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WO2001066591A1 - Nouveau polypeptide, proteine humaine 11 de reception tyrosine phosphatase, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 11 de reception tyrosine phosphatase, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001066591A1
WO2001066591A1 PCT/CN2001/000213 CN0100213W WO0166591A1 WO 2001066591 A1 WO2001066591 A1 WO 2001066591A1 CN 0100213 W CN0100213 W CN 0100213W WO 0166591 A1 WO0166591 A1 WO 0166591A1
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Prior art keywords
polypeptide
polynucleotide
protein tyrosine
tyrosine phosphatase
human receptor
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Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU42237/01A priority Critical patent/AU4223701A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide-human receptor-related protein tyrosine phosphatase 11, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Protein tyrosine phosphorylation is an important mechanism for cellular signal transduction in vivo, and is usually controlled by protein tyrosine kinase and protein tyrosine phosphatase. At present, the research on protein tyrosine kinase is more detailed, while the research on protein tyrosine phosphatase is relatively less. Studies have found that there are two different types of protein tyrosine phosphatases in the organism: soluble cytosolic enzymes and membrane-bound enzymes. These two phosphatases have similar functions in organisms, except that the sites of action are different [Jones SW, Erikson RL et al., 1989, J Biol.
  • Protein tyrosine kinases and Protein tyrosine phosphatase works synergistically in vivo. Inactivation of protein tyrosine kinase will lead to abnormal cell signaling pathways, that is, abnormal cell proliferation and deformation and deterioration of proto-oncogenes.
  • As a protein tyrosine kinase antagonist it is a tumor suppressor gene that inhibits this process. The mutation or inactivation of the phosphatase will cause the deformation of the proto-oncogene, that is, the occurrence of various tumors and cancers in the body.
  • ⁇ TPTP ⁇ has similar structure and biological activity to the above-mentioned human PTP1B protein and Drosophila DLAR and DPTP protein, which are all related to developmental disorders in the body, related tissue tumors and cancer, etc. Related to the occurrence of disease [Ne il X. Krueger, Michele Streul i et al., 1990, The EMBO Journa l, 9: 3241-3252].
  • the expression profile of the polypeptide of the present invention is very similar to the expression profile of human protein tyrosine phosphatase, so the functions of the two may be similar.
  • the present invention is named as human receptor-related protein tyrosine phosphatase 11.
  • the human receptor-related protein tyrosine phosphatase 11 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art. Identification of more human receptor-associated protein tyrosine phosphatase 11 proteins involved in these processes, especially the amino acid sequence of this protein. Isolation of the novel human receptor-related protein tyrosine phosphatase 11 protein encoding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Polynucleotide recombinant vector Polynucleotide recombinant vector.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human receptor-related protein tyrosine phosphatase 1 1.
  • Another object of the present invention is to provide a method for producing a human receptor-related protein tyrosine phosphatase 1 1.
  • Another object of the present invention is to provide an antibody against the polypeptide-human receptor-related protein tyrosine phosphatase 11 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the protein-human receptor-related protein tyrosine phosphatase 11 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of a human receptor-related protein tyrosine phosphatase 1 1.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 2, or a conservative variant, biologically active fragment, or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID NO: 1
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of a human receptor-related protein tyrosine phosphatase 11 protein, 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 disease susceptibility associated with abnormal expression of a protein tyrosine phosphatase 11 protein associated with a human receptor, comprising detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the present invention also relates to a pharmaceutical composition, which contains the polypeptide of the present invention or a mimic, activator, antagonist Antibiotics or inhibitors and pharmaceutically acceptable carriers.
  • the present invention also relates to the polypeptides and / or polynucleotides of the present invention in the preparation for the treatment of malignant tumors, hematological diseases, developmental disorders, H IV infection and immune diseases and various types of inflammation or other protein receptors related to human receptors.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • 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 bound to a human receptor-associated protein tyrosine phosphatase 11, can cause changes in the protein and thereby regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to a human receptor-related protein tyrosine phosphatase 11.
  • Antagonist refers to a type of protein that can block or regulate human receptor-associated protein tyrosine phosphatase 11 when bound to human receptor-associated protein tyrosine phosphatase 11.
  • Active or immunologically active molecule can include proteins, nucleic acids, carbohydrates or any other It binds to human receptor-related protein tyrosine phosphatase 11 molecules.
  • Regular refers to changes in the function of human receptor-related protein tyrosine phosphatase 11, including any increase or decrease in protein activity, changes in binding properties, and any Changes in other biological, functional or immune properties.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human receptor-associated protein tyrosine phosphatase 11 using standard protein purification techniques.
  • a substantially pure human receptor-associated protein tyrosine phosphatase 11 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human receptor-related protein tyrosine phosphatase 11 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 refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Mad Son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Cluster method divides each group of sequences by checking the distance between all pairs. Arranged in clusters. The clusters are then assigned in pairs or groups.
  • sequence A and sequence B The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A Number of interval residues in a sequence B
  • Nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun He in Percent identity (He in L, (1990) Me thods in emzumo l ogy 18 3: 625-645) 0 "Similarity” refers to the identity or conservation of amino acid residues at corresponding positions when the alignment of amino acid sequences is aligned. Degree of sexual substitution.
  • Amino acids used for conservative substitutions 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,? (& 1) ') 2 and? It can specifically bind to the epitope of human receptor-related protein tyrosine phosphatase 11.
  • 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 receptor-related protein tyrosine phosphatase 11 means that human receptor-related protein tyrosine phosphatase 11 is substantially free of other proteins, lipids, and sugars naturally associated with it. Or other substances. Those skilled in the art can purify human receptor-associated protein tyrosine phosphatase 11 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 receptor-related protein tyrosine phosphatase 11 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-human receptor-related protein tyrosine phosphatase 11, 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, Natural polypeptides, synthetic polypeptides, and preferably recombinant polypeptides.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human receptor-related protein tyrosine phosphatase 11.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human receptor-related protein tyrosine phosphatase 11 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 ( ⁇ ) such a type in which one or more amino acid residues are substituted with other groups to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • such fragments, 00 derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 2272 bases, and its open reading frames 486-791 encode 101 amino acids.
  • this polypeptide has a similar expression profile with human protein tyrosine phosphatase, and it can be inferred that the human receptor-related protein tyrosine phosphatase 11 is similar to human protein tyrosine phosphatase.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optionally the additional Plus coding sequences) and non-coding sequences.
  • 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 present 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 0.2 xSSC, 0.1% SDS, 60 ° C; or (2 ) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only in two sequences Crosses occur only when the identity between them 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 More than 100 nucleotides.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human receptor-related protein tyrosine phosphatase 1 1.
  • 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 receptor-related protein tyrosine phosphatase 11 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 cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or Phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes 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 the transcription of the human receptor-related protein tyrosine phosphatase 11 (4) Detecting protein products expressed by genes through immunological techniques or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human receptor-related protein tyrosine phosphatase 11 gene expression can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). .
  • 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 a human receptor-related protein tyrosine phosphate Hi11 coding sequence, and recombinant technology to produce the present invention.
  • a method of inventing the polypeptide in the present invention, a polynucleotide sequence encoding a human receptor-related protein tyrosine phosphatase 11 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Cheni. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a human receptor-related protein tyrosine phosphatase 11 and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the 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 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, tumorigenic enhancers on the late side of the origin of replication, and adenoviral 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 receptor-related protein tyrosine phosphatase 11 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a gene containing the polynucleotide or the recombinant vector.
  • Engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or Lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: E.
  • 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 s melanoma cells, etc. .
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM can be harvested after exponential growth and treated with CaCl.
  • the steps used are well known in the art.
  • the alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human receptor-related protein tyrosine phosphatase 11 (Sc ience, 1 984; 224: 14 31). Generally speaking, there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be 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 diagram of the human receptor-related protein tyrosine phosphatase 11 and human protein tyrosine phosphatase of the present invention. Comparison of gene chip expression profiles.
  • the upper graph is a graph of the expression profile of the human receptor-related protein tyrosine phosphatase 11, and the lower sequence is the graph of the expression profile of the human protein tyrosine phosphatase.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human receptor-related protein tyrosine phosphatase 11.
  • lKDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • RNA Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA I solat i on Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • a Smart cDNA cloning kit purchased from Clontech) was used to insert the 00 ⁇ fragment into the multicloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ to form a cDNA library.
  • the sequences at the 5 'and 3' ends of all clones were determined using Dye terminate cyc le react ion sequencing kit (Perk in-Eiraer) and ABI 377 automatic sequencer (Perkin-Elmer).
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one clone 0196f 01 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the 0196f 01 clone contains a full-length cDNA of 2272bp (as shown in Seq ID N0: 1), and a 306bp open reading frame (0RF) from 486bp to 791bp, encoding a new protein (such as Seq ID NO: 2).
  • This clone pBS_0196f 01 and the encoded protein was named human receptor-related protein tyrosine phosphatase 11.
  • Example 2 Cloning of a gene encoding human receptor-related protein tyrosine phosphatase 11 by RT-PCR
  • CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imer 1 5'- TCTGCGTTGCTGTGGATCCTGAAA -3 '(SEQ ID NO: 3)
  • Pr imer2 5'- GCACCTATTTTTTTATTTGTGGGAT -3 '(SEQ ID NO: 4)
  • Pr iraerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Pr inier2 is the 3′-end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 mmol / L KC1, 10 legs ol / L Tris-Cl, (pH8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol in a 50 ⁇ 1 reaction volume Primer, 1U Taq DNA polymerase (C 1 on te ch). The reaction was performed for 25 cycles on a PE 9600 DNA thermal cycler (Perkin-Elmer) under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-2272bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human receptor-related protein tyrosine phosphatase 11 gene expression: Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0 This method includes acid thiocyanate Guanidinephenol-chloroform extraction. I.e.
  • RNA precipitate was washed with 70% ethanol, dried and dissolved in water.
  • 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 ⁇ ⁇ / ⁇ 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 4 In vitro expression, isolation and purification of recombinant human receptor-related protein tyrosine phosphatase 11
  • Primer 3 5, _ CCCCATATGATGTGTGTCTCTGCGTCTTTCCTTCC -3, (Seq ID No: 5)
  • Primer4 5'- CCCAAGCTTTCATGGAACAAGTCCCCGTTTGAA -3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and Hindlll digestion sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and Hindll I restriction sites correspond to the selection on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3) Sex endonuclease site.
  • PCR was performed using the pBS-0196f01 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1, a plasmid containing 10 pg of pBS-0196f01, primers Primer-3 and Primer-4 were lOpmol, Advantage, respectively polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60. C 30s, 68 ° C 2 min, a total of 25 cycles.
  • the amplified product and plasmid pET-28 (+) were double-digested with Mel and Hindl11, respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5c by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. Selected positive clones with the correct sequence (pET- 0196f01) the recombinant plasmid by the calcium chloride method to transform E. coli BL21 (DE3) P lySs (Novagen Co.).
  • the host bacteria BL21 (pET-0196f01) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, and continued Incubate for 5 hours.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation.
  • a peptide synthesizer (product of PE) was used to synthesize the following human receptor-related protein tyrosine phosphatase 11 specific peptides:
  • NH2-Met-Cys-Pro-Cys-Val-Phe-Pro-Ser-Phe-Ser-Leu-Ser-Leu-Ser-Leu-C00H SEQ ID NO: 7
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin for the method, see: Avraraeas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned jk cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the 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.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive home-immunized serum.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method demonstrated that the purified antibody specifically binds to the human receptor-related protein tyrosine phosphatase 11.
  • 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 identified whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or Pathology Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • 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 mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt): 5'- TGTGTCCCTGCGTCTTTCCTCCCTTCTCTCTTTCGCTCTCT -3 '(SEQ ID NO: 9)
  • SEQ ID NO: 1 or its complementary fragment (41Nt): 5'- TGTGTCCCTGCGTCTTTCCTCCCTTCTCTCTTTCGCTCTCT -3 '(SEQ ID NO: 9
  • SEQ ID NO: 9 for other commonly used reagents listed below and their preparation methods, please refer to the literature: DNA PROBES GH Kel ler; MM Manak; Stockton Press, 1989 ( USA) and more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide” (U998 Second Edition) [US] Sambrook et al., Science Press.
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (10xDenhardt's; 6xSSC, 0.1 mg / ml) was added.
  • CT DNA calf thymus DNA).
  • Gene chip or gene micro-matrix (DNA Mi croarray) is a new technology that many national laboratories and large pharmaceutical companies are currently researching and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the amplified product was adjusted to a concentration of about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps have been reported in the literature in various ways. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • Reagent Cy3dUTP (5-Amino-propargy 1-2 ⁇ -deoxyuri dine 5'-triphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5 — Amino-propargy Bu 2'-deoxyuridine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech, was used to label the mRNA of specific tissues (or stimulated cell lines) in the body, and probes were prepared after purification.
  • fluorescent reagent Cy5dUTP (5 — Amino-propargy Bu 2'-deoxyuridine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and the washing solution (1 ⁇ SSC, 0.2% SDS) After washing, scan with a ScanArray 3000 scanner (purchased from Genera Scanning, USA). The scanned images are analyzed and processed with Imagene software (Biod scovery, USA), and each point is calculated. Cy3 / Cy5 ratio.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv 304 cell line, PMA-Ecv304 cell line, and non-starved L02 cell line , Arsenic stimulated L02 cell line and prostate tissue for 1 hour.
  • polypeptides of the present invention 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 tyrosine kinases and protein tyrosine phosphatases are important enzyme systems for cell signaling in vivo. Inactivation of protein tyrosine kinase will cause abnormal cell signaling pathways, that is, abnormal proliferation of cells and deformation and deterioration of proto-oncogenes, and protein tyrosine phosphatase, as an antagonist of protein tyrosine kinase, is a A tumor suppressor gene that inhibits this process. The mutation or inactivation of the phosphatase will cause the deformation of the proto-oncogene, which will lead to the occurrence of various tumors and cancers in the body.
  • the polypeptide of the invention has high homology with leukocyte antibodies and molecules related to leukocyte antibodies, and its expression profile is consistent with the expression profile of protein tyrosine phosphatase similar to human receptors And both have similar biological activities in vivo, and their abnormal expression is usually related to the development of disorders in vivo, related tissue tumors, and cancer.
  • the abnormal expression of the human receptor-related protein tyrosine phosphatase 11 of the present invention will produce various diseases, especially various tumors, embryonic developmental disorders, disorders of growth and development, inflammation, and immune diseases. These diseases include, but are not limited to:
  • Tumors of various tissues stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, colon cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma, embryonic developmental disorders Symptoms: Congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital hearing loss
  • Growth and development disorders mental retardation, brain development disorders, skin, fat and muscle hair Fertility disorders, bone and joint dysplasia, various metabolic defects, stunting, dwarfism, Cushing syndrome, sexual retardation
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • Abnormal expression of the human receptor-associated protein tyrosine phosphatase 11 of the present invention will also produce certain hereditary, hematological diseases and the like.
  • 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 various diseases, especially various tumors, embryonic development disorders, growth and development disorders, inflammation, immunity Sexual diseases, certain hereditary, blood diseases, etc.
  • protein tyrosine phosphatases have similar structural characteristics. They cooperate with protein tyrosine kinases in the body and participate in the regulation of various important intracellular signaling pathways.
  • this protein is a tumor suppressor, and its mutation or abnormal expression will cause abnormal proliferation of related tissue cells and deformation and deterioration of proto-oncogenes.
  • the protein is usually closely related to the occurrence of some developmental disorders, related tissue tumors, and cancers in the body.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human receptor-related protein tyrosine phosphatases 11.
  • Agonists enhance human receptor-related protein tyrosine phosphatase 11 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing a human receptor-related protein tyrosine phosphatase 11 can be cultured with a labeled human receptor-related protein tyrosine phosphatase 11 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human receptor-related protein tyrosine phosphatase 11 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human receptor-related protein tyrosine phosphatase 11 can bind to human receptor-related protein tyrosine phosphatase 11 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 receptor-associated protein tyrosine phosphatase 11 can be added to bioanalytical assays. The effect of this interaction is used to determine whether the compound is an antagonist. Screening compounds using the above In the same way, receptor deletions and analogs that act as antagonists can be screened. Polypeptide molecules capable of binding to human receptor-related protein tyrosine phosphatase 11 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, the human receptor-related protein tyrosine phosphatase 11 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against human receptor-related protein tyrosine phosphatase 11 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human receptor-related protein tyrosine phosphatase 11 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including It is not limited to Freund's adjuvant and the like.
  • Techniques for preparing monoclonal antibodies to human receptor-related protein tyrosine phosphatase 11 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975,
  • Antibodies against human receptor-related protein tyrosine phosphatase 11 can be used in immunohistochemical techniques to detect human receptor-related protein tyrosine phosphatase 11 in biopsy specimens.
  • Monoclonal antibodies that bind to human receptor-related protein tyrosine phosphatase 11 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 receptor-related protein tyrosine phosphatase 11 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 receptor-related protein tyrosine Phosphatase 11 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human receptor-related protein tyrosine phosphatase 11.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human receptor-related protein tyrosine phosphatase 11.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting human receptor-related protein tyrosine phosphatase 11 levels.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the human receptor-related protein tyrosine phosphatase 11 levels detected in the test can be used to explain the importance of human receptor-related protein tyrosine phosphatase 11 in various diseases and to diagnose human receptors Related diseases where protein tyrosine phosphatase 11 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human receptor-related protein tyrosine phosphatases 11 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 receptor-related protein tyrosine phosphatase 11. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human receptor-associated protein tyrosine phosphatase 11 to inhibit endogenous human receptor-associated protein tyrosine phosphatase 11 activity.
  • a variant human receptor-associated protein tyrosine phosphatase 11 may be a shortened human receptor-associated protein tyrosine phosphatase 11 lacking a signaling domain, although it may be related to downstream substrates. Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human receptor-related protein tyrosine phosphatase 11.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding a human receptor-related protein tyrosine phosphatase 11 can be used to transfer a polynucleotide encoding a human receptor-related protein tyrosine phosphatase 11 into a cell .
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human receptor-related protein tyrosine phosphatase 11 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding a human receptor-related protein tyrosine phosphatase 11 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 DM
  • ribozymes that inhibit human receptor-related protein tyrosine phosphatase 11 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense 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 RM molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DNA 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.
  • a polynucleotide encoding a human receptor-related protein tyrosine phosphatase 11 can be used in the diagnosis of diseases related to the human receptor-related protein tyrosine phosphatase 11.
  • Polynucleotides encoding human receptor-related protein tyrosine phosphatase 11 can be used to detect the expression of human receptor-related protein tyrosine phosphatase 11 or human receptor-related protein tyrosine in disease states Abnormal Expression of Phosphatase 11
  • the DM sequence encoding human receptor-related protein tyrosine phosphatase 11 can be used to hybridize biopsy specimens to determine the expression status of human receptor-related protein tyrosine phosphatase 11.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a micro array (Mic roarray) or a DNA chip (also known as a "gene chip"), and used for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • a micro array Mossel roarray
  • a DNA chip also known as a "gene chip”
  • Human receptor-associated protein tyrosine phosphatase 11 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the human receptor-related protein tyrosine phosphatase 11 transcription products.
  • Human receptor-associated protein tyrosine phosphatase 1 1 mutations include point mutations, translocations, deletions, recombinations, and any other mutations compared to the normal wild-type human receptor-associated protein tyrosine phosphatase 11 DNA sequence. Exception, etc. Mutations can be detected using existing techniques such as Southern imprinting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes can be refined in one step Perform chromosomal mapping accurately.
  • 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 observed in any normal individual, 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 receptor-related protein tyrosine phosphatase 11 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human receptor-associated protein tyrosine phosphatase 11 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 11 de réception tyrosine phosphatase, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, des troubles du développement, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine 11 de réception tyrosine phosphatase.
PCT/CN2001/000213 2000-03-07 2001-02-26 Nouveau polypeptide, proteine humaine 11 de reception tyrosine phosphatase, et polynucleotide codant pour ce polypeptide Ceased WO2001066591A1 (fr)

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CN 00111925 CN1312369A (zh) 2000-03-07 2000-03-07 一种新的多肽——人受体相关的蛋白酪氨酸磷酸酶11和编码这种多肽的多核苷酸
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5538886A (en) * 1990-07-11 1996-07-23 New York University Receptor-type phosphotyrosine phosphatase-alpha
US5888794A (en) * 1990-07-11 1999-03-30 New York University Receptor-type phosphotyrosine phosphatase-alpha

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5538886A (en) * 1990-07-11 1996-07-23 New York University Receptor-type phosphotyrosine phosphatase-alpha
US5888794A (en) * 1990-07-11 1999-03-30 New York University Receptor-type phosphotyrosine phosphatase-alpha

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. MOL. BIOL. INT., vol. 40, no. 5, 1996, pages 1025 - 1032 *
GENE, vol. 194, no. 2, 1997, pages 241 - 247 *

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