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WO2001038364A1 - Nouvelle proteine 68 regulatrice de la proteine phosphatase 1 polypeptidique-humaine, et polynucleotide codant le polypeptide - Google Patents

Nouvelle proteine 68 regulatrice de la proteine phosphatase 1 polypeptidique-humaine, et polynucleotide codant le polypeptide Download PDF

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Publication number
WO2001038364A1
WO2001038364A1 PCT/CN2000/000456 CN0000456W WO0138364A1 WO 2001038364 A1 WO2001038364 A1 WO 2001038364A1 CN 0000456 W CN0000456 W CN 0000456W WO 0138364 A1 WO0138364 A1 WO 0138364A1
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Prior art keywords
polypeptide
polynucleotide
human protein
protein phosphatase
sequence
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bioroad Gene Development Ltd
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Shanghai Bioroad Gene Development Ltd
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Priority to AU15102/01A priority Critical patent/AU1510201A/en
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    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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, a human protein phosphatase 1 regulatory protein 68, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides. Background technique
  • Protein phosphatase 1 is a gene family that has important functions in the brain. Its combination with many different regulatory proteins (regulatory subunits) determines that it can function in different subcellular environments, and has different enzyme effects. Thing. (J Comp Neurol 1999 Oct 25; 413 (3): 373-84)
  • PP-1 has a disulfide redox site in addition to its phosphorylation active site. Therefore, it is speculated that PP-1 also has the function of an oxidoreductase, but this has not been confirmed. (FASEB J 1999 Oct; 13 (13): 1866-74)
  • Phosphoproteinases can be complexed with calmodulin to regulate the fusion of bilayers of cell membranes and play an important role in exocytosis. (Science 1999 Aug 13; 285 (5430): 1084-7)
  • PP-1 can interact with a variety of regulatory proteins, of which PP-1 nuclear binding factor (NIPP1) PP-1 is a more common binding protein.
  • NIPP1 nuclear binding factor PP-1 nuclear binding factor
  • PP-1G Hepatic glucose-related regulatory protein of protein phosphatase in insulin-stimulated hepatic glucose synthesis, glucose breakdown in anaerobic conditions in skeletal muscle, etc. Play a regulatory role in the process.
  • NIPP1 has an endonuclease domain and an RNA binding domain, and this The positioning of these functional domains has been confirmed.
  • P t 1 RNA binding domain
  • the polypeptide of the present invention is inferred and identified as human protein phosphatase 1 regulatory protein 68 (HPP1 RP68), and its homologous protein is the human protein phosphatase 1 regulatory subunit 7 al pha2, and the protein number is AF 067 1 36.
  • the human protein phosphatase 1 regulatory protein 68 protein plays an important role in important functions of the body, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art to identify more human protein phosphatases involved in these processes.
  • 1 Regulatory protein 68 protein especially the amino acid sequence of this protein is identified.
  • the isolation of the new human protein phosphatase 1 regulatory protein 68 protein-coding 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 developing diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is important. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human protein phosphatase 1 regulatory protein 68.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human protein phosphatase 1 regulatory protein 68.
  • Another object of the present invention is to provide a method for producing human protein phosphatase 1 regulatory protein 68.
  • Another object of the present invention is to provide an antibody against the polypeptide-human protein phosphatase 1 regulating protein 68 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide-human protein phosphatase 1 regulating protein 68 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of human protein phosphatase 1 regulatory protein 68.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 26 to 1870 in SEQ ID NO: 1; and (b) a sequence having 1-1881 in SEQ ID NO: 1 Sequence of bits.
  • 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; 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 human protein phosphatase 1 regulating protein 68 protein activity, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human protein phosphatase 1 regulatory protein 68 protein in vitro, comprising detecting mutations in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or Detection of 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 protein phosphatase 1 regulatory protein 68.
  • 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
  • polypeptide or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants may have "conservative" changes in which the substituted amino acid 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.
  • 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 a human protein phosphatase 1 regulatory protein 6 8, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind a human protein phosphatase 1 regulatory protein 6 8.
  • Antagonist refers to a molecule that, when combined with human protein phosphatase 1 regulatory protein 68, can block or regulate the biological or immunological activity of human protein phosphatase 1 regulatory protein 68.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human protein phosphatase 1 regulatory protein 68.
  • Regular refers to a change in the function of human protein phosphatase 1 regulatory protein 68, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties, functions, or immunity of human protein phosphatase 1 regulatory protein 68 Change of nature.
  • 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 protein phosphatase 1 regulatory protein 68 using standard protein purification techniques.
  • the substantially pure human protein phosphatase 1 regulatory protein 68 can generate a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human protein phosphatase 1 regulatory protein 68 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • 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 A partially complementary sequence that at least partially inhibits the hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Northern 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 the same or similar in a 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 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, D. G. and P.M. 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 number of matching residues between sequence A and sequence B
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or by methods known in the art such as Jotun Hein. J., (1990) Methods in emzumology 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 a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of human protein phosphatase 1 regulatory protein 68.
  • 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 protein phosphatase 1 regulatory protein 68 refers to human protein phosphotransferase 1 regulatory protein 68 that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify human protein phosphatase 1 regulatory protein 68 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human protein phosphatase 1 regulatory protein 68 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-human protein phosphatase 1 regulatory protein 68, 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 human protein phosphatase 1 regulatory protein 68.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human protein phosphatase 1 regulatory protein 68 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III)
  • Such a type in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • UV a type in which the additional amino acid sequence is fused into the mature polypeptide and formed by the polypeptide sequence ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes 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 1881 bases in length and its open reading frame (26-1870) encodes 614 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 34% homology with the human protein phosphatase 1 regulatory subunit 7a lha 2. It can be concluded that the human protein phosphatase 1 regulatory protein 68 has human protein phosphatase 1 Regulatory subunits 7a lha2 have similar structures and functions.
  • 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 but having a sequence different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is 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 invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • "strict conditions” means: (1) in the lower Hybridization and elution at ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) Add denaturant during hybridization, such as 50 ° /.
  • hybridized 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 protein phosphatase 1 regulatory protein 68.
  • 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 protein phosphatase 1 regulatory protein 68 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 DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate 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.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determining the level of the human protein phosphatase 1 regulatory protein 68 transcript; (4) Detecting the protein product of gene expression by immunological technology 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. It is preferably within 1000 nucleotides.
  • the probe used here is generally 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. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human protein phosphatase 1 regulating protein 68 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 of applying a PCR technique 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, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the human protein phosphatase 1 regulatory protein 68 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding the human protein phosphatase 1 regulatory protein 68 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human protein phosphatase 1 regulatory protein 68 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Labora tory Manua l, cold Spr ing Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the l ac or trp promoter of E.
  • 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 for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a human protein phosphatase 1 regulatory protein 68 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • 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 absorbing MA can be harvested after the exponential growth phase and treated with the ( 12 method, the steps used are well known in the art.
  • MgC 12 If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Body packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human protein phosphatase 1 regulatory protein 68 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • the polynucleotide (or variant) encoding the human protein phosphatase 1 regulatory protein 68 of the present invention is used.
  • Body or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;
  • the medium used in the culture may be selected from various conventional mediums depending on the host cells used. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be 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 the amino acid sequence homology of the human protein phosphatase 1 regulatory protein 68 and the human protein phosphatase 1 regulatory subunit 7a lha2 of the present invention.
  • the upper sequence is the human protein phosphatase 1 regulatory protein 68
  • the lower sequence is the human protein phosphatase 1 regulatory subunit 7a lha2.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human protein phosphatase 1 regulatory protein 68.
  • 68kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Example 1 Cloning of human protein phosphatase 1 regulatory protein 68
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Quik mRNA I so lat ion Ki t (Qiegene product) Isolate poly (A) mRM 2ug poly (A) mRNA from total RNA and reverse transcription to form cDNA.
  • the 00 ⁇ fragment was directionally inserted into a multicloning site of a pBSK (+) vector (Clontech) using a Smart cDNA cloning kit (purchased from Clontech) to transform DH5 ⁇ , and the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • cDNA sequences at the 5 'and 3' ends of all clones Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the CDM sequence of one of the clones 0157f08 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the sequence of the human protein phosphate ⁇ 1 regulatory protein 68 of the present invention and the protein sequence encoded by the same were performed using the Blast program (Basiclocal Alignment search tool) [Altschul, SF et a 1. J. Mol. Biol. 1990; 215: 403 -10], perform homology search in databases such as Genbank Swissport.
  • the gene with the highest homology to the human protein phosphatase 1 regulatory protein 68 of the present invention is a known human protein phosphatase 1 regulatory subunit 7alha2, and the accession number of the encoded protein in Genbank is AF067136.
  • the results of protein homology are shown in Figure 1. The two are highly homologous, their identity is 26%; their similarity is 58%.
  • Example 3 Cloning of the gene encoding human protein phosphatase 1 regulatory protein 68 by RT-PCR
  • CDNA was synthesized using fetal brain 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 '-GAGAGAACCTGGACTGTCTCCTA-3' (SEQ ID NO: 3)
  • Priraer2 5-CTAGCTTTTCATTATAAAAGTC-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.
  • Amplification conditions 50 ol / L KC1, 10 mmol / L Tris-Cl, (pH8.5), 1.5mraol / L MgCl 2 200 ⁇ mol / L dNTP, lOpmol primers in a 50 ⁇ 1 reaction volume, 1U of Taq DNA polymerase (C 1 on te ch).
  • the reaction was performed on a PE 9600 DNA thermal cycler (Pe rki nElmer) 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.
  • Amplification products were purified using QIAGEN kits and TA The cloning kit was ligated to a pCR vector (Invitrogen). DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1881 bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human protein phosphatase 1 regulatory protein 68 gene expression:
  • RNA extraction in one step involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) are added. ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA - (N - morpholino) propanesulfonic acid (pH 7.0) - was electrophoresed on a 1.2% agarose gel ImM EDTA- 2.2M formaldehyde - 5mM sodium acetate. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the PCR-encoded human protein phosphatase 1 regulatory protein 68 coding region sequence (26bp to 1870bp) 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-25mM KH 2 P0 4 (pH7.4)-5 x SSC- 5 x 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 for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human protein phosphatase 1 regulatory protein 68
  • Primer3 5,-CCCCATATGATGATTCCCGGGAAATATCGCTC- 3 '(Seq ID No: 5)
  • Primer4 5,-CATGGATCCTTATAAAAGTCCTCTTGTAGTAT- 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, respectively.
  • the Ndel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • the PCR reaction was performed using the pBS-0157f08 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS- 0157f 08 plasmid, Primer-3 and Primer-4 (J is lOpmol, Advantage polymerase Mix (Clontech)) 1 ⁇ 1. Cycle parameters : 25 ° C at 94 ° C for 20s, 60 ° C for 30 min at 68 ° C for 2 min. Double digestion of the amplified product and plasmid pET-28 (+) with Ndel and BamHI, respectively, to recover large fragments and use T4 ligase ligation.
  • the ligated product was transformed into Escherichia coli DH5cx by the calcium chloride method, cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), and positive clones were selected by colony PCR method and sequenced Select positive clones with the correct sequence (pET-0157f08). Transform the recombinant plasmid into the large intestine with the calcium chloride method.
  • Bacillus BL21 (DE3) plySs (product of Novagen).
  • the host strain BL21 (pET-0157f08) was at 37. C.
  • 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. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. 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.
  • 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 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 blotting Trace method, Nor thern blot method, and copy method, etc., all are used to fix the polynucleotide sample to be tested on the filter membrane and then hybridize using basically the same steps.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • 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 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • 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 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 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. Schema, M., Cha i, A., Sha lom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DM, including the present invention
  • Polynucleotide They were respectively amplified by (as described in the examples) PCR, and the amplified product was purified to adjust its concentration to about 500ng / ul, and spotted on a glass with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance between the points on the medium is 280 ⁇ m.
  • the spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DM on the glass slides to prepare chips.
  • the specific method steps have been variously reported in the literature, and the specific method steps have been variously reported in the literature.
  • the spot post-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 coupled to Cy3 fluorescent dye (purchased from Amersham Phamacia Biotech) was used to label mRNA of normal liver tissue.
  • Cy5 fluorescent dye purchased from Amersham Phamacia Biotech
  • Specific steps refer to and methods Schena, M., Shalon, D., Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614- 10619. Schena, M., Shalon, Dari., Davis, RW (1995) Science. 270. (20): 467-480.
  • Probes from the above two tissues and 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.
  • 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.
  • human protein phosphatase 1 regulatory protein 68 The function of the polypeptide (human protein phosphatase 1 regulatory protein 68) of the present invention remains to be further studied, but in general, human protein phosphatase 1 regulatory protein 68 can be used to diagnose and treat many diseases. For example, malignant tumors, endocrine system diseases, neurological diseases, immune diseases, human acquired immune deficiency syndrome (A IDS) and so on.
  • diseases For example, malignant tumors, endocrine system diseases, neurological diseases, immune diseases, human acquired immune deficiency syndrome (A IDS) and so on.
  • a IDS human acquired immune deficiency syndrome
  • the polypeptide of the present invention can be used to treat human malignant tumors, including epithelial tissues (such as basal epithelium, squamous epithelium, mucus cells, etc.), (such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue, blood vessels and lymphatic endothelial tissue, etc.
  • epithelial tissues such as basal epithelium, squamous epithelium, mucus cells, etc.
  • fibrous tissue such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue, blood vessels and lymphatic endothelial tissue, etc.
  • Etc. hematopoietic tissue (such as B cells, T cells, histiocytes, etc.), tumors of central nervous tissue, peripheral nerve tissue, endocrine tissue, gonadal tissue, special tissue (such as dental tissue, etc.), such as gastric cancer, Liver cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, esophageal cancer, etc.
  • the polypeptide of the present invention is an immunomodulator and has an immune promoting or immunosuppressing effect.
  • the polypeptide of the present invention can be used for the treatment of diseases including non-reactivity of immune response, or abnormal immune response, or ineffective host defense.
  • the polypeptides and antibodies of the present invention also have effects on damage, defects or disorders of immune tissues, especially for hematopoietic diseases (such as malignant anemia), skin diseases (such as psoriasis), and autoimmune diseases (such as rheumatoid arthritis). ), Radiation diseases and the production and regulation of immune lymphocytes are extremely closely related.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human protein phosphatase 1 regulatory protein 68.
  • Agonists enhance human protein phosphatase 1 regulatory protein 68 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human protein phosphatase 1 regulatory protein 68 can be cultured with labeled human protein phosphatase 1 regulatory protein 68 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human protein phosphatase 1 regulatory protein 68 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human protein phosphatase 1 regulatory protein 68 can bind to human protein phosphatase 1 regulatory protein 68 and eliminate its function, or inhibit the production of the polypeptide, or with the activity of the polypeptide Site binding prevents the polypeptide from performing its biological function.
  • human protein phosphatase 1 regulatory protein 68 When screening compounds as antagonists, human protein phosphatase 1 regulatory protein 68 can be added to bioanalytical assays, and compounds can be identified by measuring the effect of the compound on the interaction between human protein phosphatase 1 regulatory protein 68 and its receptor Whether it is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human protein phosphatase 1 regulatory protein 68 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In the screening, generally, human protein phosphatase 1 regulatory protein 68 molecules should 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 human protein phosphatase 1 regulatory protein 68 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 human protein phosphatase 1 regulatory protein 68 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 human protein phosphatase 1 regulatory protein 68 include, but are not limited to, hybridoma technology (Kohler and Mil te in. Nature, 1975, 256: 495-497), triple tumor technology, human beta -Cell hybridoma technology, EBV-hybridoma technology, etc.
  • An inlay antibody combining a human constant region and a non-human-derived variable region can be produced using existing technologies (Morr i son 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 human protein phosphatase 1 regulatory protein 68.
  • Antibodies against human protein phosphatase 1 regulatory protein 68 can be used in immunohistochemical techniques to detect human protein phosphatase 1 regulatory protein 68 in biopsy specimens.
  • Monoclonal antibodies that bind to human protein phosphatase 1 regulatory protein 68 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 protein phosphatase 1 regulatory protein 68 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 human protein phosphatase 1 regulatory protein 68 positive Cell.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human protein phosphatase 1 regulatory protein 68.
  • Administration of appropriate doses of antibodies can stimulate or block the production of human protein phosphatase 1 regulatory protein 68 or Active.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human protein phosphatase 1 regulatory protein 68 levels. These tests are well known in the art and include F I SH assays and radioimmunoassays. The level of human protein phosphatase 1 regulatory protein 68 detected in the test can be used to explain the importance of human protein phosphatase 1 regulatory protein 68 in various diseases and to diagnose the role of human protein phosphatase 1 regulatory protein 68. disease.
  • 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 human protein phosphatase 1 regulatory protein 68 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 protein phosphatase 1 regulatory protein 68.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human protein phosphatase 1 regulatory protein 68 to inhibit endogenous human protein phosphatase 1 regulatory protein 68 activity.
  • a mutated human protein phosphatase 1 regulatory protein 68 may be a shortened human protein phosphatase 1 regulatory protein 68, which lacks a signaling domain.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human protein phosphatase 1 regulatory protein 68.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer the polynucleotide encoding human protein phosphatase 1 regulatory protein 68 into cells.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding a human protein phosphatase 1 regulatory protein 68 can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human protein phosphatase 1 regulatory protein 68 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 protein phosphatase 1 regulatory protein 68 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by 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 RNA molecules can be obtained by in vitro or in vivo transcription of DM sequences encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. To increase the stability of nucleic acid molecules, they can be modified in a variety of ways. For example, if the sequence length on both sides is increased, the linkage between ribonucleosides should use phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human protein phosphatase 1 regulatory protein 68 can be used for the diagnosis of diseases related to human protein phosphatase 1 regulatory protein 68.
  • the polynucleotide encoding human protein phosphatase 1 regulatory protein 68 can be used to detect the expression of human protein phosphatase 1 regulatory protein 68 or the abnormal expression of human protein phosphatase 1 regulatory protein 68 in disease states.
  • the DNA sequence encoding human protein phosphatase 1 regulatory protein 68 can be used to hybridize biopsy specimens to determine the expression of human protein phosphatase 1 regulatory protein 68.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a micro array or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human protein phosphatase 1 regulatory protein 68 specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect human protein phosphatase 1 regulatory protein 68 transcription products.
  • Human protein phosphatase 1 regulatory protein 68 gene can also be used to diagnose human protein phosphatase 1 regulatory protein 68-related diseases.
  • Human protein phosphatase 1 regulatory protein 68 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human protein phosphatase 1 regulatory protein 68 DM sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern imprinting method and Western blotting method 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.
  • the PCR primers (preferably 15-35bp) are prepared according to cDM, 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 by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct a chromosome-specific c-shiphouse.
  • Fluorescent in situ hybridization (FISH) 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.
  • 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 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 protein phosphatase 1 regulatory protein 68 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human protein phosphoprotein 1 regulatory protein 68 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 porte sur une nouvelle protéine 68 régulatrice de la protéine phosphatase polypeptidique humaine et sur le polynucléotide codant le polypeptide, ainsi que sur un procédé de production du polypeptide par des méthodes de recombinaison. L'invention porte également sur un procédé d'application du polypeptide dans le traitement de diverses pathologies telles que le cancer, l'hémopathie, l'infection par le VIH, les maladies immunes et les inflammations. L'antagoniste du polypeptide et son utilisation thérapeutique sont égalemtn décrits. L'invention porte en outre sur l'utilisation du polynucléotide codant la protéine (68) régulatrice de la protéine phosphatase 1 humaine.
PCT/CN2000/000456 1999-11-22 2000-11-20 Nouvelle proteine 68 regulatrice de la proteine phosphatase 1 polypeptidique-humaine, et polynucleotide codant le polypeptide Ceased WO2001038364A1 (fr)

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AU15102/01A AU1510201A (en) 1999-11-22 2000-11-20 A novel polypeptide - human protein phosphatase 1 regulatory protein 68 and the polynucleotide encoding said polypeptide

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CN99124054A CN1297047A (zh) 1999-11-22 1999-11-22 一种新的多肽——人蛋白磷酸酶1调控蛋白68和编码这种多肽的多核苷酸
CN99124054.5 1999-11-22

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EGLOFF M.P. ET AL.: "Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1", EMBO J., vol. 16, no. 8, 15 April 1997 (1997-04-15), pages 1876 - 1887 *

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