WO2001047972A1 - Nouveau polypeptide, proteine 9 polyisoprene synthetase, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine 9 polyisoprene synthetase, et polynucleotide codant pour ce polypeptide Download PDF

Info

Publication number
WO2001047972A1
WO2001047972A1 PCT/CN2000/000619 CN0000619W WO0147972A1 WO 2001047972 A1 WO2001047972 A1 WO 2001047972A1 CN 0000619 W CN0000619 W CN 0000619W WO 0147972 A1 WO0147972 A1 WO 0147972A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
polyisoprenyl
synthase protein
sequence
Prior art date
Application number
PCT/CN2000/000619
Other languages
English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
Original Assignee
Biowindow Gene Development Inc. Shanghai
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biowindow Gene Development Inc. Shanghai filed Critical Biowindow Gene Development Inc. Shanghai
Priority to AU19883/01A priority Critical patent/AU1988301A/en
Publication of WO2001047972A1 publication Critical patent/WO2001047972A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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 novel polypeptide, a polyisoprenyl synthase protein 9, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide. Background technique
  • isoprenoid biosynthetic pathway in eukaryotes is responsible for the synthesis of different end products, including cholesterol, long alcohol, ubiquinone, or coenzyme Q.
  • This pathway results in isoprene tRNA, isoprenoid quinone, and sugar carrier lipids in bacteria.
  • the enzymes involved in this pathway are a series of polyisoprenyl synthetases that catalyze the 1'-4 condensation reaction between 5-carbon isoprene units.
  • Protein isoprenylation is a process in which molecules are covalently modified by binding to a lipophilic isoprenoid group.
  • Isoprenoids are a family of various lipophilic molecules based on a repeating 5-carbon structure. In the human body, farnesyl diphosphate, a 15-molecule and geranyl diphosphate, a 20-carbon molecule, are isoprenoid compounds that are most relevant to protein isoprenylation. Many specific proteins are modified by isoprene groups after translation.
  • Isoprene proteins exist in different cellular regions, including the nuclear cytosol and membrane-bound organelles. Members of this family are involved in isoprenylation of various cellular proteins, including cancer-related proteins, such as the ras protein.
  • All polyisoprenyl synthetases contain regions with similar sequences. Two of these regions are rich in aspartic acid residues and are involved in catalyzing or binding substrates.
  • the conserved aspartic acid repeats help to bind to the substrate by forming a magnesium or manganese salt bridge between the substrate and the catalytic site, which is extremely important for the catalytic activity of polyisoprenyl synthase.
  • the second template contains an allyl binding site.
  • the hydrophobic amino acid fixes the binding to the substrate through the hydrocarbon tail interaction with the allyl and allyl homologous substrates.
  • other positively charged amino acids in the template are also important for enzyme activity.
  • One of the main biological functions of arginine residues is to interact with phosphorylation metabolism. Lysine residues also have this function and Acts on phosphorylated substrates.
  • Polypeptides containing the above-mentioned two conserved sequences and antagonists and antagonists of the polypeptides can be used for diagnosis, prevention and treatment of tumors and prevention of virus proliferation, control of cell morphology, inhibition of programmed cell death, and prevention of classes Like p21.
  • Sup. Ras protein expression Since the polyisoprenyl synthase protein 9 protein plays an important role in important body functions 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 polyisotopes involved in these processes.
  • the pentadienyl synthase protein 9 protein, especially the amino acid sequence of this protein is identified.
  • the isolation of the new polyisoprenyl synthase protein 9 protein-encoding gene also provides a basis for research to determine the role of this protein in health and disease states.
  • This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. It is an object of the present invention to provide an isolated novel polypeptide, polyisoprenyl synthase protein 9 and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a polyisoprenyl synthase protein 9.
  • Another object of the present invention is to provide a method for producing polyisoprenyl synthase protein 9.
  • Another object of the present invention is to provide an antibody against the polypeptide-polyisoprenyl synthase protein 9 of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polyisoprenyl synthase protein 9 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of the polyisoprenyl synthase protein 9.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • the sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID NO: 1 A sequence of positions 2469-2726; and (b) a sequence of positions 1-3022 in SEQ ID NO: 1.
  • 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.
  • 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 the polyisoprenyl synthase protein 9 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 polyisoprenyl synthase protein 9 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof 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 polyisoprenyl synthase protein 9 .
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino 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 a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the absence of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence. Missed.
  • 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 in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with polyisoprenyl synthase protein 9, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind polyisoprenyl synthase protein 9.
  • Antagonist refers to a biological or immunological activity that can block or modulate polyisoprenyl synthase protein 9 when combined with polyisoprenyl synthase protein 9.
  • Molecule Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind polyisoprenyl synthase protein 9.
  • Regular refers to a change in the function of polyisoprenyl synthase protein 9, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties of polyisoprenyl synthase protein 9. , Functional or immune properties.
  • substantially pure ' means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify polyisoprenyl synthase proteins using standard protein purification techniques. 9. A substantially pure polyisoprenyl synthase protein 9 produces a single main band on a non-reducing polyacrylamide gel. The purity of the polyisoprenyl synthase protein 9 polypeptide can be analyzed by amino acid sequence .
  • Complementary refers to the natural binding of a nucleotide 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 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. 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 the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Mad Son 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). The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun He in (He in J., (1990) Methods in emzumo logy 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 the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of polyisoprenyl synthase protein 9.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component 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 polyisoprenyl synthase protein 9 means that polyisoprenyl synthase protein 9 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify polyisoprenyl synthase protein 9 using standard protein purification techniques. Basically pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the polyisoprenyl synthase protein 9 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a polyisoprenyl synthase protein 9, 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.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of polyisoprenyl synthase protein 9.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the polyisoprenyl synthase protein 9 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 ( ⁇ ) is one in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or a UV), a polypeptide sequence in which an additional amino acid sequence is fused to a mature polypeptide (such as Leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • Polynucleotides of the invention are found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 3022 bases in length and its open reading frame 2469-2726 encodes 85 amino acids.
  • This peptide A characteristic sequence having a characteristic sequence of a polyisoprenyl synthetase can be deduced that the polyisoprenyl synthase protein 9 has the structure and function represented by the characteristic sequence of the polyisoprenyl synthetase.
  • 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 the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or it may be a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fico ll, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. 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 polyisoprenyl synthase protein 9.
  • 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 polyisoprenyl synthase protein 9 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These technologies include but are not Limitations are: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleotide fragments with common structural characteristics.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) Isolating a double-stranded DNA sequence from genomic DNA: 2) Chemically synthesizing a DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDM of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (DDNA-DNA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of the level of the transcript of polyisoprenyl synthase protein 9; (4) Detecting protein products of gene expression through immunological techniques or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of the polyisoprenyl synthase protein 9 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones 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 polyisoprenyl synthase protein 9 coding sequence, and the present invention is produced by recombinant technology Methods of the polypeptide.
  • a polynucleotide sequence encoding the polyisoprenyl synthase protein 9 may 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: a ⁇ promoter-based expression vector (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a polyisoprenyl synthase protein 9 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. Ew York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pairs of SV40 enhancer at a late stage of the replication initiation point, polyoma enhancer and adenovirus enhancer at the late stage of 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 for eukaryotic cell culture. And green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture.
  • GFP green fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding the polyisoprenyl synthase protein 9 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • 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 in the exponential growth phase were harvested, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant polyisoprenyl synthase protein 9 (Scence, 1984; 224: 1431). Generally there are the following steps-.
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromat
  • Fig. 1 is a comparison diagram of amino acid sequence homology of 42 isoforms of polyisoprenyl synthetase protein 9 of the present invention from 2 to 43 and a characteristic sequence domain of polyisoprenyl synthetase.
  • the upper sequence is the polyisoprene synthase protein 9, and the lower sequence is the characteristic sequence domain of the polyisoprene synthase.
  • ⁇ "and”: "and” ⁇ indicate that the probability of different amino acids occurring at the same position between two sequences decreases in sequence.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated polyisoprenyl synthase protein 9.
  • 9KDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0041 a 03 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the results show that the full-length cDNA contained in the 0041a03 clone is 3022bp (as shown in Seq IDNO: 1), and has a 258bp open reading frame (0RF) from 2469bp to 2726bp, which encodes a new protein (such as SeQ ID NO: 2).
  • This clone pBS-0041a03 and the encoded protein was named polyisoprenyl synthase protein 9.
  • Example 2 Domain analysis of cDNA clones
  • the sequence of the polyisoprenyl synthase protein 9 of the present invention and the protein sequence encoded by the same are used in GCG
  • the profile scan program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] was used to perform domain analysis in databases such as Prote.
  • the polyisoprenyl synthase protein 9 of the present invention has homology with the characteristic sequence of the domain polyisoprenyl synthase at 2-43. The results of the homology are shown in FIG. 1, and the homology rate is 0.21. The score is 8.80; The threshold is 8.63.
  • Example 3 Cloning of a gene encoding polyisoprenyl synthase protein 9 by RT-PCR
  • CDNA was synthesized using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction.
  • PCR amplification was performed with the following primers:
  • Primerl 5-ATGTTGGGCTTTGACTAACATTAA-3 '(SEQ ID NO: 3)
  • Primer 2 5-ACAGAGTCTCGCTCAGTCACCCAG-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer 2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50 ⁇ l of KC1, 10 mmol / L Tris-CI, (pH8.5), 1.5 mmol / 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.
  • RT-PCR set ⁇ -act in as a positive control and template blank as a negative control.
  • 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 (pH 4.0), 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 synthesized using 20 g of RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation 32 ⁇ - DNA probe labeled with oc- 32 P dATP by random priming method. The DNA probe used was the PCR amplified polyisoprenyl synthase protein 9 coding region sequence (2469bp to 2726bp) shown in FIG. 1.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 ( pH7.4)-5 x SSC- 5 x Denhardt's solution and 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, the filter Wash in 1 x SSC_0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant polyisoprenyl synthase protein 9
  • Primer3 5'— CATGCTAGCATGGGAGAAATTATTTGCCAATCA-3 '(Seq ID No: 5)
  • Primer4 5-CCCGAGCTCTTATAGCTGGACAGTGGGCATAAA-3' (Seq ID No: 6)
  • the 5 ′ ends of these two primers contain Nhel and Sacl restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are respectively followed by Nhel and Sacl restriction sites corresponding to the selective endonucleases on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Enzyme site.
  • a PCR reaction was performed using the pBS-0041a03 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of P BS- 0041a03 plasmid, primers Primer-3 and Primer- 4 points, and another 1 J was 10pmol, Advantage polymerase Mix
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following polyisoprenyl synthase protein 9-specific peptides:
  • NH2-Met-Gly-Glu-Ile-Ile-Cys-Gln-Ser-Tyr-Val-Ser-Asp-Lys-Gly-Leu-C00H SEQ ID NO: 7
  • the 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. Immunochemistry, 1969; 6: 43. With 4mg of hemocyanin Peptide complex plus complete Freund's adjuvant was used to immunize rabbits. After 15 days, hemocyanin peptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Se P harose 4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to polyisoprenyl synthase protein 9.
  • Example 7 Use of a polynucleotide fragment of the present invention as a hybridization probe
  • 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 identified whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in pathological 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, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) 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.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 (probel), 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 or its complementary fragment of SEQ ID NO: 1:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are needed for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • probes 1 3 ⁇ IProbe (0.1 OD / 10 ⁇ 1), add 2 ⁇ IKinase buffer, 8-10 uCi ⁇ - 32 P- dATP + 2U Kinase, to make up to a final volume of 20 ⁇ 1.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared after the collection solutions of the first peak are combined.
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of large numbers 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 rapid, 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 the references DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And the literature 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 amplified by PCR respectively. After purification, the amplified product was adjusted to a concentration of about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method has been reported in the literature in various ways. The post-sampling processing steps of this embodiment are:
  • Total mRNA was extracted from normal liver and liver cancer in one step, and mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5- Amino- propargy 1-2 '-deoxyuri dine 5--tr iphate cou led to Cy3 fluorescent dye (purchased from Amersham Phamacia Biotech) was used to label the inRNA of normal liver tissue, and the fluorescent reagent Cy5dUTP (5-Amino-propargy 1-2' -deoxyur i dine 5'-tr iphate coupled to Cy5 fluorescent dye (purchased from Amersham Phamacia Biotech) was used to label liver cancer tissue mRNA, and the probe was prepared after purification.
  • Cy3dUTP 5- Amino- propargy 1-2 '-deoxyuri dine 5--tr iphate cou led to Cy3 fluorescent dye (purchased from Amersham Phamacia Biotech
  • the probes from the two types of tissues and the chips were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000.
  • Scanner purchased from General Scanning Company, USA
  • the scanned image was analyzed and processed with Imagene software (Biodiscovery Company, USA), and the Cy3 / Cy5 ratio of each point was calculated.
  • the points with the ratio less than 0.5 and greater than 2 were considered Genes with differential expression.
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Isoprenoids are a family of various lipophilic molecules based on a repeating 5-carbon structure.
  • farnesyl diphosphate is the isoprenoid compound most relevant to protein isoprenylation.
  • Many specific proteins are modified by isoprene groups after translation.
  • Most isoprenylated proteins are modified by binding to 15-carbon farnesyl or 20-carbon geranyl groups, which are bound by thioether bonds. On cysteine residues.
  • Isoprenylated proteins exist in different cellular regions, including nuclear cytosol and membrane-bound organelles. Members of this family are involved in isoprenylation of various cellular proteins, including cancer-related proteins, such as the ras protein.
  • Polyisoprenyl synthase-specific conserved sequences are necessary to form its active motif. It can be seen that the abnormal expression of the specific polyisoprenyl synthetase motif will cause the present invention containing more motif. Peptide dysfunction, which leads to the abnormality of the isoprene function of various cellular proteins, further abnormalities of related proteins such as ras protein, abnormal synthesis of cholesterol, long alcohol, ubiquinone or coenzyme Q, and produce related Diseases such as tumors, embryonic developmental disorders, material metabolism disorders, cholesterol metabolism disorders, growth and development disorders, etc.
  • the abnormal expression of the polyisoprenyl synthase protein 9 of the present invention will produce various diseases, especially tumors, embryonic developmental disorders, material metabolic disorders, cholesterol metabolic disorders, growth and development disorders, These diseases include, but are not limited to:
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, Colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymic tumor, nasal cavity and sinus tumor, nose Pharyngeal cancer, Laryngeal cancer, Tracheal tumor, Fibroma, Fibrosarcoma, Lipoma, Liposarcoma, Leiomyoma
  • Embryonic disorders congenital abortion, cleft palate, limb absentness, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney disease, double ureter, crypto, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias , Bisexual deformity, Atrial septal defect, Ventricular septal defect, Pulmonary stenosis, Arterial duct occlusion, Neural tube defect, Congenital hydrocephalus, Iris defect, Congenital cataract, Congenital glaucoma or cataract, Congenital deafness
  • Material metabolism disorders isovalerate, propionate, methylmalonic aciduria, combined carboxylase deficiency, glutarate type I, phenylketonuria, albinism,
  • Cholesterol metabolism disorders fatty liver, steatosis cardiomyopathy, steatosis nephropathy, coronary atherosclerotic heart disease, hypertension
  • the polypeptide of the present invention can be directly used in the treatment of diseases, for example, it can treat various diseases, especially tumors, embryonic developmental disorders, material metabolism disorders, cholesterol metabolism disorders. , Growth and developmental disorders, certain hereditary, hematological and immune system diseases
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) polyisoprenyl synthase protein 9.
  • Agonists increase biological functions such as polyisoprenyl synthase protein 9 to stimulate cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing polyisoprenyl synthase protein 9 can be cultured with labeled polyisoprenyl synthase protein 9 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of polyisoprene synthase protein 9 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of polyisoprenyl synthase protein 9 can bind to polyisoprenyl synthase protein 9 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to The polypeptide cannot perform biological functions.
  • polyisoprenyl synthase protein 9 When screening compounds as antagonists, polyisoprenyl synthase protein 9 can be added to the bioanalytical assay, and the interaction between polyisoprenyl synthase protein 9 and its receptor can be determined by determining the compound Influence to determine if a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to the polyisoprenyl synthetase protein can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 9 molecules of polyisoprenyl synthase protein 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 directed against the polyisoprenyl synthase protein 9 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting polyisoprenyl synthase protein 9 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc.
  • Techniques for preparing monoclonal antibodies to polyisoprenyl synthase protein 9 include, but are not limited to, hybridoma technology (Kohler and Mil te in. Nature, 1975, 256: 495-497), triple tumor technology, Human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Inlay antibodies combining human constant regions and non-human variable regions can be produced using existing technologies (Morris on e t al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against polyisoprenyl synthase protein 9.
  • Antibodies against polyisoprene synthase protein 9 can be used in immunohistochemical techniques to detect polyisoprene synthase protein 9 in biopsy specimens.
  • Monoclonal antibodies that bind to polyisoprenyl synthase protein 9 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • polyisoprenyl synthase protein 9 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill the polyisoprenyl synthase protein. 9 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to polyisoprenyl synthase protein 9.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of polyisoprenyl synthase protein 9.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of polyisoprenyl synthase protein 9 levels. These tests are well known in the art and include F I SH assays and radioimmunoassays. The level of polyisoprenyl synthase protein 9 detected in the test can be used to explain the importance of polyisoprenyl synthase protein 9 in various diseases and to diagnose polyisoprenyl synthesis Diseases where Enzyme Protein 9 Works
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding the polyisoprenyl synthase protein 9 can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat abnormalities in cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of polyisoprenyl synthase protein 9. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated polyisoprene synthase protein 9 to inhibit endogenous polyisoprene synthase protein 9 activity.
  • a variant polyisoprenyl synthase protein 9 may be a shortened polyisoprenyl synthase protein 9 that lacks a signaling domain, although it can bind to downstream substrates, but lacks Signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of polyisoprenyl synthase protein 9.
  • 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 a polyisoprenyl synthase protein 9 into a cell.
  • a polynucleotide encoding a polyisoprenyl synthase protein 9 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • RNA oligonucleotides including antisense RNA and DM
  • ribozymes are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the A polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding the polyisoprenyl synthase protein 9 can be used for the diagnosis of diseases related to the polyisoprenyl synthase protein 9.
  • Polynucleotide encoding polyisoprenyl synthase protein 9 can be used to detect the expression of polyisoprenyl synthase protein 9 or abnormal expression of polyisoprenyl synthase protein 9 in disease states .
  • the DNA sequence encoding polyisoprenyl synthase protein 9 can be used to hybridize biopsy specimens to determine the expression of polyisoprenyl synthase protein 9.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microarr ay) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis of genes in tissues and genes. diagnosis.
  • a microarray Microarr ay
  • a DNA chip also known as a "gene chip”
  • Polyisoprene synthase protein 9 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the polyisoprene synthase protein 9 transcription product.
  • Polyisoprene synthase protein 9 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type polyisoprene synthase protein 9 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, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification.
  • 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 few chromosome markers based on actual sequence data (repeat polymorphisms) 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 based on cDNA, and the sequence can be mapped to 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 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, 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.
  • 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 which 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.
  • containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • instructional instructions given by government regulatory agencies that manufacture, use, or sell pharmaceuticals or biological products, which instructions reflect production, use Or a government agency that sells it allows it to be administered to humans.
  • 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.
  • Polyisoprenyl synthase protein 9 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of polyisoprenyl synthase protein 9 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un nouveau polypeptide, une protéine 9 polyisoprène synthétase, 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 protéine 9 polyisoprène synthétase.
PCT/CN2000/000619 1999-12-24 2000-12-18 Nouveau polypeptide, proteine 9 polyisoprene synthetase, et polynucleotide codant pour ce polypeptide WO2001047972A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU19883/01A AU1988301A (en) 1999-12-24 2000-12-18 A novel polypeptide-poly isoprenyl synthetase protein 9 and polynucleotide encoding said polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 99125760 CN1301731A (zh) 1999-12-24 1999-12-24 一种新的多肽——多异戊二烯基合成酶蛋白9和编码这种多肽的多核苷酸
CN99125760.X 1999-12-24

Publications (1)

Publication Number Publication Date
WO2001047972A1 true WO2001047972A1 (fr) 2001-07-05

Family

ID=5284170

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2000/000619 WO2001047972A1 (fr) 1999-12-24 2000-12-18 Nouveau polypeptide, proteine 9 polyisoprene synthetase, et polynucleotide codant pour ce polypeptide

Country Status (3)

Country Link
CN (1) CN1301731A (fr)
AU (1) AU1988301A (fr)
WO (1) WO2001047972A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1098408A (zh) * 1993-04-02 1995-02-08 布里斯托尔-迈尔斯斯奎布公司 法呢基蛋白转移酶的杂环抑制剂
CN1124142A (zh) * 1994-06-29 1996-06-12 科学研究与运用咨询公司 抑制异戊二烯基蛋白质转移酶表达的寡核苷酸
CN1151156A (zh) * 1994-03-15 1997-06-04 卫材株式会社 异戊二烯基转移酶抑制剂

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1098408A (zh) * 1993-04-02 1995-02-08 布里斯托尔-迈尔斯斯奎布公司 法呢基蛋白转移酶的杂环抑制剂
CN1151156A (zh) * 1994-03-15 1997-06-04 卫材株式会社 异戊二烯基转移酶抑制剂
CN1124142A (zh) * 1994-06-29 1996-06-12 科学研究与运用咨询公司 抑制异戊二烯基蛋白质转移酶表达的寡核苷酸

Also Published As

Publication number Publication date
AU1988301A (en) 2001-07-09
CN1301731A (zh) 2001-07-04

Similar Documents

Publication Publication Date Title
WO2001068694A1 (fr) Nouveau polypeptide, proteine humaine d'hexokinase 10, et polynucleotide codant pour ce polypeptide
WO2001047972A1 (fr) Nouveau polypeptide, proteine 9 polyisoprene synthetase, et polynucleotide codant pour ce polypeptide
WO2001048168A1 (fr) Nouveau polypeptide, gamma1-pyroline-5-hydroxy reductase 14, et polynucleotide codant pour ce polypeptide
WO2001055404A1 (fr) Nouveau polypeptide, alcool deshydrogenase humaine 39, et polynucleotide codant pour ce polypeptide
WO2002026798A1 (fr) Nouveau polypeptide, acide phosphatidique phosphatase 29.81, et polynucleotide codant ce polypeptide
WO2001055417A1 (fr) Nouveau polypeptide, proteine a f-box 65, et polynucleotide codant pour ce polypeptide
WO2001048222A1 (fr) Nouveau polypeptide, unite de repetition hexapeptidique contenant une transferase 11, et polynucleotide codant pour ce polypeptide
WO2001046250A1 (fr) Nouveau polypeptide, triose phosphate isomerase 9, et polynucleotide codant pour ce polypeptide
WO2001090376A1 (fr) Nouveau polypeptide, triose phosphate isomerase humaine 11, et polynucleotide codant ce polypeptide
WO2002020606A1 (fr) Nouveau polypeptide, la proteine humaine 38.39 liee aux defauts d'audition, et polynucleotide codant pour elle
WO2001048219A1 (fr) Nouveau polypeptide, proteine de transport phosphorylee 10, et polynucleotide codant pour ce polypeptide
WO2001055375A1 (fr) Nouveau polypeptide, agent de reduction humain et une proteine reagissant a la tunicamycine 40, et polynucleotide codant pour ce polypeptide
WO2001055379A1 (fr) Nouveau polypeptide, proteine humaine 48 d'inhibition de la croissance cellulaire, et polynucleotide codant pour ce polypeptide
WO2001075062A2 (fr) Nouveau polypeptide, hydrogenase humaine 10, et polynucleotide codant pour ce polypeptide
WO2001073046A1 (fr) Nouveau polypeptide, quinine reductase humaine 13, et polynucleotide codant pour ce polypeptide
WO2001048001A1 (fr) Nouveau polypeptide, quinone-proteine reductase 7, et polynucleotide codant pour ce polypeptide
WO2001048218A1 (fr) Nouveau polypeptide, proteine muts 13, et polynucleotide codant pour ce polypeptide
WO2001048012A1 (fr) Nouveau polypeptide, sous-unite i-11 de nadh-deshydrogenase, et polynucleotide codant pour ce polypeptide
WO2001047984A1 (fr) Nouveau polypeptide, proteine 10 de la famille des amidases, et polynucleotide codant pour ce polypeptide
WO2001048156A1 (fr) Nouveau polypeptide, hydrogenase 9, et polynucleotide codant pour ce polypeptide
WO2001048198A1 (fr) Nouveau polypeptide, proteine ribosomale s4 8, et polynucleotide codant pour ce polypeptide
WO2001047982A1 (fr) Nouveau polypeptide, support mitochondrial 30, et polynucleotide codant pour ce polypeptide
WO2001055382A1 (fr) Nouveau polypeptide, acp humaine 51, et polynucleotide codant pour ce polypeptide
WO2001047998A1 (fr) Nouveau polypeptide, proteine activant la ras gtpase 12, et polynucleotide codant pour ce polypeptide
WO2001079435A2 (fr) Nouveau polypeptide, sous-unite humaine 14 de flavoproteine, et polynucleotide codant pour ce polypeptide

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP