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WO2001055193A1 - Nouveau polypeptide, sous-unite 35 pb1 de l'arn polymerase, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, sous-unite 35 pb1 de l'arn polymerase, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001055193A1
WO2001055193A1 PCT/CN2001/000080 CN0100080W WO0155193A1 WO 2001055193 A1 WO2001055193 A1 WO 2001055193A1 CN 0100080 W CN0100080 W CN 0100080W WO 0155193 A1 WO0155193 A1 WO 0155193A1
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Prior art keywords
polypeptide
polynucleotide
subunit
rna polymerase
sequence
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Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai BioDoor Gene Technology Ltd
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Shanghai BioDoor Gene Technology Ltd
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Priority to AU2001231494A priority Critical patent/AU2001231494A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, an RNA polymerase PB1 subunit 35, and a polynucleotide sequence encoding the polypeptide. The invention also relates to the preparation method and application of the polynucleotide and polypeptide. Background technique
  • RNA polymerase is the core part of a complex transcriptional mechanism and is closely related to the transcription and replication of RNA genes. Some RNA polymerases contain the PB1 subunit. The PB1 subunit was first found in influenza virus RNA polymerase. Influenza virus RNA polymerase is a multifunctional, multi-subunit enzyme composed of three filterable bacterial P proteins. Subunit composition, namely PB1, PB2 and PA.
  • RNA-dependent RM polymerase sites of the two binding sites are located in different structural regions, but each of them is relatively conserved. Both of them are extremely closely related to RNA synthesis substrates, primer binding, and product formation.
  • PB1 protein at positions 506-659 and its amino terminus of 78 amino acids can interact with other components of the polymerase.
  • PB1 is not a structurally independent polypeptide chain. Its N-terminal and C-terminal regions are connected to the PA and PB2 subunits, respectively, to form a complex structure. Therefore, PB1 is not only an important component of RNA polymerase, but also its core member. It plays a very important catalytic function and directly affects the transcription and replication process of viral RNA genes.
  • PB1 functions as a catalytic subunit in RNA polymerase. When there are no two subunits, PB2 and PA, it can independently catalyze RNA synthesis. Under the right conditions, it can also catalyze RNA synthesis in vitro. .
  • RNA polymerase a catalytic subunit in RNA polymerase.
  • RNP nucleoprotein
  • the catalytic unit promotes the entire transcription and replication process.
  • the polypeptide of the present invention contains a structural sequence characteristic of the RNA polymerase PB1 subunit, has a biological function similar to that of the RNA polymerase PB1 subunit, and is named RM polymerase PB1 subunit xx.
  • RNA polymerase PB1 subunit 35 protein plays an important role in important functions in the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more RNA polymerase PB1 involved in these processes.
  • Subunit 35 protein, especially the amino acid sequence of this protein is identified.
  • the isolation of the novel RNA polymerase PB1 subunit 35 protein encoding gene has also been used to determine the role of this protein in health and disease states. Use provides the foundation. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding 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 an RNA polymerase PB1 subunit 35.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding an RNA polymerase PB1 subunit 35.
  • Another object of the present invention is to provide a method for producing the RNA polymerase PB1 subunit 35. '
  • Another object of the present invention is to provide resistance to the polypeptide-RNA polymerase PB1 subunit 35 of the present invention.
  • Another object of the present invention is to provide mimic compounds against the polypeptide-RNA polymerase PB1 subunit 35 of the present invention Antagonists, agonists, inhibitors.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of RNA polymerase PB1 subunit 35.
  • 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 present 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 134-1 to 093 in SEQ ID NO: 1; and (b) a sequence having 1- in SEQ ID NO: 1 1418-bit sequence.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of RNA polymerase PB1 subunit 35 protein, which comprises utilizing 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 disease susceptibility related to abnormal expression of RNA polymerase PB1 subunit 35 protein in vitro, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the present 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 RNA polymerase PB1 subunit 35.
  • 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 DM 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 a fragment or part thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acid or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the substituted amino acid has similar structural or chemical properties as the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind to specific antibodies in a suitable animal or cell.
  • RNA polymerase PB1 subunit 35 means that when combined with RNA polymerase PB1 subunit 35, a protein that causes the protein to change A molecule that regulates 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 RNA polymerase PB1 subunit 35.
  • Antagonist refers to a molecule that, when combined with the RNA polymerase PB1 subunit 35, can block or regulate the biological or immunological activity of the RNA polymerase PB1 subunit 35.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds the RNA polymerase PB1 subunit 35.
  • RNA polymerase PB1 subunit 35 refers to a change in the function of RNA polymerase PB1 subunit 35, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of RNA polymerase PB1 subunit 35. change. ⁇
  • Substantially pure '' means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify the RNA polymerase PB1 subunit 35 using standard protein purification techniques.
  • the substantially pure RNA polymerase PB1 subunit 35 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the RM polymerase PB1 subunit 35 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of a polynucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules can be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits the hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such 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 two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). The Clus ter method checks the distance between all pairs by Each group of sequences is arranged into clusters. Each cluster is 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:
  • nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art, such as Jotun Hein (Hein 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 DM or RM 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 a substitution of a hydrogen atom with a fluorenyl group, an acyl group or an amino group. Nucleic acid derivatives encode codes that retain the main biological properties 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 the RNA polymerase PB1 subunit 35.
  • 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 thing, 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 it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated RNA polymerase PB1 subunit 35 means that the RNA polymerase PB1 subunit 35 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 RNA polymerase PB1 subunit 35 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the RNA polymerase PB1 subunit 35 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-RNA polymerase PB1 subunit 35, which is basically composed of SEQ ID It consists of the amino acid sequence shown in NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, mimicry, 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 RNA polymerase PB1 subunit 35.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the RNA polymerase PB1 subunit 35 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ ⁇ )
  • 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
  • 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 protein sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence that is 1,418 bases in length, and its open reading frame 134-1093 encodes 319 amino acids.
  • This polypeptide has the characteristic sequence of the RM polymerase PB1 subunit, and it can be deduced that the RNA polymerase PB1 subunit 35 has the structure and function represented by the RNA polymerase PB1 subunit.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • 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 is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least two sequences between
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 ° /. SDS, 60 ° C; or (2) adding a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol l, 42 ° C, etc .; or ( 3) Hybridization occurs only when the identity between the two sequences is at least 95%, more preferably 97%.
  • the hybridizable polynucleotide-encoded polypeptide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding the RNA polymerase PB1 subunit 35.
  • 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 RNA polymerase PB1 subunit 35 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Kits are also commercially available (Qiagene).
  • construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spruing Harbor Laboratory. New York, 1989).
  • 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. These genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determining the level of the transcript of the RNA polymerase PB1 subunit 35; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA 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).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the RNA polymerase PB1 subunit 35 gene expression protein.
  • ELISA enzyme-linked immunosorbent assay
  • a method using PCR to amplify DNA / RNA 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 may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the RNA polymerase PB1 subunit 35 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding the RNA polymerase PB1 subunit 35 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 Lee and Nathans, J Bio Chem. 263: 3521, 1988
  • baculovirus-derived vectors expressed in insect cells In short, as long as it can replicate and stabilize in the host It is determined that 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.
  • RNA polymerase PB1 subunit 35 Methods known to those skilled in the art can be used to construct expression vectors containing a DM sequence encoding the RNA polymerase PB1 subunit 35 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecuar ar Clinging, a Labora tory Manua l, cod Spring Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the l ac 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 MA polymerase PB1 subunit 35 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or a 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 DNA uptake can be harvested after exponential growth phase, 0 & (used: Treatment 1 2, used in this step are well known in the art alternative is to use MgCl 2.
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional machine Mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant RNA polymerase PB1 subunit 35 by conventional recombinant DNA technology (Scence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be 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 the amino acid sequences of the RM polymerase PB1 subunit 35 and the RNA polymerase PB1 subunit domain of the present invention.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated RNA polymerase PB1 subunit 35.
  • 35KDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RM with Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRM was formed by reverse transcription.
  • the Smart cDNA Cloning Kit (purchased from Clontech) was used to insert the cDM fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5c.
  • the bacteria formed a cDNA library.
  • the sequences at the 5 'and 3' ends of all clones were determined using Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer).
  • the determined cDNA sequence was compared with a public DNA sequence database (Genebank). As a result, the cDNA sequence of one of the clones, 0883M 1, was found to be a new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • RNA polymerase PB1 subunit 35 of the present invention The sequence of the RNA polymerase PB1 subunit 35 of the present invention and the protein sequence encoded by the RNA polymerase PB1 of the present invention were profiled by the prog i le scan program (Basicloca l Al ignment search tool) in GCG [Al tschul, SF et a l. J. Mol Biol. 1990; 215: 403-10], domain analysis was performed in a database such as Prote.
  • the RNA polymerase PB1 subunit 35 of the present invention is homologous to the domain RM polymerase PB1 subunit, and the homology results are shown in FIG. 1.
  • Example 3 Cloning of a gene encoding RNA polymerase PB1 subunit 35 by RT-PCR
  • CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, PCR was performed using the following primers:
  • Pr iraerl 5-GGTCTCCCGGAAGACGAACTCCGC -3 '(SEQ ID NO: 3)
  • Pr imer2 5-ACATCTACTATTTATTAATTCCCC -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Pr imer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10 mol / L Tri s-Cl, (pH 8.5.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP in a reaction volume of 50 ⁇ 1 , 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.
  • RT-PCR (3-act in was used as a positive control and template blank was used as a negative control.
  • Amplification products were purified using a QIAGEN kit and cloned with TA.
  • the kit was connected to a pCR vector (Invitrogen).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1418bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of RNA polymerase PB1 subunit 35 gene expression:
  • RNA extraction in one step involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4. G), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) are added. ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • the 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RM 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 20 g / ml salmon sperm DNA. After hybridization, wash the filter in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, use Analysis and quantification using Phosphor Imager.
  • Example 5 In vitro expression, isolation and purification of recombinant RNA polymerase PB1 subunit 35
  • Primer 3 5- CCCCATATGATGGCCATCTGCCCAGAATTGGCC -3 '(Seq ID No: 5)
  • Primer4 5'- CATGGATCCTTACACATTATCATTAACAATCAC -3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, and the Ndel and BamHI restriction sites correspond to the selective endonucleases on the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865.3). Enzyme site.
  • PCR was performed using the pBS-0883bll plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ l contains 10 pg of pBS-0883M1 plasmid, primers Primer-3 and Prier-4, and 1 j is 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 pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into the colibacillus DH5ot by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 3 ( ⁇ g / ml)), positive clones were selected by colony PCR method and sequenced. The correct positive clone (pET-0883bll) was used to transform the recombinant plasmid into E. coli by calcium chloride method. BL21 (DE3) plySs (product of Novagen).
  • the host bacteria BL21 (pET-0883M1) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 to make ol / L , Continue to cultivate for 5 hours.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation.
  • the supernatant was collected by centrifugation. Chromatography was performed using an affinity chromatography column His s. Bind Quick Cartridge (product of Novagen) capable of binding to 6 histidines (6His-Tag).
  • the purified protein RNA polymerase PB1 subunit 35 was obtained.
  • a peptide synthesizer (product of PE company) was used to synthesize the following RNA polymerase PB1 subunit 35-specific peptides: -Ser-Ala-Leu-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the aforementioned hemocyanin polymorphic complex with complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects.
  • 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. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer so that the non-specific binding site of the sample on the filter is supported by the carrier and synthetic The polymer is saturated.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • 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.
  • the selection of 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, then 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 (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • Gene chip or gene micro matrix (DNA Mi croarray) is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high density arrangement of a large number of target gene fragments on glass. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature, for example, see the literature DeRi s i, J. L., Lyer, V. & Brown, P. 0.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between the points is 280 ⁇ ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps have various reports in the literature. The post-spot processing steps in this embodiment are:
  • Total mRNA was extracted from normal liver and liver cancer in one step, and mRM was purified with Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5-Araino- propargyl-2'- deoxyur idine 5'-tr iphate coupled to Cy3 f luorescent dye (purchased from Amersham Phamacia Biotech) was used to label mMA of normal liver tissue, and the fluorescent reagent Cy5dUTP (5-Araino-propargy 1-2 ⁇ -deoxyuri dine 5'-tr iphate cou led to Cy5 f luorescent dye (purchased from Amersham Phamacia Biotech) was used to label the liver cancer tissue mRNA, and the probe was prepared after purification.
  • Cy3dUTP 5-Araino- propargyl-2'- deoxyur idine 5'-tr iphate coupled to Cy3 f lu
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodi scovery, USA) to calculate the Cy3 / Cy5 ratio of each point, which was less than 0.5 Dots greater than 2 are considered genes with differential expression.
  • RNA polymerase is a core part of complex transcriptional machinery.
  • the two characteristic binding sites on the RNA polymerase PB1 subunit are extremely closely related to the RNA synthesis substrate, primer binding, and product formation. Mutations in the PB1 subunit can reduce RNA polymerase activity or stall transcription.
  • the polypeptide of the present invention is a polypeptide containing a characteristic binding site sequence of the RM polymerase PB1 subunit, and abnormal expression of the polypeptide will lead to a decrease or error in the vitality of the transcription process, and cause related diseases.
  • RNA polymerase PB1 subunit 35 of the present invention will produce various diseases, especially various tumors, embryonic developmental disorders, growth disorders, and inflammation. These diseases include, but are not limited to:
  • Tumors of various tissues stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, adrenal cancer, bladder cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymic tumor, nasal cavity and sinus tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, Fibrosarcoma, lipoma, liposarcoma
  • Fetal developmental disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, cryptorchidism, vaginal atresia, hypospadias, amphoteric deformity, atrial septal defect, neural tube defect, congenital hydrocephalus, iris defect, congenital Glaucoma or cataract, congenital deafness
  • Growth disorders mental retardation, cerebral palsy, mental retardation, mental retardation, familial cerebellar dysplasia, strabismus, skin, fat, and muscular dysplasias such as congenital skin relaxation, albinism , Alzheimer's disease, congenital keratosis, bone and joint dysplasia diseases such as cartilage dysplasia, epiphyseal dysplasia, metabolic bone disease, various metabolic defects such as various amino acid metabolic defects, dementia, dwarfism Cushing syndrome, sexual retardation
  • Inflammation allergic reaction, bronchial asthma, adult respiratory distress syndrome, sarcoidosis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, dermatomyositis, urticaria, specific dermatitis, polymyositis, Addison's disease, Graves' disease, intestinal emergency syndrome, chronic rhinitis, atrophic gastritis, chronic gastritis, systemic lupus erythematosus, myasthenia gravis, multiple spinal cord sclerosis, Guillain-Barre syndrome, Intracranial granuloma, multiple scleroderma, pancreatitis, cholecystitis, glomerulonephritis, chronic active hepatitis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, benign prostatic hyperplasia
  • RNA polymerase PB1 subunit 35 of the present invention will also produce certain hereditary, hematological and immune system diseases.
  • the polypeptides of the present invention can be directly used in the treatment of diseases. For example, they can treat various diseases, especially various tumors, embryonic development disorders, growth disorders, inflammation, Some hereditary, hematological and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) RM polymerase PB1 subunit 35. Agonists increase biological functions such as RNA polymerase PB1 subunit 35 to stimulate cell proliferation, and antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing the RNA polymerase PB1 subunit 35 can be cultured with the labeled RNA polymerase PB1 subunit 35 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of RM polymerase PB1 subunit 35 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of RNA polymerase PB1 subunit 35 can bind to RNA polymerase PB1 subunit 35 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 exert its biology Features.
  • RNA polymerase PB1 subunit 35 can be added to a bioanalytical assay to determine whether a compound is a compound by measuring the effect of the compound on the interaction between RM polymerase PB1 subunit 35 and its receptor. Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • the peptide molecules capable of binding to the RNA polymerase PB1 subunit 35 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In the screening, the RNA polymerase PB1 subunit 35 molecule 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 directed against the RNA polymerase PB1 subunit 35 epitope. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.
  • Polyclonal antibodies can be produced by direct injection of RNA polymerase PB1 subunit 35 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 Agent.
  • Techniques for preparing monoclonal antibodies to RNA polymerase PB1 subunit 35 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.
  • Chimeric antibodies that combine human constant regions and non-human-derived variable regions can be produced using existing techniques (Morri et al, PMS, 1985, 81: 6851).
  • the existing technology for producing single-chain antibodies U.S. Pat No. 4946778, can also be used to produce single-chain antibodies against RNA polymerase PB1 subunit 35.
  • Antibodies against RNA poly 'synthase PB1 subunit 35 can be used in immunohistochemistry to detect RNA polymerase PB1 subunit 35 in biopsy specimens.
  • Monoclonal antibodies that bind to RNA polymerase PB1 subunit 35 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.
  • RNA polymerase PB1 subunit 35 high affinity monoclonal antibody 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 sulfhydryl crosslinker such as SPDP and bind the toxin to the antibody by disulfide exchange.
  • This hybrid antibody can be used to kill RM polymerase PB1 subunit 35 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the RNA polymerase PB1 subunit 35.
  • Administration of an appropriate amount of antibody can stimulate or block the production or activity of RNA polymerase PB1 subunit 35.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of RNA polymerase PB1 subunit 35.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of RNA polymerase PB1 subunit 35 detected in the test can be used to explain the importance of RNA polymerase PB1 subunit 35 in various diseases and to diagnose diseases in which RNA polymerase PB1 subunit 35 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry.
  • the polynucleotide encoding the RNA polymerase PB1 subunit 35 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 RM polymerase PB1 subunit 35.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated RNA polymerase PB1 subunit 35 to inhibit endogenous RM polymerase PB1 subunit 35 activity.
  • a mutated RNA polymerase PB1 subunit 35 may be a shortened RNA polymerase PB1 subunit 35 lacking a signaling domain, although it can bind to downstream substrates, but lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of RNA polymerase PB1 subunit 35.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding the RNA polymerase PB1 subunit 35 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding the RNA polymerase PB1 subunit 35 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding the RNA polymerase PB1 subunit 35 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly 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
  • RNA that inhibit RNA polymerase PB1 subunit 35 mRNA Enzymes are also within the scope of the 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 MA to perform endonucleation.
  • Antisense RNA and DNA and ribozymes can be obtained by any conventional RNA or DNA synthesis technology. For example, solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding RNA polymerase PB1 subunit 35 can be used for the diagnosis of diseases related to RM polymerase PB1 subunit 35.
  • the polynucleotide encoding the RNA polymerase PB1 subunit 35 can be used to detect the expression of the RNA polymerase PB1 subunit 35 or the abnormal expression of RM polymerase PB1 subunit 35 in a disease state.
  • the DNA sequence encoding the RNA polymerase PB1 subunit 35 can be used to hybridize biopsy specimens to determine the expression status of the RNA polymerase PB1 subunit 35.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization.
  • RNA polymerase PB1 subunit 35 specific primers can be used to perform RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect the transcription products of RNA polymerase PB1 subunit 35.
  • RNA polymerase PB1 subunit 35 mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type RNA polymerase PB1 subunit 35 DNA sequence. Mutations can be detected using existing techniques such as Sou thern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nort Hern blotting and Western blotting can be used to indirectly determine the presence or absence of mutations in a gene.
  • 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 according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • sublocalization can be achieved by a similar method using a set of fragments from a specific chromosome or a large number of genomic clones.
  • 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 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 Iher Intance in Man (available online with Johns Hopkins University Welch Medi Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptide of the present invention can be used in combination with other therapeutic compounds.
  • RNA polymerase PB1 subunit 35 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of RNA polymerase PB1 subunit 35 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une sous-unité 35 PB1 de l'ARN polymérase, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la sous-unité 35 PB1 de l'ARN polymérase.
PCT/CN2001/000080 2000-01-28 2001-01-21 Nouveau polypeptide, sous-unite 35 pb1 de l'arn polymerase, et polynucleotide codant pour ce polypeptide Ceased WO2001055193A1 (fr)

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CN 00111588 CN1307109A (zh) 2000-01-28 2000-01-28 一种新的多肽——rna聚合酶pb1亚基35和编码这种多肽的多核苷酸

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J. BIOCHEM., vol. 122, no. 3, September 1997 (1997-09-01), TOKYO, pages 627 - 634 *
NUCLEIC ACIDS RES., vol. 24, no. 22, 15 November 1996 (1996-11-15), pages 4456 - 4463 *
VIRUS GENES, vol. 12, no. 2, 1996, pages 155 - 163 *

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