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WO2001030841A1 - Nouveau polypeptide, proteine 60 a doigt de zinc, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine 60 a doigt de zinc, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001030841A1
WO2001030841A1 PCT/CN2000/000394 CN0000394W WO0130841A1 WO 2001030841 A1 WO2001030841 A1 WO 2001030841A1 CN 0000394 W CN0000394 W CN 0000394W WO 0130841 A1 WO0130841 A1 WO 0130841A1
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Prior art keywords
polypeptide
zinc finger
polynucleotide
finger protein
seq
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bioroad Gene Development Ltd
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Shanghai Bioroad Gene Development Ltd
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Priority to AU12654/01A priority Critical patent/AU1265401A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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, zinc finger protein 60, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide.
  • Zinc finger protein is a DNA-binding protein that was first discovered in the amino acid sequence of the protein 5S rRNA gene transcription factor TF III A mediated by Xenopus RNA polymerase II. Since then, it has been found that zinc finger protein is expressed in various tissues of different organisms. If the fly cell division gene Kruppel is expressed, in humans these tissues include the nervous system such as the brain, hematopoietic cells, epidermal tissues, various tissues related to secretion and absorption, and Tumors and tissues related to immortalized cell lines. It is estimated that more than 1% of the zinc finger protein genes are in the human genome (Bellefroid et al., 1989; Pellegrino and Berg, 1991).
  • Zinc finger proteins mainly have the following zinc finger structures: C2H2 configuration, C2C2 configuration, C2HC configuration, C2HC4C configuration, C3H configuration, and C3HC4 configuration (Dai KS et al., 1998).
  • the zinc finger protein genes containing the C2H2 configuration constitute the largest family of genes in the human genome (Berker et al., 1995).
  • the C2H2 configuration of zinc finger protein is as follows:
  • the typical zinc finger structure is composed of about 28 amino acid residues in length, which is Cys-Cys ...
  • the zinc finger unit contains a relatively conserved sequence: Cys- X 2 _ 4 -Cys- X 3 -Phe- X 5 -Leu-X 2 -His-X 2 _ 4 -His (Bray, p et al., 1993).
  • Cys- X 2 _ 4 -Cys- X 3 -Phe- X 5 -Leu-X 2 -His-X 2 _ 4 -His (Bray, p et al., 1993).
  • two conservative Cys participate in the formation of an antiparallel P-fold
  • two conservative His participates in the formation of an alpha helix.
  • These two structures combine with the core Z ⁇ atom to form a compact spherical region (Pabo, CA and Sauer, R. T, 1992).
  • Such zinc finger proteins often have 1 to 37 zinc finger structures (Rhodes, D. and Klug, 1993).
  • the C2H2 conformation zinc finger protein that connects two adjacent Kruppels often, but not always, contains the conserved specific amino acid sequence TGEKP (Bray, p. Et al., 1993).
  • TGEKP conserved specific amino acid sequence
  • C2H2 conformation zinc finger proteins regulate gene transcription by binding to specific sequences on DNA and / or RNA (Engelke, D. R et al., 1980; Kadonaga, J. T et al., 1987; Stanojevic, D. et al ., 1989), some C2H2 configuration zinc finger proteins regulate gene transcription and expression through interaction with other zinc finger proteins (Lee, JS. Et al., 1993; Seto, ⁇ . Et al., 1993; Maheswaran. S.
  • the human gene protein of the present invention has 99% homology with human zinc finger protein C2H2-25 (homologous protein number U38904). After isolation and accurate mapping, the polypeptide has a zinc finger domain in the C2H2 configuration and belongs to The human zinc finger protein Kruppel gene family was named human zinc finger protein 60, and it was inferred that its domain was similar to the Kruppel family gene domain and had similar biological functions.
  • Human zinc finger proteins Kruppel gene family contains human zinc finger proteins 131-140, 142, 143, 148, 151, 154, 155 (Tommerup and Vissing, Genomics 27: 259-264, 1995).
  • Human zinc finger proteins 138, 139, and 143 are related to Williams syndrome, cracked hands and feet, and Bayesian syndrome respectively;
  • human zinc finger proteins 132, 134, 135, 137, 154, and 155 can cause solid tumors such as Thyroid adenoma
  • human zinc finger protein 151 is closely related to neuroblastoma, colon cancer, breast cancer or other tumors;
  • human zinc finger protein 139, 148, 151 can cause hematological malignancies such as leukemia, non-Hodgkin's lymph Tumors (Genomics 27: 259-264, 1995).
  • Human zinc finger protein C2H2-25 also belongs to the human zinc finger protein Kruppel gene family (Becker et al. Mammalian Genome 8: 287-289, 1997), which is a C2H2 configuration zinc finger protein regulated by human development. PCR analysis of hybridization plates for rodent sarcoma cells was located on chromosome 19. Becker et al. Isolated 118 kinds of zinc finger proteins of C2H2 configuration from human hippocampus cDNA library, including human zinc finger protein C2H2-25. A large number of different C2H2 configuration zinc finger proteins were expressed in human brain. The expression levels of these genes in the nervous system and their importance are shown (Kev in G. Becker et al., 1995).
  • human zinc finger protein 60 is related to the following but not limited to the following diseases: solid tumors such as thyroid adenoma, uterine fibroids, neurological diseases such as extrapyramidal dysfunction, Parkinson's syndrome, ataxia, neuroblastoma Glioblastomas, hematological malignancies such as leukemia, non-Hodgkin's lymphoma, Wi ll iams syndrome, cleft foot and cleft foot disease, Bezier syndrome, other tumors such as neuroblastoma, colon cancer , Breast cancer and more.
  • solid tumors such as thyroid adenoma, uterine fibroids
  • neurological diseases such as extrapyramidal dysfunction, Parkinson's syndrome, ataxia
  • neuroblastoma Glioblastomas hematological malignancies such as leukemia, non-Hodgkin's lymphoma, Wi ll iams syndrome, cleft foot and cleft foot disease, Bezier syndrome
  • other tumors such as neuroblastoma
  • the discovery of a polynucleotide encoding a zinc finger protein and its encoded zinc finger protein provides a method for studying the physiological and biochemical processes of cell differentiation and proliferation under normal and pathological conditions, as well as for diagnosis, treatment and Disorders caused by cell differentiation and proliferation disorders, including cancer, provide a new approach.
  • 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 zinc finger protein 60.
  • Another object of the invention is to provide a genetically engineered host cell containing a polynucleotide encoding a zinc finger protein 60.
  • Another object of the present invention is to provide a method for producing zinc finger protein 60.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, zinc finger protein 60.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, zinc finger protein 60.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of zinc finger protein 60. Summary of invention
  • a novel isolated zinc finger protein 60 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or its activity Fragments, or their active derivatives, analogs.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 99 nucleotides with a nucleotide sequence selected from the group consisting of % Phase Identical: (a) a polynucleotide encoding the aforementioned zinc finger protein 60; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 241-1875 in SEQ ID NO: 1; and (b) a sequence having 1-2458 in SEQ ID NO: 1 Sequence of bits.
  • Fig. 1 is a comparison diagram of amino acid sequence homology of zinc finger protein 60 of the present invention and human zinc finger protein C2H2-25.
  • the upper sequence is zinc finger protein 60
  • the lower sequence is human zinc finger protein C2H2-25.
  • Identical amino acids are represented by single character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated zinc finger protein 60.
  • 60kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • 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 zinc finger protein 60 means that zinc finger protein 60 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify zinc finger protein 60 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the zinc finger protein 60 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, the terminus protein 60, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be obtained from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammals) using recombinant technology. Cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of zinc finger protein 60.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the zinc finger protein 60 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) 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 substitution The amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ ) Such a polypeptide sequence in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an 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 protease sequence) As set forth herein, such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 2458 bases in length and its open reading frame (241-1875) encodes 544 amino acids.
  • the polypeptide has 99% homology with the human zinc finger protein C2H2-25, and it can be deduced that the zinc finger protein 60 has a similar structure and function to the human zinc finger protein C2H2-25.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include C DNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • DM can be coded or non-coded.
  • 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.
  • the term "polynucleotide encoding a polypeptide" is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) added during hybridization) Use a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 ° /.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding zinc finger protein 60.
  • 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 zinc finger protein 60 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 CDM 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 DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences. Isolate cDNA of interest
  • the standard method is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • kits are also commercially available (Qiagene).
  • 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 loss of marker gene function; (3) determination of the level of zinc finger protein 60 transcripts; (4) by Immunological techniques or assays for biological activity to detect gene-expressed protein products. 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 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).
  • the protein product of the zinc finger protein 60 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be 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 a polynucleotide of the present invention, and a vector or a direct use of the vector of the present invention.
  • the polynucleotide sequence encoding the zinc finger protein 60 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.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.
  • DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a zinc finger protein 60 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS, or Bowes s melanoma cells, etc. .
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be harvested after exponential growth phase, with (Treatment 1 2 ⁇ , with steps well known in the art.
  • Alternative is MgC l 2
  • 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 lipid Body packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant zinc finger protein 60 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immune diseases.
  • the human zinc finger protein 60 of the present invention is involved in gene transcription regulation, gene expression, regulation of RNA metabolism and may affect staining Abnormal expression in the functional aspects of qualitative packaging will cause various developmental disorders, various tumors, certain nervous system diseases, genetic diseases, blood diseases, endocrine system diseases, and immune system diseases.
  • the human zinc finger protein 60 of the present invention can be used to treat or prevent diseases caused by abnormal expression of this protein.
  • diseases include, but are not limited to, developmental disorders such as extrapyramidal dysfunction, teratosis, Wi lli ams syndrome, Al ag ille syndrome, cracked hands and feet disease, and Bayesian syndrome, etc .; various Carcinogenesis of tissues, such as leukemia, lymphoma, malignant histiocytosis, melanoma, sarcoma, myeloma, teratoma, etc., adrenal cancer, bladder cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, uterine cancer, gallbladder Cancer, liver cancer, lung cancer, thyroid tumors, uterine fibroids, thymic tumors, etc .; certain genetic diseases; neurological diseases, such as Parkinson's syndrome, ataxia, neurocytoma, glioblastoma, etc .; endocrine system diseases , Such as endocrine aden
  • the zinc finger protein 60 of the present invention is likely to play an important role in human brain development and function formation. Its gene transcription regulation, gene expression, regulation of RNA metabolism, and functions that may affect chromatin packaging are normal in the physiological functions of the nervous system. Coordinated activities of the extrapyramidal system and pathological functions, such as abnormalities caused by abnormal extrapyramidal development and tumors, will be applied.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) zinc finger protein 60.
  • Agonists increase zinc finger protein 60 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 zinc finger protein 60 can be cultured together with labeled zinc finger protein 60 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of zinc finger protein 60 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of zinc finger protein 60 can bind to zinc finger protein 60 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • screening compounds as antagonists zinc finger protein 60 can be added to a bioanalytical assay to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between zinc finger protein 60 and its receptor.
  • 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 zinc finger protein 60 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 60 molecules of zinc finger protein should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the zinc finger protein 60 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 direct injection of zinc finger protein 60 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 Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies against zinc finger protein 60 include, but are not limited to, hybridoma technology (Kohler and Milstei. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma Technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrie et al, PNAS, 1 985, 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 zinc finger protein 60.
  • Anti-zinc finger protein 60 antibodies can be used in immunohistochemical techniques to detect zinc finger protein 60 in biopsy specimens.
  • Monoclonal antibodies that bind to zinc finger protein 60 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.
  • zinc finger protein 60 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 zinc finger protein 60 positive cells.
  • the antibodies in the present invention can be used to treat or prevent diseases related to zinc finger protein 60.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of zinc finger protein 60.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of zinc finger protein 60 levels. These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of zinc finger protein 60 detected in the test can be used to explain the importance of zinc finger protein 60 in various diseases and to diagnose diseases where zinc finger protein 60 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.
  • the polynucleotide encoding zinc finger protein 60 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 zinc finger protein 60.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant zinc finger protein 60 to inhibit endogenous zinc finger protein 60 activity.
  • a variant zinc finger protein 60 may be a shortened zinc finger protein 60 lacking a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of zinc finger protein 60.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the polynucleotide encoding zinc finger protein 60 into cells.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding a zinc finger protein 60 can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding zinc finger protein 60 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 zinc finger protein 60 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of DM sequences encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding zinc finger protein 60 can be used for the diagnosis of diseases related to zinc finger protein 60.
  • a polynucleotide encoding zinc finger protein 60 can be used to detect the expression of zinc finger protein 60 or the abnormal expression of zinc finger protein 60 in a disease state.
  • the DNA sequence encoding zinc finger protein 60 can be used to hybridize biopsy specimens to determine the expression of zinc finger protein 60.
  • 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 polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Zinc finger protein 60 specific primers can also be used to detect the transcription products of zinc finger protein 60 by R-polymerase chain reaction (RT-PCR) in vitro amplification.
  • RT-PCR R-polymerase chain reaction
  • Zinc finger protein 60 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild type zinc finger protein 60 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the 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, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome locations. 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 heterozygous cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDM clones and metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckus ck, Mende l ian Inher i tance in Man (available online with Johns Hopk ins Un ivers it ty Welch Medica l L ibrary). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDM or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all of the affected individuals and the mutation is not 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 the chromosome, 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 disease-related chromosomal region 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.
  • Zinc finger protein 60 is administered in an amount effective to treat and / or prevent a particular indication.
  • the amount and range of zinc finger protein 60 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.
  • Total RM of human fetal brain was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRM was isolated from total UNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragment into the multicloning site of the pBSK (+) vector (Clontech) to transform DH5 ct. The bacteria formed a cDNA library.
  • Dye terminate cycle reaction sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the sequence of the zinc finger protein 60 of the present invention and the protein sequence encoded by the zinc finger protein 60 were subjected to a Blast program (Basic local alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], in Genbank, Swissport and other databases perform homology search.
  • the gene with the highest homology to the zinc finger protein 60 of the present invention is a known human zinc finger protein C2H2-25, and its encoded protein has an accession number of 1138904 in Genbank.
  • the protein homology results are shown in Figure 1. The two are highly homologous, with 99% identity; 99% similarity.
  • Example 3 Cloning of a gene encoding zinc finger protein 60 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.
  • PCR amplification was performed with the following primers:
  • Primerl 5'- GCAGGGCGGGACTTCTGGCGTCTTC-3 '(SEQ ID NO: 3)
  • Primer2 5'- TTAAATAATAATGCTTTTATTATTG-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting from the 1st lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions 50 mmol / L of KC1, 10 mmol / L Tris-Cl, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer, 1U in a reaction volume of 50 ⁇ 1 Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min.
  • ⁇ -actin 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.
  • DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 2458bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of zinc finger protein 60 gene expression
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) and an RNA-transferred nitrocellulose membrane were placed in a solution at 42 ° C. C hybridization overnight, the solution contains 50% formamide-25 mM KH 2 P0 4 (pH 7.4)-5 x SSC-5 x Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant zinc finger protein 60
  • Primer3 5'- CCCCATATGATGGCGACCGCACTGAGGTACCCGG -3, (Seq ID No: 5)
  • Primer4 5,-CCCGGATCCCTAAGGCTTTTCTCAATGTGAAC -3 '(Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28b ( +) (Novagen, Cat. No. 69865. 3) selective endonuclease site.
  • the pBS-0085d03 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0085d03 plasmid, 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. Ndel 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 DH5cc using the calcium chloride method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following zinc finger protein 60-specific peptides:
  • the peptide was coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • the method please refer to Avrameas, et al. I hidden chemi s try, 1969; 6: 43. Rabbits were immunized with 1 ⁇ 2 g of the hemocyanin polypeptide complex and complete Freund's adjuvant. After 15 days, the immune response was boosted once with hemocyanin polypeptide complex and incomplete Freund's adjuvant. Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate
  • the antibody titer in rabbit serum was measured by ELISA.
  • Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Seph a rose 4B column, and the anti-peptide antibody was separated from total i gG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to zinc finger protein 60.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine 60 à doigt de zinc, 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 60 à doigt de zinc.
PCT/CN2000/000394 1999-10-27 2000-10-27 Nouveau polypeptide, proteine 60 a doigt de zinc, et polynucleotide codant pour ce polypeptide Ceased WO2001030841A1 (fr)

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AU12654/01A AU1265401A (en) 1999-10-27 2000-10-27 A new polypeptide-zinc finger protein 60 and the polynucleotide encoding it

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CN99119863.8 1999-10-27
CN99119863A CN1303864A (zh) 1999-10-27 1999-10-27 一种新的多肽——锌指蛋白60和编码这种多肽的多核苷酸

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 29 June 1998 (1998-06-29), Database accession no. AC003682 *
HUM. MOL. GENET., vol. 4, no. 4, 1995, pages 685 - 691 *

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