WO2002002618A1 - Nouveau polypeptide, sous-unite 10 de nadh deshydrogenase, et polynucleotide codant ce polypeptide - Google Patents
Nouveau polypeptide, sous-unite 10 de nadh deshydrogenase, et polynucleotide codant ce polypeptide Download PDFInfo
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- WO2002002618A1 WO2002002618A1 PCT/CN2001/000991 CN0100991W WO0202618A1 WO 2002002618 A1 WO2002002618 A1 WO 2002002618A1 CN 0100991 W CN0100991 W CN 0100991W WO 0202618 A1 WO0202618 A1 WO 0202618A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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- the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a NADH dehydrogenase 51Kd subunit 10, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
- Respiratory chain NADH dehydrogenase ubiquinone oxidoreductase, also known as respiratory complex I, is the most complex enzyme system in the respiratory chain. Its role is to catalyze the transfer of 2 electrons of MDH to ubiquinone.
- Complex I spans the mitochondrial inner membrane to shift protons from the side of the matrix (M) to the side of the cytoplasm (C), so it is also a proton translocation body.
- This complex can be decomposed into water-soluble flavin protein, water-soluble iron-sulfur protein and insoluble water (including Phospholipids, iron, and sulfur that is unstable to acids).
- Fungal NADH dehydrogenase is composed of at least 30 different subunits, and mammals are composed of at least 41 subunits. No matter whether it is a fungus or mammal, its NADH dehydrogenase contains 7 most hydrophobic subunits, namely ND1 to ND6 and ND4L. These 7 subunits are encoded by mitochondrial genes and synthesized in mitochondria. Electron microscopy showed that N. Cras sad's MDH dehydrogenase exhibited an unusual L-shaped structure, and its exterior projected into the mitochondrial matrix and became a fixed component of the membrane.
- NADH dehydrogenase is an oligomycin enzyme complex located on the inner membrane of the mitochondria (and possibly in chloroplasts in cyanobacteria).
- a 51kd subunit is the second largest subunit of all subunits, and is one of the constituents of iron sulfur protein. This subunit may bind NAD, FMN and a 2Fe-2S protein cluster.
- the 51kd subunit of NADH dehydrogenase and the bacterial dehydrogenase alpha subunit contain three similar regions. One is most likely to correspond to the NAD binding site, the second to the FMN-binding site, and the third to 3 cysteine, corresponding to the iron-sulfur binding site.
- the sequence of characteristic patterns corresponding to the FMN binding site and the 2Fe-2S binding site is as follows: [1]
- NADH dehydrogenase 51Kd subunit 10 protein plays an important role in important functions in the body, Moreover, it is believed that a large number of proteins are involved in these regulatory processes, so there is a need in the art to identify more NADH dehydrogenase 51Kd subunit 10 proteins involved in these processes, especially the amino acid sequence of this protein. Isolation of the new NADH dehydrogenase 51Kd subunit 10 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its code for DM. Disclosure of invention
- Another object of the invention is to provide a polynucleotide encoding the polypeptide.
- Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a 51DH subunit 10 of NADH dehydrogenase.
- Another object of the present invention is to provide a method for producing the 51DH subunit 10 of NADH dehydrogenase.
- Another object of the present invention is to provide an antibody against the polypeptide of the present invention-NADH dehydrogenase 51M subunit 10.
- Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-NADH dehydrogenase 51Kd subunit 10.
- Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in the 51Kd subunit 10 of NADH dehydrogenase.
- the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
- the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
- the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
- sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 335-604 in SEQ ID NO: 1; and (b) a sequence having 1-2081 in SEQ ID NO: 1 Sequence of bits.
- the invention further relates to a vector, in particular an expression vector, containing a polynucleotide of the invention.
- the vector genetically engineered host cell includes a transformed, transduced or transfected host cell; a method for preparing a polypeptide of the present invention comprising culturing the host cell and recovering an expression product.
- the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
- the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit NADH dehydrogenase 51Kd subunit 10 protein activity, which comprises using the polypeptide of the invention.
- the invention also relates to compounds obtained by this method.
- the invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of MDH dehydrogenase 51Kd subunit 10 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
- the invention also relates to a pharmaceutical composition
- a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
- the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of NADH dehydrogenase 51Kd subunit 10.
- Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
- amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
- amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
- a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
- “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
- Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
- Replacement refers to a different amino acid Or nucleotides replace one or more amino acids or nucleotides.
- Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
- immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
- An "agonist” refers to a molecule that, when combined with NADH dehydrogenase 51Kd subunit 10, causes a change in the protein to regulate the activity of the protein.
- An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to the NADH dehydrogenase 51Kd subunit 10.
- Antagonist refers to a molecule that, when combined with NADH dehydrogenase 51Kd subunit 10, can block or regulate the biological or immunological activity of NADH dehydrogenase 51Kd subunit 10.
- Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to the 51DH subunit 10 of the NADH dehydrogenase.
- Regular refers to a change in the function of NADH dehydrogenase 51Kd subunit 10, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties, functions, or immunity of MDH dehydrogenase 51Kd subunit 10. Change of nature.
- Substantially pure 1 'means substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
- Those skilled in the art can purify the NADH dehydrogenase 51Kd subunit 10 using standard protein purification techniques.
- the substantially pure NADH dehydrogenase 51Kd subunit 10 can generate a single main band on a non-reducing polyacrylamide gel.
- the purity of the NADH dehydrogenase 51Kd subunit 10 polypeptide can be analyzed by amino acid sequence.
- “Complementary” or “complementary” refers to the natural binding of a nucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
- the sequence "C-T-G-A” can be combined with the complementary sequence A-C-T ".
- the complementarity between two single-stranded molecules can be partial or complete.
- the degree of complementarity between nucleic acid strands is The efficiency and strength of hybridization between nucleic acid strands has a significant effect.
- “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
- Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
- Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other specifically or selectively.
- Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.).
- the MEGALIGN program can be Methods such as the Cluster method compare two or more sequences (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244).
- the Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups.
- the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by the Clus ter method or by a method known in the art such as Jotun He in. The percent identity between nucleic acid sequences (He in J., (1990) Methods in emzumology 183: 625 -645) 0
- Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
- Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
- Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
- Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
- Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
- Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and?, Which specifically bind to the epitope of NADH dehydrogenase 51Kd subunit 10.
- a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
- isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
- a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
- Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
- isolated refers to the separation of a substance from its original environment (if natural Matter, the original environment is the natural environment).
- polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
- isolated NADH dehydrogenase 51Kd subunit 10 means that NADH dehydrogenase 51Kd subunit 10 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
- Those skilled in the art can purify the NADH dehydrogenase 51Kd subunit 10 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of NADH dehydrogenase 51Kd subunit 10 peptide can be analyzed by amino acid sequence.
- the present invention provides a new polypeptide, a NADH dehydrogenase 51Kd subunit 10, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
- the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
- the polypeptides of the invention can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
- the invention also includes fragments, derivatives, and analogs of the 51DH subunit 10 of NADH dehydrogenase.
- fragment refers to a polypeptide that substantially maintains the same biological function or activity of the NADH dehydrogenase 51Kd subunit 10 of the present invention.
- a fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably 'conservative amino acid residues), and Substituted amino acids may or may not be encoded by the genetic code; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) Such a polypeptide sequence in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) As set forth herein, such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
- the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
- the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
- the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 2081 bases, and its open reading frame (335-604) encodes 87 amino acids.
- This polypeptide has the characteristic sequence of the 51DH subunit of NADH dehydrogenase. It can be deduced that the MDH dehydrogenase 51 lKd subunit 10 has the structure and function represented by the NADH dehydrogenase 51Kd subunit.
- the polynucleotide of the present invention may be in the form of DNA or RNA.
- DNA forms include cDM, genes Group DNA or synthetic DNA.
- DNA can be single-stranded or double-stranded.
- DNA can be coding or non-coding.
- the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
- a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
- the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
- polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
- the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
- This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
- These nucleotide variants include substitution variants, deletion variants, and insertion variants.
- an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
- the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
- the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
- "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 ° /.
- the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
- nucleic acid fragments that hybridize to the sequences described above.
- a "nucleic acid fragment” contains at least 1 Q nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 nucleotides. Nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding the NADH dehydrogenase 51Kd subunit 10.
- the polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
- the specific polynucleotide sequence encoding the NADH dehydrogenase 51Kd subunit 10 of the present invention can be obtained by various methods.
- polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
- the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) the double-stranded DNA sequence separated from the group DNA; 2) the chemically synthesized DNA sequence to obtain the double-stranded DNA of the polypeptide.
- the DM of the separation group 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 the mRNA from the donor cells that are highly expressive and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene).
- the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
- Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
- the plugs of the invention can be screened from these CDM libraries using conventional methods:. These methods include (but are not limited to): (l) DNA-DNA or DM-RNA hybridization; (2) the appearance or loss of marker function; (3) determination of the level of the NADH dehydrogenase 51Kd sub-10 transcript; ( 4) Detection by immunological techniques or determination of biological activity: expressed protein products. The above methods can be used alone or in combination.
- the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
- the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
- the probes used herein are generally DNA sequences that are chemically synthesized on the basis of the present invention: sequence information. The invention itself or a fragment thereof can of course be used as a probe.
- DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
- the expressed protein products can be used immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
- a method using DNA technology to amplify DNA / RNA is preferably used to obtain the base of the present invention:. It is particularly difficult.
- the RACE method RACE-Rapid Amplification of cDNA Ends
- the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
- the amplified MA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
- polynucleotide sequence of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones 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 MDH dehydrogenase 51Kd subunit 10 coding sequence, and the recombinant technology to produce the Polypeptide method.
- the polynucleotide sequence encoding the NADH dehydrogenase 51Kd subunit 10 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.
- any plasmid and vector can be used to construct a recombinant expression vector.
- An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
- Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding the MDH dehydrogenase 51Kd subunit 10 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold 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 lac or trp promoter of E.
- 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 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
- the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
- selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
- GFP fluorescent protein
- tetracycline or ampicillin resistance for E. coli.
- a polynucleotide encoding a NADH dehydrogenase 51Kd subunit 10 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
- host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
- coli Streptomyces
- bacterial cells such as Salmonella typhimurium
- fungal cells such as yeast
- plant cells such as fly S2 or Sf 9
- animal cells such as CH0, COS or Bowes melanoma cells.
- Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
- the host is a prokaryote such as E. coli
- competent cells capable of absorbing MA can be harvested after the exponential growth phase and treated with CaCl ' ⁇ .
- the steps used are well known in the art.
- the alternative is to use MgCl 2 blanket
- transformation can also be performed by electroporation.
- the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as Microinjection, electroporation, liposome packaging, etc.
- the polynucleotide sequence of the present invention can be used to express or produce recombinant NADH dehydrogenase 51Kd subunit 10 (Scence, 1984; 224: 1431). Generally there are the following steps:
- the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
- a suitable method such as temperature conversion or chemical induction
- the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
- recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
- conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromat
- FIG. 1 is a comparison diagram of amino acid sequence homology of a total of 46 amino acids between 5 and 50 of the MDH dehydrogenase 51Kd subunit 10 of the present invention and the characteristic domain of the 51Kd subunit of NADH dehydrogenase.
- the upper sequence is the MDK dehydrogenase 51Kd subunit 10
- the lower sequence is the characteristic domain of the NADH dehydrogenase 51Kd subunit.
- Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".
- FIG. 2 is a polyacrylamide gel electrophoresis diagram (SDS-PAGE) of the isolated NADH dehydrogenase 51Kd subunit 10.
- 0 lOkDa is the molecular weight of the protein.
- the arrow indicates the isolated protein band. Best way to implement the invention '
- Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
- Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
- the Smart 00 cDNA cloning kit purchased from Clontech was used to insert the 00 fragment into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a cDNA library.
- the Dye terminate cycle reaction sequencing kit Perkin-Elmer
- ABI 377 automatic sequencer Perkin-Elmer
- the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one clone 1906f09 was new DNA.
- a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
- the 1906f09 clone contained a full-length cDNA of 2081bp (as shown in Seq IDN0: 1), and a 264bp open reading frame (0RF) from 335bp to 604bp, encoding a new protein (such as Seq ID NO: 2).
- a new protein such as Seq ID NO: 2.
- Example 2 Domain analysis of cDNA clones
- the sequence of the NADH dehydrogenase 51Kd subunit 10 of the present invention and the protein sequence encoded by the same were subjected to a profile scan program (Basiclocal Alignment search tool) in GCG [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], performing domain analysis in databases such as prosite.
- the NADH dehydrogenase 51Kd subunit 10 of the present invention is homologous with the domain NADH dehydrogenase 51Kd subunit from 5 to 50, and the homology result is shown in FIG. 1.
- the homology rate is 0.28, and the score is 12.80; the threshold value is 12.56 .
- Example 3 Cloning of a gene encoding NADH dehydrogenase 51I (d subunit 10 by RT-PCR method
- CDNA was synthesized using fetal brain cell total MA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
- Primerl 5'- CATCCTGAGAACTGAAATTGATCGC- 3 '(SEQ ID NO: 3)
- Primer2 5 -AT AAAATTTTTGAATTTATGTTCAA- 3 '(SEQ ID NO: 4)
- Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
- Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
- Amplification reaction conditions 50 ⁇ l reaction volume containing 50 mraol / L KC1, 10 crypto ol / L Tris-Cl, (pH 8.5), 1.5 ramol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer , 1U of Taq DNA polymerase (Clontech).
- the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min.
- ⁇ -actin was set as a positive control and template blank was set as a negative control.
- the amplified product was purified using a QIAGEN kit and ligated to a PCR vector (Invitrogen product) using a TA cloning kit.
- the DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as the 1-2081bp shown in SEQ ID NO: 1.
- Example 4 Northern blot analysis of the expression of NADH dehydrogenase 51Kd subunit 10 gene:
- Total RM was extracted in one step [Anal. Biochem 1987, 162, 156-159].
- This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
- a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.4) -5 x SSC- 5 x Denhardt's solution and 200 yg / ml salmon DM. After hybridization, place the filter at 1 x SSC-0.1 ° /. Wash at 5 5 ° C in 30min SDS. Then, Phosphor Imager was used for analysis and quantification.
- Example 5 In vitro expression, isolation and purification of recombinant NADH dehydrogenase 51Kd subunit 10
- Primer 3 5, — CCCCATATGATGCTCTGTCACCTTCAAAGGATGG- 3 '(Seq ID No: 5)
- Primer 4 5' — CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC- 3 '(Seq ID No: 6)
- the two ends of these two primers contain Ndel and BamHI digestion sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and BamHI restriction sites correspond to the selective endonucleases on the expression vector plasmid pET28b (+) (Novagen, Cat. No. 69865.3). Enzyme site.
- PCR was performed using the pBS-1906f09 plasmid containing the full-length target gene as a template.
- the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS_1906f 09 plasmid, Primer-3 and Primer- 4 points, and 1 J was lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1.
- Cycle parameters 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles.
- Ndel and BamHI were used to double-digest the amplified product and plasmid P ET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
- the ligated product was transformed into E. coli DH5cc using the calcium chloride method. After being cultured overnight in LB plates containing kanamycin (final concentration 3 () ⁇ ⁇ ⁇ ), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-1906f09) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In containing kanamycin (final concentration of 30 ⁇ g / ml) of LB liquid medium, host strain BL21 (P ET-1906f09) at 37. C.
- the following peptides specific to NADH dehydrogenase 51Kd subunit 10 were synthesized using a peptide synthesizer (product of PE): NH2-Met-Leu-Cys-His-Leu-Gln-Arg-Met-Val-Ser-Glu-Gln -Cys-His-Leu- C00H (SEQ ID NO: 7).
- the peptide was coupled to hemocyanin and bovine serum albumin to form a complex.
- Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
- the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
- the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
- the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method Acid sequence or a homologous polynucleotide sequence thereof.
- Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
- the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
- the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
- the unhybridized probes are removed by a series of membrane washing steps.
- This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
- the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
- the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
- the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
- oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
- the preferred range of probe size is 18-50 nucleotides
- Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
- Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
- Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence of the gene fragment or its complementary fragment (41Nt) of SEQ ID NO: 1
- step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
- NC membranes nitrocellulose membranes
- Probes 1 3 ⁇ l Probe (0.10D / 10 ⁇ 1), add 2 ⁇ IKinase buffer, 8-10 uCi y- 32 P-dATP + 2U Kinase, to make up to a final volume of 20 ⁇ 1.
- probe 1 can be used for qualitative and quantitative analysis.
- the presence and differential expression of the polynucleotide of the present invention in different tissues are analyzed.
- polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
- NADH dehydrogenase also known as respiratory complex I, is the most complex enzyme system in the respiratory chain. Its role is to catalyze the transfer of two electrons of NADH to ubiquinone. Flavin mononucleotide (FMN) and an unknown specific number of iron-sulfur protein clusters as the prosthetic group of the electron transfer pathway exist as dimers in the inner membrane of the mitochondria.
- the mammalian NADH dehydrogenase consists of at least 41 subunits, of which a 51kd subunit is the second largest subunit of all subunits and is one of the constituents of iron sulfur protein.
- This subunit may bind NAD, FMN and an iron-sulfur protein cluster.
- the characteristic pattern sequence of the NADH dehydrogenase 51Kd subunit corresponding to the binding site of FMN and iron-sulfur protein clusters is as follows: [1] G- [AM] -G- [AR] -Y- [LIVM] -CG- [ DE] (2)-[STA] (2)-[LIM] (2)-[EN] -S
- the new polypeptide of the present invention has a high degree of homology and similarity in structure and function with the human respiratory chain NADH dehydrogenase, and its amino acid sequence contains the above-mentioned conservative characteristic sequence template, which determines the normal of the respiratory chain in vivo get on.
- the abnormal expression of the above-mentioned specific conserved sequence will cause the dysfunction of the polypeptide containing the profi le of the present invention, thereby leading to the dysfunction of the NADH dehydrogenase of the respiratory chain, affecting the progress of the respiratory chain, and causing the body to various Impairment of energy supply in the process of substance metabolism (sugar, lipid, protein), leading to the occurrence of related diseases, including but not limited to:
- Hypoglycemia / hypoglycemia can cause hyper / hypoinsulinemia.
- Hyperinsulinemia can promote lipid synthesis and stimulate arterial intimal smooth muscle cell proliferation (causing vascular lumen narrowing); hypoinsulinemia can reduce lipid clearance and vascular lysosomal lipase activity and accelerate arteries Occurrence and development of atherosclerosis.
- tissue hypoxia can be aggravated, so glucose metabolism disorders can lead to atherosclerosis of large and medium blood vessels and microvessels in the whole system.
- Hyperglycemia can cause changes in aqueous osmotic pressure and promote eyeballs.
- Cardio-cerebral vessels angina pectoris, myocardial infarction, arrhythmia, coronary heart disease, metabolic cardiomyopathy, heart failure, cardiogenic shock (aorta, coronary arteries, cardiac microvessels), transient ischemic attack, cerebral infarction, Lacunar infarction, cerebral hemorrhage (intracerebral artery), etc .;
- Renal blood vessels renal artery stenosis, renal artery embolism and thrombosis, arteriolar renal sclerosis (benign, malignant), acute / chronic renal failure, etc .;
- Peripheral blood vessels of the limb occlusive arteriosclerosis (lower limb arteries), malnutrition skin ulcers (cutaneous arterioles), etc .;
- Ophthalmic diseases Metabolic cataract, refractive error, iridocyclitis, ocular motor paralysis, retinopathy (simple, proliferative), iris redness, neovascular glaucoma, etc. 3.
- Nervous system diseases Peripheral neuropathy (symmetrical distal polyneuropathy, majority mononeuropathy, autonomic neuropathy), myelopathy, hypertonic coma, hypoglycemic encephalopathy, dementia, paralysis, etc. 4.
- Protein peptide hormone dysfunction can cause the following diseases:
- Insulin and glucagon diabetes, hypoglycemia, etc .;
- Hypothalamus and pituitary hormones giant disease, dwarfism, acromegaly, cortisol syndrome (Cushing's syndrome), primary aldosteronism, secondary chronic adrenal insufficiency, hyperthyroidism Hypothyroidism (stingle disease, juvenile hypothyroidism, adult hypothyroidism male / female infertility, menstrual disorders (functional uterine bleeding, amenorrhea, polycystic ovary syndrome, premenstrual tension syndrome, menopausal syndrome) Disease), sexual development disorder, diabetes insipidus, inappropriate antidiuretic hormone secretion syndrome, abnormal lactation, etc .;
- parathyroid hormone hyperparathyroidism, hypoparathyroidism, etc .
- Gastrointestinal hormones peptic ulcer, chronic indigestion, chronic gastritis, etc .; 2.
- Disorders of amine metabolism can cause the following diseases:
- disorders of lipid metabolism can cause disorders in physiological functions of lipids, which in turn can lead to the occurrence of related conditions, including but not limited to:
- Phospholipids and cholesterol are important components of cell membrane, nuclear membrane, nerve myelin membrane, etc.
- Demyelinating peripheral neuropathy limb paralysis, limb dysfunction, respiratory palsy (intercostal, diaphragmatic paralysis), facial paralysis, medulla paralysis (hoarseness, cough), autonomic symptoms (increased sweating, skin flushing) , Tachycardia, orthostatic hypotension, urine retention), ataxia, mental disorders, etc .;
- Leukodystrophy (demyelinating) disease:, metachromatic leukodystrophy, Pei-Mei
- Phospholipid and cholesterol molecules contain hydrophilic and hydrophobic groups, so they have the effect of emulsifying triglycerides and fat-soluble vitamins, and promote their absorption and transport.
- Vi tA night blindness, dry eye, bone retardation
- Vi tE infertility, abortion, anemia, muscle wasting, neurodegeneration
- Vi tK coagulation factor II, ⁇ , IX, X Lack
- Vi tD3 child rickets, adult osteomalacia, kidney stones), etc .
- fatty deposition diseases fatty liver, fatty deposition cardiomyopathy, fatty deposition nephropathy
- related tumors lipoma, lipoblastoma, liposarcoma
- Phospholipid molecules contain many unsaturated fatty acids. Among them, linoleic acid, linolenic acid, and arachidonic acid are essential fatty acids in the human body and are indispensable for maintaining normal life activities. For example, arachidonic acid is the raw material for prostaglandin synthesis.
- Bile acid metabolism disorders steatosis (fat malabsorption can cause fat-soluble vitamin deficiency), gallbladder gallstones, biliary cirrhosis, etc .;
- Glucocorticoid cortisol: high / low blood sugar, muscle wasting, osteoporosis, delayed wound healing, infection, concentric obesity, water poisoning (headache, convulsions, coma), mental disorders, etc .;
- Mineralocorticoids aldosterone: Edema, hypertension, high / low blood sodium (headache, convulsions, coma), high / low blood potassium (muscle paralysis, arrhythmia, renal failure, paralytic intestinal obstruction, drowsiness, Coma) etc.
- c sex hormones testosterone, progesterone: abnormal sexual development, abortion, etc .;
- the three major metabolic disorders of sugar, lipid, and protein are causal and affect each other, and their specific clinical manifestations need to be considered in conjunction with the patient's original physical condition.
- the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) the NADH dehydrogenase 51Kd subunit 10.
- Agonists increase biological functions such as NADH dehydrogenase 51Kd subunit 10 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 NAM dehydrogenase 5 d subunit 10 and labeled NADH dehydrogenase 51Kd subunit 10 can be cultured in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
- NADH dehydrogenase 51Kd subunit 10 antagonists include antibodies, compounds, receptor deletions, and the like that have been screened.
- the antagonist of NADH dehydrogenase 51I (d subunit 10 can bind to NADH dehydrogenase 51Kd subunit 10 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the Peptides cannot perform biological functions.
- NADH dehydrogenase 51Kd subunit 10 When screening compounds as antagonists, NADH dehydrogenase 51Kd subunit 10 can be added to the bioanalytical assay, and the compound can be identified by measuring the effect of the compound on the interaction between NADH dehydrogenase 51Kd subunit 10 and its receptor. Whether it is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to MDH dehydrogenase 51Kd subunit 10 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 10 molecules of NADH dehydrogenase 51Kd subunit should generally be 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 invention also provides antibodies against the MDK dehydrogenase 51Kd subunit 10 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
- Polyclonal antibodies can be produced by injecting NAM dehydrogenase 51Kd subunit 10 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, etc.
- Techniques for preparing monoclonal antibodies to MDH dehydrogenase 51Kd subunit 10 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta cells Hybridoma technology, EBV-hybridoma technology, etc.
- Antibodies against NADH dehydrogenase 51Kd subunit 10 can be used in immunohistochemical techniques to detect NADH dehydrogenase 51I (d subunit 10 in biopsy specimens).
- Monoclonal antibodies that bind to NADH dehydrogenase 51Kd subunit 10 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.
- NADH dehydrogenase 51Kd subunit 10 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 an antibody with a thiol cross-linking agent such as SPDP, and bind the toxin to the antibody through the exchange of disulfide bonds.
- This hybrid antibody can be used to kill the MDH dehydrogenase 51Kd subunit 1 0 Positive cells.
- the antibodies of the present invention can be used to treat or prevent diseases related to the MDH dehydrogenase 51Kd subunit 10.
- Administration of an appropriate dose of antibody can stimulate or block the production or activity of NADH dehydrogenase 5 lKd subunit 10.
- the invention also relates to diagnostic test methods for the quantitative and localized detection of NADH dehydrogenase 51I (d subunit 10 levels. These tests are well known in the art and include FISH assays and radioimmunoassays.
- the level of catalase 51Kd subunit 10 can be used to explain the importance of NADH dehydrogenase 51Kd subunit 10 in various diseases and to diagnose diseases in which MDH dehydrogenase 51Kd subunit 10 functions.
- 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. Analysis.
- the polynucleotide encoding the NADH dehydrogenase 51Kd subunit 10 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 NADH dehydrogenase 51Kd subunit 10.
- Recombinant gene therapy vectors (such as viral vectors) can be designed to express the mutated NADH dehydrogenase 51Kd subunit 10 to inhibit endogenous MDH dehydrogenase 51Kd subunit 10 activity.
- a variant of the MDH dehydrogenase 51I (cl subunit 1 0 may be a shortened NADH dehydrogenase 51Kd subunit 10 that lacks a signaling domain, although it can bind to downstream substrates, but lacks Signal transduction activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by NADH dehydrogenase 51Kd subunit 10 expression or abnormal activity.
- Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, Herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding NADH dehydrogenase 51Kd subunit 10 into cells.
- a recombinant viral vector carrying a polynucleotide encoding NADH dehydrogenase 51Kd subunit 10 The method can be found in the existing literature (Sarabrook, eta l.).
- the recombinant polynucleotide encoding the NADH dehydrogenase 51Kd subunit 10 can be packaged into liposomes and transferred into cells.
- Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
- a vector such as a virus, phage, or plasmid
- Oligonucleotides including antisense RNA and DNA
- ribozymes that inhibit the 51DH subunit 10 mRNA of NADH dehydrogenase are also within the scope of the present invention.
- a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RM. 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 using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
- Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
- the polynucleotide encoding NADH dehydrogenase 51Kd subunit 10 can be used for the diagnosis of diseases related to MDH dehydrogenase 51Kd subunit 10.
- the polynucleotide encoding the NADH dehydrogenase 51 Kd subunit 10 can be used to detect the expression of NADH dehydrogenase 51Kd subunit 10 or the abnormal expression of NADH dehydrogenase 51Kd subunit 10 in disease states.
- the DNA sequence encoding the NADH dehydrogenase 51Kd subunit 10 can be used to hybridize biopsy specimens to determine the expression status of the NADH dehydrogenase 51Kd subunit 10.
- Hybridization techniques include Sout hern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. Part or all of the polynucleotides of the invention can be used as probes It is fixed on a micro array (Mi croarray) or DM chip (also known as a "gene chip"), and is used to analyze differential expression analysis and gene diagnosis of genes in tissues.
- Mi croarray Micro array
- DM chip also known as a "gene chip”
- NADH dehydrogenase 51Kd subunit 10 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the NADH dehydrogenase 51Kd subunit 10 transcription products.
- NADH dehydrogenase 51Kd subunit 10 Detection of mutations in the NADH dehydrogenase 51Kd subunit 10 gene can also be used to diagnose NADH dehydrogenase 51Kd subunit 10-related diseases.
- the NADH dehydrogenase 51Kd subunit 10 mutant forms include point mutations, translocations, deletions, weights, groups, and any other abnormalities compared to the normal wild-type NADH dehydrogenase 51Kd subunit 10 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
- the sequences of the invention are also valuable for chromosome identification.
- the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
- specific sites for each gene on the chromosome need to be identified.
- only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
- an important first step is to locate these DNA sequences on a chromosome.
- PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
- PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes.
- oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
- Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDM libraries.
- Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
- FISH Fluorescent in situ hybridization
- the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Wetch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
- the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If at A mutation is observed in some or all of the affected 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 disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping) Resolving power and each 20kb corresponds to a gene).
- the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
- suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
- the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
- the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
- a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
- these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
- the polypeptides of the invention can be used in combination with other therapeutic compounds.
- the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
- NADH dehydrogenase 51 Kd subunit 10 is administered in an amount effective to treat and / or prevent a specific indication.
- the amount and range of NADH dehydrogenase 51Kd subunit 10 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.
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AU93626/01A AU9362601A (en) | 2000-06-19 | 2001-06-18 | A novel polypeptide - nadh dehydrogenase 51 kd subunit 10 and a polynucleotide encoding the same |
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CN 00116593 CN1330145A (zh) | 2000-06-19 | 2000-06-19 | 一种新的多肽——NADH脱氢酶51Kd亚基10和编码这种多肽的多核苷酸 |
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PCT/CN2001/000991 WO2002002618A1 (fr) | 2000-06-19 | 2001-06-18 | Nouveau polypeptide, sous-unite 10 de nadh deshydrogenase, et polynucleotide codant ce polypeptide |
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Publication number | Priority date | Publication date | Assignee | Title |
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US5919686A (en) * | 1997-08-08 | 1999-07-06 | Incyte Pharmaceuticals, Inc. | NADH dehydrogenase subunits |
US5925543A (en) * | 1997-09-12 | 1999-07-20 | Incyte Pharmaceuticals, Inc. | Isolated polynucleotide sequence encoding NADH dehydrogenase B17 subunit |
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2000
- 2000-06-19 CN CN 00116593 patent/CN1330145A/zh active Pending
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2001
- 2001-06-18 WO PCT/CN2001/000991 patent/WO2002002618A1/zh active Application Filing
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5919686A (en) * | 1997-08-08 | 1999-07-06 | Incyte Pharmaceuticals, Inc. | NADH dehydrogenase subunits |
US5925543A (en) * | 1997-09-12 | 1999-07-20 | Incyte Pharmaceuticals, Inc. | Isolated polynucleotide sequence encoding NADH dehydrogenase B17 subunit |
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