WO2001040283A1 - NOUVEAU POLYPEPTIDE, ACYL-CoA DESHYDROGENASE HUMAINE 11 CONTENANT UN DOMAINE DE LIAISON ATP/GTP, ET POLYNUCLEOTIDE CODANT POUR CE POLYPEPTIDE - Google Patents

NOUVEAU POLYPEPTIDE, ACYL-CoA DESHYDROGENASE HUMAINE 11 CONTENANT UN DOMAINE DE LIAISON ATP/GTP, ET POLYNUCLEOTIDE CODANT POUR CE POLYPEPTIDE Download PDF

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WO2001040283A1
WO2001040283A1 PCT/CN2000/000495 CN0000495W WO0140283A1 WO 2001040283 A1 WO2001040283 A1 WO 2001040283A1 CN 0000495 W CN0000495 W CN 0000495W WO 0140283 A1 WO0140283 A1 WO 0140283A1
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polypeptide
polynucleotide
acyl
coa dehydrogenase
binding domain
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PCT/CN2000/000495
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Bioroad Gene Development Ltd. Shanghai
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Priority to AU16876/01A priority Critical patent/AU1687601A/en
Publication of WO2001040283A1 publication Critical patent/WO2001040283A1/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.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, an acyl-CoA dehydrogenase 11, which contains an ATP / GTP binding domain, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • the respiratory chain in living organisms consists of the following types of molecules: pyridine nucleotide-linked dehydrogenases, flavin-related dehydrogenases (ie, flavin protein), iron sulfur protein, coenzyme Q, and cytochromes. Flavin-associated dehydrogenases are the main components of the respiratory chain. There are two common types: NADH dehydrogenases with flavin single nucleotides (FMNs) as co-groups and flavin adenine dinucleotides (FAD). ) Is a prosthetic succinate dehydrogenase. FMN and FAD molecules participate in hydrogenation and dehydrogenation reactions in living organisms, and are hydrogen transfer bodies that transfer two hydrogen atoms.
  • FMNs flavin single nucleotides
  • FAD flavin adenine dinucleotides
  • Acyl-CoA dehydrogenase is a flavin protein that catalyzes ⁇ and ⁇ dehydrogenation reactions of acyl-CoA esters in the body and transports electrons to the electron transport protein ETF. So far, many members of the acyl-CoA dehydrogenase family have been cloned, including long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and isovaleryl-CoA dehydrogenase.
  • Abnormal expression of the dehydrogenase family will cause abnormal function of the respiratory chain, resulting in abnormal function of the energy conversion system of the organism, and triggering Various diseases related to it.
  • the abnormal function of isovaleryl coenzyme A dehydrogenase is the direct cause of isovaleric acidemia [Tanaka K., Ikeda et al., 1987, Enzyme, 38: 91-107].
  • acyl dehydrogenase protein family each contain two conserved sequence fragments, the first in the central region of the enzyme and the second in the C-terminal region of the protein. These two sequence fragments are sequence 1: [GAC]-[LIVM]-[ST] -EX (2)-[GSAN] -G- [ST] -DX (2)-[GSA] and sequence 2: [ QDE] -X (2) _G_ [GS]-XG- [LIVMFY] -X (2)-[DEN] -X (4)-[KR] -X (3)-[DEN]].
  • Both members contain these two conserved sequence fragments, of which conserved sequence 1 may be the central region of the protein to perform normal physiological functions.
  • ATP or GTP binding motif is a ubiquitous domain in various ATP / GTP binding proteins. Some ATP or GTP binding proteins all contain this conserved mot if sequence. This motif has conservative characteristics. Sequence fragment: [AG]-X (4)-G- K- [ST] (where X is any amino acid residue). The most conservative of these mot i f is the g l yc ine enrichment region, which forms a flexible loop between the ⁇ line and the ⁇ helix. This curved loop usually reacts with a phosphate group on the nucleotide. The motif sequence is often referred to as the "A" consensus sequence or the P-loop.
  • This motif is usually responsible for binding with ATP and GTP in the body, providing the energy required for the protein to perform biological functions and stabilizing the structure of the protein in the body, assisting the protein to complete its normal physiological function. Its abnormal expression will directly lead to the abnormal expression and dysfunction of some proteins, thus causing various related diseases.
  • the new human ATP / GTP binding domain-containing acyl dehydrogenase 11 also contains two conserved sequence fragments of the acyl dehydrogenase family as described above, and contains an ATP / GTP binding domain, which is human A new member of the acyl dehydrogenase family, and similar to other members of the family, has similar biological functions, and is involved in the occurrence of various metabolic disorders related to energy metabolism in the body.
  • the human acyl-CoA dehydrogenase 11 protein containing the ATP / GTP binding domain 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 more needs to be identified in the art Humans involved in these processes have an acyl-CoA dehydrogenase 11 protein containing an ATP / GTP binding domain, and in particular the amino acid sequence of this protein was identified. Isolation of the acyl-CoA dehydrogenase 1 1 protein containing the ATP / GTP binding domain in newcomers also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the acyl-CoA dehydrogenase 11 containing the ATP / GTP binding domain of 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 in human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID No. 2 or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1579-1869 in SEQ ID NO: 1; and (b) a sequence having 1-2218 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 present invention also relates to a method for screening a compound that mimics, activates, antagonizes or inhibits the activity of a human acyl-CoA dehydrogenase 11 protein containing an ATP / GTP binding domain, which comprises using the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of a human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 1 1 protein, which comprises detecting the polypeptide or its encoding in a biological sample. A mutation in a polynucleotide sequence, or the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • 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 abnormal expression of the polypeptide and / or polynucleotide of the present invention for treating metabolic disorders associated with energy metabolism or other human acyl-CoA dehydrogenase 1 1 containing ATP / GTP binding domains. Use of medicines that cause disease.
  • Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.
  • 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 acid or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • 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 acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain, can cause the protein to change and thereby regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds to an acyl-CoA dehydrogenase 1 1 containing a human ATP / GTP binding domain.
  • Antagonist refers to an acyl-CoA that blocks or regulates a human ATP / GTP binding domain when bound to a human ATP / GTP-binding domain containing acyl-CoA dehydrogenase 11.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds to an acyl-CoA dehydrogenase 11 containing a human ATP / GTP binding domain.
  • “Regulation” refers to a change in the function of human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain, This includes increased or decreased protein activity, changes in binding characteristics, and any other biological, functional, or immune changes in human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • 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 human ATP / GTP binding domains using standard protein purification techniques.
  • Acyl-CoA dehydrogenase 11 Basically pure human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain produces a single main band on a non-reducing polyacrylamide gel. Humans contain ATP / GTP The purity of the acyl-CoA dehydrogenase 11 polypeptide of the binding domain can be analyzed by amino acid sequence.
  • 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. 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 specifically or selectively.
  • 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 percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence B
  • ⁇ ' the number of residues in A-the number of spacer residues in sequence
  • A-the number of spacer residues in sequence B can also be determined by Clus ter method or using methods known in the art such as Jot un He in Sex percentage (He in 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 may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to 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,? (& 1) ') 2 and? ⁇ It can specifically bind to human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 epitopes.
  • 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 vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not a component 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 human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 1 1 refers to human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 1 1 that is substantially free of natural Related to other proteins, lipids, sugars or other substances.
  • Those skilled in the art can purify human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of a human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 polypeptide can be analyzed by amino acid sequence analysis.
  • the present invention provides a new polypeptide-a human acyl-CoA dehydrogenase containing an ATP / GTP binding domain 11, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • fragment refers to substantially retaining the same biological function or activity of the human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 of the present invention Of peptides.
  • a fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) is one in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such One, in which the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide) consisting essentially of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention 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 polynucleotide sequence with a total length of 2218 bases, and its open reading frame (1579-1869) encodes 96 amino acids.
  • This polypeptide has the characteristic sequence of acyl-CoA dehydrogenase and ATP / GTP binding domain. It can be deduced that the acyl-CoA dehydrogenase 11 containing the ATP / GTP-binding domain has the representative of acyl-CoA dehydrogenase Structure and function.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or it may be 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.
  • the term "polynucleotide encoding a polypeptide" is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding 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 (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, 60 ° C; or (2) Add a denaturant during hybridization, such as 50 ° / «(v / v) formamide, 0.1» /. Calf serum / 0.1 ° / »Fico l l, 42 ° C, etc .; or (3) hybridization occurs only when the identity between the two sequences is at least 95% or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 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 acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • 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 a human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DM 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 cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DNA-DM or DM-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of acyl-CoA dehydrogenation in humans containing an ATP / GTP binding domain The level of the transcript of enzyme 11; (4) The protein product of gene expression is detected by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human acyl-CoA dehydrogenase 11 gene containing ATP / GTP binding domain can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay. (ELISA) and so on.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay. (ELISA) and so on.
  • a method using PCR to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • 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 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 a host cell that is genetically engineered using the vector of the present invention or directly using a human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 coding sequence, and A method for producing a polypeptide according to the present invention by recombinant techniques.
  • a polynucleoside encoding human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain The acid sequence can be inserted into a vector to constitute a recombinant vector containing a 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 human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DM technology, DNA synthesis technology, in vivo recombination technology (Sambroook, eta 1. Mo l ecul ar C l on ing, a Labora tory Manua l, co ld Spr ing Harbor Labora tory. New York, 1989 ).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: the l ac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, and the early and late SV40 promoters Promoters, retroviral LTRs, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. Expression vectors also include ribosome binding sites and transcription terminators for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription.
  • Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide or the like.
  • Genetically engineered host cells for recombinant vectors 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. Cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S 2 or Sf 9
  • animal cells such as CH0, COS or Bowes 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 DNA uptake can be harvested after exponential growth phase, with (: Treatment 1 2 ', used in the step are well known in the art alternative is to use MgC l 2.
  • 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 lipid Plastid packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 (Sc ience, 1984; 224: 1431), Generally there are the following steps:
  • polynucleotide (or variant) of the present invention encoding a human-human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 1 1 or transform it with a recombinant expression vector containing the polynucleotide or Transduce suitable host cells;
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be 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 an acyl-CoA dehydrogenase 1 containing an ATP / GTP binding domain of the present inventor 1 1 in 1-69 in total 6 9 Comparison of the amino acid sequence homology of an amino acid with an acyl-CoA dehydrogenase domain.
  • the upper sequence is a human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain, and the lower sequence is an acyl-CoA dehydrogenase domain.
  • "I” and ":” and ".” Indicate that the probability of the occurrence of different amino acids at the same position between two sequences decreases in sequence.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of an isolated human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • llkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0120F02 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the results showed that the full-length cDNA contained in the 0120F02 clone was 2218 bp (as shown in Seq ID NO: 1), and there was a 291 bp open reading frame (0RF) from 1579 bp to 1869 bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • CDNA was synthesized using fetal brain cell 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,-GAATAAAGGAATGTATTCATTTCC -3, (SEQ ID NO: 3)
  • Primer2 5,-GTTAAAAGCACTTTATTGATTACA -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions reaction volume containing 5 0 ⁇ 1 in 50mmol / L KC1, 10mmol / L Tris- CI, (pH8.5), 1.5mmol / L MgCl 2, 200 ⁇ mol / L dNTP, lOpmol primer, 1U of 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.
  • ⁇ -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.
  • the DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as the 1-2222bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 gene expression:
  • RNA extraction in one step involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • the Ndel and BamHI restriction sites correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • the PCR reaction was performed using the pBS-0120F02 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS- 0120F02 plasmid, primers Pr imer-3 and Pr imer- 4 points; j is lOpmol, Advantage polymerase Mix
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation.
  • An affinity column His Bind Quick Cartridge capable of binding to 6 histidines (6His-Tag) was used.
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. ⁇ Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • unhybridized probes are removed by a series of membrane washes.
  • This embodiment uses 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
  • 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 known genomic sequences and their complements Region for homology comparison, if the homology with non-target molecular region is greater than 85% After 15 consecutive bases are identical, the primary probe should not be used in general;
  • 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 2 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 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • pre-hybridization solution 10xDenhardfs; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA).
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat metabolic disorders related to energy metabolism and the like.
  • Acyl-CoA dehydrogenase is a flavin protein that catalyzes the oc and P dehydrogenation reactions of acyl-CoA esters in vivo and transports electrons to the electron transport protein ETF. They directly regulate the respiratory chain in the body, and its abnormal expression will lead to the abnormal function of the respiratory chain, which will lead to the abnormal function of the energy conversion system of the organism, and cause various metabolic disorders related to energy and material metabolism. Causes growth and development disorders.
  • the abnormal expression of the acyl-CoA dehydrogenase 1 1 containing the ATP / GTP binding domain of the present invention will produce various diseases, especially metabolic disorders related to energy and material metabolism, and disorders of growth and development. These diseases include, but are not limited to:
  • Organic acidemia isovalerate, propionate, methylmalonic aciduria, combined carboxylase deficiency, glutarate type I, etc.
  • Amino acid metabolism defects phenylketonuria, tyrosine metabolism defects such as albinism, sulfur amino acid metabolism defects, tryptophan metabolism defects such as tryptophanemia, branch amino acid metabolism defects, glycine metabolism defects such as Glycineemia, Hypersarcosineemia, Proline and Hydroxyproline Metabolism Defects, Glutamate Metabolism Defects, Urea Cycle Metabolism Defects, Histidine Metabolism Defects, Lysine Metabolism Defects , And other amino acid metabolism defects.
  • Mucopolysaccharidosis and other marginal diseases Mucopolysaccharidosis ⁇ ⁇ ⁇ type, Mucopolysaccharidosis marginal diseases such as rheumatoid mucopolysaccharidosis and mucolipid storage disease.
  • Purine and Pyrimidine Metabolism Defects Abnormal purine metabolism such as Ray-niney syndrome, xanthineuria, abnormal pyrimidine metabolism such as orotic aciduria, and adenosine deaminase deficiency.
  • Lipid metabolism abnormalities hyperlipoproteinemia, familial hyperoc-lipoproteinemia, familial P-lipoproteinemia, familial hypobeta-lipoproteinemia, familial lecithin-cholesterol acetyltransferase Deficiency.
  • Glucose metabolism defects Congenital sugar digestion and absorption defects such as congenital lactose intolerance, hereditary fructose intolerance, monosaccharide metabolism defects such as galactosemia, fructose metabolism defects, glycogen metabolism diseases such as glycogen storage Backlog.
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, familial cerebral nucleus dysplasia syndrome, skin, fat and muscular dysplasias such as congenital skin relaxation, premature aging, congenital horn Poor metabolism, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism, sexual retardation
  • Abnormal expression of the human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 1 1 will also produce Cause certain tumors, 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, for example, it can treat various diseases, especially metabolic disorders related to energy and material metabolism, and disorders of growth and development. These tumors, some hereditary, hematological and immune system diseases.
  • the invention also provides screening compounds to identify human compounds that increase (agonist) or suppress (antagonist)
  • Method for ATP / GTP-binding domain of acyl-CoA dehydrogenase 1 1 Agonists enhance human acyl-CoA dehydrogenases containing ATP / GTP binding domains to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a membrane preparation of an acyl-CoA dehydrogenase 11 expressing human ATP / GTP-binding domain and mammalian cells or a human ATP / GTP-binding domain-containing acyl-CoA can be removed in the presence of a drug.
  • Catalase 1 1 is cultivated together. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain include selected antibodies, compounds, receptor deletions, and the like.
  • Human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 11 antagonists can bind to human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 11 and eliminate its function, or inhibit the production of the polypeptide Or, in combination with the active site of the polypeptide, the polypeptide cannot perform biological functions.
  • human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 11 may be added to the bioanalytical assay, and by measuring the compounds against human ATP / GTP-binding domain-containing acyl-CoA The effect of the interaction between dehydrogenase 11 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.
  • Polypeptides capable of binding to human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, human acyl-CoA dehydrogenase 11 molecules containing ATP / GTP binding domains 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 human acyl-CoA dehydrogenase 11 epitopes containing an ATP / GTP binding domain. 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 ATP / GTP binding domain-containing acyl-CoA dehydrogenase 1 1 to immunized animals (eg, home immunity, mice, rats, etc.).
  • immunized animals eg, home immunity, mice, rats, etc.
  • a variety of adjuvants can be used to enhance immunity
  • the reaction includes, but is not limited to, Freund's adjuvant and the like.
  • Preparation of human ATP / GTP binding domain containing acyl Monoclonal antibody technologies for enzyme A dehydrogenase 11 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV-hybridization Tumor technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • Antibodies against acyl-CoA dehydrogenase 11 containing ATP / GTP-binding domains can be used in immunohistochemistry to detect human acyl-CoA dehydrogenase 11 containing ATP / GTP-binding domains in biopsy specimens.
  • Monoclonal antibodies that bind to human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain can bind covalently to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human ATP / GTP binding domain-containing Acyl-CoA dehydrogenase 11 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • Administration of an appropriate dose of the antibody can stimulate or block human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 production or activity.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 detected in the test can be used to explain the importance of human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 11 in various diseases.
  • Sexual and diagnostic diseases in which acyl-CoA dehydrogenase 11 with human ATP / GTP binding domains plays a role.
  • 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 human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat cell proliferation, development or metabolic heterogeneity caused by non-expression or abnormal / inactive expression of acyl-CoA dehydrogenase 11 containing human ATP / GTP binding domain Often.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 to inhibit endogenous human ATP / GTP binding domain-containing acyl groups Coenzyme A dehydrogenase 11 activity.
  • a variant human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 1 1 may be a shortened human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of acyl-CoA dehydrogenase 11 containing human ATP / GTP binding domain.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain Transfer into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain 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 acyl-CoA dehydrogenase 1 1 mRNA containing an ATP / GTP binding domain are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA 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 using any existing RM or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DNA sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified by various methods, such as increasing the sequence length on both sides, and the linkage between ribonucleosides should use phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • a polynucleotide encoding a human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 11 can be used for diagnosis of diseases related to human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 11.
  • a polynucleotide encoding a human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 1 1 can be used to detect the expression of human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 1 1 or a disease state Abnormal expression of acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain.
  • the DNA sequence encoding human acyl-CoA dehydrogenase 1 1 containing ATP / GTP binding domain can be used to hybridize biopsy specimens to determine the expression of human acyl-CoA dehydrogenase 11 containing ATP / GTP binding domain.
  • Hybridization techniques include Southern blotting, Nort hern blotting, and in situ hybridization. These technical methods are publicly mature technologies, 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 tissue.
  • Human ATP / GTP binding domain-containing acyl-CoA dehydrogenase 11 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human ATP / GTP binding domain-containing acyl-CoA dehydrogenase Transcript of catalase 11.
  • Detecting mutations in the acyl-CoA dehydrogenase 11 gene containing human ATP / GTP-binding domains can also be used to diagnose human acyl-CoA dehydrogenase 11-related diseases containing ATP / GTP-binding domains.
  • Human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 11 mutant forms include point mutations and translocations compared to normal wild-type human ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 11 DNA sequences , Deletions, reorganizations, and any other abnormalities. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine if genes and genes have been mapped to chromosomal regions Relationship between diseases.
  • the cDNA or genomic sequence differences between the affected and the affected 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 individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping) Resolving power 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 acyl-CoA dehydrogenase 11 containing an ATP / GTP binding domain is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of the ATP / GTP-binding domain-containing acyl-CoA dehydrogenase 1 1 to be administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une acyl-CoA déshydrogénase humaine 11 contenant un domaine de liaison ATP/GTP, 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 l'acyl-CoA déshydrogénase humaine 11 contenant un domaine de liaison ATP/GTP.
PCT/CN2000/000495 1999-11-29 2000-11-27 NOUVEAU POLYPEPTIDE, ACYL-CoA DESHYDROGENASE HUMAINE 11 CONTENANT UN DOMAINE DE LIAISON ATP/GTP, ET POLYNUCLEOTIDE CODANT POUR CE POLYPEPTIDE WO2001040283A1 (fr)

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AU16876/01A AU1687601A (en) 1999-11-29 2000-11-27 A novel polypeptide - human atp/gtp binding domain containing acyl-coa dehydrogenase 11 and the polynucleotide encoding said polypeptide

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CN 99124150 CN1298003A (zh) 1999-11-29 1999-11-29 一种新的多肽——人含atp/gtp结合结构域的酰基辅酶a脱氢酶11和编码这种多肽的多核苷酸
CN99124150.9 1999-11-29

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCHUELKE M. ET AL.: "Cloning of the human mitochindrial 51kDa subunit (NDUFV1) reveal a 100% antisense homology of its 3' UTR with the 5' UTR of the gamma-interferon induvicle protein(IP-30) precursor: is this a link between mitochondrial myopathy and inflammation", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 245, no. 2, April 1998 (1998-04-01), pages 599 - 606 *

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