostate, vas deferens, uterus, skin, brain, heart, thymus, pancreas, lung and spleen.
  • peripheral tissues such as adipose tissue, breast, kidney, Liver, prostate, vas deferens, uterus, skin, brain, heart, thymus, pancreas, lung and spleen.
  • Beta-HSD-deficient male newborns have adrenal dysplasia with pseudohermaphroditism; female newborns are less affected.
  • Deficiency of another 3 beta-HSD causes deficiency of steroid 21-hydroxylase.
  • the scientific function is named human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase XX. Since the human 3-beta hydroxysterol dehydrogenase / 5-ene-4 -ene isomerase 57 protein plays an important role in important functions in the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, the art There has been a need to identify more people involved in these processes. 3-beta hydroxyalcohol dehydrogenase / 5-ene-4-ene isomerase 57 egg In particular, the amino acid sequence of this protein is identified.
  • Isolation of the novel 3-beta hydroxysterol dehydrogenase / 5-en-4-ene isomerase 57 protein encoding gene also provides a basis for the study 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 isolating its coding DNA is very important. Disclosure of invention
  • Another object of the present 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 human 3-beta hydroxydentanol dehydrogenase / 5-ene-4-ene isomerase 57.
  • Another object of the present invention is to provide a method for producing human 3-betahydroxylitol dehydrogenase / 5-ene-4-ene isomerase 57.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, which is human 3-beta hydroxyl alcohol dehydrogenase / 5 -en-4-ene isomerase 57. . '
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of 3-beta hydroxylitol dehydrogenase / 5-en-4-ene isomerase 57 against the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human 3-betahydroxydenthol dehydrogenase / 5-en-4-ene isomerase 57.
  • 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 99-1646 in SEQ ID NO: 1; and (b) a sequence having positions 1-1754 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing a polynucleotide of the invention.
  • the vector genetically engineered host cell includes a transformed, transduced or transfected host cell; a method for preparing a polypeptide of the present invention comprising culturing the host cell and recovering an 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 human 3-betahydroxysterol dehydrogenase / 5-en-4-ene isomerase 57 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 the abnormal expression of human 3-betahydroxydentanol dehydrogenase / 5-ene-4-ene isomerase 57 protein in vitro, which comprises detecting all diseases in a biological sample. Mutations in said polypeptide or its encoding polynucleotide sequence, or detecting the amount or biological activity of the polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the invention also relates to the polypeptides and / or polynucleotides of the invention in the preparation for the treatment of cancer, developmental or immune diseases or other due to human 3-betahydroxylol dehydrogenase / 5-ene-4-ene Use of a medicine for diseases caused by abnormal expression of enzyme 57.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or 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.
  • Insert refers to a change in the amino acid sequence or nucleotide sequence that results in Compared to the molecule, one or more amino acids or nucleotides are increased.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human 3-betahydroxyl alcohol dehydrogenase / 5-ene-4 -ene isomerase 57, causes the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human 3-betahydroxyl alcohol dehydrogenase / 5-en-4-ene isomerase 57.
  • an “antagonist” or “inhibitor” is a compound that blocks or regulates human 3-betahydroxylitol when combined with human 3-betahydroxylol dehydrogenase / 5-ene-4-ene isomerase 57.
  • Dehydrogenase / 5-ene-4-ene isomerase 57 is a biologically or immunologically active molecule.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human 3-betahydroxysterol dehydrogenase / 5-en-4-ene isomerase 57.
  • Regular refers to changes in the function of human 3-betahydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57, including an increase or decrease in protein activity, changes in binding characteristics, and human 3-beta hydroxyl Alteration of any other biological, functional, or immunological properties of sterol dehydrogenase / 5-ene-4-ene isomerase 57.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human 3-beta hydroxysterol dehydrogenase / 5-ene-4 -ene isomerase 57 using standard protein purification techniques.
  • Substantially pure human 3-betahydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 polypeptide can be analyzed by amino acid sequence analysis.
  • Complementary refers to the natural binding of a nucleotide 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. The 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 bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). 0 The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244) 0 Clus ter method by examining the distances between all pairs of each set of sequence arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art, such as Jotun He in (Hein J., (1990) Methods in emzumology 183: 625-645).
  • 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. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of human 3-be tadritol dehydrogenase / 5-ene-4-ene isomerase 57.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • 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 with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not in its natural environment Ingredients, they are still separate.
  • 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 human 3-beta hydroxydentanol dehydrogenase / 5-ene-4-ene isomerase 57 refers to human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene iso Constitutive enzyme 57 is essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 polypeptide can be analyzed by amino acid sequence analysis.
  • the present invention provides a new polypeptide, human 3-betahydroxylol dehydrogenase / 5-en-4-ene isomerase 57, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2 of.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, 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.
  • polypeptides of the invention may be glycosylated, or 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 human 3-beta hydroxysterol dehydrogenase / 5-en-4-ene isomerase 57.
  • fragment As used in the present invention, the terms “fragment”, “derivative” and “analog” refer to the human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 which is substantially the same as the present invention 57.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (II) a type in which a group on one or more amino acid residues is substituted with another group to include a substituent; or ( ⁇ ⁇ ) A type in which 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 type in which the additional amino acid sequence is fused into the mature polypeptide Sequences (such as leader sequences or secretory sequences or sequences used to purify this polypeptide or protease sequences) As set forth herein, 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 of SEQ ID NO: 1 Sequence.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1754 bases in length and its open reading frame 99-1646 encodes 515 amino acids.
  • This peptide has the characteristic sequence of the 3 beta-HSD family, and it can be deduced that the human 3-beta hydroxydentanol dehydrogenase / 5-ene-4 -ene isomerase 57 has the structure and function represented by the 3 beta-HSD family .
  • the polynucleotide of the present invention may be in the DM form or the MA form.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • 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.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • 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 (having at least 50%, preferably 70% identity between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 ° / »SDS, 60 ° C; or ( 2) A denaturant is added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol l, 42.
  • 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, 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 human 3-beta hydroxysterol dehydrogenase / 5-en-4-ene isomerase 57.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence of the present invention encoding human 3-betahydroxylitol dehydrogenase / 5-en-4-ene isomerase 57 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 DM sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DM 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 cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDM library.
  • mRM plasmid or phage cDM library.
  • kits are also commercially available (Q i agene;).
  • the construction of cDNA library is also a common method (Sambrook, et al., Moleculoar Cloning, A Labora tory Manua, Collspring Harbor Labora tory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human 3-betahydroxylol dehydrogenase / 5-ene- The level of the transcript of 4-ene isomerase 57; (4) detecting the protein product of the gene expression by immunological techniques or 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 Is at least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 gene expression can be detected by immunological techniques such as Western blotting and radioimmunoprecipitation Method, enzyme-linked immunosorbent assay (ELISA), etc.
  • a method (Sa iki, et al. Sc ience 1985; 230: 1350-1 354) using PCR technology to amplify DNA / RM is preferred for obtaining the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers for PCR can be appropriately selected according to the polynucleotide sequence information of the present invention disclosed herein, and can be synthesized by 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 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 the polynucleotide of the present invention, and genetically engineered using the vector of the present invention or directly using human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 coding sequence.
  • a polynucleotide sequence encoding human 3-be tadritol dehydrogenase / 5-en-4-ene isomerase 57 may be inserted into a vector to constitute a gene containing the polynucleotide of the present invention.
  • Recombinant vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, etal.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually incorporate a replication origin, a promoter, a marker gene, and translational regulatory elements.
  • 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 large About 10 to 300 base pairs act on promoters to enhance gene transcription. Examples include 100 to 270 base pair SV40 enhancers on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human 3-beta hydroxydentate dehydrogenase / 5-ene-4-ene isomerase 57 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to A genetically engineered host cell comprising the polynucleotide or a recombinant vector is constructed.
  • 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 insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes s melanoma cells, etc. .
  • 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 01 12 method, the steps used are well known in the art.
  • the alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 (Scence, 1984 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. When the host cell has 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 separated 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 chromatography
  • FIG. 1 is a comparison diagram of the amino acid sequences of the functional domains of the 3-beta hydroxydentanol dehydrogenase / 5-ene-4-ene isomerase 57 and 3 be ta-HSD family proteins of the present inventors.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human 3-betahydroxyl alcohol dehydrogenase / 5-ene-4-ene isomerase 57.
  • 57KDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction.
  • PCR amplification was performed with the following primers:
  • Primerl 5'- GGAGTGAAGAGAGCGCGACGGCGG -3 '(SEQ ID NO: 3)
  • Primer 2 5'_ CTAAGATGGTTTAATGTCTTGGCA -3 '(SEQ ID NO: 4)
  • Priraerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50ramol / L KCl, 10mmol / L Tris-Cl, (pH8.5), 1.5mmol / L MgCl 2 , 200 mol / L dNTP, 'lOpmol primer, 1U in a 50 ⁇ 1 reaction volume Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • RT-PCR set beta-actin as a positive control and template blank as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Ir itrogeii) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1754bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human 3-betahydroxylitol dehydrogenase / 5-en-4-ene isomerase 57 gene expression:
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.4)-5xSSC-5xDenhardt, s solution and 200 g / ffll salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 was designed based on the sequence of the coding region shown in SEQ ID NO: 1 and FIG. 1 A pair of specific amplification primers was generated, and the sequence is as follows:
  • Primer 3 5'- CATGCTAGCATGGTTTCAATCCCAGAATACTAT -3 '(Seq ID No: 5)
  • Priraer4 5'- CCCGAGCTCTCAGTATCTCATAGTGCTGGATGC —3' (Seq ID No: 6)
  • Nhel and Sacl restriction sites correspond to the selectivity on the expression vector plasmid pET 28b (+) (Nova gen, Cat. No. 69865.3). Endonuclease site.
  • the PCR reaction was performed using pBS-0391g06 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ l contains 10 pg of pBS- 0391g06 plasmid, primers Primaer-3 and PI ⁇ imer-4, and J is 10 pmol, Advantage polymerase Mix (Clofitech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60. C 30s, 68 ° C 2 min, a total of 25 cycles. Nhel and Sacl were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into coliform bacteria DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0391g06) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the host bacteria BL21 (pET-0391g06) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 to make ol / L. , Continue to cultivate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation. An affinity chromatography column His. Bind Quick Cartridge capable of binding to 6 histidines (6His-Tag) was used.
  • NH2-Met-Val-Ser-I le-Pro-Glu-Tyr-Tyr-Glu-Gly-Lys-Asn-Val-Leu-Leu- COOH SEQ ID NO: 7
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avraraeas, et al. Imraunochemi s try, 1969; 6: 43. Rabbits were immunized with the hemocyanin peptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin peptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Se P ha r0 Se 4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human 3-betahydroxydentanol dehydrogenase / 5-ene-4-ene isomerase 57.
  • Example 7 Use of a polynucleotide fragment of the present invention as a hybridization probe
  • the 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 the genome or CDM library of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • 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 Off.
  • 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 from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other unknown genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 1 which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of fast, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, refer to the literature DeRi si, JL, Lyer, V.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500ng / ul after purification. The spots were spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps have been reported in the literature in various ways. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from normal liver and liver cancer in one step, and mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5- Amino- propargyl-2'- deoxyur idine 5 '-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech company) labeled mRNA of normal liver tissue, using a fluorescent reagent Cy5dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy5 f luorescent dye (purchased from Amersham Phamacia Biotech) was used to label liver cancer tissue mRNA, and the probe was prepared after purification.
  • Cy3dUTP 5- Amino- propargyl-2'- deoxyur idine 5 '-tr iphate coupled to Cy3 f luorescent dye
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChera) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned image was analyzed and processed with Imagene software (Biodi scovery, USA) to calculate the Cy3 / Cy5 ratio of each point, which was less than 0.5 Dots greater than 2 are considered genes with differential expression.
  • the poly'peptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infection and immune diseases, etc. .
  • the human adrenal glands can secrete a large amount of precursor sex steroids (PSS) and dehydroisoandrostenone (DHEA).
  • PSS precursor sex steroids
  • DHEA dehydroisoandrostenone
  • Adrenal PSS requires 3-beta hydroxydentate dehydrogenase / 5-ene-4-ene isomerase (3 beta-HSD), 17-beta hydroxysterol dehydrogenase ⁇ -ene-4 4-ene isomerase, 5 Alpha reductase and / or aromatase activity to synthesize the male hormone dihydrotestosterone or estrogen 17 beta estradiol and androstene glycol.
  • 3 beta-HSD catalyzes the sterol precursors of 5-ene-3-beta-hydroxypregnene and 5-ene-hydroxyandrostene as the corresponding 4-ene-sterones. The latter are important for the synthesis of various steroid hormones. necessary.
  • beta-HSD is widely found in the gonads and adrenals, and many other peripheral tissues such as adipose tissue, breast, kidney, liver, prostate, vas deferens, uterus, skin, brain, heart, thymus, pancreas, lung, and spleen .
  • the deficiency of beta-HSD will cause CAHI I, a serious congenital adrenal hyperplasia, which will reduce the synthesis of steroids in the adrenal glands and gonads, which will cause adrenal dysplasia in male newborns with pseudohermaphroditism, and female newborns will suffer Less affected.
  • Deficiency of another 3 beta-HSD causes deficiency of steroid 21-hydroxylase.
  • the characteristic sequence of the 3-beta hydroxydentanol dehydrogenase / 5-ene-4-ene isomerase protein family is necessary for its biological activity.
  • the polypeptide of the present invention is a polypeptide containing a characteristic sequence of this protein family. Abnormal expression of the polypeptide will lead to abnormal biosynthesis and metabolism of steroid hormones, and may also affect the metabolism of prostaglandins and fats, and cause related diseases. ,
  • steroid hormones precocious puberty, delayed sexual development, disorders of sexual differentiation, other defects in external genital development, hyperadrenal diseases such as Cushing syndrome, aldosteronism, and adrenal insufficiency
  • Prostaglandin metabolism disorders dystocia, dystocia, persistent luteal, vasomotor diastolic disorder, related cardiovascular disease, bronchospasm, gastric ulcer
  • Adipose disorder fatty liver, steatosis cardiomyopathy, steatosis nephropathy, coronary atherosclerotic heart disease, hypertension
  • Certain tumors lipoma, lipoblastoma, liposarcoma, breast cancer, endometrioma
  • Abnormal expression of the human 3-betahydroxyl alcohol dehydrogenase / 5-ene-4-ene isomerase 57 of the present invention will also produce certain hereditary, hematological and immune system 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.
  • it can treat various diseases, especially steroid hormone metabolism disorder, prostaglandin metabolism disorder, adipose disorder, bile acid disorder, related tumors, certain hereditary, blood diseases and immune system diseases Wait.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human 3-betahydroxysteol dehydrogenase / 5-en-4-ene isomerase 57.
  • Agonists enhance human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 can be delabeled with labeled human 3-beta hydroxydentanol in the presence of a drug.
  • Catalase / 5-en-4-ene isomerase 57 is cultivated together. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 can bind to human 3-beta hydroxy alcohol dehydrogenase / 5-ene-4-ene isomerase 57 and Eliminating its function, or inhibiting the production of the polypeptide, or binding to the active site of the polypeptide prevents the polypeptide from performing its biological function.
  • human 3-betahydroxylol dehydrogenase / 5-ene-4 -ene isomerase 57 can be added to the bioanalytical assay, and the compound can be tested for human 3-betahydroxylol The effect of the interaction between dehydrogenase / 5-ene-4-ene isomerase 57 and its receptor to determine whether a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human 3-beta hydroxydentanol dehydrogenase / 5-ene-4-ene isomerase 57 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase . In screening, the molecule of human 3-betahydroxyl alcohol dehydrogenase / 5-ene-4-ene isomerase 57 should generally 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 the human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by direct injection of human 3-betahydroxydentanol dehydrogenase / 5-ene-4-ene isomerase 57 into immunized animals (such as rabbits, mice, rats, etc.).
  • Adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.
  • Techniques for preparing monoclonal antibodies to human 3-betahydroxyl alcohol dehydrogenase / 5-ene-4-ene isomerase 57 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions 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 human 3-betahydroxydentate dehydrogenase / 5-ene-4-ene isomerase 57.
  • Antibodies against human 3-betahydroxylitol dehydrogenase / 5-ene-4-ene isomerase 57 can be used in immunohistochemistry for detection of human 3-betahydroxylitol dehydrogenase / 5 in biopsy specimens -Ene-4-ene isomerase 57.
  • Monoclonal antibodies that bind to human 3-beta hydroxyl alcohol dehydrogenase / 5-ene-4-ene isomerase 57 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis. Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, human 3-beta hydroxysterol dehydrogenase / 5-en-4-ene isomerase 57.
  • High-affinity monoclonal antibodies can co-exist with bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.) ⁇ ⁇ Price combination.
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human 3-betahydroxyl alcohol dehydrogenation.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human 3-betahydroxylitol dehydrogenase / 5-en-4-ene isomerase 57.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57.
  • diagnostic test method for quantitatively and locally detecting the level of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57.
  • the level of human 3-betahydroxydentanol dehydrogenase / 5-ene-4-ene isomerase 57 detected in the test can be used to explain human 3-betahydroxydentanol dehydrogenase / 5-ene-4-
  • 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.
  • the polynucleotide encoding human 3-betahydroxyl alcohol dehydrogenase / 5-ene-4-ene isomerase 57 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57.
  • Recombinant gene therapy vectors can be designed to express variant human 3-betahydroxysterol dehydrogenase / 5-en-4-ene isomerase 57 to inhibit endogenous human 3-beta hydroxyl Sterol dehydrogenase / 5-ene-4-ene isomerase 57 activity.
  • a variant of human 3-betahydroxyl alcohol dehydrogenase / 5-ene-4-ene isomerase 57 It may be a shortened human 3-betahydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 lacking a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human 3-betahydroxylol dehydrogenase / 5-ene-4-ene isomerase 57.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to encode human 3-betahydroxylol dehydrogenase / 5-ene-4-ene isomerase
  • the 57 polynucleotide is transferred into the cell.
  • a recombinant viral vector carrying a polynucleotide encoding human 3-betahydroxylol dehydrogenase / 5-ene-4-ene isomerase 57 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human 3-beta hydroxysterol dehydrogenase / 5-en-4-ene isomerase 57 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human 3-betahydroxyl alcohol dehydrogenase / 5-ene-4-ene isomerase 57 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 MA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology of solid phase phosphate amide synthesis of 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 vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in various ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides should use phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • a polynucleotide encoding human 3_beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 can be used in combination with human 3-beta hydroxydentanol dehydrogenase / 5-ene-4-ene isomerase 57 Diagnosis of related diseases.
  • a polynucleotide encoding human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 can be used to detect human 3-beta hydroxy alcohol dehydrogenase / 5-ene-4-ene isomerase 57 Expression or Abnormal Expression of Human 3-beta Hydroxyl Alcohol Dehydrogenase / 5-ene-4-ene Isomerase 57 in Disease Status.
  • the DNA sequence encoding human 3-beta hydroxylol dehydrogenase / 5-ene-4-ene isomerase 57 can be used to hybridize biopsy specimens to determine human 3-betahydroxysterol dehydrogenase / 5-ene- Expression of 4-ene isomerase 57.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • RNA-polymerase chain reaction (RT-PCR) amplification of the primers can also be used to detect the transcription product of human 3-beta hydroxysterol dehydrogenase / 5-en-4-ene isomerase 57.
  • Human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 mutant forms include a normal wild-type human 3-beta hydroxysterol dehydrogenase / 5-ene 4-ene isomerase 57 DNA sequence compared to point mutations, translocations, deletions, recombinations and any other abnormalities.
  • Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, 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 based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization 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. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Wetch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals, and the mutation is not observed in any normal individual, The mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human 3-beta hydroxy alcohol dehydrogenase / 5-ene-4-ene isomerase 57 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of doses of human 3-beta hydroxysterol dehydrogenase / 5-ene-4-ene isomerase 57 administered to a patient will depend on many factors, such as the mode of administration, the health conditions and diagnosis of the person to be treated Doctor's judgment.

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Abstract

L'invention concerne un nouveau polypeptide, une 3-béta-hydroxystéroïde déshydrogénase/5-ène-4-ène isomérase 57, 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 3-béta-hydroxystéroïde déshydrogénase/5-ène-4-ène isomérase 57.
PCT/CN2001/000077 2000-01-28 2001-01-21 Nouveau polypeptide, 3-beta-hydroxysteroide deshydrogenase/5-ene-4-ene isomerase 57, et polynucleotide codant pour ce polypeptide Ceased WO2001055380A1 (fr)

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AU2001231491A AU2001231491A1 (en) 2000-01-28 2001-01-21 A novel polypeptide - 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase 57 and the polynucleotide encoding said polypeptide

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CN00111574.X 2000-01-28
CN 00111574 CN1307113A (zh) 2000-01-28 2000-01-28 一种新的多肽——人3－beta羟甾醇脱氢酶/5－烯－4－烯异构酶57和编码这种多肽的多核苷酸

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4337234A4 (fr) * 2021-05-12 2025-12-03 Biomica Ltd Microorganisme du microbiome et ses utilisations

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