WO2002040520A1 - Nouveau polypeptide, phosphatase humaine riche en striatum 14.85, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, phosphatase humaine riche en striatum 14.85, et polynucleotide codant ce polypeptide Download PDF

Info

Publication number
WO2002040520A1
WO2002040520A1 PCT/CN2001/001048 CN0101048W WO0240520A1 WO 2002040520 A1 WO2002040520 A1 WO 2002040520A1 CN 0101048 W CN0101048 W CN 0101048W WO 0240520 A1 WO0240520 A1 WO 0240520A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
phosphatase
human striatum
enriched
Prior art date
Application number
PCT/CN2001/001048
Other languages
English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
Original Assignee
Shanghai Biowindow Gene Development Inc.
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 Shanghai Biowindow Gene Development Inc. filed Critical Shanghai Biowindow Gene Development Inc.
Priority to AU2002214905A priority Critical patent/AU2002214905A1/en
Publication of WO2002040520A1 publication Critical patent/WO2002040520A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • 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 ⁇ ⁇ striatum-enriched phosphatase 14. 85, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides.
  • the dephosphorylation process is a molecular switch that regulates vital life activities such as cell division, differentiation, and signaling in the body. Phosphorylation is controlled by a kinase, which transfers high-energy phosphate from adenosine triphosphate to a special protein; while phosphatase removes high-energy phosphate from the protein.
  • protein kinases can activate metabolic enzymes, regulatory factors, receptors, cytoskeletal proteins, ion channels, and transcription factors.
  • Protein tyrosine phosphatase can terminate the signaling pathway activated by tyrosine protein kinase, clear the previous stimulus information, and enable this pathway to receive the next signal. It is now known that eight protein tyrosine phosphatases and tyrosine protein kinases jointly regulate cell activity, proliferation, and differentiation.
  • the striatum-enriched phosphatase (str iatum enr iched phospha ta se, STEP) was first discovered in the rat brain striatum cDM library. This is a nervous system-specific protein. 3kb and 4.4kb mRNAs were found in different parts of the brain. The 3kb raRNA is mainly distributed in the striatum, so STEP is named, while the 4.4kb mRNA is mainly distributed in the cerebral cortex, and it is rarely distributed in the striatum. These two mRNAs are two spliced forms of the same gene [Proc Nat l Acad Sc i U S A 1991 Aug 15; 88 (16): 7242-6].
  • This gene has also been found in humans.
  • This is a protein tyrosine phosphatase.
  • the enzyme has a single phosphatase catalytic region at the C-terminus and contains an amino acid domain [I / V] HCXAGXXR [S / T] GX [F / Y] that is highly conserved among all tyrosine phosphatases.
  • Two N-terminal proline-rich regions may contain SH3 binding domains [Genomics 28: 442-449, 1995].
  • bladder cancer construct cell EJ bladder cancer side, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunum adenocarcinoma, cardia cancer, the expression profile of the polypeptide of the present invention and human striatum
  • the expression profiles of enriched phosphatases are very similar, so the functions of the two may be similar.
  • the present invention is named human striatum-enriched phosphatase 14.85.
  • the human striatum is enriched in phosphatase 14.85 protein, which plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so identification in the art has been required More human striatum involved in these processes are enriched for the phosphatase 14.85 protein, especially the amino acid sequence of this protein is identified. Newcomer striatum-enriched phosphatase 14. The isolation of the 85 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 the development of diagnostic and / or therapeutic drugs for diseases, so isolation of its coding DNA is important. Object of the 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 striatum-enriched phosphatase 14.85.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human striatum-enriched phosphatase 14.85.
  • Another object of the present invention is to provide a method for producing human striatum-enriched phosphatase 14.85.
  • Another object of the present invention is to provide a human striatum-enriched phosphatase directed against the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human striatum-enriched phosphatase 14.85.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human striatum enriched phosphatase 14.85. Summary of invention
  • 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 291-698 in SEQ ID NO: 1; and (b) having a sequence of 1-1849 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 striatum-enriched phosphatase 14.85 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 in vitro detection of a disease or susceptibility to disease associated with abnormal expression of human striatum-enriched phosphatase 14.85 protein, comprising detecting the polypeptide in a biological sample or its coding polynucleotide sequence. Mutates, or detects 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 preparation of a polypeptide and / or polynucleotide of the present invention for the treatment of cancer, developmental disease or immune disease or other drugs caused by abnormal expression of human striatum-enriched phosphatase 14.85. use.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human striatum-enriched phosphatase 14.85 and human striatum-enriched phosphatase of the present invention.
  • the upper figure is a graph of the human striatum-enriched phosphatase 14.85, and the lower figure is the human striatum-enriched phosphatase expression.
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of isolated human striatum-enriched phosphatase 14.85. 14. 85kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RM, 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 protein related to the protein molecule. Amino acid.
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human striatum-enriched phosphatase 14.85, causes a change in the protein to regulate the activity of the protein.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecule that binds to the human striatum-enriched phosphatase 14.85.
  • Antagonist refers to a biological activity or immunity that can block or regulate human striatum-enriched phosphatase 14.85 when combined with human striatum-enriched phosphatase 14.85 Academic activity Child.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to the human striatum-enriched phosphatase 14.85.
  • Regular refers to changes in the function of human striatum-enriched phosphatase 14.85, including any increase or decrease in protein activity, changes in binding characteristics, and human striatum-enriched phosphatase 14.85 in any other organism Changes in nature, function, or immunity.
  • 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 striatum-enriched phosphatase 14.85 using standard protein purification techniques.
  • Substantially pure human striatum is enriched in phosphatase 14. 85 produces a single main band on a non-reducing polyacrylamide gel.
  • Human striatum-enriched phosphatase 14. 85 The purity of the peptide 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 refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to target sequences 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., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method will check the distance between all pairs by Groups of sequences are arranged in clusters. 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 Clus ter method or by methods known in the art such as Jotun Hein (He in J., (1990) Methods in enzymology 183: 625-645) 0 "similarity" means The amino acid residues in the corresponding positions are the same when the amino acid sequences are aligned. Or the extent of conservative substitution.
  • Amino acids used for conservative substitution 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; aspartamide and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to HFP or a chemical modification of its nucleic acid. 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? , which can specifically bind to the human striatum-enriched phosphatase 14.85 epitope.
  • 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 striatum-enriched phosphatase 14.85 means human striatum-enriched phosphatase 14.85 is substantially free of other proteins, lipids, sugars, or other substance. Those skilled in the art can purify human striatum-enriched phosphatase 14.85 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Human striatum-enriched phosphatase 14. 85 The purity of the peptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-human striatum-enriched phosphatase 14.85, 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 obtained from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects and Mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives, and analogs of the human striatum-enriched phosphatase 14.85.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human striatum-enriched phosphatase 14.85 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by the genetic code; or ( ⁇ ) such that a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ )
  • One, in which the mature polypeptide is fused to another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • such a polypeptide sequence in which the additional amino acid sequence is fused into the 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.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1849 bases, and its open reading frame 291-698 encodes 135 amino acids.
  • this peptide has a similar expression profile to human striatum-enriched phosphatase, and it can be inferred that the human striatum-enriched phosphatase 14.85 has similar human striatum-enriched phosphatase. Functions.
  • the polynucleotide of the present invention may be in the IA form or the RNA form.
  • DM forms include cDM, genomic DM, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • the DM can be a coding chain or a non-coding chain.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant” refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide means including a polynucleotide encoding the polypeptide and including additional Coding and / or non-coding polynucleotides.
  • 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 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 denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • 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 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 striatum-enriched phosphatase 14.85.
  • the polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human striatum-enriched phosphatase 14.85 of the present invention can be obtained by various methods.
  • the polynucleotide can be isolated by 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 multinucleated clones with common scab characteristics Nucleotide fragments.
  • the DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the DM of the genome; 2) chemically synthesizing the DM 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 CDM library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes 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 marker gene functions; (3) determination of human striatum-enriched phosphatase 14.85 transcripts (4) Detecting the protein product of gene expression by 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 herein 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 human striatum-enriched phosphatase 14.85 gene expression protein product can be detected using 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 for amplifying DNA / RNA by PCR 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 DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be measured 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 needs to be repeated. Sometimes it is necessary to determine the cDM sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly enriched with human striatum to a phosphatase 14.85 coding sequence, and the present invention is produced by recombinant technology Methods of the polypeptide.
  • a polynucleotide sequence encoding human striatum-enriched phosphatase 14.85 can be inserted into a vector to form 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 adenovirus, Retrovirus or other vector.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human striatum-enriched phosphatase 14.85 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DM technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Labora tory Manua 1, Coll Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in the 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 expressed by DM, 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 and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human striatum-enriched phosphatase 14.85 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 cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • 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 DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human striatum-enriched phosphatase 14. 85 (Sc ience, 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. If necessary, the recombinant protein can be isolated and purified by various separation methods using its 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
  • 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.
  • Protein phosphorylation and dephosphorylation are molecular switches that regulate vital life activities such as cell division, differentiation, and signaling in the body. Phosphorylation is controlled by a kinase, which transfers high-energy phosphate from adenosine triphosphate to a special protein; while phosphatase removes high-energy phosphate from the protein. Through the phosphorylation process, protein kinases can activate metabolic enzymes, regulatory factors, receptors, cytoskeleton proteins, ion channels, and transcription factors.
  • Protein tyrosine phosphatase can terminate the signaling pathway activated by tyrosine protein kinase, clear the last stimulus information, and enable this pathway to receive the next signal. It is currently unknown that eight protein tyrosine phosphatases and tyrosine protein kinases jointly regulate cell activity, proliferation, and differentiation.
  • Human striatum-enriched phosphatase (str iatum enr iched phosphatas e, STEP) is a neural system-specific protein tyrosine phosphatase, and its 3kb and 4.4kb mRNAs were found in different parts of the brain. 3kb mRNA is mainly distributed in the striatum, and STEP is named for it, while 4.4kb mRNA is mainly distributed in the cerebral cortex and is rarely distributed in the striatum. These two mRMs are two spliced forms of the same gene. Phosphatase enrichment in the human striatum in vivo regulates the physiological functions of the striatum and the cerebral cortex.
  • Abnormal expression can affect the normal physiological functions of the striatum and the cerebral cortex, and then lead to the occurrence of related diseases.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human striatum-enriched phosphatase protein, and both have similar biological functions.
  • the polypeptide of the present invention regulates the physiological functions of the striatum and the cerebral cortex in vivo, and its abnormal expression can affect the normal physiological functions of the striatum and the cerebral cortex, thereby leading to the occurrence of related diseases, including but not limited to:
  • Sensory analysis dysfunction physical dysfunction, visceral dysfunction (taste, nausea, defecation), visual impairment, hearing impairment, olfactory taste dysfunction, etc.
  • Dysregulation of physical movements central paralysis (facial muscles, tongue muscles, skeletal muscles of limbs), etc .
  • Dysregulation of visceral activity respiratory depression / acceleration, blood pressure drop / rise, arrhythmia, pupil dilation / shrinking, etc.
  • the striatum is a major component of the extrapyramidal system, and its dysfunction can cause various extrapyramidal diseases, including but not limited to:
  • polypeptide of the present invention and its antagonists, agonists and inhibitors can be directly used for the treatment of various diseases, especially cerebral cortical dysfunction diseases, extrapyramidal diseases and the like.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or inhibit (antagonist) human striatum-enriched phosphatase 14.85.
  • Agonists increase human striatum-enriched phosphatase 14.85 to stimulate cell proliferation and other biological functions, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or human striatum can be enriched for phosphatase in the presence of drugs 14.
  • membrane preparations and labeled human striatum are enriched in phosphatase 14. 85 together. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of S5 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • Antagonist of human striatum-enriched phosphatase 14.85 can bind to human striatum-enriched phosphatase 14.85 and eliminate its function, or inhibit the production of the polypeptide, or with the active site of the polypeptide Binding prevents the polypeptide from functioning biologically.
  • human striatum-enriched phosphatase 14.85 When screening compounds as antagonists, human striatum-enriched phosphatase 14.85 can be added to the bioanalytical assay, and by measuring the compounds, human striatum-enriched phosphatase 14.85 and its receptors interact with each other. Effect to determine whether 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. Peptide molecules capable of binding to human striatum-enriched phosphatase 14. 85 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally the human striatum-enriched phosphatase 14.85 molecule 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 against the human striatum-enriched phosphatase 14.85 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by enriching human striatum with phosphatase 14.85 by direct injection in immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not Limited to Freund's adjuvant and the like.
  • Techniques for preparing monoclonal antibodies to human striatum-enriched phosphatase 14.85 include, but are not limited to, hybridoma technology (Kohler and Mistein. 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 variable region can be produced using conventional techniques (Morrison et 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 striatum-enriched phosphatase 14.85.
  • Monoclonal antibodies that bind to human striatum-enriched phosphatase 14. 85 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 striatum is enriched in phosphatase 14.
  • High affinity monoclonal antibodies can interact with bacterial or plant toxins (such as diphtheria toxin, castor egg White, ormosine, etc.) covalently bind.
  • 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 striatum-enriched phosphatase 14 . 85 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human striatum-enriched phosphatase 14.85. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human striatum-enriched phosphatase 14.85.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human striatum-enriched phosphatase 14.85 levels. These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the human striatum-enriched phosphatase 14.85 level detected in the test can be used to explain the importance of human striatum-enriched phosphatase 14.85 in various diseases and to diagnose human striatum-enriched Diseases that collect phosphatase 14.85.
  • 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 striatum-enriched phosphatase 14. 85 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 human striatum-enriched phosphatase 14.85. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human striatum-enriched phosphatase 14.85 to inhibit endogenous human striatum-enriched phosphatase 14.85 activity.
  • a mutated human striatum-enriched phosphatase 14.85 may be shortened and a human striatum-enriched phosphatase 14.85 lacking a signaling domain, although it can bind to downstream substrates, However, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human striatum-enriched phosphatase 14.85.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the polynucleotide encoding human striatum-enriched phosphatase 14.85 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human striatum-enriched phosphatase 14.85 can be found in the existing literature (Sambrook. E t a l.).
  • a recombinant polynucleotide encoding human striatum-enriched phosphatase 14.85 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense MA and DM
  • ribozymes that inhibit human striatum-enriched phosphatase 14.85 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA that specifically breaks down specific MAs Molecules, whose mechanism of action is endonucleation after specific hybridization of ribozyme molecules with complementary target RNAs.
  • Antisense MA, 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 a DM sequence encoding the RNA. This DM sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human striatum-enriched phosphatase 14.85 can be used for the diagnosis of diseases related to human striatum-enriched phosphatase 14.85.
  • the polynucleotide encoding human striatum-enriched phosphatase 14.85 can be used to detect the expression of human striatum-enriched phosphatase 14.85 or human striatum-enriched phosphatase 14.85 in a disease state. Abnormal expression.
  • the DM sequence encoding human striatum-enriched phosphatase 14.85 can be used to hybridize biopsy specimens to determine the expression of human striatum-enriched phosphatase 14.85.
  • Hybridization techniques include Southern blotting, or thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are available commercially.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • 85 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human striatum-enriched phosphatase 14. 85 transcription products.
  • Human striatum-enriched phosphatase 14.85 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human striatum-enriched phosphatase 14.85 DNA sequence. Mutations can be detected using well-known 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.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DM sequences on a chromosome.
  • a P primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments. Somatic hybrid cell PGR mapping is a fast method for mapping DNA to specific chromosomes. Using the 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 chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDM 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 diseased and diseased 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 striatum is enriched for phosphatase 14. 85 is administered in an amount effective to treat and / or prevent a specific indication.
  • Human striatum-enriched phosphatase 14.85 administered to patients The amount and dosage range 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. Examples
  • RNA Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRM was isolated from total RM using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA forms cDM by reverse transcription. Smart cDNA cloning kit (purchased from Clontech
  • Dye terminate cycle reaction sequencing ki t Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elraer
  • the determined cDNA sequences were compared with the existing public DM sequence database (Genebank). By comparison, the cDNA sequence of one of the clones 1014f 09 was found to be new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragment of the clone in both directions.
  • Example 2 Cloning and encoding of human striatum-enriched phosphorus by RT-PCR method
  • Primer2 5,-CAATAAGTAGTCATTTAATTAAAG -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer 2 is the 3 'terminal reverse sequence of SEQ ID NO: 1.
  • a reaction volume of 50 ⁇ 1 contains 50 mmol / L KCl, 10 mraol / L Tris-HCl pH 8. 5, 1. 5 and ol / L MgCl 2 , 20 ( ⁇ mol / L dNTP, 1 Opmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2rain 0 At the same time, set ⁇ -act in as a positive control and template blank as a negative control during RT-PCR.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector using a TA cloning kit (Invitrogen).
  • DM sequence analysis results showed that the DM sequence of the PCR product was exactly the same as l-18 4 9bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human striatum-enriched phosphatase 14.85 gene expression Total MA was extracted in one step [Anal.
  • This method includes acid guanidinium thiocyanate- Chloroform extraction: The tissue is homogenized with 4M guanidine isothiocyanate-25raM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl are added. Alcohol (49: 1), mixed and centrifuged. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RM precipitate.
  • RNA precipitate with 70% ethanol, dry and dissolve in water Using 20 ⁇ ⁇ RNA on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (H7. 0)-5 mM sodium acetate-ImM EDTA-2. 2M formaldehyde electrophoresis then transferred to nitrocellulose by a -. 32 P dATP Preparation 32 P- DNA probe labeled by the random primer SYSTEM DNA probes used for PCR amplification shown in FIG striatum. 14.85 enriched phosphatase coding sequence (291bp to 698bp).
  • the 32P- labeled probes (about 2 ⁇ 10 6 cpffl / ml) and RNA was transferred to a nitrocellulose membrane in a solution After overnight hybridization at 42 C, the solution containing 50% formamide -25mM H (pH7. 4) -5 ⁇ SSC-5 ⁇ Denhardt, s solution and 200 ⁇ g / ml salmon sperm DNA 2 P0 4. Hybridization, the filter Wash in 1 x SSC-0. 1 ° /. SDS at 55 ° C for 30rain. Then, analyze and quantify with Phosphor Imager.
  • Example 4 In vitro expression and isolation of recombinant human striatum enriched phosphatase 14.85 And purification According to the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers was designed, the sequence is as follows:
  • Pr imer 3 5, — CCCCATATGATGCCTTCTGCATCCAGTGGTGAA- 3, (Seq ID No: 5)
  • Pr imer 4 5'-CATGGATCCTCAGGTTCTCCTTGTTCTCCTCAG-3- '-(Seq ID No: 6)
  • the 5' ends of these two primers contain Mel and BamHI, respectively Enzymatic digestion sites, followed by coding sequences for the 5 ,, and 3 'ends of the target gene, respectively.
  • the Ndel and BamHI digestion sites correspond to the expression vector plasmid pET- 2 8b (+) (Novagen, Cat. No. 69865). 3) Selective endonuclease sites.
  • the PCR reaction was performed using the pBS-1014f 09 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-1014f 09 plasmid, Primer-3 and Primer-4 primers were 1 Opmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively. Do not recover large fragments and ligate with T4 ligase. The ligation product was transformed into E.
  • the host bacteria BL21 (pET-1014f 09) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, Continue incubation for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. Chromatography was performed using an affinity chromatography column His s. Bind Quick Cartridge (product of Novagen) capable of binding to 6 histidines (6His-Tag). 85. The purified target protein human striatum is enriched in phosphatase 14.85.
  • a peptide synthesizer (product of PE company) was used to synthesize the following human striatum-enriched phosphatase 14. 85-specific peptides:
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as EUSA to determine the antibody titer in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody could specifically bind to human striatum-enriched phosphatase 14.85.
  • Example 6 Application of the 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 example is to select a suitable oligonucleoside from the polynucleotide SEQ ID NO: 1 of the present invention
  • the acid fragment is used as a hybridization probe, and the membrane hybridization method is used to identify whether some tissues contain the polynucleotide sequence of the present invention or a homologous polynucleotide sequence.
  • Filter hybridization methods include dot blotting, Southern blotting, Nor thern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is 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 from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (right) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • PBS phosphate buffered saline
  • step 14 Resuspend the DM pellet in a small volume of TE or water. Vortex at low speed or suck with a dropper while gradually increasing TE, mix until DM is fully dissolved, and add approximately 1 ul per 1-5 ⁇ 10 6 cells.
  • steps 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 required 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.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P- ⁇ ) is prepared.
  • the sample membrane was placed in a plastic bag, and 3 to 10 mg of prehybridization solution (lOxDen ardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution lOxDen ardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • Gene chip or gene microarray is a new technology 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 genetic diseases .
  • the specific method steps have been reported in the literature. For example, see DeRis i, JL, Lyer, V.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500 ng / ul after purification, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were dried to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRM was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified with Ol igotex mRNA Midi Kit (purchased from QiaGen).
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5--triphate coupled to Cy3 f luorescent dye, purchased from Araersham Phamacia Biotech) was used to label mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino-propargyl-2 '-deoxyuridine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Amershara Phamacia Biotech Company, labeled the specific tissue (or stimulated cell line) mRNA of the body, and purified the probe to prepare a probe.
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5--triphate coupled to Cy3 f luorescent dye, purchased
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Iraagene software (Biodi scovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line, thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scar formation fc Growth factor stimulation, 1013HT, scar into fc without growth factor stimulation, 1013HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, cardia cancer. Based on these 18 Cy3 / Cy5 ratios, a histogram is drawn (Figure 1). It can be seen from the figure that the expression profile of human striatum-enriched phosphatase 14.85 and human striatum-enriched phosphatase according to the present invention are very similar.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un nouveau polypeptide, une phosphatase humaine riche en striatum 14.85, et un polynucléotide codant 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 la phosphatase humaine riche en striatum 14.85.
PCT/CN2001/001048 2000-06-26 2001-06-25 Nouveau polypeptide, phosphatase humaine riche en striatum 14.85, et polynucleotide codant ce polypeptide WO2002040520A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002214905A AU2002214905A1 (en) 2000-06-26 2001-06-25 A novel polypeptide, a human striatum enriched phosphatase 14.85 and the polynucleotide encoding the polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 00116719 CN1331327A (zh) 2000-06-26 2000-06-26 一种新的多肽——人纹状体富集磷酸酶14.85和编码这种多肽的多核苷酸
CN00116719.7 2000-06-26

Publications (1)

Publication Number Publication Date
WO2002040520A1 true WO2002040520A1 (fr) 2002-05-23

Family

ID=4586119

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2001/001048 WO2002040520A1 (fr) 2000-06-26 2001-06-25 Nouveau polypeptide, phosphatase humaine riche en striatum 14.85, et polynucleotide codant ce polypeptide

Country Status (3)

Country Link
CN (1) CN1331327A (fr)
AU (1) AU2002214905A1 (fr)
WO (1) WO2002040520A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058514A1 (fr) * 1998-05-12 1999-11-18 American Home Products Corporation Derives de l'acide (2-acylaminothiazole-4-yl)acetique
US6020179A (en) * 1996-10-03 2000-02-01 Incyte Pharmaceuticals, Inc. Nucleic acids encoding human tyrosine phosphatases

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6020179A (en) * 1996-10-03 2000-02-01 Incyte Pharmaceuticals, Inc. Nucleic acids encoding human tyrosine phosphatases
WO1999058514A1 (fr) * 1998-05-12 1999-11-18 American Home Products Corporation Derives de l'acide (2-acylaminothiazole-4-yl)acetique

Also Published As

Publication number Publication date
AU2002214905A1 (en) 2002-05-27
CN1331327A (zh) 2002-01-16

Similar Documents

Publication Publication Date Title
WO2002020795A1 (fr) Nouveau polypeptide, hexokinase proteine 9.68, et polynucleotide codant ce polypeptide
WO2001090378A1 (fr) Nouveau polypeptide, proteine humaine hexokinase 11, et polynucleotide codant ce polypeptide
WO2002040520A1 (fr) Nouveau polypeptide, phosphatase humaine riche en striatum 14.85, et polynucleotide codant ce polypeptide
WO2002012314A1 (fr) Nouveau polypeptide, proteine-1 humaine associee a la dihydropyrimidinase (drp-1) 9.68, et polynucleotide codant ce polypeptide
WO2001074876A1 (fr) Nouveau polypeptide, phosphatidylinositol-3 kinase humaine 14, et polynucleotide codant pour ce polypeptide
WO2001066578A1 (fr) Nouveau polypeptide, adn polymerase 10, et polynucleotide codant pour ce polypeptide
WO2001087943A1 (fr) Protease a serine 13 atp-dependante, polypeptide humain, et polynucleotide le codant
WO2001088158A1 (fr) Nouveau polypeptide, triose phosphate isomerase humaine 10, et polynucleotide codant pour ce polypeptide
WO2001094401A1 (fr) Nouveau polypeptide, proteine npat humaine 15, et polynucleotide codant pour ce polypeptide
WO2001070785A1 (fr) Nouveau polypeptide, serine proteinase humaine atp-dependante 13, et polynucleotide codant pour ce polypeptide
WO2002012297A1 (fr) Nouveau polypeptide, proteine humaine 9 de liaison a la tropomoduline, et polynucleotide codant ce polypeptide
WO2001072986A1 (fr) Nouveau polypeptide, serine hydrolase humaine atp-dependante 10, et polynucleotide codant pour ce polypeptide
WO2001092315A1 (fr) Nouveau polypeptide, proteine humaine d'epissage 10.56, et polynucleotide codant ce polypeptide
WO2001075048A2 (fr) Nouveau polypeptide, proteine ribosomale humaine s11 23, et polynucleotide codant pour ce polypeptide
WO2002006471A1 (fr) Nouveau polypeptide, nucleophosmine 9.68, et polynucleotide codant ce polypeptide
WO2002012315A1 (fr) Nouveau polypeptide, proteine mid1 10.56 du gene de type « ring finger », et polynucleotide codant ce polypeptide
WO2001094402A1 (fr) Nouveau polypeptide, proteine npat humaine 12, et polynucleotide codant pour ce polypeptide
WO2001079508A1 (fr) Nouveau polypeptide, proteine porteuse mitochondriale humaine 18, et polynucleotide codant pour ce polypeptide
WO2002006336A1 (fr) Nouveau polypeptide, proteine humaine drim 13.09, et polynucleotide codant ce polypeptide
WO2002012490A1 (fr) Nouveau polypeptide, proteine membranaire de type ii 12.65, et polynucleotide codant ce polypeptide
WO2001083758A2 (fr) Nouveau polypeptide, dihydroorotase humaine 8, et polynucleotide codant pour ce polypeptide
WO2001090178A1 (fr) Nouveau polypeptide, dihydroorotase humaine 11, et polynucleotide codant ce polypeptide
WO2001087963A1 (fr) Proteine s18-12 ribosomale, polypeptide humain, et polynucleotide la codant
WO2001087967A1 (fr) Proteine s18-26 ribosomale, polypeptide humain, et polynucleotide la codant
WO2002026971A1 (fr) Nouveau polypeptide, facteur de transcription humain lcr-f112.1, et polynucleotide codant 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 CO 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
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