WO2001066586A1 - Nouveau polypeptide, arn helicase humaine 13, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, arn helicase humaine 13, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001066586A1
WO2001066586A1 PCT/CN2001/000202 CN0100202W WO0166586A1 WO 2001066586 A1 WO2001066586 A1 WO 2001066586A1 CN 0100202 W CN0100202 W CN 0100202W WO 0166586 A1 WO0166586 A1 WO 0166586A1
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polypeptide
polynucleotide
rna helicase
human rna
sequence
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PCT/CN2001/000202
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU44046/01A priority Critical patent/AU4404601A/en
Publication of WO2001066586A1 publication Critical patent/WO2001066586A1/fr

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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
    • C12N9/90—Isomerases (5.)
    • 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, human RNA helicase 13, 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 structure In many activities of the cell, the regulation of RNA structure, such as splicing of precursor RNA, assembly of spliceosome, protein translation, etc. are all necessary. These processes are usually regulated by a variety of different RNA helicases. RNA helicases from different sources constitute a large family of proteins, and their presence can be detected in many biological systems where RNA plays an important role. They are widely distributed in various tissues and organs from prokaryotes (including viruses) to lower and higher organisms. They are involved in cell and mitochondrial division, RNA editing, rRNA processing, transcription initiation, nuclear mRNA transport and mRNA. Degradation and other processes.
  • RNA helicases are considered important factors in cell development and differentiation, and some of them also play a role in the transcription and replication of viral single-stranded RNA [Arri Eisen, John C. Lucchesi, Bioessays, 1998, 20: 634 -641] 0 which, diagnose neurological diseases and immune system diseases, prevention and treatment provides an effective means for cancer in vivo.
  • RNA helicases are divided into different subfamilies, subfamily I and subfamily II based on their structural characteristics. All helicases contain two Walker-type NTP binding domains, the A domain (or ATPase A domain) and the B domain (or ATPase B domain). Members of subfamily I contain the conserved ATPase A domain: GXXXXGKT. It was later discovered that there are mutations in the ATPase domain A of some RNA helicases at some amino acid positions, and the conserved sequence of the ATPase A domain is: AXXGXGKT, so these proteins are divided into one class, namely subfamily .
  • DEAD box protein In subfamily II, some proteins contain a conserved DEAD box in the ATPase domain B. It was discovered that members of this subfamily can be divided into three different subclasses according to their DEAD boxes, namely DEAD boxes. Protein, DEAH box protein and DEXH box protein [Angelika Luking, Ulf Stahl et al., 1998, Crit. Rev. Biochem. Mol. Biol., 33: 259-296].
  • RNA helicase A has both RNA helicase activity and DNA helicase activity in vivo.
  • RNA helicase protein family containing the DEAD box play an important biological function in the transcriptional regulation of RNA.
  • the abnormal expression of this type of protein will cause some metabolic disorders caused by abnormal transcriptional regulation.
  • the human RNA helicase 13 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need to identify more involved in these processes
  • the human RNA helicase 13 protein, especially the amino acid sequence of this protein was identified. Isolation of the new human RNA helicase 13 protein encoding gene also provides a basis for research to determine the role of the 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 coding DNA. 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 human RNA helicase 13.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human RNA helicase 13.
  • Another object of the present invention is to provide a method for producing human RNA helicase 13.
  • Another object of the present invention is to provide an antibody against the polypeptide-human RNA helicase 13 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human RNA helicase 13.
  • Another object of the present invention is to provide diagnosis and treatment of diseases related to abnormalities of human RNA helicase 13 Method.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 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) having SEQ ID D NO: 1
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; "a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; The method for preparing a polypeptide of the present invention by describing a host cell and recovering an expressed product is described.
  • 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 human RNA helicase 13 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 susceptibility to disease associated with abnormal expression of human RM helicase 1 3 protein in vitro, comprising 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 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 human RNA helicase 1 3.
  • 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 “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insert or “addition” means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to the molecule present.
  • 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 in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human RNA helicase 13, 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 binds human RNA helicase 1 3.
  • Antagonist refers to a molecule that blocks or regulates the biological or immunological activity of human RNA helicase 13 when combined with human RNA helicase 13.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human RNA helicase 13.
  • RNA helicase 13 refers to a change in the function of human RNA helicase 13, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human RNA helicase 13 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 human RNA helicase 1 3 using standard protein purification techniques.
  • the substantially pure human RNA helicase 1 3 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human RNA helicase 1 3 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to polynucleotides that naturally bind through 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 may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • 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 percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.).
  • the MEGALIGN program can compare two or more sequences according to different methods, such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Clus ter method groups groups of sequences by checking the distance between all pairs. Arranged in clusters. The clusters are then assigned in pairs or groups.
  • sequence A and sequence B 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 Number of interval residues in a sequence B
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art, such as Jotim Hein (Hein J., (1990) Methods in erazumology 183: 625-645).
  • 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 a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to an intact antibody molecules and fragments thereof, such as Fa, F (a b ') 2 and F V, which specifically binds to human RNA helicase 13 antigen determinant.
  • 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 is naturally occurring).
  • 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 certain vector, or such a polynucleotide or polypeptide may be part of a certain 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 in the natural state .
  • isolated human RNA helicase 1 3 means that human RNA helicase 1 3 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
  • Those skilled in the art can purify human RM helicase 1 3 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the human RM helicase 1 3 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human RNA helicase 1 3, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the 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 human RNA helicase 1 3.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human RNA helicase 1 3 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 the genetic codon; or ( ⁇ ) a type in which one or more amino acid residues A group is substituted by another group to include a substituent; or (in) a type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or
  • polypeptide sequence such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protein sequence
  • a polypeptide sequence formed by fusing additional amino acid sequences into a mature polypeptide.
  • fragments, 00 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 of 3116 bases in length and its open reading frame 2608-2970 encodes 120 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile to human RNA helicase 95, and it can be deduced that the human RNA helicase 13 has a similar function to human RNA helicase 95.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence 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 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least 501 ⁇ 2, preferably 70% identity, between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • "strict conditions” means: a) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) hybridization L% Ficoll , 42 ⁇ When adding a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42.
  • hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. 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 human RM helicase 13.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human RM helicase 13 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 DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • 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.
  • genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DM-DM or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of human RNA helicase 13 transcripts; ( 4) ) Detection of protein products expressed by genes through immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein is 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 human RNA helicase 13 gene.
  • a method of applying a PCR technique to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-cDM terminal rapid amplification method
  • the primers for PCR may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein.
  • 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 cDM 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 a human RNA helicase 13 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .
  • a polynucleotide sequence encoding human RNA helicase 13 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • RNA sequence encoding human RNA helicase 13 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 DM 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 and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pairs of SV40 enhancer at a later stage of the replication initiation point, polyoma enhancer and adenovirus enhancer at the late side of the replication initiation point.
  • 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 human RNA helicase 13 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 the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as insect cells such as Fly S2 or Sf9
  • 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 DM may be harvested after exponential growth phase, with (: Treatment 1 2, steps well known in the art used 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 mechanical methods such as microinjection, electroporation, and liposomes Packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human RNA helicase 1 (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. When host cells grow to proper After inducing the cell density, the appropriate promoter (such as temperature conversion or chemical induction) is used to induce the selected promoter, and the cells are cultured for a period of time.
  • the appropriate promoter such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • 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
  • Fig. 1 is a comparison diagram of gene chip expression profiles of human RNA helicase 13 and human RNA helicase 95 of the present invention.
  • the upper figure is a graph of the expression profile of human RNA helicase 13 and the lower sequence is the graph of the expression profile of human RNA helicase 95.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human RNA helicase 13 isolated. 13kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • MRNA is formed by reverse transcription cDNA Quik mRNA Isolation Kit (Qiegene Co.) isolated from the total RNA poly (A) mRNA 0 2ug poly ( A) used.
  • the Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5cc.
  • the bacteria formed a cDNA library.
  • 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 one of the clones was found.
  • the cDNA sequence of 0421a04 is new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'- GAGAAACTAGAAAAAGTCATTCTA- 3, (SEQ ID NO: 3)
  • Primer2 5,-CTTTTTCCTTTTTTTTTTGTCCAACC-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 mmol / L KC1, 10 mmol / L Tris-CI, (pH 8.5), 1.5 ramol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers in a 50 ⁇ 1 reaction volume, 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 0 set ⁇ -actin as a positive control and template blank as a negative control at the same time during RT-PCR.
  • 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 that of 1-3116bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human RNA helicase 13 gene expression:
  • This method involves acid guanidinium thiocyanate phenol-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 ), 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.
  • RNA was electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the PCR amplified human RNA helicase 13 coding region sequence (2608bp to 2970bp) shown in FIG. 1.
  • a 32P-labeled probe (about 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-25m KH 2 P0 4 (pH7.4) -5 ⁇ SSC-5 ⁇ Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filters were placed in 1 x SSC-0.1% SDS at 55. C Wash for 30min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human RNA helicase 13
  • Primer3 5,-CCCGAATTCATGGATCCCCTAAGACACCCATCA- 3, (Seq ID No: 5)
  • Primer4 5, — CCCGAGCTCTTATCTTTCAAAAGGTTTTTGCTT-3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene
  • the Ndel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • PCR was performed using the pBS-0421a04 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0421a04 plasmid, primers P ⁇ imer-3 and Prime ⁇ -4 were included in a total volume of 50 ⁇ 1; J was 10 pmol, 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 E. coli DH5a by the calcium chloride method.
  • RNA helicase 13 was purified. After SDS-PAGE electrophoresis, a single band was obtained at 13 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2.
  • RNA helicase 13 The following peptides specific to human RNA helicase 13 were synthesized using a peptide synthesizer (product of PE):
  • polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Immunocheniistry, 1969; 6: 43. Use 4mg of the above blood blue eggs 1/66586
  • 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.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • 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) 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 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 mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment: ⁇ 5-TGGATCCCCTAAGACACCCACCAAAATCCACCAGTGGGATT-3 '(SEQ ID NO: 9)
  • SEQ ID NO: 9 Please refer to the literature for other commonly listed reagents and their preparation methods related to the following specific experimental procedures: DNA PROBES G, H. Kel ler; ⁇ ⁇ ⁇ ⁇ Manak; Stockton Press, 1989 (USA) and more commonly used molecular clones (Experimental manual books such as "Molecular Cloning Experiment Guide” U998 Second Edition) [US] Sambrook waiting, Science Press.
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • probes 1 3 ⁇ IProbe (0.1 IOD / ⁇ 1), add 2 ⁇ IKinase buffer, 8-10 uCi ⁇ - 32 P- dATP + 2U Kinase, to make up to a final volume of 20 ⁇ 1.
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Gene chip or DNA microarray 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, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of fast, efficient, and 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 DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And Helle, RA, Schema, M. , Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
  • 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 respectively amplified by PCR. 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), between the points. The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the DNA was fixed on the glass slide to prepare a chip. The specific method steps have been variously reported in the literature. The post-spotting processing steps of this embodiment are:
  • Probes from the above two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution at room temperature— (1 ⁇ SSC, 0.2% SDS), and then scanned with ScanArray.
  • the 3000 scanner purchased from General Scanning, USA was used for scanning.
  • the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Based on these 13 Cy3 / Cy5 ratios, a bar graph is drawn. (figure 1 ) . It can be seen from the figure that the expression profiles of human RNA helicase 13 and human RNA helicase 95 according to the present invention are very similar. Industrial applicability
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • RNA and DNA helicases are expressed in cancer tissues, brain and nerve tissues, and tissues associated with inflammation and immune responses.
  • RNA helicases play an important role in cancer, nervous system diseases, and immune system diseases.
  • the expression of RNA helicase is significantly increased in cancer and immune system diseases; in nervous system-related diseases, its expression level or activity is reduced.
  • the enzyme and its fragments or derivatives can be used to diagnose and treat diseases related to the nervous system, including but not limited to the following, sedation panic disorder, premature senility Dementia, anterograde memory forgetting, amyotrophic lateral sclerosis, bipolar neurocytic disorders Disorder, tension, brain tumor, dementia, depression, delayed dyskinesia, dystonia, epilepsy, hereditary chronic chorea, multiple lateral sclerosis, neurofibromatosis, Parkinson's disease, paranoid neuropathy, Schizophrenia, Tourette's disease, etc.
  • diseases related to the nervous system including but not limited to the following, sedation panic disorder, premature senility Dementia, anterograde memory forgetting, amyotrophic lateral sclerosis, bipolar neurocytic disorders Disorder, tension, brain tumor, dementia, depression, delayed dyskinesia, dystonia, epilepsy, hereditary chronic chorea, multiple lateral sclerosis, neurofibromatosis, Parkinson's disease
  • novel helicases and fragments or derivatives thereof of the present invention can also be used to diagnose and treat some cancers, including but not limited to the following, adenoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, etc .; Of cancer that includes these tissues, thyroid, bladder, bone, bone marrow, brain, breast, cervix, tendon sheath cyst, heart, kidney, lung, liver, muscle, ovary, pancreas, parathyroid gland, prostate, uterus, salivary glands , Skin, spleen, testis, penis, thymus, gallbladder, gastrointestinal tract, etc.
  • novel human helicases and fragments or derivatives thereof of the present invention can also be used to diagnose and treat diseases related to the immune system, including but not limited to the following, rheumatoid arthritis, chronic active hepatitis, primary Sjogren's syndrome, ankylosing spondylitis, hemochromatosis, immune complex glomerulonephritis, mycocarditis after gonococcal infection, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, polymyositis, mouth Xerophthalmia syndrome, nodular polyarteritis, Wegener's granulomatosis, myasthenia gravis, Guillain-Barre syndrome, autoimmune hemolytic anemia, immune thrombocytopenic purpura, insulin autoimmune syndrome, autoimmune Immune thyroid disease, autoimmune heart disease, Down syndrome, short limb dwarfism, hereditary transcobalamin II deficiency with hypogammaglob
  • novel human helicases and fragments or derivatives thereof of the present invention can also be used to diagnose and treat diseases related to the immune system, including but not limited to the following, pulmonary eosinophilia, sarcoidosis, rheumatism Arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immune complex glomerulonephritis, acute anterior uveitis, osteoporosis, dermatomyositis, urticaria, Specific dermatitis, hemochromatosis, polymyositis, Addison's disease, Graves' disease, chronic active hepatitis, emergency bowel syndrome, atrophic gastritis, systemic lupus erythematosus, myasthenia gravis, cerebral spinal cord Multiple sclerosis, Guillain-Barre syndrome, intracranial granuloma, Wegener's granulomatosis,
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human RM helicase 13.
  • Agonists increase biological functions such as human RNA helicase 13 to stimulate cell proliferation, and antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human RNA helicase 13 is cultured together with the labeled human RNA helicase 13 in the presence of. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human RNA helicase 13 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human RNA helicase 13 can bind to human RNA helicase 13 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human RNA helicase 13 When screening compounds as antagonists, human RNA helicase 13 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human RNA helicase 13 and its receptor. . 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 human RNA helicase 13 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human RNA helicase 13 molecules should generally be labeled. ⁇
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human RNA helicase 13 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human RM helicase 13 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Wait.
  • Techniques for preparing monoclonal antibodies to human RNA helicase 13 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV- Hybridoma technology, etc.
  • Chimeric antibodies that combine human constant regions with non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). 0
  • Existing techniques for producing single-chain antibodies can also be used to produce single-chain antibodies against human RNA helicase 13.
  • Antibodies to human RNA helicase 13 can be used in immunohistochemical techniques to detect human RNA helicase 13 in biopsy specimens.
  • Monoclonal antibodies that bind to human RNA helicase 13 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human RNA helicase 13 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of the antibody with a thiol crosslinker such as SPDP. The exchange of sulfur bonds binds toxins to antibodies.
  • This hybrid antibody can be used to kill human RNA helicase 13+ positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human RNA helicase 1 3.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of human RNA helicase 1 3.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human RNA helicase 13 levels. These tests are well known in the art and include F I SH assays and radioimmunoassays.
  • the level of human RNA helicase 1 3 detected in the test can be used to explain the importance of human RNA helicase 1 3 in various diseases and to diagnose diseases in which human RNA helicase 1 3 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.
  • Polynucleotides encoding human RNA helicase 1 3 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human RNA helicase 1 3.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human RNA helicase 1 3 to inhibit endogenous human RNA helicase 13 activity.
  • a mutated human RNA helicase 1 3 may be a shortened human RNA helicase 1 3 lacking a signaling domain, and although it can bind to a downstream substrate, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human RNA helicase 13.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human RNA helicase 1 3 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human RNA helicase 1 3 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding human RNA helicase 1 3 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 human RNA helicase 1 3 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense NA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • Antisense RM 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 vector's RNA polymerase promoter. To increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as Adding sequence lengths on both sides, the linkage between ribonucleosides uses phosphothioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human RNA helicase 13 can be used for the diagnosis of diseases related to human RNA helicase 13.
  • the polynucleotide encoding human RNA helicase 13 can be used to detect the expression of human RNA helicase 13 or the abnormal expression of human RM helicase 13 in a disease state.
  • the DNA sequence encoding human RNA helicase 13 can be used to hybridize biopsy specimens to determine the expression of human RNA helicase 13.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and so on. These techniques and methods are publicly available and mature, and related kits are available commercially.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human RNA helicase 13 specific primers can also be used to detect transcribed products of human RNA helicase 13 using RM-polymerase chain reaction (RT-PCR) in vitro amplification.
  • RT-PCR RM-polymerase chain reaction
  • -Detection of mutations in the human RNA helicase 13 gene can also be used to diagnose human RNA helicase 13-related diseases.
  • Human RNA helicase 13 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human RM helicase 13 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, Northern 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 DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones to metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the 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, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • 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. Based on the resolution capabilities of current physical mapping and gene mapping technologies, cDNAs that are accurately mapped to disease-related chromosomal regions can be one of 50 to 500 potentially pathogenic genes (assuming
  • 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.
  • Human RNA helicase 13 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human RNA helicase 13 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 ARN hélicase humaine 13, 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 l'ARN hélicase humaine 13.
PCT/CN2001/000202 2000-03-07 2001-02-26 Nouveau polypeptide, arn helicase humaine 13, et polynucleotide codant pour ce polypeptide WO2001066586A1 (fr)

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CN 00111900 CN1312378A (zh) 2000-03-07 2000-03-07 一种新的多肽——人rna解旋酶13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
IMAMURA O. ET AL.: "Cloning and characterization of a putative human RNA helicase gene of the DEAH-box protein family", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 240, no. 2, 17 November 1997 (1997-11-17), pages 335 - 340 *
MATSUDA S. ET AL.: "Molecular cloning and characterization of a novel human gene (HERNA) which encodes a putative RNA-helicase", BIOCHEM. BIOPHYS. ACTA, vol. 1490, no. 1-2, 31 January 2000 (2000-01-31), pages 163 - 169 *
ZHANG S. ET AL.: "Molecular cloning of the gene encoding nuclear DNA helicase II. A bovine homologue of human RNA helicase A and drosophila Mle protein", J. BIOL. CHEM., vol. 270, no. 27, 7 July 1995 (1995-07-07), pages 16422 - 16427 *

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