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WO2001032701A1 - Nouveau polypeptide, sous-unite 15 de la synthase humaine f1f0 atp, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, sous-unite 15 de la synthase humaine f1f0 atp, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001032701A1
WO2001032701A1 PCT/CN2000/000403 CN0000403W WO0132701A1 WO 2001032701 A1 WO2001032701 A1 WO 2001032701A1 CN 0000403 W CN0000403 W CN 0000403W WO 0132701 A1 WO0132701 A1 WO 0132701A1
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Prior art keywords
polypeptide
human
polynucleotide
atp synthase
synthase subunit
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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 AU12658/01A priority Critical patent/AU1265801A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/03—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; catalysing transmembrane movement of substances (3.6.3)
    • C12Y306/03014—H+-transporting two-sector ATPase (3.6.3.14), i.e. F1 ATPase
    • 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
    • C12N9/14—Hydrolases (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 novel polypeptide—human FIFO ATP synthetase subunit 15 and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides.
  • mitochondrial ATP synthase can act as a driving force, which can be hydrolyzed by proton pump ATP synthase.
  • Mitochondrial ATP synthase is a multi-subunit complex, which is easily divided into two sub-complexes, one is called F0, which crosses the mitochondrial membrane, and the other is called Fl, which is connected to F0 through a thin handle on the inner side of the mitochondrial membrane.
  • F1 is the catalytic site, and F0 is responsible for converting the membrane's electrochemical proton gradient energy to Fl 'through the fine-stem configuration change.
  • the synthesis substrate of ATP includes phosphoric acid, ADP, and membrane electrochemical proton gradient.
  • ATP synthetase corresponds to the maximum rate of ATP synthesis (Kathryn F et al., 1992).
  • the hydrophobic catalytic region of ATP synthase, F1 has three large ⁇ subunits, three large cc subunits, and three simple repeating subunits ⁇ , ⁇ , and ⁇ subunits.
  • the catalytic region of ATP synthase F1 is like a "proton hole" where hydrogen ions are used. Its energy is not directly used to form the P-0 bond, but is used to change the binding configuration of ATP.
  • the site that binds adenine nucleotides on F1 can bind mitochondrial subunit particles with ADP and phosphate to form ATP in a tightly catalyzed region. The release of ATP on the catalytic area can cause ATP or P to exchange.
  • an ATP synthetase F1 has three specific catalytic sites, and the synthesis and hydrolysis of ATP occur at such catalytic sites.
  • the fine handle of ATP synthase F1 contains subunits ⁇ , ⁇ , ⁇ , 0SCP, F-6 and b and d subunits, and its length is about 40-50 angstroms (Ha iming Chen et al., 1995).
  • ATP synthase is a multimeric complex composed of at least 16 different polypeptides, two of which are encoded on the mitochondria and the rest are encoded by nuclear genes. It can be repressed by cyanide, and it can be regulated by calcium (Das AM et al., 1998).
  • Lieber's disease (retroglobular optic neuritis, LH0N) and NARP (neurogenesis, ataxia, pigmented optic neuritis) syndrome are caused by mutations in the 6 subunit of FIFO ATP synthase (Majander A, et al. 1997). Insufficient FIFO ATP synthase can cause Alzheimer's disease (Schagger H, et al. 1995) e
  • F1 has three large p subunits, three large ⁇ subunits, and three simple repeating subunits ⁇ , ⁇ , and ⁇ subunits.
  • the catalytic region is located in the P subunit or the interface of an ⁇ subunit ⁇ subunit.
  • An ATP synthetase F1 has three specific catalytic sites.
  • the fine handle of the ATP synthase F1 contains subunits ⁇ , ⁇ , ⁇ , 0SCP, F-6, and b and d subunits; the FO region of the ATP synthase is involved in the catalysis and inhibition of proton shifts.
  • the human polypeptide of the present invention has a characteristic structure of the FIFO ATP synthetase subunit d. Therefore, the new gene of the present invention is considered to be a gene encoding the FIFO ATP synthetase gene family, and is named human FIFO-ATP synthase subunit 15. It is inferred that it has similar biological functions as the FIFO ATP synthase gene family.
  • 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 FIFO ATP synthase subunit 15.
  • Another object of the present invention is to provide a method for producing human FIFO ATP synthase subunit 15. Another object of the present invention is to provide antibodies against the human FIFO ATP synthase subunit 15 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention, human FIFO ATP synthase subunit 15.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in the human FIFO ATP synthase subunit 15.
  • a novel isolated human FIFO ATP synthetase subunit 15 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a conservative variant polypeptide thereof , Or an active fragment thereof, or an active derivative thereof, or the like.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human FIFO ATP synthase subunit 15; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a sequence having SEQ ID Polypeptide with the amino acid sequence shown in NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 47-460 in SEQ ID NO: 1; and (b) a sequence having 1-531 in SEQ ID NO: 1 Sequence of bits.
  • Figure 1 is a comparison diagram of the amino acid sequence homology of the inventor's FIFO ATP synthase subunit 15 and the homologous protein FIFO ATP synthase subunit d.
  • the upper sequence is human FIFO ATP synthase subunit 15, and the lower sequence is homologous human FIFO ATP synthase subunit d.
  • Identical amino acids are represented by a single character amino acid between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 is a polyacrylamide gel electrophoresis image of the isolated human FIFO ATP synthase subunit 15 (SDS- PAGE) neuriteda is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • 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 in the natural state .
  • isolated human FIFO ATP synthase subunit 15 means that human FIFO ATP synthase subunit 15 is substantially free of other proteins, lipids, sugars, or other substances that are naturally associated with it. Those skilled in the art can purify the human FIFO ATP synthase subunit using standard protein purification techniques 15. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human FIFO ATP synthase subunit 15 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human FIFO ATP synthase subunit 15, 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 polypeptides of the invention may be glycosylated or 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 FIFO ATP synthase subunit 15.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human FIFO ATP synthase subunit 15 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are 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 ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ )
  • Such a type in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • UV a type in which the additional amino acid sequence is fused into the mature polypeptide and formed by the polypeptide sequence ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cD library of human fetal brain tissue. It contains a polynucleotide sequence of 531 bases in length and its open reading frame (47-460) encodes 137 amino acids. Based on the amino acid sequence homology comparison, it was found that this polypeptide has 62% homology with the homologous protein human FIFO ATP synthase subunit d. It can be inferred that the human FIFO ATP synthase subunit 15 has homologous protein human FIFO ATP synthesis Enzyme subunit d has a similar structure and function.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DM forms include cDNA, genomic D or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but having a sequence 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 comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide 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 invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the 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 ° /.
  • hybridizable polynucleotide (V / v) formamide, 0.1% calf serum / 0.1% F ico ll, 42 ° C, etc .; or (3) Hybridization occurs only when the identity between the two sequences is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores.
  • nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human FIFO ATP synthase subunit 15.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human FIFO ATP synthase subunit 15 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the D-sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Mol ecu lar Cling, A Labora tory Manua l, Cold 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.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DM-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determining the level of the transcript of human FIFO ATP synthase subunit 15; (4) Detecting the protein product of gene expression by immunological technology or measuring biological activity. The above methods can be used alone 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 nucleosides Acid, 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 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 FIFO ATP synthase subunit 15 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method for amplifying DNA / RNA using PCR technology 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 e t al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. To obtain the full-length cD sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using a human FIFO ATP synthase subunit 15 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding the human FIFO ATP synthase subunit 15 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct a gene encoding human FIFOFOATP
  • the D sequence and an expression vector with appropriate transcriptional / translational regulatory elements include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Mo l ecu lar Cloning, a Labora tory Manua l, Col d Harbor Harbora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a human FIFO ATP synthase subunit 15 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing D may be in the exponential growth phase were harvested, treated with CaC l 2 method used in steps 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 DNA transfection methods can be used: calcium phosphate co-precipitation method, Or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human FIFO ATP synthase subunit 15 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be 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.
  • Mitochondrial respiratory chain complex V FIFO ATP synthase
  • Lieber's disease (retroglobular optic neuritis, LH0N) and RP (neurogenesis, ataxia, pigmented optic neuritis) syndrome are caused by mutations in the 6th subunit of FIFO ATP synthase. Insufficient FIFO ATP synthetase can cause Al zhe imer's disease.
  • the abnormal expression of the human FIFO-ATP synthase subunit 15 of the present invention will produce various diseases, such as DOWN syndrome, growth retardation in the uterus, delayed fibroblast DCloid lipofuscinosis in young children, BATTEN disease Disease, Lieber's disease, Alzhe imer's disease, etc., and is closely related to energy and substance metabolism disorders.
  • diseases include, but are not limited to:
  • Carbohydrate metabolism diseases glycogen storage disease, galactosemia, hereditary fructose intolerance, primary lactic acidosis
  • Amino acid metabolism diseases Phenylketonuria, tyrosine metabolism defects such as transient hypertyrosinemia in newborns, acute tyrosine subacute and chronic tyrosinemia, albinism, sulfur amino acid metabolism deficiency diseases, Tryptophan metabolism deficiency disease, glycine metabolism deficiency disease, glutamate metabolism deficiency disease, metabolic deficiency disease of the urea cycle, histidine metabolism deficiency disease, lysine metabolism deficiency disease, and other amino acid metabolism deficiency diseases.
  • congenital abortion spina bifida, craniocerebral fissure, cerebral bulge, foramen malformations, congenital hydrocephalus, aqueduct malformation, dwarfism of cartilage hypoplasia, spinal epiphyseal dysplasia, pseudochondral hypoplasia Disease, gonad hypoplasia, congenital adrenal hyperplasia, upper urethra, cryptorchidism, congenital glaucoma or cataract, congenital blepharoplasia, retinal dysplasia, congenital optic nerve atrophy, congenital sensorineural hearing loss, split hand cleft Podiatry, Teratosis, Wi ll iams Syndrome, Bezier Syndrome
  • the polypeptides of the present invention can be directly used in the treatment of diseases, for example, can treat a variety of diseases, especially disorders of energy metabolism and material metabolism disorders, such as DOWN syndrome, intrauterine Growth retardation, late-onset fibroblast-like lipofuscinosis in young children, BATTEN disease, Lieber's disease, Alzhe imer's disease and other carbohydrate metabolism diseases, abnormal lipid metabolism diseases, amino acid metabolism diseases, development disorders, Certain tumors, certain hereditary diseases, nervous system diseases, blood diseases, and immune system diseases.
  • the invention also discloses an antagonist against the polypeptide and its therapeutic effect.
  • the invention also discloses the use of a polynucleotide encoding this new human FIFO ATP synthase subunit 15.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) the human FIFO ATP synthase subunit 15.
  • Agonists enhance human FIFO ATP synthase subunit 15 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human FIFO ATP synthase subunit 15 can be cultured with labeled human FIFO ATP synthase subunit 15 in the presence of drugs. The ability of the drug to increase or suppress this interaction is then determined.
  • Antagonists of human FIFO ATP synthase subunit 15 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human FIFO ATP synthase subunit 15 can be combined with human FIFO ATP The enzyme-forming subunit 15 binds and eliminates its function, either inhibits the production of the polypeptide, or binds to the active site of the polypeptide so that the polypeptide cannot perform a biological function.
  • human FIFO ATP synthase subunit 15 When screening compounds as antagonists, human FIFO ATP synthase subunit 15 can be added to a bioanalytical assay, and the compound can be identified by measuring the effect of the compound on the interaction between human FIFO ATP synthase subunit 15 and its receptor. Whether it 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 that bind to human FIFO ATP synthase subunit 15 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, the human FIFO ATP synthase subunit 15 molecule 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 against human FIFO ATP synthase subunit 15 epitopes. 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 injecting human FIFO ATP synthase subunit 15 directly into immunized animals (eg rabbits, mice, rats, etc.).
  • immunized animals eg rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's Adjuvant, etc.
  • Techniques for preparing monoclonal antibodies to human FIFO ATP synthase subunit 15 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma Technology, EBV-hybridoma technology, etc.
  • Incorporeal antibodies combining human constant regions and non-human variable regions can be produced using existing technologies (Morrison et al., PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human FIFO ATP synthase subunit 15.
  • Antibodies against human FIFO ATP synthase subunit 15 can be used in immunohistochemical techniques to detect human FIFO ATP synthase subunit 15 in biopsy specimens.
  • Monoclonal antibodies that bind to human FIFO ATP synthase subunit 15 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 FIFO ATP synthase subunit 15 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the ammonia of the antibody with a thiol crosslinker such as SPDP. Group, through the exchange of disulfide bonds, toxins are bound to antibodies, this hybrid antibody can be used to kill human FIFO
  • the antibodies of the present invention can be used to treat or prevent diseases related to the human FIFO ATP synthase subunit 15. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human FIFO ATP synthase subunit 15.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human FIFO ATP synthase subunit 15 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human FIFO ATP synthase subunit 15 detected in the test can be used to explain the importance of human FIFO ATP synthase subunit 15 in various diseases and to diagnose the role of human FIFO ATP synthase subunit 15 disease.
  • 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 FIFO ATP synthase subunit 15 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 FIFO ATP synthase subunit 15.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human FIFO ATP synthase subunit 15 to inhibit endogenous human FIFO ATP synthase subunit 15 activity.
  • a variant human FIFO ATP synthase subunit 15 may be a shortened human FIFO ATP synthase subunit 15 that lacks a signaling functional domain.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human FIFO ATP synthase subunit 15.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses can be used to transfer polynucleotides encoding human FIFO ATP synthase subunit 15 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human FIFO ATP synthase subunit 15 can be found in the existing literature (Sambrook, et al.).
  • recombinant polynucleotide encoding human FIFO ATP synthase subunit 15 can be packed 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) that inhibit human FIFO ATP synthetase E3 ⁇ 4 15 raRNA 3 ⁇ 43 ⁇ 4 ribozyme Within the scope of the Ming.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis 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 RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human FIFO ATP synthase subunit 15 can be used for the diagnosis of diseases related to human FIFO ATP synthase subunit 15.
  • the polynucleotide encoding human FIFO ATP synthase subunit 15 can be used to detect the expression of human FIFO ATP synthase subunit 15 or the abnormal expression of human FIFO ATP synthase subunit 15 in a disease state.
  • the DNA sequence encoding human FIFO ATP synthase subunit 15 can be used to hybridize biopsy specimens to determine the expression of human FIFO ATP synthase subunit 15. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a micro array or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human FIFO ATP synthase subunit 15 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human FIFO ATP synthase subunit 15 transcription products.
  • Human FIFO ATP synthase subunit 15 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human FIFO ATP synthase subunit 15 DNA sequence. 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, the Nor thern imprinting method and Wes tern blotting method 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 (repeating polymorphisms) are available for labeling chromosome positions.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers preferably 15-35 bp
  • 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 the chromosome 3 ⁇ 4 include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct a chromosome-specific cDNA library.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the 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 difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • containers there can be medicines manufactured, used or sold by Instructions given by the government regulatory agency for the product or biological product, which reflects the permission of the government regulatory agency for production, use, or sale to be administered to the human body.
  • the polypeptide of the present invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human FIFO ATP synthase subunit 15 to effectively treat and / Or prevent the amount of specific indications to be administered.
  • the amount and range of human FIFO ATP synthase subunit 15 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 diagnosing physician.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Quik mRNA Isolat ion Kit product of Qiegene was used to isolate poly (A) mRNA from total RNA. 2ug poly (A) mRNA was reverse transcribed to form cDNA.
  • the Smar t cDNA cloning kit purchased from Clontech
  • the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public D-sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0223 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the sequence of the human FIFO ATP synthase subunit 15 of the present invention and the protein sequence encoded by the FIFO ATP synthase 15 were coded using the Blas t program (Basic loca l al ignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990 215: 403-10], to perform homology search in databases such as Genbank, Switzerland, and so on.
  • the gene with the highest homology to the human FIFO ATP synthetase subunit 15 of the present invention is a known human FIFO ATP synthase subunit d, and its encoded protein has the accession number 'AF070650 in Genbank.
  • the protein homology results are shown in Figure 1.
  • Example 3 Cloning of a gene encoding human FIFO ATP synthetase subunit 15 by RT-PCR method The total RNA from fetal brain cells was used as a template, and oligo-dT was used as a primer for reverse transcription reaction to synthesize cDNA. , Using the following primers for PCR amplification:
  • Pr imer 1 5'- GGCTGCGGAGGACCGTGGGCAGC -3 '(SEQ ID NO: 3)
  • Pr imer2 5'- ACCGTATAATTATTATTTTTAAT -3 '(SEQ ID NO: 4)
  • Pr imerl 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 50 mmol / L KC1, 10 mmol / L Tri s-CI, (pH 8.5.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, l in 50 ⁇ 1 reaction volume Opmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min.
  • RT-PCR set ⁇ -act in as a positive control and template blank as a negative control.
  • Amplification products were purified using a QIAGEN kit, and the TA cloning kit was used to connect to a PCR vector (Unvi trogen).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the l-531bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human FIFO ATP synthase subunit 15 gene expression Total RNA was extracted in one step [Ana l. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate ( PH 4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49 : 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • 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- 2 5mM KH 2 P0 4 (pH7. 4) -5 ⁇ SSC-5 ⁇ Denhardt's solution and 200 ⁇ g / ml 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 FIFO ATP Synthase Subunit 15
  • Pr iraer 3 5,-CCCCATATGATGGCTGGGCGAAAACTTGCTCTA -3 '(Seq ID No: 5)
  • Pr imer4 5'- CCCCTCGAGTTTATAAATTCTCAATTGGTTGGT -3' (Seq ID No: 6)
  • the Ndel and Xhol restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • PCR was performed using the pBS-0023 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 P BS-0023 plasmid, primers Primer-3 and Primer-4, and j is l Opmol, Advantage po lymerase Mix (Clontech) 1 ⁇ 1.
  • Cycle parameters 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Ndel and Xhol 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 E.
  • coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 ⁇ g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0023) 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-0023) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to the final concentration lmraol / L, Continue incubation for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. An affinity chromatography column His s. Bind Quick Cartr idge (product of Novagen) was used which could bind 6 histidines (6His-Tag). Chromatography was performed to obtain a purified human protein FIFO ATP synthase subunit 15.
  • 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. Immunochemi s try, 1969; 6:43. Rabbits were immunized with 4 mg of the above L cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the 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. Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit sera.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody specifically binds to human FIFO ATP synthase subunit 15.

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Abstract

L'invention concerne un nouveau polypeptide, une sous-unité 15 de la synthase humaine F1F0 ATP, 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 sous-unité 15 de la synthase humaine F1F0 ATP.
PCT/CN2000/000403 1999-10-29 2000-10-30 Nouveau polypeptide, sous-unite 15 de la synthase humaine f1f0 atp, et polynucleotide codant pour ce polypeptide Ceased WO2001032701A1 (fr)

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CN 99119920 CN1302868A (zh) 1999-10-29 1999-10-29 一种新的多肽——人f1f0 atp合成酶亚基15和编码这种多肽的多核苷酸
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WO1999036526A1 (fr) * 1998-01-19 1999-07-22 Shanghai Second Medical University Cbmajc02: gene semblable au complexe de synthase bovine f1f0-atp de f-sous-unite a membrane f¿0?
US5962646A (en) * 1997-03-17 1999-10-05 Incyte Pharmaceuticals, Inc. ATP synthase Fo subunit

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5962646A (en) * 1997-03-17 1999-10-05 Incyte Pharmaceuticals, Inc. ATP synthase Fo subunit
WO1999036526A1 (fr) * 1998-01-19 1999-07-22 Shanghai Second Medical University Cbmajc02: gene semblable au complexe de synthase bovine f1f0-atp de f-sous-unite a membrane f¿0?

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HIGUTI T. ET AL.: "The complete amino acid sequence of subunit of rat liver mitochondrial H(+)- ATP synthase", J. BIOCHEM., vol. 114, no. 5, 1993, pages 714 - 717 *

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