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WO2001048160A1 - Nouveau polypeptide, proteine tyrosine phosphatase 12, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine tyrosine phosphatase 12, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001048160A1
WO2001048160A1 PCT/CN2000/000709 CN0000709W WO0148160A1 WO 2001048160 A1 WO2001048160 A1 WO 2001048160A1 CN 0000709 W CN0000709 W CN 0000709W WO 0148160 A1 WO0148160 A1 WO 0148160A1
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Prior art keywords
polypeptide
tyrosine
polynucleotide
protein phosphatase
specific protein
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PCT/CN2000/000709
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU21463/01A priority Critical patent/AU2146301A/en
Publication of WO2001048160A1 publication Critical patent/WO2001048160A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, a tyrosine-specific protein phosphatase 12, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • Tyrosine-specific protein phosphatase is an enzyme that catalyzes the attachment of phosphate groups to tyrosine residues. This enzyme plays an important role in controlling cell growth, proliferation, differentiation, and transformation. PTPase is a large family of enzymes, which can be classified into two types according to their differences in form and characteristics: one is soluble PTPase, and the other is a transmembrane receptor protein that contains PTPase regions.
  • PTPase consists of 300 amino acid residues and has two conserved Cys residues, of which the second Cys has a very important role in the activity of the entire enzyme. More interestingly, many conserved residues in its immediate vicinity have also shown a very important role. The sequence of this amino acid residue is as follows: [LIVMF] — H— C— X (2) _G—X (3) — [STC] — [STAGP] — X— [LIVMFY]. Structurally, all receptor PTPases consist of extracellular regions of different lengths, a transmembrane region, and a C-terminal region located in the cytoplasm.
  • Some extracellular regions of the receptor PTPase include repeating fibronectin III (FN-I II), an immunoglobulin-like region, a MAM region, or a carbon dehydratase-like region.
  • the cytoplasmic region usually contains two copies of the PTPase region. The first one has enzyme activity, and the other one has no enzyme activity, but affects the first enzyme. Substrate specificity. In these regions, catalytically active Cys residues are usually more conserved, but others are not.
  • the PTPase family has very high enzyme activity, which is about 10-1000 times higher than that of ordinary tyrosine kinases, so it can effectively control the amount of phosphoryl tyrosine in cells. In vivo, they can dephosphorylate tyrosine hydrazone residues and participate in signal transduction and cell cycle regulation.
  • PTPase acts as a tumor suppressor in tissues.
  • the activity of PTPase is very low in normal human breasts, while its activity is significantly increased in patients with breast cancer.
  • it has similar functions in many organ and tissue cancers of many people, including rectal cancer, epithelial cell cancer, pancreatic cancer, glial cancer, prostate cancer, lung cancer, and cellular leukemia.
  • Many hormone and growth factor receptors and some retroviruses also have significant PTPase activity.
  • the tyrosine-specific protein phosphatase 12 protein plays an important role in important functions of the body. It is believed that a large number of proteins are involved in these regulatory processes, so there is a need in the art to identify more tyrosine-specific protein phosphatase 12 proteins involved in these processes, especially the amino acid sequence of this protein.
  • the isolation of the novel tyrosine-specific protein phosphatase 12 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA.
  • 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 tyrosine-specific protein phosphatase 12.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a tyrosine-specific protein phosphatase 12.
  • Another object of the present invention is to provide a method for producing tyrosine-specific protein phosphatase 12.
  • Another object of the present invention is to provide a tyrosine-specific protein phosphatase directed against the polypeptide of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed against the polypeptide of the present invention, tyrosine-specific protein phosphatase 12.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of tyrosine-specific protein phosphatase 12.
  • 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 807-11 in SEQ ID NO: 1; and (b) having a sequence in SEQ ID NO: 1 in 1- 21 36-bit sequences.
  • the invention further relates to a vector, in particular an expression vector, containing a polynucleotide of the invention.
  • the vector genetically engineered host cell includes a transformed, transduced or transfected host cell; a method for preparing a polypeptide of the present invention comprising culturing the host cell and recovering an 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 screen for mimicking, activating, antagonizing or inhibiting tyrosine-specific protein phosphatase.
  • a method of a 12-protein-active compound comprising utilizing a 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 tyrosine-specific protein phosphatase 12 protein, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention for the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of tyrosine-specific protein phosphatase 12.
  • Fig. 1 is a comparison diagram of amino acid sequence homology of tyrosine-specific protein phosphatase 12 of the present invention between 64 and 22 amino acids at 22-85 and a characteristic sequence fragment of tyrosine-specific protein phosphatase.
  • the upper sequence is the tyrosine-specific protein phosphatase 12 and the lower sequence is the characteristic sequence of the tyrosine-specific protein phosphatase. "I" and ":” and ".” Indicate that the probability that the same amino acid appears between the two sequences decreases in order.
  • Figure 2 is an isolated specific tyrosine protein phosphatase polyacrylamide gel electrophoresis of FIG. 12 (SDS - PAGE) 0 12kDa molecular weight proteins.
  • the arrow indicates the isolated protein band.
  • 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 be related to the protein molecule Of complete natural amino acids.
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the 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 tyrosine-specific protein phosphatase 12, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to a tyrosine-specific protein phosphatase 12.
  • Antagonist refers to a molecule that, when combined with tyrosine-specific protein phosphatase 12, blocks or regulates the biological or immunological activity of tyrosine-specific protein phosphatase 12.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to tyrosine-specific protein phosphatase 12.
  • Regular refers to a change in the function of tyrosine-specific protein phosphatase 12, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of tyrosine-specific protein phosphatase 12. Or changes in immune properties.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify tyrosine-specific protein phosphatase 12 using standard protein purification techniques.
  • a substantially pure tyrosine-specific protein phosphatase 12 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the tyrosine-specific protein phosphatase 12 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence CGA
  • GACT complementary sequence
  • 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.
  • Inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in 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 (Higgins, D. G. and P. M. Sharp (1988)
  • the Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:
  • the 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 (Hein J., (1990) Methods in enzymology 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or RM sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “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, F (ab ') 2 and Fv, which can specifically bind to the epitope of tyrosine-specific protein phosphatase 12.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated tyrosine-specific protein phosphatase 12 means that tyrosine-specific protein phosphatase 12 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify tyrosine-specific protein phosphatase 12 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the tyrosine-specific protein phosphatase 12 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, tyrosine-specific protein phosphatase 12, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude initial methionine residues.
  • the invention also includes fragments, derivatives and analogs of tyrosine-specific protein phosphatase 12.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the tyrosine-specific protein phosphatase 12 of the invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type 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 substituted
  • 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 ( ⁇ ⁇ ) like this
  • the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protein sequence).
  • a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protein sequence such as a leader sequence or a secreted
  • 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 a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 2136 bases in length and its open reading frame 807-1130 encodes 107 amino acids.
  • This peptide has a characteristic sequence of a tyrosine-specific protein phosphatase, and it can be deduced that the tyrosine-specific protein phosphatase 12 has the structure and function represented by a tyrosine-specific protein phosphatase characteristic sequence fragment.
  • the polynucleotide of the present invention may be in the D form or the RNA form.
  • DNA forms include cDM, genomic DM, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • D can be a coding or 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 refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60'C; or (2) Add a denaturant during hybridization, such as 50 " ⁇ / ⁇ ) formamide, 0.1 lj, bovine serum / 0.1% Ficol 1, 42.
  • 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 tablets A 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 nucleotides.
  • Nucleic acid fragments are also Nucleic acid amplification techniques such as PCR can be used to identify and / or isolate a polynucleotide encoding a tyrosine-specific protein phosphatase 12.
  • 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 tyrosine-specific protein phosphatase 12 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 D of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecuar ar Clinging, A Laboratory Manua, Co. Harbor Harbor Labora tory. 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 screened from these cDM 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 level of tyrosine-specific protein phosphatase transcripts; (4) Detecting the protein product of gene expression by immunological technology or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1 000 nucleotides.
  • the probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the tyrosine-specific protein phosphotrans 12 gene.
  • a method of amplifying DM / RM using PCR technology (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • RACE-rapid amplification of cDNA ends can be preferably used.
  • 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 D / 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 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 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 that is genetically engineered using the vector of the present invention or directly using a tyrosine-specific protein phosphatase 12 coding sequence, and to produce the present invention by recombinant technology Said method of polypeptide.
  • a polynucleotide sequence encoding a tyrosine-specific protein phosphatase 12 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a D sequence encoding a tyrosine-specific protein phosphatase 12 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • Expression vector also includes a nucleus for translation initiation Glycosome binding sites and transcription terminators. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and 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 tyrosine-specific protein phosphatase 12 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing 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 eukaryotic organism, 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 tyrosine-specific protein phosphatase 12 by conventional recombinant DNA technology (Scence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. When the host cell has 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 can be directly used in the treatment of diseases, for example, they can be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immunological diseases.
  • Tyrosine-specific protein phosphatase is an enzyme that catalyzes the attachment of phosphate groups to tyrosine residues. This enzyme plays an important role in controlling cell growth, proliferation, differentiation, and transformation.
  • the PTPase family has high enzyme activity, which is about 10-1000 times higher than that of ordinary tyrosine kinases, so it can effectively control the amount of phosphoryl tyrosine in cells. In vivo, they can dephosphorylate tyrosine residues and participate in signal transduction and cell cycle regulation.
  • PTPase acts as a tumor suppressor in tissues.
  • PTPase activity is very low in normal human breasts, while its activity is significantly increased in breast cancer patients.
  • it has similar functions in many organ and tissue cancers of many people, including rectal cancer, epithelial cell cancer, pancreatic cancer, glial cancer, prostate cancer, lung cancer, and cellular leukemia.
  • Many hormone and growth factor receptors also have significant PTPase activity with some retroviruses.
  • a tyrosine-specific protein phosphatase-specific conserved sequence is required to form its active mot if. It can be seen that the abnormal expression of the specific tyrosine-specific protein phosphatase mot if will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, thereby causing the receptor signal pathway of tyrosine protein kinase activity to be abnormal.
  • the signal transduction and cell cycle regulation are abnormal, and related diseases such as tumors, embryonic developmental disorders, growth and development disorders, inflammation, etc. occur.
  • the abnormal expression of the tyrosine-specific protein phosphatase 12 of the present invention will produce various diseases, especially various tumors, embryonic developmental disorders, growth and development disorders, and inflammation.
  • diseases include, but are not limited to: Tumors: breast cancer, rectal cancer, epithelial cancer, pancreatic cancer, glial cancer, prostate cancer, lung cancer, leukemia, stomach cancer, liver cancer, esophageal cancer, lymphoma, thyroid tumor, uterine fibroids, Neuroblastoma, astrocytoma, ependymal tumor, colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, Colon cancer, thymic tumor, nasal cavity and sinus tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma,
  • Embryonic disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney, double ureter, cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, suburethral Fissure, hermaphroditism, atrial septal defect, ventricular septal defect, pulmonary stenosis, arterial duct occlusion, neural tube defect, congenital hydrocephalus, iris defect, congenital cataract, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus dysplasia syndrome, strabismus, skin, fat and muscular dysplasia such as congenital skin laxity, premature aging Disease, congenital keratosis, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism, sexual retardation
  • Inflammation allergic reaction, bronchial asthma, allergic pneumonia, adult respiratory distress syndrome, sarcoidosis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immune complex Types of glomerulonephritis, dermatomyositis, urticaria, atopic dermatitis, polymyositis, chronic active hepatitis, emergency bowel syndrome, atrophic gastritis, systemic lupus erythematosus, myasthenia gravis, cerebral spinal cord multiple Sclerosis, Guillain-Barre Syndrome, intracranial granuloma, pancreatitis, myocarditis, and infectious inflammation
  • the abnormal expression of the tyrosine-specific protein phosphatase 12 of the present invention will also produce certain hereditary, blood diseases, and immune system diseases.
  • 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 tumors, embryonic development disorders, growth and development disorders, inflammation, certain heredity, blood Sexually transmitted diseases and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) tyrosine-specific protein phosphatase 12.
  • Agonists increase tyrosine-specific protein phosphatase 12 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing tyrosine-specific protein phosphatase 12 can be cultured with labeled tyrosine-specific protein phosphatase 12 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of tyrosine-specific protein phosphatase 12 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of tyrosine-specific protein phosphatase 12 can bind to tyrosine-specific protein phosphatase 12 and eliminate its function, or inhibit the production of the polypeptide, or interact with the polypeptide The active site binding prevents the polypeptide from performing biological functions.
  • tyrosine-specific protein phosphatase 12 can be added to the bioanalytical assay by measuring the effect of the compound on the interaction between tyrosine-specific protein phosphatase 12 and its receptor Determine if the compound is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to tyrosine-specific protein phosphatase 12 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, the tyrosine-specific protein phosphatase 12 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 against a tyrosine-specific protein phosphatase 12 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting tyrosine-specific protein phosphatase 12 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to 'S adjuvant and so on.
  • Techniques for preparing monoclonal antibodies to tyrosine-specific protein phosphatase 12 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta- Cell hybridoma technology, EBV-hybridoma technology, etc.
  • An inlay antibody combining a human constant region and a non-human variable region can be produced using existing techniques (Morr i son e t al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against tyrosine-specific protein phosphatase 12.
  • Antibodies against tyrosine-specific protein phosphatase 12 can be used in immunohistochemistry to detect tyrosine-specific protein phosphatase 12 in biopsy specimens.
  • Monoclonal antibodies that bind to tyrosine-specific protein phosphatase 12 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.
  • tyrosine-specific protein phosphatase 12 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 disulfide exchange.
  • This hybrid antibody can be used to kill tyrosine-specific protein phosphatase 12 Positive cells.
  • the antibodies of the present invention can be used to treat or prevent tyrosine-specific protein phosphatase 12. Disease. Administration of appropriate doses of antibodies can stimulate or block the production or activity of tyrosine-specific protein phosphatase 12.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of tyrosine-specific protein phosphatase 12 levels.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of tyrosine-specific protein phosphatase 12 detected in the test can be used to explain the importance of tyrosine-specific protein phosphatase 12 in various diseases and to diagnose tyrosine-specific protein phosphatase 12 A working disease.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding tyrosine-specific protein phosphatase 12 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 non-expression or abnormal / inactive expression of tyrosine-specific protein phosphatase 12.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated tyrosine-specific protein phosphatase 12 to inhibit endogenous tyrosine-specific protein phosphatase 12 activity.
  • a variant tyrosine-specific protein phosphatase 12 may be a shortened tyrosine-specific protein phosphatase 12 lacking a signaling domain.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of tyrosine-specific protein phosphatase 12.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding a tyrosine-specific protein phosphatase 12 into a cell.
  • a polynucleotide encoding a tyrosine-specific protein phosphatase 12 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit tyrosine-specific protein phosphatase 12 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA and D and ribozymes can be obtained by any existing RNA or DNA synthesis technology. For example, the technology for the synthesis of oligonucleotides by solid-phase phosphate amide chemical synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector's RNA Downstream of the 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 tyrosine-specific protein phosphatase 12 can be used for the diagnosis of diseases related to tyrosine-specific protein phosphatase 12.
  • a polynucleotide encoding tyrosine-specific protein phosphatase 12 can be used to detect the expression of tyrosine-specific protein phosphatase 12 or the abnormal expression of tyrosine-specific protein phosphatase 12 in a disease state.
  • the D sequence encoding tyrosine-specific protein phosphatase 12 can be used to hybridize biopsy specimens to determine the expression of tyrosine-specific protein phosphatase 12.
  • Hybridization techniques include Sou thern blotting, or thern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available. 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 known as
  • RNA-polymerase chain reaction in vitro amplification with tyrosine-specific protein phosphatase 12 specific primers can also detect tyrosine-specific protein phosphatase 12 transcription products.
  • Detection of mutations in the tyrosine-specific protein phosphatase 12 gene can also be used to diagnose tyrosine-specific protein phosphatase 12-related diseases.
  • Forms of tyrosine-specific protein phosphatase 12 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type tyrosine-specific protein phosphatase 12 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. 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 DNA sequences on a chromosome.
  • a PCR 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 heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosome localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybrids to construct chromosome-specific cDNA library.
  • Fluorescent in situ hybridization (FISH) of cD clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Tyrosine-specific protein phosphatase 12 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of tyrosine-specific protein phosphatase 12 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. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA forms CDM by reverse transcription.
  • the Smart cDNA Cloning Kit purchased from Clontech was used to insert the cDM fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5cc. The bacteria formed a cDNA library.
  • the Dye terminate cycle reaction sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public D sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0465B03 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the sequence of the tyrosine-specific protein phosphatase 12 of the present invention and the protein sequence encoded by the tyrosine-specific protein phosphatase 12 of the present invention were used in a profile scan program (Basic local alignment search tool) in GCG [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], performing domain analysis in databases such as Prote.
  • the tyrosine-specific protein phosphatase 12 of the present invention is homologous with a domain tyrosine-specific protein phosphatase characteristic sequence fragment at 22-85, and the homology result is shown in FIG. 1.
  • the homology rate is 0.12. Is 7.28; the threshold is 7.26.
  • Example 3 Cloning of the gene encoding tyrosine-specific protein phosphatase 12 by RT-PCR method.
  • Total fetal brain cells were used as a template, and oligo-dT was used as a primer for reverse transcription to synthesize cDNA.
  • primers for PCR amplification Using the following primers for PCR amplification:
  • Primerl 5-TCCTGGGCCAGCCATGGAAGGAAA-3 '(SEQ ID NO: 3)
  • Primer2 5-CCCCCCCTAAGCTTCGTCTTCTCG-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions 50mmol / L KC1, 10mraol / L Tris-HCl, pH8.5, 1.5raraol / L MgCl 2 , 200 ⁇ ol / L dNTP, lOpmol primer, 1U Taq DNA in 50 ⁇ 1 reaction volume Polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min solicitAt the same time, set ⁇ -actin as a positive control and template blank as a negative control at the time of RT-PCR.
  • Amplification products were purified using QIAGEN's kit and connected with TA cloning kit To the pCR vector by Unvitrogen).
  • the DNA sequence analysis results show that the D sequence of the PCR product is exactly the same as the 1 to 2136bp shown in SEQ ID NO: 1.
  • Example 4 Northern Typing Analysis of Tyrosine-Specific Protein Phosphatase 12 Gene expression was extracted by a one-step method of total RM [Anal. Biochem 1987, 162,156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • RNA containing 20mM 3- (N- morpholino) propanesulfonic acid (pH 7 .0) electrophoresis on a 1.2% agarose gel of -5raM sodium acetate-IraM EDTA-2.2M formaldehyde Is then transferred to a nitrocellulose membrane.
  • the DNA probes used for PCR amplification of FIG. 1 specific tyrosine Sequence of protein phosphatase 12 coding region (807bp to 1130bp).
  • 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with nitrocellulose membrane to which RNA was transferred in a solution at 42 ° C overnight, This solution contains 501 ⁇ 2 formamide-25ra KH 2 P0 4 (pH7.4)-5 xSSC-5 xDenhardt, s solution and 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, the filter membrane is at 1 X SSC-0.1% Wash the SDS at 55 ° C for 30 min. Then, analyze and quantify with Phosphor Imager.
  • Example 5 In vitro expression, isolation and purification of recombinant tyrosine-specific protein phosphatase 12 according to SEQ ID NO: 1 and Figure 1 A pair of specific amplification primers were designed for the coding region sequence, and the sequence is as follows:
  • Priraer3 5-CATGCTAGCATGCCACATGCAGGATTTAAAAAA-3 '(Seq ID No: 5)
  • Primer4 5'-CATGGATCCTTAACTTCTCCGTGACTTCATTTT-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Nhel and BamHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are respectively followed by Nhel and BamHI restriction sites corresponding to the selective endonucleases on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Enzyme site.
  • the PBS-0465B03 plasmid of the target gene was used as a template for PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0465B03 plasmid, primers Primer-3 and Primer-4 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. Nhel and BamHI were used to double-digest the amplified product and plasmid pET_28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E.
  • coli DH5a by the calcium chloride method, and cultured overnight on LB plates containing kanamycin (final concentration 3 ( ⁇ g / ml)), and positive clones were selected by colony PCR method and sequenced. Positive sequence correct clone (PET-0465B03) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method. Cultured in LB liquid containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1) In the medium, the host bacteria BL21 (pET-0465B03) was cultured at 37 ° C.
  • a peptide synthesizer (product of PE company) was used to synthesize the following tyrosine-specific protein phosphatase 12-specific peptides:
  • NH 2 -Met-Pro-His-Ala-Gly-Phe-Lys-Lys-Lys-Ser-Pro-Phe-Phe-Phe-Leu-COOH SEQ ID NO: 7
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin for methods, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 1 ⁇ 2 g of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit 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.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to tyrosine-specific protein phosphatase 12.
  • the probes can be used to hybridize to the genome or CDM library of normal tissue or pathological tissue from different sources.
  • the probe may further be used to detect the polynucleotide sequence of the present invention or a homologous polynucleotide sequence thereof in normal tissue or Whether the expression in pathological tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • 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
  • 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, the primary probe should not be used;
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt) 5 '-TGCCACATGCAGGATTTAAAAAAAAGTCTCCCTTCTTTTTC -3' (SEQ ID NO: 8)
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutation sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41N:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • Two NC membranes are required for each probe, so that it can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (10xDenhardt's; 6xSSC, 0.1 mg / ml CT DM (calf thymus DM)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution 10xDenhardt's; 6xSSC, 0.1 mg / ml CT DM (calf thymus DM)
  • Gene microarray or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass. , 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 Targeting DNA for gene chip technology for high-throughput research on new gene functions; finding and screening new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases. The specific method steps have been reported in the literature.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution 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 mRNA was extracted from normal liver and liver cancer in one step, and mRNA was purified with Oligotex mRM Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5-Amino-propargy 1-2 ' -deoxyur i dine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech company) labeled mRNA of normal liver tissue, using a fluorescent reagent Cy5dUTP (5-Amino-propargyl-2 , -deoxyuridine 5'-tr iphate Coupled to Cy5 f luorescent dye (purchased from Amersham Phamacia Biotech) was used to label liver cancer tissue mRNA, and the probe was prepared after purification. For specific steps and methods, see

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Abstract

L'invention concerne un nouveau polypeptide, une protéine tyrosine phosphatase 12, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine tyrosine phosphatase 12.
PCT/CN2000/000709 1999-12-27 2000-12-25 Nouveau polypeptide, proteine tyrosine phosphatase 12, et polynucleotide codant pour ce polypeptide WO2001048160A1 (fr)

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CN99125389A CN1301828A (zh) 1999-12-27 1999-12-27 一种新的多肽——酪氨酸特异性蛋白磷酸酶12和编码这种多肽的多核苷酸
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994018309A1 (fr) * 1993-02-10 1994-08-18 New York University Nouvelle phosphotyrosine phosphatase-gamma de type recepteur
WO1994018308A1 (fr) * 1993-02-10 1994-08-18 New York University NOUVELLE PHOSPHOTYROSINE PHOSPHATASE-β DE TYPE RECEPTEUR
WO1997006262A1 (fr) * 1995-08-10 1997-02-20 Incyte Pharmaceuticals, Inc. Tyrosine phosphatase proteique derivee de basophiles / mastocytes humains

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994018309A1 (fr) * 1993-02-10 1994-08-18 New York University Nouvelle phosphotyrosine phosphatase-gamma de type recepteur
WO1994018308A1 (fr) * 1993-02-10 1994-08-18 New York University NOUVELLE PHOSPHOTYROSINE PHOSPHATASE-β DE TYPE RECEPTEUR
WO1997006262A1 (fr) * 1995-08-10 1997-02-20 Incyte Pharmaceuticals, Inc. Tyrosine phosphatase proteique derivee de basophiles / mastocytes humains

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LOFTUS B.J. ET AL.: "Genome duplications and other features in 12 Mb of DNA sequence from human chromosome 16p and 16q", GENOMICS, vol. 60, no. 3, 1999, pages 295 - 308 *
SULSTON J.E. AND WATERSTON R.: "Toward a complete human genome sequence", GENOME RES., vol. 8, no. 11, 1998, pages 1097 - 1108 *

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