WO2001030836A1 - Novel human atpases and encoding sequences thereof - Google Patents
Novel human atpases and encoding sequences thereof Download PDFInfo
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- WO2001030836A1 WO2001030836A1 PCT/CN2000/000371 CN0000371W WO0130836A1 WO 2001030836 A1 WO2001030836 A1 WO 2001030836A1 CN 0000371 W CN0000371 W CN 0000371W WO 0130836 A1 WO0130836 A1 WO 0130836A1
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Definitions
- the present invention belongs to the field of biotechnology and genetic engineering. Specifically, the present invention relates to a novel polypeptide-human ATI) synthase (Nove) Human ATPase ("Bi oATPase”), and a polynucleotide encoding the polypeptide. sequence. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
- ATP provides energy for the demand response of any cell.
- ⁇ synthase is a membrane-intrinsic protein, a unit of biofilm energy conversion, and is involved in the key steps of oxidative phosphorylation of the mitochondrial respiratory chain. It is a multi-component complex with a three-part structure, namely the head, handle, and membrane.
- the head (F1 coupling factor) is composed of five kinds of 10 peptides, including a nucleic acid domain (binding ATP) and a thermostable small protein molecule, which has a catalytic function and can synthesize or hydrolyze ATP.
- the handle contains several Other proteins, one of which is sensitive to oligomycin.
- the membrane part (F0 coupling factor) is composed of a hydrophobic polypeptide chain and lipoprotein, which is embedded in the membrane lipid bilayer and has a proton conductor function.
- ATP synthase is coupled with Ca 2+ pump or Na + -k + pump, and participates in the transport of Ca 2+ or Na + -k ⁇ and the hydrolysis of ATP.
- the Na k ion pump can promote the conformational transformation of A ⁇ P synthase in E1 and E2 types.
- the ion-binding site of E1 faces the inner moon and has a high affinity with Na ⁇ ; the ion-binding site of E2 faces extracellularly and has a low affinity.
- the bound ATP synthase can pump Ca 2T out of the cytoplasm, keep the Ca 2+ concentration low in the cytoplasm, and maintain basic cell functions [Arrondo JL, et al .. J Biol Chem 1987; 262: 9037-43].
- Ca can be transported in the sarcoplasmic reticulum : from the cytoplasm to the sarcoplasmic reticulum, Ca 2+ is stored in the sarcoplasmic reticulum. The release of Ca 2+ from the sarcoplasmic reticulum into the myocyte fluid causes contraction.
- the polypeptide of the present invention can be deduced to be a new human ATP synthase, which contains characteristic domains of the ATP synthase family and has similar biological functions.
- ATP synthase and its encoded polynucleotides have been linked to diseases such as neurological degenerative diseases, muscle diseases, immune disorders, and cancer. Therefore, research and development of human ATP synthase for therapeutic purposes is of great significance. Public open
- Another object of the present invention is to provide a polynucleotide encoding the Bi oATPase polypeptide.
- Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding Bi oATPase. Cell.
- Another object of the present invention is to provide a method for producing BioATPase.
- Another object of the present invention is to provide an antibody against the BioATPase polypeptide of the present invention.
- Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the BioATPase polypeptide of the present invention.
- Another object of the present invention is to provide a method for diagnosing and treating a disease associated with an abnormality of BioATPase.
- a novel and isolated human ATP synthase (BioATPase) is provided.
- the polypeptide is of human origin. It 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, and the like.
- the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a derivative thereof having no more than 5% amino acid variation.
- 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 above Bi oATPa.se; (b) a polynucleotide complementary to the polynucleotide (a).
- the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
- sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence of positions 97-1 to 137 in SEQ ID NO: 1; and (b) a sequence of 1-in SEQ ID NO: 1 :-! A sequence of S4n bits.
- 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. of.
- isolated BioATPase protein or polypeptide means that BioATPase is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can use standard protein purification techniques to purify BioATPase. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of BioATPase polypeptide can be analyzed by amino acid sequence.
- the present invention provides a new polypeptide, BioATPase polypeptide, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
- the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
- Polypeptides of the invention may be naturally purified products or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using a set of 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 BioATPase.
- fragment As used in the present invention, the terms “fragment”, “derivative” and “analog” refer to essentially maintaining the same biological function of the natural Bi oATPase of the present invention
- peptide, a fragment, derivative or analog of the peptide of the present invention may be: (1) such a species' One or more amino acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted amino acid may or may not be encoded by a genetic codon; or (II) such a, One of the one or more amino acid residues is substituted with another group to include a substituent; or (in) a type in which the mature polypeptide is associated with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene Diol) fusion; or (IV) a polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) in which an additional amino acid sequence is fused into a mature polypeptide.
- such fragments, derivatives and analogs are considered to be within the knowledge of those skilled
- the invention also provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ: NO: 2.
- polynucleotide sequence of the present invention has a nucleotide sequence of SEQ ID NU: 1.
- the polynucleotide of the present invention may be in the form of DNA or RNA.
- DNA forms include cDNA, genomic DNA, or synthetic DNA.
- DNA can be single-stranded or double-stranded.
- DNA can be coding or non-coding.
- the coding region sequence of the encoded 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: but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
- the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
- polynucleotide encoding a polypeptide refers to a polynucleotide 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 a replacement form of a polynucleotide, which may be one or more than 1, a nucleotide substitution, deletion, or insertion, but does not substantially change the polypeptide encoded by it Functions.
- the invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least 50%, preferably 70% identity between the two sequences).
- the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
- "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.
- the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID I) NU: 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 nucleosides. Ii preferably at least ion t nucleosides Acid above. Nucleic acid fragments can also be amplified using f nucleic acid (such as
- polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
- polynucleotide sequence encoding the BioATPa.se 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 cDA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect polynuclear clones with common structural characteristics Nucleotide fragments.
- the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) Isolating a double-stranded DNA sequence from genomic DNA: 2) Chemically synthesizing a DNA 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.
- Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
- the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (D DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of Bi oATPase transcripts; (4) through immunological techniques Or measure the biological activity to detect the protein product of gene expression. 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 has a length of at least 10 nucleotides, preferably at least 3 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
- the length of the probe is usually within 2,000 nucleotides, preferably within 1000 nucleotides.
- the probe used here is generally a IA ⁇ 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 BioATPase 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 cDNA end rapid amplification method
- the primers 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 the de-stranding method (Sanger et ul. PNAS, 1977, 74: 5463-5467). 3 ⁇ 4'i nucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
- the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the Bi oATPa.se coding sequence, and a method for producing the peptide of the present invention by recombinant technology.
- a polynucleotide sequence encoding Bi oATPa.se 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 expressed in bacteria (Rosenberg, et al.
- pMSXND expression vectors expressed in mammalian cells e an d Nathans, J Bio Chem. 263: 3521, 1988
- baculovirus-derived vectors expressed in insect cells e an d Nathans, J Bio Chem. 263: 3521, 1988
- insect cells e an d Nathans, J Bio Chem. 263: 3521, 1988
- any plasmid and vector can be used to construct a recombinant expression vector.
- An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
- Methods known to those skilled in the art can be used to construct expression vectors containing a wake sequence encoding BioATPa.se and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et ul. Molecular Cloning, a Laboratory Manual, co. Spring Harbor Laboratory. 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: the l ac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter,
- HSV thymidine kinase promoter HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
- the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. 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 30 () base pairs that act on promoters to enhance gene transcription. Examples include 100 to 270 base pair SV40 enhancers on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
- the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
- selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
- GFP fluorescent protein
- tetracycline or ampicillin resistance for E. coli.
- a polynucleotide encoding Bi oATPase or a recombinant vector containing the polynucleotide is transformed or introduced 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.
- host cells are: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf9 : animal fine I H0, U) S or Bowes melanin Tumor cells and so on. Transformation of a host cell with a warship sequence or a recombinant vector containing the DNA sequence of the present invention can be performed by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing NA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. It can also be performed with M S C 1 2 .
- 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 liposome packaging.
- the polynucleotide sequence of the present invention can be used to express or produce recombinant Biatotiase (Science, 1984; 224: 1431). Generally there are the following steps:
- the medium used in the culture may be selected from various conventional culture media depending on the host cell 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. If necessary, 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, osmosis, 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. Brief explanation of attached picture
- FIG. 1 is a comparison diagram of the amino acid sequence homology between human ATP synthase Bi oATPa.se and nematode ATP synthase (AF099920). Identical amino acids are represented by single-character amino acids between the two sequences, similar amino acids are represented by "+", and industrial applicability
- polypeptides of the present invention can be directly used in the treatment of diseases.
- BioATPase protein or polypeptides can be used as drugs to treat diseases caused by low or loss of ATP synthase function.
- B i uAT ast 'antagonists can be used to treat or prevent immune disorders, including but not limited to: glomerulonephritis, lupus erythematosus, Graves' disease, diabetes, anemia, emphysema, pancreatitis , Atrophic gastritis, Sjogren's syndrome, Addison's disease, osteoarthritis, gout, polymyositis, myasthenia gravis, atopic dermatitis, asthma, bronchitis, autoimmune thyroiditis, etc.
- Antibodies that specifically bind to B o oATPase can be used directly as antagonists, Or indirectly, agents are brought into cells or tissues expressing BioATPase by targeting or delivery mechanisms.
- Antagonists or fragments or derivatives of ioATPase can be used to treat or prevent degenerative neurological diseases, including (but not limited to): Alzheimer's disease, Huntington's disease, Parkinson's disease, epilepsy, Down syndrome, multiple sclerosis, etc.
- Antagonists or fragments or derivatives of BioATPase can be used to treat or prevent muscle disorders including (but not limited to): progressive peripheral ophthalmoplegia, Kearns-Sayre syndrome, myoclonus, encephalopathy, myocarditis, Lactic acidosis and so on.
- BioATPase antagonists or fragments or derivatives can be used to treat or prevent cancer.
- Cancers include (but are not limited to): adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, etc .; especially kidney, bladder, pancreas, bone, Brain, breast, uterus, gallbladder, liver, lung, thyroid, esophagus, testis, skin, mesentery and other cancer-related antibodies that specifically bind to BwATPase can be used directly as antagonists, or indirectly through the targeting or delivery mechanism of the agent Taken into cells or tissues that express BioATPase. Antagonists or fragments or derivatives of BioATPase can be used to treat or prevent cancer immune disorders.
- the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) BioATPase.
- Agonists enhance biological functions such as BioATPase to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
- mammalian cells or membrane preparations expressing BinATPa.se are cultured with labeled BioATPase in the presence of a drug, and then the ability of the drug to increase or suppress this interaction is determined to identify an agonist or antagonist.
- BioATPase antagonists include antibodies, compounds, receptor deletions, and analogs that have been screened. BioATPase antagonists can bind to BioATPase and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
- BioATPase When screening compounds as antagonists, BioATPase can be added to the bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between BioATPase and its receptor. Using the same method of screening compounds described above, Receptor deletions and analogues that act as antagonists are screened out. Polypeptide molecules that can bind to BioATPase can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally respond BioATPase molecules are labeled.
- the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen. These antibodies can be polyclonal or monoclonal antibodies.
- the present invention also provides
- Antibodies to the BioATPase epitope include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fah fragments, and fragments generated from Fab expression libraries.
- Polyclonal antibodies can be produced by injecting BioATPase directly into immunized animals (such as rabbits, mice, rats, etc.).
- immunized animals such as rabbits, mice, rats, etc.
- adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
- Monoclonal antibodies include (but are not limited to): Hybridoma technology (Kohler and i lst t , i ..
- Anti-BioATPase antibodies can be used in immunohistochemical techniques to detect BioATP in biopsy specimens ; ise. Monoclonal antibodies that bind to BioATPase 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.
- BioATPase high-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
- a common method is to attack the amino group of the antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
- This hybrid antibody can be used to kill BioATPase positive cells.
- the antibodies of the present invention can be used to treat or prevent diseases associated with BioATPase. Administration of appropriate doses of antibodies can stimulate or block BioATPase production or activity.
- the invention also relates to a diagnostic test method for quantitative and localized detection of BioATPase levels. These tests are well known in the art and include FISH and radioimmunoassays. The level of BioATPa.se measured in the test can be used to explain the importance of BioATPa.se in various diseases and to diagnose diseases where BioATPase works.
- 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.
- BioATPase 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 Bi oATPa.se.
- Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated BioATPase to inhibit endogenous BioATPas activity.
- a mutated BioATPase can be a shortened B ioATPase that lacks the signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of BioATPase.
- Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer polynucleotides encoding BioATPase into cells.
- the method of constructing a recombinant viral vector carrying a polynucleotide encoding BioATPase can be found in the existing literature (Sambrook, et al.).
- the recombinant polynucleotide encoding BioATPase can be packaged into liposomes and transferred into cells.
- Methods for introducing polynucleotides into tissues or cells include: Injecting the polynucleotides directly into tissues in the body: Or, first introduce the polynucleotides into cells through a vector (such as a virus, phage, or plasmid) in vitro, and then transfer the cells. Pull into the body and so on.
- a vector such as a virus, phage, or plasmid
- Oligonucleotides including antisense RNA and DNA
- ribozymes that inhibit BioATPase mRNA are also within the scope of the present invention.
- a ribozyme is an enzyme-like RNA molecule that can specifically decompose a 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 using ⁇ : Ho KNA or UNA synthesis techniques, such as 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. This IAA sequence has been integrated downstream of the RNA polymerase promoter of the vector. To increase the stability of nucleic acid molecules, It can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
- the polynucleotide encoding BioATPase can be used for the diagnosis of diseases related to BioATPase.
- Polynucleotides encoding BioATPase can be used to detect the expression of BioATPase or abnormal expression of BioATPas in disease states.
- the DNA sequence encoding BioATPase can be used to hybridize biopsy specimens to determine the expression of BioATPase.
- Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and so on. These techniques and methods are publicly available and mature, and related kits are commercially available.
- polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (hereinafter referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
- BioATPas specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect BioATPase transcripts.
- BioATPase mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type BioATPa.se DNA sequences. 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, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
- sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position of a human chromosome and can hybridize with it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
- PCR primers (preferably 15-35bp) are prepared based on 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 hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
- PCR localization of somatic hybrid cells is a quick way to locate 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 chromosome-specific cDNA libraries.
- Fluorescent in situ hybridization of cDNA clones to metaphase chromosomes allows precise chromosomal localization in one step.
- FISH Fluorescent in situ hybridization
- the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If in some or A mutation is observed in all diseased individuals, and the mutation is not observed in any normal individuals, then the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
- the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier (pharmaceutically acceptable carrier).
- a suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
- the composition comprises a safe and effective amount of a polypeptide or antagonist of the present invention and a carrier and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
- the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
- a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
- these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that manufacture, use, or sell them.
- the polypeptides of the invention can be used in combination with other therapeutic compounds.
- the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
- BioATPa.se is administered in an amount effective to treat and / or prevent a specific indication.
- the amount and range of BioATPa.se administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.
- an isolated polynucleotide which encodes a mature polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
- the polynucleotide was found from a cDNA library of human fetal brain tissue.
- the polynucleotide sequence is 3845 bases in length and its open reading frame (97-1 137) encodes 346 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 41% homology with the ATP synthase of Caenorhabdi tis el egans, and it can be inferred that the new human BiOATPase of the present invention has a similar structure to the ATP synthase gene family. and function.
- the cDNA, oligonucleotides, peptides, and antibodies of the human BioATPase provided by the present invention are useful for studying the role of ATP synthase in different tissues and cells, diagnosing diseases related to ATP synthase disorders, screening inhibitors or drug treatment. These diseases have important value.
- the best way to implement the invention is useful for studying the role of ATP synthase in different tissues and cells, diagnosing diseases related to ATP synthase disorders, screening inhibitors or drug treatment. These diseases have important value. The best way to implement the invention
- Isolation of total human fetal brain RNA with guanidine isothiocyanate / phenol / chloroform-isocyanate method o Quik mRNA I so l at i on K it (QieKen Company)
- the Smart. CDNA cloning kit purchased from Clontech was used to insert the cDNA fragments into the multicloning site of pBSK (+> vector (Gk tech)) to transform the DH5a bacteria to form a cDNA library.
- the dye was used to terminate the cycle reaction sequencing reagent Cassette (Perkin nE 1 mer company) and AB I 377 automatic sequencing protocol (Perkin-E 1 mt 'r company) to determine the sequence of the 5' and 3 'ends of all clones.
- the determined cDNA sequence was compared with Some public I) NA sequence databases (Genebank) were compared and found that the cDNA sequence of one of the clones (0504g02) was new DNA.
- a series of primers were synthesized for the bidirectional determination of the inserted cDNA fragments contained in the clone.
- the sequence of the human BioATPase gene of the present invention and the protein sequence encoded by the bioATPase gene of the present invention were performed using the B 1 as t program (Has ic 1 oca J Alignment search tool) [Altschul, SF et al. ; 215: 403-10] for homology search in databases such as Genbank and Swissport.
- the gene with the highest homology to the human BioATPase gene of the present invention is a known nematode Caenorhabdi ti s e3 ⁇ 4 ", s') ATP synthase gene, and the accession number encoded by the protein in Genbank is AF099920.
- the results of the source comparison are shown in Figure 1.
- CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.
- Primer 1 5 '-GGGGGTCCCGGCTGCTCGGC -3' (SEQ ID NO.3)
- Primer 2 5 '-TGCAGTAATTTAATTTAATA -3' (SEQ ID NO.4)
- Primer 1 is the forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1; Primer 2 is the reverse sequence of the: ') end in SEQ ID NO: 1.
- set ⁇ -actin as a positive control and template blank as a negative control at the time of RT-PCR.
- the amplification product was purified using a QIAGEN kit and connected to a pCR vector using a TA clone kit (Invitrogen product). ).
- the DNA sequence analysis results show that the [) NA sequence of the PCR product is exactly the same as the 1-3845bp shown in SEQ ID NO: 1.
- Example 4 Analysis of BioATPase gene expression by Northern blotting:
- This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49 : 1), centrifuge after mixing. The aqueous phase was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RNA precipitate. The resulting precipitate was washed with 70% ethanol, dried and dissolved in water.
- Primer 4 5 '-CCCGCGGCCGCAACTCACTGTGTCGCCCCAT -3' (SEQ ID No 6)
- the 5 'ends of these two primers contain BamHI and Ncol restriction sites, respectively, followed by the coding sequences of the 5 and 3' ends of the target gene, respectively.
- the BamHI and Ncol restriction sites correspond to the expression vector plasmid pET_28b (+) ( Novagen product, Cat. No. 69865. 3).
- the PCR reaction was performed using the pBS 0504 ⁇ 2 plasmid containing the full-length target gene as a template. PCR reaction conditions were: total volume of 50 ⁇ 1 containing pBS_0504g () 2 Plasmid 10pg, Wo port bow Bow I was 3 I was 4 points to another lj l Opmmol, Advantage polymerase Mi x ( Cl ontech Products) ⁇ ⁇ 1.
- Cycle parameters 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
- the amplified product and plasmid pET-28 (+) were double-digested with BamH I and Nco l, respectively, and large fragments were recovered and ligated with T4 ligase.
- the ligation product was transformed into E. coli Dh5a using the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 3 ( ⁇ g / ml)), positive clones were screened by colony PCR method and sequenced. Correct positive clone (pET-0504 g 02) The recombinant plasmid was transformed into E.
- the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
- hemocyanin and bovine serum albumin For the method, see: Avr ameiiS , et aJ. Immunochemistry, 1969; 6:43. Rabbits were immunized with 1 ⁇ 2 g of the hemocyanin-peptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
- the titer of antibody in rabbit serum was determined by ELISA using a titer plate coated with 15 ⁇ K ⁇ / ml bovine serum albumin polypeptide complex.
- Total protein Ig was isolated from antibody-positive rabbit serum using protein A-Sepharo.se.
- the peptide was bound to a cyanogen bromide-activated Sepharose 4B column, and anti-peptide antibodies were isolated from total IgG by affinity chromatography.
- the immunoprecipitation method proved that the purified antibody could specifically bind to BioATPase.
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Abstract
The invention discloses a novel polypeptide, human ATPase (for short, 'BioATPase'), the polynucleotide encoding the polypeptide, and the method of production thereof by DNA recombinant techniques. Methods of using the polypeptide and the polynucleotide for treating diseases such as nerve retrograde affection, muscle disease, immune disorder, cancer, and the like are also disclosed. Furthermore, the invention discloses the antagonist of the polypeptide and the effects thereof. Using of the polynucleotide encoding the novel BioATPase is also disclosed.
Description
说 明 书 Explanation book
新的人 ATP合酶及其编码序列 技 术 领 域 New human ATP synthase and its coding sequence technology field
本发明属于生物技术领域和遗传工程领域, 具体地说, 本发明涉及了一种新的多 肽一人 ATI) 合酶(Nove ] Human ATPase,简称 " Bi oATPase " ), 以及编码此多肽的多核 苷酸序列。 本发明还涉及此多核苷酸和多肽的制备方法和应用。 背 景 技 术 The present invention belongs to the field of biotechnology and genetic engineering. Specifically, the present invention relates to a novel polypeptide-human ATI) synthase (Nove) Human ATPase ("Bi oATPase"), and a polynucleotide encoding the polypeptide. sequence. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
ATP 为任何一个细胞的需能反应提供能量。 ΑΓΡ 合酶是膜内在蛋白, 是生物膜能量 转换单位, 参与线粒体呼吸链的氧化磷酸化关键步骤。 它是个多组分的复合物, 分三部 分结构, 即头部, 柄部和膜部。 头部 (F1偶联因子) 由五种 10条多肽构成, 包含一个核 酸结构域 (结合 ATP ) 和一个热稳定小蛋白分子, 具催化作用, 并能合成或水解 ATP ., 柄部包含几种其他蛋白, 其中有一种对寡霉素敏感的蛋白。 膜部 (F0 偶联因子) 由疏水 性的多肽链和脂蛋白组成, 嵌入膜脂双分子层, 具有质子导体作用。 ATP provides energy for the demand response of any cell. ΑΓΡ synthase is a membrane-intrinsic protein, a unit of biofilm energy conversion, and is involved in the key steps of oxidative phosphorylation of the mitochondrial respiratory chain. It is a multi-component complex with a three-part structure, namely the head, handle, and membrane. The head (F1 coupling factor) is composed of five kinds of 10 peptides, including a nucleic acid domain (binding ATP) and a thermostable small protein molecule, which has a catalytic function and can synthesize or hydrolyze ATP. The handle contains several Other proteins, one of which is sensitive to oligomycin. The membrane part (F0 coupling factor) is composed of a hydrophobic polypeptide chain and lipoprotein, which is embedded in the membrane lipid bilayer and has a proton conductor function.
ATP合酶与 Ca2+泵或 Na+- k+泵偶合, 参与 Ca2+或 Na+-kτ的转运及 ATP的水解。 Na k 离子泵可促使 ΑΓΡ合酶的二级结构以 E1型, E2型实现构象转变。 E1 的离子结合位点面 向月包内, 与 Na^有高亲和性; E2的离子结合位点面向胞外, 与 有低亲和性。 大多数 ATP 水解反应循环中, 在胞内 Na^与 ATP合酶 E1 构型结合, 触发 E1 的磷酸化, 以至离子结 合位点翻转朝外成 E2构型, 并释放 Na+在胞外, 而后结合 以至触发 E2的去磷酸化 转为 E1构型 [Gasset M,et al. J Biol Chem 1997;272: 1608-14] Na - k ATP合酶的正常功 能对维持物质逆浓度梯度的主动运输是非常必要的。 ATP synthase is coupled with Ca 2+ pump or Na + -k + pump, and participates in the transport of Ca 2+ or Na + -k τ and the hydrolysis of ATP. The Na k ion pump can promote the conformational transformation of AΓP synthase in E1 and E2 types. The ion-binding site of E1 faces the inner moon and has a high affinity with Na ^; the ion-binding site of E2 faces extracellularly and has a low affinity. In most ATP hydrolysis reaction cycles, intracellular Na ^ binds to the ATP synthase E1 configuration, triggering the phosphorylation of E1, so that the ion-binding site flips outwards into the E2 configuration, and releases Na + outside the cell, and then Binding to trigger E2 dephosphorylation into the E1 configuration [Gasset M, et al. J Biol Chem 1997; 272: 1608-14] The normal function of Na-k ATP synthase is active to maintain the reverse concentration gradient of the substance very necessary.
结合的 ATP合酶能将 Ca2T泵出细胞质, 使 Ca2+浓度在细胞质中维持低水平, 维持 细胞基本功能 [Arrondo JL,et al.. J Biol Chem 1987;262:9037-43]。如肌质网膜内能运输 Ca: 从细胞质到肌质网内, 在肌质网内储存 Ca2+。 从肌质网释放 Ca2+到肌细胞液引起收缩。 The bound ATP synthase can pump Ca 2T out of the cytoplasm, keep the Ca 2+ concentration low in the cytoplasm, and maintain basic cell functions [Arrondo JL, et al .. J Biol Chem 1987; 262: 9037-43]. For example, Ca can be transported in the sarcoplasmic reticulum : from the cytoplasm to the sarcoplasmic reticulum, Ca 2+ is stored in the sarcoplasmic reticulum. The release of Ca 2+ from the sarcoplasmic reticulum into the myocyte fluid causes contraction.
本发明的多肽根据同源比较, 可推断为新的人 ATP 合酶, 含 ATP 合酶家族的特征性 结构域, 并具有相似的生物学功能。 研究发现, ATP合酶及其编码的多核苷酸与神经系统 退行性疾病、 肌肉疾病、 免疫紊乱、 癌症等疾病有关。 因此, 为治疗目的研究和开发 人 ATP合酶有重要意义。 发 明 的 公 开 Based on the homology comparison, the polypeptide of the present invention can be deduced to be a new human ATP synthase, which contains characteristic domains of the ATP synthase family and has similar biological functions. Studies have found that ATP synthase and its encoded polynucleotides have been linked to diseases such as neurological degenerative diseases, muscle diseases, immune disorders, and cancer. Therefore, research and development of human ATP synthase for therapeutic purposes is of great significance. Public open
本发明的一个目的是提供分离的新的多肽——人 ATP 合酶(简称为 " Bi oATPase " ) 以及其片段、 类似物和衍生物。 It is an object of the present invention to provide an isolated novel polypeptide, human ATP synthase (referred to as "Bi oATPase") and fragments, analogs and derivatives thereof.
本发明的另一个目的是提供编码该 Bi oATPase多肽的多核苷酸。 Another object of the present invention is to provide a polynucleotide encoding the Bi oATPase polypeptide.
本发明的另一个目的是提供含有编码 BioATPase的多核苷酸的重组载体。 It is another object of the present invention to provide a recombinant vector containing a polynucleotide encoding a BioATPase.
本发明的另一个目的是提供含有编码 Bi oATPase 的多核苷酸的基因工程化宿 ΐ细
胞。 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding Bi oATPase. Cell.
本发明的另一个目的是提供生产 BioATPase的方法。 Another object of the present invention is to provide a method for producing BioATPase.
本发明的另一个目的是提供针对本发明的 BioATPase多肽的抗体。 Another object of the present invention is to provide an antibody against the BioATPase polypeptide of the present invention.
本发明的另一个目的是提供了针对本发明 BioATPase 多肽的模拟化合物、 拮抗剂、 激动剂、 抑制剂。 Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the BioATPase polypeptide of the present invention.
本发明的另一个目的是提供诊断和治疗与 BioATPase异常相关的疾病的方法,, 在本发明的第一方面, 提供新颖的分离出的人 ATP合酶(BioATPase) , 该多肽是人 源的, 它包含: 具有 SEQ ID NO: 2 氨基酸序列的多肽、 或其保守性变异多肽、 或其 活性片段、 或其活性衍生物、 类似物。 较佳地, 该多肽是具有 SEQ ID NO : 2 氨基酸 序列的多肽或其氨基酸变异不超过 5 %的衍生物。 Another object of the present invention is to provide a method for diagnosing and treating a disease associated with an abnormality of BioATPase. In a first aspect of the present invention, a novel and isolated human ATP synthase (BioATPase) is provided. The polypeptide is of human origin. It 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, and the like. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a derivative thereof having no more than 5% amino acid variation.
在本发明的第二方面, 提供编码分离的这些多肽的多核苷酸, 该多核苷酸包含一 核苷酸序列, 该核苷酸序列与选自下组的一种核苷酸序列有至少 70%相同性: (a)编码 上述 Bi oATPa.se 的多核苷酸; (b)与多核苷酸(a)互补的多核苷酸。 较佳地, 该多核苷 酸编码具有 SEQ ID NO : 2 所示氨基酸序列的多肽。 更佳地, 该多核苷酸的序列是选 自下组的一种: (a)具有 SEQ ID NO : 1中 97- 1 137位的序列; 和(b)具有 SEQ Ιϋ NO : 1 中 l -:-!S4n位的序列。 In a second aspect of the present invention, there is provided 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 above Bi oATPa.se; (b) a polynucleotide complementary to the polynucleotide (a). Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2. More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence of positions 97-1 to 137 in SEQ ID NO: 1; and (b) a sequence of 1-in SEQ ID NO: 1 :-! A sequence of S4n bits.
在本发明的第三方面, 提供了含有上述多核苷酸的载体, 以及被该载体转化或转 导的宿主细胞或者被上述多核苷酸直接转化或转导的宿主细胞。 In a third aspect of the present invention, there are provided a vector containing the above-mentioned polynucleotide, and a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above-mentioned polynucleotide.
本发明的其它方面因本文的技术公开, 对本领域的技术人员而言是显而易见的。 如本发明所用, "分离的" 是指物质从其原始环境中分离出来(如果是天然的物质, 原始环境即是天然环境)。 例如, 活体细胞内的天然状态下的多聚核苷酸和多肽是没 有分离纯化的, 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分 开, 则为分离纯化的。 Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein. As used herein, "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). For example, 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. of.
如本文所用, "分离的 BioATPase蛋白或多肽" 是指 BioATPase基本上不含天然与 其相关的其它蛋白、 脂类、 糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯 化技术纯化 BioATPase 基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主 带。 BioATPase多肽的纯度能用氨基酸序列分析。 As used herein, "isolated BioATPase protein or polypeptide" means that BioATPase is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can use standard protein purification techniques to purify BioATPase. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of BioATPase polypeptide can be analyzed by amino acid sequence.
本发明提供了一种新的多肽—— BioATPase多肽, 其基本上是由 SEQ ID NO : 2所 示的氨基酸序列组成的。 本发明的多肽可以是重组多肽、 天然多肽、 合成多肽, 优 选重组多肽。 本发明的多肽可以是天然纯化的产物, 或是化学合成的产物, 或使用 £组技术从原核或真核宿主(例如, 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞) 中产生。 根据重组生产方案所用的宿主, 本发明的多肽可以是糖基化的, 或可以是 非糖基化的。 本发明的多肽还可包括或不包括起始的甲硫氨酸残基。 The present invention provides a new polypeptide, BioATPase polypeptide, which basically consists of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. Polypeptides of the invention may be naturally purified products or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using a set of 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.
本发明还包括 BioATPase的片段、 衍生物和类似物。 如本发明所用, 术语 "片段"、 "衍生物" 和 "类似物" 是指基本上保持本发明天然的 Bi oATPase 相同的生物学功能 The invention also includes fragments, derivatives and analogs of BioATPase. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to essentially maintaining the same biological function of the natural Bi oATPase of the present invention
^T; '^肽,, 本发明 肽的片段、 衍生物或类似物可以是: ( 1)这样 ·种' 其屮
个或多个氨基酸残基被保守或非保守氨基酸残基(优选的是保守氨基酸残基)取代, 并 且取代的氨基酸可以是也可以不是由遗传密码子编码的; 或者(I I)这样一种, 其中一 个或多个氨基酸残基上的某个基团被其它基团取代包含取代基; 或者(i n)这样一种, 其中成熟多肽与另一种化合物(比如延长多肽半衰期的化合物, 例如聚乙二醇)融合; 或者(I V)这样一种, 其中附加的氨基酸序列融合进成熟多肽而形成的多肽序列(如前 导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列)。 通过本文的阐述, 这样 的片段、 衍生物和类似物被认为在本领域技术人员的知识范围之内。 ^ T ; '^ peptide, a fragment, derivative or analog of the peptide of the present invention may be: (1) such a species' One or more amino acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted amino acid may or may not be encoded by a genetic codon; or (II) such a, One of the one or more amino acid residues is substituted with another group to include a substituent; or (in) a type in which the mature polypeptide is associated with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene Diol) fusion; or (IV) a polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) in which an additional amino acid sequence is fused into a mature polypeptide. As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
本发明还提供了分离的核酸(多核苷酸), 该多核苷酸基本由编码具有 SEQ II) NO : 2 氨基酸序列的多肽的多核苷酸组成。 较佳地, 本发明的多核苷酸序列具有 SEQ ID NU: 1的核苷酸序列。 The invention also provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ: NO: 2. Preferably, the polynucleotide sequence of the present invention has a nucleotide sequence of SEQ ID NU: 1.
本发明的多核苷酸可以是 DNA形式或是 RNA形式。 DNA形式包括 cDNA、 基因组 DNA 或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或非编码链。 编 码成熟多肽的编码区序列可以与 SEQ ID N0 : 1 所示的编码区序列相同或者是简并的变 异体。 如本发明所用, "简并的变异体"在本发明中是指编码具有 SEQ ID NO : 的蛋 白质或多肽, 但与 SEQ ID N0: 1所示的编码区序列有差别的核酸序列。 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence of the encoded mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
编码 SEQ ID N0 : 2 的成熟多肽的多核苷酸包括: 只有成熟多肽的编码序列; 成熟 多肽的编码序列和各种附加编码序列; 成熟多肽的编码序列(和任选的附加编码序列) 以及非编码序列。 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.
术语 "编码多肽的多核苷酸" 是指包括编码此多肽的多核苷酸和包括附加编码和 /或非编码序列的多核苷酸。 The term "polynucleotide encoding a polypeptide" refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
本发明还涉及上述描述多核苷酸的变异体, 其编码与本发明有相同的氨基酸序列 的多肽或多肽的片断、 类似物和衍生物。 此多核苷酸的变异体可以是天然发生的等位 变异体或非天然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺失变异体和插 入变异体。 如本领域所知的, 等位变异体是一种多核苷酸的替换形式, 它可能是一个 或多 1、核苷酸的取代、 缺失或插入, 但不会从实质上改变其编码的多肽的功能。 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. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be one or more than 1, a nucleotide substitution, deletion, or insertion, but does not substantially change the polypeptide encoded by it Functions.
本发明还涉及与以上所描述的序列杂交的多核苷酸(两个序列之间具有至少 50%, 优选具有 70%的相同性)。 本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交 的多核苷酸。 在本发明中, "严格条件" 是指: (1)在较低离子强度和较高温度下的杂 交和洗脱, 如 0. 2xSSC, 0. 1%SDS,60°C ;或(2)杂交时加用变性剂, 如 50% (v/v)甲酰胺, 0. 小卞血淸 /0. l %F i co l I , 等;或(3)仅在两条序列之间的相同性至少在 9 以」:, 更好是 97%以上时才发生杂交。 并且, 可杂交的多核苷酸编码的多肽与 SEQ I I) NU: 2 所示的成熟多肽有相同的生物学功能和活性。 The invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least 50%, preferably 70% identity between the two sequences). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "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 denaturing agent during hybridization, such as 50% (v / v) formamide, 0. small blood pupa / 0.1% F i co l I, etc .; or (3) only the same between the two sequences Sex is at least 9 ":, and more preferably 97% or more. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID I) NU: 2.
本发明还涉及与以上所描述的序列杂交的核酸片段。 如本发明所用, "核酸片段" 的长度至少含 10 个核苷酸, 较好是至少 20-30 个核苷酸, 更好是至少 50-60 个核苷 . ii 好是至少 ion t核苷酸以上。 核酸片段也可用 f核酸的扩增技术(如 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleosides. Ii preferably at least ion t nucleosides Acid above. Nucleic acid fragments can also be amplified using f nucleic acid (such as
定和 或分离编码 BioAT ase的多核苷酸。
本发明中的多肽和多核苷酸优选以分离的形式提供, 更佳地被纯化至均质。 Identify and or isolate a polynucleotide encoding a BioAT ase. The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
本发明的编码 BioATPa.se 的特异的多核苷酸序列能用多种方法获得。 例如, 用本 领域熟知的杂交技术分离多核苷酸。 这些技术包括但不局限于: 1 )用探针与基因组或 cD A 文库杂交以检出同源的多核苷酸序列, 和 2)表达文库的抗体筛选以检出具有共 同结构特征的克隆的多核苷酸片段。 The specific polynucleotide sequence encoding the BioATPa.se of the present invention can be obtained by various methods. For example, 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 cDA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect polynuclear clones with common structural characteristics Nucleotide fragments.
本发明的 DNA片段序列也能用下列方法获得: 1 )从基因组 DNA分离双链 DNA序列: 2)化学合成 DNA序列以获得所述多肽的双链 DNA。 The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) Isolating a double-stranded DNA sequence from genomic DNA: 2) Chemically synthesizing a DNA sequence to obtain the double-stranded DNA of the polypeptide.
上述提到的方法中, 分离基因组 DNA最不常用。 DNA序列的直接化学合成是经常 选用的方法。 更经常选用的方法是 cDNA序列的分离。 分离感兴趣的 cDNA的标准方法 是从高表达该基因的供体细胞分离 mRNA并进行逆转录, 形成质粒或噬菌体 cDNA文库。 提取 mRXA的方法已有多种成熟的技术, 试剂盒也可从商业途径获得(Qiagene 而构 建 cDNA文库也是通常的方法(Sambrook, e t al. , Molecular Cloning, A Laboratory Manual , Col d Spring Harbor Laboratory. New York, 1989)。 还可得到商业供应的 cDNA文库, 如 Clontech公司的不同 cDNA文库。 当结合使用聚合酶反应技术时, 即使 极少的表达产物也能克隆。 Of the methods mentioned above, 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. There are a variety of mature methods for extracting mRXA, and kits are also commercially available (Qiagene and cDNA libraries are also common methods (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
可用常规方法从这些 cDNA文库中筛选本发明的基因。 这些方法包括(但不限于): (D DNA-DNA或 DNA-RNA杂交; (2)标志基因功能的出现或丧失; (3)测定 Bi oATPase的 转录本的水平; (4)通过免疫学技术或测定生物学活性, 来检测基因表达的蛋白产物。 上述方法可单用, 也可多种方法联合应用。 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (D DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of Bi oATPase transcripts; (4) through immunological techniques Or measure the biological activity to detect the protein product of gene expression. The above methods can be used alone or in combination.
在第(1 )种方法中, 杂交所用的探针是与本发明的多核苷酸的任何一部分同源, 其 长度至少 10个核苷酸, 较好是至少 3个核苷酸, 更好是至少 50个核苷酸, 最好是至 少 100 个核苷酸。 此外, 探针的长度通常在 2000个核苷酸之内, 较佳的为 1000 个核 苷酸之内。 此处所用的探针通常是在本发明的基因序列信息的基础上化学合成的 ΙΑΛ 序列。 本发明的基因本身或者片段当然可以用作探针。 DNA 探针的标记可用放射性同 位素, 荧光素或酶(如碱性磷酸酶)等。 In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and has a length of at least 10 nucleotides, preferably at least 3 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2,000 nucleotides, preferably within 1000 nucleotides. The probe used here is generally a IAΛ 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).
在第(4)种方法中,检测 BioATPase基因表达的蛋白产物可用免疫学技术如 Western 印迹法, 放射免疫沉淀法, 酶联免疫吸附法(ELISA)等。 In the (4) method, the protein product of BioATPase gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
应用 PCR技术扩增 DNA/RNA的方法(Saiki , et al. Science 1985; 230: 1350-1.354) 被优选用于获得本发明的基因。 特别是很难从文库中得到全长的 cDNA 时, 可优选 用 RACE法(RACE- cDNA末端快速扩增法), 用于 PCR的引物可根据本文所公开的本发明 的多核苷酸序列信息适当地选择, 并可用常规方法合成。 可用常规方法如通过凝胶电 泳分离和纯化扩增的 DNA/RNA片段。 A method for amplifying DNA / RNA using PCR technology (Saiki, et al. Science 1985; 230: 1350-1.354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-rapid cDNA end rapid amplification method) can be preferably used, and the primers 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.
如上所述得到的本发明的基因, 或者各种 DNA 片段等的多核苷酸序列可用常规方 法如 脱¾链终止法(Sanger et ul. PNAS , 1977 , 74: 5463- 5467)测定。 ¾ ' i 苷酸序列测定也可用商业测序试剂盒等。 为了获得全长的 cDNA 序列, 测序需反复进 行。 有时需要测定多个克隆的 cDNA序列, 才能拼接成全长的 cDNA序列。
本发明也涉及包含本发明的多核苷酸的载体, 以及用本发明的载体或直接用 Bi oATPa.se 编码序列经基因工程产生的宿主细胞, 以及经重组技术产生本发明所述 肽的方法。 The 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 the de-stranding method (Sanger et ul. PNAS, 1977, 74: 5463-5467). ¾'i nucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence. The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the Bi oATPa.se coding sequence, and a method for producing the peptide of the present invention by recombinant technology.
本发明中, 编码 Bi oATPa.se 的多核苷酸序列可插入到载体中, 以构成含有本发明 所述多核苷酸的重组载体。 术语 "载体"指本领域熟知的细菌质粒、 噬菌体、 酵母质 粒、 植物细胞病毒、 哺乳动物细胞病毒如腺病毒、 逆转录病毒或其它载体。 在本发明 中适用的载体包括但不限于: 在细菌中表达的基于 T7 启动子的表达载体(Rosenberg, et al . Gene, 1987, 56 : 125): 在哺乳动物细胞中表达的 pMSXND 表达载体( e an d Nathans, J Bi o Chem. 263 : 3521 , 1988)和在昆虫细胞中表达的来源于杆状病毒的载 体。 总之, 只要能在宿主体内复制和稳定, 任何质粒和载体都可以用于构建重组表达 载体。 表达载体的一个重要特征是通常含有复制起始点、 启动子、 标记基因和翻译调 控元件。 In the present invention, a polynucleotide sequence encoding Bi oATPa.se can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "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 expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125): pMSXND expression vectors expressed in mammalian cells ( e an d Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, 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.
可用本领域的技术人员熟知的方法来构建含编码 BioATPa.se的醒序列和合适的 转录 /翻译调控元件的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成技术、 体 内重组技术等 (Sambroook, et ul . Mo l ecular Clon ing, a Laboratory Manual , co 1 d Spri ng Harbor Laboratory. New York, 1989)。 所述的 DNA 序列可有效连接到表达 载体中的适当启动子上, 以指导 mRNA 合成。 这些启动子的代表性例子有: 大肠杆菌 的 l ac或 trp启动子; λ噬菌体的 PL启动子; 真核启动子包括 CMV立即早期启动子、 Methods known to those skilled in the art can be used to construct expression vectors containing a wake sequence encoding BioATPa.se and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et ul. Molecular Cloning, a Laboratory Manual, co. Spring Harbor Laboratory. 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: the l ac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter,
HSV胸苷激酶启动子、 早期和晚期 SV40启动子、 反转录病毒的 LTRs和其它一些己知 的可控制基因在原核细胞或真核细胞或其病毒中表达的启动子。 表达载体还包括翻译 起始用的核糖体结合位点和转录终止子等。 在载体中插入增强子序列将会使其在高等 真核细胞中的转录得到增强。增强子是 DNA表达的顺式作用因子,通常大约有 10到 30() 个碱基对, 作用于启动子以增强基因的转录。 可举的例子包括在复制起始点晚期一侧 的 100 到 270 个碱基对的 SV40增强子、 在复制起始点晚期一侧的多瘤增强子以及腺 病毒增强子等。 HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. 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 30 () base pairs that act on promoters to enhance gene transcription. Examples include 100 to 270 base pair SV40 enhancers on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
此外, 表达载体优选地包含一个或多个选择性标记基因, 以提供用于选择转化的 宿主细胞的表型性状, 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗性以及绿色荧 光蛋白(GFP), 或用于大肠杆菌的四环素或氨苄青霉素抗性等。 In addition, 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. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
本领域一般技术人员都清楚如何选择适当的载体 /转录调控元件(如启动子、 增强 子等)和选择性标记基因.. Those of ordinary skill in the art will know how to select the appropriate vector / transcription control element (such as promoter, enhancer, etc.) and selectable marker gene ..
本发明中, 编码 Bi oATPase的多核苷酸或含有该多核苷酸的重组载体^转化或 导入宿主细胞, 以构成含有该多核苷酸或重组载体的基因工程化宿主细胞。 术语 "宿 主细胞"指原核细胞, 如细菌细胞; 或是低等真核细胞, 如酵母细胞; 或是高等真核 细胞, 如哺乳动物细胞。 宿主细胞的代表性例子有: 大肠杆菌, 链霉菌属; 细菌细胞 如鼠伤寒沙门氏菌; 真菌细胞如酵母; 植物细胞; 昆虫细胞如果蝇 S2或 Sf9 : 动物细 I H0 , U)S或 Bowes黑素瘤细胞等。
用本发明所述的舰序列或含有所述 DNA序列的重组载体转化宿主细胞可用本领 域技术人员熟知的常规技术进行。 当宿主为原核生物如大肠杆菌时, 能吸收 【)NA 的感 受态细胞可在指数生长期后收获, 用 CaCl 2法处理, 所用的步骤在本领域众所周知。 也可用 MSC 12进行。 如果需要, 转化也可用电穿孔的方法进行。 当宿主是真核生物, 可选用如下的 DNA 转染方法: 磷酸钙共沉淀法, 或者常规机械方法如显微注射、 电穿 孔、 脂质体包装等。 In the present invention, a polynucleotide encoding Bi oATPase or a recombinant vector containing the polynucleotide is transformed or introduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples of host cells are: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf9 : animal fine I H0, U) S or Bowes melanin Tumor cells and so on. Transformation of a host cell with a warship sequence or a recombinant vector containing the DNA sequence of the present invention can be performed by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing NA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. It can also be performed with M S C 1 2 . If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
通过常规的重组 DNA技术, 利用本发明的多核苷酸序列可用来表达或生产重组的 B i oATI'ase (Sc i ence , 1984; 224: 1431 )。 一般来说有以下步骤: By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant Biatotiase (Science, 1984; 224: 1431). Generally there are the following steps:
( 1 ) .用本发明的编码人 B i oATPase 的多核苷酸(或变异体), 或用含有该多核苷 酸的重组表达载体转化或转导合适的宿主细胞; (1) transforming or transducing a suitable host cell with the polynucleotide (or variant) encoding human BioATPase of the present invention, or with a recombinant expression vector containing the polynucleotide;
(2) .在合适的培养基中培养宿主细胞; (2) culturing host cells in a suitable medium;
从培养基或细胞中分离、 纯化蛋白质。 Isolate and purify proteins from culture media or cells.
在歩骤(2)中, 根据所用的宿主细胞, 培养中所用的培养基可选自各种常规培养 基。 在适于宿主细胞生长的条件下进行培养。 当宿主细胞生长到适当的细胞密度后, 用合适的方法(如温度转换或化学诱导)诱导选择的启动子, 将细胞再培养一段时间。 In step (2), the medium used in the culture may be selected from various conventional culture media depending on the host cell 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.
在步骤(3)中, 重组多肽可包被于细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如果需要, 可利用其物理的、 化学的和其它特性通过各种分离方法分离和纯化重组的 蛋白。 这些方法是本领域技术人员所熟知的。 这些方法包括但并不限于: 常规的复性 处理、 蛋白沉淀剂处理(盐析方法)、 离心、 渗透破菌、 超声波处理、 超离心、 分子筛 层析(凝胶过滤)、 吸附层析、 离子交换层析、 高效液相层析 (HPLC)和其它各种液相层 析技术及这些方法的结合。 附 图 的 简 要 说 明 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, 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, osmosis, 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. Brief explanation of attached picture
下列附图用于说明本发明的具体实施方案, 而不用于限定由权利要求书所界定 的本发明范围。 The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.
图 1是本发明的人 ATP合酶 Bi oATPa.se和线虫的 ATP合酶(AF099920)的氨基酸序列 同源性比较图。 相同氨基酸在两个序列间用单字符氨基酸表示, 相似氨基酸用 " + " 表示', 工 业 应 用 性 FIG. 1 is a comparison diagram of the amino acid sequence homology between human ATP synthase Bi oATPa.se and nematode ATP synthase (AF099920). Identical amino acids are represented by single-character amino acids between the two sequences, similar amino acids are represented by "+", and industrial applicability
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗 B i oATPase蛋白或多肽可做为药物治疗 ATP合酶功能低下或丧失所致的疾病。 B i uAT ast' 的拮抗剂可用来治疗或预防免疫紊乱, 免疫紊乱包括但(不局限于): 肾小球性肾炎、 红斑狼疮、 格雷夫氏病、 糖尿病、 贫血、 肺气肿、 胰腺炎、 萎缩性胃炎、 Sjogren's综合症、 阿迪森氏症、 骨关节炎、 痛风、 多发性肌炎、 重症肌无力、 遗传性过敏性皮炎、 哮喘、 支气管炎、 自身免疫甲状腺炎等。 与 B i oATPase 特异性结合的抗体可直接用作拮抗剂,
或间接以靶向或传递机制将药剂带到表达 BioATPase的细胞或组织中。The polypeptides of the present invention, as well as the antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases. BioATPase protein or polypeptides can be used as drugs to treat diseases caused by low or loss of ATP synthase function. B i uAT ast 'antagonists can be used to treat or prevent immune disorders, including but not limited to: glomerulonephritis, lupus erythematosus, Graves' disease, diabetes, anemia, emphysema, pancreatitis , Atrophic gastritis, Sjogren's syndrome, Addison's disease, osteoarthritis, gout, polymyositis, myasthenia gravis, atopic dermatitis, asthma, bronchitis, autoimmune thyroiditis, etc. Antibodies that specifically bind to B o oATPase can be used directly as antagonists, Or indirectly, agents are brought into cells or tissues expressing BioATPase by targeting or delivery mechanisms.
ioATPase 的拮抗剂或片段或衍生物可用来治疗或预防神经系统退行性疾病, 神 经系统退行性疾病包括(但并不局限于): 老年痴呆症、 亨廷顿氏症、 帕金森氏症、 癫 痫病、 唐氏综合症、 多样性硬化症等。 Antagonists or fragments or derivatives of ioATPase can be used to treat or prevent degenerative neurological diseases, including (but not limited to): Alzheimer's disease, Huntington's disease, Parkinson's disease, epilepsy, Down syndrome, multiple sclerosis, etc.
BioATPase 的拮抗剂或片段或衍生物可用来治疗或预防肌肉疾病, 肌肉疾病包括 (但并不局限于): 进行性外周眼肌瘫痪、 Kearns- Sayre 综合症、 肌阵挛症、 脑病、 心 肌炎、 乳酸中毒症等。 Antagonists or fragments or derivatives of BioATPase can be used to treat or prevent muscle disorders including (but not limited to): progressive peripheral ophthalmoplegia, Kearns-Sayre syndrome, myoclonus, encephalopathy, myocarditis, Lactic acidosis and so on.
BioATPase的拮抗剂或片段或衍生物可用来治疗或预防癌症, 癌症包括(但并不局 限于): 腺癌、 白血病、 淋巴瘤、 黑色素瘤、 肉瘤等; 尤其是肾、 膀胱、 胰腺、 骨、 脑、 乳腺、 子宫、 胆囊、 肝、 肺、 甲状腺、 食道、 睾丸、 皮肤、 肠系膜等部位有关的癌症 与 BwATPase 特异性结合的抗体可直接用作拮抗剂, 或间接以靶向或传递机制将药剂 带到表达 BioATPase 的细胞或组织中。 BioATPase 的拮抗剂或片段或衍生物可用来治 疗或预防癌症免疫紊乱。 BioATPase antagonists or fragments or derivatives can be used to treat or prevent cancer. Cancers include (but are not limited to): adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, etc .; especially kidney, bladder, pancreas, bone, Brain, breast, uterus, gallbladder, liver, lung, thyroid, esophagus, testis, skin, mesentery and other cancer-related antibodies that specifically bind to BwATPase can be used directly as antagonists, or indirectly through the targeting or delivery mechanism of the agent Taken into cells or tissues that express BioATPase. Antagonists or fragments or derivatives of BioATPase can be used to treat or prevent cancer immune disorders.
本发明也提供了筛选化合物以鉴定提高(激动剂)或阻遏(拮抗剂) BioATPase 的药 剂的方法。 激动剂提高 BioATPase 刺激细胞增殖等生物功能, 而拮抗剂阻止和治疗与 细胞过度增殖有关的紊乱如各种癌症。 例如, 在药物的存在下, 将哺乳动物细胞或表 达 BinATPa.se 的膜制剂与标记的 BioATPase—起培养, 然后测定药物提高或阻遏此相 互作用的能力, 从而鉴别出激动剂或拮抗剂。 The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) BioATPase. Agonists enhance biological functions such as BioATPase to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing BinATPa.se are cultured with labeled BioATPase in the presence of a drug, and then the ability of the drug to increase or suppress this interaction is determined to identify an agonist or antagonist.
BioATPase 的拮抗剂包括筛选出的抗体、 化合物、 受体缺失物和类似物等。 BioATPase 的拮抗剂可以与 BioATPase 结合并消除其功能, 或是抑制该多肽的产生, 或是与该多肽的活性位点结合使该多肽不能发挥生物学功能。 BioATPase antagonists include antibodies, compounds, receptor deletions, and analogs that have been screened. BioATPase antagonists can bind to BioATPase and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
在筛选作为拮抗剂的化合物时, 可以将 BioATPase加入生物分析测定中, 通过测 定化合物对 BioATPase和其受体之间相互作用的影响来确定化合物是否是拮抗剂„ 用 上述筛选化合物的同样方法, 可以筛选出起拮抗剂作用的受体缺失物和类似物。 能与 BioATPase结合的多肽分子可通过筛选由各种可能组合的氨基酸结合于固相 物组成的 随机多肽库而获得。 筛选时, 一般应对 BioATPase分子进行标记。 When screening compounds as antagonists, BioATPase can be added to the bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between BioATPase and its receptor. Using the same method of screening compounds described above, Receptor deletions and analogues that act as antagonists are screened out. Polypeptide molecules that can bind to BioATPase can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally respond BioATPase molecules are labeled.
本发明提供了用多肽, 及其片段、 衍生物、 类似物或它们的细胞作为抗原以生产 抗体的方法。 这些抗体可以是多克隆抗体或单克隆抗体。 本发明还提供了针对 The present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen. These antibodies can be polyclonal or monoclonal antibodies. The present invention also provides
BioATPase抗原决定簇的抗体。 这些抗体包括 (但不限于): 多克隆抗体、 单克隆抗体、 嵌合抗体、 单链抗体、 Fah片段和 Fab表达文库产生的片段。 Antibodies to the BioATPase epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fah fragments, and fragments generated from Fab expression libraries.
多克隆抗体的生产可用 BioATPase直接注射免疫动物(如家兔, 小鼠, 大鼠等)的 方法得到, 多种佐剂可用于增强免疫反应, 其中包括但不限于弗氏佐剂等。 制备 BiuAri' st. 的单克隆抗体的技术包括(但不限于): 杂交瘤技术(Kohler and i lstt,i„. Nature, 1975, 256:495-497) , 三瘤技术, 人 Β-细胞杂交瘤技术, EBV-杂交瘤技术等., 将人恒定区和非人源的可变区结合的嵌合抗体可用已有的技术生产(Morrison el a J , NAS, 1985, 81 :6851)„ 而已有的生产单链抗体的技术(U. S. Pat No.4%77H)也可
用于生产抗 BioATPase的单链抗体。 Polyclonal antibodies can be produced by injecting BioATPase directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Techniques for preparing BiuAri 'st. Monoclonal antibodies include (but are not limited to): Hybridoma technology (Kohler and i lst t , i .. Nature, 1975, 256: 495-497), triple tumor technology, human beta cells Hybridoma technology, EBV-hybridoma technology, etc., chimeric antibodies that combine human constant regions with non-human variable regions can be produced using existing technologies (Morrison el a J, NAS, 1985, 81: 6851). The existing technology for producing single chain antibodies (US Pat No. 4% 77H) is also available. Used to produce single chain antibodies against BioATPase.
抗 BioATPase的抗体可用于免疫组织化学技术中, 检测活检标本中的 BioATP;ise。 与 B i oATPase 结合的单克隆抗体也可用放射性同位素标记, 注入体内可跟踪其位置和 分布。 这种放射性标记的抗体可作为一种非创伤性诊断方法用于肿瘤细胞的定位和判 断是否有转移。 Anti-BioATPase antibodies can be used in immunohistochemical techniques to detect BioATP in biopsy specimens ; ise. Monoclonal antibodies that bind to BioATPase 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.
抗体还可用于设计针对体内某一特殊部位的免疫毒素。 如 BioATPase 高亲和性的 单克隆抗体可与细菌或植物毒素(如白喉毒素, 蓖麻蛋白, 红豆碱等)共价结合。 一种 通常的方法是用巯基交联剂如 SPDP, 攻击抗体的氨基, 通过二硫键的交换, 将毒素结 合于抗体上, 这种杂交抗体可用于杀灭 BioATPase阳性的细胞。 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, BioATPase high-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of the antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill BioATPase positive cells.
本发明中的抗体可用于治疗或预防与 BioATPase相关的疾病。 给予适当剂量的抗 体可以刺激或阻断 BioATPase的产生或活性。 The antibodies of the present invention can be used to treat or prevent diseases associated with BioATPase. Administration of appropriate doses of antibodies can stimulate or block BioATPase production or activity.
本发明还涉及定量和定位检测 BioATPase水平的诊断试验方法。 这些试验是本领 域所熟知的, 且包括 FISH 测定和放射免疫测定。 试验中所检测的 BioATPa.se 水平, 可以用作解释 BioATPa.se 在各种疾病中的重要性和用于诊断 BioATPase 起作用的疾 病。 The invention also relates to a diagnostic test method for quantitative and localized detection of BioATPase levels. These tests are well known in the art and include FISH and radioimmunoassays. The level of BioATPa.se measured in the test can be used to explain the importance of BioATPa.se in various diseases and to diagnose diseases where BioATPase works.
本发明的多肽还可用作肽谱分析, 例如, 多肽可用物理的、 化学或酶进行特异性 切割, 并进行一维或二维或三维的凝胶电泳分析,更好的是进行质谱分析。 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, 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.
编码 BioATPase 的多核苷酸也可用于多种治疗目的。 基因治疗技术可用于治疗由 于 Bi oATPa.se 的无表达或异常 /无活性表达所致的细胞增殖、 发育或代谢异常。 重组 的基因治疗载体(如病毒载体)可设计用于表达变异的 BioATPase , 以抑制内源性的 Bi oATPas 活性。 例如, 一种变异的 BioATPase 可以是缩短的、 缺失了信号传导功能 域的 B ioATPase , 虽可与下游的底物结合, 但缺乏信号传导活性。 因此重组的基因治 疗载体可用于治疗 BioATPase 表达或活性异常所致的疾病。 来源于病毒的表达载体如 逆转录病毒、 腺病毒、 腺病毒相关病毒、 单纯疱疹病毒、 细小病毒等可用于将编码 BioATPase 的多核苷酸转移至细胞内。 构建携带编码 BioATPase 的多核苷酸的重组病 毒载体的方法可见于己有文献(Sambrook, et al. )„ 另外重组编码 BioATPase 的多核 苷酸可包装到脂质体中转移至细胞内。 The polynucleotide encoding BioATPase 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 Bi oATPa.se. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated BioATPase to inhibit endogenous BioATPas activity. For example, a mutated BioATPase can be a shortened B ioATPase that lacks the signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of BioATPase. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer polynucleotides encoding BioATPase into cells. The method of constructing a recombinant viral vector carrying a polynucleotide encoding BioATPase can be found in the existing literature (Sambrook, et al.). In addition, the recombinant polynucleotide encoding BioATPase can be packaged into liposomes and transferred into cells.
多核苷酸导入组织或细胞内的方法包括: 将多核苷酸直接注入到体内组织中: 或 在体外通过载体(如病毒、 噬菌体或质粒等)先将多核苷酸导入细胞中, 再将细胞移拉 到体内等。 Methods for introducing polynucleotides into tissues or cells include: Injecting the polynucleotides directly into tissues in the body: Or, first introduce the polynucleotides into cells through a vector (such as a virus, phage, or plasmid) in vitro, and then transfer the cells. Pull into the body and so on.
抑制 BioATPase mRNA 的寡核苷酸(包括反义 RNA和 DNA)以及核酶也在本发明的 范围之内。 核酶是一种能特异性分解特定 RNA的酶样 RNA分子, 其作用机制是核酶分 子与互补的靶 RNA特异性杂交后进行核酸内切作用。 反义的 RNA和 DNA及核酶可用已 ^的 ίί:何 ΚΝΑ或 UNA 合成技术获得, 如固相磷酸酰胺化学合成法合成寡核苷酸的技 已广泛应用。 反义 RNA分子可通过编码该 RNA的 DNA序列在体外或体内转录获得。 这 种 IAA序列已整合到载体的 RNA聚合酶启动子的下游。 为了增加核酸分子的稳定性,
可用多种方法对其进行修饰, 如增加两侧的序列长度, 核糖核苷之间的连接应用磷酸 硫酯键或肽键而非磷酸二酯键。 Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit BioATPase mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a 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 using ^: Ho KNA or UNA synthesis techniques, such as 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. This IAA sequence has been integrated downstream of the RNA polymerase promoter of the vector. To increase the stability of nucleic acid molecules, It can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
编码 BioATPase 的多核苷酸可用于与 BioATPase 的相关疾病的诊断。 编码 BioATPase 的多核苷酸可用于检测 BioATPase 的表达与否或在疾病状态下 BioATPas 的异常表达。 如编码 BioATPase 的 DNA 序列可用于对活检标本进行杂交以判断 BioATPase 的表达状况。 杂交技术包括 Southern 印迹法, Northern 印迹法、 原位杂 交等。 这些技术方法都是公开的成熟技术, 相关的试剂盒都可从商业途径得到。 本发 明的多核苷酸的一部分或全部可作为探针固定在微阵列(Microarray)或 DNA 芯片(乂 称为"基因芯片")上, 用于分析组织中基因的差异表达分析和基因诊断。用 BioATPas 特异的引物进行 RNA-聚合酶链反应(RT- PCR)体外扩增也可检测 BioATPase 的转录产 物。 The polynucleotide encoding BioATPase can be used for the diagnosis of diseases related to BioATPase. Polynucleotides encoding BioATPase can be used to detect the expression of BioATPase or abnormal expression of BioATPas in disease states. For example, the DNA sequence encoding BioATPase can be used to hybridize biopsy specimens to determine the expression of BioATPase. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and so on. These techniques and methods are publicly available and mature, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (hereinafter referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. BioATPas specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect BioATPase transcripts.
检测 BioATPase基因的突变也可用于诊断 BioATPase相关的疾病。 BioATPase突 变的形式包括与正常野生型 BioATPa.se DNA 序列相比的点突变、 易位、 缺失、 重组和 其它任何异常等。 可用已有的技术如 Southern 印迹法、 DNA序列分析、 PCR和原位杂 交检测突变。 另外, 突变有可能影响蛋白的表达, 因此用 Northern 印迹法、 Western 印迹法可间接判断基因有无突变。 Detection of mutations in the BioATPase gene can also be used to diagnose BioATPase-related diseases. BioATPase mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type BioATPa.se DNA sequences. 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, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人染色 体具体位置且并可以与其杂交。 目前, 需要鉴定染色体上的各基因的具体位点。 现 在, 只有很少的基于实际序列数据(重复多态性)的染色体标记物可用于标记染色体 位置。 根据本发明, 为了将这些序列与疾病相关基因相关联, 其重要的第一步就是 将这些 DNA序列定位于染色体上。 The sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position of a human chromosome and can hybridize with it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
简而言之, 根据 cDNA制备 PCR引物(优选 15- 35bp), 可以将序列定位于染色体 上。 然后, 将这些引物用于 PCR 筛选含各条人染色体的体细胞杂合细胞。 只有那些 含有相应于引物的人基因的杂合细胞会产生扩增的片段。 In short, PCR primers (preferably 15-35bp) are prepared based on 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 hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
体细胞杂合细胞的 PCR 定位法, 是将 DNA 定位到具体染色体的快捷方法。 使用 本发明的寡核苷酸引物, 通过类似方法, 可利用一组来自特定染色体的片段或大量 基因组克隆而实现亚定位。 可用于染色体定位的其它类似策略包括原位杂交、 用标 记的流式分选的染色体预筛选和杂交预选, 从而构建染色体特异的 cDNA库。 PCR localization of somatic hybrid cells is a quick way to locate DNA to specific chromosomes. Using the 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 chromosome-specific cDNA libraries.
将 cDNA克隆与中期染色体进行荧光原位杂交 (FISH), 可以在一个步骤中精确地 进行染色体定位。此技术的综述,参见 Ve進等, Human Chromosomes: a Manual o Has i Techniques, Pergamon Press, New York(1988)。 Fluorescent in situ hybridization (FISH) of cDNA clones to metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Ve Jin et al., Human Chromosomes: a Manual o Has i Techniques, Pergamon Press, New York (1988).
一旦序列被定位到准确的染色体位置, 此序列在染色体上的物理位置就可以与 基因图数据相关联。 这些数据可见于例如, V. Mckusick.Mendelian Inheritance in Man (::|] "jfiii - J Johns Hopkins University Welch Medical Library 联机获得),' 然 后可通过连锁分析, 确定基因与业己定位到染色体区域上的疾病之间的关系。 Once the sequence is located at the exact chromosomal location, 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 (:: |] "jfiii-J Johns Hopkins University Welch Medical Library available online) 'Then, linkage analysis can be used to determine genes and genes that have been mapped to chromosomal regions Relationship between diseases.
接着, 需要测定患病和未患病个体间的 cDNA或基因组序列差异。 如果在一些或
所有的患病个体中观察到某突变, 而该突变在任何正常个体中未观察到, 则该突变 可能是疾病的病因。 比较患病和未患病个体, 通常涉及首先寻找染色体中结构的变 化, 如从染色体水平可见的或用基于 cDNA序列的 PCR可检测的缺失或易位。 根据目 前的物理作图和基因定位技术的分辨能力, 被精确定位至与疾病有关的染色体区域 的 cDNA , 可以是 50至 500个潜在致病基因间之一种(假定 1 兆碱基作图分辨能力和 每 20kb对应于一个基因)。 Next, the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If in some or A mutation is observed in all diseased individuals, and the mutation is not observed in any normal individuals, then the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
可以将本发明的多肽、 多核苷酸及其模拟物、 激动剂、 拮抗剂和抑制剂与合适的 药物载体(药学上可接受的载体)组合后使用。 这些载体可以是水、 葡萄糖、 乙醇、 盐 类、 缓冲液、 甘油以及它们的组合。 组合物包含安全有效量的本发明多肽或拮抗剂以 及不影响药物效果的载体和赋形剂。 这些组合物可以作为药物用于疾病治疗。 The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier (pharmaceutically acceptable carrier). These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of a polypeptide or antagonist of the present invention and a carrier and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
本发明还提供含有一种或多种容器的药盒或试剂盒, 容器中装有一种或多种本发 明的药用组合物成分。 与这些容器一起, 可以有由制造、 使用或销售药品或生物制品 的政府管理机构所给出的指示性提示, 该提示反映出生产、 使用或销售的政府管理机 构许可其在人体上施用。 此外, 本发明的多肽可以与其它的治疗化合物结合使用。 The invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that manufacture, use, or sell them. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.
药物组合物可以以方便的方式给药, 如通过局部、 静脉内、 腹膜内、 肌内、 皮下、 鼻内或皮内的给药途径。 BioATPa.se 以有效地治疗和 /或预防具体的适应症的量来给 药。 施用于患者的 Bi oATPa.se 的量和剂量范围将取决于许多因素, 如给药方式、 待治 疗者的健康条件和诊断医生的判断。 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. BioATPa.se is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of BioATPa.se administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.
在本发明的一个实例中, 提供了一种分离的多核苷酸, 它编码具有 SEQ ID NO : 2 所示氨基酸序列的成熟多肽。 该多核苷酸是从人胎脑组织的 cDNA 文库中发现的, 多 核苷酸序列全长为 3845 个碱基, 其开放读框(97-1 137)编码了 346个氨基酸。 根据氨 基酸序列同源比较发现, 此多肽与线虫(Caenorhabdi t i s el egans)的 ATP 合酶有 41 % 的同源性, 由此推断本发明新的人 B i oATPase 具有 ATP合酶基因家族相似的结构和功 能。 In one example of the present invention, an isolated polynucleotide is provided, which encodes a mature polypeptide having the amino acid sequence shown in SEQ ID NO: 2. The polynucleotide was found from a cDNA library of human fetal brain tissue. The polynucleotide sequence is 3845 bases in length and its open reading frame (97-1 137) encodes 346 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 41% homology with the ATP synthase of Caenorhabdi tis el egans, and it can be inferred that the new human BiOATPase of the present invention has a similar structure to the ATP synthase gene family. and function.
本发明所提供的人 BioATPase 的 cDNA、 寡聚核苷酸、 多肽及抗体等, 对于研究不 同组织和细胞中 ATP合酶的作用、 诊断 ATP合酶失调的相关性疾病、 筛选抑制剂或药物 治疗这些疾病有重要价值。 实 现 本 发 明 的 最 佳 方 式 The cDNA, oligonucleotides, peptides, and antibodies of the human BioATPase provided by the present invention are useful for studying the role of ATP synthase in different tissues and cells, diagnosing diseases related to ATP synthase disorders, screening inhibitors or drug treatment. These diseases have important value. The best way to implement the invention
下面结合具体实施例, 进一步阐述本发明。 应理解, 这些实施例仅用于说明本发 明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法, 通常按照 常规条件如 Sambrook等人, 分子克隆: 实验室手册(New York : Co l d Spri ng Harbor Laborat ory Press, 1989)中所述的条件, 或按照制造厂商所建议的条件。 雄'例 1: Bi oATPase d)NA的克隆 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Laborat ory Press, 1989), or Follow the conditions recommended by the manufacturer. Male 'Example 1: Bi oATPase d) Cloning of NA
用异硫氰酸胍 /酚 /氯仿一歩法提取人胎脑总 RNA o 用 Qu ik mRNA I so l at i on K i t
(QieKen 公司产品)从总 RNA 中分离 poly (A) mRNA。 2ug poly (A) mRNA 经逆转录形 成 cDNA。 用 Smart. cDNA克隆试剂盒(购自 Clontech)将 cDNA片段定向插入到 pBSK (+> 载体(Gk tech公司产品)的多克隆位点上, 转化 DH5a细菌形成 cDNA文库。 用染料终 止循环反应测序试剂盒(P er k i n-E 1 mer公司产品)和 AB I 377 自动测序议(P er k i n -E 1 m t' r 公司)测定所有克隆的 5'和 3'末端的序列。 将测定的 cDNA 序列与已有的公共 I)NA 序 列数据库(Genebank)进行比较, 结果发现其中一个克隆(0504g02)的 cDNA 序列为新的 DNA。 通过合成一系列引物对该克隆所含的插入 cDNA 片段进行双向测定。 结果表明, 0n04ff0 克隆所含的全长 cDNA为 3845bp (如 SEQ ID N0:1 所示), 从第 97bp至 l 7hp 有一个 1041bp 的开放阅读框架(0RF), 编码一个新的蛋白质(如 SEQ ID NO: 2 所示) 此克隆被命名为 PBS-0504g02, 其编码的蛋白质命名为人 ATP 合酶(简称为 "BioATPase" )。 实施例 2: cDNA 克隆的同源检索 Isolation of total human fetal brain RNA with guanidine isothiocyanate / phenol / chloroform-isocyanate method o Quik mRNA I so l at i on K it (QieKen Company) Isolate poly (A) mRNA from total RNA. 2ug poly (A) mRNA is reverse transcribed to form cDNA. The Smart. CDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragments into the multicloning site of pBSK (+> vector (Gk tech)) to transform the DH5a bacteria to form a cDNA library. The dye was used to terminate the cycle reaction sequencing reagent Cassette (Perkin nE 1 mer company) and AB I 377 automatic sequencing protocol (Perkin-E 1 mt 'r company) to determine the sequence of the 5' and 3 'ends of all clones. The determined cDNA sequence was compared with Some public I) NA sequence databases (Genebank) were compared and found that the cDNA sequence of one of the clones (0504g02) was new DNA. A series of primers were synthesized for the bidirectional determination of the inserted cDNA fragments contained in the clone. The results show that the 0n04 ff 0 clone contains a full-length cDNA of 3845bp (as shown in SEQ ID N0: 1), from 97bp to 17hp has a 1041bp open reading frame (0RF), which encodes a new protein (such as (SEQ ID NO: 2) This clone was named P BS-0504g02, and its encoded protein was named human ATP synthase (referred to as "BioATPase"). Example 2: Homologous search of cDNA clones
将本发明的人 B i oATPase基因的序列及其编码的蛋白序列, 用 B 1 as t程序(Has i c 1 oca J Alignment search tool) [Altschul, SF et al. .]. Mo I . Biol. 1990;215:403-10] , 在 Genbank、 Swissport等数据库进行同源检索。 与本发 明的人 BioATPase基因同源性最高的基因是一种己知的线虫 Caenorhabdi ti s e¾ ",s')ATP合酶的基因, 其编码的蛋白在 Genbank的准入号为 AF099920。 蛋白质同 源比较结果示于图 1, 两者高度同源, 其相同性为 41% ; 相似性为 59%。 这表明, 本 发明的新多肽具有 ATP合酶的结构和功能。 实施例 用 RT-PCR方法克隆 BioATPase基因 The sequence of the human BioATPase gene of the present invention and the protein sequence encoded by the bioATPase gene of the present invention were performed using the B 1 as t program (Has ic 1 oca J Alignment search tool) [Altschul, SF et al. ; 215: 403-10] for homology search in databases such as Genbank and Swissport. The gene with the highest homology to the human BioATPase gene of the present invention is a known nematode Caenorhabdi ti s e¾ ", s') ATP synthase gene, and the accession number encoded by the protein in Genbank is AF099920. The results of the source comparison are shown in Figure 1. The two are highly homologous, with an identity of 41% and a similarity of 59%. This indicates that the novel polypeptide of the present invention has the structure and function of ATP synthase. RT-PCR is used in the examples Methods to clone BioATPase gene
用胎脑细胞总 RNA为模板, 以 oligo-dT为引物进行逆转录反应合成 cDNA,用 CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.
Q gt'ne的试剂盒纯化后,用下列引物进行 PCR扩增: After purification of Q gt'ne's kit, PCR amplification was performed with the following primers:
引物 1: 5' - GGGGGTCCCGGCTGCTCGGC -3' (SEQ ID NO.3) Primer 1: 5 '-GGGGGTCCCGGCTGCTCGGC -3' (SEQ ID NO.3)
引物 2: 5' - TGCAGTAATTTAATTTAATA -3' (SEQ ID NO.4) Primer 2: 5 '-TGCAGTAATTTAATTTAATA -3' (SEQ ID NO.4)
引物 1为位于 SEQ ID NO: 1的 5'端的第 lbp开始的正向序列; 引物 2为 SEQ ID NO: 1 中的:' ) '端反向序列。 Primer 1 is the forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1; Primer 2 is the reverse sequence of the: ') end in SEQ ID NO: 1.
扩增反应的条件: 在 50μ1的反应体积中含有 50mmol/L KC1, 10mmol/L Tris- Cl, (pHH. n), 1.5mmol/L MgC12, 200μπιο1/ί dNTP, lOpmol引物, 1U的 Taq DNA聚合酶 ((Ίπη ι L'uh ^司产品)„ 在 PE%00型 DM热循环仪 (Perkin- Elmer公司)上按下列条件 应 25个周期: 94°C 30sec; 55°C, 30sec; 72°C 2min。 在 RT-PCR时同时设 β-肌动蛋白 为阳性对照和模板空白为阴性对照。 扩增产物用 QIAGEN公司的试剂盒纯化, 用 ΤΑ克隆 试剂盒连接到 pCR载体上(Invitrogen公司产品)。 DNA序列分析结果表明, PCR产物的 [)NA 序列与 SEQ ID NO: 1所示的 1- 3845bp完全相同。
实施例 4 : Northern 印迹法分析 BioATPase基因的表达: Amplification reaction conditions: 50μ1 reaction volume containing 50mmol / L KC1, 10mmol / L Tris-Cl, (pHH. N), 1.5mmol / L MgC12, 200μπιο1 / ί dNTP, lOpmol primer, 1U Taq DNA polymerization Enzyme ((Ίπη ι L'uh ^ company products) „25 cycles on the PE% 00 DM thermal cycler (Perkin-Elmer) under the following conditions: 94 ° C 30sec; 55 ° C, 30sec; 72 ° C 2min. At the same time, set β-actin as a positive control and template blank as a negative control at the time of RT-PCR. The amplification product was purified using a QIAGEN kit and connected to a pCR vector using a TA clone kit (Invitrogen product). ). The DNA sequence analysis results show that the [) NA sequence of the PCR product is exactly the same as the 1-3845bp shown in SEQ ID NO: 1. Example 4: Analysis of BioATPase gene expression by Northern blotting:
用一步法提取总 RNA [Anal. Biochem 1987, 162, 156-159]。 该法包括酸性硫氰 酸胍苯酚-氯仿抽提。 即用 4M 异硫氰酸胍 -25mM 柠檬酸钠, 0. 2M 乙酸钠(pH4. 0)对组 织进地匀浆, 加入 1 倍体积的苯酚和 1 /5体积的氯仿-异戊醇(49: 1), 混合后离心。 吸出水相层, 加入异丙醇(0. 8体积)并将混合物离心得到 RNA沉淀。 将得到的 醒沉 淀用 70%乙醇洗涤, 干燥并溶于水中。 Total RNA was extracted in one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49 : 1), centrifuge after mixing. The aqueous phase was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RNA precipitate. The resulting precipitate was washed with 70% ethanol, dried and dissolved in water.
用 20μ RNA, 在含 20mM 3- (N-吗啉代)丙磺酸(pH7. 0) -5mM乙酸钠 - ImM EDTA-2. 2M 甲醛的 1. 2%琼脂糖凝胶上进行电泳。 然后转移至硝酸纤维素膜上。 用 α_32Ρ dATP通过 随机引物法制备 32P-标记的 DNA探针。 所用的 DNA探针为 SEQ 1 所示的 PCK扩增的 Bi oATPase编码区序列(97bp至 1 137bp)。 将 32P-标记的探针(约 2x 106cpm/m] )与转移 了 RNA 的硝酸纤维素膜在溶液中于 42°C 杂交过夜, 该溶液包含 50%甲酰胺 - 25mM KH,,P()_; (pH7. 4) -5xSSC-5xDenhardt' s溶液和 200 g/ml鮭精 DNA。 杂交之后, 将滤膜在 l xSSOO. 1 %SDS中于 55。C洗 30min。 然后, 用 Phosphor Imager进行分析和定量。 实施例 5: 重组 BioATPase的体外表达、 分离和纯化 Using 20 μ RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2. 2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation 32 P- DNA probe labeled with α_ 32 Ρ dATP by random priming method. The DNA probe used was the PCO amplified Bi oATPase coding region sequence (97bp to 1137bp) shown in SEQ. Hybridize a 32 P-labeled probe (approximately 2 x 10 6 cpm / m) with an RNA-transferred nitrocellulose membrane at 42 ° C overnight in a solution containing 50% formamide-25mM KH, P () _ ; (pH7.4.) -5xSSC-5xDenhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filters were placed in 1 x SSOO. 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 BioATPase
根据 SEQ ID N0 : 1 和图 1 所示的编码区序列, 设计出一对特异性扩增引物, 序列 如下: According to SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers was designed, the sequence is as follows:
引物: 3 : 5, - CCCGGATCCCGGGCGGAGGGGCTGGGGAC -3' (SEQ ID No 5) Primers: 3: 5,-CCCGGATCCCGGGCGGAGGGGCTGGGGAC -3 '(SEQ ID No 5)
引物 4 : 5' - CCCGCGGCCGCAACTCACTGTGTCGCCCCAT -3' (SEQ ID No 6) Primer 4: 5 '-CCCGCGGCCGCAACTCACTGTGTCGCCCCAT -3' (SEQ ID No 6)
此两段引物的 5'端分别含有 BamHI和 Ncol酶切位点, 其后分别为目的基因 5端 和 3'端的编码序列, BamHI和 Ncol酶切位点相应于表达载体质粒 pET_28b (+) (Novagen 公司产品, Cat. No. 69865. 3)上的选择性内切酶位点。 以含有全长目的基因的 pBS 0504ίΐϋ2质粒为模板, 进行 PCR反应。 PCR反应条件为: 总体积 50μ1中含 pBS_0504g()2 质粒 10pg、 弓 I物 3禾口弓 I物 4分另 lj为 l Opmmol 、 Advantage polymerase Mi x (Cl ontech 公司产品) ΐμ1。 循环参数: 94 °C 20s, 60 °C 30s, 68 °C 2 min, 共 25个循环。 用 BamH I 和 Nco l分别对扩增产物和质粒 pET-28 (+)进行双酶切, 分别回收大片段,并用 T4连接 酶连接。 连接产物转化用氯化钙法大肠杆细菌 Dh5a, 在含卡那霉素(终浓度 3(^g/ml ) 的 LB 平板培养过夜后, 用菌落 PCR 方法筛选阳性克隆, 并进行测序。 挑选序列正确 的 阳 性 克 隆 (pET-0504g02) 用 氯 化 钙 法 将 重 组 质 粒 转 化 大 肠 杆 菌 BL21 (I)E3) pl ySs (Novagen公司产品)。 在含卡那霉素(终浓度 30 g/ml)的 LB液体培养 基中, 宿主菌 BL21 (pET-0504g02)在 37 Ό培养至对数生长期, 加入 IPTG 至终浓度 1瞧 υ Ι Λ, 继续培养 5小时。 离心收集菌体, 经超声波破菌, 离心收集上清, 用能 Π 个组氨酸(6Hi s-Tag)结合的亲和层析柱 His. Bind Quick Cartridge (Novagen 公司产 品)进行层析, 得到了纯化的目的蛋白 BioATPase。 经 SDS- PAGE 电泳, 在 38kl)a处得 到一单一的条带。 将该条带转移至 PVDF膜上用 Edams水解法进行 N_端氨基酸序列分 析, 结果 N-端 15个氨基酸与 SEQ ID N0: 2所示的 N-端 15个氨基酸残基完全相同。
实施例 6: 抗 BioATPa.se抗体的产生 The 5 'ends of these two primers contain BamHI and Ncol restriction sites, respectively, followed by the coding sequences of the 5 and 3' ends of the target gene, respectively. The BamHI and Ncol restriction sites correspond to the expression vector plasmid pET_28b (+) ( Novagen product, Cat. No. 69865. 3). The PCR reaction was performed using the pBS 0504ίΐϋ2 plasmid containing the full-length target gene as a template. PCR reaction conditions were: total volume of 50μ1 containing pBS_0504g () 2 Plasmid 10pg, Wo port bow Bow I was 3 I was 4 points to another lj l Opmmol, Advantage polymerase Mi x ( Cl ontech Products) ΐ μ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. The amplified product and plasmid pET-28 (+) were double-digested with BamH I and Nco l, respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E. coli Dh5a using the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 3 (^ g / ml)), positive clones were screened by colony PCR method and sequenced. Correct positive clone (pET-0504 g 02) The recombinant plasmid was transformed into E. coli BL21 (I) E3) pl ySs (product of Novagen) by the calcium chloride method. In a LB liquid medium containing kanamycin (final concentration 30 g / ml), the host strain BL21 (pET-0504 g 02) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 υ Ι Ι Λ, continue to cultivate for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain chromatograms. Purified the target protein BioATPase. After SDS-PAGE electrophoresis, a single band was obtained at 38 kl) a. The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 6: Production of anti-BioATPa.se antibodies
用多肽合成仪(PE公司产品)合成下述 BioATPase特异性的多肽: The following peptides specific to BioATPase were synthesized using a peptide synthesizer (product of PE):
Met-Lys-Cys-Thr-Ala-Val-Leu-Cys-Cys-Arg-Met-Ala-Pro-Leu-Gln (SEQ ID NO :7)。 将该多 肽分别与血蓝蛋白和牛血清白蛋白耦合形成复合, 方法参见: AvrameiiS, et aJ. Immunochemistry, 1969; 6:43。 用 ½g 上述血蓝蛋白多肽复合物加上完全弗氏佐剂免 疫家兔, 15 天后再用血蓝蛋白多肽复合物加不完全弗氏佐剂加强免疫一次。 采用经 15μΚ^/ ml 牛血清白蛋白多肽复合物包被的滴定板做 ELISA 测定兔血清中抗体的滴度。 用蛋白 A-Sepharo.se 从抗体阳性的家兔血清中分离总 Ig (;。 将多肽结合于溴化氰活化 的 Sepharose 4B 柱上, 用亲和层析法从总 IgG 中分离抗多肽抗体。 免疫沉淀法证明 纯化的抗体可特异性地与 BioATPase结合。 序 歹 ί 表 Met-Lys-Cys-Thr-Ala-Val-Leu-Cys-Cys-Arg-Met-Ala-Pro-Leu-Gln (SEQ ID NO: 7). The polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively. For the method, see: Avr ameiiS , et aJ. Immunochemistry, 1969; 6:43. Rabbits were immunized with ½ g of the hemocyanin-peptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. The titer of antibody in rabbit serum was determined by ELISA using a titer plate coated with 15μK ^ / ml bovine serum albumin polypeptide complex. Total protein Ig was isolated from antibody-positive rabbit serum using protein A-Sepharo.se. The peptide was bound to a cyanogen bromide-activated Sepharose 4B column, and anti-peptide antibodies were isolated from total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to BioATPase.
(1)一般信息: (1) General information:
(i)发明名称: 新的人 ATP合酶及其编码序列 (i) Title of invention: New human ATP synthase and its coding sequence
(ii)序列数目: 7 (ii) Number of sequences: 7
(2) SEQ ID NO: 1的信息: (2) Information of SEQ ID NO: 1:
(i)序列特征: (i) Sequence characteristics:
(A)长度: 3845bp (A) Length: 3845bp
(B)类型: 核酸 (B) Type: Nucleic acid
(C)链性: 双链 (C) Chain: double stranded
(D)拓扑结构: 线性 (D) Topological structure: linear
(ii)分子类型: cDNA (ii) Molecular type: cDNA
(iii)序列描述: SEQ ID NO: 1: (iii) Sequence description: SEQ ID NO: 1:
4 I (; L 'CCAGGTGTCI^'ATC'rGACTTGGGG'rGTGCrGCAGGAAAGAG'rGATGACCCGCAGC'rA
4 I (; L 'CCAGGTGTCI ^'ATC'rGACTTGGGG'rGTGCrGCAGGAAAGAG'rGATGACCCGCAGC'rA
(3) SEQ ID NO: 2的信息: (3) Information of SEQ ID NO: 2:
(i)序列特征: (i) Sequence characteristics:
(A)长度: 34S个氨基酸 (A) Length: 34S amino acids
(B)类型: 氨基酸 (B) Type: Amino acid
([))拓扑结构: 线性 (()) Topology: Linear
(ii)分子类型: 多肽 (ii) Molecular type: peptide
(iii)序列描述: SE(J ID NO: 2: (iii) Sequence description: SE (J ID NO: 2:
1 Met Lys Cys Thr Ala Val Leu Cys Cys Arg Met Ala Pro Leu GJn6 L.ys Ala Gin He Val Arg Met Val Lys Asn Leu Lys Gly Ser Pro1 J l Thr Leu Ser J J e Gly Asp Gly Ala Asn Asp Val Ser Mel lieB L,eu Glu Ser His Val Gly lie Gly He Lys Gly Lys Glu (; J y Ar«1 (iln Ala Ala Ar^ Asn Ser Asp Tyr Ser Val Pro Lys Phe Lys His
76 Leu Lys Lys Leu Leu Leu Ala His Gly His Leu Tyr Tyr Val Aru1 Met Lys Cys Thr Ala Val Leu Cys Cys Arg Met Ala Pro Leu GJn6 L.ys Ala Gin He Val Arg Met Val Lys Asn Leu Lys Gly Ser Pro1 J l Thr Leu Ser JJ e Gly Asp Gly Ala Asn Asp Val Ser Mel lieB L, eu Glu Ser His Val Gly lie Gly He Lys Gly Lys Glu (; J y Ar «1 (iln Ala Ala Ar ^ Asn Ser Asp Tyr Ser Val Pro Lys Phe Lys His 76 Leu Lys Lys Leu Leu Leu Ala His Gly His Leu Tyr Tyr Val Aru
91 lie Ala His Leu Val Gin Tyr Phe Phe Tyr Lys Asn Leu Cys Phe91 lie Ala His Leu Val Gin Tyr Phe Phe Tyr Lys Asn Leu Cys Phe
106 H Leu Pro Gin Phe Leu Tyr Gin Phe Phe Cys Gly Phe Ser Gin106 H Leu Pro Gin Phe Leu Tyr Gin Phe Phe Cys Gly Phe Ser Gin
121 Gin Pro Leu Tyr Asp Ala Ala Tyr Leu Thr Met Tyr Asn lie Cys121 Gin Pro Leu Tyr Asp Ala Ala Tyr Leu Thr Met Tyr Asn lie Cys
1 6 Phe Thr Ser Leu Pro lie Leu Ala Tyr Ser Leu Leu Glu Gin His1 6 Phe Thr Ser Leu Pro lie Leu Ala Tyr Ser Leu Leu Glu Gin His
151 He Asn lie Asp Thr Leu Thr Ser Asp Pro Arg Leu Tyr Met Lys151 He Asn lie Asp Thr Leu Thr Ser Asp Pro Arg Leu Tyr Met Lys
166 He Ser Gly Asn Ala Met Leu Gin Leu Gly Pro Phe Leu Tyr Trp166 He Ser Gly Asn Ala Met Leu Gin Leu Gly Pro Phe Leu Tyr Trp
181 Thr Phe Leu Ala Ala Phe Glu Gly Thr Val Phe Phe Phe Gly Thr181 Thr Phe Leu Ala Ala Phe Glu Gly Thr Val Phe Phe Phe Gly Thr
196 Tyr Phe Leu Phe Gin Thr Ala Ser Leu Glu Glu Asn Gly Lys Val196 Tyr Phe Leu Phe Gin Thr Ala Ser Leu Glu Glu Asn Gly Lys Val
211 Tyr Gly Asn Trp Thr Phe Gly Thr lie Val Phe Thr Val Leu Val211 Tyr Gly Asn Trp Thr Phe Gly Thr lie Val Phe Thr Val Leu Val
226 Phe Thr Val Thr Leu Lys Leu Ala Leu Asp Thr Arg Phe Trp Thr226 Phe Thr Val Thr Leu Lys Leu Ala Leu Asp Thr Arg Phe Trp Thr
241 Trp He Asn His Phe Val lie Trp Gly Ser Leu Ala Phe Tyr V J241 Trp He Asn His Phe Val lie Trp Gly Ser Leu Ala Phe Tyr V J
256 Phe Phe Ser Phe Phe Trp Gly Gly lie lie Trp Pro Phe Leu Lys256 Phe Phe Ser Phe Phe Trp Gly Gly lie lie Trp Pro Phe Leu Lys
271 Gin Gin Arg Met Tyr Phe Val Phe Ala Gin Met Leu Ser Ser Val271 Gin Gin Arg Met Tyr Phe Val Phe Ala Gin Met Leu Ser Ser Val
286 Ser Thr Trp Leu Ala lie lie Leu Leu lie Phe lie Ser Leu Phe286 Ser Thr Trp Leu Ala lie lie Leu Leu lie Phe lie Ser Leu Phe
301 Pro Glu lie Leu Leu lie Val Leu Ly-s Asn Val Arg Arg Arg Ser301 Pro Glu lie Leu Leu lie Val Leu Ly-s Asn Val Arg Arg Arg Ser
316 Ala Arg Val Thr Lys Arg Leu Pro Ser Ser Gly Thr Ser Ala He316 Ala Arg Val Thr Lys Arg Leu Pro Ser Ser Gly Thr Ser Ala He
331 Phe Met Leu Ser Gin Thr Ser Ser Asn His Ser Phe Ser Trp Ser331 Phe Met Leu Ser Gin Thr Ser Ser Asn His Ser Phe Ser Trp Ser
Glu Glu
(4)SEQ ID NO: 3的信息 (4) Information of SEQ ID NO: 3
(i)序列特征 (i) Sequence characteristics
(A)长度: 20个碱基 (A) Length: 20 bases
(B)类型: 核酸 (B) Type: Nucleic acid
(C)链性: 单链 (C) Chain: single chain
(D)拓扑结构: 线性 (D) Topological structure: linear
(ii)分子类型: 寡核苷酸 (ii) Molecular type: Oligonucleotide
(iii)序列描述: SEQ ID 3: (iii) Sequence description: SEQ ID 3:
(; GGGGTCCCGGCTGCTCGGC 20 (; GGGGTCCCGGCTGCTCGGC 20
(5) SEQ ID NO: 4的信息 (5) Information of SEQ ID NO: 4
(i)序列特征 (i) Sequence characteristics
(A)长度: 20个碱基 (A) Length: 20 bases
(B)类型: 核酸 (B) Type: Nucleic acid
(C)链性: 单链 (C) Chain: single chain
(1))拓扑结构: 线性
(ii)分子类型: 寡核苷酸 (1)) Topological structure: linear (ii) Molecular type: Oligonucleotide
(iii)序列描述: SEQ ID NO 4: (iii) Sequence description: SEQ ID NO 4:
TGCAGTAATTTAATTTAATA 20 TGCAGTAATTTAATTTAATA 20
(6) SEQ ID NO: 5的信息 (6) Information of SEQ ID NO: 5
(i)序列特征 (i) Sequence characteristics
(A)长度: 29个碱基 (A) Length: 29 bases
(B)类型: 核酸 (B) Type: Nucleic acid
(C)链性: 单链 (C) Chain: single chain
(D)拓扑结构: 线性 (D) Topological structure: linear
(Π)分子类型: 寡核苷酸 (Π) Molecular type: Oligonucleotide
(iii)序列描述: SEQ ID NO (iii) Sequence description: SEQ ID NO
CC ; (; ATCCCGGGCGGAGGGGCTGGGGAC CC; (; ATCCCGGGCGGAGGGGCTGGGGAC
(7) SEQ ID NO: 6的信息 (7) Information of SEQ ID NO: 6
(i)序列特征 (i) Sequence characteristics
(A)长度: 31 个碱基 (A) Length: 31 bases
(B)类型: 核酸 (B) Type: Nucleic acid
(C)链性: 单链 (C) Chain: single chain
(D)拓扑结构: 线性 (D) Topological structure: linear
(ii)分子类型: 寡核苷酸 (ii) Molecular type: Oligonucleotide
(iii)序列描述: SEQ ID NO : 6 (iii) Sequence description: SEQ ID NO: 6
CCaiCGGCCGCAACTCACTGTGTCGCCCCAT CCaiCGGCCGCAACTCACTGTGTCGCCCCAT
(H)SEg ID NO :7的信息: Information of (H) SEg ID NO: 7:
(i)序列特征: (i) Sequence characteristics:
(A)长度: 15个氨基酸 (A) Length: 15 amino acids
(B)类型: 氨基酸 (B) Type: Amino acid
(1))拓扑结构: 线性 (1)) Topological structure: linear
(Π)分子类型: 多肽 (Π) Molecular type: Polypeptide
(iii)序列描述: SEQ Ιϋ NO: 7: (iii) Sequence description: SEQ ID NO: 7:
Met-Lys-Cys-Thr-Ala-Val-Leu-Cys-Cys-Arg-Met-Ala-Pro-Leu-Gln 15
Met-Lys-Cys-Thr-Ala-Val-Leu-Cys-Cys-Arg-Met-Ala-Pro-Leu-Gln 15
Claims
权 利 要 求 书 Request for Rights
1、 一种分离的人 BioATPase多肽, 其特征在于, 它是具有: SEQ ID NO. 2所示的氨基 酸序列的多肽、 或其多肽的片段、 类似物或衍生物。 1. An isolated human BioATPase polypeptide, characterized in that it is a polypeptide having the amino acid sequence shown in SEQ ID NO. 2, or a fragment, analog, or derivative of a polypeptide thereof.
2、 如权利要求 1 所述的多肽, 其特征在于, 它是具有 SEQ ID NO. 2 所示的氨基酸序 列的多肽或其氨基酸变异不超过 5 % 的衍生物。 2. The polypeptide according to claim 1, characterized in that it is a polypeptide having the amino acid sequence shown in SEQ ID NO. 2 or a derivative thereof with an amino acid variation not exceeding 5%.
3、 如权利要求 2 所述的多肽, 其特征在于, 它是具有 SEQ ID NO. 2 所示的氨基酸序 列的多肽。 3. The polypeptide according to claim 2, characterized in that it is a polypeptide having the amino acid sequence shown in SEQ ID NO.
4、 一种分离的多核苷酸, 其特征在于, 所述多核苷酸是选自下组: 4. An isolated polynucleotide, characterized in that said polynucleotide is selected from the group consisting of:
(a)编码具有 SEQ ID NO. 2 所示氨基酸序列的多肽或其片段、 类似物、 衍生物的 多核苷酸; ( a ) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or a fragment, analog, or derivative thereof;
(h)与多核苷酸(a)互补的多核苷酸; (h) a polynucleotide complementary to polynucleotide (a);
( )与 (a)或(h)有至少 70%相同性的多核苷酸。 () A polynucleotide that is at least 70% identical to (a) or (h).
5、 如权利要求 4所述的多核苷酸, 其特征在于, 所述多核苷酸是编码具有 SEQ 二' 所示氨基酸序列的多核苷酸。 5. The polynucleotide according to claim 4, wherein the polynucleotide is a polynucleotide encoding an amino acid sequence represented by SEQ ′ ′.
6、如权利要求 4所述的多核苷酸,其特征在于,所述多核苷酸的序列是具有 SEQ ID NO. 1 中 97- 1 Π7位的序列或具有 SEQ ID NO. 1中 1 -3845位的序列。 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide is a sequence having positions 97-1 to Π7 in SEQ ID NO. 1 or 1 to 3845 in SEQ ID NO. 1. Sequence of bits.
7、 一种含有外源多核苷酸的重组载体, 其特征在于, 它是由要求要求 4 所述多核苷 酸与质粒、 病毒或运载体表达载体构建而成的重组载体。 7. A recombinant vector containing an exogenous polynucleotide, characterized in that it is a recombinant vector constructed from the polynucleotide according to claim 4 and a plasmid, virus or carrier expression vector.
8、 一种含有外源多核苷酸的遗传工程化宿主细胞, 其特征在于, 它是选自于下列一 种宿主细胞: 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from the following host cells:
(a) 用权利要求 7所述的重组载体转化或转导的宿主细胞; (a) a host cell transformed or transduced with the recombinant vector of claim 7;
(b) 用权利要求 4所述多核苷酸转化或转导的宿主细胞。 (b) a host cell transformed or transduced with the polynucleotide of claim 4.
、 一种具有 BioATPa.se活性的多肽的制备方法, 其特征在于, 所述方法包括: A method for preparing a polypeptide having BioATPa.se activity, characterized in that the method includes:
(u) 在适合表达 BioATPase条件下, 培养权利要求 8所述的工程化宿主细胞; (b) 从培养物中分离出具有 BioATPase活性的多肽。 (u) culturing the engineered host cell of claim 8 under conditions suitable for expression of BioATPase; (b) isolating a polypeptide having BioATPase activity from the culture.
10、 一种能与多肽结合的抗体,其特征在于, 所述抗体是能与 BioATPase 特异性结合 的抗体。 10. An antibody capable of binding to a polypeptide, characterized in that said antibody is an antibody capable of specifically binding to BioATPase.
1 1、 模拟或调节多肽活性或表达的化合物, 其特征在 f, 它是模拟 Bi A'I'pM s 的 活性化合物, 或促进 BioATPa.se的活性的化合物, 或拮抗 BioATPase的活性的化合物, 或抑制 Bi oATPase的活性的化合物。 1 1. A compound that mimics or regulates the activity or expression of a polypeptide, characterized by f, which is an active compound that mimics Bi A'I'pM s, or a compound that promotes the activity of BioATPa.se, or a compound that antagonizes the activity of BioATPase, Or a compound that inhibits the activity of Bi oATPase.
12、 如权利要求 1 1 所述的化合物, 其特征在于, 它是 SEQ ID NO . 1 所示的多核苷酸 序列或其片段的反义序列。 12. The compound according to claim 11, characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO.1.
1 :;、 种权利要求 1 1所述化 物的应用,其特征在于,所述化合物用于调节 B i nATP s 在体内、 体外活性的方法。 1: The application of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of BinATPs in vivo and in vitro.
14、 一种检测与权利要求 1 所述的 Bi oATPa.se 多肽相关的疾病或疾病易感性的方法,
其特征在于, 是通过选自下组的方法来检测相关的疾病或疾病的易感性: (a)间接或直接检测所述多肽表达量是否异常; 14. A method for detecting a disease or disease susceptibility related to the Bi oATPa.se polypeptide according to claim 1, It is characterized by detecting the related disease or susceptibility of the disease by a method selected from the group consisting of: (a) indirectly or directly detecting whether the expression level of the polypeptide is abnormal;
(h)间接或直接检测所述多肽活性是否异常; (h) detecting indirectly or directly whether the activity of the polypeptide is abnormal;
(c)直接或间接检测多核苷酸中引起所述多肽表达量或活性异常的核苷酸变异 1 5、 如权利要求 1所述多肽的应用, 其特征在于, 它应用于筛选 Bi oATPase的模拟物、 激动剂、 拮抗剂或抑制剂; 或者用于肽指纹图谱鉴定。 (c) directly or indirectly detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide 15 5. The use of the polypeptide according to claim 1, characterized in that it is used to screen a simulation of Bi oATPase Substances, agonists, antagonists or inhibitors; or for identification of peptide fingerprints.
1 、 如权利要求 4所述的核酸分子的应用, 其特征在于它作为引物用于核酸扩增反应, 或者作为探针用于杂交反应, 或者用于制造基因芯片或微阵列。 1. The use of a nucleic acid molecule according to claim 4, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or used for manufacturing a gene chip or a microarray.
1 7、 一种药物组合物, 其特征在于, 它含有安全有效量的权利要求 1 所述的多肽和 / 或权利要求 4所述的多核苷酸以及药学上可接受的载体。 17. A pharmaceutical composition, characterized in that it contains a safe and effective amount of the polypeptide according to claim 1 and / or the polynucleotide according to claim 4 and a pharmaceutically acceptable carrier.
1 «、 如权利要求 1 所述的多肽的应用, 其特征在于, 用所述多肽制备用于治疗神经系统 退行性疾病、 肌肉疾病、 免疫紊乱、 癌症等疾病的药物。
1 «The use of the polypeptide according to claim 1, characterized in that the polypeptide is used to prepare a medicament for treating diseases such as degenerative diseases of the nervous system, muscle diseases, immune disorders, cancer and the like.
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ES2432653R1 (en) * | 2012-05-23 | 2014-09-29 | Centro De Investigacion Biomedica En Red De Enfermedades Raras | A PROCEDURE AND KIT FOR THE DIFFERENTIAL DIAGNOSIS OF A DISEASE THAT COURSES WITH MUSCLE AFFECTION. |
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Non-Patent Citations (3)
Title |
---|
KURDI HAIDAR B. ET AL.: "Immunohistochemical analysis of the distribution of the human ATPase (hASNA-1) in normal tissues and its overexpression in breast adenomas and carcinomas", J. HISTOCHEM. CYTOCHEM., vol. 46, no. 11, November 1998 (1998-11-01), pages 1243 - 1248 * |
MATEO JESUS ET AL.: "Functional expression of a cDNA encoding a human ecto-ATPase", BRITISH JOURNAL OF PHARMACOLOGY, vol. 128, no. 2, September 1999 (1999-09-01), pages 396 - 402 * |
MOURO I. ET AL.: "Cloning, expression and chromosomal mapping of a human ATPase II gene, member of the third subfamily of T-type ATPases and orthologous to the presumed bovine and murine aminophospholipid translocase", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 257, no. 2, 13 April 1999 (1999-04-13), pages 333 - 339 * |
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ES2432653R1 (en) * | 2012-05-23 | 2014-09-29 | Centro De Investigacion Biomedica En Red De Enfermedades Raras | A PROCEDURE AND KIT FOR THE DIFFERENTIAL DIAGNOSIS OF A DISEASE THAT COURSES WITH MUSCLE AFFECTION. |
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