WO2001092329A1 - Nouveau polypeptide, sous-unite $g(a) d'atp-synthetase 9.9, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, sous-unite $g(a) d'atp-synthetase 9.9, et polynucleotide codant ce polypeptide Download PDF

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WO2001092329A1
WO2001092329A1 PCT/CN2001/000843 CN0100843W WO0192329A1 WO 2001092329 A1 WO2001092329 A1 WO 2001092329A1 CN 0100843 W CN0100843 W CN 0100843W WO 0192329 A1 WO0192329 A1 WO 0192329A1
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polypeptide
polynucleotide
subunit
atp synthase
sequence
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PCT/CN2001/000843
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU81684/01A priority Critical patent/AU8168401A/en
Publication of WO2001092329A1 publication Critical patent/WO2001092329A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention belongs to the field of biotechnology, and specifically, the present invention describes a new polypeptide-"ATP synthase alpha subunit 9.9, and a polynucleotide sequence encoding the polypeptide.
  • the present invention also relates to the polynucleotide And polypeptide preparation method and application.
  • Mitochondrial ATP synthetase synthesizes ATP by using H ion transmembrane concentration gradients [Mi tchel l, P. 1979. Science 206, 1148-1159] [Hatef i, Y. 1985. Annu. Rev. Biochem. 54, 1015 -1069] 0 Mitochondrial ATP synthase is composed of 14 and different peptides, which make up two main units: F. Is embedded in the inner membrane of mitochondria; Fn is located in the stromal region. F. It is a hydrophobic complex and is a channel for protons through the inner membrane. It consists of 5 subunits (oc, ⁇ , ⁇ , ⁇ , and ⁇ ).
  • the composition ratio of these 5 subunits is roughly 3: 3: 1: 1.
  • the ⁇ ⁇ complex is the catalytic core.
  • the 6 and 8 subunits of F fl are encoded by the mitochondrial genome, while the other subunits are encoded by nuclear genes.
  • Subunits and other proteins required for ATP synthesis are coordinated in several cases [Kagawa, Y. and Ohta, S. 1990. Int. J. Biochem, 22, 219- 229], which include muscle activity.
  • F l 0 t subunit gene is 14 kbp in length and has 12 exons and 11 introns. All introns have GT and AG consensus nucleotides at the 5 'and 3' ends. Exon 1 contains a 5 'untranslated region and directs the enzyme to the mitochondrial region that encodes a 20 amino acid residue. Upstream region of the gene encoding the transcription initiation site F l C subunit has several characteristic features.
  • TATA box and CAAT box which is similar to the common sequence of the start region of several mammalian genes, including human subunits.
  • One is a binding domain that contains several cis-elements in the 5 'flanking region. These cis-elements play a very important role in regulating some nuclear genes encoding mitochondrial proteins.
  • Another is that the CS2 and CS3 sequences are the binding sites of nuclear factors [Shuichi Akiyama, Hitoshi Endo, Naohiro Inohara et al., 1994. Biochim. Biophys. Ac ta 1219 (1), 129-140].
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding the ATP synthase ⁇ subunit 9.9.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide "ATP synthase alpha subunit 9.9" of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with ATP synthase ⁇ subunit 9.9 abnormalities.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 81 to 353 in SEQ ID NO: 1; and (b) a sequence having 1-1714 in SEQ ID NO: 1 Sequence of bits.
  • 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 detecting a disease or susceptibility to disease associated with abnormal expression of ATP synthase ⁇ subunit 9.9 protein in vitro, comprising detecting a mutation in the polypeptide or a coding polynucleotide sequence thereof in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • the present invention also relates to the use of the polypeptides and / or polynucleotides of the present invention in the preparation of a medicament for the treatment of cancer, 'developmental or immune disease, or other diseases caused by abnormal expression of ATP synthase ⁇ subunit 9.9. .
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the ATP synthase oc subunit 9.9 and the ATP synthase ⁇ subunit of the present invention.
  • the upper graph is a graph of the expression profile of the ATP synthase ⁇ subunit 9.9
  • the lower graph is the graph of the expression profile of the ATP synthase ⁇ subunit.
  • 1-bladder mucosa 2-PMA + Ecv304 cell line, 3-LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5- Fibrob las t, growth factor stimulation, 1024NT, 6-scar into fc Growth factor stimulation, 1013HT, 7-scar into fc without growth factor stimulation, 1013HC, 8-bladder cancer construct cell EJ, 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12-liver cancer cell line, 13 -Fetal skin, 14- spleen, 15- prostate cancer, 16-jejunal adenocarcinoma, 17 cardiac cancer.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated ATP synthase ⁇ subunit 9.9.
  • OkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • Antagonist refers to a molecule that, when combined with the ATP synthase ct subunit 9.9, can block or regulate the biological or immunological activity of the ATP synthase alpha subunit 9.9.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to the ATP synthase alpha subunit 9.9.
  • Regulation refers to a change in the function of ATP synthase ⁇ subunit 9.9, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of ATP synthase c subunit 9.9. Or changes in immune properties.
  • Substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify the ATP synthase alpha subunit 9.9 using standard protein purification techniques.
  • Substantially pure ATP synthase ct subunit 9. 9 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the ATP synthase ⁇ subunit 9.9 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules 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 imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction. '
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods, such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: Number of residues matching between sequences X blue
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (He in L, (1990) Methods in enzyraology 183: 625-645). 0
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ,) 2 and? ⁇ It can specifically bind to the ATP synthase ⁇ subunit 9.9 epitope.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • 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 ( ⁇ ) such a type in which one or more amino acid residues are substituted with other groups to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide ( Such as leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences).
  • 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 CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 1,714 bases in length, and its open reading frames 81-353 encode 90 amino acids.
  • this peptide has a similar expression profile to the ATP synthase cc subunit, and it can be inferred that the ATP synthase ⁇ subunit 9.9 has similar functions to the ATP synthase ⁇ subunit.
  • the polynucleotide of the present invention may be in the form of MA or RNA.
  • DM forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DM can be coded or non-coded.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • 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 .
  • 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 the cDNA of interest is to isolate the 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 Manua, Cold Spruing 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.
  • genes of the present invention can be screened from these cDM libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DM-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determining the level of the ATP synthase ⁇ subunit 9.9 transcript (4) Detecting protein products expressed by genes through immunological techniques or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably 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 herein 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. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • a method for amplifying DM / RNA by PCR 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 measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDM sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDM sequences of multiple clones in order to splice into full-length cDM sequences.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the ATP synthase ⁇ subunit 9.9 coding sequence, and recombinant technology to produce the present invention Said method of polypeptide.
  • 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.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the ⁇ 01 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposomes Packaging, etc.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant ATP synthase alpha subunit 9.9 (Sc ience, 1984; 224: 1431). Generally there are the following
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • the AT enzyme complex is -F. Coupling factors play a coupling role in the process of oxidative phosphorylation. During ATP synthesis, the head, 0SCP handle, and? . Factor membranes work together. among them ? It consists of 5 subunits ⁇ , ⁇ , ⁇ , ⁇ and ⁇ . The ⁇ ⁇ complex is the catalytic core. The alpha subunit is of great significance in the energy metabolism of mitochondria.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human ATP synthase c subunit, both of which have Similar biological functions.
  • the polypeptide of the present invention plays a coupling role in oxidative phosphorylation during the energy metabolism of cell mitochondria, and its abnormal expression is usually associated with some related disorders of substance metabolism disorders, protein metabolism disorders and related tissue tumors and cancers It is closely related and produces related diseases.
  • the abnormal expression of the ATP synthase ⁇ subunit 9.9 of the present invention will produce various diseases, especially various tumors, embryonic development disorders, growth disorders, inflammation, and immune diseases. These diseases include But not limited to:
  • Tumors of various tissues stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma
  • Fetal developmental disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, brain development disorders, skin, fat and muscle dysplasia, bone and joint dysplasia, various metabolic defects, stunting, dwarfism, Cushing syndrome Sexual retardation
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • 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 various tumors, embryonic development disorders, growth and development disorders, inflammation, and immunity. Sexual diseases, certain hereditary, blood diseases, etc.
  • ATP synthase ⁇ subunit 9.9 When selecting a compound as an antagonist, ATP synthase ⁇ subunit 9.9 can be added to the bioanalytical assay, and the effect of the compound on the interaction between ATP synthase ⁇ subunit 9.9 and its receptor can be determined. To determine if the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to ATP synthase ⁇ subunit 9.9 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the ATP molecule ⁇ subunit 9.9 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 the ATP synthase alpha subunit 9.9 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Embedding 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 U.S. Pat No. 4946778, can also be used to produce single chain antibodies against the ATP synthase ⁇ subunit 9.9.
  • Antibodies against ATP synthase ⁇ subunit 9.9 can be used in immunohistochemistry to detect ATP synthase ⁇ subunit 9.9 in biopsy specimens.
  • Monoclonal antibodies that bind to ATP synthase ⁇ subunit 9.9 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.
  • immunotoxins such as ATP synthase ⁇ subunit 9.9 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. 9, through the exchange of disulfide bonds, toxins are bound to antibodies, this hybrid antibody can be used to kill ATP synthase ⁇ subunit 9.9 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the ATP synthase ⁇ subunit 9.9. Administration of appropriate doses of antibodies can stimulate or block the production or activity of ATP synthase ⁇ subunit 9.9.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of the level of ATP synthase cc subunit 9.9.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of ATP synthase ⁇ subunit 9.9 detected in the test can be used to explain the importance of ATP synthase ot subunit 9.9 in various diseases and to diagnose the ATP synthase ⁇ subunit 9.9. A working 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.
  • Polynucleotides encoding the ATP synthase alpha subunit 9.9 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 ATP synthase ⁇ subunit 9.9.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated ATP synthase a subunit 9.9 to inhibit endogenous ATP synthase ⁇ subunit 9.9 activity.
  • a variant ATP synthase ⁇ subunit 9.9 may be a shortened ATP synthase ⁇ subunit 9.9 lacking a signaling domain, although it can bind to downstream substrates, but lacks signal transduction. active.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of ATP synthase ⁇ subunit 9.9.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, parvoviruses, and the like can be used to transfer polynucleotides encoding the ATP synthase ⁇ subunit 9.9 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding the ATP synthase ⁇ subunit 9.9 can be found in the existing literature (Sambrook, eta l.).
  • a polynucleotide encoding the ATP synthase ⁇ subunit 9.9 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 RM and DM
  • ribozymes that inhibit the ATP synthase alpha subunit 9.9 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target MA to perform endonucleation.
  • Antisense RNA and DNA and ribozymes can be obtained by any conventional RNA or DNA synthesis technology. For example, solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. RNA polymerase Promoter downstream. 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 the ATP synthase ⁇ subunit 9.9 can be used for the diagnosis of diseases related to the ATP synthase ⁇ subunit 9.9. Coder?
  • the polynucleotide of the synthetase 01 subunit. 9.9 can be used to detect the expression of the ATP synthase ⁇ subunit 9.9 or the abnormal expression of the ATP synthase ⁇ subunit 9.9 in a disease state.
  • a DNA sequence encoding ATP synthase ⁇ subunit 9.9 can be used to hybridize biopsy specimens to determine the expression status of ATP synthase ⁇ subunit 9.9.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like.
  • Detection of mutations in the ATP synthase ⁇ subunit 9.9 gene can also be used to diagnose ATP synthase ⁇ subunit 9.9 related diseases.
  • ATP synthetase ⁇ subunit 9. 9 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type ATP synthase a subunit 9.9 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, MA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct a chromosome-specific CDM library.
  • Fluorescent in situ hybridization (FISH) of cDNA clones to metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH fluorescent in situ hybridization
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Quik raRNA Isolat ion Kit product of Qiegene was used to isolate poly (A) mRNA from 2 ⁇ g poly (A) mRM by reverse transcription to form cDNA.
  • Smart cDM cloning kit purchased from Clontech was used to insert the cDM fragment into the multicloning site of pBSK (+) vector (Clontech) to transform DH5c. The bacteria formed a cDM library.
  • the 0645A12 clone contains a full-length cDNA of 1714bp (as shown by Seq ID NO: l), and has a 272bp open reading frame (0RF) from 81bp to 353bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0645A12 and the encoded protein was named ATP synthase ct subunit 9.9.
  • Example 2 Cloning of a gene encoding the ATP synthase ⁇ subunit 9.9 using RT-PCR
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • the amplified product was purified with a QIAGEN kit and connected to a PCR vector with a TA cloning kit (Invi trogen). DNA sequence analysis The results showed that the DNA sequence of the PCR product was identical to l-1714bp shown in SEQ ID NO: 1.
  • 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 the genome or cD library of normal tissues or pathological tissues 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 sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps Off.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecule region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • 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:
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32P-dATP) is prepared.
  • the sample membrane was placed in a plastic bag, and 3-1 Omg pre-hybridization solution (1 OxDenhardt's; 6xSSC, 0.1 lrag / ml CT DM (calf thymus DM)) was added. After sealing the bag, shake at 68 ° C for 2 hours.
  • 3-1 Omg pre-hybridization solution (1 OxDenhardt's; 6xSSC, 0.1 lrag / ml CT DM (calf thymus DM)
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotides of the present invention. They were amplified by PCR respectively. After purification, the amplified product was adjusted to a concentration of about 500ng / ul, and spotted on a glass medium with a Cartesian 7500 spotting instrument (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DM on the glass slide to prepare chips. Its specific method steps There are many reports in the literature. The sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Ol igotex mRNA Midi Kit (purchased from QiaGen), and separated by reverse transcription.
  • L The fluorescent reagent Cy3dUTP (5-Araino-propargyl-2'-deoxyuridine 5 '-triphate coupled to Cy3 f luorescent dye, purchased from Amershara Pharaacia Biotech) was used to label mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino- propargyl -2'-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech Company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Cy3dUTP 5-Araino-propargyl-2'-deoxyuridine 5 '-triphate coupled to Cy3
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 10 2 4NT, scar-like fc growth Factor stimulation, 1013HT, scar into fc without stimulation with growth factor, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunum adenocarcinoma, cardia cancer .

Abstract

L'invention concerne un nouveau polypeptide, une sous-unité α d'ATP-synthétase 9.9, et un polynucléotide codant 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, des troubles du développement, 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 la sous-unité α d'ATP-synthétase 9.9.
PCT/CN2001/000843 2000-05-24 2001-05-21 Nouveau polypeptide, sous-unite $g(a) d'atp-synthetase 9.9, et polynucleotide codant ce polypeptide WO2001092329A1 (fr)

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AU81684/01A AU8168401A (en) 2000-05-24 2001-05-21 A novel polypeptide - alpha-subunit 9.9 of atp synthase and a polynucleotide encoding the same

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CN00115847.3 2000-05-24
CN 00115847 CN1324930A (zh) 2000-05-24 2000-05-24 一种新的多肽——ATP合成酶α亚基9.9和编码这种多肽的多核苷酸

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US20110236389A1 (en) * 2007-05-22 2011-09-29 Human Antibodomics Development (Sip) Limited Human Angiostatin Interacting and Tumor Metastasis Involving Protein Variants and Uses Thereof

Citations (3)

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Publication number Priority date Publication date Assignee Title
US5786150A (en) * 1997-03-11 1998-07-28 Incyte Pharmaceuticals, Inc. F0 ATP synthase 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?
US5962646A (en) * 1997-03-17 1999-10-05 Incyte Pharmaceuticals, Inc. ATP synthase Fo subunit

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Publication number Priority date Publication date Assignee Title
US5786150A (en) * 1997-03-11 1998-07-28 Incyte Pharmaceuticals, Inc. F0 ATP synthase subunit
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?

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TOMIHIKO HIGUTI, J. BIOCHEM., vol. 114, no. 5, 1993, pages 714 - 717, XP002905319 *

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